ROCK2 and Its Alternatively Spliced Isoform ROCK2m Positively Control the Maturation of the Myogenic Program

Cell culture and RNA interference.C2C12 mouse myoblasts were maintained in Dulbecco's modified Eagle's medium (DMEM) with 20% fetal bovine serum (growth medium [GM]). To induce differentiation, cells were shifted to DMEM with 2% horse serum (differentiation medium [DM]). All media, sera, and complements for cell cultures were from Gibco-Invitrogen. Small interfering RNA (siRNA) duplex target sequences were designed by QIAGEN Gene Silencing. The siRNA target sequences were 5′-CAGAAGCGTTGTCTTATGCAA-3′ (exon 20) or 5′-TTGGATAAACATGGACATCTA-3′ (exon 5) for mouse ROCK2 and 5′-CTCTCTCAGTATTGCCACTAA-3′ (exon 27′) for mouse ROCK2m. These siRNAs against ROCK2 or ROCK2m produced consistent biological effects, thereby minimizing the possibility of off-target effects (see Results).

Undifferentiated C2C12 cells in GM at 70% of confluence plated in six-well dishes were transfected with 200 pmol/well of ROCK2 siRNA, ROCK2m siRNA, or nonsilencing (nonspecific) control siRNA (QIAGEN), using 20 μl/well of RNAi-Fect transfection reagent (QIAGEN). Eighteen hours after transfection, cells were shifted in DM or in DM containing 50 nM IGF-1 (R&D Systems) or 130 nM insulin (Sigma). Cells were harvested at 18, 42, 66, or 90 h after transfection, and total proteins were extracted for Western blotting analysis, or total RNA was extracted for quantitative PCR analysis.

Protein extraction and immunoblots.C2C12 cells were washed with phosphate-buffered saline and lysed with a buffer containing 150 mM NaCl, 20 mM Tris-HCl (pH 7.4), 1% wt/vol sodium dodecyl sulfate (SDS), 50 mM NaF, 1 mM 2-glycerophosphate, 1 mM sodium orthovanadate, 10 mM Na₄P₂O₇, 2 mM EGTA, and a protease inhibitor cocktail (Complete Mini; Roche). Cells were sonicated and cleared by centrifugation (14,000 × g for 10 min at 4°C), and the whole-cell lysate supernatant was collected. Proteins were resolved on SDS-polyacrylamide gel electrophoresis (PAGE), transferred onto Protran nitrocellulose membrane (Schleicher & Schuell), and immunoblotted under standard conditions with primary antibodies, as follows: ROCK2 and ROCK1 (BD Biosciences); desmin, cyclin D1, cyclin D3, and Cdkn1a (Santa Cruz); MyoD1 clone 5.8A (DakoCytomation); myogenin (a kind gift from Giulio Cossu); α-tubulin (Sigma); phospho-p42/p44 MAPK, total p42/p44 MAPK, phospho-p90RSK Thr-359/Ser-363, phospho-p90RSK Ser-380, total p90RSK, and eEF2 Thr56 (Cell Signaling). Anti-mouse or anti-rabbit immunoglobulin G horseradish peroxidase linked F(ab′)₂ secondary antibodies and an enhanced chemiluminescence detection kit were from Amersham.

RNA preparation from mouse tissues and Northern blotting analysis.Total RNA was extracted from tissues of 15-week-old wild-type C57BL/6J mice and from whole embryos (at embryonic day 11.5 [E11.5], E15.5, and E17.5) by RNA-TRIzol extraction (Invitrogen). Total RNA (15 μg) was size fractionated by electrophoresis on 1% agarose-formaldehyde gels, subjected to Northern blotting, and hybridized as previously described (26). cDNA probes for Northern blotting analysis were prepared by reverse transcription-PCR (Access RT-PCR system; Promega) using mouse liver total RNA as the template for ROCK1 and ROCK2 cDNA. The primers used in the RT-PCR for ROCK1 and for ROCK2 Northern blotting probes were the same as those described by Nakagawa et al. (28). The PCR fragments were subcloned into a pGEM-T Easy Vector (Promega), and the inserted cDNA was confirmed by sequencing. The excised probes (QIAGEN gel extraction kit) were purified and radioactively labeled by random priming (Amersham).

PCR amplifications and sequencing of ROCK2 isoforms. (i) Mouse isoforms.Total RNA (1 μg) from each mouse tissue or cell line was subjected to reverse transcription-PCR by the Access RT-PCR system (Promega). The primers were designed according to a mouse ROCK2 genomic sequence (GenBank reference sequence NM_009072) as follows: “a” (sense), 5′-AGCTGGAGATCAAAGAGATGATGG-3′ (in the exon 23 of ROCK2); “b” (antisense), 5′-AGTGTTGTTGCGCACAGGCAAT-3′ (in the exon 28 of ROCK2); “c” (sense), 5′-CCATATCACTCAGTCACACAC-3′ (in the intron 27 of ROCK2); and “d” (antisense), 5′-CCCACTGGTTCCACTGGAAAC-3′ (in the exon 31 of ROCK2).

(ii) Human and rat ROCK2m isoforms.A panel of first-strand cDNA from different human (Caucasian) or rat (Sprague-Dawley) tissues (BD Biosciences) was used as the template for PCR amplification. The primers were designed according to the human ROCK2 genomic sequence (GenBank reference sequence NM_004850), as follows: “c” (sense), as described above; and “e” (antisense), 5′-CCAACTGGCTCCACTGGAAAT-3′ (in exon 31 of the ROCK2 human sequence).

(iii) PCR amplification of rat ROCK2m isoforms.The primers were designed according to a rat ROCK2 genomic sequence (reference sequence NM_013022), as follows: “c” (sense), as described above; and “f” (antisense), 5′-AGTGTTGTTCCGCACAGGCAAC-3′ (in the exon 28 of the ROCK2 rat sequence). PCR fragments were sequenced in forward and reverse directions using BigDye Terminator chemistry and an ABI 3100 DNA sequencer (Applied Biosystems).

