extfieldsofstudy: Medicine Biology

s2fieldsofstudy: Biology Chemistry

text:
Specific Interaction with Transcription Factor IIA and Localization of the Mammalian TATA-binding Protein-like Protein (TLP/TRF2/TLF)*

TBP-like protein (TLP) is structurally similar to the TATA-binding protein (TBP) and is thought to have a transcriptional regulation function. Although TLP has been found to form a complex with transcription factor IIA (TFIIA), the in vivo functions of TFIIA for TLP are not clear. In this study, we analyzed the interaction between TLP and TFIIA. We determined the biophysical properties for the interaction of TLP with TFIIA. Dissociation constants of TFIIA versus TLP and TFIIA versus TBP were 1.5 and 10 nm, respectively. Moreover, the dissociation rate constant of TLP and TFIIA (1.2 × 10–4/m·s was significantly lower than that of TBP (2.1 × 10–3/m·s). These results indicate that TLP has a higher affinity to TFIIA than does TBP and that the TLP-TFIIA complex is much more stable than is the TBP-TFIIA complex. We found that TLP forms a dimer and a trimer and that these multimerizations are inhibited by TFIIA. Moreover, TLP mutimers were more stable than a TBP dimer. We determined the amounts of TLPs in the nucleus and cytoplasm of NIH3T3 cells and found that the molecular number of TLP in the nucleus was only 4% of that in the cytoplasm. Immunostaining of cells also revealed cytoplasmic localization of TLP. We established cells that stably express mutant TLP lacking TFIIA binding ability and identified the amino acids of TLP required for TFIIA binding (Ala-32, Leu-33, Asn-37, Arg-52, Lys-53, Lys-78, and Arg-86). Interestingly, the level of TFIIA binding defective mutant TLPs in the nucleus was much higher than that of the wild-type TLP and TFIIA-interactable mutant TLPs. Immunostaining analyses showed consistent results. These results suggest that the TFIIA binding ability of TLP is required for characteristic cytoplasmic localization of TLP. TFIIA may regulate the intracellular molecular state and the function of TLP through its property of binding to TLP.

TATA-binding protein (TBP) 1 plays a central role in all three classes of transcription (1) and is thought to be a unique universal transcription factor (2). In the case of RNA polymerase II genes, TBP binds to the TATA-box element found in genes transcribed by RNA polymerase II and then triggers the preinitiation complex assembly (3). TBP directly binds to the general transcription factor IIA (TFIIA). TFIIA facilitates binding of TBP to DNA via several distinct mechanisms and thus activates TBP-dependent transcription of class II genes. TFIIA promotes stabilization of TBP on the TATA box by inducing a conformational change of TBP (4,5). Furthermore, TFIIA acts as an anti-repressor against co-repressors that inhibit DNA binding and TFIIB binding of TBP (6,7). TBP or TFIID forms a transcription-inactive homodimer in solution, and dissociation of a TBP dimer is induced by direct interaction of TFIIA (8,9,11,12). Those processes concerning TFIIA functions are further influenced by distinct classes of TFIIA-interactive regulatory factors. Because some transcriptional activations are dependent on TFIIA/TBP and TFIIA/activator interactions (6,(13)(14)(15)(16)(17)(18)(19)(20), TFIIA is thought to be responsible for promoter selectivity for the RNA polymerase II transcription machinery via interactions with both regulatory factors and TBP.
Higher eukaryotes have TBP family genes. A TBP-related factor involved in neurogenesis, TBP-related factor 1 (TRF1), has been identified in Drosophila (21). TRF1 forms a multiprotein complex with novel proteins (22), binds to promoter DNA bearing a non-traditional TATA box, and directs embryogenesis-related expression of the tudor gene (23). A recent study has also demonstrated that TRF1 rather than TBP formed a complex with TAFs of TFIIIB and directs the expression of class III genes (24). Thus, TRF1 is thought to behave as a promoterselective factor like bacterial factors (25), although it is still a mystery why TRF1 has been identified only in Drosophila.
At least one other TBP-like protein (TLP) exists in metazoa (26 -28). It has been shown that defects in functional TLP in several animals resulted in developmental abnormalities accompanying decreased expression levels of particular genes (29 -34). Recently, we reported that a promoter-bound TLP and free TLP functioned as transcriptional activators (35,36) and that TLP affected levels of transcription of some cell cyclerelated genes in chicken cells (37). The PCNA gene was identified as a native target of TRF2/TLP in Drosophila (38). These findings suggest that TLP plays a role as a positive regulatory factor for some genes in a different way from that of TBP. It has been demonstrated that TLP is associated with TFIIA and TFIIB but does not bind to TATA-DNA in solution (39,40) and that TLP purified from nuclear extracts of HeLa cells is associated with TFIIA (41). Although TLP is not able to be substituted with TBP in in vitro transcriptional activation, the addition of TLP into in vitro transcription and in vivo reporter gene assay systems inhibited TATA box-dependent RNA polymerase II transcription by a model of TLP-mediated titration out of TFIIA (41,42). However, the molecular mechanism underlying the role of vertebrate TLP in transcriptional activation has not been elucidated.
To elucidate the function of TLP distinct from that of TBP, molecules that have specific affinity to TLP and affect localization and interaction with the general transcription machinery of TLP must be identified. In this regard, the fact that TLP is transiently translocated to mouse spermatocytes and stressed vertebrate cells is interesting (37,43). It is known that TLP, but not TBP, is stably associated with TFIIA in cells. Hence, it is mostly probable that, among the TLP-interactable proteins, TFIIA is one critical regulator that gives TLP unique properties distinct from those of TBP. In this study, we investigated in detail the biochemical properties of interaction between TLP and TFIIA in vivo and in vitro. We found that TLP has a high affinity to TFIIA and that the pre-formed TFIIA-TLP complex is more stable than is the TFIIA-TBP complex. We also found that TLP forms TFIIA-sensitive dimers and trimers. Last, we found that mutant TLPs lacking TFIIA binding ability do not exhibit a clear cytoplasmic distribution pattern. These results imply that TFIIA negatively regulates TLP function as a potential nuclear transcription factor by inducing cytoplasmic localization of TLP. We discuss in this paper how TFIIA works for dynamism and function of TLP in a cell.

Cell Culture and Establishment of Mouse TLP-expressing Cells-
The open reading frame of mouse TLP (mTLP) with FLAG and oligohistidine tags at its amino terminus (F/H-mTLP) was subcloned into the pCI-neo vector (Promega). Mutant TLPs (Fig. 5A) were made by substitution of one or two residues of F/H-mTLP/pCI-neo by a standard procedure. NIH3T3 cells were maintained in a low-glucose Dulbecco's modified Eagle's medium (Sigma) and transfected with F/H-mTLP and mutant F/H-mTLPs in the pCI-neo vector by using LipofectAMINE PLUS reagent (Invitrogen) according to the manufacturer's instructions. The stable transformants were isolated by a standard procedure in a medium containing 400 g/ml G418 (Carbiochem).
Preparation of Extracts and Immunoprecipitation-Nuclear extracts, cytoplasmic extracts, and whole cell extracts (WCE) were prepared as described previously (44). F/H-protein in WCE prepared by the method of Dignam (44) was precipitated by M2-agarose (Sigma). Bound materials were eluted by an addition of 0.25 mg/ml FLAG peptide.
Purification of Bacterially Expressed Recombinant Proteins-The open reading frames of F/H-mTLP and F/H-hTBP were subcloned into the pET3a vector (Novagen). The open reading frames of hTFIIA␣␤ and histidine-tagged hTFIIA␥ were subcloned into the pET16b vector (Novagen). The open reading frames of the human TFIIA␣␤, mouse TLP, and human TBP were subcloned into the pGEX4T-1 vector (Amersham Biosciences). All constructs were introduced into Escherichia coli BL21(DE3)pLysS strain (Stratagene).
Expression of F/H-proteins in E. coli cells were induced according to the manufacturer's instructions. Soluble F/H-proteins were purified by nickel-nitrilotriacetic acid-agarose (Qiagen) (45). F/H-hTBP in eluates from nickel-agarose was further purified by Mono-Q HR5/5 column (Amersham Biosciences) followed by Mono-S HR5/5 column (Amersham Biosciences). F/H-mTLP in eluates from nickel-agarose was further purified by Mono-S HR5/5 column (Amersham Biosciences). Recombinant hTFIIA␣␤ and oligohistidine-tagged hTFIIA␥ (His-hTFIIA␥) were purified under a denaturing condition and renatured as described previously (46). Expressions of recombinant GST-hTBP, GST-mTLP, and GST-hTFIIA␣␤ were induced by the same procedure as that used for F/H fusion proteins. GST fusion proteins were purified by using glutathione-Sepharose beads (Amersham Biosciences) according to the manufacturer's instructions. Eluates containing GST-hTFIIA␣␤ were subjected to SDS-PAGE and renatured as previously described (46). Eluates of GST-hTBP and GST-mTLP were loaded onto a Superdex-200 column to remove glutathione. All recombinant proteins were finally dialyzed against a buffer used for each assay.
Western Blotting, Immunostaining, and Antibodies-SDS-PAGE and Western blotting were performed by standard procedures (45). Horseradish peroxidase-conjugated protein A (Amersham Biosciences), horseradish peroxidase-conjugated anti-mouse IgG (New England Biolabs), or horseradish peroxidase-conjugated anti-rabbit IgG (Cell Signaling Technology) and an ECL-Plus system (Amersham Biosciences) or alkaline phosphatase-conjugated anti-rabbit IgG and a 5-bromo-4-chloro-3indolyl phosphate/nitro blue tetrazolium substrate (Promega) were used for Western blotting. Indirect immunofluorescent staining of NIH3T3 cells on coverslips was carried out as described previously (37).
Determination of Molecular Numbers of Proteins-The molecular number in subcellular fractions were determined by Western blotting of the fractions together with concentration-known recombinant proteins as described previously (48).
Surface Plasmon Resonance Analysis-Surface plasmon resonance measurements were performed using a Biacore 2000 (Biacore). All experiments were performed in a running buffer (10 mM Hepes-KOH, 150 mM NaCl, 0.1 mM EDTA, 5 mM MgCl 2 , 1 mM dithiothreitol, 0.05% Tween 20, pH 7.9) at 25°C. Recombinant proteins that were Ͼ95% pure were excessively dialyzed against the running buffer. At first, F/H-mTLP and F/H-hTBP were immobilized on a sensor chip and TFIIA was used as an analyte. However, a specific interaction was not observed because TFIIA non-specifically bound to the matrix of the sensor chip. Moreover, TFIIA could not be immobilized on a sensor chip. Thus, GST-TFIIA␣␤ was indirectly immobilized on a sensor chip as described below.
To capture GST-TFIIA␣␤, anti-GST antibodies (Biacore) were immobilized on the surface of a CM5 sensor chip (Biacore) by the aminecoupling procedure according to the manufacturer's instructions. GST-hTFIIA␣␤ (1000 RU) was bound to the anti-GST antibody on the sensor chip. An equal molar amount of GST (Biacore) (400 RU) against GST-hTFIIA␣␤ was also bound to the anti-GST antibody on another sensor chip. His-hTFIIA␥ was then injected and ϳ250 RU of His-hTFIIA␥ was bound to the GST-hTFIIA␣␤-bound flow cell. Based on molecular masses of GST-hTFIIA␣␤ (68 kDa) and His-hTFIIA␥ (15 kDa), the amount (250 RU) of His-hTFIIA␥ that had bound to GST-hTFIIA␣␤ (1000 RU) indicated that most of the hTFIIA␣␤ bound to hTFIIA␥. Then F/H-hTBP or F/H-mTLP was injected into a flow cell pre-bound with GST or GST-hTFIIA␣␤/␥ complex and washed with running buffer to monitor the dissociation phase at a flow rate of 30 l/min. The surfaces were regenerated with 15 l of 2 M NaCl-containing running buffer. For kinetic analysis, various concentrations of analytes were injected. Each sensorgram of the flow cell immobilized with GST-hTFIIA␣␤ was corrected with that of GST, and kinetic parameters were calculated from hTFIIA-specific responses (49). It was necessary to reduce the concentrations of analytes, and the obtained response was relatively weak because the amount of indirectly immobilized TFIIA as a ligand was only ϳ1300 RUs.
Analysis of Multimerized Proteins and Chemical Cross-linking-The multimerized forms of proteins were analyzed by glycerol density gradient centrifugation (GDGC) as described previously (47) with slight modifications. In this case, the buffer was replaced with that used for BMH (1,6-bismaleimidohexane) cross-linking (BMH buffer). The fractions including sedimented proteins were subjected to SDS-PAGE followed by silver staining and Western blotting. Relative band intensities of recombinant proteins were determined using ImageMaster one-dimensional software (Amersham Biosciences). Chemical cross-linking with BMH (Pierce) and GST pull-down were performed as described previously (12). Double-strand DNA containing a TATA sequence of the adenovirus major late promoter from Ϫ41 to Ϫ15 (50) was synthesized. Double-strand DNA containing a GAGA sequence instead of the TATA sequence was also prepared.

