Activation of DNA Damage Checkpoint Pathways During Skeletal Myoblast Differentiation and Apoptosis

A subset of skeletal myoblasts undergo apoptosis rather than differentiation when cultured in differentiation media (DM: absence of growth factors). While the muscle regulatory transcription factor MyoD is known to control the process of differentiation, our lab has recently discovered that MyoD is also controlling the apoptotic process in response to culture in DM by direct up-regulation of the pro-apoptotic Bcl2 family member PUMA. We similarly discovered that MyoD plays a role in the increased expression of PUMA and apoptosis in response to the DNA damaging agent, etoposide. This led to the hypothesis that culture in DM may lead to stalled replication forks during DNA synthesis that are “recognized” as DNA damage. We are testing our hypothesis by determining if culture in DM results in the activation of pathways known to respond to DNA damage. We have determined that p38, p53, and c-abl are all up-regulated in response to culture in DM. Next, we will determine the significance of MyoD to the increased expression of these molecules.https://engagedscholarship.csuohio.edu/u_poster_2014/1027/thumbnail.jp

Basal Signalling Through Death Receptor 5 and Caspase 3 Activates p38 Kinase To Regulate Serum Response Factor (SRF)-Mediated Myod Transcription

We have previously reported that stable expression of a dominant negative Death Receptor 5 (dnDR5) in skeletal myoblasts results in decreased basal caspase activity and decreased mRNA and protein expression of the muscle regulatory transcription factor MyoD in growth medium (GM), resulting in inhibited differentation when myoblasts are then cultured in differentiation media (DM). Further, this decreased level of MyoD mRNA was not a consequence of altered message stability, but rather correlated with decreased acetylation of histones in the distal regulatory region (DRR) of the MyoD extended promoter known to control MyoD transcription. As serum response factor (SRF) is the transcription factor known to be responsible for basal MyoD expression in GM, we compared the level of SRF binding to the non-canonical serum response element (SRE) within the DRR in parental and dnDR5 expressing myoblasts. Herein, we report that stable expression of dnDR5 resulted in decreased levels of serum response factor (SRF) binding to the CArG box in the SRE of the DRR. Total SRF expression levels were not affected, but phosphorylation indicative of SRF activation was impaired. This decreased SRF phosphorylation correlated with decreased phosphorylation-induced activation of p38 kinase. Moreover, the aforementioned signaling events affected by expression of dnDR5 could be appropriately recapitulated using either a pharmacological inhibitor of caspase 3 or p38 kinase. Thus, our results have established a signaling pathway from DR5 through caspases to p38 kinase activation, to SRF activation and the basal expression of MyoD

Mitogen-Activated Protein Kinase Kinase (MEK) Activity Is Required for Inhibition of Skeletal Muscle Differentiation by Insulin-Like Growth Factor 1 or Fibroblast Growth Factor 2

Both insulin-like growth factor 1 (IGF-1) and fibroblast growth factor 2 (FGF-2) are key modulators of skeletal myoblast differentiation. The critical signaling pathways used by either IGF-1 or FGF-2 to inhibit differentiation have not been determined. In this study, we show that both IGF-1 and FGF-2 inhibit the differentiation of 23A2 myoblasts and that both stimulate signaling through mitogen-activated protein kinase (MAPK) kinase (MEK) to MAPK roughly 8-fold in 23A2 myoblasts. We used the selective chemical inhibitor of MEK, PD 098059, to determine if signaling by MEK is required by IGF-1 or FGF-2 to inhibit differentiation. PD 098059 did not affect the ability of 23A2 myoblasts to differentiate. Addition of PD 098059 to the culture medium 10 min before the addition of IGF-1 or FGF-2 completely blocked the signal from MEK to MAPK and restored the ability of the 23A2 myoblasts to differentiate in the presence of either IGF-1 or FGF-2. The peak of signaling through MEK to MAPK in response to either IGF-1 or FGF-2 occurred within the first hour with maximal activation observed after 10 min. This signal remained elevated (at roughly 70% above basal) for at least 48 h, PD 098059 was added to the culture 60 min after IGF-1 or FGF-2 to test whether this initial peak of signaling was sufficient for the inhibition of differentiation. The restoration of myogenic potential seen when cells were preincubated with PD 098059 was essentially identical to that seen when PD 098059 was added to cultures after the initial peak of signaling from MEK to MAPK, suggesting that persistent signaling through MEK is required for the inhibition of differentiation by either IGF-1 or FGF-2

Oncogenic Ras-Induced Secretion of a Novel Inhibitor of Skeletal Myoblast Differentiation