Real-time quantitative PCR.Total RNA was extracted from C2C12 cells using RNeasy mini columns (QIAGEN) and treated with RNase-free DNase. First-strand cDNA was generated from 1 μg of total RNA by using a T-primed first-strand kit Ready-To-Go (Amersham). Gene expression by real-time quantitative PCR was performed on an SDS-ABI Prism 7700 (Applied Biosystems) using premade 6-carboxyfluorescein (FAM)-labeled TaqMan assays for α-tubulin, ROCK1, ROCK2, MRF4 (gene alias, Myf6), Mef2c, myogenin, MyoD1, Cdkn1a, cyclin D1, cyclin D3, and desmin (Applied Biosystems). For the housekeeping gene, ubiquitin-B, and for the ROCK2m isoform, we designed two specific TaqMan assays (Applied Biosystems) consisting of sense primer 5′-CTCTGAAGAAGATTCCTTGCCTTACT-3′, antisense primer 5′-ACAGGCAATGACAACCATCCTT-3′, and FAM-labeled probe 5′-CCTATTAGTACAGAATCAAGATTAG-3′ for ROCK2m; and sense primer 5′-ACCTGGTCCTCCGTCTGA-3′, antisense primer 5′-TGGCTAGAGTGCAGAGTAATGC-3′, and FAM-labeled probe 5′-CCAGTGGGCAGTGATG-3′ for ubiquitin-B. Changes in gene expression (n-fold) were calculated by the 2^−ΔΔCT method, using the lower expressor as the calibrator (expression = 1).

Bioinformatics analysis.Exon-intron prediction analysis was performed by Genscan software (http://genes.mit.edu/GENSCAN.html ) (5). Conservation scores analysis for ROCK2 gene exonic and intronic structures from 17 different vertebrate species was performed by Vertebrate Multiz Alignment/Conservation at UCSC (http://genome.ucsc.edu ). Prediction of potential phosphorylation sites was performed by NetPhos server (http://www.cbs.dtu.dk/services/NetPhos/ ) software (3).

Generation of the ROCK2m-expressing vector.The expression plasmid pCAG-Myc-ROCK2 (generated by S. Narumiya, Kyoto University [28]) was cut with EcoRV restriction enzyme, and the resulting 2,800-bp fragment, containing the ROCK2 cDNA sequence from nucleotide 1665 to the end of the 3′ untranslated region of the insert, was ligated into the EcoRV site of vector pGEM-5Zf(+) (Promega) to generate the plasmid pGEM5-EcoRV-ROCK2. Moreover, a cDNA library from skeletal muscle was subjected to PCR amplification using the primers forward-25 (5′-GCTGTGAATAAGTTGGCGGAGATC-3′), located in the exon 25, and reverse-32 (5′-CCGACTAACCCATTTCTGCTG-3′), located in the exon 32 of ROCK2, and high-fidelity DNA polymerase AccuPrime-Pfx (Invitrogen). Two PCR products were obtained, corresponding to the cDNA fragments from exon 25 to exon 32 of ROCK2 alternative spliced forms, a 930-bp fragment from ROCK2 and an 1,100-bp fragment from ROCK2m cDNA. The blunt-ended 1,100-bp PCR fragment was purified, subjected to 3′-dATP tailing with Taq polymerase (Promega), and cloned into pGEM-Teasy vector (Promega). The resulting plasmid pGEM-T-(25-32)ROCK2m was cut with AflII and XbaI restriction enzymes, and an 826-bp AflII-XbaI fragment was purified and ligated in the plasmid pGEM5-EcoRV-ROCK2, previously cut with AflII and XbaI, to generate a pGEM5-EcoRV-ROCK2m construct. The plasmid pGEM5-EcoRV-ROCK2m was cut with ScaI to fragment the scaffold plasmid and with EcoRV to generate a 3,000-bp fragment that was purified and ligated into the final destination vector pCAG-Myc-ROCK2, previously cut with EcoRV, to finally obtain the pCAG-Myc-ROCK2m expression plasmid. All the constructs and PCR fragments were verified by restriction analysis and direct sequencing.

Transfections, immunoprecipitation, and kinase assay.C2C12 and HEK293T cells were transiently transfected with pCAG-Myc-ROCK2 and pCAG-Myc-ROCK2m or with the empty vector by Lipofectamine 2000 (Invitrogen). Thirty-six hours after transfection, cells were lysed in 1% Nonidet P-40-containing buffer and immunoprecipitated by protein G-Sepharose with a monoclonal anti-Myc tag antibody, 9E10 (BD Pharmingen). ROCK2 and ROCK2m catalytic activity levels were assessed in vitro by incubating anti-Myc immunoprecipitates for 5 min at 37°C in 25 mM Tris-HCl (pH 7.5), 50 mM NaCl, 10 mM MgCl₂, 0.1 mM Na₃VO₄, 500 μM dithiothreitol, 33 ng/ml myelin basic protein (Sigma) as a substrate, and 5 μM [γ-P³²]ATP (specific activity 10 mCi/μmol). Samples were resolved in SDS-PAGE and transferred onto nitrocellulose membranes. Radiolabeled bands were visualized by autoradiography and quantitated by using ImageQuant software after scanning in a PhosphorImager (Amersham).

