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B transcription factor
Department of Medicine, Baylor College of Medicine, Houston, Texas, USA
1Correspondence: Department of Medicine, Pulmonary and Critical Care Section, Suite 520B, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA. E-mail: axkumar{at}bcm.tmc.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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B) transcription factor and the expression of NF-B-regulated genes, cyclin D1 and IL-6, were augmented in response to mechanical strain. Cyclic strain also increased the activity of Rho GTPases, especially Rac-1. The inhibition of Rho GTPases activity, by overexpression of Rho GDP dissociation inhibitor (Rho-GDI), inhibited the strain-induced activation of NF-B in C2C12 cells. Overexpression of either NF-B inhibitory protein IB
N (a degradation resistant mutant IB) or Rho-GDI blocked the strain-induced proliferation of C2C12 cells. Furthermore, overexpression of FRNK, a dominant negative mutant of focal adhesion kinase (FAK), inhibited the strain-induced proliferation of C2C12 cells. Our study demonstrates that cyclic mechanical strain inhibits myogenesis through the activation of FAK, Rac-1, and NF-B.—Kumar, A., Murphy, R., Robinson, P., Wei, L., Boriek, A. M. Cyclic mechanical strain inhibits skeletal myogenesis through activation of focal adhesion kinase, Rac-1 GTPase, and NF-B transcription factor.
Key Words: skeletal myogenesis • C2C12 cells • cyclic stretch • FAK • Rac-1 • NF-kappa B
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3)
. Myogenesis is required for growth, maintenance, and repair of injured muscle fibers (4)
. In recent years, research has led to significant understanding of the complexity of specification and differentiation of skeletal muscle cells in mammals (3
, 5
6
7
8)
. Accumulating evidence suggests a significant role of physical forces in the development and maintenance of skeletal muscle and in the onset and perpetuation of several myofiber diseases (9
, 10)
.
Mechanical stress is recognized as an important extracellular stimulus that promotes cellular growth and survival, influences metabolic processes (including gene expression), and governs tissue architecture in various cell types (11
12
13)
. Skeletal muscles are quite responsive to mechanical forces. This is evident by the fact that expression of several genes and cellular size of skeletal muscles are modulated in response to applied external mechanical stress (14
15
16)
. Although the precise mechanism(s) by which mechanical stress modulates muscle function remain(s) enigmatic, there is a growing list of signaling pathways that are activated in response to mechanical stress in skeletal muscle cells. These include protein kinase C (17)
, mitogen-activated protein kinases (18
19
20)
, phosphatidylinositol-3 kinase/Akt (21)
, NF-B (15
, 22)
, calcineurin (23)
, and nitric oxide synthase (24
, 25)
. There is also evidence that Rho family GTPases play a central role in mediating mechanical strain-induced cellular responses in skeletal muscles (26)
. We recently showed that the activation of extracellular signal-regulated kinases 1/2 (ERK1/2) in response to constant mechanical strain is dependent on the direction of applied forces (27)
. Furthermore, we demonstrated that mechanical stress can activate proinflammatory transcription factors such as activator protein-1 (AP-1) and nuclear-factor-kappa B (NF-B) in skeletal myofibers (15
, 27
, 28)
.
Despite the significant progress in our understanding of mechanical stress-induced cellular responses, the effect of mechanical forces on myogenic differentiation remains unclear. A recent report by Akimoto et al. suggested that application of cyclic mechanical stretch inhibits the mRNA level of MyoD and myocyte nuclear factor- (MNF-) in C2C12 myoblasts (29)
. However, Akimoto et al. did not investigate whether cyclic stretch can regulate the expression of muscle-specific proteins such as myosin heavy chain (MHC) and the terminal differentiation of myoblasts into multinucleated myotubes (29)
. Furthermore, the molecular mechanisms responsible for the modulation of myogenic differentiation in response to cyclic mechanical stretch remain unknown.
In the present report, we investigated the role of cyclic mechanical stretch in the proliferation and differentiation of C2C12 myoblasts. Our data demonstrate that cyclic stretch acts as a potent mitogen that induces the proliferation of C2C12 cells and inhibits their differentiation into myotubes. Our study provides strong evidence that cyclic strain inhibits myogenic differentiation through activation of a signaling pathway that involves focal adhesion kinase (FAK), Rac-1 GTPase, and NF-B transcription factor.
