Post-transcriptional regulation of satellite cell quiescence by TTP-mediated mRNA decay
Skeletal muscle satellite cells ( SCs ) are maintained as a quiescent stem cell population ( Schultz , 1976 ) possessing minimal cytoplasm , condensed chromatin , and few ribosomes ( Wakayama et al . , 1979 ) . Upon muscle injury , SCs activate and then enter S-phase to generate a proliferating myoblast population that differentiates , repairs skeletal muscle tissue , and self-renews to replenish the stem cell pool ( Cornelison et al . , 2001 ) . Induction of MyoD , a myogenic regulatory transcription factor , is a critical component of this activation cascade . Prior to MyoD induction , and immediately upon satellite cell activation , the p38α/β MAPK pathway is activated , serving as a molecular switch ( Jones et al . , 2005 ) . In a subset of SCs , p38α/β is asymmetrically activated during cell division to promote SC self-renewal and re-acquisition of quiescence ( Troy et al . , 2012 ) . Moreover , aged SCs exhibit an intrinsic self-renewal deficit that arises in part from a failure to asymmetrically activate p38α/β MAPK ( Bernet et al . , 2014 ) . Importantly , post-transcriptional gene regulatory mechanisms , including translational repression , contribute to SC activation and play a key role in the maintenance of the quiescent SC state ( Crist et al . , 2012 ) . TTP ( Tristetraprolin ) , a target of the p38α/β MAPK pathway , binds to AU-rich elements within the 3′ untranslated region ( 3′ UTR ) of target transcripts and recruits deadenylases , initiating mRNA decay ( Lai et al . , 2000 ) . Rates of mRNA decay , which fluctuate depending on cellular conditions , are controlled by specialized mRNA binding proteins ( RBPs ) that bind target mRNAs and activate cellular mRNA decay enzymes ( Parker and Song , 2004 ) . TTP is the best studied of the four member Tis11 gene family , which is comprised of the genes: Zfp36 ( TTP ) , Zfp36l1 , Zfp36l2 and Zfp36l3 . In macrophages , TTP suppresses the inflammation response by destabilizing proinflammatory mRNAs until extracellular stimuli activate p38α/β MAPK ( Stumpo et al . , 2010 ) . The p38α/β MAPK signaling pathway then phosphorylates TTP , promoting association with 14-3-3 adaptor proteins and blocking the recruitment of deadenylases , thereby stabilizing transcripts with TTP 3′ UTR binding sites ( Clement et al . , 2011 ) . TTP loss-of-function has profound effects on overall organismal health and is associated with a severe inflammatory syndrome caused by chronic over-expression of Tumor Necrosis Factor ( TNF ) α mRNA , as loss of TNF receptor function largely rescues TTP KO phenotypes in TTP/TNFR1/2 triple knockout mice ( Carballo et al . , 2001 ) . Transcripts encoding TTP family members are among the fastest declining and dynamically regulated mRNAs following SC activation , where the transcripts initially decline and then are re-expressed ( Farina et al . , 2012 ) . Consistent with this observation , we identified the MyoD 3′ UTR as a TTP substrate , and show that p38α/β MAPK-mediated phosphorylation of TTP regulates MyoD mRNA decay , thereby regulating SC activation . Therefore our data , combined with recent reports of miRNA-mediated regulation of SC quiescence ( Cheung et al . , 2012; Crist et al . , 2012 ) , identify post-transcriptional gene regulation as an effective and potent means of maintaining SC homeostasis in adult skeletal muscle .
Transitioning from a quiescent SC to an activated SC involves large-scale changes in mRNA expression as well as changes in miRNAs ( Farina et al . , 2012 ) and subsequent events that permit SCs to begin DNA synthesis and proliferate as myoblasts ( Jones et al . , 2005 ) . To identify regulators involved in SC activation and myoblast commitment , we compared restricted gene expression profiles of FACS-enriched SCs isolated from uninjured muscle with expression profiles from SCs isolated 12 hr post-injury from wild type and Syndecan 4 ( Sdc4 ) −/− mice ( Farina et al . , 2012 ) . Syndecan-4 null SCs are incapable of muscle repair , exhibit severely delayed activation , aberrant MyoD expression , poor growth kinetics , fail to self-renew ( Cornelison et al . , 2004; Hall et al . , 2010 ) and thus , transcripts differentially expressed between wildtype and Sdc4−/− SCs may represent genes involved in SC activation and commitment . Genes unique to wild type SCs that change expression 12 hr post muscle injury but do not change in Sdc4−/− SC are referred to as the ‘WT-S4 gene list’ ( Figure 1A , Supplemental file 1A , 1B ) . An unexpectedly high percentage of genes in this list decreased in expression during SC activation ( 47% ) , with ‘nucleic acid binding: RNA binding , ’ ( GO:0003723; p-value = 8 . 7e-05 ) as the most significantly overrepresented gene ontology ( GO ) term ( Figure 1B ) . Among these rapidly down-regulated transcripts are genes encoding mRNA destabilizing proteins and genes implicated in cell cycle entry of quiescent C2C12 cells following methylcellulose-induced G0 arrest ( Sachidanandan et al . , 2002 ) . One of these decay factors , Zfp36 , which encodes TTP , declines precipitously upon wild type SC activation ( Figure 1C , D ) . Additionally , transcripts for two other TTP family members , Zfp36l1 and Zfp36l2 , which encode Brf1 and Brf2 , respectively , also decrease during SC activation ( Figure 1—figure supplement 1 ) . Among the minority of transcripts that are up-regulated upon SC activation are genes with known roles in myogenesis , including Elavl1 , which encodes HuR , a MyoD mRNA-stabilizing protein ( Figure 1E , F ) ( Figueroa et al . , 2003 ) , consistent with the induction of MyoD , which occurs as early as 3 hr following isolation of myofiber-associated SCs ( Cornelison et al . , 2004; Jones et al . , 2005 ) . 10 . 7554/eLife . 03390 . 003Figure 1 . Transcripts encoding RNA binding proteins are differentially regulated during SC activation . ( A ) Venn diagram illustrating subtraction strategy used to obtain the WT-S4 gene list . A total of 5162 and 2236 unique genes changed when comparing satellite cells ( SCs ) isolated from uninjured muscle and muscle 12 hr post-injury in wild type and Syndecan-4 ( Sdc4 ) null SCs , respectively . Genes common to both ( 1069 ) were subtracted from the wild type gene list yielding 4093 unique genes that changed after injury in wild type but not in Sdc4−/− SCs . ( B ) Heat map depicting transcripts filtered from the WT-S4 gene list annotated with the Molecular Function Gene Ontology ( GO ) term: RNA binding . Transcripts annotated as RNA binding were highly enriched in the WT-S4 gene list ( p-value = 8 . 7e-05 ) and 70% decreased in wild type SCs but did not change in Sdc4−/− SCs 12 hr post injury . Total RNA binding genes: 152; decreased: 107 ( 70% ) . ( C ) The relative expression of Zfp36 decreased in wild type SCs but not in Sdc4−/− SCs during the first 12 hr post-injury . ( D ) Zfp36 transcripts decline rapidly following culture of freshly isolated SCs . ( E ) The relative expression of Elavl1 increased in wild type SCs but did not change in Sdc4−/− SCs during the first 12 hr post-injury . ( F ) Freshly isolated SCs increase Elavl1 expression upon culture . ( C and E values from SCs isolated by FACS and analyzed on Affymetrix MOE430A gene chips; D and F determined by QT-PCR where one representative of two experiments is shown , n = 5 mice; * p-value < 0 . 