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Localization of MMR proteins on meiotic chromosomes in mice indicates distinct functions during prophase I

Mammalian MutL homologues function in DNA mismatch repair (MMR) after replication errors and in meiotic recombination. Both functions are initiated by a heterodimer of MutS homologues specific to either MMR (MSH2–MSH3 or MSH2–MSH6) or crossing over (MSH4–MSH5). Mutations of three of the four MutL homologues (Mlh1, Mlh3, and Pms2) result in meiotic defects. We show herein that two distinct complexes involving MLH3 are formed during murine meiosis. The first is a stable association between MLH3 and MLH1 and is involved in promoting crossing over in conjunction with MSH4–MSH5. The second complex involves MLH3 together with MSH2–MSH3 and localizes to repetitive sequences at centromeres and the Y chromosome. This complex is up-regulated in Pms2 − / − males, but not females, providing an explanation for the sexual dimorphism seen in Pms2 − / − mice. The association of MLH3 with repetitive DNA sequences is coincident with MSH2–MSH3 and is decreased in Msh2 − / − and Msh3 − / − mice, suggesting a novel role for the MMR family in the maintenance of repeat unit integrity during mammalian meiosis.

The mammalian meiotic MMR complex, consisting of MSH4, MSH5, MLH1, and MLH3, has been implicated in the processing of DNA double strand breaks (DSB) through the double Holliday junction (dHJ) recombination intermediate repair pathway that results in reciprocal crossover events between parental homologous chromosomes (Ross-Macdonald and Roeder, 1994;Hollingsworth et al., 1995;Novak et al., 2001;Borner et al., 2004;Hoffmann and Borts, 2004;Surtees et al., 2004). Studies in mouse Santucci-Darmanin et al., 2000;Moens et al., 2002) and human germ cells (Lenzi et al., 2005) show that MSH4-MSH5 appear at recombination intermediates, as part of the multiprotein meiotic nodule (MN), early in prophase I and accumulate at frequencies that far exceed the eventual number of crossover sites . In vitro biochemical studies suggest that this heterodimer forms a sliding clamp that associates with dHJ structures at zygonema (Snowden et al., 2004) a subset of which may be stabilized by interactions with MLH1-MLH3 heterodimers at pachynema. It is these sites that will eventually become the crossovers that ensure accurate chromosomal segregation at the first meiotic division. The additional MSH4-MSH5 sites that are not stabilized by MLH1-MLH3 are then repaired through noncrossover mechanisms.
The temporal dynamics of MMR protein association with MNs raises the question of how MLH1 and MLH3 selects only a subset of MSH4-MSH5 sites to become crossover events. Previous studies have demonstrated that MLH3-only foci are apparent during pachynema of prophase I, whereas MLH1 appearance at crossovers is dependent on MLH3 (Lipkin et al., 2002). This suggests an MLH3-driven mechanism for dHJ selection. Alternatively, the involvement of a third MutL homologue, PMS2, has been suggested, at least in males, by the observation that Pms2 Ϫ / Ϫ males are sterile, whereas females remain fertile (Baker et al., 1995). By contrast, mutation of Mlh1 or Mlh3 results in sterility of both males and females (Baker et al., 1996;Edelmann et al., 1996;Woods et al., 1999;Lipkin et al., 2002). Interestingly, however, although germline crossover rates are decreased to ف 10% in male and female mice lacking Mlh1 (Baker et al., 1996;Woods et al., 1999), they appear to be unaffected in male mice lacking Pms2 (Qin et al., 2002). Because MLH1 is capable of heterodimerizing with both MLH3 and PMS2, this prompted us to explore the interactions between these MutL homologues at the chromosomal level in spermatocytes from wild-type mice, and from Mlh1 -, Mlh3 -, and Pms2null animals.
Using a variety of cytological and molecular techniques, we present genetic evidence that MLH3 associates with complexes involving both MSH2-MSH3 and MSH4-MSH5 in mammalian meiocytes, and that only the latter is necessary for reciprocal recombination. A second complex, consisting of MutS ␤ (MSH2-MSH3) together with MLH3, associates with regions of the genome that are rich in repetitive sequences, such as the major and minor satellites of the centromere region and the Y chromosome, suggesting a new role for MMR complexes in the mammalian germline.

Temporal dynamics of MutL association with MN during recombination
The synaptonemal complex (SC) is the tripartite protein structure that forms a continuous filament structure along homologous chromosomes and tethers them together during prophase I (Page and Hawley, 2004). The SC also serves as the docking site for key recombinational proteins of the MN.
To assess the dynamics of MutL homologue accumulation through prophase I, MLH1 and MLH3 were localized to SCs of mouse spermatocytes. MLH3 foci are found on the SC of wild-type spermatocytes from early pachynema (Fig. 1, A  and B), but MLH1 does not associate with MLH3 until midpachynema (Fig. 1, A and B). The early MLH3 foci probably account for the few instances of MLH3-positive, MLH1-negative foci reported in our previous studies (Lipkin et al., 2002), that we suggest then recruit MLH1 in mid-pachynema. MLH1 and MLH3 persist through to late pachynema and are rarely seen at diplonema. The number of MLH1-MLH3 foci correlates well with the predicted number of crossovers in mouse spermatocytes (Anderson et al., 1999), suggesting that MLH1-MLH3 marks most, if not all, of the reciprocal recombination sites during mouse meiosis, and making it less likely that MLH1-PMS2 heterodimers are marking another subset of such sites.
The MLH1-MLH3 localization pattern does not exclude the possibility of other PMS2 effects on meiotic progression. However, despite trying at least eight different anti-PMS2 antibodies, generated by our laboratory and others, it has not been possible to localize PMS2 in meiotic chromosome preparations. Instead, we examined the distribution of MLH1 and MLH3 on SCs from Pms2 Ϫ / Ϫ spermatocytes and found that both localize to the SCs at EM-defined MNs, with near normal temporal ( Fig. 1, C and D) and spatial frequency. MLH1 accumulates slightly earlier in the absence of PMS2 than in spermatocytes from wild-type males ( Fig. 1 D) but is still always found in combination with MLH3. The eventual number of MLH1 and MLH3 observed at late pachynema in Pms2 Ϫ / Ϫ spermatocytes is slightly (and, in the case of MLH1, significantly) higher than that seen in wild-type spermatocytes (Fig. 1, D and E; P ϭ 0.033 and 0.077 by Mann-Whitney U test, for MLH1 and MLH3, respectively). The normal accumulation of MLH1 and MLH3 on SCs of Pms2 Ϫ / Ϫ males is associated with the persistence of MN structures, as observed at the EM level ( Fig. 1 C), and with normal chiasma counts at metaphase I in these mice (unpublished data).
Our previous studies demonstrated that MLH1 fails to localize to SCs in spermatocytes from Mlh3 Ϫ / Ϫ males and that this was associated with loss of MNs at pachynema and absence of crossovers at metaphase (Lipkin et al., 2002). In view of the temporal dissociation of MLH1 and MLH3 accumulation observed in the current study, we hypothesized that MLH3 foci might localize independently of MLH1 on SCs from Mlh1 Ϫ / Ϫ spermatocytes. Indeed, immunogold-labeled SCs from Mlh1 Ϫ / Ϫ spermatocytes revealed large residual MNs that were loaded with MLH3 ( Fig. 1 F). Quantitation of MLH3-and MLH1-containing foci (by immunofluorescence microscopy) on SCs from spermatocytes of all MMR genotypes confirmed this observation, demonstrating that accumulation of MLH3 in Mlh1 Ϫ / Ϫ spermatocytes occurs at a significantly lower frequency than that in wild-type spermatocytes (Fig. 1 E; P Ͻ 0.0001 by Mann-Whitney U test), whereas MLH1 fails to localize at all on SCs of Mlh3 Ϫ / Ϫ spermatocytes, as described previously (Fig. 1 E;Lipkin et al., 2002). In view of the apparently normal recombination frequency in spermatocytes of Pms2 Ϫ / Ϫ males ( Fig. 1; Qin et al., 2002), we investigated whether other MMR-related functions might be disrupted in these animals. Examination of the SCs from Pms2 Ϫ / Ϫ males by EM revealed an accumulation of both MLH1 and MLH3 close to the centromere (Fig. 2 A), a region that is usually not subject to crossing over (Broman et al., 1998;Froenicke et al., 2002) and is therefore not expected to be positive for this MutL complex. Examination of the centromere regions from other MMR-deficient mouse strains revealed similar, but reduced frequency of, localization of MLH3 in spermatocytes from wild-type (Fig. 2 B) and Mlh1 Ϫ / Ϫ males ( Fig. 2 C), but not from Mlh3 Ϫ / Ϫ males (Fig. 2 D). In wild-type and Mlh1 Ϫ / Ϫ males, MLH3 associated with ف 15-25% of all spermatocyte autosomal centromeres. Little if any MLH1 was associated with centromere regions in wild-type, Mlh1 Ϫ / Ϫ , or Mlh3 Ϫ / Ϫ mice (Fig. 2, B-E). The localization of MLH3 at centromeres increased to include Ͼ 85% of centromere regions in spermatocytes from Pms2 Ϫ / Ϫ males, and colocalized at most of these sites ( Ͼ 65% of total centromeres) with MLH1 ( Fig. 2 E). The EM images of immunogold-labeled spermatocyte spread preparations were supported by similar localization at the immunofluorescence level ( Fig. S1 available at http://www. jcb.org/cgi/content/full/jcb.200506170/DC1), but was much weaker using fluorescently-labeled secondary antibodies (which are monoclonal antibodies) than using gold-labeled secondaries (polyclonal antibodies). The EM analysis involves a higher concentration of primary antibody, coupled with DNaseI digestion of heterochromatin to improve accessibility of the antibodies to the centromere sites, perhaps explaining the difference in staining intensity.
Considering mouse centromeres are telocentric, we examined whether the accumulation of MLH1-MLH3 at the centromere region in Pms2 Ϫ / Ϫ spermatocytes was due to the centromere per se or the closely-adjacent telomere. We did not observe elevated MLH1-MLH3 accumulation at the distal telomere of Pms2 Ϫ / Ϫ males (Fig. 2 F). Moreover, the Y chromosome, which has a larger expanse between the proximal telomere and the centromere, showed specific accumulation to the centromeric region (Fig. 3), leading us to conclude that the accumulation of MLH3 at these regions was associated specifically with centromere-associated sequences. The presence of MLH3 at centromere regions in wild-type and Mlh1 Ϫ / Ϫ spermatocytes indicates that MLH3 is able to localize to the chromosome independently of MLH1, as it does at early MNs (Fig. 1, B and C).