Confocal immunofluorescence.Transfected C2C12 or HEK293T cells were fixed in formaldehyde and permeabilized by 0.5% Triton X-100. Anti-Myc tag (71D10) rabbit monoclonal antibody (MAb; Cell Signaling) and RhoA (26C4) mouse MAb (Santa Cruz) were incubated overnight at 4°C. Alexa-488 or Alexa-633-conjugated secondary antibodies (Molecular Probes) and 4′,6-diamidino-2-phenylindole (DAPI; 1 μg/ml) were used prior to mounting in Vectashield (Vector Laboratories). Omitting any of the primary antibodies resulted in a loss of the immunostaining, demonstrating the lack of the cross-reactivity in the double labeling. Cells were imaged using a confocal laser microscope (Leica TCS-SP5 system) with a Leica ×63 HCX plan-apochromat 1.4-numerical-aperture objective. Whole-cell volumes were collected at 1-μm Z steps, and images were projected.

ROCK2 knockout mouse embryo generation.Previously generated and described ROCK2^+/− heterozygous mice from the C57BL/6J genetic background (41) were intercrossed to produce ROCK2^−/− offspring. Genotyping was performed by PCR, according to conditions previously described (41), using genomic DNA prepared from tails of mice or visceral yolk sacs from embryos.

β-Galactosidase staining and immunohistochemistry.Dissected embryos were fixed in 2% formaldehyde for 30 min and stained for β-galactosidase as described previously (41). After color development, embryos were dehydrated and embedded in paraffin. Transverse sections (8 μm thick) were cut for histological observation. Antibody staining of sections was performed using antigen retrieval with 10 mM Tris buffer (pH 8.0). The antibodies antidesmin (Santa Cruz), antilaminin (Sigma), anti-phospho-p42/p44 MAPK (Cell Signaling), and anti-p42/p44 MAPK (Santa Cruz) were used. Anti-ROCK2m antiserum production was performed by immunization of New Zealand rabbits, with the peptide SHTLLDFDSEEDSLP conjugated to keyhole limpet hemocyanin. Immunohistochemical analysis of the myosin heavy chain, as shown in Fig. 8, was performed as previously described (27).

Nucleotide sequence accession number.DNA sequences were deposited in the GenBank database under accession number DQ864977.

ROCK2 is highly expressed in skeletal muscle.Rho has been implicated in the control of skeletal muscle differentiation, and ROCK1 and ROCK2 are among the downstream effectors of Rho. However, in vivo and in vitro, the specific functions of each ROCK isoform in muscle differentiation have not been elucidated. Accordingly, we performed a detailed analysis of expression and regulation of ROCK isoforms during muscle differentiation. Quantitative PCR analysis of a variety of adult mouse tissues revealed the highest expression of ROCK2 in heart, brain, and lung and in most skeletal muscles (Fig. 1B). ROCK1 and ROCK2 were expressed at high levels in heart and lung, whereas ROCK2 predominated in the brain and skeletal muscles (Fig. 1A and B). Northern blotting analysis of ROCK1 and ROCK2 expression was consistent with the results of the quantitative PCR (see Fig. 1C and reference 28). Consistent with the report by Nakagawa et al. (28), Northern blotting experiments showed one transcript of approximately 6.6 kb for ROCK1, whereas ROCK2 generated three major transcripts of approximately 7.6, 6.6, and 5.6 kb. The difference in tissue distribution of ROCK1 and ROCK2 raises the possibility that these kinases play distinct functions and that ROCK2 is likely to be more functionally relevant during muscle differentiation.

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FIG. 1.

ROCK2 is the predominant isoform in mouse skeletal muscles. (A and B) Quantitative PCR analysis of ROCK1 and ROCK2 expression in adult mouse tissues. First-strand cDNA was generated from 1 μg of total RNA. The expression data were normalized for the expression of ubiquitin-B. Results are expressed as means ± standard deviations. (C) Northern blotting analysis of isoforms ROCK1 and ROCK2 in adult mouse tissues. Northern blots of total RNA (15 μg/lane) were hybridized with ³²P-labeled cDNA probes specific for ROCK1 or for ROCK2; ethidium bromide (EtBr) staining of the 18S and 28S rRNA was used to verify equal amounts of loading and integrity of the RNA samples.

ROCK2m is a novel splicing isoform of ROCK2, evolutionarily conserved and predominantly expressed in skeletal muscle.We performed a bioinformatic analysis of the mouse ROCK2 gene sequence, using a predictive program to detect putative exonic and intronic regions in genomic sequences (5). This analysis predicted the presence of a potentially alternatively spliced exon located in an evolutionarily conserved region of intron 27 of the mouse ROCK2 gene (Fig. 2A). To confirm this in silico result, we performed a reverse-transcription PCR analysis using RNA from different mouse tissues and two primers located in exon 23 and exon 28 of the mouse ROCK2 gene (Fig. 2A and B, left panel). The PCR products from brain, lung, liver, spleen, and intestine showed the amplification of a single band of the expected size (655 bp), whereas the PCR products from four skeletal muscles (quadriceps, diaphragm, soleus, and gastrocnemius) showed amplification of the expected band of 655 bp plus an additional band of about 850 bp (Fig. 2B, left panel). This latter band was sequenced, and it revealed the inclusion of an extra exon between exon 27 and exon 28 (Fig. 2A). This extra exon, which was called exon 27′, encodes an open reading frame (ORF) of 171 bp (Fig. 2A). This ORF is in frame with the coding sequence of ROCK2 and generates a novel splicing variant (Fig. 2A), which we named ROCK2m (GenBank accession number DQ864977).

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FIG. 2.