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MATERIAL AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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B consensus oligonucleotides and luciferase assay kit were purchased from Promega (Madison, WI, USA). Poly dI·dC was obtained from Amersham Biosciences (Arlington Heights, IL, USA). [
-32P]ATP (specific activity, 3000 (111 TBq) Ci/mmol) was obtained from Perkin-Elmer Life Sciences (Boston, MA, USA).
Plasmids
The pCMV4-FLAGIBN plasmid was a kind gift from Prof. Dean Ballard of Vanderbilt University School of Medicine (Nashville, TN, USA). FLAGIBN gene was subcloned at HindIII and EcoRV sites of pcDNA3 plasmid. pNF-B-Luc plasmid was purchased from Clontech (Palo Alto, CA, USA). The pEF-GDI vector has been described in our previous work (30)
. The cDNA constructs for full-length avian FAK and the truncated COOH-terminal FAK (FRNK) were cloned into the pCMV-Myc vector and were kindly provided by Dr. J. Thomas Parsons (31)
.
Cell culture, transfection, and differentiation
C2C12 cells were obtained from American Type Culture Collection (Rockville, MD, USA). The cells were grown in DMEM (Invitrogen, Carlsbad, CA, USA) supplemented with 20% fetal bovine serum (Invitrogen) at 37°C in a CO2 incubator. Differentiation of C2C12 cells was induced by replacing the media with differentiation medium (DMEM supplemented with 2% horse serum). The C2C12 cells (
70–80% confluence in 100 mm culture plates) were stably transfected with 30 µg of indicated plasmids along with 3 µg pBABE-puro plasmids (1:10 ratio) using a standard calcium phosphate DNA precipitation method. The cells were incubated with the transfection mixture for 16 h, washed with phosphate-buffered saline (PBS), the medium of the cells was changed, and the cells were incubated for an additional 24 h in a CO2 incubator. The cells were split and selected in the presence of puromycin (1.9 µg/mL) for 7 to 8 days. The positive colonies overexpressing desired proteins were isolated and grown for further experimentation.
Mechanical stimulation using cyclic strain
C2C12 were plated onto type I collagen-coated flexible-bottom plates (BioFlex plates, Flexcell International, McKeesport, PA, USA) and incubated at 37°C in a CO2 incubator for 24 h before applying mechanical strain. The cells were subjected to cyclic strain at 1 Hz (0.5 s of 17% stretch alternating with 0.5 s of relaxation) for different intervals using a computer-controlled vacuum stretch apparatus (FX-4000T Tension Plus System, FlexCell International) with a vacuum pressure that is sufficient to generate 17% mechanical strain. Replicate control samples were maintained under static conditions with no applied cyclic strain.
Immunohistochemistry
To determine the expression of MHC fast twitch (MHCf) protein in C2C12 cultures, immunohistochemistry was performed by the avidin-biotin method as described previously with slight modifications (32)
. Cells were fixed with 3.7% paraformaldehyde in phosphate buffered saline (PBS) for 5 min and permeabilized with 0.1% Triton X-100 in PBS for 5–10 min. The endogenous peroxidase activity in the cells was quenched by treatment of the cells for 15 min with 3% hydrogen peroxide. To reduce nonspecific binding of the primary antibody, the cells were first blocked for 30 min at room temperature with CAS block (Zymed Laboratories, San Francisco, CA, USA). The CAS block was drained off, and the cells were treated with 1:50 dilution of MF20 antibody (against MHCf) for 30 min. The cells were washed thoroughly with PBS (3 times; each wash for 3 min) and treated with horse anti-mouse biotinylated conjugate (1:100 in PBS; 30 min). The cells were then washed with PBS (3 times; each wash for 3 min), and were treated with 2 drops of avidin-biotin complex (Vector Laboratories, Burlingame, CA, USA) for 30 min, followed by a thorough washing with PBS (3 times, each wash for 3 min). Finally, the cells were stained for 30 s with freshly prepared solution of DAB (3,3'-diamino benzidine) according to the instructions outlined in the kit (Vector Laboratories). The cells were rinsed in running water and finally counter stained with hematoxylin for 1 min to visualize nuclei. The cells were again washed in running water, dried, and observed under light microscopy. Cells were considered to be positive if brown stain was noted within the cytoplasm of cells above the level of nonspecific signal. The percentage of positive cells was determined by counting MHCf positive cells in the total cells at several places on the membrane.