Kinetics of Interactions between TBP and TFIIA and TLP
and TFIIA-Results of previous studies have shown that TLP binds to TFIIA (41). However, because the specificity of the interaction between TLP and TFIIA had not been precisely determined, we investigated the kinetic parameters of TBP-TFIIA and TLP-TFIIA interactions using a Biacore system to find differences between TBP-TFIIA and TLP-TFIIA interactions. As stated under "Experimental Procedures," we indirectly immobilized GST-hTFIIA␣␤ to a sensor chip mediated by an anti-GST antibody. After binding of GST-hTFIIA␣␤ to antibodies on the sensor chip, oligohistidine-tagged hTFIIA␥ (His-hTFIIA␥) was subsequently injected to form a TFIIA␣␤/␥ complex. This complex was not dissociated by a continuous flow under our experimental conditions (data not shown). F/H-hTBP or F/H-mTLP as a ligand was then injected. TLP of various concentrations was applied over a flow cell immobilized with GST-hTFIIA␣␤/␥ or control GST, and typical sensorgrams were obtained (Fig. 1A). The obtained data indicated a specific interaction between TLP and TFIIA. Kinetic parameters were calculated from appropriate parts of the sensorgrams. Residual plots of obtained sensorgrams against fitted curves (Fig. 1, B and C) were less than ϩ2 resonance response units, indicating that the sensorgrams of TLP-TFIIA interaction fit well to a 1:1 binding model. From the ideal curve, the association rate constant (k a ) between TLP and TFIIA was calculated to be 7.66 ϩ 0.02 ϫ 10 4 /M⅐s and the dissociation rate constant (k d ) was calculated to be 1.16 ϩ 0.02 ϫ 10 Ϫ4 /s; thereby, the K D value was calculated to be 1.52 ϫ 10 Ϫ9 M (Fig. 1G). The interaction between TBP and TFIIA was similarly analyzed (Fig. 1, D-F). The k a , k d , and K D values were calculated to be 2.00 Ϯ 0.03 ϫ 10 5 /M⅐s, 2.07 Ϯ 0.02 ϫ 10 Ϫ3 /s, and 1.04 ϫ 10 Ϫ8 M, respectively (Fig. 1G). These data demonstrated that TLP has a one-order higher affinity to TFIIA than does TBP. Notably, it was also found that the k d value of TLP from TFIIA was significantly lower (approximately one-seventeenth) than that of TBP from TFIIA, whereas k a of TBP was slightly higher (ϳ3 times) than that of TLP. This means that a pre-formed TLP-TFIIA complex is much more stable than is a TBP-TFIIA complex.
TFIIA-sensitive Multimer of TLP-One of the unique properties of TBP is its formation of a transcriptionally negative dimer, and it has been shown that TFIIA induces dissociation of the dimer and promotes DNA binding of TBP (9, 10). Thus, we carried out experiments to determine whether TLP has the same nature as that of TBP and clarify the multimerization of TLP. We analyzed the molecular states of recombinant F/H-hTBP and F/H-mTLP by GDGC under a physiological condition. Because, as is well known, TBP is a sticky protein, TLP was also thought to be a sticky protein. Therefore, we avoided using solid supports for analysis of a high-molecular weight state of TLP. When F/H-hTBP was analyzed by GDGC, F/H-hTBP was mainly detected in fractions around 43 kDa, and a minor population was found in fractions lighter than 150 kDa ( Fig. 2A), indicating that a certain population of TBP was polymerized (probably dimerized) as previously reported (8). On the other hand, a peak of F/H-mTLP was detected in a dimer position but not in a monomer position (Fig. 2B). Moreover, a considerable amount of TLP was also detected in 67-158-kDa fractions. These results suggested that TLP exists as a multimer (at least a dimer) like TBP in a physiological condition. Because monomeric TLP was minor (Fig. 2B) and a significant population of TBP was present in monomer fractions ( Fig. 2A), it seems that a TLP dimer is more stable than is TBP.
Next, we performed chemical cross-linking of TLP using BMH to detect stable multimers. As shown in Fig. 2D (lanes 2  and 3), TBP was cross-linked efficiently to form dimers but not larger multimers, as was previously reported (8). When F/H-mTLP was cross-linked in the same way, dimers as well as trimers were clearly detected (Fig. 2E, lanes 2 and 3). These results suggest that the high-molecular weight TLPs observed in GDGC fractions of 67-158 kDa (Fig. 2B) are mainly trimers. It is known that TBP dimerization is inhibited by TFIIA and TATA DNA and that the dimerization and TATA binding are mutually exclusive (9). These phenomena were reproducibly observed in our experiments, because cross-linking of the TBP dimer was inhibited by TFIIA and TATA DNA (Fig. 2D, lanes 4 -6 and 10 -12, respectively) but not by bovine serum albumin or mutant TATA DNA (lanes 7-9 and 13-15, respectively). When TFIIA, bovine serum albumin, TATA DNA, and mutant TATA DNA were preincubated with F/H-mTLP, formation of cross-linked TLP dimers and trimers was inhibited only when increased amounts of TFIIA were added (Fig. 2E, lanes 4 -6). The fact that TFIIA-TLP binding was detected by immunoprecipitations in the same buffer conditions (Fig. 2C, lanes 6 and 8) suggests that the TFIIA-mediated inhibition is because of direct binding of TFIIA and TLP. The finding that TATA DNA did not inhibit TLP dimerization is consistent with the previously reported inability of TLP to stably bind to TATA DNA (39,40).
There is a possibility that TFIIA-mediated interference of TLP dimerization is just because of inhibition of the crosslinking reaction by TFIIA. We performed a pull-down assay to detect direct interaction between TLP molecules in the presence of TFIIA (Fig. 2F). GST proteins and F/H proteins were mixed and subjected to pull-down experiments with glutathione beads. In the absence of TFIIA, F/H-proteins were precipitated with GST-mTLP (Fig. 2F, lane 10) and GST-hTBP (lane 4) but not with GST (lanes 2 and 8). Co-precipitations of F/Hproteins with GST fusion proteins were drastically decreased by TFIIA (Fig. 2F, lanes 5, 6, 11, and 12). These results suggested that TLP is dimerized and trimerized in solutions and that this multimerization is inhibited by binding of TFIIA like TBP.

