The Experts below are selected from a list of 8994 Experts worldwide ranked by ideXlab platform
Ravi Kambadur - One of the best experts on this subject based on the ideXlab platform.
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Myostatin signals through pax7 to regulate satellite cell self renewal
Experimental Cell Research, 2008Co-Authors: Craig Mcfarlane, Alex Hennebry, Erin Plummer, Mark Thomas, Ravi Kambadur, Nicholas Ling, Mridula SharmaAbstract:Myostatin, a Transforming Growth Factor-beta (TGF-β) super-family member, has previously been shown to negatively regulate satellite cell activation and self-renewal. However, to date the mechanism behind Myostatin function in satellite cell biology is not known. Here we show that Myostatin signals via a Pax7-dependent mechanism to regulate satellite cell self-renewal. While excess Myostatin inhibited Pax7 expression via ERK1/2 signaling, an increase in Pax7 expression was observed following both genetic inactivation and functional antagonism of Myostatin. As a result, we show that either blocking or inactivating Myostatin enhances the partitioning of the fusion-incompetent self-renewed satellite cell lineage (high Pax7 expression, low MyoD expression) from the pool of actively proliferating myogenic precursor cells. Consistent with this result, over-expression of Pax7 in C2C12 myogenic cells resulted in increased self-renewal through a mechanism which slowed both myogenic proliferation and differentiation. Taken together, these results suggest that increased expression of Pax7 promotes satellite cell self-renewal, and furthermore Myostatin may control the process of satellite cell self-renewal through regulation of Pax7. Thus we speculate that, in addition to the intrinsic factors (such as Pax7), extrinsic factors both positive and negative in nature, will play a major role in determining the stemness of skeletal muscle satellite cells.
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Myostatin signals through pax7 to regulate satellite cell self renewal
Experimental Cell Research, 2008Co-Authors: Craig Mcfarlane, Alex Hennebry, Erin Plummer, Mark Thomas, Ravi Kambadur, Nicholas Ling, Mridula SharmaAbstract:Myostatin, a Transforming Growth Factor-beta (TGF-β) super-family member, has previously been shown to negatively regulate satellite cell activation and self-renewal. However, to date the mechanism behind Myostatin function in satellite cell biology is not known. Here we show that Myostatin signals via a Pax7-dependent mechanism to regulate satellite cell self-renewal. While excess Myostatin inhibited Pax7 expression via ERK1/2 signaling, an increase in Pax7 expression was observed following both genetic inactivation and functional antagonism of Myostatin. As a result, we show that either blocking or inactivating Myostatin enhances the partitioning of the fusion-incompetent self-renewed satellite cell lineage (high Pax7 expression, low MyoD expression) from the pool of actively proliferating myogenic precursor cells. Consistent with this result, over-expression of Pax7 in C2C12 myogenic cells resulted in increased self-renewal through a mechanism which slowed both myogenic proliferation and differentiation. Taken together, these results suggest that increased expression of Pax7 promotes satellite cell self-renewal, and furthermore Myostatin may control the process of satellite cell self-renewal through regulation of Pax7. Thus we speculate that, in addition to the intrinsic factors (such as Pax7), extrinsic factors both positive and negative in nature, will play a major role in determining the stemness of skeletal muscle satellite cells.
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Myostatin induces cachexia by activating the ubiquitin proteolytic system through an nf κb independent foxo1 dependent mechanism
Journal of Cellular Physiology, 2006Co-Authors: Craig Mcfarlane, Alex Hennebry, Erin Plummer, Mark Thomas, Nicholas Ling, Mridula Sharma, Murray Ashby, Heather K Smith, Ravi KambadurAbstract:Myostatin, a transforming growth factor-beta (TGF-beta) super-family member, has been well characterized as a negative regulator of muscle growth and development. Myostatin has been implicated in several forms of muscle wasting including the severe cachexia observed as a result of conditions such as AIDS and liver cirrhosis. Here we show that Myostatin induces cachexia by a mechanism independent of NF-kappaB. Myostatin treatment resulted in a reduction in both myotube number and size in vitro, as well as a loss in body mass in vivo. Furthermore, the expression of the myogenic genes myoD and pax3 was reduced, while NF-kappaB (the p65 subunit) localization and expression remained unchanged. In addition, promoter analysis has confirmed Myostatin inhibition of myoD and pax3. An increase in the expression of genes involved in ubiquitin-mediated proteolysis is observed during many forms of muscle wasting. Hence we analyzed the effect of Myostatin treatment on proteolytic gene expression. The ubiquitin associated genes atrogin-1, MuRF-1, and E214k were upregulated following Myostatin treatment. We analyzed how Myostatin may be signaling to induce cachexia. Myostatin signaling reversed the IGF-1/PI3K/AKT hypertrophy pathway by inhibiting AKT phosphorylation thereby increasing the levels of active FoxO1, allowing for increased expression of atrophy-related genes. Therefore, our results suggest that Myostatin induces cachexia through an NF-kappaB-independent mechanism. Furthermore, increased Myostatin levels appear to antagonize hypertrophy signaling through regulation of the AKT-FoxO1 pathway.