Immunoprecipitation and cdk2 assay
The activity of cdk2 kinase was measured by immunoprecipitation and in vitro kinase assay using histone H1 as substrate as described previously (27)
.
Small GTPase activity assay
The activities of RhoA, Cdc42, and Rac-1 were measured using coprecipitation assays as described (33
, 34)
. Active Cdc42-GTP and Rac1-GTP interact with the PAK1 binding domain (PBD) fused to glutathione S-transferase bound to glutathione-coupled agarose beads, whereas GDP-bound Cdc42 and Rac1 do not bind PBD. The C2C12 cells were washed with PBS and lysed in lysis buffer (25 mM Tris-Cl (pH 7.4), 200 mM NaCl, 50 mM NaF, 0.3% NP-40, 10 µg/mL leupeptin, 10 µg/mL aprotinin, 1 mM phenylmethylsulfonyl fluoride, 0.5 µg/mL benzamidine, 1 mM dithiothreitol, 1 mM sodium orthovanadate, and 10 mM ß-glycerophosphate). The lysates were clarified by centrifugation at 15,000 g for 4 min at 4°C. The protein content in supernatants was measured using BioRad Protein Assay Reagent. PAK-1 PBD agarose (10 µg; Upstate Biotechnology, Lake Placid, NY, USA) was added to the 600 µg clarified lysate and the samples were incubated for 45 min on ice with occasional mixing. The agarose beads were pelleted at 4°C, washed three times with lysis buffer, and resuspended in 20 µL of SDS-PAGE loading buffer. Samples were then subjected to 15% SDS-PAGE and Western blot for antibodies with Rac-1 or cdc42. Active Rho (GTP-bound) was detected in a similar fashion as described for Cdc42-GTP and Rac1-GTP. Since GTP-bound RhoA (but not RhoA-GDP) interacts with the rhoketin Rho binding domain (RBD), the clarified lysate were coprecipitated with 20 µg of RBD-agarose (Upstate Biotechnology, Lake Placid, NY, USA). Detection of active RhoA was done by Western blot using RhoA antibodies. All other experimental conditions were identical to those described for Cdc42 and Rac-1 above. The total protein content of RhoA, Rac1 and cdc42 was determined by Western blot without coprecipitation.
Electrophoretic mobility shift assay (EMSA)
The DNA binding activity of NF-B transcription factors was determined by EMSA as described previously (35
, 36)
.
RNA isolation and reverse transcription-polymerase chain reaction (RT-PCR)
RNA isolation and RT-PCR analysis were performed as described previously (15
, 35)
. Primer sequences used to amplify mouse IL-6 and ß-actin were as follows:
Statistical analysis
All experiments were repeated at least three times unless otherwise indicated. Results are expressed as mean ± SD. Statistical analysis used Student’s t test or ANOVA to compare quantitative data populations with normal distribution and equal variance. A value of P < 0.05 was considered statistically significant unless specified otherwise.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 38)
.
Cyclic stretch induces proliferation and inhibits expression of MHCf in C2C12 cells
We first studied the effects of cyclic stretch on the proliferation of C2C12 cells. The C2C12 cells were plated on collagen I-coated BioFlex plates and the medium of the cells was replaced with differentiation medium (DM). The cells were then subjected to cyclic stretch (17% strain, 1 Hz) for 1 h, followed by incubation of the cells in a CO2 incubator for 24 h. The proliferation of C2C12 cells was measured by counting the number of viable cells using Trypan blue dye in a counting chamber. As shown in Fig. 1
A, incubation of the C2C12 cells in DM inhibited their proliferation. However, application of cyclic stretch significantly increased the proliferation of C2C12 cells in DM. Because enhanced proliferation of the cells is antagonist to their differentiation, we next investigated the effect of cyclic mechanical stretch on MHCf expression, an important marker of skeletal muscle differentiation. The C2C12 cells incubated in DM were subjected to cyclic stretch for 1 h every 24 h and the expression of MHCf at different times was measured by Western blot. As shown in Fig. 1B
, the expression of MHCf was significantly blocked in the mechanically stretched C2C12 cells. The level of ß-actin, an unrelated protein, was not affected by either mechanical stretch or during the differentiation of C2C12 cells (Fig. 1C
).