ROCK2m is a novel splicing isoform of ROCK2, expressed predominantly in skeletal muscle. (A, upper panel) Exon-intron prediction analysis of part of the ROCK2 mouse gene sequence, performed by Genscan software (http://genes.mit.edu/GENSCAN.html ). Arrows represent primers, as described in Materials and Methods. (Middle panel) The alternatively spliced exon 27′ is located in a highly evolutionarily conserved part of intron 27 of the ROCK2 gene. The histogram shows evolutionary conservation scores for ROCK2 genes from 17 different vertebrate species obtained by Vertebrate Multiz Alignment/Conservation UCSC (http://genome.ucsc.edu ). (Lower panel) Schematic representation of the predicted functional domains of ROCK2/ROCK2m proteins (28). Amino acids are numbered. (B) PCR analysis of mouse ROCK2/ROCK2m. (Left panel) RT-PCR analysis of total RNA from mouse tissues, using primers “a” in exon 23 and “b” in exon 28. (Right panel) RT-PCR analysis of ROCK2m expression, using primers “c” in exon 27′ and “d” in exon 30 of the ROCK2 gene. (C) Quantitative PCR analysis of ROCK2m in mouse tissues. Results are expressed as means ± standard deviations. (D) Relative expression of ROCK2m in mouse tissues. Total ROCK2 and ROCK2m levels of expression were detected by quantitative PCR analysis. Numbers and white bars represent the percentages of ROCK2m expression with respect to those of the total ROCK2. (E) Immunohistochemical detection of ROCK2m in back muscles of an E18.5 mouse embryo. A specific anti-ROCK2m polyclonal antibody was raised, using an antigenic peptide corresponding to part of the mouse amino acid sequence of exon 27′. (F) PCR analysis of ROCK2m expression in human and rat tissues. cDNA panels from human and rat tissues were subjected to PCR amplification using primer “c” located in exon 27′ and primer “e” (located in the human exon 31) or primer “f” (located in the rat exon 28). (G) ROCK2 and ROCK2m kinase activity. HEK293T cells were transfected with the empty vector pCAG or with the same plasmid encoding Myc-ROCK2 or Myc-ROCK2m. The immunoprecipitated kinases were subjected to an in vitro kinase assay in the presence of [γ-³²P]ATP and in the presence (lanes 1, 2, and 3) or in the absence (lanes 4, 5, and 6) of the exogenous substrate MBP and developed by autoradiography (left panel) and PhosphorImager analysis. The amount of protein kinase in each immunoprecipitated sample was determined by immunoblotting (right panel, lanes 7, 8, and 9) and densitometry. The experiment shown is representative of three independent determinations. (H) Cellular localization of ROCK2 and ROCK2m. Immunofluorescent cells were imaged using a confocal laser microscope. Panels A and B show C2C12 cells transfected with ROCK2 or ROCK2m and stained with an anti-Myc antibody (red); DAPI was used to stain the DNA (blue). Panels C and D show HEK293T cells transfected with ROCK2 or ROCK2m and double stained with an anti-Myc antibody (red) and with an anti-RhoA antibody (green); DAPI was used to stain the DNA (blue). White bar = 25 μm in panels A and B and 10 μm in panels C and D.

Expression of the ROCK2m isoform was tested using two primers located in exon 27′ and in exon 30 of the mouse ROCK2 gene (Fig. 2B, right panel). ROCK2m was expressed in all the adult skeletal muscles and in both the proliferating myoblast and the differentiated myotubes of the mouse myogenic cell line C2C12 (Fig. 2B, right panel). ROCK2m transcripts were also detected in heart and skin (Fig. 2B, right panel). However, quantitative analysis revealed that ROCK2m is only minimally expressed in these two latter tissues (Fig. 2C). Notably, ROCK2m was not present in brain and lung (Fig. 2B), although ROCK2 expression was found to be very high in these tissues (Fig. 1B). Moreover, ROCK2m was also undetectable in tissues rich in smooth muscle (i.e., stomach and intestine, see Fig. 2B) where ROCK plays an important physiological role in the process of Ca²⁺ sensitization (38).

Evaluation of relative ROCK2m expression levels was performed by quantitative real-time PCR assay, developing a specific TaqMan probe (Fig. 2C and D). Quantitative PCR measurements of ROCK2m transcripts showed a high expression of this isoform in skeletal muscles (with the exception of the latissimus dorsi), whereas very low levels of ROCK2m were detected in heart and skin (Fig. 2C). ROCK2m mRNA was undetectable by quantitative PCR in brain, liver, intestine, stomach, lung, testis, kidney, and spleen (Fig. 2C and data not shown). The ratio between the ROCK2m versus total ROCK2 expression level was obtained by the real-time PCR comparative C_T method and showed that ROCK2m accounts for 20 to 50% of the total ROCK2 in all the adult mouse muscles tested, whereas in heart and skin, ROCK2m accounts for only 3% and 9% of the total ROCK2, respectively (Fig. 2D).

To evaluate the evolutionary conservation of ROCK2m, we performed PCR experiments with cDNA panels from different human and rat tissues, using ortholog DNA primers. ROCK2m was detected in heart and skeletal muscles, whereas it was absent from the other tissues tested (Fig. 2F). Thus, the mechanism and pattern of alternative splicing of mouse ROCK2/ROCK2m are conserved in other mammalian species.

An anti-peptide polyclonal antibody specifically recognizing the ROCK2m isoform was unable to recognize ROCK2m in Western blotting experiments but was effective in the immunohistochemical detection of ROCK2m in muscle (Fig. 2E).

The amino acidic sequence encoded by the exon 27′ ORF revealed an extremely high content of Ser/Thr (one-third of the total amino acid residues), half of which was predicted in silico as potential phosphorylation sites (3). The amino acidic sequence of the exon 27′ is located in the carboxyl-terminal region of ROCK2, flanking the pleckstrin homology domain (Fig. 2A). The carboxyl-terminal region of ROCK has been demonstrated to function as an autoregulatory inhibitor domain of the amino-terminal kinase region (1). Therefore, we can speculate that the insertion of an extra domain in such regulatory position could have potential effects on the conformation and the activity of the kinase.