Localization of MLH3 and MLH1 at centromeric regions is associated with major and minor satellite repeat sequences
The mammalian centromere is a highly specialized structure consisting of protein-associated centromeric sequences and ad-jacent minor satellite DNA flanked by pericentric major satellite DNA (Guenatri et al., 2004). Major/minor satellites are regions of highly repetitive heterochromatin-forming sequences that are important for centromere function associated with spindle attachment and sister chromatid cohesion. In mice, the major satellite consists of tandem repeats of 234-bp units, of which there are ف 700,000 copies per cell, reflecting an average of 35,000 copies per chromosome (totaling 8 MB in length; Horz and Altenburger, 1981). The minor satellite binds histone H3 and CENPA/B, is present in 50-100,000 copies, and appears to share common ancestry with the major satellite (Silver, 1995). In humans, similar repeats of ف 171 bp are present but are termed ␣ satellites (Muro et al., 1992).
The localization of MLH3 (and in some cases, MLH1) at the centromere prompted us to investigate the specificity of this association with centromere-associated repeats. We performed chromatin immunoprecipitation (ChIP) experiments using either crude germ cell extracts (consisting of mixed populations of germ cells) or isolated pachytene spermatocytes (estimated to be Ͼ 95% pure). MLH3-containing complexes were immunoprecipitated using an anti-MLH3 antibody, followed by amplification using degenerate oligonucleotide PCR (Telenius et al., 1992) and subsequent slot-blot analysis using PCR-generated probes specific to mouse major and/or minor satellite sequences (Garagna et al., 2002). A B2 SINE element probe was used as a loading control (Serdobova and Kramerov, (C) C 500 nm. All SCs are counterstained with phosphotungstic acid. (E) Quantitation of MMR protein localization to centromeres from Mlh1 ϩ/ϩ , Mlh1 Ϫ/Ϫ , Mlh3 ϩ/ϩ , Mlh3 Ϫ/Ϫ , Pms2 ϩ/ϩ , and Pms2 Ϫ/Ϫ spermatocytes. Counts were obtained from immunogold-EM micrographs exemplified in A-D and represent those centromeres showing more than four gold grains for each antibody staining. At least 100 centromeres were counted for each genotype from three mice. Percentages represent pooled counts from all these mice. MLH3 (striped bars) immunolocalizes to 15-25% of wild-type and Mlh1 Ϫ/Ϫ , but not Mlh3 Ϫ/Ϫ , spermatocyte centromeres. MLH1 (black bars) does not localize in any appreciable amount to centromeres in wild-type, Mlh1 Ϫ/Ϫ or Mlh3 Ϫ/Ϫ spermatocytes. CREST (white bars) localizes to 100% of the centromeres. Pms2 Ϫ/Ϫ spermatocyte centromeres show a statistically significant increase in localization of both MLH1 and MLH3 in comparison with the Pms2 ϩ/ϩ and all other mouse strains observed. (F) Quantitation of distal (noncentromeric) telomere localization (more than four gold grains) indicates that the localization of MutL homologues is specific to the centromere. (G) EM micrograph of MLH1 (6 nm gold grains) and MLH3 (12 nm gold grains) on SCs from Pms2 Ϫ/Ϫ oocytes indicates that, whereas MLH3 and MLH1 localize normally to MNs (bottom inset), these MutL homologues fail to localize to the centromere (cen), labeled with CREST (18 nm gold grains; top inset). Bar,200 nm. 1998), representing a ubiquitous genome-wide repeat element, and showing that MLH3 does not bind to all repeat sequences throughout the genome.
ChIP of mixed germ cell extracts from Pms2 Ϫ/Ϫ males with the anti-MLH3 antibody enriched centromeric major/minor satellite sequences above control levels at comparable rates to that seen for polyclonal CREST serum, which recognizes a variety of CENPs (Fig. 4, A and B). The enrichment of centromere repeats in Pms2 Ϫ/Ϫ spermatocytes with anti-MLH3 antibody or with CREST was statistically elevated compared with mock controls (unpaired t tests, P Ͻ 0.0001). No enrichment of similar regions was found in ChIP extracts from Mlh1 Ϫ/Ϫ and Mlh3 Ϫ/Ϫ males, although specificity of enrichment was confirmed by PCR of random genomic sequences (␤-actin) using the same immunoprecipitates ( Fig. S2 available at http:// www.jcb.org/cgi/content/full/jcb.200506170/DC1).
To examine the specific contribution of major and minor satellite sequences to the observed MLH3 association, purified populations of Pms2-null spermatocytes were subjected to ChIP using anti-MLH3 antibody, followed by PCR to amplify banded sequences specific to each satellite. MLH3 associates specifically with both minor and major satellite sequences (Fig. 4 C, lanes 2 and 6), along with CREST immunoserum (Fig. 4 C, lanes 3 and 7). The enrichment of major and minor sequences by MLH3 or CREST immunosera was significantly raised above mock (no antibody) controls, although was greater for the MLH3 enrichment of minor satellites than either the MLH3 or CREST enrichment of major satellite sequences. CREST enrichment of minor sequences was barely visible because of the extent of the dilution required to optimize the MLH3 signal.

Association of MLH3 on the nonpseudoautosomal region of the Y chromosome
To investigate the association of the MMR machinery with other repetitive sequences in the genome, we explored the localization of MLH3 across the Y chromosome, which in humans and mice contains large palindromic repeat sequences (Bishop et al., 1985;Singh et al., 1994;Rozen et al., 2003;Skaletsky et al., 2003) and which, like the centromere, is refractory to reciprocal recombination except at the X-Y pseudoautosomal region (PAR). MLH3 localizes in dense foci along the Y chromosome in some pachytene spermatocytes from wild-type, Pms2 Ϫ/Ϫ and Mlh1 Ϫ/Ϫ males (Fig. 5 A, Pms2 Ϫ/Ϫ example shown), but the localization is not consistent among spermatocyte populations. In total, of the more than 500 nuclei examined from Pms2 Ϫ/Ϫ males, %01ف exhibited the Y chromosome accumulation of MLH3. No MLH1 association was found along the Y chromosome in wild-type or in Pms2 Ϫ/Ϫ spermatocytes (unpublished data). ChIP analysis confirmed the localization of MLH3 (Fig. 5, B and C), and the absence of MLH1 (unpublished data), on Y chromosome sequences from Pms2 Ϫ/Ϫ males. centromere ChIP experiments, expressed as a percentage of input DNA ϩ SD. Statistical analysis was performed using unpaired t-tests (***P Ͻ 0.0001). (C) ChIP assay followed by PCR using primers against minor and major satellite sequences performed on purified pachytene cells from Pms2 Ϫ/Ϫ testes. Lanes (from left to right): lane 1, molecular weight marker; lanes 2-5, minor satellite PCR; lanes 6-9, major satellite PCR. M, 100 bp marker; Input, input DNA. 10-fold dilutions of ChIP pulldown substrate DNA was used for minor satellite PCR, DNA for major satellite PCR was undiluted. Immunogold-EM localization of MLH1 (6 nm gold grains, green arrows), MLH3 (12 nm gold grains, red arrows), and CREST (18 nm gold grains, blue arrows) on meiotic chromosome cores of Pms2 Ϫ/Ϫ sex chromosomes. The p-arm of the Y chromosome is cytologically distinct from the q-arm, which contains the PAR that synapses with the PAR of the X chromosome. Due to the length of the p-arm, the Y chromosome centromere (top inset) can be differentiated from the terminally located telomere and both MLH1 and MLH3 localize to both the X (bottom inset) and the Y chromosome centromere. SCs are counterstained with phosphotungstic acid. Bar, 500 nm.