Molecular Numbers of TLP, TBP, and TFIIA in Nuclear and
Cytoplasmic Fractions-Although TLP was originally identified as a protein similar to nuclear-concentrated TBP, vertebrate TLP has been demonstrated to be basically a cytoplasmic protein, the localization of which is dynamically regulated depending on the cell states (37,43). Actually, vertebrate TLP has no apparent nuclear localization signal. These facts suggest that the considerably high level of TFIIA-TLP affinity is related to the TLP-specific localization pattern. Before we investigated the effects of TFIIA binding to TLP in vivo, we first precisely quantified the molecular numbers of TLP in subcellular fractions of NIH3T3 cells. The cell fractionation procedure used was sufficient for our purpose, because the marker nuclear protein c-Fos (51) and cytoplasmic protein ␤-tubulin (45) were mostly detected in nuclear and cytoplasmic fractions, respectively (Fig. 3A).
We determined the molecular numbers of three transcription factors in subcellular fractions of NIH3T3 cells by Western blotting (Fig. 3, B and C). The number of TBP molecules in the nuclear fraction was calculated to be 1.34 Ϯ 0.04 ϫ 10 5 /cell and that in the cytoplasmic fraction was calculated to be 1.0 Ϯ 0.2 ϫ 10 5 /cell, indicating that TBP is abundant in the nucleus, being consistent with the generally accepted notion (Fig. 3B). The sum of molecular numbers of TBP in the nucleus and cytoplasm is comparable with that reported previously (48). Molecular numbers of TFIIA␣ were 4.4 Ϯ 0.7 ϫ 10 3 /cell in the nucleus and 1.7 Ϯ 0.4 ϫ 10 4 /cell in the cytoplasm (Fig. 3B). Molecular numbers of TFIIA␣␤ precursor were 5.8 Ϯ 1.3 ϫ 10 3 /cell in the nucleus and 1.9 Ϯ 0.2 ϫ 10 4 /cell in the cytoplasm (Fig. 3B). Moreover, TFIIA␤ and -␥ were also enriched in the cytoplasmic fraction like TFIIA␣ (Fig. 3D, top and bottom panels, respectively). We concluded that holo-TFIIA is concentrated in the cytoplasm.
The molecular number of TLP was also determined (Fig. 3, B and C). Molecular numbers of TLP in the nucleus and cytoplasm were calculated to be 3.2 Ϯ 1.1 ϫ 10 3 /cell and 8.9 Ϯ 0.3 ϫ 10 4 /cell, respectively (Fig. 3B). TLP in the nuclear fraction was only 3.5% of that in the cytoplasmic fraction and only 2% of TBP in the nuclear fraction (Fig. 3B). These results demonstrated that TLP molecules are highly concentrated in the cytoplasmic fraction, as was confirmed by results of cytochemical analysis (Fig. 3E). Consistent results were also obtained using HeLa cell extracts (data not shown). Based on these results, it was concluded that TLP is a cytoplasmic protein in normally dividing NIH3T3 cells in contrast to TBP even though TLP and TBP are structurally similar.
TLP-TFIIA Association and Localization of TLP-We examined the relationship between TFIIA association ability of TLP and its localization. First, we established cell lines that expressed different mutant TLPs to find TFIIA-binding defective mutant TLPs (Fig. 4A). These mutants were designed on the basis of results of crystallographic analysis of the TBP-TFIIA complex and results of radical mutation analysis of TBP (6,52,53). Eight mutants (AL32/33EA, N37E, R52E, K53E, R55E, EE74/75RA, K78E, and R86S) had substitutions of one or two residues in the NH 2 -terminal tandem repeat in which some of these corresponding residues of TBP were involved in TFIIA contact. Two other mutants (E133R and L134E) had substituted residues in the COOH-terminal tandem repeat. We investigated the TFIIA binding of each mutant TLP by immunoprecipitation of WCE from established cell lines that express exogenous F/H-mTLP at a level comparable with that of endogenous TLP (50 -100% of endogenous TLP, Fig. 5, A and C). F/H-mTLP-R55E, -L134E, and -E133R associated with comparable amounts of TFIIA like F/H-mTLP (Fig. 4B, lanes 2, 7, 11, and 12). F/H-mTLP-EE74/75RA bound a 2-fold greater amount of TFIIA than F/H-mTLP did (Fig. 4B, lane 8). On the other hand, F/H-mTLP-AL32/33EA, -N37E, -R52E, and -R86S lost their TFIIA binding ability (Fig. 4B, lanes 3-5 and 10). F/H-mTLP-K53E and -K78E showed slight impairment in TFIIA binding ability (Fig. 4B, lanes 6 and 9). These results are consistent with those obtained for TFIIA/TBP interaction.

DISCUSSION
High Affinity of TLP to TFIIA-TFIIA was identified as the main TLP-interacting partner (41). The K D of TLP to TFIIA was 1.5 nM and that of TBP to TFIIA was 10 nM (Fig. 1). In this study, TLP was shown to have a higher affinity to TFIIA than that of TBP. Moreover, we found that the dissociation rate constant (k d ) of TLP from TFIIA was significantly lower than that of TBP (1/20-fold), indicating that a TLP-TFIIA complex is more stable than is a TBP-TFIIA complex. In an in vitro transcription assay, the addition of a three times molar excess of TLP than TBP inhibited TATA-dependent transcription (41). A TFIIA interception model has been proposed to explain this phenomenon. Our present data are consistent with the proposed model. TFIIA interacts with various activators that stabilize TBP/DNA interaction synergistically with TFIIA (4,5,54). If there is a potent TFIIA-interactive activator on a promoter, TFIIA acts as a mediator for TBP recruitment even though the promoter lacks a TATA box. On the other hand, TLP artificially bound to a proximal promoter region has been demonstrated to be able to activate transcription (35). There-fore, when the amount of nuclear TLPs is increased, TFIIA recruits TLP rather than TBP to a promoter because of the low K D of TLP/TFIIA interaction. The promoter-recruited TLP may remain for a long period on a promoter because of its low k d and participate in transcriptional regulation. This situation is equivalent to that of Drosophila TRF2, which is recruited to the PCNA promoter via DREF (38). On a TATA-less promoter, to which a powerful TFIIA-interacting activator is recruited, TLP may initiate transcription more efficiently through TFIIA binding than does TBP. Some genes whose expression levels were drastically decreased in functional TLP-deficient mice did not have a TATA box (31,34). If the promoter has a TATA box, increasing amounts of nuclear TLP may inhibit such transcriptional activation.
Formation of TFIIA-sensitive Dimers and Trimers of TLP-Although we clarified that TLP forms a TFIIA-sensitive dimer as does TBP, several differences were found between a TLP dimer (or multimers) and a TBP dimer. First, TLP formed both dimers and trimers, whereas TBP predominantly formed dimers (Fig. 1). Although a TLP multimer larger than a trimer was indistinguishable by GDGC, the results of chemical crosslinking assays clearly demonstrated the existence of only trimers/dimers as stable multimers, whereas a TBP trimer was not detected (Fig. 2, D and E). Second, a TLP dimer is more stable than a TBP dimer because TBP was mainly detected as monomers, whereas the peak of TLP was detected in dimer- containing fractions in GDGC (Fig. 2, A and B). Third, the TLP dimer and TLP trimer were not dissociated by TATA-DNA (Fig.  2E). This finding is consistent with the finding that TLP does not bind to traditional TATA-DNA (39,40). The rate of dissociation of TBP dimers is significantly low, whereas TBP monomers generated by a dynamic equilibrium can rapidly bind to TATA-DNA (9). The finding that TLP forms a dimer/trimer that is more stable than is a TBP dimer implies that a TLP dimer acts as a potent rate-limiting element for TLP-dependent transcription if TLP functions as a monomer and is more dependent on TFIIA than is a TBP dimer.
TFIIA-binding Region of TLP-From studies on the relationship between localization and TFIIA binding of TLP, we identified TLP mutations that impair TFIIA binding (Fig. 4, A and  B). Results of crystallographic and mutant analyses of TBP revealed that Ala-184/Leu-185 (86/87), Asn-189 (91), and Arg-205 (107) in the ␣-helix 1, ␤-sheet 2, and COOH terminus adjacent to the ␤-sheet 3 region of the tandem repeat of human TBP (yeast TBP) made contact with amino acids of TOA2 and were required for TFIIA binding (6,52,53). Basic amino acids in the ␣-helix 2 region have been proposed to be involved in TBP-DNA-TFIIA complex formation (55,56). Mutation of basic amino acids at Arg-231 and Arg-239 in this region of human TBP diminished the binding ability to the TFIIA column (6). Ala-32/Leu-33, Asn-37, Arg-52, Lys-78, and Arg-86 of mTLP, which are equivalent to Ala-184/Leu-185, Asn-189, Arg-205, Arg-231, and Arg-239 of hTBP, respectively, are highly conserved among TLPs in different species and have been shown to be defective in TFIIA binding ability (Fig. 4B). Taken together with the fact that mutations in other regions of TLP (Arg-55 in the ␤-sheet 4 and Glu-133 and Leu-134 in the COOH terminus) did not affect the TFIIA interaction like TBP (6), it seems that the mechanism by which TLP binds to TFIIA is similar to that of TBP. However, although the substitution of Glu-228 of hTBP impaired the TBP-DNA-TFIIA complex formation (56), the equivalent Glu-75 of TLP did not affect TFIIA binding (Fig. 4B). Moreover, K53E of mutant TLP exhibited a slightly decreased level of TFIIA binding ability (Fig. 4B), although a mutated amino acid at the corresponding position in TBP had no effect on TFIIA binding (6). These differences are thought to be responsible for the higher degree of stability of the TLP-TFIIA complex than that of the TBP-TFIIA complex.
Localization and Interaction with TFIIA of TLP-Because TLP is thought to be a transcriptional regulator, it has been thought that TLP would be normally localized in the nucleus. Although it has been reported that TLP is localized in the nuclei of particular stages of mouse spermatocytes and stressed cells (37,43), our present study clearly demonstrated that TLP is ordinarily a cytoplasmic protein (Fig. 4B). Because vertebrate TLPs do not have an apparent nuclear localization signal, cytoplasmic localization of mammalian TLP seems to be reasonable, at least for normally dividing cells. The cytoplasmic localization of TLP is a feature of TLP that makes TLP essentially different from TBP. If a particular regulation mechanism required for TLP localization does not exist in a cell, TLP  3 and 7), and M2-41 cells (lanes 4 and 8) were subjected to Western blotting using anti-TLP antibody. Positions of endogenous mTLP and exogenous F/H-mTLP (or F/H-mTLP-AL32/33EA) are indicated. B, summary of a population of nuclear TLPs. Molarity in each extract was calculated from Western blotting (panel A) as described in the legend to Fig. 3, and the proportion of TLP in the nucleus to that in WCE was determined. C, detection of endogenous and exogenous TLPs in extracts of NIH3T3 cells that stably express mutant TLP proteins. WCEs (10 g, indicated with W) and nuclear extracts (20 g, indicated with N) were subjected to Western blotting using anti-TLP antibody. Names of mutant F/H-mTLPs and cell lines are indicated below and above the panels, respectively. Positions of endogenous mTLP and exogenous F/H-mTLP are indicated by asterisks and arrowheads, respectively. D, summary of localization phenotype of exogenous F/H-mTLPs. Molarity in each extract was calculated from Western blotting (panel C) as described in the legend to Fig. 3, and the ratio of exogenous F/H-mTLP to endogenous TLP in WCEs was calculated and compared with that in nuclear extracts. Ϫ means that the ratio was not different between WCEs and nuclear extracts and that exogenous F/H-mTLP was localized in the cytoplasm as was endogenous TLP. Ϫ/ϩ, ϩ, and ϩϩ mean that the ratio in nuclear extracts was higher (Ͻ2-fold, 2-3-fold, and Ͼ3-fold, respectively) than that in WCEs. should homogeneously exist throughout a cell by simple diffusion because the molecular mass of a TLP monomer is small enough for it to pass through the nuclear pore (57). Therefore, the cytoplasmic localization of TLP implies the existence of an active mechanism for TLP localization. The localization profile of TLP was basically correlated with its TFIIA binding ability (Fig. 5). The diffused localization pattern of TLP was not related to its TFIIB binding ability (data not shown). This ability was thought to have no effect on the multimerization of TLP, because the AL32/33EA mutant exhibited a degree of dimerization kinetics comparable with that of F/H-mTLP (data not shown). Therefore, the results of the present study suggest that TFIIA is involved in the regulation of TLP localization.
There are two possible mechanisms by which TFIIA regulates cytoplasmic localization of TLP. First, TLP may be carried to the cytoplasm by TFIIA. Vertebrate TFIIA has not been demonstrated to have a obvious nuclear localization signal, and nuclear localization of TFIIA in NIH3T3 cells was not so evident (Fig. 3). Because it is known that the nuclear import/ export pathway of some transcription factors is modified by intracellular signaling (57,58), the nuclear import/export of TFIIA in mammalian cells would be regulated depending on the cell state and thus influence TLP localization. Second, TLP multimerization may be related to the intracellular localization of TLP. It is known that a stress-induced p53 tetramer is retained in the nucleus and that its dissociation results in export of p53 (59). Inactivated monomeric/dimeric HSF3, which is normally localized in the cytoplasm, is converted into a transcriptional active trimer depending on the degree of nuclear localization upon stress (60). These observations indicate that the dynamic change in molecular states, including multimerization and dissociation, is one of the regulation pathways for intracellular localization of proteins. Because TLP forms a TFIIA-sensitive dimer/trimer (Fig. 2) and the ratio of TLPs to TFIIAs in the nucleus is much smaller than that in the cytoplasm (Fig. 3), it is thought that the proportion of TLP multimers in the nucleus is less than that in the cytoplasm. Thus, a multimer-to-monomer transition of TLP induced by TFIIA could be related to the cytoplasmic localization of TLP.
The idea that TFIIA regulates the cytoplasmic localization of TLP seems to be inconsistent with another speculation that TFIIA potentiates transcriptional regulatory functions of TLP as described above. However, these apparent inconsistent actions of TFIIA on TLP are thought to be related to a unique role of TLP in transcriptional regulation that is different from that of TBP. When nuclear TLP is increased in a particular situation (37,43) by an unknown TFIIA-independent mechanism, tight association between TLP and TFIIA generates the potential role of TLP in transcriptional regulation as described above and inhibits TBP-dependent transcription. When cells do not need TLP for their activity, TLP is exported from the nucleus by TFIIA association so that TLP does not accumulate in the nucleus and affect transcription of target genes.