In order to address the structure/function of ROCK2m, we cloned the full-length cDNA of ROCK2m (see Materials and Methods), and we evaluated ROCK2m kinase activity, cellular localization, and interaction with RhoA. HEK293T and C2C12 cells were transfected with plasmids expressing N-terminally Myc-tagged ROCK2 or ROCK2m. Immunofluorescence microscopy revealed that about 80% of the HEK293T cells and about 10% of the C2C12 cells were efficiently transfected by the ROCK2- or the ROCK2m-expressing plasmid (data not shown).

As shown in Fig. 2G, when anti-Myc immunoprecipitates from transiently transfected HEK293T cells were subjected to an in vitro kinase assay using myelin basic protein (MBP) as the exogenous substrate, ROCK2m displayed a kinase activity on MBP not significantly different than that of ROCK2 (Fig. 2G). Indeed, after normalization for the amount of immunoprecipitated proteins (Fig. 2G, right panel), the ratio between the catalytic activity of ROCK2m and the catalytic activity of ROCK2 on MBP was found to be 0.84 ± 0.31 (mean ± standard deviation [SD], n = 3). In the presence of MBP in the kinase assay, both of the immunoprecipitated ROCK2 isoforms underwent autophosphorylation, whereas in the absence of the exogenous substrate, we could not observe any significant autophosphorylation (Fig. 2G).

Notably, ROCK2m displayed an autophosphorylation level significantly lower than that of ROCK2 (Fig. 2G). After normalization for the amount of immunoprecipitated proteins, the ratio between the autophosphorylation level of ROCK2m and the autophosphorylation level of ROCK2 was found to be 0.42 ± 0.20 (n = 3). Under the conditions of immunoprecipitation employed, we did not see any differential phosphorylated band coprecipitating with ROCK2 or with ROCK2m (Fig. 2G).

Cellular localization of ROCK2 isoforms was analyzed with C2C12 myocytes and HEK293T cells. ROCK2 and ROCK2m showed cellular distributions that were similar: both isoforms were mainly diffused, with some localization at the cell margins (Fig. 2H). This cell distribution was similar to that previously observed for ROCK2 (19, 21).

It has been reported that ROCK2 could be partly recruited to the plasma membrane by RhoA (19, 21). We addressed the functional interaction between RhoA and ROCK2m by a colocalization study. We were able to efficiently stain endogenous RhoA in HEK293T cells, but the same antibodies were not efficient in C2C12 cells (data not shown). Using confocal immunofluorescence microscopy, we observed that ROCK2 and the endogenous RhoA partly colocalize in HEK293T cells (Fig. 2H, panel C), consistent with previous observations (19). However, in HEK293T cells, we could not observe the same colocalization between the ROCK2m splicing isoform and the endogenous RhoA (Fig. 2H, panel D). These data suggest that ROCK2m may constitute a specialized pool of the cellular ROCK2 not directly recruited at the plasma membrane by RhoA. Further biochemical studies are needed to uncover the distinct properties of ROCK2m and to explore whether the insertion of the extra domain can modulate its binding capacity to RhoA and its autophosphorylation properties.

ROCK2 and ROCK2m are progressively expressed during muscle differentiation.To study the dynamics of ROCK1, ROCK2, and ROCK2m expression during muscle differentiation, we utilized C2C12 cells as an in vitro myogenic differentiation model system which allows a detailed characterization of molecular events, such as expression of cell cycle regulators and myogenic markers (36). Using quantitative real-time PCR, we observed that in C2C12 cells, ROCK1 mRNA was less abundant compared to that of ROCK2 (Fig. 3A). Consistently, ROCK1 expression remained relatively constant during the course of the differentiation, whereas ROCK2 expression was up-regulated more than threefold during the process of myotube formation and maturation (Fig. 3A). ROCK2m accounted for about 50% of the total ROCK2 in every stage of differentiation of C2C12 cells, with kinetics of expression similar to those of ROCK2 (Fig. 3B). The dynamics of ROCK isoform expression levels during C2C12 myogenic differentiation were compared to markers of myogenic differentiation (Fig. 3C to F). Expression levels of MyoD, myogenin, MEF-2C, desmin, cyclin D1, cyclin D3, and Cdkn1a were consistent with the progressive transition from the proliferative stage to the cell cycle withdrawal and myogenic differentiation (36). While MyoD, myogenin, Cdkn1a, and desmin expression increased early after cell shifting in DM and decreased at later stages of differentiation, MEF-2C, cyclin D3, and Mrf4 showed a more sustained expression level, and cyclin D1 was progressively and dramatically down-regulated (Fig. 3C to F). These analyses established the relative dynamics of ROCK expression and provided a model for detailed analysis of ROCK isoform functions during muscle differentiation.

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FIG. 3.

ROCK2 and ROCK2m are progressively expressed during myogenic differentiation. (A through F) C2C12 undifferentiated myoblasts were maintained in GM; differentiation was induced by switching subconfluent cells to DM for 1 to 3 days. RNA was extracted, and first-strand cDNA was generated from 1 μg of total RNA. The expression data from quantitative real-time PCR analysis were normalized for the expression of ubiquitin-B. Results are expressed as means ± standard deviations.

ROCK2 expression during mouse embryonic skeletal muscle development.Analysis of ROCK isoform expression was performed with RNA isolated from total mouse embryos at different developmental stages. As seen in Fig. 4, ROCK1 expression remained relatively constant from E11.5 to E17.5, whereas ROCK2 expression was up-regulated approximately twofold in the same developmental window (Fig. 4A and B). ROCK1 mRNA was consistently less abundant than ROCK2 mRNA (data not shown). ROCK2m expression was progressively up-regulated by about 70-fold from E11.5 to E17.5 (Fig. 4C), and at E17.5, ROCK2m accounted for about 20% of the total ROCK2 expressed by the whole embryo (Fig. 4D).

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FIG. 4.