MutS␤ (MSH2-MSH3) but independent of MutS␥ (MSH4-MSH5)
The observations outlined above indicate that MLH3 localizes to centromere repetitive sequences and the Y chromosome in spermatocytes from wild-type mice, and hyperlocalizes to centromeric regions, along with MLH1, when PMS2 is absent. That both of these sites do not usually undergo reciprocal exchange suggests that the DNA-binding MutS partners for MLH3 at these sites are not the MSH4-MSH5 heterodimers that are usually only associated with DSB-induced recombination intermediates in meiotic cells. Therefore, we hypothesized that another MSH2-containing MutS heterodimer could be associated with these sites. In view of yeast genetic data demonstrating that Mlh3 associates with MutS␤ (MSH2-MSH3) heterodimers (and not MutS␣, MSH2-MSH6) to effect repair (Flores-Rozas and , this complex was the most obvious choice for investigation. Immunofluorescence analysis of chromosome preparations from wild-type spermatocytes demonstrated that MSH2 and MSH3 localize strongly to the centromere (Fig. 6, A-F) and on the sex chromosomes (Fig. 6, C and F, arrows), and that this localization is as frequent in wild-type spermatocytes as it is in Pms2 Ϫ/Ϫ males (Fig. 7, A-C and J). These observations indicate that the association of MSH2-MSH3-containing complexes appear to be a feature of normal prophase I and are not specifically up-regulated in the absence of PMS2. No association of MSH2 with these genomic regions is observed in spermatocytes from Msh3 Ϫ/Ϫ males either by immunofluorescence or EM (Fig. 6 G and Fig. 7, D-F), indicating that MSH3 is required for MSH2 localization to these sites. By contrast, immunofluorescence and immunogold localization of MSH2 at the centromere is not affected in spermatocytes from Msh4 Ϫ/Ϫ , Msh5 Ϫ/Ϫ , or Msh6 Ϫ/Ϫ males (Fig. 6, H and I, and Fig. 7, G-J). MSH2, and to a lesser degree MSH3, localizes along the chromosomes, but at an intensity that is far lower than that seen at the centromere (Fig. 6, A and D, arrowheads; Fig. 7, A and G, arrowheads). This interstitial localization could be the result of canonical MMR activity.
Quantitation of MLH1 and MLH3 association with centromere regions by immunogold-EM in spermatocytes from Msh2 Ϫ/Ϫ , Msh3 Ϫ/Ϫ , Msh5 Ϫ/Ϫ , and Msh6 Ϫ/Ϫ males confirmed the requirement for MSH2-MSH3, and not MSH2-MSH6 or MSH4-MSH5, to localize MLH3 to these sites (Fig. 6 J). In Msh2 Ϫ/Ϫ males, the localization of MLH3 at sites of reciprocal exchange is normal, but there is a statistically significant decrease in the localization of MLH3 at the centromere, from the usual 15-25% frequency seen in wild-type pachytene spermatocytes to near undetectable levels (Fig. 6 J; chi-square analysis P Ͻ 0.0002). Similarly, significantly decreased centromere localization of MLH3 is observed in spermatocytes from Msh3 Ϫ/Ϫ animals (Fig. 6, G and J; 2 analysis P Ͻ 0.0002), but not in Msh5 Ϫ/Ϫ or Msh6 Ϫ/Ϫ males (Fig. 6, H-J; 2 analysis P ϭ 0.81 and P ϭ 0.04, respectively). Thus, the association of MLH3 with repeat sequences uses the MSH2-MSH3 complex, whereas MLH1-MLH3 association at MNs uses MSH4-MSH5. Combined antibodies against MSH2-MSH3 were used in ChIP assays and were shown to enrich sequences within the centromeric (unpublished data) and Y chromosome repeat domains (Fig. 5, B and C). This MSH2-MSH3 enrichment of repetitive sequences is observed in spermatocyte extracts from wild-type males as well as in Pms2 Ϫ/Ϫ extracts, whereas MLH3 enrichment is only observed in the absence of PMS2. These data confirm the immunolocalization observations indicating that MSH2-MSH3 is constitutively associated with centromeric repetitive sequences and the Y chromosome. This repeat association appears to be meiosis-specific as no centromeric localization of MSH2-MSH3 or MLH3 was observed in somatic cells in vitro (unpublished data).

Analysis of Msh2 ؊/؊ Pms2 ؊/؊ double-null males suggests that PMS2 is not acting directly at the centromere region
The meiotic failure observed in Pms2 Ϫ/Ϫ males together with the hyperlocalization of MLH1-MLH3 at the spermatocyte centromeres in these mice indicated a role for PMS2 at centromere repeats, possibly dependent on MSH2-MSH3. Confounding this hypothesis, however, is the normal fertility in Msh2 Ϫ/Ϫ males. One possible explanation is that MSH2-MSH3 heterodimers act as a repeat tract surveillance mechanism that, under normal circumstances, signals to a PMS2-containing complex to effect repeat-specific processes. In the absence of PMS2, the inability of MSH2-MSH3 to signal downstream events would result in checkpoint-induced apoptotic removal of the germ cells, and subsequent infertility, but the absence of MSH2 would prevent checkpoint activation and hence allow meiotic progression even in the absence of PMS2. If true, we hypothesized that the removal of MSH2 in the Pms2 Ϫ/Ϫ males would restore spermatogenesis. To test this, we generated double-null males for Msh2 and Pms2, and counted epididymal sperm in adult males. Sperm numbers in Msh2 Ϫ/Ϫ Pms2 ϩ/Ϫ males were close to that found in double heterozygous males ( Table I). As expected, sperm numbers in Msh2 ϩ/Ϫ Pms2 Ϫ/Ϫ males was reduced to %5.3ف of normal, but swimming sperm were observed, albeit at a very reduced frequency (Table I). Sperm numbers in Msh2 Ϫ/Ϫ Pms2 Ϫ/Ϫ were similar to that seen in Pms2 Ϫ/Ϫ single nulls, with a few (8 out of a total of 1,102 sperm counted) swimming sperm. Thus, deletion of Msh2 does not restore the fertility of Pms2 Ϫ/Ϫ males. During the course of these experiments, we sought to identify the basis for the sexually dimorphic meiotic phenotypes exhibited by male and female Pms2 Ϫ/Ϫ mice. Interestingly, we observed no increase in MLH1-MLH3 association with centromeric repetitive sequences in prophase I oocytes from Pms2 Ϫ/Ϫ females (Fig.  2 G). This suggests that, although hyperaccumulation of MLH proteins at male centromeres in the absence of PMS2 causes apoptosis, the absence of such accumulation in females does not trigger apoptosis. Thus, oocytes from Pms2 Ϫ/Ϫ females are viable and the mice are fertile. Alternatively, in view of the hyperlocalization of the MMR proteins on the Y chromosome, we hypothesized that loss or destabilization of Y chromosome genes critical for spermatogenesis might result in spermatogenic failure specifically in males. However, microarray analysis of germ cell RNA through prophase I from Pms2 ϩ/ϩ and Pms2 Ϫ/Ϫ spermatocytes, using the 34,000 gene Affymetrix gene chips, revealed no specific changes in expression profiles of Y chromosome genes in the absence of PMS2, suggesting that the failure of spermatogenesis in Pms2 Ϫ/Ϫ males is unlikely to be due to misexpression of spermatogenic genes residing on the Y chromosome. Instead, our data point to differential effects on MMR protein accumulation and/or functioning at centromere-associated repetitive sequences in male versus female Pms2-null mice. , and CREST (empty bars) on spermatocyte centromeres from wild-type males, and males that are homozygous for mutations in Msh2, Msh3, Msh5, and Msh6. MLH3 localization is significantly decreased compared with wild type on centromeres from Msh2 Ϫ/Ϫ and Msh3 Ϫ/Ϫ spermatocytes, but not on centromeres from Msh5 Ϫ/Ϫ or Msh6 Ϫ/Ϫ spermatocytes. 2 analysis of MLH1 and MLH3 localization at spermatocyte centromeres reveals statistically significant differences between wild-type and both Msh2 Ϫ/Ϫ and Msh3 Ϫ/Ϫ males (***P Ͻ 0.0002), but not between wild-type and Msh5 Ϫ/Ϫ males (not significant, n.s., P ϭ 0.81). MLH3 levels were slightly increased at the centromeres of Msh6 Ϫ/Ϫ spermatocytes (*P ϭ 0.043).