01 ) . See also Supplemental file 1A , 1B . DOI: http://dx . doi . org/10 . 7554/eLife . 03390 . 00310 . 7554/eLife . 03390 . 004Figure 1—figure supplement 1 . Transcripts encoding the RNA binding proteins Brf1 and Brf2 decrease during satellite cell activation . ( A ) and ( B ) Abundance of Zfp36l1 and Zfp36l2 transcripts , which encode Brf1 and Brf2 , respectively , decreased in wild type satellite cells but did not change in Sdc4−/− satellite cells . The level of gene expression as determined by microarray analysis of wild type ( WT ) and Sdc4−/− satellite cells isolated from uninjured muscle ( UI ) and muscle 12 hr post-injury ( 12 hr ) . ( C ) and ( D ) Brf1 and Brf2 mRNA decreased as satellite cells activated in culture as determined by QT-PCR analysis ( means from one representative experiment out of two , each experiment contained five mice; Graphs plotted ± standard deviation . *p-value < 0 . 01 ) . DOI: http://dx . doi . org/10 . 7554/eLife . 03390 . 004 Unphosphorylated , active TTP protein is short-lived as TTP targets its own mRNA for rapid decay , resulting in low levels of unphosphorylated TTP that are technically challenging to detect ( Brook et al . , 2006 ) . Any skeletal muscle perturbation such as induced injury or immediate dissection following sacrifice , activates SCs and p38α/β MAPK ( Jones et al . , 2005 ) , which in other cell types , phosphorylates and stabilizes TTP ( Tchen et al . , 2004 ) . Mice perfused with paraformaldehyde to fix the tissue in situ prior to muscle dissection revealed Sdc4-marked SCs immunoreactive for TTP ( Figure 2A ) . Similarly , rapid dissection of the TA muscle followed by immediate fixation also revealed TTP staining in SCs ( Figure 2B ) . Upon scoring for TTP positive SCs , we found no quantitative differences in the numbers of TTP+ SCs when the muscle was pre-fixed by perfusion or rapidly dissected ( Figure 2A , B ) . In uninjured muscle , 50% of the Sdc4+ SCs were immunoreactive for TTP and located underneath the basal lamina ( Figure 2A , B , E ) . Since steady-state levels of active TTP are very low and difficult to detect due to decay of TTP mRNA by active TTP , the lack of detectable immunoreactivity in all SCs is likely due to TTP protein detection limits as opposed to the absence of TTP ( Brook et al . , 2006 ) . 10 . 7554/eLife . 03390 . 005Figure 2 . TTP positive SCs decrease 1 hr following muscle injury . ( A ) Uninjured perfused TA muscle sections or ( B ) rapidly dissected uninjured muscle sections were stained for Sdc4 ( red ) and laminin ( white ) to identify SCs ( carets ) and for-TTP ( green ) . ( C ) and ( D ) TA muscle sections 1 hr following BaCl2 induced injury were stained as in A and B . ( For A–D , F–G , DAPI is blue; * denotes extralaminar Sdc4+/TTP+ cells ) . ( E ) The number of Sdc4+ and Sdc4+/TTP+ cells in uninjured TA muscle sections or TA muscle sections 1 hr following BaCl2 induced injury were plotted for two independent experiments . ( F , G ) TA muscle sections stained for mouse IgG ( green ) to assess necrotic fibers in ( F ) uninjured muscle or in ( G ) muscle 30 min following a BaCl2 induced injury . Scale bars = 50 µm ( A–D ) , 100 µm ( F , G ) . DOI: http://dx . doi . org/10 . 7554/eLife . 03390 . 00510 . 7554/eLife . 03390 . 006Figure 2—figure supplement 1 . p38α/β MAPK inhibition prevents TTP phosphorylation and reduces HuR protein levels . ( A ) Uninjured TA muscle sections harvested and stained as described in Figure 3 . Intraperitoneal injection of mice with the p38α/β MAPK inhibitor SB203580 and inclusion of SB203580 during SC isolation reduces TTP phosphorylation and HuR induction . ( B ) Experimental schematic . ( C ) SCs isolated from treated and untreated mice stained for phospho-TTP ( p-TTP , red ) , HuR ( white ) , and Sdc4 ( green ) . ( D ) The normalized intensity averaged and plotted for phospho-TTP , HuR and Sdc4 in isolated SCs ( Student's two-tailed t-test: phospho-TTP and HuR; p < 0 . 001 . n = 4 independent experiments ) . DOI: http://dx . doi . org/10 . 7554/eLife . 03390 . 00610 . 7554/eLife . 03390 . 007Figure 2—figure supplement 2 . phospho-TTP expression in myofiber-associated satellite cells . Myofiber-associated SCs isolated from wildtype mice were fixed for immunofluorescence at 0 , 3 , 6 , 12 , and 24 hr post isolation . The 0 hr timepoint corresponds to ∼1 hr 50 min post-hindlimb dissection . Myofiber-associated SCs were subsequently stained for Pax7 to mark SCs ( green ) , phospho-TTP ( red ) , and DAPI ( blue ) to mark nuclei . The white dotted lines highlight the myofiber . Scale bars = 20 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 03390 . 007 In contrast to the uninjured state , 1 hr post-injury we found the majority of TTP+/Syndecan-4+ cells were located outside of the basal lamina or encased within the basal lamina ( Figure 2C , E ) with a minority present in the SC niche underneath the basal lamina ( Figure 2D , E ) . This observation is consistent with substantial muscle damage , as evidenced by serum IgG permeable necrotic myofibers , observed in injured tissue 30 min following a BaCl2 induced injury ( Weller et al . , 1990 ) ( Figure 2F , G ) . Upon TTP phosphorylation by p38α/β MAPK , TTP accumulates rapidly as a result of increased TTP message and protein stability arising from TTP inactivation ( Tchen et al . , 2004 ) . Therefore , following an induced muscle injury we expect the majority of TTP to be phosphorylated . Unfortunately , the anti-phospho-TTP antibody proved unreliable for detection of phospho-TTP in muscle sections ( not shown ) and thus , we queried SCs in injured muscle for a direct target of p38α/β MAPK phosphorylation , MAPKAPK2 ( MK2 ) , which phosphorylates TTP ( Brooks and Blackshear , 2013 ) . 30 min post-injury , 45% of the Syndecan-4+ SCs were phospho-MK2 immunoreactive ( Figure 3A , B ) , while uninjured muscle SCs were phospho-MK2 negative ( Figure 2—figure supplement 1A ) . Consistent with MK2 activation by p38α/β MAPK , phospho-MK2 was significantly reduced if mice were pre-treated with an intraperitoneal ( IP ) injection of SB203580 , a p38α/β MAPK inhibitor ( Figure 3A , B ) . 10 . 7554/eLife . 03390 . 008Figure 3 . TTP is phosphorylated by p38α/β MAPK and regulates MyoD expression in primary SCs . ( A ) TA muscle sections harvested 30 min following BaCl2 injury from either vehicle injected or SB203580 injected mice were stained with anti-Sdc4 antibodies ( green ) and anti-Laminin antibodies ( white ) to identify SCs ( carets ) and anti-phospho-MK2 antibodies ( red; * denotes myonuclei positive for p-MK2 ) . ( B ) Percentage of phospho-MK2 positive SCs was decreased in the presence of SB203580 following injury . ( ± SEM * p-value < 0 . 01 for n = 3 ) . ( C ) PLA in primary , 24 hr cultured SCs using an anti-phospho-MK2 antibody and either an anti-TTP antibody ( left column ) , an anti-HuR antibody ( middle column ) , or an anti-CUGBP1 antibody ( right column ) . ( D ) Box-and-whisker plots depicting cumulative PLA results from three independent experiments in primary SCs and in the MM14 myoblast cell line ( * p-value < 0 . 