ROCK2 expression during mouse embryonic development. (A to D) Expression dynamics of ROCK1, ROCK2, and ROCK2m during mouse embryonic development. Total RNA was isolated from total mouse embryos at E11.5, E15.5, and E17.5, and first-strand cDNA was generated. The expression data from quantitative PCR analysis were equalized for the expression of ubiquitin-B. Results are expressed as means ± standard deviations. (E) Phenotype of the E11.5 offspring obtained by crossing ROCK2^+/− with ROCK2^+/−; results are shown for E11.5 ROCK2^+/+ (left bars), E11.5 ROCK2^+/− (center bars), and E11.5 ROCK2^−/− (right bars) littermates. ROCK2 expression was revealed by X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) staining of the LacZ reporter gene. Genotyping was performed by PCR with embryonic visceral yolk sacs. (F to I) Transverse sections of E11.5 ROCK2^+/− embryo at the trunk level. Dashed lines indicated by g, h, and i in panel F refer, respectively, to the sections shown in panels G, H, and I. dm, dermomyotome; s, somite; mp, limb muscle precursors; sc, anterior horn of the spinal cord.

To monitor the expression pattern of ROCK2 during embryonic development, we took advantage of a mouse line previously generated and described by Narumiya and collaborators, in which the ROCK2 gene was disrupted by the insertion of the lacZ reporter cassette into exon 3, in frame with the start codon (41). In this previous analysis, the majority of the ROCK2^−/− embryos died in utero in the late stage of pregnancy, and growth retardation was observed in the small number of surviving ROCK2^−/− embryos and pups (41). Statistical analysis of genotype distribution in utero revealed that until E15.5, ROCK2^−/− embryos were viable (41). Moreover, until E15.5, no growth retardation and no gross anatomical abnormalities were observed (Fig. 4E and reference 41). We therefore restricted our analysis to stages earlier than E15.5. Since the secondary wave of muscle differentiation begins approximately at E15.5 (17), our muscle phenotypic analysis was restricted to initial myofiber formation.

LacZ reporter expression in ROCK2^+/− heterozygous mice (Fig. 4E to I) recapitulated the spatiotemporal expression of ROCK2 during mouse embryogenesis (41). Migrating muscle precursor cells delaminate from the lateral dermomyotome, a derivative of the somite, to generate the muscles of limbs (30). At E11.5, LacZ expression was high in the somites (Fig. 4I), in the dermomyotome and in the limb muscle precursors (Fig. 4G and H) as well as in multiple neuronal precursor populations (particularly in the anterior horn of the spinal cord, see Fig. 4H). Similar patterns of LacZ expression in the dermomyotome and in migrating muscle precursors cells were also observed in E11.5 ROCK2^−/− embryos (not shown), implying that the total absence of ROCK2 protein did not impair its own transcription in these embryonic structures, nor did it impair the process of delamination/migration of the muscle precursor cells. Analysis of skeletal muscles (distal forelimb and hind limb muscles, hypaxial musculature at the trunk level, diaphragm, back muscles, and intrinsic tongue muscles) at E13.5 and E15.5 in ROCK2^−/− did not show any gross muscle alteration (not shown), suggesting that ROCK2 is dispensable for the proper execution of the primary wave of myogenesis.

ROCK2 and ROCK2m positively regulate myogenic differentiation.In previous studies, the synthetic inhibitor Y-27632 or the forced expression of the dominant active form of ROCK1 has been used to investigate the possible functions of ROCK isoforms during myogenic differentiation (7, 29). However, Y-27632 cannot distinguish isoform-specific effects and can inhibit kinases other than ROCK isoforms (32, 41). Moreover, the overexpression of a dominant active truncated form of ROCK could have generalized effects on actin cytoskeleton, thus complicating the interpretation of muscle-specific effects of ROCK isoforms (18, 32).

We addressed the isoform-specific role of ROCK2 and ROCK2m in muscle differentiation. We selectively knocked down the endogenous ROCK2 or ROCK2m, using siRNA.

Specific knockdown of ROCK2 in C2C12 cells by siRNA reduced the total endogenous ROCK2 and ROCK2m transcripts (Fig. 5A). Transfection of C2C12 cultures with siRNA specific for the ROCK2m isoform resulted in a dramatic decrease of ROCK2m transcripts (Fig. 5B). ROCK2m transcripts account for about 50% of the total ROCK2 in C2C12 cells (Fig. 3). Indeed, knockdown of ROCK2m also resulted in a significant reduction in the levels of total ROCK2 (Fig. 5B).

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FIG. 5.

ROCK2 and ROCK2m positively regulate myogenic differentiation. Undifferentiated C2C12 cells in GM were transfected with ROCK2 siRNA, ROCK2m siRNA, or control (nonsilencing) siRNA and after 18 h were switched into DM. (A and B) Quantitative PCR analysis of myogenic differentiation factors in siRNA-transfected C2C12 cells. siRNA-transfected cells were harvested after 2 days in DM; total RNA was extracted, and first-strand cDNA was generated from 1 μg of total RNA. Expression data were normalized for the expression of ubiquitin-B. Results are expressed as means ± standard deviations. (C) Western blotting analysis of siRNA-transfected C2C12 cells. siRNA-transfected cells were harvested in GM or after 1 or 2 days in DM, and total protein was extracted. Total cell lysate (70 μg) was subjected to Western blotting analysis with the indicated specific antibodies.

Microscopy examination of the general morphology of the transfected C2C12 cells showed that siRNA-mediated knock-down of ROCK2 or of ROCK2m did not cause any obvious impairment in the fusion process or any significant alteration of myotube formation and maintenance during the culture time examined (up to 4 days in DM; results not shown).