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Cyclic stretch inhibits myotubes formation in C2C12 cultures
The differentiation of C2C12 cells into myotubes was also analyzed by the immunohistochemistry technique. The C2C12 cells were incubated in DM and subjected to 1 h of cyclic strain every 24 h for 5 days. The cells were then fixed with paraformaldehyde and subjected to immunohistochemical analysis for MHCf protein using MF-20 antibody. Representative micrograph data of two independent studies shown in Fig. 2
A demonstrate that cyclic stretch significantly inhibits the formation of myotubes (brown-colored) in C2C12 cultures. We also measured the percentage of cells positive for MHCf protein in the C2C12 cultures subjected to cyclic stretch. Significantly fewer MHCf positive cells were observed in stretched C2C12 cultures than in those incubated under static conditions (Fig. 2B
). These data indicate that cyclic mechanical stretch induces the proliferation of C2C12 cells and inhibits their differentiation.
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Cyclic stretch augments the activation of cdk2 in C2C12 cells
The withdrawal of myogenic cells from the cell cycle is a prerequisite for their differentiation into myotubes (39)
. Since cyclic stretch induced the proliferation of C2C12 and inhibited their differentiation, we next investigated the effects of cyclic stretch on the activity of cdk2, an important cell cycle regulator. The activation of cdk2 promotes progression of cells from G1 to S phase of the cell cycle (40
, 41)
. C2C12 cells were subjected to 1 h of cyclic mechanical stretch (17% strain, 1 Hz) every 24 h, the cells were collected at different intervals and the activation of cdk2 was measured by immunoprecipitation and in vitro kinase assay using histone H1 as substrate. The cellular level of cyclin A was determined by Western blot. The data in Fig. 3
show that the level of cyclin A and cdk2 activity was considerably high in C2C12 cells incubated in normal growth medium (day 0), which is typical of proliferating cells. Incubation of C2C12 cells in DM significantly inhibited the activity of cdk2 and reduced the cellular level of cyclin A within 24 h. In contrast, application of cyclic mechanical stretch led to an increased activation of cdk2 in C2C12 cells. Although cdk2 activity was decreased after 48 h, the cellular level of cyclin A remained significantly higher in stretched C2C12 cells than in unstretched cells, indicating that cyclic mechanical stretch inhibits withdrawal of cells from the cell cycle on incubation in DM (Fig. 3B
).
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Cyclic stretch activates NF-B transcription factor in C2C12 cells
NF-B transcription factor has been known to induce the expression of a plethora of genes that are involved in various cellular responses, including proliferation and differentiation of myogenic cells (42
43
44)
. To understand the mechanisms by which cyclic mechanical stretch induces the proliferation of C2C12, we investigated the effects of cyclic mechanical stretch on the activation of NF-B. C2C12 cells were plated on BioFlex plates and subjected to cyclic mechanical stretch for different time periods ranging from 0 to 60 min. The nuclear extracts of cells were made and analyzed for activation of NF-B by EMSA. As shown in Fig. 4
A, the application of cyclic mechanical stretch to C2C12 cells increased the NF-B/DNA binding activity in a time-dependent manner (Fig. 4A
).
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To assay the level of NF-B-dependent transcriptional activity in C2C12 cells in response to cyclic mechanical stretch, a luciferase reporter assay was used. The C2C12 cells were transiently transfected with pNF-B-Luc plasmid (Clontech), which contains multiple kappa enhancer elements upstream of the luciferase gene. The cells were then subjected to 1 h cyclic mechanical stretch. After 24 h of application of mechanical stretch, luciferase activity in the cells was measured using a luciferase assay kit (Promega, Madison, WI, USA) and Turner Designs Luminometer (model TD 20/20). Data depicted in Fig. 4B
show that cyclic stretch augments the NF-B transcriptional activity.