01 compared to CUGBP1 ) . ( E ) Myofiber-associated SCs were cultured in the presence of 10 μM MK2 inhibitor III ( MK2i ) or a vehicle control ( DMSO ) for 24 hr , fixed and stained for Sdc4 to mark SCs ( red ) , phospho-TTP ( green ) , and MyoD ( white ) . ( F–H ) Myofiber-associated SCs transfected 6 hr post isolation with an eGFP plasmid as a transfection marker and either a control plasmid or a plasmid expressing TTP-AA-myc . Explanted SCs were cultured for an additional 24 hr , fixed and stained for Sdc4 ( red ) , eGFP ( green ) , and MyoD ( white ) . ( H ) The percentage of transfected SCs scored for MyoD plotted for three independent experiments from a minimum 25 myofibers per condition . Scale bars = 10 µm ( A ) , 4 µm ( C ) , 30 µm ( E ) , and 10 µm ( F , G ) . DOI: http://dx . doi . org/10 . 7554/eLife . 03390 . 00810 . 7554/eLife . 03390 . 009Figure 3—figure supplement 1 . TTP over-expression does not affect HuR protein levels . Myofiber-associated SCs transfected 6 hr post isolation with a plasmid expressing ß-galactosidase as a transfection marker and either a control plasmid ( A ) or a plasmid expressing TTP-AA-myc ( B ) were cultured for an additional 24 hr , fixed and stained for Sdc4 ( red ) , ß-galactosidase ( green ) , and HuR ( white ) . ( C ) The percentage of transfected SCs scored for HuR plotted for two independent experiments from a minimum of 25 myofibers per condition . Scale bar = 5 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 03390 . 009 Dissection of SCs from whole muscle activates p38α/β MAPK ( Jones et al . , 2005 ) and as expected , SCs are phospho-TTP+ following isolation ( Figure 2—figure supplement 1B , C; Figure 2—figure supplement 2 ) . If TTP phosphorylation is mediated via the p38α/β MAPK pathway , then inhibition of p38α/β MAPK should prevent TTP phosphorylation . To test this hypothesis , we injected SB203580 intraperitoneally ( IP ) , isolated SCs in the presence of SB203580 , and examined isolated SCs for phospho-TTP to identify activated SCs and examined SCs for HuR induction , which occurs rapidly upon SC activation ( Figure 1—figure supplement 1B , C ) . Phospho-TTP and HuR were significantly reduced in SCs isolated from SB203580 treated mice compared to SCs isolated in the absence of the p38α/β MAPK inhibitor , while Syndecan-4 staining remained constant ( Figure 2—figure supplement 1C , D ) . Thus , in SCs , TTP appears to be a p38α/β MAPK target and pretreating skeletal muscle with a p38α/β MAPK inhibitor reduces TTP phosphorylation . MAPKAPK2 ( MK2 ) is a downstream p38α/β MAPK target regulating TTP in murine macrophage cells ( Mahtani et al . , 2001; Tiedje et al . , 2012; Brooks and Blackshear , 2013 ) . We employed a Duolink in situ proximity ligation assay ( PLA ) ( Pisconti et al . , 2010; Troy et al . , 2012 ) to determine if phospho-MK2 was associated with TTP . As a positive control , we performed PLA using phospho-MK2 and HuR primary antibodies as HuR is a well-established p38/MK2 target ( Kim et al . , 2010 ) . Duolink signals detected in phospho-MK2::HuR samples ( Figure 3C ) , were significantly greater than phospho-MK2 PLA using an unrelated RNA binding protein ( CUGBP1 ) antibody where ≤3 complexes per cell were observed ( Figure 3C ) above background . A robust signal for phospho-MK2::TTP association using PLA was observed ( Figure 3C ) and when quantified , was significant compared to contols ( Figure 3D ) , indicating that TTP is a bona-fide p38/MK2 target in SCs and MM14 cells . If TTP is a functional target of MK2 , then inhibition of MK2 should reduce or eliminate phospho-TTP expression . Primary myofiber-associated SCs were cultured for 24 hr in the presence or absence of an MK2 inhibitor ( MK2 Inhibitor III , CAS: 1186648-22-5 ) then fixed , stained and scored for Syndecan-4 , phospo-TTP , and MyoD . Wild-type , vehicle treated myofiber-associated SCs ( Syndecan 4+ ) uniformly expressed MyoD and were phospho-TTP positive at 24 hr post-isolation , while phospho-TTP was barely detectable in the presence of 10uM MK2 inhibitor III ( MK2i , Figure 3E ) . We observed concurrent downregulation of MyoD in the MK2i treated SCs , suggesting that TTP may play a role in p38α/β MAPK-mediated MyoD regulation . The rapid p38α/β MAPK-dependent phosphorylation ( inactivation ) of TTP upon SC activation suggests that TTP plays a role in maintaining SC quiescence . Furthermore , TTP inactivation preceding MyoD up-regulation is consistent with the hypothesis that TTP actively suppresses pro-myogenic mRNA targets . We therefore transfected myofiber-associated SCs with a constitutively active TTP mutant , TTPS52AS178A ( TTP-AA-myc ) , to determine if direct manipulation of TTP influenced MyoD protein levels . TTP-AA-myc is unresponsive to p38α/β MAPK signaling , binds target transcripts , recruits mRNA decay factors , and reduces target mRNA stability ( Stoecklin et al . , 2004 ) . Myofiber-associated SCs transfected with either a control ( pcDNA3 . 1 ) or a TTP-AA-myc expression plasmid immediately following explant were fixed and scored for Syndecan-4 and MyoD immunoreactivity after 30 hr in culture . The majority ( 70% ) of control transfected cells were MyoD immunoreactive ( Figure 3F , H ) , while only 30% of the TTP-AA-myc transfected cells were MyoD immunoreactive ( Figure 3G , H ) , demonstrating that TTP reduces myogenic commitment . Importantly , 15% of SCs were MyoD immunoreactive on a subset of myofibers removed immediately following transfection at 6 hr post-explant ( dotted line , Figure 3H ) . Thus , ectopic expression of TTP-AA-myc blocked virtually all cells from inducing MyoD post-transfection ( Figure 3H ) . MyoD mRNA in skeletal muscle cell lines is stabilized by HuR ( van der Giessen et al . , 2003 ) and induction in SCs is concomitant with the loss of TTP ( see Figure 1 ) . Moreover , TTP may regulate HuR since a polyadenylation variant of HuR mRNA contains an AU-rich element ( Al-Ahmadi et al . , 2009 ) and blocking p38α/β MAPK signaling prevents HuR induction ( Figure 2—figure supplement 1C , D ) . Therefore , HuR is a potential TTP target that may stabilize MyoD mRNA in activated SCs promoting MyoD induction . If correct , HuR expression should be reduced by the TTP-AA-myc mutant similar or to a greater extent then that observed for MyoD . To test this , SCs on intact myofibers were transfected with either a control or TTP-AA-myc mutant plasmid , fixed ( as described above ) 30 hr post explant and then stained for Syndecan-4 ( to identify SCs ) and HuR ( Figure 3—figure supplement 1A , B ) . Scoring the percentage of Syndecan-4+ cells that were HuR immunoreactive showed that there were no detectable differences in HuR protein expression in control transfected SCs as compared to TTP-AA-myc transfected SCs ( Figure 3—figure supplement 1C ) . Thus , it is unlikely that HuR represents a primary TTP target mRNA in activating SCs and may function to control mRNA stability via an alternative signaling pathway . TTP does not appear to regulate MyoD mRNA by preventing HuR induction and thus , we asked if TTP directly regulated MyoD transcript stability . Bioinformatic analysis of the MyoD 3′ UTR identified a highly conserved TTP binding site ( Figure 4—figure supplement 1A ) . To verify TTP binding to the MyoD 3′ UTR , we inserted the full length MyoD 3′ UTR into a Tet-Off β-Globin reporter ( referred to as β-MyoD ) and performed RNA-immunoprecipitation assays . HEK293T cells were co-transfected