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decorin binds Myostatin and modulates its activity to muscle cells
Biochemical and Biophysical Research Communications, 2006Co-Authors: Takayuki Miura, Alex Hennebry, Ravi Kambadur, Jun-ichi Wakamatsu, Mridula Sharma, Akihito Hattori, Carole Berry, Yasuhiro Kishioka, Takanori NishimuraAbstract:Myostatin, a member of TGF-beta superfamily of growth factors, acts as a negative regulator of skeletal muscle mass. The mechanism whereby Myostatin controls the proliferation and differentiation of myogenic cells is mostly clarified. However, the regulation of Myostatin activity to myogenic cells after its secretion in the extracellular matrix (ECM) is still unknown. Decorin, a small leucine-rich proteoglycan, binds TGF-beta and regulates its activity in the ECM. Thus, we hypothesized that decorin could also bind to Myostatin and participate in modulation of its activity to myogenic cells. In order to test the hypothesis, we investigated the interaction between Myostatin and decorin by surface plasmon assay. Decorin interacted with mature Myostatin in the presence of concentrations of Zn(2+) greater than 10microM, but not in the absence of Zn(2+). Kinetic analysis with a 1:1 binding model resulted in dissociation constants (K(D)) of 2.02x10(-8)M and 9.36x10(-9)M for decorin and the core protein of decorin, respectively. Removal of the glycosaminoglycan chain by chondroitinase ABC digestion did not affect binding, suggesting that decorin could bind to Myostatin with its core protein. Furthermore, we demonstrated that immobilized decorin could rescue the inhibitory effect of Myostatin on myoblast proliferation in vitro. These results suggest that decorin could trap Myostatin and modulate its activity to myogenic cells in the ECM.
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Myostatin auto regulates its expression by feedback loop through smad7 dependent mechanism
Journal of Cellular Physiology, 2006Co-Authors: Davanea Forbes, Mark Thomas, Ravi Kambadur, Mark Jackman, Amy Bishop, Mridula SharmaAbstract:Myostatin, a secreted growth factor, is a member of the TGF-β superfamily and an inhibitor of myogenesis. Previously, we have shown that Myostatin gene expression is regulated at the level of transcription and that Myostatin is a downstream target gene of MyoD. Here we show that Myostatin gene expression is auto-regulated by a negative feedback mechanism. Northern blot analysis indicated that there are relatively higher levels of Myostatin mRNA in the biceps femoris muscle of cattle that express a non- functional Myostatin allele (Belgian Blue) as compared to normal cattle. In contrast, addition of exogenous Myostatin decreases endogenous Myostatin mRNA. Consistent with this result, wild type Myostatin protein is able to repress Myostatin promoter activity via Activin type IIb receptor (ActRIIB) and ALK5 (P < 0.001). However, non-functional Myostatin (Piedmontese) failed to repress the Myostatin promoter suggesting that Myostatin auto-regulates its promoter by negative feedback inhibition. Auto-regulation by Myostatin appears to be signaled through Smad7, since the expression of the inhibitory Smad7 is induced by Myostatin and the over-expression of Smad7 in turn inhibits the Myostatin promoter activity (P < 0.001). In contrast down regulation of Smad7 by siRNA results in increased Myostatin mRNA indicating that Smad7 is a negative regulator of Myostatin gene expression. Consistent with these results, a decrease in Smad7 mRNA and concomitant increase in Myostatin expression is seen in myotubes that express non functional Myostatin. In addition, interference with Myostatin signaling prevents the induction of Smad7 promoter activity by Myostatin. Based on these results, we propose that Myostatin auto-regulates its gene expression through a Smad7 dependent mechanism in myogenic cells. © 2005 Wiley-Liss, Inc.
Mridula Sharma - One of the best experts on this subject based on the ideXlab platform.
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Myostatin signals through pax7 to regulate satellite cell self renewal
Experimental Cell Research, 2008Co-Authors: Craig Mcfarlane, Alex Hennebry, Erin Plummer, Mark Thomas, Ravi Kambadur, Nicholas Ling, Mridula SharmaAbstract:Myostatin, a Transforming Growth Factor-beta (TGF-β) super-family member, has previously been shown to negatively regulate satellite cell activation and self-renewal. However, to date the mechanism behind Myostatin function in satellite cell biology is not known. Here we show that Myostatin signals via a Pax7-dependent mechanism to regulate satellite cell self-renewal. While excess Myostatin inhibited Pax7 expression via ERK1/2 signaling, an increase in Pax7 expression was observed following both genetic inactivation and functional antagonism of Myostatin. As a result, we show that either blocking or inactivating Myostatin enhances the partitioning of the fusion-incompetent self-renewed satellite cell lineage (high Pax7 expression, low MyoD expression) from the pool of actively proliferating myogenic precursor cells. Consistent with this result, over-expression of Pax7 in C2C12 myogenic cells resulted in increased self-renewal through a mechanism which slowed both myogenic proliferation and differentiation. Taken together, these results suggest that increased expression of Pax7 promotes satellite cell self-renewal, and furthermore Myostatin may control the process of satellite cell self-renewal through regulation of Pax7. Thus we speculate that, in addition to the intrinsic factors (such as Pax7), extrinsic factors both positive and negative in nature, will play a major role in determining the stemness of skeletal muscle satellite cells.