To test whether the knockdown of ROCK2/ROCK2m could induce general changes in C2C12 differentiation, we analyzed the expression profile of MyoD, myogenin, MEF-2C, Mrf4, and desmin in cells transfected with siRNA for ROCK2 or for ROCK2m (Fig. 5A and B). Inhibition of the expression of ROCK2 or ROCK2m caused a reduction of desmin, Mrf4, and MyoD expression, while no changes were induced in levels of myogenin and MEF-2C transcripts (Fig. 5A and B). Notably, the knockdown of ROCK2m resulted in an even greater down-modulation of myogenic markers compared to that of total ROCK2. Considering that ROCK2m comprises up to 50% of the total ROCK2 in C2C12 cells, the interference of ROCK2m expression had a more significant effect in myogenic differentiation compared to the interference of total ROCK2.

To test whether effects of the siRNA-mediated knockdown of ROCK2/ROCK2m were also reproducible at the protein level, the total cell lysates from C2C12 cells transfected with siRNA for ROCK2 or for ROCK2m were analyzed by Western blotting (Fig. 5C). The knockdown of ROCK2 or ROCK2m caused a significant decrease in the protein levels of total ROCK2, MyoD, and desmin, whereas myogenin, α-tubulin, and ROCK1 (Fig. 5C and data not shown) were not significantly affected. By using anti-ROCK2 antibody, we detected a proportional decrease of the protein levels of total ROCK2 after ROCK2m knockdown (Fig. 5C).

We investigated whether ROCK2/ROCK2m may regulate cell cycle-related markers. We monitored the protein levels of cyclin D1 and cyclin D3, two well-established mitogenic sensors that couple cell cycle withdrawal to muscle differentiation (36, 44, 45). Cyclin D1 is down-regulated, whereas expression of cyclin D3 is up-regulated during the process of normal differentiation of C2C12 cells (Fig. 3). Notably, no major changes were induced in the protein levels of both cyclins by ROCK2 siRNA or by ROCK2m siRNA during myogenic differentiation (Fig. 5C).

The acquisition of apoptosis resistance by myogenic precursor cells is also a critical event during differentiation (44). Cdkn1a, which promotes cell cycle exit, also confers apoptosis resistance to the differentiating myoblasts (44, 45). The knock-down of ROCK2 or ROCK2m did not perturb the expression of Cdkn1a (Fig. 5C).

ROCK2 and ROCK2m positively control the activation of p42/p44 MAPK and of p90RSK.p42/p44 MAPK have a key role in myogenesis in that sustained p42/p44 MAPK activation is required at the onset of differentiation in myogenic cells (2, 15, 35). To determine whether signals arising from ROCK2/ROCK2m can influence p42/p44 MAPK activity, we performed Western blotting analysis with differentiating C2C12 cells in the presence of ROCK2 or ROCK2m siRNA. Normal (control) C2C12 differentiating myocytes strongly induced phospho-active p42/p44 MAPK compared to proliferating cells in growth medium (Fig. 6A and B). In the differentiation window explored, p44 MAPK activation was largely predominant over that of p42 MAPK (Fig. 6A and B). In C2C12 cells transfected with siRNA targeting ROCK2 or ROCK2m, significant decreases in the activation of both p42 and p44 MAPK were observed (Fig. 6A and B). The total amounts of p42 and p44 MAPK proteins during differentiation were not affected by ROCK2 or ROCK2m siRNA (Fig. 6A). Analogously, the activation and the total amount of p38 and AKT, two kinases important for myogenic growth and differentiation (20, 43, 50), were not affected by ROCK2 or by ROCK2m siRNA (not shown).

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FIG. 6.

ROCK2 and ROCK2m positively control the activation of p42/p44 MAPK and of p90RSK during myogenic differentiation. Undifferentiated C2C12 cells in GM were transfected with ROCK2 siRNA, ROCK2m siRNA, or control (nonsilencing) siRNA and after 18 h were switched into DM. (A) Western blotting analysis of siRNA-transfected C2C12 cells. siRNA-transfected cells were harvested in GM or after 1 or 2 days in DM, and total protein was extracted. Total cell lysates (70 μg) were subjected to Western blotting analysis with the indicated specific antibodies. (B) Quantitation by densitometry of the phospho-p42/p44MAPK immunoblot bands shown in panel A.

The p90RSK is activated by p42/p44 MAPK in vivo and in vitro via phosphorylation of specific sites (10, 51). Once activated, p90RSK phosphorylates elongation factor 2 kinase (eEF2K), thus inhibiting eEF2K activity (Fig. 8C and reference 46). Inhibition of eEF2K activity results in a decrease in the inhibitory phosphorylation at the Thr56 residue of eEF2, thus activating eEF2 (Fig. 8C and 46). Indeed, only the dephosphorylated form of eEF2 can mediate the translocation step of elongation and promote protein synthesis (46). Using antibodies specific for activated forms of p90RSK, we observed that p90RSK undergoes full activation during the normal progression of C2C12 myogenic differentiation, concomitantly to p42/p44 MAPK activation (Fig. 6A). Specific down-modulation of ROCK2 or ROCK2m caused a dramatic decrease in the levels of phospho-active p90RSK during C2C12 differentiation, thus increasing the level of phospho-Thr56 eEF2 (Fig. 6A). These results suggest that in myogenic cells, ROCK2/ROCK2m can positively regulate differentiation-induced protein synthesis.

To explore the relevance of these findings in vivo, we compared equivalent histological cross-sections of E13.5 and E15.5 distal forelimbs from ROCK2^−/− and ROCK2^+/− (control) embryos (Fig. 7). At these stages of limb development, skeletal muscle cells already express muscle-specific proteins such as desmin and laminin, which were visualized by immunohistochemistry. In the limb cross-sections from ROCK2^−/− embryos, both extensor and flexor groups of muscles were normally developed, and compared with sections from control heterozygous embryos, there were no significant reductions in the size of the muscle fibers and in the number of laminin-positive cells. However, at E13.5 and E15.5, there was a significant reduction in the number of desmin-positive cells and, notably, in the level of activated phospho-p42/p44 MAPK (Fig. 7). These in vivo results mirror the effects of ROCK2 knock-down in C2C12 myogenic cells (Fig. 5 and Fig. 6) and indicate that although the absence of ROCK2 or ROCK2m protein did not alter the number of muscle fibers and the gross anatomy of limb muscles, it reduced the steady-state levels of desmin protein and perturbed the phosphorylation/activation of p42/p44 MAPK.