Discussion
The principal observations in this report include the following. First, the MLH1-MLH3 heterodimer appears to account for all the MNs found in late pachytene spermatocytes, a situation similar to that seen in yeast and their frequency is slightly elevated in the absence of PMS2. Second, MLH3 localizes to these sites before MLH1 and can associate with other regions of the genome independently of MLH1. Localization of MLH3 throughout the genome is substantially reduced but not eliminated in spermatocytes from Mlh1 Ϫ/Ϫ males and is up-regulated in spermatocytes from Pms2 Ϫ/Ϫ males. Third, MLH3 associates with repeat sequences around the centromere and on the Y chromosome during pachynema in spermatocytes from wild-type and Mlh1 Ϫ/Ϫ males. This localization appears to differ from the localization at the MN sites because the pattern of distribution is not limited to chromosome cores, but instead appears diffuse and heterochromatic in its distribution. In addition, these sites do not appear to accumulate other DSB repair machinery components (such as RAD51; unpublished data), suggesting that DSBs are not forming at these sites. Fourth, the association of MLH3 with repeat sequences at the centromere is significantly increased, with almost all centromeres now being positive for MLH3. In addition, in the absence of PMS2, MLH1 associates with these MLH3 sites at repeat sequences. Fifth, the association of MLH3 (and MLH1) with repeat sequences at the centromere and on the Y chromosome during prophase I is dependent on MSH2 and MSH3, but not on MSH4, MSH5 or MSH6. Moreover, MSH2 localizes to these sites of repetitive sequence in all spermatocytes, both wild-type and Pms2 null, and this localization is dependent on MSH3 (but not MSH4, 5, or 6).
Taken together these observations suggest that MLH1-MLH3 is the sole MutL heterodimer responsible for processing Values are means Ϯ SD. ND, not determined (assumed to be greater than 95%. Normal sperm samples had to be formalin fixed in order to be counted). of meiotic crossovers and rule out a direct function for PMS2 in crossing over, paralleling the situation in Saccharomyces cerevisiae (Wang et al., 1999). In addition, these data demonstrate the existence of an additional meiotic MMR complex that associates specifically with sites of repetitive DNA at sites that are presumed to be refractory to reciprocal recombination events. It was not surprising, therefore, to find an MMR complex driven by MSH2-MSH3 instead of MSH4-MSH5. Also of significance is the fact that MLH3 can associate with chromosome regions independently of MLH1, suggesting the existence of a new MutL complex that is either stabilized by MLH1 or that can function independently of other MutL homologues. The MLH3-only complex could be inactive in terms of MMR activity, but could instead trigger alternative downstream events that would result in genome stabilization. The up-regulation of centromere-associated MMR complexes in Pms2 Ϫ/Ϫ males suggests that the absence of PMS2 facilitates the accumulation of repeat-associated MMR complexes, but does not necessarily point to any direct role for PMS2 at these sites. Msh2 Ϫ/Ϫ •Pms2 Ϫ/Ϫ males are also sterile while Msh2 Ϫ/Ϫ males are fertile, arguing against a role for PMS2 at the centromere. Instead, our results show that the loss of PMS2 results in an accumulation of the MSH2-MSH3-MLH1-MLH3 complex at sites of repetitive DNA as a secondary effect. We propose that PMS2 is working at other sites during spermatogenesis, in combination with MSH2, and that the loss of PMS2 at these sites leads to a redistribution of MMR complexes through the genome, resulting in increased association of MLH1-MLH3 with centromeric sequences (and, to a lesser extent, with sites of DSB processing). Alternatively, or in addition, the removal of PMS2 increases the need for MMR complexes at sites of repetitive DNA because of the general increase in genome instability in Pms2 Ϫ/Ϫ mice. Moreover, we cannot rule out the possibility that the effect of Pms2 deletion occurs at an earlier point in spermatogenesis, possibly during postreplicative repair at premeiotic S-phase, and that the meiotic failure is secondary to these earlier events. Indeed, Richardson et al. (2000) demonstrated high levels of mRNA and protein expression in mouse spermatogonia (Richardson et al., 2000), suggesting perhaps that PMS2 may be functioning at this earlier time in spermatogenesis.
Previous studies have suggested that tandem repeats, such as those found in human ␣ satellite centromeric DNA, are susceptible to mispairing and other genome destabilizing events. They are conserved by gene conversion (Greig et al., 1993;Warburton et al., 1993;Schindelhauer and Schwarz, 2002), as are palindromic repeat units on the Y (Rozen et al., 2003;Skaletsky et al., 2003). Given that localization of MMR proteins to these sites is determined by MSH2-MSH3, our data support a function in noncrossover and/or repair events at these tandem repeat units, rather than a function in MSH4-MSH5driven reciprocal recombination. In addition, the antirecombinational role of MSH2-MSH3 (Lin et al., 2004) could be beneficial at sites such as the centromere to prevent deleterious crossover events during prophase I, and may explain the high degree of conservation amongst centromere repeat regions.
The dynamics of MMR complex association with repetitive sequences at the centromere and on the Y chromosome dur-ing meiosis is reminiscent of the dynamics of MMR involvement in the expansion of simple trinucleotide repeat (TNR) sequences that give rise to complex disorders such as Huntington disease  and myotonic dystrophy (van den Broek et al., 2002;Nag, 2003). The secondary structures that arise as a result of TNR misalignments are substrates for MSH2 (Pearson et al., 1997), and mutations in Msh2 prevent CAG•CTG repeat expansion at the Huntington disease Hdh locus (Manley et al., 1999). In somatic cells, these events involve MSH3 because TNR expansion in myotonic dystrophy knock-in mice is abolished on a Msh3-null background (van den Broek et al., 2002). These MutS␤-dependent events, both in the germ line and in somatic cells, could involve canonical MMR and/or recombination processes or, as has been suggested, could instead result from the stabilization and protection from repair of CAG•CTG hairpin structures (Manley et al., 1999; resulting from misalignment and looping of tandem triplet repeats (Moore et al., 1999).
Secondary looping structures could also play roles in MMR-driven events during meiosis. Heteroduplexes formed during recombination in yeast can include insertions as large as 5.6 kb in size (Kearney et al., 2001) and are targeted specifically by Msh2-Msh3 (Kearney et al., 2001). These substrates may induce very different biochemical events to the traditional MMR pathway and may therefore initiate a different cascade of downstream events. Moreover, long repeat tracts, involving triplet CAG motifs or large palindromes, induce DSB formation during yeast meiosis Nasar et al., 2000;Nag, 2003) that could give rise to recombinational repair events. This raises the possibility that the repeat tracts and palindromes in the centromere regions and on the Y chromosome could present similar recombinational substrates during murine meiosis, inducing DSBs in late pachynema through the action of SPO11, which is still present during that time (Keeney et al., 1999;Romanienko and Camerini-Otero, 1999), that then need to be repaired by recombinational events, with a preference for noncrossover mechanisms. However, this possibility is tempered by the failure to detect other DSB repair machinery components (RAD51) at these sites (unpublished data).
In conclusion, our results suggest a novel function for the MMR machinery during mammalian meiosis, whereby MSH2-MSH3 are present as a surveillance mechanism, to restore repeat frequency, through stabilization/maintenance of hairpin structures present within the context of tandem repeats, and/or through suppression of reciprocal recombination events resulting from secondary structure-induced DSB formation, that could result in chromosomal translocations, enlarged loops, and other cytogenetic errors. Should repeat units become misaligned or out of register, a resulting single stranded loop structure would be detected by the surveillance complex and would activate downstream (repair) events involving, at the very least, MLH3, and that would ultimately effect the restoration of unit numbers.