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Myostatin signals through pax7 to regulate satellite cell self renewal
Experimental Cell Research, 2008Co-Authors: Craig Mcfarlane, Alex Hennebry, Erin Plummer, Mark Thomas, Ravi Kambadur, Nicholas Ling, Mridula SharmaAbstract:Myostatin, a Transforming Growth Factor-beta (TGF-β) super-family member, has previously been shown to negatively regulate satellite cell activation and self-renewal. However, to date the mechanism behind Myostatin function in satellite cell biology is not known. Here we show that Myostatin signals via a Pax7-dependent mechanism to regulate satellite cell self-renewal. While excess Myostatin inhibited Pax7 expression via ERK1/2 signaling, an increase in Pax7 expression was observed following both genetic inactivation and functional antagonism of Myostatin. As a result, we show that either blocking or inactivating Myostatin enhances the partitioning of the fusion-incompetent self-renewed satellite cell lineage (high Pax7 expression, low MyoD expression) from the pool of actively proliferating myogenic precursor cells. Consistent with this result, over-expression of Pax7 in C2C12 myogenic cells resulted in increased self-renewal through a mechanism which slowed both myogenic proliferation and differentiation. Taken together, these results suggest that increased expression of Pax7 promotes satellite cell self-renewal, and furthermore Myostatin may control the process of satellite cell self-renewal through regulation of Pax7. Thus we speculate that, in addition to the intrinsic factors (such as Pax7), extrinsic factors both positive and negative in nature, will play a major role in determining the stemness of skeletal muscle satellite cells.
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Myostatin induces cachexia by activating the ubiquitin proteolytic system through an nf κb independent foxo1 dependent mechanism
Journal of Cellular Physiology, 2006Co-Authors: Craig Mcfarlane, Alex Hennebry, Erin Plummer, Mark Thomas, Nicholas Ling, Mridula Sharma, Murray Ashby, Heather K Smith, Ravi KambadurAbstract:Myostatin, a transforming growth factor-beta (TGF-beta) super-family member, has been well characterized as a negative regulator of muscle growth and development. Myostatin has been implicated in several forms of muscle wasting including the severe cachexia observed as a result of conditions such as AIDS and liver cirrhosis. Here we show that Myostatin induces cachexia by a mechanism independent of NF-kappaB. Myostatin treatment resulted in a reduction in both myotube number and size in vitro, as well as a loss in body mass in vivo. Furthermore, the expression of the myogenic genes myoD and pax3 was reduced, while NF-kappaB (the p65 subunit) localization and expression remained unchanged. In addition, promoter analysis has confirmed Myostatin inhibition of myoD and pax3. An increase in the expression of genes involved in ubiquitin-mediated proteolysis is observed during many forms of muscle wasting. Hence we analyzed the effect of Myostatin treatment on proteolytic gene expression. The ubiquitin associated genes atrogin-1, MuRF-1, and E214k were upregulated following Myostatin treatment. We analyzed how Myostatin may be signaling to induce cachexia. Myostatin signaling reversed the IGF-1/PI3K/AKT hypertrophy pathway by inhibiting AKT phosphorylation thereby increasing the levels of active FoxO1, allowing for increased expression of atrophy-related genes. Therefore, our results suggest that Myostatin induces cachexia through an NF-kappaB-independent mechanism. Furthermore, increased Myostatin levels appear to antagonize hypertrophy signaling through regulation of the AKT-FoxO1 pathway.
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decorin binds Myostatin and modulates its activity to muscle cells
Biochemical and Biophysical Research Communications, 2006Co-Authors: Takayuki Miura, Alex Hennebry, Ravi Kambadur, Jun-ichi Wakamatsu, Mridula Sharma, Akihito Hattori, Carole Berry, Yasuhiro Kishioka, Takanori NishimuraAbstract:Myostatin, a member of TGF-beta superfamily of growth factors, acts as a negative regulator of skeletal muscle mass. The mechanism whereby Myostatin controls the proliferation and differentiation of myogenic cells is mostly clarified. However, the regulation of Myostatin activity to myogenic cells after its secretion in the extracellular matrix (ECM) is still unknown. Decorin, a small leucine-rich proteoglycan, binds TGF-beta and regulates its activity in the ECM. Thus, we hypothesized that decorin could also bind to Myostatin and participate in modulation of its activity to myogenic cells. In order to test the hypothesis, we investigated the interaction between Myostatin and decorin by surface plasmon assay. Decorin interacted with mature Myostatin in the presence of concentrations of Zn(2+) greater than 10microM, but not in the absence of Zn(2+). Kinetic analysis with a 1:1 binding model resulted in dissociation constants (K(D)) of 2.02x10(-8)M and 9.36x10(-9)M for decorin and the core protein of decorin, respectively. Removal of the glycosaminoglycan chain by chondroitinase ABC digestion did not affect binding, suggesting that decorin could bind to Myostatin with its core protein. Furthermore, we demonstrated that immobilized decorin could rescue the inhibitory effect of Myostatin on myoblast proliferation in vitro. These results suggest that decorin could trap Myostatin and modulate its activity to myogenic cells in the ECM.