with the ß-MyoD construct and a negative control ß-globin sequence that does not bind TTP ( β-GAP ) , along with wild type TTP or an RNA binding-deficient TTP mutant ( F126N ) ( Lai et al . , 2002 ) , both containing a flag epitope tag . Following transfection , cell lysates were immunoprecipitated with an anti-flag epitope tag antibody . Immunoprecipitation of wild type flag-TTP efficiently co-immunoprecipitated β-MyoD when compared with the negative control β-globin reporter ( β-GAP ) ( Figure 4A , lane 1 ) , whereas immunoprecipitation of the flag-TTPF162N mutant failed to co-immunoprecipitate β-MyoD ( Figure 4A , lane 2 ) . As an internal positive control , wild type flag-TTP co-immunoprecipitated a known target of TTP , the 3′ UTR sequence of Granulocyte Macrophage Colony Stimulating Factor ( GM-CSF ) , ( Figure 4A , lane 1 ) that was not co-immunoprecipitated with the flag-TTPF126N mutant ( Figure 4A , lane 2 ) . Anti-flag immunoprecipitation of cell extracts expressing an unrelated RNA binding protein , the MS2 viral coat protein , failed to co-immunoprecipate any of the reporter RNAs ( Figure 4A , lane 3 ) . These data demonstrate that wild type TTP directly binds the MyoD 3′ UTR . 10 . 7554/eLife . 03390 . 010Figure 4 . TTP binds and regulates the 3′ UTR of MyoD mRNA . ( A ) Northern blots of co-immunoprecipitation assays show TTP binding to the MyoD 3′ UTR in HEK293T cells . Assays were performed with cells co-expressing FLAG-tagged wild type TTP ( WT , lanes 1 and 4 ) , an RNA binding mutant of TTP ( F126N , lanes 2 and 5 ) , or an unrelated non-RNA binding protein , ( MS2 , lanes 3 and 6 ) together with the reporter β-globin mRNA containing the MyoD 3′ UTR ( β-MyoD ) or the GM-CSF 3′ UTR ( β-GM-CSF ) . Cells were extracted , immunoprecipitated with the indicated flag-tagged protein and associated mRNAs detected by Northern Blot with radioactive anti-sense β-globin probes . A β-globin reporter with no putative TTP binding sites served as an internal negative control ( β-GAP ) . Pellet ( lanes 1–3 ) and 10% input ( lanes 4–6 ) fractions were loaded as indicated . Percentage bound normalized to input RNA indicated ( ND-not detectable ) . ( B ) A schematic of the tetracycline responsive ( TRE ) β-globin reporter constructs containing the 684 bp MyoD 3′ untranslated region ( UTR ) . Shown are the U-rich HuR binding sites , the putative TTP binding sequence ( WTß-MyoD , bold ) , and the mutated sequence ( MUTß-MyoD , bold ) . ( C ) TTP reduces the half-life of a WTß-MyoD reporter mRNA . Tet-off Hela cells transfected with either pcTET2-WTß-MyoD or pcTET2-MUTß-MyoD and CMV-β-Globin , a tetracycline unresponsive loading and transfection control , were subjected to pulse-chase mRNA decay assays and extracts separated by Northern blotting and probed with radioactive anti-sense β-globin probes in the presence of an empty vector control ( – ) or TTP . ( D ) The calculated β-MyoD mRNA half-life determined from the average ± SEM plotted from at least three independent experiments . Student's two-tailed t-test; * β-MyoD + TTP , p = 0 . 012 . DOI: http://dx . doi . org/10 . 7554/eLife . 03390 . 01010 . 7554/eLife . 03390 . 011Figure 4—figure supplement 1 . The MyoD 3′ UTR TTP binding site is conserved and stabilized by constitutive MAPKAPK-2 signaling . ( A ) The MyoD 3′ UTR TTP binding sequence ( TATTTAT ) is identical in seven mammals as depicted in the UCSC genome browser . ( B ) TTP reduces the half-life of a WTß-MyoD reporter mRNA but is stabilized in the presence of constitutively active MAPKAPK-2 . Tet-off Hela cells transfected with pcTET2-β-MyoD and CMV-β-Globin , a tetracycline unresponsive loading and transfection control , were subjected to pulse-chase mRNA decay assays and extracts separated by Northern blotting were probed with radioactive anti-sense β-globin probes in the presence of an empty vector control , TTP , or TTP and a constitutively active MK2 ( TTP+MK2-EE ) . The calculated β-MyoD mRNA half-life was determined from the average ± SEM plotted of at least 3 independent experiments . Student's two-tailed t-test; * β-MyoD + TTP , p = 0 . 012; β-MyoD + TTP + MK2EE compared to empty vector control p < 0 . 01 . DOI: http://dx . doi . org/10 . 7554/eLife . 03390 . 011 Active TTP may regulate SC activation by targeting mRNAs for rapid decay , similar to TTP-mediated regulation of inflammation via targeting of proinflammatory mRNAs in macrophages ( Stumpo et al . , 2010 ) . To test whether TTP regulates MyoD mRNA stability , we utilized a Hela cell line stably transfected with the Tetracycline trans-repressor ( Tet-Off Hela ) to express a β-Globin-MyoD 3′ UTR construct ( referred to as β-MyoD ) containing a tetracycline responsive element ( TRE ) upstream of the promoter ( Figure 4B ) . β-MyoD transcription is constitutive until tetracycline addition terminates transcription . At various time points post-chase ( following transcription termination ) , β-MyoD mRNA was quantified and the half-life calculated ( Clement and Lykke-Andersen , 2008 ) . WTß-MyoD mRNA half-life was 250 min when expressed in Tet-Off Hela cells ( Figure 4C , D ) , and co-transfection of a construct expressing TTP destabilized the WTß-MyoD mRNA , decreasing mRNA half-life by nearly twofold ( Figure 4C , D ) . Furthermore , TTP-mediated decay of the ß-MyoD mRNA was sensitive to p38α/β MAPK signaling as co-transfection of the TTP construct with a constitutively active MK2 kinase ( MK2-EE ) increased β-MyoD mRNA stability to ≥350 min ( Figure 4—figure supplement 1B ) . MyoD mRNA decay was dependent on the predicted TTP binding sequence as mutation of the putative TTP binding sequence ( MUTβ-MyoD; Figure 4B ) rendered TTP incapable of destabilizing MUTβ-MyoD mRNA ( Figure 4C , D ) . TTP may be required for SC quiescence as ( 1 ) TTP is present in quiescent SCs , ( 2 ) TTP is rapidly inhibited by p38α/β MAPK signaling within minutes of SC activation , and ( 3 ) active TTP reduces the half-life of MyoD mRNA . Therefore , loss of TTP from skeletal muscle tissue should precociously activate SCs , promoting inappropriate MyoD expression , and aberrant cell cycle entry . To test this prediction , we analyzed whole tibialis anterior ( TA ) muscle tissue from uninjured TTP-knockout mice ( Taylor et al . , 1996 ) and from uninjured TTP/TNFR1/2 triple knockout mice ( 3KO ) ( Carballo et al . , 2001 ) . The triple knockout mice were included to rule out potential muscle phenotypes arising as a secondary consequence of increased inflammation due to systemic stabilization of TNFα mRNA in the absence of TTP ( Stumpo et al . , 2010 ) . Neither the TTP knockout mice nor 3KO mice exhibited any detectable gross morphological differences in muscle sections immunostained with laminin ( Figure 5A ) . Moreover , we did not observe Pax7+ cells outside of the basal lamina ( Figure 5—figure supplement 1A ) nor any significant quantitative differences in Pax7+ SCs between wild type and knock out muscle sections when scoring for Pax7 immunoreactivity ( Figure 5—figure supplement 1B; additional quantification in Supplementary file 2A ) . In contrast , extralaminar and sublaminar Ki67+ cells were increased in TTP knockout mice and 3KO mice , suggesting that SCs exit the quiescent G0 state in the absence of TTP ( Figure 5—figure supplement 1C ) . Some sublaminar Ki67+ cells are Pax7 negative , suggesting these cells are myoblasts committed to myogenesis and undergoing terminal differentiation ( Figure 5—figure supplement 1A ) . Consistent with increased myogenic commitment of SCs , we observe twofold greater numbers of sublaminar MyoD+ cells in TTP knockout mice and in 3KO muscle compared to wild type mice ( Figure 5A , B ) . More centrally located nuclei ( CLN ) that are indicative of SC fusion into myofibers and a hallmark of muscle injury and repair , were present in TTP knockout mice and 3KO mice compared to controls ( Figure 5C ) . Thus , global loss of TTP appears sufficient to drive a low-level injury response in skeletal muscle in the absence of an overt physical or chemical insult . 