Animals
The generation of mouse mutants for Pms2, Mlh1,Mlh3,Msh2,Msh3,Msh4,Msh5, and Msh6 have been described previously, along with genotyping strategies for each strain (Baker et al., 1995;Edelmann et al., 1996Edelmann et al., , 1999Edelmann et al., , 2000Kneitz et al., 2000;Lipkin et al., 2002). All strains were housed in the Albert Einstein College of Medicine Animal Institute on standardized light, heat, and feeding schedules. All strains were maintained on similar backgrounds by backcrossing heterozygote fathers with C57Bl/6J mothers.

Chromosome preparations
The standard chromosome spreading protocol has been described previously for our laboratory (Lenzi et al., 2005). The nuclear contents of whole-mount spermatocytes (or oocytes) were displayed by drying down a cell suspension, in hypotonic buffer, from either testis, or ovary, in 1% paraformaldehyde containing 0.15% Triton X-100 (Peters et al., 1997). Whole testes or ovaries were incubated on ice for 60 min in hypotonic extraction buffer (HEB; 30 mM Tris, pH 8.2, 50 mM sucrose, 17 mM trisodium citrate dihydrate, 5 mM EDTA, 0.5 mM DTT, and 0.5 mM PMSF). Either a one-inch length of tubule, or a whole ovary, were placed in a 20-l drop of 100 mM sucrose, pH 8.2, the tissue was macerated, and a second 20-l drop of sucrose solution was added and the cell suspension was pipetted up and down several times. Remnant pieces of tubule were removed. Cleaned slides were dipped in the paraformaldehyde and Triton X-100 solution, and most liquid was drained off, such that only enough liquid remained to coat the slide. 20 l of the cell suspension was added in one corner and the cells were slowly dispersed, first in a horizontal direction and then vertical. The remaining 20 l of cell suspension was used to make a second slide and both were placed in a humid chamber to dry slowly at RT for 2 h. The slides were washed three times for 1 min in 0.4% Kodak Photo-Flo 200 and air dried for at least 15 min. For EM preparations, to make the SCs accessible to immunogold grains, the slides were DNaseI treated (1 l/ml of DMEM) before being air dried (Moens et al., 2002). The slides were washed and blocked (three times for 10 min each) in PBS and incubated in primary antibodies overnight at RT in a humid chamber. Primary antibodies were used at varying concentrations, and generally a 10-fold higher concentration was used for EM than immunofluorescence. After washes, slides were incubated in secondary antibodies, conjugated to either fluorochrome or colloidal gold (Jackson ImmunoResearch Laboratories), for 2 h at 37ЊC. After washes the slides were mounted with ProLong Antifade (Invitrogen) for fluorescence microscopy. Images were captured on a Olympus IX81 microscope attached to a 12bit Cooke Sensicam CCD instrument and sent to IP Lab software.
For EM, slides were incubated in 4% alcoholic phosphotungstic acid for 15 min, followed by three 1-min washes in 95% ethanol, to enhance visualization of MNs. Slides were air dried and then dipped in 0.25% formvar (Electron Microscopy Sciences) and air dried under glass. The plastic was scored, treated with 25% hydrofluoric acid, and floated off in water with attached cells. Plastic was transferred to EM grids and used for transmission EM (JEOL 1200EX).

Quantitation and statistical analysis
Immunofluorescence chromosome counts and cell staging were performed by at least three independent observers, compiled using Excel, and then analyzed using the statistical software package, Prism 3.0 (GraphPad Software). Staging of prophase I cells was performed using well-defined criteria for SC morphology, XY synapsis, and double dense body (DDB) and sex body status (Ashley and Plug, 1998;Moens et al., 2002). For such studies, we used previously published criteria (Dresser et al., 1987;Allen et al., 1988;Backer et al., 1988). Pachynema was divided into early, mid, and late stages on the basis of chromosome appearance, degree of compaction, XY status, and DDB formation. In early pachynema, the XY is synapsed along most of the length of the PAR and much of the X chromosome appears synapsed. Small regions of the autosomes may not be synapsed at this point. By mid-pachynema, the autosomes are completely synapsed and further compacted, whereas the XY is synapsed up to %02ف of the length of the X chromosomes. The DDB appears at this time and the sex body becomes apparent. By late pachynema, only the tip of the PAR remains synapsed, the sex body is much more distinct, and the DDB is still present. Note that the MLH3 (and MLH1) signal found at the centromere was not included in our MN focus counts depicted in Fig. 1 and that these populations are, we believe, distinct from one another.
Gold grain counts of centromere-associated proteins were counted at the EM level. For each staining preparation, counts were made from spermatocytes of at least three animals, with no fewer than 200 centromeres counted per animal, including up to 19 autosomes from a single cell being counted. In some cases fewer autosomes were counted in cells whose total chromosome count was not completely visible. Equivalent telomere counts were performed for each preparation as matched controls. In all cases, the number of centromeres/telomeres having one, two or three, or more than four gold grains were tabulated, but the threshold for specificity was set at more than four gold grains.

Chromatin immunoprecipitation
Adult testes were decapsulated, placed in a drop of cell culture media, minced, and seminiferous tubules squeezed to release germ cells. Alternatively, pure populations of pachytene spermatocytes were obtained by sedimentation velocity gradient ("STA-PUT"; Romrell et al., 1976). Germ cells were taken up in 1 ml of cell culture media and cross-linking was performed at RT for 5 min by addition of formaldehyde to 1% concentration. Cross-linking was stopped by addition of glycine to 0.125 M. Cells were collected by centrifugation, washed in PBS, and lysed in 1 ml of FA lysis buffer (50 mM Hepes, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, pH 7.5) with addition of protease inhibitors. Cell lysates were sonicated eight times for 20 s each at 20% output setting, and lysates were cleared of cell debris by centrifugation. For each immunoprecipitation 1/10 of the total testis lysate volume was diluted 10 times in FA buffer and precleared with 2 g sheared salmon sperm DNA, 10 l of preimmune serum and protein A/G-Sepharose slurry for 1 h at 4ЊC. Immunoprecipitation was performed overnight at 4ЊC with specific antibodies. After immunoprecipitation, protein A/G-Sepharose slurry was added and the incubation was continued for 1 h. Precipitates were washed sequentially for 5 min each in FA buffer, FA buffer with 0.5 M NaCl, LiCl wash buffer (10 mM TrisCl, 0.25 M LiCl, 1 mM EDTA, 0.5% Nonidet P-40, 0.5% sodium deoxycholate), and TE buffer. The immune complexes were eluted from the beads by addition of 50 mM Tris, 10 mM EDTA, 1% SDS. Cross-linking was reversed by incubation at 65ЊC for at least 6 h DNA fragments were purified by phenolchloroform-isoamyl alcohol extraction and ethanol precipitation. Equal aliquots from each immunoprecipitation were amplified using degenerate oligonucleotide PCR method of Telenius et al. (1992). Products of amplification were analyzed by slot-blot hybridization, using "North2South" hybridization kit (Pierce Chemical Co.). Probes for hybridization were generated by PCR according to published sequences for the mouse minor and major satellite probe (Garagna et al., 2002), B2 SINE element probe (Serdobova and Kramerov, 1998), and mouse Y chromosome-specific LINE-1 probe (DYzEms10 locus; Navin et al., 1996).