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Myostatin auto regulates its expression by feedback loop through smad7 dependent mechanism
Journal of Cellular Physiology, 2006Co-Authors: Davanea Forbes, Mark Thomas, Ravi Kambadur, Mark Jackman, Amy Bishop, Mridula SharmaAbstract:Myostatin, a secreted growth factor, is a member of the TGF-β superfamily and an inhibitor of myogenesis. Previously, we have shown that Myostatin gene expression is regulated at the level of transcription and that Myostatin is a downstream target gene of MyoD. Here we show that Myostatin gene expression is auto-regulated by a negative feedback mechanism. Northern blot analysis indicated that there are relatively higher levels of Myostatin mRNA in the biceps femoris muscle of cattle that express a non- functional Myostatin allele (Belgian Blue) as compared to normal cattle. In contrast, addition of exogenous Myostatin decreases endogenous Myostatin mRNA. Consistent with this result, wild type Myostatin protein is able to repress Myostatin promoter activity via Activin type IIb receptor (ActRIIB) and ALK5 (P < 0.001). However, non-functional Myostatin (Piedmontese) failed to repress the Myostatin promoter suggesting that Myostatin auto-regulates its promoter by negative feedback inhibition. Auto-regulation by Myostatin appears to be signaled through Smad7, since the expression of the inhibitory Smad7 is induced by Myostatin and the over-expression of Smad7 in turn inhibits the Myostatin promoter activity (P < 0.001). In contrast down regulation of Smad7 by siRNA results in increased Myostatin mRNA indicating that Smad7 is a negative regulator of Myostatin gene expression. Consistent with these results, a decrease in Smad7 mRNA and concomitant increase in Myostatin expression is seen in myotubes that express non functional Myostatin. In addition, interference with Myostatin signaling prevents the induction of Smad7 promoter activity by Myostatin. Based on these results, we propose that Myostatin auto-regulates its gene expression through a Smad7 dependent mechanism in myogenic cells. © 2005 Wiley-Liss, Inc.
Mark Thomas - One of the best experts on this subject based on the ideXlab platform.
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Myostatin signals through pax7 to regulate satellite cell self renewal
Experimental Cell Research, 2008Co-Authors: Craig Mcfarlane, Alex Hennebry, Erin Plummer, Mark Thomas, Ravi Kambadur, Nicholas Ling, Mridula SharmaAbstract:Myostatin, a Transforming Growth Factor-beta (TGF-β) super-family member, has previously been shown to negatively regulate satellite cell activation and self-renewal. However, to date the mechanism behind Myostatin function in satellite cell biology is not known. Here we show that Myostatin signals via a Pax7-dependent mechanism to regulate satellite cell self-renewal. While excess Myostatin inhibited Pax7 expression via ERK1/2 signaling, an increase in Pax7 expression was observed following both genetic inactivation and functional antagonism of Myostatin. As a result, we show that either blocking or inactivating Myostatin enhances the partitioning of the fusion-incompetent self-renewed satellite cell lineage (high Pax7 expression, low MyoD expression) from the pool of actively proliferating myogenic precursor cells. Consistent with this result, over-expression of Pax7 in C2C12 myogenic cells resulted in increased self-renewal through a mechanism which slowed both myogenic proliferation and differentiation. Taken together, these results suggest that increased expression of Pax7 promotes satellite cell self-renewal, and furthermore Myostatin may control the process of satellite cell self-renewal through regulation of Pax7. Thus we speculate that, in addition to the intrinsic factors (such as Pax7), extrinsic factors both positive and negative in nature, will play a major role in determining the stemness of skeletal muscle satellite cells.
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Myostatin signals through pax7 to regulate satellite cell self renewal
Experimental Cell Research, 2008Co-Authors: Craig Mcfarlane, Alex Hennebry, Erin Plummer, Mark Thomas, Ravi Kambadur, Nicholas Ling, Mridula SharmaAbstract:Myostatin, a Transforming Growth Factor-beta (TGF-β) super-family member, has previously been shown to negatively regulate satellite cell activation and self-renewal. However, to date the mechanism behind Myostatin function in satellite cell biology is not known. Here we show that Myostatin signals via a Pax7-dependent mechanism to regulate satellite cell self-renewal. While excess Myostatin inhibited Pax7 expression via ERK1/2 signaling, an increase in Pax7 expression was observed following both genetic inactivation and functional antagonism of Myostatin. As a result, we show that either blocking or inactivating Myostatin enhances the partitioning of the fusion-incompetent self-renewed satellite cell lineage (high Pax7 expression, low MyoD expression) from the pool of actively proliferating myogenic precursor cells. Consistent with this result, over-expression of Pax7 in C2C12 myogenic cells resulted in increased self-renewal through a mechanism which slowed both myogenic proliferation and differentiation. Taken together, these results suggest that increased expression of Pax7 promotes satellite cell self-renewal, and furthermore Myostatin may control the process of satellite cell self-renewal through regulation of Pax7. Thus we speculate that, in addition to the intrinsic factors (such as Pax7), extrinsic factors both positive and negative in nature, will play a major role in determining the stemness of skeletal muscle satellite cells.