10 . 7554/eLife . 03390 . 012Figure 5 . TTP loss-of-function promotes promiscuous SC activation . ( A–C ) Uninjured TA muscle sections from wild type ( WT ) , TTP knockout , and TTP/TNFR1/2 triple knockout mice ( 3KO ) were stained for MyoD ( red ) to identify cells committed to myogenesis . Loss of TTP or combined loss of TTP/TNFR1/2 in muscle results in significant increases in MyoD+ cells ( A , B ) and centrally located nuclei ( quantified in C ) , both hallmarks of actively regenerating muscle . ( D–F ) Pax7+ SCs were infected with mCherry-tagged shRNAs and analyzed 3 weeks post-infection to examine the effect of TTP knockdown on SC activation ( Figure 5—figure supplement 2B ) . TA muscle cross sections were stained with either Pax7 ( green ) or MyoD ( green ) and laminin ( white ) to determine the activation status of infected ( mCherry+ ) SCs . Shown in ( E ) and ( F ) are bar graphs quantifying the fraction of mCherry+ cells immunoreactive for Pax7 or MyoD , respectively . N = 3 mice for each experimental condition . For all images , nuclei are labeled blue with DAPI and muscle fibers are depicted in white using an anti-laminin antibody; scale bars = 50 µm . In bar graphs , ( * ) denotes a p-value < 0 . 05 , ( ** ) p < 0 . 01 , ( *** ) p < 0 . 001 as determined by student's two tailed t-tests . DOI: http://dx . doi . org/10 . 7554/eLife . 03390 . 01210 . 7554/eLife . 03390 . 013Figure 5—figure supplement 1 . TTP loss-of-function promotes muscle activation . Uninjured TA muscle sections from wild type ( WT ) , TTP knockout , and TTP/TNFR1/2 triple knockout mice ( 3KO ) were stained for ( A ) Pax7 ( red ) and laminin ( white ) to identify SCs ( carets ) , and-Ki67 ( green ) to mark non-quiescent , cycling cells ( asterisks ) . ( A–B ) When compared to WT muscle sections , the number of Pax7+ SCs were similar in TTP KO and 3KO muscle sections . ( C ) TTP loss-of-function results in a significant increase in sublaminar Ki67+ cells . Scale bar = 50 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 03390 . 01310 . 7554/eLife . 03390 . 014Figure 5—figure supplement 2 . SC-specific TTP knockdown breaks quiescence in uninjured muscle . ( A ) A graphic image depicting the breeding scheme employed to express tva ( RCAS receptor ) on Pax7+ SCs . ( B ) Experimental timeline for RCAS-based studies presented in ( C–D ) and in Figure 5 . Briefly , concentrated RCAS was injected via tail vein on 3 consecutive days into recombined Pax7CreERT2;Rosa26LSL-tva-lacZ recipient mice and muscle tissue was harvested for analysis 3 weeks following the final RCAS injection . ( C ) Representative montage reconstructions of TA muscle cross-sections from mice infected with control ( left ) or TTP ( right ) shRNAs . Dashed boxes highlight areas of mCherry+ fibers resulting from activation and eventual fusion of mCherry infected Pax7+ SCs . ( D ) Bar graph depicting the activation index ( mCherry+ fiber/single cell ratio ) of control vs TTP knockdown muscle cross-sections 3 weeks post-infection . ( ** ) p < 0 . 01 . N = 3 mice for each experimental condition . Scale bar = 200 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 03390 . 014 The relative contributions of non cell autonomous and cell autonomous effects on precocious SC activation cannot be resolved with the global knockout mice and thus , we employed an alternative approach to determine the effects of SC-specific TTP loss-of-function in vivo . We expressed the receptor for RCAS ( A ) virus , avian leukosis virus receptor ( subgroup A ) , or tva , in SCs and infected skeletal muscle tissue with RCAS modified to express U6-driven TTP shRNAs or control shRNAs with a fluorescent CMV-driven mCherry reporter ( Hughes , 2004; Harpavat and Cepko , 2006; Seidler et al . , 2008; von Werder et al . , 2012 ) . Since mammalian cells lack tva we generated Pax7CreERT2;Rosa26LSL-tva-lacZ mice that express tva on Pax7+ SCs upon tamoxifen-mediated Cre recombinase activation to evaluate RNAi-mediated TTP knockdown in quiescent adult muscle SCs ( Figure 5—figure supplement 2A ) . Tamoxifen-treated Pax7CreERT2;Rosa26LSL-tva-lacZ mice were injected intravenously with RCAS-shRNA-mCherry viruses to infect quiescent SCs ( Figure 5—figure supplement 2B ) . Myofibers were not mCherry+ 72 hr post-infection , ruling out direct infection by RCAS viruses ( not shown ) . 3 weeks post infection with a control shRNA ( shScramble ) , we observed mCherry expression in individual cells and in a small percentage of muscle fibers ( Figure 5—figure supplement 2C , left panel ) , demonstrating in vivo SC infection and subsequent fusion of infected SCs into myofibers ( mCherry+ fibers ) . A marked increase in mCherry+ fibers was observed in TTP shRNA ( shTTPmix ) infected muscle compared to muscle infected with scrambled shRNA expressing virus ( Figure 5—figure supplement 2C , right panel , Figure 5—figure supplement 2D ) . Muscle sections from control infected and TTP knockdown infected mice were then visualized for Pax7 and MyoD immunoreactivity and scored to quantify SC activation ( Figure 5D–F; additional quantification in Supplementary file 2B ) . The mCherry+ cells in control muscles infected with the scrambled shRNA expressing RCAS were >95% Pax7+ with fewer than 5% of the mCherry+ cells immunoreactive for MyoD ( Figure 5D–F ) . In contrast , a >threefold increase in MyoD+ immunoreactivity was observed mCherry+ cells in shTTPmix-infected muscle ( Figure 5D , F ) . Thus , SC-specific TTP knockdown breaks SC quiescence , promoting precocious SC activation and enhancing cell fusion with existing myofibers in the absence of muscle injury .