extfieldsofstudy: Biology Medicine

text:
Transforming Growth Factor β1 Is Up-regulated by Activated Raf in Skeletal Myoblasts but Does Not Contribute to the Differentiation-defective Phenotype*

The Raf/MEK/MAPK signaling module elicits a strong negative impact on skeletal myogenesis that is reflected by a complete loss of muscle gene transcription and differentiation in multinucleated myocytes. Recent evidence indicates that Raf signaling also may contribute to myoblast cell cycle exit and cytoprotection. To further define the mechanisms by which Raf participates in cellular responses, a stable line of myoblasts expressing an estrogen receptor-Raf chimeric protein was created. The cells (23A2RafERDD) demonstrate a strict concentration-dependent increase in chimeric Raf protein synthesis and downstream phosphoMAPK activation. Initiation of low-level Raf activity in these cells augments contractile protein expression and myocyte fusion. By contrast, induction of high level Raf activity in 23A2RafERDD myoblasts inhibits the formation of myocytes and muscle reporter gene expression. Interestingly, treatment of myoblasts with conditioned medium isolated from Raf-repressive cells inhibits all of the aspects of myogenesis. Closer examination indicates that the transforming growth factor-β1 (TGF-β1) gene is up-regulated in Raf-repressive myoblasts. The cells also direct elevated levels of Smad transcriptional activity, suggesting the existence of a TGF-β1 autocrine loop. However, extinguishing the biological activity of TGF-β1 does not restore the myogenic program. Our results provide evidence for the involvement of Raf signal transmission during myocyte formation as well as during inhibition of myogenesis.

The Raf/MEK/MAPK signaling module elicits a strong negative impact on skeletal myogenesis that is reflected by a complete loss of muscle gene transcription and differentiation in multinucleated myocytes. Recent evidence indicates that Raf signaling also may contribute to myoblast cell cycle exit and cytoprotection. To further define the mechanisms by which Raf participates in cellular responses, a stable line of myoblasts expressing an estrogen receptor-Raf chimeric protein was created. The cells (23A2RafER DD ) demonstrate a strict concentration-dependent increase in chimeric Raf protein synthesis and downstream phos-phoMAPK activation. Initiation of low-level Raf activity in these cells augments contractile protein expression and myocyte fusion. By contrast, induction of high level Raf activity in 23A2RafER DD myoblasts inhibits the formation of myocytes and muscle reporter gene expression. Interestingly, treatment of myoblasts with conditioned medium isolated from Raf-repressive cells inhibits all of the aspects of myogenesis. Closer examination indicates that the transforming growth factor-␤ 1 (TGF-␤ 1 ) gene is up-regulated in Raf-repressive myoblasts. The cells also direct elevated levels of Smad transcriptional activity, suggesting the existence of a TGF-␤ 1 autocrine loop. However, extinguishing the biological activity of TGF-␤ 1 does not restore the myogenic program. Our results provide evidence for the involvement of Raf signal transmission during myocyte formation as well as during inhibition of myogenesis.
Myogenesis, the formation of contractile-competent skeletal muscle cells, is tightly regulated by the presence of several critical growth factors in the extracellular environment. As an example, local production of insulin-like growth factor-I (IGF-I) 1 is a strong permissive growth factor for both morphological and biochemical differentiation of myoblasts (1)(2)(3)(4). Ectopic IGF-I delivery to skeletal muscle cells increases muscle mass and morphometric measures of strength in birds and mice (5,6). On the contrary, several laboratories have documented the repressive effects of transforming growth factor ␤ 1 (TGF-␤ 1 ) and members of the fibroblast growth factor family, chiefly FGF2 (for review see Refs. 7 and 8). The mechanism by which these soluble inhibitory proteins impede skeletal myogenesis involves the activation of specific intracellular signaling cascades. In the case of TGF-␤ 1 , a requisite induction of Smad3 protein phosphorylation and nuclear translocation occurs that interferes with muscle gene transcription (9). The precise mechanism for FGF2-mediated inhibition of myogenesis is less understood but may involve downstream signaling events that are controlled through G-proteins (10).
Overexpression studies and application of specific kinase inhibitors has led to the identification of several essential signaling pathways in skeletal myoblasts. It is firmly established that phosphatidylinositol 3-kinase and p38, a stress-activated mitogen-activated protein kinase (MAPK), both are necessary for the formation of mature myocytes (11)(12)(13)(14)(15). Removal of downstream signaling events by the respective kinases through the use of chemical inhibitors abolishes myogenesis. By contrast, initiation of downstream signaling modules by constitutively active Ras, a membrane-localized GTPase that participates in numerous receptor tyrosine kinase-initiated signaling events, results in a severe reduction in muscle gene transcription and myoblast fusion (16,17). Activation of Raf, the downstream target of Ras, also abolishes the myogenic program (18 -21). Interestingly, the means by which Ras and Raf inhibit muscle gene expression does not appear to be directly dependent upon the subsequent phosphorylation and activation of extracellular signal-regulated kinase 1 and 2 (ERK1/2). Inhibition of MEK/ERK signal transduction does not reinstate the full complement of muscle gene transcription and myoblast fusion to myoblasts expressing constitutively active Ras or Raf (17,22).
Raf kinase is a serine/threonine kinase whose activation precludes ERK1/2 (MAPK) phosphorylation and a concomitant alteration in gene transcription (23,24). Signaling through the Raf/MEK/ERK pathway is a common mitogenic response for many cell types. Recently, several groups have reported differential cellular responses as a consequence of Raf/MAPK signal intensity. At lower levels of MAPK activity, epithelial and fibroblasts demonstrate increased proliferative rates (25,26). By contrast, high levels of Raf/MAPK result in cell cycle arrest and senescence (26,27). The Raf-mediated responses are attributed to signaling through MEK/MAPK as well as non-MEKdependent signaling events. Regulation of Raf activity indicates that the protein physically associates with other kinases, regulatory proteins, and scaffolding proteins (for review see Ref. 28). Indeed, it has become increasingly apparent that Raf kinase can control cellular transcriptional responses through mechanisms that are independent of the archetypical MEK/ ERK module. A Raf kinase allele that fails to interact with MEK and cause ERK1/2 phosphorylation retained the ability to activate NFB-directed transcription and promote neuronal differentiation (29). These results argue that Raf participates in several downstream signaling cascades. Differential utilization of these pathways may be reflected in the decision of the cell to undergo proliferation, differentiation, apoptosis, and senescence.
Because many growth factors exhibit contrasting effects on skeletal myogenesis yet utilize many of the same intracellular signaling pathways, it is likely that signal intensity plays a critical role in the decision to complete terminal differentiation. To this end, myoblasts that express an inducible activated Raf allele were created. Our results indicate that low level Raf activity promotes myogenesis, whereas high level Raf activity inhibits muscle formation. Coincident with repression of differentiation by activated Raf is an increase in TGF-␤ 1 gene expression and Smad-directed transcriptional activity. However, the removal of TGF-␤ 1 from the extracellular environment of the Raf-repressive myoblasts does not reinstate the differentiation program. In summary, Raf signaling modules are both positive and negative mediators of myogenesis that is a direct reflection of signal strength.