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Myostatin induces cachexia by activating the ubiquitin proteolytic system through an nf κb independent foxo1 dependent mechanism
Journal of Cellular Physiology, 2006Co-Authors: Craig Mcfarlane, Alex Hennebry, Erin Plummer, Mark Thomas, Nicholas Ling, Mridula Sharma, Murray Ashby, Heather K Smith, Ravi KambadurAbstract:Myostatin, a transforming growth factor-beta (TGF-beta) super-family member, has been well characterized as a negative regulator of muscle growth and development. Myostatin has been implicated in several forms of muscle wasting including the severe cachexia observed as a result of conditions such as AIDS and liver cirrhosis. Here we show that Myostatin induces cachexia by a mechanism independent of NF-kappaB. Myostatin treatment resulted in a reduction in both myotube number and size in vitro, as well as a loss in body mass in vivo. Furthermore, the expression of the myogenic genes myoD and pax3 was reduced, while NF-kappaB (the p65 subunit) localization and expression remained unchanged. In addition, promoter analysis has confirmed Myostatin inhibition of myoD and pax3. An increase in the expression of genes involved in ubiquitin-mediated proteolysis is observed during many forms of muscle wasting. Hence we analyzed the effect of Myostatin treatment on proteolytic gene expression. The ubiquitin associated genes atrogin-1, MuRF-1, and E214k were upregulated following Myostatin treatment. We analyzed how Myostatin may be signaling to induce cachexia. Myostatin signaling reversed the IGF-1/PI3K/AKT hypertrophy pathway by inhibiting AKT phosphorylation thereby increasing the levels of active FoxO1, allowing for increased expression of atrophy-related genes. Therefore, our results suggest that Myostatin induces cachexia through an NF-kappaB-independent mechanism. Furthermore, increased Myostatin levels appear to antagonize hypertrophy signaling through regulation of the AKT-FoxO1 pathway.
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Myostatin auto regulates its expression by feedback loop through smad7 dependent mechanism
Journal of Cellular Physiology, 2006Co-Authors: Davanea Forbes, Mark Thomas, Ravi Kambadur, Mark Jackman, Amy Bishop, Mridula SharmaAbstract:Myostatin, a secreted growth factor, is a member of the TGF-β superfamily and an inhibitor of myogenesis. Previously, we have shown that Myostatin gene expression is regulated at the level of transcription and that Myostatin is a downstream target gene of MyoD. Here we show that Myostatin gene expression is auto-regulated by a negative feedback mechanism. Northern blot analysis indicated that there are relatively higher levels of Myostatin mRNA in the biceps femoris muscle of cattle that express a non- functional Myostatin allele (Belgian Blue) as compared to normal cattle. In contrast, addition of exogenous Myostatin decreases endogenous Myostatin mRNA. Consistent with this result, wild type Myostatin protein is able to repress Myostatin promoter activity via Activin type IIb receptor (ActRIIB) and ALK5 (P < 0.001). However, non-functional Myostatin (Piedmontese) failed to repress the Myostatin promoter suggesting that Myostatin auto-regulates its promoter by negative feedback inhibition. Auto-regulation by Myostatin appears to be signaled through Smad7, since the expression of the inhibitory Smad7 is induced by Myostatin and the over-expression of Smad7 in turn inhibits the Myostatin promoter activity (P < 0.001). In contrast down regulation of Smad7 by siRNA results in increased Myostatin mRNA indicating that Smad7 is a negative regulator of Myostatin gene expression. Consistent with these results, a decrease in Smad7 mRNA and concomitant increase in Myostatin expression is seen in myotubes that express non functional Myostatin. In addition, interference with Myostatin signaling prevents the induction of Smad7 promoter activity by Myostatin. Based on these results, we propose that Myostatin auto-regulates its gene expression through a Smad7 dependent mechanism in myogenic cells. © 2005 Wiley-Liss, Inc.