Cyclic stretch increases the expression of interleukin-6 and cyclin D1 in C2C12 cells
IL-6 is a proinflammatory cytokine that causes proliferation of myocytes/myoblasts and might inhibit their differentiation (45
, 46)
. The promoter/enhancer region of IL-6 gene contains multiple NF-B binding sites (42)
. We investigated whether cyclic stretching ofC2C12 cells increases IL-6 expression. The C2C12 cells were subjected to cyclic stretch for 1 h and IL-6 expression was measured by RT-PCR after 8–10 h. The data shown in Fig. 5
A suggest that cyclic stretch induces the expression of IL-6 in these cells. Another possible mechanism by which activated NF-B promotes cell proliferation is through augmenting the expression of cyclin D1 (47)
. We also investigated the effects of mechanical stretch on the level of cyclin D1 in C2C12 cells. C2C12 cells were incubated in DM, then subjected to 1 h of cyclic stretch. After 24 h of the application of mechanical stretch, the cellular level of cyclin D1 was measured by Western blot. As shown in Fig. 5B
, the level of cyclin D1 was higher in the cells subjected to cyclic mechanical stretch. Together, these data indicate that the application of cyclic mechanical stretch increases the expression of IL-6 and cyclin D1 in C2C12 cells.
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Rho GTPases are involved in stretch-induced NF-B activation in C2C12 cells
Rho family GTP binding proteins such as RhoA, Rac-1, and cdc42 play an important role in the activation of many signal transduction pathways (48
, 49)
. It has been shown that the activation of Rac-1 is required for the activation of NF-B in response to various stimuli (50)
. We first studied the effects of cyclic mechanical stretch on the activation of Rho GTPases in C2C12 cells. The C2C12 cells were subjected to cyclic stretch for different time periods, and GTP-bound RhoA, Rac-1, and cdc42 were measured by coprecipitation as described in Materials and Methods. As shown in Fig. 6
A, cyclic stretch strongly activated Rac-1 in C2C12 cells. Activation of RhoA was only marginally increased and activation of cdc42 was unaffected by cyclic stretch. The total cellular level of RhoA, Rac-1, and cdc42 was not altered by the application of cyclic mechanical stretch (Fig. 6A
).
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We investigated the role of Rho GTPases in the activation of NF-B in response to cyclic strain. Rho GDP dissociation inhibitor (GDI) protein is an endogenous inhibitor of the activity of RhoA, Rac-1, and cdc42 (51)
. The C2C12 cells were stably transfected with either pEF vector alone or pEF-GDI. Control or GDI-expressing positive clone of C2C12 cells were then subjected to cyclic mechanical stretch for different time intervals. The activation of NF-B in the nuclear extracts was measured by EMSA. The data presented in Fig. 6B
show that the inhibition of Rho GTPases by overexpressing GDI reduced cyclic stretch-induced activation of NF-B in C2C12 cells.
Rho GTPases and NF-B are involved in stretch-induced proliferation of C2C12 cells
To investigate whether activation of Rho GTPases was required for mechanical stretch-induced proliferation of C2C12 cells, the GDI-expressing C2C12 cells were incubated in DM and subjected to 1 h of cyclic mechanical stretch. The proliferation of C2C12 cells was measured after 24 h of the application of mechanical stretch by counting the number of viable cells using Trypan blue dye in a counting chamber. The overexpression of GDI completely inhibited the proliferation of C2C12 cells in response to cyclic mechanical stretch. Because mechanical stretch activated NF-B and GDI inhibited the activation of NF-B in response to cyclic stretch (as described above), we next investigated the role of NF-B in stretch-induced proliferation of C2C12 cells. C2C12 cells were transfected with human IBN cDNA (a dominant negative form NF-B inhibitory protein IB) and the proliferation of the cells in response to cyclic stretch was measured. Similar to Rho-GDI, the overexpression of IBN in C2C12 cells completely inhibited the stretch-induced proliferation of C2C12 cells (Fig. 7
). Overexpression of Rho-GDI alone did not affect the proliferation of C2C12 cells in normal growth medium whereas overexpression of IBN reduced the proliferation of C2C12 cells (data not shown). These data strongly suggest that the activation of Rho GTPases and NF-B is involved in the proliferation of C2C12 cells in response to cyclic mechanical stretch.