MATERIALS AND METHODS
Cell Culture, Plasmids, and Transfections-23A2RafER DD myoblasts were created by transduction of 23A2 myoblasts with a retrovirus encoding the fusion protein RafER DD (30). RafER DD is comprised of the kinase domain of human c-Raf-1 fused in-frame with the estrogen receptor ligand binding domain. Following infection, the cells were selected in puromycin and clones were isolated by limiting dilution. 23A2 and 23A2RafER DD myoblasts were cultured on gelatinized tissue culture grade plasticware in Dulbecco's modified Eagle medium containing 15% fetal bovine serum (BioWhittaker), 1% penicillin-streptomycin, and 0.5% Geneticin (Invitrogen). Differentiation was induced in confluent cultures by continuous culture in Dulbecco's modified Eagle medium supplemented with 2% horse serum, 1% penicillin-streptomycin, and 0.5% Geneticin. For the measurement of muscle-specific reporter gene activity, myogenic cells cultured in 6-well tissue plasticware were transiently transfected with 1 g of troponin I luciferase (TnI-Luc), the minimal E-box reporter plasmid, 4Rtk-Luc, or a multimerized AP-1 binding site reporter (AP-1-Luc) and 50 ng of pRL-tk, a Renilla luciferase plasmid as a monitor of transfection efficiency, using standard calcium phosphate methods (16,22). The cells were maintained in differentiation media for 48 h in the presence or absence of varying concentrations of 4-hydroxytamoxifen (4HT, Sigma) prior to lysis and measurement of luciferase activities (Dual-luciferase, Promega, Madison, WI). Inhibition of MEK activity was achieved by supplementation of differentiation medium with 20 M PD98059. The amount of corrected luciferase activity generated by 23A2 or 23A2RafER DD myoblasts treated with vehicle was set to 100%. Each experiment was replicated a minimum of three times. For conditioned medium experiments, the culture medium was removed from myoblasts treated for 48 h with Me 2 SO or varying amounts of 4HT. The medium was centrifuged to remove debris and brought to a final concentration of 2% horse serum. The resulting medium was applied to confluent 23A2 myoblasts, and the cells were maintained for an additional 48 h prior to fixation. Heat inactivation of the conditioned medium was performed as described previously (31).
RNA Isolation, Northern Blots, and RT-PCR-Total RNA was isolated with STAT60-denaturing solution per the manufacturer's recommendations (Tel-Test, Friendswood, TX). Twenty micrograms of total RNA were separated through 1% agarose gels containing 2% formaldehyde, transferred to nitrocellulose membrane, and irreversibly crosslinked to the medium by UV. The blots were hybridized with 32 P-labeled cDNA probes generated by random hexamer priming (DecaPrime, Ambion, Houston, TX). The probes corresponded to regions contained within the TGF-␤ 1 and glyceraldehydes 3-phosphate dehydrogenase (GAPDH) gene-coding regions. Northern blot analysis was performed using Ultra-Hyb (Ambion) at 42°C overnight according to the manufacturer's recommendations. Blots were washed with 2ϫ SSC, 0.1% SDS once at room temperature and twice with 1ϫ SSC, 0.1% SDS at 42°C. Blots were exposed to phosphorimaging screens for the visualization and quantification of message levels. For the semi-quantitative assessment of mRNA levels, 1 g of total RNA was reverse-transcribed with Moloney murine leukemia virus reverse transcriptase. Amplification of GAPDH and TGF-␤ 1 was performed using Taq polymerase (Fisher Scientific, Pittsburgh, PA), gene-specific primers, and thermocycle conditions of 95°C for 45 s, 55°C for 45 s, and 72°C for 90 s for a total of 35 cycles. To ensure that quantification was accomplished in the linear range of amplification, an aliquot was removed from each reaction after 25, 30, and 35 cycles. GDF-8 was amplified as described previously (32). Amplicons were separated through 2% agarose gels containing ethidium bromide, visualized under UV, and photographed.
Western Blot and Scanning Densitometry-23A2RafER DD myoblasts were differentiated in the presence or absence of 4HT. After 48 h, the cells were lysed in 4ϫ SDS-PAGE sample buffer and an aliquot was removed for protein quantification. Equal amounts of protein were electrophoretically separated through denaturing gels and transferred to nitrocellulose membrane. The blots were incubated with 5% nonfat dry milk in TBST (10 mM Tris, pH 8.0, 150 mM NaCl, 0.1% Tween 20) to remove nonspecific binding sites. Primary antibodies were diluted in blocking buffer, and the blots were incubated overnight at 4°C with shaking. Antibodies and dilutions included the following: anti-myogenin (F5D ascites, Developmental Hybridoma Bank, University of Iowa, 1:5000); anti-myosin heavy chain (MF20 hybridoma supernatant, Developmental Hybridoma Bank, 1:5); anti-ERK1/2 and anti-phos-phoERK1/2 (Cell Signaling); and anti-estrogen receptor (Santa Cruz Biotechnology, Santa Cruz, CA, 1:300). After extensive washing with TBST, the blots were reacted with the appropriate peroxidase secondary antibody for 45 min at room temperature. Visualization of protein bands was accomplished by chemiluminescence and autoradiography. Multiple exposures to x-ray film were used to ensure that the linear range of densitometry was maintained. Autoradiograms were scanned on a Storm 860 phosphorimaging system (Molecular Dynamics, Amersham Biosciences).
Immunocytochemistry-23A2 and 23A2RafER DD myocytes were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 10 min at room temperature. The cells were washed with PBS, and nonspecific antigen sites were blocked by incubation with 5% horse serum in PBS containing 0.1% Triton X-100 for 20 min at room temperature. Cultures were incubated with anti-myosin heavy chain (MF20) for 60 min at room temperature. After exhaustive washing with PBS, the cells were reacted with donkey anti-mouse fluorescein isothiocyanate (Vector Laboratories, Burlingame, CA, 1:200) for 45 min at room temperature. Cultures were washed with PBS and counterstained with 4,6-diamidino-2-phenylindole (DAPI). Immunofluorescent detection was accomplished using a Nikon TE200 inverted phase microscope equipped with epifluorescence. Representative photomicrographs were captured to slide film and assembled in Adobe Photoshop.

Creation and Characterization of an Inducible Raf Myogenic
Cell Line-Previous work has clearly demonstrated that overexpression of activated alleles of Raf are inhibitory to myocyte formation and muscle gene expression (17,18,21). More importantly, MAPK-dependent repression appears to be a function of Raf signaling intensity in avian myoblasts (18). To extend these observations, an activated Raf kinase that is responsive to the estrogen analog, 4-hydroxytamoxifen (4HT), was stably expressed in 23A2 mouse myoblasts (23A2RafER DD ). Confluent cultures of 23A2RafER DD myoblasts were differentiated in the presence or absence of increasing concentrations of 4HT. After 48 h, the cells were lysed for Western analysis of chimeric Raf (␣-estrogen receptor), activated MAPK (␣-phosphoMAPK), and total MAPK (␣-MAPK) expression. As shown in Fig. 1, a dosedependent increase in the relative amount of RafER DD protein is found with amounts 4HT from 0.25 nM to 2.5 M (lanes 2-6). Coincident with the increase in chimeric Raf protein is an elevation in the amounts of phosphorylated MAPK. Phosphorylated ERK2 is preferentially activated at 4HT concentrations as low as 0.25 nM, whereas activated ERK1 is first apparent at 4HT concentrations of 25 nM or greater. No differences in the amounts of total MAPK (ERK1/2) were evident.
Activation of ERK1/2 signal transmission leads to an increase in AP-1-directed transcriptional activity in many cell types including skeletal myoblasts. To ensure that the Raf/ ERK pathway was functional in 23A2RafER DD myoblasts, semiconfluent cells were transiently transfected with a multimerized AP-1 reporter plasmid and pRL-tk, a plasmid encoding Renilla luciferase as a marker of transfection efficiency. Subsequently, the cells were treated with increasing amounts of 4HT for 48 h prior to lysis and measurement of luciferase activities. Control 23A2 myoblasts were treated in an analogous fashion. AP-1-Luc reporter activity was normalized to Renilla luciferase activity. Results demonstrate a dose-dependent increase in AP-1-Luc activity with increasing amounts of 4HT (Fig. 2). At concentrations of 4HT greater than 250 nM, a decline in AP-1 luciferase activity was evident. Moreover, the response is directly attributed to the induction of RafER activity as 23A2 myoblasts failed to initiate AP-1-transcriptional activity upon treatment with 4HT. These results indicate that the Raf/ERK signaling axis is intact and functional in the myogenic cells.
As final confirmation of the integrity of the Raf signaling system in 23A2RafER DD myoblasts, confluent cultures of cells were treated for 48 h in differentiation medium containing vehicle only (Me 2 SO) or 1 M 4HT. Subsequently, cells were fixed and immunostained for myosin heavy chain (␣-MyHC). Control 23A2RafER DD myocytes treated with vehicle readily differentiate into large multinucleated myofibers that express copious amounts of the contractile protein, MyHC (Fig. 3). By contrast, activation of the Raf/ERK signaling pathway leads to a severe reduction in both myofiber number and MyHC protein expression. These results directly reflect published reports of the effects of activated Raf on skeletal myogenesis. Thus, 23A2RafER DD myoblasts represent a myogenic cell line that retains its muscle features in the absence of Raf activity and is differentiationdefective in the presence of Raf signal transmission.
Contrasting Effects of Raf Signal Transduction on Skeletal Myoblasts-To understand the effects of signal intensity of myogenesis, 23A2RafER DD myoblasts were differentiated in the presence of increasing amount of 4HT. After 48 h, the cells were lysed and equal amounts of total cellular proteins were analyzed by Western blot for the expression of muscle proteins (anti-MyHC, anti-myogenin). Scanning densitometry was performed on the chemiluminescent autoradiograms. Muscle protein expression was normalized to total ERK2 expression, an internal marker for protein loading, transfer, and detection efficiency. In the absence of 4HT, 23A2RafER DD myoblasts differentiate into myocytes that express both MyHC and myo-genin (Fig. 4). Unexpectedly, low level induction of Raf activity using concentrations of 4HT (0.25 or 2.5 nM) causes an increase in the amount of contractile and regulatory protein expression. Supplementation of differentiation medium with 25 nM 4HT or greater causes the predicted loss of MyHC and myogenin protein expression. The biphasic response of 23A2RafER DD myoblasts to increasing 4HT concentrations is further reflected in muscle-specific reporter gene activity. 23A2 and 23A2RafER DD myoblasts were transiently transfected with TnI-Luc and pRLtk. After 48 h in differentiation medium supplemented with 4HT, the cells were lysed and luciferase activities were measured. TnI luciferase enzymatic activity did not vary as a function of 4HT treatment in control 23A2 myoblasts, indicating that the estrogen analog does not significantly alter basal muscle gene expression (Fig. 5). The levels of Raf activity directed in response to 25 nM 4HT or greater are sufficient for a reduction in TnI-Luc activity. However, low level Raf activity (0.25-2.5 nM 4HT) directs only a slight increase in muscle reporter gene activity. It is likely that a larger portion of the TnI regulatory region is necessary to obtain a substantial increase in reporter gene transcription. Our results clearly demonstrate that Raf has contrasting effects on muscle formation that are intensity-dependent.
Previously, we have documented the existence of MEK-independent signaling in response to activated Raf (18,33). To determine the role of MEK in the RafER-imposed block to myogenesis, confluent 23A2RafER DD myoblasts were transiently transfected with TnI-Luc and pRL-tk. The myoblasts were treated for 48 h with increasing amounts of 4HT and 20 M PD98059 or vehicle only. Cell lysates were collected, and luciferase activities were measured. The amount of muscle reporter gene activity directed in the absence of 4HT and the chemical MEK inhibitor was set to 1. As expected, the selected concentrations of 4HT caused a reduction in the amount of TnI-Luc reporter activation (Fig. 6). Suppression of MEK function restored muscle gene transcription as evidenced by an increase in TnI-Luc activity to levels comparable to controls. However, strong stimulation of Raf signaling initiated by 2.5 M 4HT was refractile to the effects of the MEK inhibitor. Western analysis of replica samples demonstrates that treatment with PD98059 reinstates myosin heavy chain expression to levels Ͻ10% of controls (data not shown). These results duplicate previous observations using constitutive Raf alleles of various signaling intensities (21,33).