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Myostatin inhibits rhabdomyosarcoma cell proliferation through an rb independent pathway
Oncogene, 2004Co-Authors: Mark Thomas, Craig Mcfarlane, Mridula Sharma, Brett Langley, Stewart Gilmour, Ravi KambadurAbstract:Rhabdomyosarcoma (RMS) tumors are the most common soft-tissue sarcomas in childhood. In this investigation, we show that Myostatin, a skeletal muscle-specific inhibitor of growth and differentiation is expressed and translated in the cultured RMS cell line, RD. The addition of exogenous recombinant Myostatin inhibits the proliferation of RD cells cultured in growth media, consistent with the role of Myostatin in normal myoblast proliferation inhibition. However, unlike normal myoblasts, upregulation of p21 was not observed. Rather, Myostatin signalling resulted in the specific downregulation of both Cdk2 and its cognate partner, cyclin-E. The analysis of Rb reveals that there was no change in its phosphorylation status with Myostatin treatment, consistent with D-type-cyclin-Cdk4/6 complexes being active in the absence of p21. Moreover, the activity of Rb appeared to be unchanged between treated and nontreated RD cells, as determined by the ability of Rb to bind E2F1. The examination of NPAT, a substrate of cyclin-E-Cdk2 involved in the transcriptional activation of replication-dependent histone gene expression, revealed that it undergoes a loss of phosphorylation with Myostatin treatment. Supporting this, a downregulation in H4-histone gene expression was observed. These results suggest that Myostatin could potentially be used as an inhibitor of RMS proliferation and define a previously uncharacterized, Rb-independent mechanism for the inhibition of muscle precursor cell proliferation by Myostatin.
Brett Langley - One of the best experts on this subject based on the ideXlab platform.
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Myostatin inhibits rhabdomyosarcoma cell proliferation through an rb independent pathway
Oncogene, 2004Co-Authors: Mark Thomas, Craig Mcfarlane, Mridula Sharma, Brett Langley, Stewart Gilmour, Ravi KambadurAbstract:Rhabdomyosarcoma (RMS) tumors are the most common soft-tissue sarcomas in childhood. In this investigation, we show that Myostatin, a skeletal muscle-specific inhibitor of growth and differentiation is expressed and translated in the cultured RMS cell line, RD. The addition of exogenous recombinant Myostatin inhibits the proliferation of RD cells cultured in growth media, consistent with the role of Myostatin in normal myoblast proliferation inhibition. However, unlike normal myoblasts, upregulation of p21 was not observed. Rather, Myostatin signalling resulted in the specific downregulation of both Cdk2 and its cognate partner, cyclin-E. The analysis of Rb reveals that there was no change in its phosphorylation status with Myostatin treatment, consistent with D-type-cyclin-Cdk4/6 complexes being active in the absence of p21. Moreover, the activity of Rb appeared to be unchanged between treated and nontreated RD cells, as determined by the ability of Rb to bind E2F1. The examination of NPAT, a substrate of cyclin-E-Cdk2 involved in the transcriptional activation of replication-dependent histone gene expression, revealed that it undergoes a loss of phosphorylation with Myostatin treatment. Supporting this, a downregulation in H4-histone gene expression was observed. These results suggest that Myostatin could potentially be used as an inhibitor of RMS proliferation and define a previously uncharacterized, Rb-independent mechanism for the inhibition of muscle precursor cell proliferation by Myostatin.
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Myostatin inhibits myoblast differentiation by down regulating myod expression
Journal of Biological Chemistry, 2002Co-Authors: Brett Langley, Mark Thomas, Mridula Sharma, Stewart Gilmour, Amy Bishop, Ravi KambadurAbstract:Abstract Myostatin, a negative regulator of myogenesis, is shown to function by controlling the proliferation of myoblasts. In this study we show that Myostatin is an inhibitor of myoblast differentiation and that this inhibition is mediated through Smad 3.In vitro, increasing concentrations of recombinant mature Myostatin reversibly blocked the myogenic differentiation of myoblasts, cultured in low serum media. Western and Northern blot analysis indicated that addition of Myostatin to the low serum culture media repressed the levels of MyoD, Myf5, myogenin, and p21 leading to the inhibition of myogenic differentiation. The transient transfection of C2C12 myoblasts with MyoD expressing constructs did not rescue Myostatin-inhibited myogenic differentiation. Myostatin signaling specifically induced Smad 3 phosphorylation and increased Smad 3·MyoD association, suggesting that Smad 3 may mediate the Myostatin signal by interfering with MyoD activity and expression. Consistent with this, the expression of dominant-negativeSmad3 rescued the activity of a MyoD promoter-reporter in C2C12 myoblasts treated with Myostatin. Taken together, these results suggest that Myostatin inhibits MyoD activity and expression via Smad 3 resulting in the failure of the myoblasts to differentiate into myotubes. Thus we propose that Myostatin plays a critical role in myogenic differentiation and that the muscular hyperplasia and hypertrophy seen in animals that lack functional Myostatin is because of deregulated proliferation and differentiation of myoblasts.