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Focal adhesion kinase is involved in cyclic stretch-induced proliferation of C2C12 cells
The process of mechanosensing and mechanotransduction in many cell types has been shown to involve nonreceptor protein tyrosine kinases (PTK) such as FAK and src family kinases (52
, 53)
. Our preliminary experiments suggested an increase in total protein tyrosine phosphorylation in C2C12 cells in response to cyclic mechanical stretch (data not shown). Using phospho-specific antibodies, we investigated whether cyclic stretch activates FAK and src kinases in C2C12 cells. As shown in Fig. 8
A, the application of cyclic stretch to C2C12 cells increased phosphorylation of FAK at Tyr-576/577, which leads to its activation (54)
. Furthermore, the phosphorylation level of src at Tyr-527 was decreased by cyclic stretch, indicating the activation of src kinases (55)
. The application of cyclic stretch did not alter the total cellular level of FAK or src as determined by immunoblotting with antibodies that recognized both normal and phosphorylated proteins (data not shown).
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To investigate whether FAK is involved in the stretch-induced proliferation of C2C12 cells, C2C12 cells were stably transfected with a truncated mutant of FAK known as FAK-related nonkinase, or FRNK. FRNK contains the COOH-terminal domain of FAK without the kinase domain and has been shown to act as a dominant negative inhibitor of FAK (31)
. Overexpression of FRNK significantly reduced the stretch-induced proliferation of C2C12 cells (Fig. 8B
). Furthermore, pretreatment of C2C12 cells with Genestein (a PTK inhibitor) completely inhibited the stretch-induced proliferation, suggesting the involvement of PTK in cell proliferation (Fig. 8B
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 56
, 57)
. Studies using cultured myoblasts have further demonstrated that myogenesis and differentiation are coupled with cell cycle exit, primarily due to overexpression of cyclin-dependent kinase (cdk) inhibitors (CKIs) such as p21, p18, and p57 (58
, 59)
. These CKIs inhibit the activity of cdks resulting in the hypophoshorylation of retinoblastoma protein and therefore cell cycle arrest in G0/G1 phase (60)
. Once differentiated, muscle fibers lack the ability to re-enter the cell cycle and proliferate. However, it has become clear that normal undamaged adult skeletal muscle contain few myogenic precursor cells that are quiescent (61)
. These myogenic precursor cells (usually beneath the basal lamina) provide extra nuclei for postnatal growth and are involved in repair and regeneration after local injury of muscle fibers (62
, 63)
. Despite these advancements, our understanding of myogenesis is still limited. In particular, the nature of several stimuli and the signaling mechanisms that lead to induction of mitotic activity of quiescent myogenic cells in vivo have remained unknown.
Our data in this study suggest that cyclic mechanical strain acts as a potent mitogen to induce the proliferation of C2C12 myoblasts and inhibits their differentiation (Fig. 1)
. The increased proliferation of C2C12 cells in response to cyclic stretch was associated with higher activity of cell cycle regulators such as cdk2 and cyclin A, suggesting that cyclic stretch inhibits the withdrawal of C2C12 cells from the cell cycle upon incubation in DM (Fig. 3)
. This is further supported by our observation that chronic application of cyclic mechanical stretch to the differentiating C2C12 cells inhibited the expression of MHCf protein in these cells (Fig. 1B
). Furthermore, a few differentiated myotubes formed in the presence of cyclic stretch were relatively smaller in size compared with the myotubes formed under static culture conditions (Fig. 2A
). The inhibition of differentiation of C2C12 cells observed in the presence of cyclic mechanical stretch was not a result of possible myotube wasting. This is because application of cyclic mechanical stretch to differentiated C2C12 myotubes using the same protocol did not cause any reduction in the expression of MHCf or total protein content (data not shown). These data thus suggest that cyclic stretch interferes with the process of myogenic differentiation.