Raf Inhibition of Myogenesis Involves Secretion of a Soluble
Factor-It has been reported that the inability of myoblasts to differentiate in the presence of activated Raf is a product of sequestration of MEF2 in the cytoplasm (21). However, forced expression of the transcription factor in avian myoblasts does not reinstate the muscle gene program, arguing that additional factors are involved (33). Moreover, rhabdomyosarcoma cells secrete bioactive TGF-␤ 1 that acts in an autocrine manner to suppress differentiation (34). To further characterize the differentiation-defective phenotype of myoblasts directing extreme levels of Raf/ERK signal, conditioned medium was collected from 23A2RafER DD myoblasts that were treated with Me 2 SO, 2.5 nM 4HT, or 2.5 M 4HT in differentiation medium. Cell debris was removed by centrifugation, and the supernatants were supplemented with horse serum to a final concentration of 2%. An aliquot of conditioned medium isolated from the 2.5 M of treatment group was heat-inactivated prior to supplementation with horse serum (31). Confluent cultures of 23A2 myoblasts were allowed to differentiate for 48 h in the conditioned medium followed by fixation and immunostaining for MyHC. Control cultures were treated with Me 2 SO, 2.5 nM 4HT, or 2.5 M 4HT and analyzed as described. Conditioned medium from 23A2RafER DD myoblasts differentiated in the presence or absence of 2.5 nM 4HT did not alter the ability of myoblasts to fuse into MyHC-positive myofibers (Fig. 7). By contrast, medium harvested from 23A2RafER DD myoblasts treated with 2.5 M 4HT dramatically inhibited myocyte formation. Ͻ1% of the myoblasts were able to fuse into myosinexpressing myofibers. Control myoblasts were not affected by 2.5 M 4HT to the same extent. The complete absence of differentiated cells strongly argues that a soluble factor is secreted from Raf-expressing myoblasts that contributes to the loss of myofiber formation. Moreover, the secreted factor probably is a protein because heat-denatured conditioned medium is unable to inhibit 23A2 myocyte formation.
TGF-␤ 1 Does Not Contribute to the Raf-imposed Block to Myogenesis-Differentiation-defective rhabdomyosarcoma cells secrete TGF-␤ 1 into the culture medium that contributes to the loss of myogenic potential. To determine whether a TGF-␤-like factor may be responsible for the inhibition of muscle formation in myoblasts treated with Raf-repressive conditioned medium, 23A2 myoblasts were transiently transfected with 3TP-Lux, a reporter plasmid whose activity is modulated by TGF-␤ proteins, and pRL-tk. Cells were treated for 48 h in conditioned medium harvested from 23A2RafER DD myoblasts that were treated with Me 2 SO, 2.5 nM 4HT, or 2.5 M 4HT. Subsequently, the cells were lysed and luciferase activities were measured. Myoblasts that are capable of fusing and expressing contractile proteins (Me 2 SO, 2.5 nM 4HT) do not direct a significant amount of 3TP-Lux activity (Fig. 8). However, myoblasts that are unable to express the differentiated phenotype demonstrate a 6-fold increase in TGF-␤-responsive re- FIG. 4. Low-level Raf activity enhances skeletal myogenesis. 23A2RafER DD myoblasts were cultured in differentiation medium supplemented with increasing amounts of 4HT. After 48 h, total cellular lysates were prepared and analyzed by Western blot for MyHC, myogenin, and ERK1/2 protein expression. Relative amounts of proteins were quantified by scanning densitometry. MyHC and myogenin protein amounts were normalized to the amount of ERK2 protein. Mean Ϯ S.E. were Ͻ5% for each measurement.

FIG. 5. Raf activation alters two distinct phases of myogenesis.
23A2 and 23A2RafER DD myoblasts transiently transfected with TnI-Luc and pRL-tk were cultured in differentiation medium supplemented with increasing concentrations of 4HT. TnI-Luc was normalized to Renilla luciferase activity. The amount of reporter activity directed by 23A2 myocytes in the absence of 4HT was set to 100%. Low level Raf activity causes a slight increase in muscle reporter gene activity, whereas high level Raf activity inhibits myogenesis. Data represent the mean Ϯ S.E. of at least three independent experiments. RLU, relative light units. porter gene function. Therefore, we conclude that a TGF-␤-like factor is present in the medium harvested from 23A2RafER DD myoblasts transmitting high level Raf signals.
To determine whether the soluble factor is TGF-␤ 1 , genespecific primers for the growth factor were designed for the amplification of messages by RT-PCR. In brief, total RNA was harvested from 23A2 and 23A2RafER DD myoblasts treated for 48 h in differentiation supplemented with 2.5 M 4HT. Equal amounts of RNA were reversed-transcribed and amplified using primers specific for the TGF-␤ 1 and GAPDH. The cDNAs were amplified with Taq polymerase, and an aliquot of each reaction was removed after 25, 30, and 35 cycles (see "Materials and Methods" for conditions). Reaction products were separated through 2% agarose gels containing ethidium bromide. By semi-quantitative PCR, a cycle-dependent increase in GAPDH was evident in both control and Raf-repressive samples (Fig. 9). No amplicons for TGF-␤ 1 were detected in RNA isolated from control myocytes, but gene products for the growth factor were evident after 30 and 35 cycles in 23A2RafER DD myoblasts treated with 4HT (Raf-repressive) RNA samples. Northern blot analysis confirmed the increase in TGF-␤ 1 mRNA in Raf-expressing myoblasts (Fig. 9B).
Our results indicate that TGF-␤ 1 , a potent inhibitor of muscle differentiation, is produced by 23A2RafER DD myoblasts exhibiting extreme levels of Raf signaling and propose that this protein may be responsible for the block to muscle formation. Therefore, removal of secreted TGF-␤ may reverse the inhibi-tion of myogenesis in myoblasts with high level Raf signal intensity. To this end, cultures of 23A2RafER DD myoblasts treated with Me 2 SO or 500 nM 4HT were incubated for 48 h in differentiation medium supplemented with a soluble inhibitor of TGF-␤. The TGF-␤ inhibitor (␤SRI) is a chimera of the TGF-␤ receptor ligand binding domain fused to human Fc portion of IgG that prevents TGF-␤ 1 , TGF-␤ 2 , and TGF-␤ 3 from interacting with cell surface receptors (36,37). After 48 h, the cells were fixed and immunostained for MyHC and numbers of immunopositive cells were measured. 23A2RafER DD myoblasts in the absence of 4HT readily fuse into large multinucleated, MyHC-immunopositive fibers (Fig. 10). Treatment of the cells with TGF-␤ 1 (2 ng/ml) suppressed myocyte formation to Ͻ25% of control, indicating that the cells respond to the growth factor in a manner analogous to parental 23A2 skeletal myocytes. This level of inhibition also is attained by treatment of 23A2RafER DD myoblasts with 500 nM 4HT. However, supplementation of the culture medium with ␤SRI at a concentration that effectively restores myogenesis to TGF-␤ 1 -inhibited myoblasts does not reinstate the differentiation program to 23A2RafER DD myoblasts directing elevated Raf signaling. Thus, we conclude that TGF-␤ 1 inhibits the differentiation of 23A2RafER DD myoblasts and that the cells produce TGF-␤ 1 in response to elevated Raf but the establishment of an autocrine TGF-␤ 1 loop in these cells does not significantly contribute to the differentiation-defective phenotype-imposed Raf signaling.
GDF-8 Is Up-regulated in Response to Activated Raf and Can Inhibit Myogenesis-GDF-8, a member of the TGF-␤ superfamily, inhibits myogenesis through a Smad-dependent signaling pathway (38,39). Total RNA was isolated from 23A2 and 23A2RafER DD myoblasts treated for 48 h in differentiation media supplemented with 1 M 4HT and analyzed by RT-PCR for GDF-8, GAPDH, and RafER DD mRNA expression. As shown in Fig. 11, 23A2 myofibers do not contain detectable amounts of GDF-8 mRNA. By contrast, message levels for the gene are up-regulated in differentiation-defective 23A2RafER DD myoblasts, which also express abundant levels of RafER DD mRNA. No differences were found in GAPDH mRNA expression between control and Raf-repressive myoblasts. Thus, the increase in GDF-8 gene expression may contribute to the Raf-imposed block to myogenesis.