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single cysteine to tyrosine transition inactivates the growth inhibitory function of piedmontese Myostatin
American Journal of Physiology-cell Physiology, 2002Co-Authors: Mark Thomas, Mridula Sharma, Carole Berry, Brett Langley, Ravi KambadurAbstract:Myostatin, a member of the transforming growth factor-beta superfamily, is a secreted growth factor that is proteolytically processed to give COOH-terminal mature Myostatin and NH2-terminal latency-associated peptide in myoblasts. Piedmontese cattle are a heavy-muscled breed that express a mutated form of Myostatin in which cysteine (313) is substituted with tyrosine. Here we have characterized the biology of this mutated Piedmontese Myostatin. Northern and Western analyses indicate that there is increased expression of Myostatin mRNA and precursor Myostatin protein in the skeletal muscle of Piedmontese cattle. In contrast, a decrease in mature Myostatin was observed in Piedmontese skeletal muscle. However, there is no detectable change in the circulatory levels of mature Myostatin in Piedmontese cattle. Myoblast proliferation assay performed with normal and Piedmontese Myostatin indicated that mature wild-type Myostatin protein inhibited the proliferation of C2C12 myoblasts. Piedmontese Myostatin, by contrast, failed to inhibit myoblast proliferation. In addition, when added in molar excess, Piedmontese Myostatin acted as a potent "competitive inhibitor" molecule. These results indicate that, in Piedmontese Myostatin, substitution of cysteine with tyrosine results in the distortion of the "cystine knot" structure and a loss of biological activity of the Myostatin. This mutation also appears to affect either processing or stability of mature Myostatin without altering the secretion of Myostatin.
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Myostatin a negative regulator of muscle growth functions by inhibiting myoblast proliferation
Journal of Biological Chemistry, 2000Co-Authors: Mark Thomas, Mridula Sharma, Carole Berry, Brett Langley, John J Bass, Sonnie P Kirk, Ravi KambadurAbstract:Myostatin, a member of the transforming growth factor-beta (TGF-beta) superfamily, has been shown to be a negative regulator of myogenesis. Here we show that Myostatin functions by controlling the proliferation of muscle precursor cells. When C(2)C(12) myoblasts were incubated with Myostatin, proliferation of myoblasts decreased with increasing levels of Myostatin. Fluorescence-activated cell sorting analysis revealed that Myostatin prevented the progression of myoblasts from the G(1)- to S-phase of the cell cycle. Western analysis indicated that Myostatin specifically up-regulated p21(Waf1, Cip1), a cyclin-dependent kinase inhibitor, and decreased the levels and activity of Cdk2 protein in myoblasts. Furthermore, we also observed that in myoblasts treated with Myostatin protein, Rb was predominately present in the hypophosphorylated form. These results suggests that, in response to Myostatin signaling, there is an increase in p21 expression and a decrease in Cdk2 protein and activity thus resulting in an accumulation of hypophosphorylated Rb protein. This, in turn, leads to the arrest of myoblasts in G(1)-phase of cell cycle. Thus, we propose that the generalized muscular hyperplasia phenotype observed in animals that lack functional Myostatin could be as a result of deregulated myoblast proliferation.
John J Bass - One of the best experts on this subject based on the ideXlab platform.
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the Myostatin gene is a downstream target gene of basic helix loop helix transcription factor myod
Molecular and Cellular Biology, 2002Co-Authors: Michael P Spiller, Mark Thomas, Ravi Kambadur, John J Bass, Ferenc Jeanplong, J A K Martyn, Mridula SharmaAbstract:Myostatin is a negative regulator of myogenesis, and inactivation of Myostatin leads to heavy muscle growth. Here we have cloned and characterized the bovine Myostatin gene promoter. Alignment of the upstream sequences shows that the Myostatin promoter is highly conserved during evolution. Sequence analysis of 1.6 kb of the bovine Myostatin gene upstream region revealed that it contains 10 E-box motifs (E1 to E10), arranged in three clusters, and a single MEF2 site. Deletion and mutation analysis of the Myostatin gene promoter showed that out of three important E boxes (E3, E4, and E6) of the proximal cluster, E6 plays a significant role in the regulation of a reporter gene in C2C12 cells. We also demonstrate by band shift and chromatin immunoprecipitation assay that the E6 E-box motif binds to MyoD in vitro and in vivo. Furthermore, cotransfection experiments indicate that among the myogenic regulatory factors, MyoD preferentially up-regulates Myostatin promoter activity. Since MyoD expression varies during the myoblast cell cycle, we analyzed the Myostatin promoter activity in synchronized myoblasts and quiescent “reserve” cells. Our results suggest that Myostatin promoter activity is relatively higher during the G1 phase of the cell cycle, when MyoD expression levels are maximal. However, in the reserve cells, which lack MyoD expression, a significant reduction in the Myostatin promoter activity is observed. Taken together, these results suggest that the Myostatin gene is a downstream target gene of MyoD. Since the Myostatin gene is implicated in controlling G1-to-S progression of myoblasts, MyoD could be triggering myoblast withdrawal from the cell cycle by regulating Myostatin gene expression.