Our data are consistent with a recent report by Akimoto et al., which demonstrated that the application of 20% cyclic mechanical stretch inhibits the mRNA level of MyoD and MNF- (additional markers of skeletal muscle differentiation) in C2C12 cells (29)
. Whereas Akimoto et al. applied cyclic stretch continuously for 24 h (29)
, we found that applying cyclic stretch for 1 h every 24 h was sufficient to induce the proliferation of C2C12 cells and inhibit their differentiation into myotubes. Although we used 17% of mechanical strain at 1 Hz frequency, it is important to recognize that various cellular responses such as activation of different signaling molecules could also depend on the magnitude of applied mechanical strain, the frequency at which strain is applied, and the duration of the application of mechanical strain (19)
. Recently, Craig et al. investigated the effect of the magnitude of mechanical strain on the activation of GTPase proteins in C2C12 myoblasts and concluded there is a threshold of strain-dependent activation. For example, when the strain rate was held at a constant 20%/s a 25 kDa GTPase was activated at strains of 10% and 20%, but not at strains of 2% (38)
.
To understand the biochemical mechanisms responsible for the enhanced proliferation of C2C12 cells in response to stretch, we investigated the role of the NF-B transcription factor. The association between normal growth and NF-B activation has been noted in many cells and tissues. Enhanced activation of NF-B is apparent during G0/G1 transition in fibroblasts (64)
and is induced by mitogenic stimuli, including serum in G0-arrested 3T3 fibroblasts (65)
. Consistent with this, overexpression of IB protein into mouse embryonic fibroblast cells retards their growth (47)
. A higher activation of NF-B had been previously reported in cultured fibroblasts (66)
and osteoblasts (67)
in response to cyclic mechanical stretch. Similar to these reports, we observed that cyclic mechanical stretch activates NF-B in C2C12 cells (Fig. 4)
. Activation of NF-B appears to be essential for the stretch-induced proliferation of C2C12 cells because overexpression of the dominant negative IB mutant (IBN) completely blocked the mechanical stretch-induced proliferation of C2C12 cells (Fig. 7)
.
How the activation of NF-B in response to mechanical stretch leads to the proliferation of C2C12 cells is not completely understood. NF-B has been shown to regulate the expression of a number of exogenous and endogenous growth factors that are known to promote cellular proliferation (68)
. Multiple NF-B binding sites have been reported in the promoter region of cyclin D1, an important cell cycle regulator that, upon association with cdk4 and cdk6, promotes the progression of cells from G0/G1 to S phase (47)
. We observed a higher level of cyclin D1 in mechanically stretched C2C12 cells compared with control cells (Fig. 5)
, suggesting that NF-B could induce proliferation of these cells through the enhanced expression of cyclin D1. Furthermore, expression of the IL-6 gene, which contains consensus B sites in its promoter region, was also increased by cyclic mechanical stretch (Fig. 5)
. Indeed, IL-6 is considered as a potent inducer for the proliferation of myocytes (45)
. Taken together, these results suggest that mitogenic effects of cyclic stretch on C2C12 myoblasts are mediated through the activation of NF-B and possibly through the increased expression of NF-B-regulated genes such as cyclin D1 and IL-6.
Accumulating evidence suggests that besides their primary role in actin cytoskeletal reorganization, Rho family GTPases such as RhoA, Rac-1, and cdc42 are involved in numerous other cellular functions, including cell growth and cell cycle progression (69
70
71)
, and in the activation of numerous cell signaling pathways (72
, 73)
. Rho GTPases have been shown to activate serum-responsive factor and NF-B transcription factors (74
, 75)
. Several recent studies suggest that mechanical stretch activates Rho GTPases in various cell types (76
77
78)
. We observed that cyclic mechanical stretch strongly activates Rac-1 GTPase in C2C12 cells (Fig. 6A
). The activation of Rho GTPases seems to be essential for stretch-induced activation of NF-B and proliferation of C2C12. This is because overexpression of GDI, which inhibits the activity of Rho family GTPases, significantly reduced NF-B activation (Fig. 6B
) and the stretch-induced proliferation of C2C12 (Fig. 7)
. Indeed, Rac-1 has been implicated in the activation of NF-B in response to various stimuli and in myogenesis (50
, 79
80
81
82)
.