DISCUSSION
Activated Raf signal transmission represents a powerful disruptor of normal myocyte formation and muscle gene expression. The exact mechanism by which activated Raf alleles accomplish this feat remains largely unknown. Several contributing factors to the loss of myogenic capacity include inhibition of myogenin gene expression and cytoplasmic sequestration of MEF2 protein (21,33). Recently, we have demonstrated that forms of activated Raf that initiate activation of differential downstream signaling intermediates cause varying responses on myogenin gene expression that may be correlated to overall signal strength (33). 23A2RafER DD myoblasts were created to provide additional insight into the effects of Raf signal intensity on myocyte formation. In the absence of Raf activity, the cells readily form muscle in a manner analogous to parental 23A2 myoblasts. Interestingly, the introduction of a low level Raf-mediated signal stimulates an increase in the numbers of myocytes formed and enhances their ability to transcribe muscle genes. This is an intriguing finding in light of the fact that medium and high level Raf activities lead to a reduction in markers of muscle differentiation. Using stable clones of mouse myoblasts that direct differing levels of Raf kinase activity, DeChant et al. (40) reported that suboptimal Raf signal transmission leads to an increase in muscle reporter gene transcription and myocyte formation. The increase in myogenic capacity is not a reflection of diminished apoptosis because the low level Raf clone and parental myoblasts did not differ in the extent of DNA fragmentation. Therefore, it is likely that enhanced muscle formation in response to nominal Rafmediated signaling occurs independent of enhanced cell survival. Others have suggested that Raf inhibits myogenesis by maintaining the cell in a proliferative state (20). L6 myoblasts expressing an estrogen-inducible allele of activated Raf fail to express muscle genes or fuse into myocytes upon initiation of Raf signaling. In addition, these cells demonstrate an increased mitotic index in response to the kinase. 23A2RafER DD myoblasts and primary avian myoblasts expressing activated Raf are refractile to the mitogenic effects of Raf/MAPK (data not shown) (18). These contrasting results may reflect differences in the absolute amounts of Raf signaling. More importantly, these differences in myogenic responses directed by Raf signaling may explain the opposing effects of various growth factors on skeletal myogenesis. For example, both IGF-I and FGF2 initiate signaling through the Raf/MEK/ERK pathway, yet IGF-I promotes differentiation and FGF2 inhibits myocyte formation. Its possible that low level stimulation of ERK via Raf leads to the positive effects of IGF-I while a strong sustained ERK function may contribute to repression of myogenesis.
The ability of Raf signaling to inhibit myogenesis occurs through both MEK-dependent and independent signaling events. Low to moderate activation of Raf suppresses muscle gene transcription and myocyte formation, but these repressive effects are countered by inclusion of a chemical MEK inhibitor. Interestingly, severe amounts of Raf activity produce a condition that is refractile to the MEK inhibitor, suggesting the existence of a MEK-independent function. The MEK-independent signaling probably involves downstream kinases or phosphatases and is not a product of altered gene transcription because the addition of PD98059 was coincident with 4HT. Hence, Raf signaling was initiated simultaneously with MEK inhibition. The existence of Raf-controlled, MEK-independent signaling pathways have been described in fibroblasts recently (29). A constitutive Raf mutant that fails to associate with MEK and activate ERK1/2 can duplicate some of the effects of activated Raf including NFB transcriptional activation. Preliminary studies in our laboratory demonstrate that this Raf mutant abolishes the transcriptional activation properties of MyoD. 2 Thus, Raf-initiated, MEK-independent signaling pathways are present in myogenic cells and probably participate in the suppression of skeletal myogenesis.
High intensity Raf signaling causes cell cycle arrest and senescence in epithelial and fibroblast cells (25,27,30,41). Coincident with mitotic arrest, Raf initiates an increase in TGF-␤ 1 gene expression, leading to the creation of an autocrine loop that directly participates in the growth inhibition (42). Similarly, TGF-␤ I is up-regulated in rhabdomyosarcoma cells and may play a role in the block to myogenic differentiation (34). Our results indicate that elevated Raf signaling does elicit an increase in TGF-␤ 1 gene expression; however, this negative regulator of myogenesis does not contribute significantly to the differentiation-defective phenotype of Raf-expressing myoblasts. 23A2RafER DD myoblasts are inhibited by TGF-␤ 1 to the same extent as parental cells, arguing that the Raf-expressing cells are not refractile to the inhibitory actions of the growth factor and that the cells synthesize a functional TGF-␤ receptor. It is possible that TGF-␤ is secreted and retained in the extracellular matrix of the muscle cells, thus leaving it unavailable for sequestration by the soluble TGF-␤ inhibitor. This scenario is doubtful as retention of the TGF-␤ 1 by the extracel-2 X. Wang and S. E. Johnson, unpublished data. Total RNA was isolated and reverse-transcribed. PCR amplification was performed using RafER DD , GAPDH, and GDF-8 gene-specific primers. Five microliters of the amplification reaction (50 l total) was separated through a 2% agarose gel impregnated with ethidium bromide. lular matrix would inactivate the growth factor and would lend itself to the promotion of myogenesis, a condition contradictory to those observed. Thus, it is most likely that TGF-␤ 1 production in response to activated Raf does not play a significant role in the suppression of muscle formation.
Treatment of parental myoblasts with conditioned medium from Raf-repressive myoblasts causes a significant increase in the amount of Smad-driven reporter gene transcription. This finding endorses the synthesis and release of a TGF-␤-like soluble factor by Raf-expressing cells. The identity of the factor remains unknown. Of the TGF-␤ superfamily members, GDF-8 (also referred to as myostatin) represents a strong candidate for the putative secreted factor. GDF-8 myoblast differentiation in vitro and animals lacking or carrying a dysfunctional GDF-8 gene exhibit myocyte hypertrophy (38,(43)(44)(45)(46). The growth factor signals through the activin receptor, a membrane-bound receptor that transmits information through the Smad proteins (39,47). Previously, our group reported an inhibition of DNA synthesis in avian myoblasts transduced with a highly active allele of Raf (18). GDF-8 not only inhibits myogenesis, but the growth factor also causes a noticeable reduction in proliferation rates in myoblasts (35,48). These two pieces of information lend support to the notion that GDF-8 acts through an autocrine loop to synergize with Raf signaling and more effectively inhibit myogenesis. However, 23A2RafER DD myoblasts treated with 1 M 4HT fail to display a mitotic index that differs significantly from control myoblasts (8 versus 13%, respectively). Moreover, the concentrations of GDF-8 required to inhibit differentiation of 23A2 myoblasts is ϳ10-fold higher than the obligatory concentration of TGF-␤ 1 (data not shown). These results suggest that GDF-8 is not as effective as TGF-␤ 1 for suppression of the myogenic program. Alternatively, it is possible that a novel TGF-␤ family member contributes to the Raf-induced block to muscle formation.
In summary, Raf signal transmission directs contrasting effects on skeletal myoblasts that is commensurate with intensity level. Low level Raf/MAPK signaling elicits a positive effect on myogenesis that is reflected by enhanced regulatory and contractile protein synthesis. By contrast, sustained and extreme levels of Raf signaling leads to the prototypical reduction in myocyte formation and muscle protein expression. Coincident with repression of myogenesis by activated Raf is an increased expression of TGF-␤ 1 and GDF-8 and release of a soluble TGF-␤-like factor into the medium. Although the identity of the secreted protein remains unknown, it likely is not TGF-␤ 1 because incubation of Raf-repressive myoblasts with an inhibitor of the growth factor does not reinstate the differentiation program.


s2fieldsofstudy: Biology Medicine

text:
Expression of inwardly rectifying potassium channels (GIRKs) and beta-adrenergic regulation of breast cancer cell lines

Background Previous research has indicated that at various organ sites there is a subset of adenocarcinomas that is regulated by beta-adrenergic and arachidonic acid-mediated signal transduction pathways. We wished to determine if this regulation exists in breast adenocarcinomas. Expression of mRNA that encodes a G-protein coupled inwardly rectifying potassium channel (GIRK1) has been shown in tissue samples from approximately 40% of primary human breast cancers. Previously, GIRK channels have been associated with beta-adrenergic signaling. Methods Breast cancer cell lines were screened for GIRK channels by RT-PCR. Cell cultures of breast cancer cells were treated with beta-adrenergic agonists and antagonists, and changes in gene expression were determined by both relative competitive and real time PCR. Potassium flux was determined by flow cytometry and cell signaling was determined by western blotting. Results Breast cancer cell lines MCF-7, MDA-MB-361 MDA-MB 453, and ZR-75-1 expressed mRNA for the GIRK1 channel, while MDA-MB-468 and MDA-MB-435S did not. GIRK4 was expressed in all six breast cancer cell lines, and GIRK2 was expressed in all but ZR-75-1 and MDA-MB-435. Exposure of MDA-MB-453 cells for 6 days to the beta-blocker propranolol (1 μM) increased the GIRK1 mRNA levels and decreased beta2-adrenergic mRNA levels, while treatment for 30 minutes daily for 7 days had no effect. Exposure to a beta-adrenergic agonist and antagonist for 24 hours had no effect on gene expression. The beta adrenergic agonist, formoterol hemifumarate, led to increases in K+ flux into MDA-MB-453 cells, and this increase was inhibited by the GIRK channel inhibitor clozapine. The tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a high affinity agonist for beta-adrenergic receptors stimulated activation of Erk 1/2 in MDA-MB-453 cells. Conclusions Our data suggests β-adrenergic receptors and GIRK channels may play a role in breast cancer.

Background
Breast cancer is the leading cancer in women [1] and estrogen receptor (ER)(-) breast cancers have a poorer prognosis than ER(+) cancers [2,3]. Smoking is a controversial risk factor for the development of these malignancies [4][5][6][7]. However, increases in pulmonary metastatic disease and lung cancer have been seen in smokers with breast cancer [8,9]. The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) causes cancer of the oral cavity, esophagus, respiratory tract and pancreas, but no breast cancer in laboratory animals [10] and has not been implicated in breast carcinogenesis to date.
Recent studies in human cancer cell lines or in animal models have shown that the growth of adenocarcinomas of the lungs, pancreas and colon are under β-adrenergic control [11][12][13][14][15]. Studies in a cohort of 2442 men found an inverse association between risk of incident adenocarcinomas of the prostate and use of antihypertensive medication, including beta-blockers [16]. The tobacco-specific carcinogenic nitrosamine NNK has recently been identified as a high affinity β-adrenergic agonist that stimulated the growth of pulmonary and pancreatic adenocarcinomas in vitro and in animal models [11,13,15]. The expression of β-adrenergic receptors has been correlated with the over-expression of the arachidonic acid-metabolizing enzymes cyclooxygenase-2 (COX-2) and lipoxygenases (LOX) in adenocarcinomas of lungs [17], colon [18], prostate [19], and pancreas [15]. Inhibitors of these enzymes have been identified as cancer preventive agents in animal models of these cancers [13,[20][21][22]. Collectively, these findings suggest that among the superfamily of adenocarcinomas at various organ sites, there is a subset of malignancies that is regulated by β-adrenergic and arachidonic acid-mediated signal transduction pathways.
The majority of breast cancers are also adenocarcinomas and many of them over express COX-2 and/or LOX [23]. This raises the possibility that comparable to findings in adenocarcinomas of the lungs, pancreas, colon and prostate, a subset of breast cancers may also be under betaadrenergic control. In support of this hypothesis, studies have demonstrated that three estrogen-responsive and three non-estrogen responsive human cell lines derived from breast adenocarcinomas demonstrated a significant reduction in DNA synthesis in response to beta-blockers or inhibitors of the arachidonic acid-metabolizing enzymes COX-2 and 5-LOX [24]. In addition, analysis by reverse transcription polymerase chain reaction (RT-PCR) revealed expression of β 2 -adrenergic receptors in all six breast cancer cell lines tested (MDA-MB-361, ZR-75-1, MCF-7, MDA-MB-453, MDA-MB-468, MDA-MB-435S), whereas β 1 receptors were not found in two estrogen non-responsive cell lines (MDA-MB-435S, MDA-MB-453) [24].
Expression of mRNA that encodes a G-protein coupled inwardly rectifying potassium channel (GIRK1) has been shown in tissue samples from approximately 40% of primary human breast cancers tested [25], and this expression of GIRK1 was associated with a more aggressive clinical behavior. Increases in GIRK currents by betaadrenergic stimulation have been reported in adult rat cardiomyocytes and in Xenopus laevis oocytes coexpressing β 2 -adrenergic receptors and GIRK1/GIRK4 subunits [26].
In addition, in rat atrial myocytes transiently transfected with β 1 or β 2 adrenergic receptors, the beta-adrenergic agonist isoproterenol stimulated GIRK currents, whereas this stimulation was not seen in non-transfected cells [27]. The current investigations test the hypothesis that GIRK1 channels in human breast cancers are correlated with beta-adrenergic control.

Relative competitive RT-PCR
Preliminary experiments were done with MDA-MB-453 cells to determine a cycle number of PCR amplification that is within the linear range, which is critical for meaningful results to compare expression levels between samples and to determine the mixture of 18S primers/18S competimers (Ambion-Classic II). The 18S ribosomal RNA primers/competimers are used as an invariant internal control, which allows correction for sample variation. Results indicated this was 31 cycles of PCR and a 1:9 18S primer/competimers ratio. For experimental treatments, as described before [33], cDNA was made and PCR performed except reactions were spiked with 5 µCi [α-32 P]-dCTP (3000 Ci/mmole, Dupont-NEN, Boston, MA). Reactions were run with the following conditions: 1 cycle of 2 min. at 94°C, then 31 cycles of 94°C, 30 sec; 55°C, 30 sec; 72°C, 45 sec. A 10 µl sample of each PCR reaction was heated at 95°C for 3 min., then loaded into a 5% TBEurea Ready Gel (Bio-Rad, Hercules, CA). This underwent electrophoresis at 200 V in TBE buffer until the xylene cyanol dye front reached the bottom of the gel. The gel was transferred to filter paper, dried and exposed to film or imaged on a Molecular Dynamics 445 SI phosphoimager (Sunnyvale, CA). A 100 bp DNA ladder (Invitrogen-Life Technologies) was exchange labeled with T4 polynucleotide kinase and 30 µCi [γ-32 P] ATP (3000 Ci/mM, Dupont-NEN).