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Myostatin a negative regulator of muscle growth functions by inhibiting myoblast proliferation
Journal of Biological Chemistry, 2000Co-Authors: Mark Thomas, Mridula Sharma, Carole Berry, Brett Langley, John J Bass, Sonnie P Kirk, Ravi KambadurAbstract:Myostatin, a member of the transforming growth factor-beta (TGF-beta) superfamily, has been shown to be a negative regulator of myogenesis. Here we show that Myostatin functions by controlling the proliferation of muscle precursor cells. When C(2)C(12) myoblasts were incubated with Myostatin, proliferation of myoblasts decreased with increasing levels of Myostatin. Fluorescence-activated cell sorting analysis revealed that Myostatin prevented the progression of myoblasts from the G(1)- to S-phase of the cell cycle. Western analysis indicated that Myostatin specifically up-regulated p21(Waf1, Cip1), a cyclin-dependent kinase inhibitor, and decreased the levels and activity of Cdk2 protein in myoblasts. Furthermore, we also observed that in myoblasts treated with Myostatin protein, Rb was predominately present in the hypophosphorylated form. These results suggests that, in response to Myostatin signaling, there is an increase in p21 expression and a decrease in Cdk2 protein and activity thus resulting in an accumulation of hypophosphorylated Rb protein. This, in turn, leads to the arrest of myoblasts in G(1)-phase of cell cycle. Thus, we propose that the generalized muscular hyperplasia phenotype observed in animals that lack functional Myostatin could be as a result of deregulated myoblast proliferation.
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Myostatin a transforming growth factor β superfamily member is expressed in heart muscle and is upregulated in cardiomyocytes after infarct
Journal of Cellular Physiology, 1999Co-Authors: Mridula Sharma, Ravi Kambadur, Kenneth G Matthews, Wayne G Somers, Gerard Devlin, John V Conaglen, Peter J Fowke, John J BassAbstract:Myostatin is a secreted growth and differentiating factor (GDF-8) that belongs to the transforming growth factor-beta (TGF-β) superfamily. Targeted disruption of the Myostatin gene in mice and a mutation in the third exon of the Myostatin gene in double-muscled Belgian Blue cattle breed result in skeletal muscle hyperplasia. Hence, Myostatin has been shown to be involved in the regulation of skeletal muscle mass in both mice and cattle. Previous published reports utilizing Northern hybridization had shown that Myostatin expression was seen exclusively in skeletal muscle. A significantly lower level of Myostatin mRNA was also reported in adipose tissue. Using a sensitive reverse transcription-polymerase chain reaction (RT-PCR) technique and Western blotting with anti-Myostatin antibodies, we show that Myostatin mRNA and protein are not restricted to skeletal muscle. We also show that Myostatin expression is detected in the muscle of both fetal and adult hearts. Sequence analysis reveals that the Belgian Blue heart Myostatin cDNA sequence contains an 11 nucleotide deletion in the third exon that causes a frameshift that eliminates virtually all of the mature, active region of the protein. Anti-Myostatin immunostaining on heart sections also demonstrates that Myostatin protein is localized in Purkinje fibers and cardiomyocytes in heart tissue. Furthermore, following myocardial infarction, Myostatin expression is upregulated in the cardiomyocytes surrounding the infarct area. Given that Myostatin is expressed in fetal and adult hearts and that Myostatin expression is upregulated in cardiomyocytes after the infarction, Myostatin could play an important role in cardiac development and physiology. J. Cell. Physiol. 180:1–9, 1999. © 1999 Wiley-Liss, Inc.
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Myostatin a transforming growth factor beta superfamily member is expressed in heart muscle and is upregulated in cardiomyocytes after infarct
Journal of Cellular Physiology, 1999Co-Authors: Mridula Sharma, Ravi Kambadur, Kenneth G Matthews, Wayne G Somers, Gerard Devlin, John V Conaglen, Peter J Fowke, John J BassAbstract:Myostatin is a secreted growth and differentiating factor (GDF-8) that belongs to the transforming growth factor-beta (TGF-β) superfamily. Targeted disruption of the Myostatin gene in mice and a mutation in the third exon of the Myostatin gene in double-muscled Belgian Blue cattle breed result in skeletal muscle hyperplasia. Hence, Myostatin has been shown to be involved in the regulation of skeletal muscle mass in both mice and cattle. Previous published reports utilizing Northern hybridization had shown that Myostatin expression was seen exclusively in skeletal muscle. A significantly lower level of Myostatin mRNA was also reported in adipose tissue. Using a sensitive reverse transcription-polymerase chain reaction (RT-PCR) technique and Western blotting with anti-Myostatin antibodies, we show that Myostatin mRNA and protein are not restricted to skeletal muscle. We also show that Myostatin expression is detected in the muscle of both fetal and adult hearts. Sequence analysis reveals that the Belgian Blue heart Myostatin cDNA sequence contains an 11 nucleotide deletion in the third exon that causes a frameshift that eliminates virtually all of the mature, active region of the protein. Anti-Myostatin immunostaining on heart sections also demonstrates that Myostatin protein is localized in Purkinje fibers and cardiomyocytes in heart tissue. Furthermore, following myocardial infarction, Myostatin expression is upregulated in the cardiomyocytes surrounding the infarct area. Given that Myostatin is expressed in fetal and adult hearts and that Myostatin expression is upregulated in cardiomyocytes after the infarction, Myostatin could play an important role in cardiac development and physiology. J. Cell. Physiol. 180:1–9, 1999. © 1999 Wiley-Liss, Inc.