Online supplemental material
Two figures are presented as supplemental data items. Fig. S1 shows MLH3 localization at the centromere of spermatocytes from Pms2 Ϫ/Ϫ males. Fig. S2 provides validation of ChIP experiments used in this study. Online supplemental materials are available at http://www.jcb.org/cgi/ content/full/jcb.200506170/DC1.


extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Medicine

text:
Platelet-derived growth factor modulates rat vascular smooth muscle cell responses on laminin-5 via mitogen-activated protein kinase-sensitive pathways

Background A treatment to remove vascular blockages, angioplasty, can cause damage to the vessel wall and a subsequent abnormal wound healing response, known as restenosis. Vascular smooth muscle cells (VSMC) lining the vessel wall respond to growth factors and other stimuli released by injured cells. However, the extracellular matrix (ECM) may differentially modulate VSMC responses to these growth factors, such as proliferation, migration and adhesion. Our previous reports of low-level expression of one ECM molecule, laminin-5, in normal and injured vessels suggest that laminin-5, in addition to growth factors, may mediate VSMC response following vascular injury. To elucidate VSMC response on laminin-5 we investigated-the role of platelet-derived growth factor (PDGF-BB) in activating the mitogen-activated protein kinase (MAPK) signaling cascade as a possible link between growth-factor initiated phenotypic changes in vitro and the ECM. Results Using a system of in vitro assays we assessed rat vascular smooth muscle cell (rVSMC) responses plated on laminin-5 to the addition of exogenous, soluble PDGF-BB. Our results indicate that although laminin-5 induces haptotactic migration of rVSMC, the addition of PDGF-BB significantly increases rVSMC migration on laminin-5, which is inhibited in a dose-dependent manner by the MAPK inhibitor, PD98059, and transforming growth factor (TGF-β1). In addition, PDGF-BB greatly reduces rVSMC adhesion to laminin-5, an effect that is reversible by MAPK inhibition or the addition of TGF-β1. In addition, this reduction in adhesion is less significant on another ECM substrate, fibronectin and is reversible using TGF-β1 but not MAPK inhibition. PDGF-BB also strongly increased rVSMC proliferation on laminin-5, but had no effect on rVSMC plated on fibronectin. Finally, plating rVSMC on laminin-5 did not induce an increase in MAPK activation, while plating on fibronectin or the addition of soluble PDGF-BB did. Conclusion These results suggest that rVSMC binding to laminin-5 activates integrin-dependent intracellular signaling cascades that are different from those of fibronectin or PDGF-BB, causing rVSMC to respond more acutely to the inhibition of MAPK. In contrast, our results suggest that fibronectin and PDGF-BB may activate parallel, reinforcing intracellular signaling cascades that converge in the activation of MAPK and are therefore less sensitive to MAPK inhibition. These results suggest a partial mechanism to explain the regulation of rVSMC behaviors, including migration, adhesion, and proliferation that may be responsible for the progression of restenosis.

Background
Angioplasty is a procedure designed to treat vascular stenosis, blockage(s), or atherosclerotic lesions, but it may also, simultaneously, cause damage to the integrity of the blood vessel wall. Restenosis is the subsequent narrowing and occlusion of the blood vessel in response to the injury or damage sustained during angioplastic procedures such as balloon dilation [1]. During restenosis, vascular smooth muscle cells (VSMC) from the injured blood vessel wall migrate into the lumen of the vessel, creating a new or neointima. The subsequent proliferation of these neointimal VSMC can lead to a thickening of this neointimal layer and re-occlusion of the vessel [1].
The characteristic response of VSMC, endothelial cells, platelets, and macrophages at the site of injury is the release of specific soluble growth factors which include platelet-derived growth factor (PDGF), transforming growth factor (TGF), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF) [2][3][4]. VSMC of the vessel wall respond to these factors by secreting proteolytic matrix metalloproteinases that degrade the extracellular matrix (ECM) and stimulate deposition of new ECM proteins such as collagen, elastin, fibronectin, and laminin in the neointima [5][6][7]. These ECM modulate VSMC integrin-dependent behaviors such as transluminal migration, adhesion, and proliferation [8][9][10].
To date, the precise molecular mechanisms that link growth factor-initiated intracellular signaling to ECMmediated adhesion, migration, and proliferation of VSMC are still unknown. Our previous reports of low-level laminin-5 expression in the intima of normal vasculature and an increased expression of laminin-5 in the neointima of injured vessels suggest that laminin-5, in addition to PDGF and TGF, may mediate VSMC responsiveness following vascular injury [11][12][13].
To further elucidate VSMC response to growth factors and the intracellular signaling cascades that may be linked to ECM-mediated adhesion, we used in vitro assays to study the role of laminin-5 in modulating these behaviors in rat vascular smooth muscle cells (rVSMC). We report here that PDGF induces differential responses in rVSMC behaviors on laminin-5, but not on fibronectin. In addition, we find that the PDGF-induced responses on laminin-5 are inhibited in a dose-dependent manner by an inhibitor of the mitogen-activated protein kinase (MAPK) pathway, PD98059, but not on fibronectin.
These differences in MAPK-sensitive rVSMC responses in vitro may be the result of different signaling pathways that are initiated by integrin-mediated adhesion to laminin-5. We suggest that rVSMC binding to laminin-5 may initially activate a MAPK-independent signaling cascade that may make these cells more responsive to MAPK inhibition. In contrast, rVSMC binding to fibronectin may activate a signaling pathway shared by PDGF that ultimately converges in MAPK activation. These results suggest that a complex interaction of ECM and growth factors may closely regulate VSMC behavior following vascular injury and these studies may directly identify define molecular targets that may reduce the incidence of restenosis following angioplasty.

Migration
We have previously reported that laminin-5 expression by rat vascular smooth muscle cells (rVSMC) is upregulated by platelet-derived growth factor (PDGF-BB) and that laminin-5 specifically enhances PDGF-BB-stimulated rVSMC migration [12]. In addition, our previous results suggested that PD98059 (a specific inhibitor of MEK and a general inhibitor of the mitogen-activated protein kinase ERK1/2 pathway) blocked PDGF-BB-stimulated migration on laminin-5 [13]. The current study characterizes this synergistic relationship between PDGF-BB and laminin-5 in the modulation of rVSMC cell behaviors, including migration.
The level of rVSMC migration was observed over 18 hours in transwell migration filters in the presence or absence of laminin-5, with and without PDGF-BB or serum. Maximal cell migration (chemotaxis) was obtained using cell migration media containing ten (10) percent fetal calf serum (FCS). Similar to our previous reports, laminin-5coated wells induced a greater than four-fold increase in rVSMC migration over that measured in naked plastic controls in three independent experiments as shown in Figure 1 (n = 24, p < 0.05). Furthermore, PDGF-BB, tested over a biologically relevant range of 5 -50 ng/mL, stimulated a dose-dependent increase in rVSMC migration on laminin-5 (between 5 -25 ng/mL), increasing migration fifteen (15) to sixty (60) percent over laminin-5-stimulated migration alone (n = 24, p < 0.005). This PDGF-BBstimulated increase in migration peaked at 25 ng/mL and did not significantly increase over the range of 25 -50 ng/ mL (data not shown).
The mitogen-activated protein kinase (MAPK) pathway is known to be activated by PDGF-BB-stimulation in VSMC [14]. To explore the mechanism of increased rVSMC migration on laminin-5 via PDGF-BB-stimulation, we used PD98059 which is a specific inhibitor of MEK1 and MEK2, and a general inhibitor of MAPK activation. The addition of PD98059 in concentrations between 10 -50 ng/mL reduced PDGF-BB-stimulated migration on laminin-5 (25 ng/mL) in a dose-dependent manner, reaching a maximal reduction of sixty (60) to seventy (70) percent over the range of 20 -40 ng/mL, as shown in Figure 1 (n = 24, p < 0.005). This reduction in PDGF-BB-stimulated migration on laminin-5 was maintained at all concentrations of PDGF-BB tested (5 -25 ng/mL).
To determine if this modulation is restricted to MEK/ MAPK-inhibition, we tested the effects of adding exogenous transforming growth factor (TGF-β1) for its ability to PDGF-BB increased rVSMC migration on laminin-5 in vitro Figure 1 PDGF-BB increased rVSMC migration on laminin-5 in vitro. The addition of PDGF-BB increased rVSMC migration on laminin-5, in a dose-dependent manner, over rVSMC haptotactic migration induced by the presence of laminin-5. rVSMC migration on laminin-5 was inhibited by both PD98059 (MEK1 inhibitor) and TGF-β1, in a dose-dependent manner.
modulate the PDGF-stimulated increase in rVSMC migration on laminin-5. Our results indicated that the addition of TGF-β1 was able to reduce laminin-5-stimulated rVSMC migration to levels approximating the levels observed in naked plastic controls over all tested ranges (5 -50 ng/mL), although the most statistically distinct reduction was found over the range of 5 -25 ng/mL, as shown in Figure 1. In addition, TGF-β1 was able to reduce maximal PDGF-BB-stimulated migration of rVSMC (25 ng/mL) on laminin-5 by approximately fifty (50) to sixty (60) to percent, over the range of concentrations from 20 to 40 ng/mL.