Measurement of potassium flux
We determined inward potassium flux in these cells by flow cytometry via the method of Krjukova et al. [30]. The negatively charged fluorescent dye bis-(1,3-dibutylbarbituric acid)trimethine oxonol (DiBaC 4 (3)) (Molecular Probes, Eugene, OR) was added to MDA-MB-453 breast cancer cell line suspensions of 1 × 10 6 cells at a final concentration of 150 × 10 -9 M. Fluorescence intensity measurement after treatment of the cells was obtained from a FACS Vantage/SE Cell Sorter (San Jose, CA).

Analysis of protein expression by western blots
Following incubation with agents as detailed in the Figure legends, cells were washed twice with phosphate buffered saline and lysed with cold RIPA lysis buffer containing protease inhibitors (50 mM Tris pH 7.4, 150 mM NaCl, 1% NP-40, 1% Triton × 100, 0.1% SDS, 1% sodium deoxycholate, 1 mM EDTA, 50 mM NaF, 10 mM sodium pyrophosphate, 0.5 mM DTT). Cell lysates were collected from culture plates using a rubber policeman, and protein collected by centrifugation. Protein concentrations were determined by BCA protein assay (Pierce, Rockford, IL). Aliquots of 20 µg protein were boiled in 2x loading buffer (0.1 M Tris-Cl, pH 6.8, 4% SDS, 0.2% Bromophenyl blue, 20% glycerol) for 4 minutes, then loaded onto 10% Tris-HCl-Polyacrylamide gels (Biorad, Hercules, CA), and transferred electrophoretically to nictrocellulose membranes. Membranes were incubated with primary antibodies (phospho-Erk; Cell Signaling, Beverly, MA) and appropriate secondary antibodies (Cell Signaling or Rockland, Gilbertsville, PA or Molecular Probes, Eugene OR). In all western blots, membranes were additionally probed with an antibody for actin (Sigma) to ensure equal loading of protein between samples. The antibody-protein complexes were detected as previous described [28] or by the LiCor Odyssey infrared imaging system (Lincoln, NE).

Results
The  (Figure 1). GIRK1 is also not expressed in the normal breast epithelial cell line MCF 10A (data not shown). The PCR product from the MDA-MB-453 cell line was sequenced to verify the integrity of the PCR process and found to be homologous to the published sequence (data not shown). The PCR primers were designed to span exon 1 and 2 of GIRK1 [29]. In addition PCR amplification of negative control reactions (without the reverse transcriptase enzyme, data not shown) indicated that this was actually representative of mRNA expression and there was no contaminating genomic DNA. Since the GIRK1 potassium channels work as heterotetramers, we needed to determine which other GIRK channels were expressed in these breast cancer cell lines. As determined by RT-PCR, GIRK4 was expressed in all six breast cancer cell lines (Figure 2), and GIRK2 was expressed in four of the six cell lines. GIRK2 was not expressed in ZR-75-1 or MDA-MB-435S cell lines ( Figure 2).
To determine if GIRK channels are functionally linked with β-adrenergic receptors in breast cancer cells expressing this ion channel, we decided to investigate the ER(-) cell line MDA-MB-453. This ER (-) cell line, which was the only ER(-) cell line tested that expressed GIRK1, was used for further experiments due to the fact that ER(-) breast cancers have a poorer prognosis than ER(+) cancers [2,3]. In addition, previous research in our laboratories indicated that this cell line expressed the β 2 adrenergic receptor but not the β 1 receptor [24]. MDA-MB-453 were continuously exposed to the beta-blocker propranolol (1 µM) for 6 days. Previous results from our laboratories indicated that maximal inhibition of breast cancer cell proliferation was at 1 µM propranolol [24]. Using relative RT-PCR, we saw a significant increase in GIRK1 channel mRNA expression (1.6 fold, Figure 3) after 6 days of con-tinuous exposure to propranolol (p < 0.0001 by t-test). In these experiments, propranolol was added fresh each day. We also saw a significant decrease (1.5 fold) in β 2 -adrenergic receptor mRNA (p < 0.0079 by t-test) (data not shown).
Using the same cDNA samples, we performed a real-time RT-PCR assay using GIRK1 primers designed for real-time PCR and a TaqMan probe. We also saw a significant increase of GIRK1 mRNA using this method ( Figure 4) and no change in the control, beta-actin values. Threshold values (C T ) were calculated for each sample, which will be lower for samples with more mRNA expression. C T values for GIRK 1 expression were significantly (p  Figure 6A &6B). Dye alone added to cells had no effect (data not shown). The GIRK inhibitor clozapine [31] (50 µM) added just prior to dye and formoterol addition completely blocked the effect of the beta-adrenergic agonist formoterol, ( Figure 6C) indicating that blockage of the GIRK channel inhibited potassium flux, and that effects of beta-adrenergic agents on this breast cancer cell line are indeed mediated by GIRK channels.
We also determined signaling events in MDA-MB-453 cells that are affected by beta-adrenergic agents. Increased activation of Erk 1/2 was seen in MDA-MB-453 cells after treatment with 100 pM NNK at times ranging from 15-150 minutes (Figure 7). The concentration of NNK used by us is within the range of systemic NNK concentrations found in smokers. In addition, an experiment in Patas monkeys [32] has shown blood levels of 1.6 pg/ml (7.72 × 10 -12 M) after exposure to a dose of tritiated NNK equivalent to the amount of NNK found in two packs of cigarettes. Stimulation of Erk 1/2 was also seen using 1 µM of the beta-adrenergic agonist formoterol, but only at 150 minutes (data not shown).

Discussion
Our data demonstrate expression of the G-protein inwardly rectifying potassium channel 1 (GIRK1) in 67% of the breast cancer cell lines tested, with higher levels in ER(+) cell lines. Approximately 40% of primary human breast cancers were found to express GIRK1 and expression of GIRK1 was not found to be correlated with ER status [25]. These differences in our studies may be due to the subset of breast cancer cell lines tested. We also found that the normal breast epithelial cell line MCF 10A lacked GIRK1 expression (data not shown). GIRK1 cannot form functional channels by itself, other GIRK channels are needed [33]. All six breast cancer cell lines tested express either GIRK2 or GIRK4 indicating that functional GIRK Comparison of GIRK1 mRNA expression levels by relative competitive RT-PCR in MDA-MB-453 cells treated with pro-pranolol for 30 minutes daily for 7 days The majority of experiments in the present study were done with the ER(-) cell line MDA-MB-453 since it was the only ER(-) cell line tested that expressed GIRK1, and because ER(-) breast cancers have a poorer prognosis than ER(+) cancers [2,3]. We saw a significant increase in GIRK1 channel mRNA expression after 6 days of continuous exposure to propranolol in MDA-MB-453 cells. It is clear that at least six days of continuous exposure to the beta-blocker propranolol is necessary to effect gene expression. Gene expression of β 2 -adrenergic mRNA was decreased by the same treatment (data not shown). Addition of propranolol for 7 days for only 30 minutes daily had no effect on GIRK1 gene expression. Treatment for a shorter period of time (24 hours) also had no effect on GIRK1gene expression in our studies. The 6 day continuous exposure to propranolol caused a barely detectable decrease in GIRK2 mRNA expression and no change in GIRK4 mRNA expression levels. Longer treatment times may be necessary for gene expression changes in GIRK2 or GIRK4 similar to gene expression changes that are seen in GIRK1.
Although there were no short-term effects of beta-adrenergic agents on GIRK gene expression, we detected other cellular effects. The beta-adrenergic agonist formoterol hemifumarate stimulated potassium influx in MDA-MB-453 cells, and this influx was prevented by the GIRK channel inhibitor clozapine. NNK, a high affinity agonist for beta-adrenergic receptors [11] increased activation of Erk 1/2 in MDA-MB-453 breast cancer cells. Formoterol also increased activation of Erk 1/2, but to a lesser degree (data not shown). Previous studies indicated that the betaadrenergic agonist isoproterenol stimulates growth [24]. GIRK currents have been shown to be increased in cells stimulated with the beta-adrenergic agonist isoproterenol in rat atrial myocytes transfected with β 1 or β 2 receptors [27]. Heterologous facilitation of GIRK currents by βadrenergic stimulation was also seen in rat cardiomyocytes [26]. Two polymorphisms in the β 2 and β 3 adrenergic receptors were found to be correlated with a decreased risk for breast cancer [34], suggesting an important role of this receptor family in the genesis of breast cancer. In previous work, we demonstrated mRNA expression by RT-PCR of the β 2 adrenergic receptor in the six breast cancer cell lines used in this study, but expression of β 1 in all the estrogen responsive cell lines but not Figure 7 Levels of ERK activation in MDA-MB-453 cells as assessed by western blot analysis. Activation was determined after indicated times after exposure to 100 pM NNK using a phospho-specific antibody.

Levels of ERK activation in MDA-MB-453 cells as assessed by western blot analysis
in two ER(-) cell lines (MDA-MB-435S and MDA-MB-453) [24]. Further studies are needed to determine how GIRK1(+) and ER(-) breast cancers are regulated and if GIRK channel agonists and antagonists have effect on proliferation in breast cancer. It also remains to be determined if this same regulation is present in GIRK1(+) and ER(+) breast cancer malignancies. This is of particular importance since a recent report indicated that 17-β-estradiol can modulate GIRK channel activation in the brain [35]. Future studies are also needed to determine if GIRK3 is involved in breast cancer. However, we think this unlikely because one of the functions of GIRK3 is to inhibit plasma membrane expression of other GIRK subunits [36].

Conclusions
All six breast cancer cell lines tested express either GIRK2 or GIRK4 indicating that functional GIRK potassium channels are possible in these breast cancer cell lines. This is the first report that implicates β-adrenergic receptors and G-protein inwardly rectifying potassium channels 1 (GIRK1) in the regulation of human breast cancer cells and suggests a potential role of the tobacco nitrosamine NNK in breast cancers expressing these regulatory pathways. Beta-adrenergic antagonists have both long term effects on gene expression and beta-adrenergic agonists have short term effect on potassium flux and cellular signaling pathways.