SC homeostasis , the maintenance of quiescence and re-acquisition of quiescence following asymmetric or symmetric cell division is not fully understood . Relatively constant SC numbers are maintained in healthy adult mice prior to and following single or multiple induced muscle injuries ( Cornelison et al . , 2004; Pisconti et al . , 2010 ) . Elegant experiments have established roles for maintenance of quiescence by microRNAs , where miR-489 represses Dek expression to maintain quiescence , miR-31 represses Myf-5 translation to maintain quiescence , and alternative polyadenylation regulates Pax3 ( Boutet et al . , 2012; Cheung et al . , 2012; Crist et al . , 2012 ) . Here , we propose that SC quiescence is actively maintained by mRNA decay , an additional post-transcriptional regulatory mechanism , via the Tis11 family of RNA binding proteins ( Figure 6 ) . Together , these studies suggest that a host of post-transcriptional regulatory mechanisms maintain the quiescent state of skeletal muscle stem cells and represent an efficient and rapid method for tissue-specific stem cell responses . 10 . 7554/eLife . 03390 . 015Figure 6 . Inhibition of TTP by p38α/β MAPK initiates a feed-forward circuit that commits SCs to the myogenic lineage . Muscle regeneration requires SCs to rapidly exit from quiescence but to also renew the quiescent SC population upon completion of regeneration . We propose that TTP suppresses transcripts for SC activation as a mechanism for immediate exit from quiescence . Upon muscle injury , p38α/β MAPK is rapidly activated resulting in phosphorylation of TTP . Phospho-TTP no longer suppresses activation-associated transcripts resulting in rapid induction and SC activation . One such activation-associated transcript targeted by TTP is MyoD , which is rapidly induced due to inactivation of TTP by p38α/β MAPK concurrent with increased MyoD mRNA stability mediated by HuR and transcriptional upregulation of the MyoD gene locus . Since MyoD up-regulates its own transcription , muscle injury would trigger a rapid feed-forward loop resulting in MyoD expression driving SCs to commit to myoblasts . Additionally , we propose that TTP may be involved in down regulation of MyoD mRNA and other TTP-target transcripts expressed by myoblasts , during the initial formation of a quiescent SC population or in reacquisition of quiescence following an injury . DOI: http://dx . doi . org/10 . 7554/eLife . 03390 . 015 Gene chip analysis of SCs isolated from uninjured and injured skeletal muscle and gene chip analysis of quiescent SCs isolated following injection of p38α/β MAPK inhibitors , identified a cohort of transcripts rapidly down-regulated upon SC activation , prior to cell cycle entry ( Farina et al . , 2012 ) . Transcripts encoding proteins that directly regulate mRNAs , including mRNA binding proteins involved in skeletal muscle diseases were highly represented , supporting a critical role for post-transcriptional regulation of RNA in skeletal muscle homeostasis ( Farina et al . , 2012 ) . The involvement of p38α/β MAPK signaling in SC activation ( Jones et al . , 2005 ) , asymmetric SC division ( Troy et al . , 2012 ) and loss of SC function during aging ( Bernet et al . , 2014 ) identifies p38α/β MAPK targets as likely mediators of these cellular processes . Therefore , we focused on TTP , which is a p38α/β MAPK signaling target ( Stumpo et al . , 2010 ) and was down-regulated to the greatest extent during SC activation . The Tis11/Zfp36 gene family is comprised of TTP and three related genes , of which TTP is the best characterized , containing a tandem zinc finger domain that binds AU rich sequences in the 3′ UTR of mRNAs and targets the mRNAs for sequestration or decay ( Brooks and Blackshear , 2013 ) . Potential TTP mRNA targets in SCs would most likely include positive regulators of myogenic commitment . One probable TTP target , HuR , is reported to stabilize MyoD and Myogenin transcripts in myogenic cell lines ( Figueroa et al . , 2003 ) . Although we found no evidence that TTP regulates HuR during SC activation , HuR is rapidly induced upon SC activation , suggesting that critical mediators of myogenic commitment , including the myogenic transcription factors MyoD and Myf5 , are regulated by multiple post-transcriptional regulatory pathways . For example , Myf5 is sequestered from translation in quiescent SCs by miR-33 ( Crist et al . , 2012 ) . MyoD is a likely candidate for post-transcriptional regulation as MyoD mRNA is present at low but detectable levels in freshly isolated SCs ( Supplemental file 1C ) ( Cornelison et al . , 2001; Liu et al . , 2013 ) . MyoD protein is induced within 3h of SC activation ( Jones et al . , 2005 ) and requires p38α/β MAPK activity ( Jones et al . , 2005 ) . Asymmetric p38α/β MAPK is required for commitment of one daughter cell to myogenesis during asymmetric SC division ( Troy et al . , 2012 ) and presumably , the TTP binding site in the 3′ UTR of MyoD would permit TTP-mediated mRNA decay in daughter cells lacking activated p38α/β MAPK . The behavior of SCs from aged mice is consistent with p38α/β MAPK-mediated regulation of MyoD as these SCs predominately commit to myogenesis with an accompanying reduction in self-renewal , consistent with their elevated p38α/β MAPK activity ( Bernet et al . , 2014; Cosgrove et al . , 2014 ) . Although MyoD mRNA appears to be a TTP target , TTP likely regulates other mRNAs with AU-rich sites involved in maintenance of SC quiescence . Furthermore , the Tis11 family members Brf1 and Brf2 were also down-regulated upon SC activation . Ongoing work is focused on identification and characterization of additional TTP/Tis11 targets and their involvement in maintenance of SC quiescence . In the absence of muscle injury , SC-specific TTP knockdown increased MyoD+ cells , presumably at the expense of Pax7+ progenitor cells . The increase in MyoD+ cells was accompanied by an increase in mCherry+ myofibers when compared to controls where SCs were infected with scrambled shRNAs . Thus , TTP inhibition breaks SC quiescence in uninjured muscle , providing strong evidence for the involvement of TTP in regulating SC homeostasis . Similarly , post-transcriptional regulation of mRNAs by miR-489 maintains SC quiescence , suggesting Dek and potentially other miR-489 targets regulate SC homeostasis ( Cheung et al . , 2012 ) . As we do not know the extent of functional diversity within the Pax7+ SC population it is possible that TTP loss-of-function and miR-489 preferentially affect sub-populations of Pax7+ SCs . Additional experiments will resolve whether SC quiescence is coordinately regulated by multiple post-transcriptional mechanisms or whether subsets of SCs are uniquely responsive to distinct skeletal muscle repair or maintenance requirements . SC quiescence is maintained by multiple post-transcriptional regulatory mechanisms including miRNA-mediated inhibition of Myf5 translation ( Crist et al . , 2012 ) and Dek translation ( Cheung et al . , 2012 ) . Here , we show that in addition to Myf5 and Pax3 ( Boutet et al . , 2012 ) , MyoD mRNA is regulated post-transcriptionally via a distinct regulatory mechanism whereby MyoD mRNA is targeted for rapid decay in quiescent SCs . Post-transcriptional regulatory circuitry is required for SC homeostasis and appears to maintain SCs in a primed state , permitting a rapid response to skeletal muscle injury . Moreover , post-transcriptional gene regulation likely facilitates SC self-renewal by asymmetric division and may regulate SC homeostasis via multiple mechanisms . In other tissues undergoing rapid turnover , similar post-transcriptional regulatory networks may ensure that a primed stem cell pool is available to repair and maintain tissue function .