Adhesion
To determine whether or not the PDGF-BB-stimulated increase in rVSMC migration correlates with a reduction in rVSMC adhesion to laminin-5, thirty-minute in vitro adhesion assays were performed. In the absence of exogenous growth factor stimulation, laminin-5-and fibronectin-coated wells (20 µg/mL) sustained an approximate two and a half-fold increase in rVSMC adhesion compared with negative controls, as shown in Figure 2 (n = 24, p < 0.05). The addition of PDGF-BB, at the maximal migration-stimulating dose of 25 ng/mL, decreased the adhesion of rVSMC on laminin-5 by more than sixty-five (65) percent (n = 24, p < 0.005). The addition of exogenous PDGF-BB (25 ng/mL), however, decreased rVSMC adhesion on fibronectin by less than thirty (30) percent.
To investigate if the relationship between the PDGF-BBstimulated increase in migration and corresponding decrease in adhesion of rVSMC on laminin-5 may be related to MAPK activation, cells were pre-treated with PD98059 (40 ng/mL) for twenty (20) minutes prior to assay and the adhesion media was supplemented with PD98059 (40 ng/mL). The addition of exogenous PD98059 (40 ng/mL) restored the PDGF-BB-stimulated reduction (25 ng/mL PDGF-BB) in rVSMC adhesion to laminin-5, to approximately eighty-five (85) percent of laminin-5 controls (n = 24, p < 0.05). The addition of PD98059, however, did not restore the thirty (30) percent PDGF-BB-stimulated reduction in rVSMC adhesion on fibronectin.
To determine if this modulation is restricted to MEK/ MAPK-inhibition, we tested the effects of adding exogenous transforming growth factor (TGF-β1). Our results indicated that the addition of TGF-β1 (25 ng/mL) was able to restore the PDGF-BB-stimulated reduction in rVSMC adhesion on laminin-5 by roughly the same level as PD98059, eighty two (82) percent versus eighty five (85) percent, respectively. In contrast to PD98059, the addition of TGF-β1 (25 ng/mL) was sufficient to restore the PDGF-BB-stimulated reduction in rVSMC adhesion on fibronectin.

Proliferation
Based upon our observations of rVSMC migration and adhesion, we performed in vitro proliferation assays to determine the relative effects of the extracellular matrix (ECM) and exogenous growth factors described above. First, to test the effects of the ECM substrate on rVSMC proliferation in the absence of exogenous growth factors, quiescent cells were plated in cell-culture plates either coated with or without laminin-5 or fibronectin, in serum-free Dulbecco's Modified Eagle's Medium (DMEM) for one (1) to six (6) days.
To induce quiescence, rVSMC were incubated for forty eight (48) hours without mitogen (0% FCS, FBS) at 37°C and quiescence was verified by proliferation controls, cultured for forty eight (48) hours at 37°C with mitogen stimulus as shown in Figure 3a and 3b. Previous studies with VSMC report that greater than 95% of cells incubated in low-serum media (0.4% FCS or less) were arrested in G 0 (G 1 ) between forty eight (48) and seventy two (72) hours as determined by flow cytometry and determination of [3H] thymidine-labeled nuclei [15].
Our results suggest that culture of rVSMC plated on exogenous laminin-5 (coated at a concentration of 20 µg/mL) did not significantly increase cellular proliferation compared with naked plastic controls over a period of four (4) days, as shown in Figure 3a (n = 24, p < 0.02). Culturing of rVSMC plated on exogenous fibronectin (coated at a concentration of 20 µg/mL) however, did significantly increase cellular proliferation over a period of four (4) days by nearly two-fold, as shown in Figures 3a and 3b.
Next, to test the modulating effects of exogenous growth factors on rVSMC proliferation when cultured on these ECM substrates, quiescent cells were plated in cell-culture plates either naked or coated with laminin-5 or fibronectin, in the presence of TGF-β1 or PDGF-BB, both at a concentration of 25 ng/mL, from one (1) to six (6) days. Our results indicated that the addition of exogenous TGF-β1 (25 ng/mL) to the cell culture medium was sufficient to induce a suppressing effect on rVSMC proliferation on naked plastic, as well as laminin-5 and fibronectin, to levels approximating the quiescent growth controls.
The addition of PDGF-BB was sufficient to induce an increase in rVSMC proliferation on laminin-5 of nearly one-half (46%) over laminin-5 DMEM (no serum, no mitogen) controls (n = 24, p < 0.01). However, our results indicate that the addition of exogenous PDGF-BB (25 ng/ mL) did not have a statistically significant effect on cells plated on fibronectin. The effect of growth factors on rVSMC proliferation in vitro Figure 3 The effect of growth factors on rVSMC proliferation in vitro. The addition of PDGF-BB stimulated proliferation of rVSMC on laminin-5 and to some extent on naked plastic, but not on fibronectin. The presence of fibronectin alone was able to stimulate proliferation of rVSMC. The proliferative response of rVSMC to presence of laminin-5 with PDGF-BB or to fibronectin was suppressed by the addition of TGF-β1. Figure 3b The ECM induces differential rVSMC proliferation responses in vitro. The plating of rVSMC on fibronectin, but not laminin, induced increases in proliferation over four days. The addition of PDGF-BB, however, increased rVSMC migration on laminin-5, but not on fibronectin. The addition of TGF-β1 was sufficient to suppress rVSMC proliferation on both laminin-5 and fibronectin.

Mitogen Activated Protein Kinase (MAPK) Western Blot
To examine MAPK activation, rVSMC were plated onto laminin-5-or fibronectin-coated plates, then lysed after thirty minutes and prepared for immunoblotting. Our results indicated that laminin-5 did not induce a detectable increase in MAPK activation over a thirty (30) minute time interval, as shown in Figure 4. rVSMC plated on fibronectin did exhibit a significant increase in MAPK levels after thirty (30) minutes.
The addition of growth factors, such as PDGF-BB and TGF-β1, have been associated with increases in p44/p42 (ERK1/2) or MAPK activation [16][17][18]. To determine the effects of PDGF-BB and TGF-β1 on rVSMC, subconfluent cell cultures were pre-treated with FCS (10%), PDGF-BB (25 ng/mL) or TGF-β1 (25 ng/mL) for thirty (30) minutes, then lysed and prepared for immunoblotting. p44/p42 phosphorylation levels were not detectable in DMEMtreated control cells, as shown in Figure 4. However, the The MAPK pathway in rVSMC is activated by different stimuli Figure 4 The MAPK pathway in rVSMC is activated by different stimuli. The addition of FCS or PDGF-BB or the plating of rVSMC on fibronectin was sufficient to induce measurable increases in p44/p42 activation over 30 minutes. However, the addition of TGF-β1 or the plating of rVSMC on laminin-5 was not sufficient to induce MAPK activation.
addition of PDGF-BB, but not TGF-β1, was sufficient to induce measurable increases in MAPK activation levels.

Discussion
PDGF-BB is an in vitro VSMC mitogen and may be responsible for initiating the phenotypic changes in VSMC migration and proliferation during restenosis in vivo [19,20]. Our recent reports of low-level laminin-5 expression in the intima of normal arteries and increased expression in the neointima of injured arteries suggest that laminin-5, in conjunction with soluble growth factors and mitogens, may determine VSMC phenotype during restenosis [11][12][13].
The current study augments the body of evidence that suggests VSMC growth is influenced by an ECM-VSMC interaction [21] and that VSMC proliferation in different species respond differently to growth factor stimulus [15]. More specifically, this study explores evidence that laminin-5 and PDGF-BB may have combined and synergistic effects in determining rVSMC phenotype in vitro [6,8,10]. More specifically, our studies suggest that PDGF-BB strongly influences rVSMC behaviors such as migration. In addition, PDGF-BB significantly alters other cellular behaviors such as adhesion and proliferation on laminin-5, but only to a lesser extent on fibronectin.
Because PDGF-BB stimulation increases the overall levels of intracellular MAPK, as well as MAPK phosphorylation, we sought to explore this signaling cascade to determine its role in modulating these rVSMC behaviors on laminin-5 [22]. Specifically, the ERK1/2 form of MAPK mediates signaling by PDGF-AA, PDGF-AB, and PDGF-BB in VSMC, as well as signaling through laminin-5 binding integrins, and is therefore the most likely signaling molecule to modulate these cellular behaviors [23][24][25]. Our results suggest that rVSMC behaviors in vitro, driven by PDGF-BB responsiveness, can be blocked by MAPK inhibition (via MEK1 inhibitor: PD98059) on laminin-5, but not on fibronectin.
An additional signaling regulator, TGF-β1, has been implicated in the negative regulation and decreased rate of proliferation of VSMC stimulated with serum or PDGF [26][27][28]. Our results from this study indicate that TGF-β1, unlike the MEK1-inhibitor PD98059, was sufficient to block rVSMC behaviors on both laminin-5 and fibronectin. Specifically, the addition of TGF-β1 was able to reduce PDGF-BB-stimulated migration and proliferation of rVSMC on both ECM substrates and was also able to restore PDGF-BB-stimulated reductions in VSMC adhesion on these ECM.
Several lines of evidence now suggest that the antimitogenic effects of TGF-β1 may be dissociated from inhi-bition of ERK1/2 signaling pathways [17,18]. These reports suggest that TGF-β1 inhibition of PDGF-BB may be temporally independent of other early signaling pathways, such as MAPK, and is more likely to block VSMC behaviors, such as proliferation, by inhibiting events later in the G 1 phase of mitosis.
Although our previous reports linked ERK1/2 to rVSMC adhesion and migration, these studies did not examine the possibility for differential signaling initiated by rVSMC binding to laminin-5 or fibronectin [12,13]. Expanding our original analysis of growth factor stimulation of MAPK to include ECM binding reveals that integrin binding of rVSMC to fibronectin strongly increases detectable MAPK activation levels, as does FCS and PDGF-BB stimulation, whereas binding to laminin-5 does not. These differences may help to explain the differing effects on cellular behaviors of binding to these ECM ligands, as fibronectin and PDGF-BB may act in unison to activate intracellular signaling cascades that converge in MAPK activation, while laminin-5 may not.