Expression of oncogenic H-Ras in 23A2 myoblasts (A2:H-Ras cells) is sufficient to induce both a transformed phenotype and a differentiation-defective phenotype. Because oncogenic Ras is known to induce the secretion of several different growth factors in involved in maintaining the transformed phenotype of both fibroblast and epithelial cells, we explored the possibility that expression of oncogenic Ras in 23A2 myoblasts might lead to the secretion of a factor which inhibits differentiation. The differentiation of 23A2 myoblasts was inhibited (i) by coculture with an equal number of A2:H-Ras cells, (ii) by culture with an equal number of A2:H-Ras cells in the same tissue culture medium on an insert which allowed equilibration of molecules smaller than 1 micron, and (iii) by culture in media previously conditioned by A2:H-Ras cells. Similar results were obtained when 23A2 myoblasts expressing oncogenic N-Ras were substituted for A2:H-Ras cells in each assay. No inhibition of differentiation was observed, however, when differentiation-defective E1A-expressing 23A2 cells or C3H10T1/2 fibroblasts were substituted for A2:H-Ras cells. The differentiation inhibitor(s) in media conditioned by A2:H-Ras cells is heat stable, larger than 3 kD, and sensitive to the non-specific growth factor antagonist, suramin. Western analyses failed to detect either FGF-2 or TGFβ (the known inhibitors of myoblast differentiation) in media conditioned by A2:H-Ras cells. Furthermore, while FGF-2 is a potent activator of MAP kinase and TGFβ is a potent inhibitor of mink lung epithelial cell (CCL64) growth, conditioned media from A2:H-Ras cells does not activate MAP kinase and does not inhibit the growth of CCL64 cells. These results indicate that expression of oncogenic Ras induces the secretion of a novel inhibitor of skeletal myoblast differentiation. Furthermore, these results are the first to implicate an autocrine/paracrine mechanism in the inhibition of differentiation by oncogenic Ras

Chapter 8 Ras: Processor of Vital Signals

The pivotal role of Ras as a transducer of vital signals is well established. As such, aberrant Ras signaling is associated with cancer, neurofibromatosis, and Alzheimer\u27s disease. This realization could make detection of mutated ras a useful diagnostic and prognostic tool. Exploiting the biochemical properties of Ras has made interfering with Ras activity a potential clinical option and dissection of the relative contribution of each signaling pathway emanating from Ras will certainly provide additional targets for clinical intervention. © 1996 Elsevier Inc. All rights reserved

Transient Blockage of Proliferative Signalling: A Novel Strategy for Protective Chemotherapy

An intact proliferative signalling pathway is essential to the growth of all normal cells, but is often not required by tumor cells. This fact was used to devise a protective chemotherapeutic protocol potentially applicable to all tissues. Four treatments were chosen to temporarily disrupt proliferative signalling. They acted either upstream, at, or downstream of cellular ras activity. As expected, the cell cycle progression of normal cells was temporarily interrupted, while those cells transformed by tumor genes, or tumor cells themselves often were not affected. During these cell cycle blocking treatments the cells were exposed to the topoisomerase inhibitor m-AMSA. This anti-cancer drug is selectively toxic to cycling cells. In each case the tumor cells were selectively killed as judged either by their ability to incorporate labeled thymidine, replate, or grow. These studies suggest new ways to utilize current drugs or search for new ones

Raf-Induced Effects on the Differentiation and Apoptosis of Skeletal Myoblasts Are Determined by the Level of Raf Signaling: Abrogation of Apoptosis by Raf Is Downstream of Caspase 3 Activation

We examined the effect of a constitutively active Raf protein (Raf-CAAX) on the differentiation and the coincident apoptosis of skeletal myoblasts. We found that a low level of Raf signaling leads to accelerated differentiation when compared to parental myoblasts, while a higher level of Raf signaling induces a transformed morphology and abrogates both differentiation and the coincident apoptosis. Raf signaling abrogates apoptosis without blocking the activation of caspase 3 and the subsequent cleavage of caspase 3 substrates. Eliminating the signal from Raf through MEK does not restore the ability to differentiate or to undergo apoptosis in the myoblasts with a high level of Raf signal, nor does it abrogate the accelerated differentiation observed in myoblasts with lower levels of Raf signal. Constitutive signaling through MEK is required, however, to maintain a transformed morphology. These results indicate that the effect of Raf on the differentiation and apoptosis of skeletal myoblasts is dictated by the level of Raf signaling, and that Raf signaling sufficient to abrogate the apoptosis coincident with differentiation does so downstream of caspase 3 signaling

Increased Expression of the Pro-Apoptotic Bcl2 Family Member PUMA and Apoptosis by the Muscle Regulatory Transcription Factor MyoD in Response to a Variety of Stimuli

We have previously reported that the level of MyoD expression correlates with the level of apoptosis that occurs in a subpopulation of skeletal myoblasts induced to differentiate by serum withdrawal. Herein we document that MyoD expression contributes to the level of apoptosis in myoblasts and fibroblasts in response to a variety of apoptotic stimuli. Specifically, re-expression of MyoD in skeletal myoblasts rendered defective for both differentiation and apoptosis by the expression of oncogenic Ras restores their ability to undergo both differentiation and apoptosis in response to serum withdrawal. Further, using a fibroblast cell line expressing an estrogen receptor:MyoD fusion protein, we have determined that addition of estrogen sensitizes these fibroblasts to apoptosis induced by serum withdrawal, or by treatment with etoposide or thapsigargin. RNAi mediated silencing of MyoD in either 23A2 or C2C12 myoblasts renders these cells resistant to apoptosis induced by serum withdrawal, or by treatment with etoposide or thapsigargin. Finally, MyoD mediated regulation of the apoptotic response to these various stimuli, in both myoblasts and fibroblasts, correlates with the level of induction of the pro-apoptotic Bcl2 family member PUMA. © 2009 Springer Science+Business Media, LLC