Mice were bred and housed according to National Institutes of Health ( NIH ) guidelines for the ethical treatment of animals in a pathogen-free facility at the University of Colorado ( Wildtype and Syndecan-4 null line ) or at the at the National Institutes of Environmental Health Sciences ( TTP and TTP;TNFR1/2 null lines ) . All animal protocols were approved by the local IACUC . Wild-type mice were C57Bl/6xDBA2 ( B6D2F1; Jackson Labs , ME , USA ) ; Syndecan-4 null ( Cornelison et al . , 2004 ) , TTP null ( Taylor et al . , 1996 ) , and TTP;TNFR1/2 null ( Carballo et al . , 2001 ) mice are previously described . Cells , myofibers and TA muscles were harvested from female mice that were 3–6 months old . For TTP null and TTP;TNFR1/2 null analysis , TA muscle tissues were collected from male and female litermates that were 3–6 months old . Isolation and analysis of primary SCs by microarray and QT-PCR analysis was as previously described ( Farina et al . , 2012 ) . CEL files from three replicate gene chips were imported directly into Spotfire ( TIBCO , MA , USA ) and normalized by GCRMA . Using a 99% confidence threshold ( p-value ≤ 0 . 01 ) , probe sets that changed ≥twofold in relative expression between Sdc4−/− uninjured SCs and Sdc4−/− SCs 12 hr post injury were subtracted from probe sets that changed ≥twofold between wild type uninjured SCs and wild type SCs 12 hr post injury . This analysis resulted in the WT-S4 gene list ( Supplemental file 1A , B ) . Gene ontology analysis was performed using Ingenuity Pathway Analysis , CA , USA ( Farina et al . , 2012 ) . For in vitro activation , SCs were isolated from five wild type mice , pooled , and cultured for 0 hr ( freshly isolated ) , 2 hr , 4 hr , 8 hr and 12 hr in F12C containing 15% Horse serum and 2 nM FGF-2; data were collected for two independent experiments . Superscript III RT ( Invitrogen , CA , USA ) was used for reverse transcription of RNA into cDNA . Fast SYBR Green master mix ( Applied Biosystems ) was used according to manufacturer's instructions to amplify target transcripts using primers spanning exon/exon junctions for Elavl1 and Zfp36 , Zfp36l1 and Zfp36l2 . 18S rRNA was used as a reference gene and samples were analyzed in triplicate . Primer sequences: elavl1 Exon1/2; ( FOR: 5′ GCTTATTCGGGATAAAGTAGCAGGA; REV: 5′ TTCACAAAACCGTAGCCCAAG ) . Zfp36 Exon1/2; ( FOR: 5′ GCCATCTACGAGAGCCTCCA; REV: 5′ CGTGGTCGGATGACAGGTC ) . Zfp36l1 Exon1/2; ( FOR: 5′ CGAAGTTTTATGCAAGGGTAA; REV: 5′ GCGCTGGGAGTGCTGTAGTT ) . Zfp36l2 Exon1/2; ( FOR 5′ CGACCACACTTCTGTCACCCT; REV 5′ GGATTTCTCCGTCTTGCACAA ) . RCASBP ( A ) vectors containing Gateway cloning sites were obtained from Addgene ( MA , USA ) ( Harpavat and Cepko , 2006 ) . Control and TTP shRNA constructs ( TRC1/1 . 5 , Sigma–Aldrich , MO , USA ) were subcloned into RCAS vectors along with the sequence encoding the florescent reporter mCherry protein . RCAS viruses were generated in DF-1 chicken fibroblast cells using standard protocols and were concentrated and stored at −80C until use ( Flanagan-Steet et al . , 2000 ) . For in vivo studies , Tamoxifen-inducible Pax7CreERT2 Cre-driver mice ( provided by Dr Gabrielle Kardon ) were crossed with a conditional Tva receptor line ( ROSA26LSL-tva-lacZ ) ( Seidler et al . , 2008 ) to generate mice that upon tamoxifen administration express the Tva viral receptor on Pax7+ SCs . 3 days following a standard tamoxifen regimen , concentrated RCAS virus was injected ( on 3 consecutive days ) via tail vein into recipient mice to introduce shRNA and reporter gene constructs into uninjured Pax7+ SCs . 3 weeks post infection , TA muscle was harvested and processed/analyzed as described for immunofluorescence . All mice were housed in a pathogen-free facility , and the Institutional Animal Care and Use Committee at the University of Colorado approved all protocols . Whole TA muscles were either perfused or fixed for 2 hr on ice with 4% Paraformaldehyde following dissection from the limb . Muscles were sunk in 30% sucrose and mounted in Tissue Tek O . C . T . mounting media . Cryosections were post-fixed onto slides for 5 min in 4% PFA , permeablized for 5 min with 0 . 5% TritonX-100 in Phosphate Buffered Saline ( PBS ) , and blocked for 1 hr at room temperature with 5% BSA+0 . 2% Tx-100 . Primary antibodies were used at the following dilutions: 1:250 mouse anti-Pax7 ( Developmental Hybridoma Bank , Iowa University ) , 1:500 rabbit anti-MyoD ( Santa Cruz [C-20] sc-304 , CA , USA ) , rabbit anti-Ki67 ( ab15580 , Abcam , MA , USA ) , 1:300 Rat anti-Laminin ( 4HB-2 , Sigma-Aldrich , MO , USA ) , 1:1000 anti-TTP ( Cao et al . , 2004 ) , 1:1000 Chicken anti-Syndecan-4 , 1:50 anti-Phospho-MAPKAPK-2 ( Thr334 ) ( Cat#3041 , Cell Signaling ) . Sections were stained for 2 hr at room temperature or overnight at 4° , washed with PBS+0 . 2% Tx-100 and stained with Alexa Fluor secondary antibodies ( anti-rabbit 488 , anti-chicken 555 , anti-Rat 647 , and for mouse IgG detection anti-mouse 488 ) at a dilution of 1:500 for 1 hr at room temperature . Primary antibody concentrations for Duolink PLA were as follows: 1:200 anti-phospho-MAPKAPK-2 ( Thr334 ) ( Cat#3041 , Cell Signaling , MA , USA ) , 1:100 anti-CUGBP1 ( sc-20003 , Santa Cruz , CA , USA ) , 1:100 anti-HuR ( sc-5261 , Santa Cruz , CA , USA ) , and 1:100 anti-TTP ( sc-14030 , Santa Cruz , CA , USA ) . PLA was otherwise performed according to the manufacturer's protocol ( Olink Biosciences , Sweden ) . All immunostaining samples were counterstained with 4′ , 6-diamidino-2-phenylindole ( DAPI ) and sections were mounted with Vectashield ( Vector Labs , CA , USA ) . Primary SCs were dried down on gelatin coated-coverslips in 10 µl PBS in a laminar flow hood and fixed with 4% Paraformadehyde for 10 min at room temperature . Syndecan-4 staining for primary SCs was done as described for myofibers . Primary antibodies were used at the following dilutions: 1:500 mouse anti-HuR , provided by Dr Joan Steitz from Yale University , and 1:200 Rabbit anti-phospho-TTP antibody provided by Dr Georg Stoecklin at the University of Heidelberg in Germany . For scoring fluorescence intensity values , the samples were blinded and masks overlapping Syndecan-4 staining were made . The mean intensity for all fluorescence channels was calculated and background staining was subtracted using Slidebook software ( Intelligent Imaging Innovations , CO , USA ) . Muscle was dissected from hind limbs and digested for 1 . 5 hr at 37°C in 400U/ml Type I Collagenase ( Worthington , NJ , USA ) . The muscle slurry was placed into tissue culture dishes containing 15% horse serum in F12C media . Myofibers were teased apart from the muscle using pulled glass pipets and placed into fresh media . Fifty myofibers were transferred to six-well plates containing 2 ml F12C with 15% horse serum and 2 nM FGF-2 . Fibers were transfected with 2 . 75 µg of the pcDNA3-TTP-AA-myc plasmid and 0 . 25 µg eGFP-N1 plasmid with Lipofectamine 2000 ( Invitrogen , CA , USA ) according to the manufacturer's instructions for 4 hr . Transfection efficiencies ranged from 25% to 29% . The myofibers were then washed with F12C containing 15% horse serum and 2 nM FGF-2 and incubated for a total of 30 hr in culture . Myofibers were washed with PBS and fixed with 4% paraformaldehyde for 10 min , incubated with 10% goat serum for 1 hr at room temperature , then incubated overnight with 1:1000 chicken anti-Syndecan-4 antibody . The myofibers were washed 3 times with PBS and incubated for 1 hr at room temperature with anti-chicken 1:1000 AlexaFluor 555 and were post-fixed . To detect internal epitopes , myofibers were then permeabilized with PBS containing 0 . 