Conclusions
Our results indicate that laminin-5 activates different intracellular signaling pathways from those of fibronectin and PDGF-BB in rVSMC and that binding to laminin-5 may modulate rVSMC behaviors that are distinctive from those modulated by fibronectin. Although binding of rVSMC to laminin-5 may not cause an initial increase in MAPK activation levels or proliferation, laminin-5 can augment PDGF-BB-stimulated proliferation and migration of rVSMC in vitro. Based upon these findings, we postulate that PDGF-BB and laminin-5 binding may initially activate different intracellular signaling cascades, causing rVSMC to be more responsive to the inhibition of MEK1 and MAPK on laminin-5 than those activated on fibronectin, as outlined in Figure 5.
In contrast to laminin-5, fibonectin and PDGF-BB may have parallel, reinforcing roles in MAPK activation. Our analysis of the effects of TGF-β1 demonstrated that TGF-β1 does not strongly activate MAPK in rVSMC, but rather strongly inhibits the effects of fibronectin and PDGF-BBstimulation on laminin-5. Our results support the previous findings that TGF-β1 may inhibit mitogenesis and other VSMC behaviors via mechanisms independent of MAPK activation.
These results suggest that a clear understanding of the roles and contributions of each ECM may provide new insights into the mechanisms of regulating rVSMC cell behaviors, including migration, adhesion, and proliferation. Further analysis of the events that trigger and sustain the underlying cellular mechanisms of rVSMC behaviors Integrins and growth factors activate intracellular signaling cascades in rVSMC Figure 5 Integrins and growth factors activate intracellular signaling cascades in rVSMC. Intracellular signaling pathways that converge through MEK and ERK activation, may be initiated in rVSMC by the addition of PDGF-BB, as well as binding to laminin-5 or fibronectin. The MEK1 inhibitor PD98059 blocks MEK1 activation and alters rVSMC responses to PDGF-BB on laminin-5, but not fibronectin. The addition of TGF-β1, which may block later events in the cell cycle, is sufficient to block PDGF-BB induced responses of rVSMC on both laminin-5 and fibronectin. may help aid in the design of more effective therapies for the treatment of restenosis.

Cell culture
Cells were maintained in 100 mm × 20 mm Corning tissue-culture dishes (Plainfield, NJ) at 37°C and 5% CO 2 in humidified chambers. Cells were maintained in DMEM High Glucose, supplemented with 10% fetal bovine serum and 1% L-glutamine (29.2 mg/mL), penicillin G (10,000 U/mL), and streptomycin sulfate (10,000 mcg/ mL) (GPS) from Irvine Scientific (Santa Ana, CA). Rat aortic smooth muscle cell explants were a gift from RC Smith and were isolated and passaged as previously described [29]. Although greater than 95% quiescence, G 0 (G 1 ) arrest can routinely be induced by incubation of cells for 72 h. in low-mitogen (0.5% FBS) medium [15,30], these authors suggest that incubation of VSMC for 48 h. without mitogen (0% FBS) is sufficient to induce quiescence. This was verified by proliferation control cells, cultured for 48 h. at 37°C with and without mitogen stimulus.

Migration assays
Cell migration assays were performed in Costar transwell filter plates either coated with purified matrix (laminin-5 or fibronectin) at a protein concentration of 20 µg/mL for one hour (60 min.) at room temperature, 25°C, and washed twice with phosphate-buffered saline 0.2% Tween-20 and 5% skim milk (PBST) prior to assay as previously described [31,32]. Cells were seeded at a concentration of 1.2 × 10 5 in each of 96-transwell chamber filters (100 µL of 1.2 × 10 6 cells/mL solution) with and without ECM in the presence or absence of PDGF-BB at the indicated concentrations (5-25 ng/mL) and allowed to migrate for 18 hours at 37°C. Where applicable, the medium was supplemented with PD98059 (MEK1-inhibitor) at the indicated concentration. Cells were counted at the end of an 18-hour interval as indicated, quantified with the following modification. 30 minutes prior to measuring migration, 5 µM calcein AM from Molecular Probes (Eugene, OR) was added to the migration wells at 37°C. To quantitate migration, cells were removed from the top of the filter with cotton-tipped applicators and fluorescence of the incorporated calcein was measured from the bottom of the filter with a fluorescence plate reader. Relative fluorescence values for each experimental condition are expressed relative to controls and untreated samples.

Adhesion assays
Cell adhesion assays were performed as previously described [31,32] using Costar 96-well cell culture cluster plates, coated with either laminin-5 or fibronectin solution at a protein concentration of 20 µg/mL for 1 hour (60 min.) at room temperature, 25°C. Wells were then washed twice with PBST prior to assay. Cells were seeded at a concentration of 1.2 × 10 5 in each of 96-transwell chamber filters (100 µL of 1.2 × 10 6 cells/mL solution) with and without ECM-coating (described above) in the presence or absence of PDGF-BB (25 ng/mL), TGF-β1 (25 ng/mL), or both, and allowed to attach for 30 minutes at 37°C. Where applicable, cells were first incubated for 20 minutes with PD98059 (40 ng/mL), a MEK1 inhibitor from New England Biolabs (Beverly, MA) at 37°C and the adhesion assay culture medium was supplemented with PD98059 at 40 ng/mL. Following adhesion, non-adherent cells were removed by suspending plates upside down in a rotating tank of PBS for 10 minutes at room temperature, 25°C. Adherent cells were then fixed and stained and the relative absorbance was measured using a TECAN-SPECTRAFluor spectrophotometer (TECAN, Durham, NC) at 595 nm.

Proliferation assays
Tissue culture plates were coated with purified fibronectin from Calbiochem (La Jolla, CA) or laminin-5 from Demos (La Jolla, CA) at a 20 µg/mL protein concentration for 1 hour (60 min.) at room temperature, 25°C as previously described [31]. Cells were seeded at a concentration of 1.2 × 10 5 in 100 mm 2 cell culture plates with and without fibronectin or laminin-5 and allowed to attach overnight (12 h.) at 37°C. Cells were then starved in serumfree DMEM for 48 hours to induce quiescence at 37°C, as outlined in Cell Culture Methods below. The medium was then replaced with fresh medium containing 25 ng/mL of TGF-β1 or PDGF-BB obtained from Calbiochem (La Jolla, CA) and incubated at 37°C. Cells were removed from culture wells with trypsin/EDTA and counted using trypan blue stain from Gibco Life Technologies (Rockville, MD) and a VWR Scientific Counting Chamber (Plainfield, NJ) at 24 hour intervals, from 1 -6 days.

Western Blot analysis
Quiescent VSMC were pre-treated with culture medium containing 10% FCS, PDGF-BB (25 ng/mL), TGF-β1 (25 ng/mL), or plated on laminin-5-or fibronectin-coated tissue culture plates with DMEM for 30 minutes at 37°C. Subconfluent cell cultures were then lysed using ice-cold, 1X lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM EDTA, 1.0% Triton X-100, pH 7.5) and boiled in SDS Boiling Buffer prior to analysis as previously described [33]. Proteins were separated by 7.5% SDS-PAGE and transferred to Immobilon-P transfer membranes from Millipore (Bedford, MA), incubated with primary antibody (1:200 dilution at 4°C overnight, 12 h.), secondary antibody (1:2000 dilution at 25°C for 1 h.), and then exposed to NEN CDP-Star chemiluminescence reagent (5-7 min.) and developed on Kodak X-OMAT LS scientific imaging film for subsequent analysis. Antibodies used for this analysis were anti-phospho ERK (p42/p44) Publish with Bio Med Central and every scientist can read your work free of charge