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FIG. 7.

ROCK2 affects the levels of desmin and the activation of p42/p44 MAPK in mouse forelimbs. Muscle-specific proteins were examined by immunohistochemistry in equivalent histological cross-sections at the level of lower forelimbs of E13.5 and E15.5 ROCK2^−/− and ROCK2^+/− (control) mouse embryos. Panels a, b, a′, and b′ are higher magnifications (×100) of the muscle groups indicated by arrows in the lower magnification panel (×20). Note the radius (R) and ulna (U). ROCK2^+/− or ROCK2^−/− E13.5 and E15.5 embryos were obtained by crossing ROCK2^+/− with ROCK2^+/− mice, and genotyping of the offspring was performed by PCR using embryonic visceral yolk sacs.

ROCK2 and ROCK2m positively control the hypertrophic effect elicited by insulin and IGF-1.We demonstrated that in myogenic cells, ROCK2 and ROCK2m positively control the activation of p42/p44 MAPK, p90RSK, and eEF2. There is ample evidence showing that in muscle, insulin and IGF-1 stimulation activate the components of the kinase cascade including p42/p44 MAPK and p90RSK, leading to eEF2 activation and sustaining the machinery of terminal differentiation (9, 33, 46). Moreover, we and others demonstrated that IGF-1 and insulin promote the maturation of the myogenic program through induction of myotube hypertrophy (9, 26, 27). Collectively, these data suggest the hypothesis that in muscle, ROCK2/ROCK2m may be involved in the regulation the hypertrophic pathways activated by IGF-1 and/or insulin. To test this hypothesis, we explored the ability of the myotubes to sustain the biological response to IGF-1/insulin during myogenic differentiation, under the knockdown conditions of ROCK2/ROCK2m.

C2C12 myoblasts were transfected with siRNA for ROCK2 or ROCK2m. Eighteen hours after transfection, the cells were switched to DM in order to permanently withdraw them from their cell cycles and to commit them to myogenic differentiation (day zero). The day after, the cells were switched to DM containing IGF-1 or insulin and allowed to differentiate for 2 days. Cells were subjected to morphological and biochemical analysis (Fig. 8).

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FIG. 8.

ROCK2 and ROCK2m positively control the hypertrophic effect elicited by IGF-1 and insulin. C2C12 cells in GM were transfected with ROCK2 siRNA, ROCK2m siRNA, or control (nonsilencing) siRNA and after 18 h were switched to DM in order to withdraw them from the cell cycle and to commit them to myogenic differentiation. After 24 h in DM, the cells were switched to DM containing 50 nM IGF-1 or 130 nM insulin. After 48 h, cells were analyzed by morphologic examination and immunoblotting. (A) Transfected and differentiated cell cultures, after 2 days in DM plus IGF-1 or DM plus insulin, were immunostained with an antibody against the embryonic isoform of myosin heavy chain. Panels 1, 2, 5 and 6 show cells transfected with control (nonsilencing) siRNA. Panels 3 and 7 show cells transfected with ROCK2 siRNA. Panels 4 and 8 show cells transfected with ROCK2m siRNA. (B) Transfected and differentiated cell cultures, after 2 days in DM plus IGF-1 or in DM plus insulin, were subjected to Western blotting analysis. Cells were harvested, and total protein was extracted; total cell lysate (70 μg) was subjected to Western blotting analysis with the indicated specific antibodies. (C) A schematic model of the signaling connections identified in this study, showing the control of ROCK2/ROCK2m on the IGF-1/insulin-p42/p44MAPK-p90RSK-eEF2 axis.

It has been previously reported that during in vitro myogenic differentiation, IGF-1-induced hypertrophy is morphologically characterized not only by the increased size of the single myotubes but also by the progressive fusion of adjacent myotubes (26, 27). Indeed, as previously reported (26, 27), myogenic differentiation in the presence of IGF-1 or insulin induced significant morphological hypertrophy of myotubes in the control (Fig. 8A). On the contrary, microscopy examination of the general morphology of C2C12 myotubes derived from myoblasts transfected with siRNA against ROCK2 or ROCK2m revealed a significant reduction in the hypertrophic response elicited by IGF-1 and insulin (Fig. 8A). It is notable that the myosin expression was not significantly affected (Fig. 8A and data not shown), indicating that down-regulation of ROCK2/ROCK2m does not dramatically affect the early phase of muscle differentiation. It is more probable that ROCK2 and ROCK2m have a greater effect on the control of the latter induction of hypertrophy (Fig. 8A) (26, 27).

Western blotting analysis revealed that differentiation of C2C12 cells in the presence of IGF-1 or insulin caused a significant increase in the levels of MyoD, desmin, phospho-active p42/p44 MAPK, and phospho-active p90RSK and the dephosphorylation of eEF2, activating its role in promoting protein synthesis (Fig. 8B). On the contrary, myotubes derived from myoblasts transfected with siRNA against ROCK2 or ROCK2m revealed a dramatic reduction in the insulin- or IGF-1-induced up-regulation of MyoD, desmin, phospho-active p42/p44 MAPK, phospho-active p90RSK, and active eEF2 (Fig. 8B). These data strongly suggest that in myogenic cells, ROCK2 and ROCK2m are positive regulators of the p42/p44MAPK-p90RSK-eEF2 intracellular signaling pathway and thereby positively regulate the hypertrophic effect elicited by insulin and IGF-1 (Fig. 8C).