5% Triton X-100 . Following a 1 hr block with 10% goat serum , fibers were incubated with primary antibodies ( 1:50 mouse anti-MyoD or 1:500 mouse anti-HuR ) overnight at 4°C . Fibers were then incubated with secondary anti-mouse Alexa Fluor 647 ( 1:500 ) antibodies for 1 hr at room temperature before being mounted on slides with Vectashield containing DAPI . In vivo inhibition of p38α/β MAPK was performed using a dosage of 15 mg SB203580 ( Alexis Biochemicals , NY , USA ) per kg of body weight . SB203580 was resuspended in DMSO and diluted using saline ( 0 . 9% NaCl ) . 1 hr prior to muscle tissue harvest , mice were injected intraperitoneally with 10 µl/g of 1 . 5 mg/ml SB203580 or a corresponding volume of diluted DMSO as a control . Hind limb muscle tissue was dissected from the leg and immediately placed in 25 μM SB203580 or the DMSO carrier as a control . Primary SCs were isolated as previously described ( Cornelison et al . , 2004 ) . Briefly , muscle tissue was minced using No . 10 scalpels for 5 min and then incubated for 1 hr at 37°C in 400U/ml Type I Collagenase ( Worthington , NJ , USA ) . The muscle slurry was diluted with F12-C media containing 15% horse serum and sequentially passed through 70 µm and 40 µm filters yielding a single cell suspension . SCs in suspension were fixed immediately in 4% paraformaldehyde for 10 min . The entire 3′ UTR of MyoD was amplified by PCR from C2C12 myoblast cDNA using the following primers: FOR: 5′ GCATCCATGCGGCCGCGGATGGTGTCCCTGGTTCTT; REV 5′ GCAATCATGCGGCCGCGCGTCTTTATTTCCAACACCT . The PCR product was cloned into NotI sites of the Tetracycline responsive β-Globin reporter construct as previously described ( Lykke-Andersen et al . , 2000 ) . Quikchange from Agilent ( CA , USA ) was used to mutate the WTß-MyoD construct according to the manufacturer's instructions using the following primers: FOR: 5′ GGGAGCCCCTTGGGCTAGAGTCATCTCCCAGGCATGCT; REV 5′ AGCATGCCTGGGAGATGACTCTAGCCCAAGGGGCTCCC . Tet-off Hela cells were co-transfected in six-well plates with 2 . 5 µg of either a wild type or mutant pcTET2-β-MyoD plasmid , 0 . 05 µg of a CMV-β-Globin plasmid , a tetracycline unresponsive loading and transfection control , and either 0 . 05 µg of a CMV-wtTTP plasmid , 0 . 05 μg CMV-wtTTP and 0 . 5 μg Flag-MK2EE or a pcDNA3 control plasmid . Transcription was pulsed for 6 hr by removal of tetracycline from the media . Following addition of 1 μg/ml tetracycline , RNA was harvested at indicated time points using Trizol according to manufacture's instructions . RNA was separated on a 1 . 2% Agarose Formaldehyde gel , transferred to a Nylon membrane , probed for β-globin , and the half-life was calculated as previously described ( Clement and Lykke-Andersen , 2008 ) . HeLa Tet-off cells in 10-cm plates were transiently transfected with 2 . 0 µg of the mouse TTP expression plasmids , pcDNA3-Flag-mTTP or pcDNA3-Flag-mTTPF126N ( previously described [Lai et al . , 2002] or 2 . 0 µg of an expression plasmid containing the N-terminus of the MS2 viral coat protein ( previously described [Lykke-Andersen et al . , 2001] ) together with 1 . 0 µg of a pwtGAP3UAC plasmid ( previously described [Clement and Lykke-Andersen , 2008] ) , which lacks a TTP binding site and either 8 . 0 µg of a pcTET2MyoD plasmid or 9 . 0 µg of a pcTET2 ATG-GMCSF plasmid . Cells were lysed in 800 µl of RNA immunoprecipitation buffer containing 10 mM Tris pH 7 . 5 , 200 mM NaCl , 2 mM EDTA , 0 . 1% Triton X-100 , 1 µg/ml FLAG Peptide ( Sigma-Aldrich , MO , USA ) , and protease inhibitors 0 . 5 mM PMSF , 2 µg/ml each of aprotinin and leupeptin . Lysates were incubated with 40 µl ( bead volume ) anti-FLAG M2 agarose ( Sigma ) at 4°C for 2 hr . Complexes were washed 8 times with Net-2 ( 50 mM Tris-HCl pH 7 . 5 , 200 mM NaCl , 0 . 1% Triton X-100 ) , eluted in 1 ml of Trizol reagent ( Invitrogen ) and stored at −20°C . Pellet and 10% input fractions were separated on a 1 . 2% agarose formaldehyde gel , transferred to a nylon membrane , and probed for β-Globin ( previously described ( Clement and Lykke-Andersen , 2008 ) ) .
Skeletal muscle satellite cells in their niche are quiescent and upon muscle injury , exit quiescence , proliferate to repair muscle tissue , and self-renew to replenish the satellite cell population . To understand the mechanisms involved in maintaining satellite cell quiescence , we identified gene transcripts that were differentially expressed during satellite cell activation following muscle injury . Transcripts encoding RNA binding proteins were among the most significantly changed and included the mRNA decay factor Tristetraprolin . Tristetraprolin promotes the decay of MyoD mRNA , which encodes a transcriptional regulator of myogenic commitment , via binding to the MyoD mRNA 3′ untranslated region . Upon satellite cell activation , p38α/β MAPK phosphorylates MAPKAP2 and inactivates Tristetraprolin , stabilizing MyoD mRNA . Satellite cell specific knockdown of Tristetraprolin precociously activates satellite cells in vivo , enabling MyoD accumulation , differentiation and cell fusion into myofibers . Regulation of mRNAs by Tristetraprolin appears to function as one of several critical post-transcriptional regulatory mechanisms controlling satellite cell homeostasis .
When muscles are damaged , they can repair themselves to some extent by making new muscle cells . These develop from groups of cells called satellite cells , which are found near the surface of muscle fibers . Once the muscle is injured , the satellite cells are activated and can divide to form two cells with different properties . One remains a satellite cell , while the other forms a ‘myoblast’ that eventually fuses into a mature muscle fiber . Under normal conditions the satellite cells remain in a dormant state and do not divide , but it is not clear how they maintain this dormant state . To create a protein , the gene that encodes it is first ‘transcribed’ to produce a molecule called mRNA , which is then used as a template to build the protein . A protein called Tristetraprolin ( TTP ) can bind to mRNA molecules and cause them to break down or decay , and so TTP can prevent the mRNA from being used to make a protein . Hausburg , Doles et al . analyzed satellite cells from uninjured muscle and compared them with those from injured tissue . This revealed that when injured , the satellite cells reduced the abundance of several mRNAs , including TTP . Further investigation found that in satellite cells from uninjured tissue , TTP causes the decay of mRNA molecules that are used to produce a protein called MyoD . As MyoD helps the satellite cells to specialize , this decay therefore prevents the formation of myoblasts and keeps the satellite cells in a dormant state . In contrast , damage to the muscle tissue activates a signaling pathway that ultimately inactivates TTP . This enables more of the MyoD protein to be made and the myoblast population to expand . When Hausburg , Doles et al . experimentally reduced the levels of TTP inside satellite cells , the cells developed into myoblasts even when the tissue was uninjured . Thus , TTP is an important regulator that allows satellite cells to remain in a dormant state . In dormant adult stem cells , regulation of protein availability by RNA binding proteins , such as TTP , may co-ordinate rapid changes in metabolic state to promptly repair injured tissue . A major challenge will be to identify the group of proteins involved and determine the precise mechanisms involved in regulating their availability .