Several recent studies have emphasized that FAK, a nonreceptor PTK localized at focal adhesion, plays an obligatory role in mechanotransduction (53
, 83
84
85)
and in other integrin- and actin-based cellular processes, including cell cycle progression, adhesion, and migration (86
, 87)
. It has been suggested that the binding of integrins to extracellular matrix ligands induces integrin clustering and recruitment of FAK. This results in FAK autophosphorylation and in the creation of a binding site for the Src-homology 2 (SH2) domain of Src (52
, 88)
. FAK-Src association increases Src PTK activity and promotes Src-dependent phosphorylation of additional tyrosine residues on FAK and FAK-associated proteins, such as Cas and paxillin (54
, 88)
. This leads to the binding of other SH2 domain proteins, which in turn activates many downstream signaling pathways, including Rac-1/JNK (89
90
91)
. Since we observed a higher activation of FAK and src kinase in response to mechanical strain (Fig. 8A
), it is possible that cyclic stretch-induced proliferation of C2C12 cells is mediated, at least in part, through the activation of these kinases. This is supported by our data that overexpression of FRNK, a dominant negative mutant of FAK, significantly reduced the proliferation of C2C12 cells in response to mechanical stretch (Fig. 8B
). The role of PTK in stretch-induced proliferation of C2C12 cells was further confirmed by our observation that pretreatment of cells with PTK inhibitor Genestein completely suppressed the stretch-induced proliferation of C2C12 (Fig. 8B
). These data thus suggest that FAK and src family PTK might be the potential upstream signaling kinases that lead to the increased proliferation of C2C12 cells in response to mechanical stretch.
Based on the data presented here we propose that the activation of a signaling pathway that involves FAK, Rac-1, and NF-B is responsible for the increased proliferation of C2C12 cells in response to cyclic mechanical strain (Fig. 9
). It is important to point out that the proposed pathway in Fig. 9
could be the principal de novo signaling pathway that is responsible for proliferation of C2C12 cells in response to mechanical stretch. A different signaling pathway might be operating for the proliferation of myoblasts before the addition of differentiation medium (DM), because overexpression of either Rho-GDI or FRNK did not affect the proliferation of C2C12 cells in normal growth medium. Furthermore, overexpression of IBN only slowed down the proliferation of C2C12 cells in normal growth medium (data not shown), but completely blocked the stretch-induced proliferation of C2C12 cells in DM (Fig. 7)
. It is therefore possible that mechanical stretch activates FAK and Rac-1, which leads to the activation of NF-B (supported by our data in Fig. 6B
) that ultimately leads to the increased proliferation of C2C12. The importance of a distinct pathway in mechanical stretch-induced C2C12 proliferation is further confirmed by our observation that the inhibition of MAP kinases (e.g., ERK1/2, JNK1, and p38) activity using their specific inhibitors reduced the proliferation of C2C12 cells in normal growth conditions (data not shown). Although cyclic mechanical stretch increased the activity of MAP kinases, the inhibition of MAP kinase activity had no effect on the proliferation of C2C12 cells in response to mechanical stretch (data not shown).
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Although our study provides fundamental information regarding the regulatory mechanisms by which cyclic mechanical strain modulates proliferation and differentiation of myoblasts in 2-dimensional cell culture systems, it remains to be established whether mechanical stretch modulates myogenesis in a similar fashion in 3-dimensional settings that mimic more closely in vivo conditions. Many recent studies have shown that cells might behave quite differently when cultured on 3-dimensional matrices than when grown on 2-dimensional flat tissue culture substrates (92
93
94)
. For example, unlike the well-studied tyrosine phosphorylated forms of FAK in focal adhesions, the FAK in 3-dimensional matrix adhesion is poorly phosphorylated at Tyr-397 residue. This discrepancy in signaling in 2-dimensional vs. 3-dimensional contexts is specific in that paxillin is equally phosphorylated at Tyr-31 in both 3-dimensional matrix adhesion and focal adhesion (93)
. Differences in signaling mechanisms have also been reported in cells suspended in 3-dimensional collagen gels vs. 2-dimensional cultures (95
, 96)
.
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ACKNOWLEDGMENTS |
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BN plasmid. This work was supported by Muscular Dystrophy Association grants (to A.K. and A.M.B.) and by a RO1 grant HL63134 from the National Institutes of Health (to A.M.B.). Received for publication May 25, 2004. Accepted for publication June 30, 2004.
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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