Crystal M. Weyman

Apoptosis coincident with the differentiation of skeletal myoblasts is delayed by caspase 3 inhibition and abrogated by MEK-independent constitutive Ras signaling

We demonstrate that during 23A2 skeletal myoblast differentiation, between 30–35% of the population apoptose. Both differentiation and apoptosis are controlled by the variables of cell density and time and these variables are inversely related. In response to conditions that permit both differentiation and apoptosis of parental 23A2 myoblasts, myoblasts rendered differentiation-defective by constitutive Ras signaling (A2:H-Ras myoblasts) do not apoptose. This is not merely a consequence of their differentiation-defective phenotype since myoblasts rendered differentiation-defective by expression of E1A (A2:E1A myoblasts) still apoptose. Although signaling through MEK is important to the survival of proliferating parental 23A2 myoblasts, constitutive signaling through MEK is not responsible for the survival of A2:H-Ras myoblasts. Finally, we demonstrate that caspase 3 is activated and that pharmacological inhibition of caspase 3 activity delays apoptosis without affecting differentiation. Abrogating apoptosis without affecting differentiation could be a useful approach to improve the efficacy of myoblast transfer in the treatment of muscular dystrophies.

Distinct signaling pathways regulate transformation and inhibition of skeletal muscle differentiation by oncogenic Ras.

Expression of oncogenic Ras in 23A2 skeletal myoblasts is sufficient to induce both a transformed phenotype and a differentiation-defective phenotype, but the signaling pathways activated by oncogenic Ras in these cells and their respective contribution to each phenotype have not been explored. In this study, we investigated MAP kinase activity in control 23A2 myoblasts and in 23A2 myoblasts rendered differentiation-defective by the stable expression of an oncogenic (G12V)Ha-ras gene (Ras9 cells). The MAP kinase immunoprecipitated from Ras9 cells was 30 – 40% more active than that from control 23A2 cells. To establish if this elevated MAP kinase activity is essential to the maintenance of the oncogenic Ras-induced phenotype, we utilized the selective MAP kinase kinase 1 (MEK1) inhibitor, PD 098059. PD 098059 decreased the MAP kinase activity in Ras9 cells to the level found in 23A2 cells. PD 098059 did not affect the ability of 23A2 myoblasts to differentiate. PD 098059 reverted the transformed morphology of Ras9 cells but did not restore the ability of these cells to express the muscle-specific myosin heavy chain gene or to form muscle fibers. Treatment with PD 098059 also did not affect the ability of oncogenic Ha-Ras to establish a non-myogenic phenotype in C3H10T1/2 cells co-expressing MyoD. These results demonstrate that the activation of MAP kinase is necessary for the transformed morphology of Ras9 cells but is not required by oncogenic Ras to establish or to maintain a differentiation-defective phenotype. While these data do not rule out the possibility that constitutive signaling by MEK1 or MAP kinase could inhibit myoblast differentiation, they clearly demonstrate that other pathways activated by oncogenic Ras are sufficient to inhibit differentiation.

Increased expression of the pro-apoptotic Bcl2 family member PUMA is required for mitochondrial release of cytochrome C and the apoptosis associated with skeletal myoblast differentiation

We have previously shown that when skeletal myoblasts are cultured in differentiation medium (DM), roughly 30% undergo caspase 3-dependent apoptosis rather than differentiation. Herein, we investigate the molecular mechanism responsible for the activation of caspase 3 and the ensuing apoptosis. When 23A2 myoblasts are cultured in DM, caspase 9 activity is increased and pharmacological abrogation of caspase 9 activation impairs caspase 3 activation and apoptosis. Further, we detect a time dependent release of mitochondrial cytochrome C into the cytosol in roughly 30% of myoblasts. Inclusion of cycloheximide inhibits the release of cytochrome C, the activation of caspase 9 and apoptosis. These data indicate that the mitochondrial pathway plays a role in this apoptotic process and that engagement of this pathway relies on de novo protein synthesis. Through RT-PCR and immunoblot analysis, we have determined that the expression level of the pro-apoptotic Bcl2 family member PUMA is elevated when 23A2 myoblasts are cultured in DM. Further, silencing of PUMA inhibits the release of cytochrome C and apoptosis. Signaling by the transcription factor p53 is not responsible for the increased level of PUMA. Finally, myoblasts rescued from apoptosis by either inhibition of elevated caspase 9 activity or silencing of PUMA are competent for differentiation. These results indicate a critical role for PUMA in the apoptosis associated with skeletal myoblast differentiation and that a p53-independent mechanism is responsible for the increased expression of PUMA in these cells.

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.

Signaling through the TRAIL receptor DR5/FADD pathway plays a role in the apoptosis associated with skeletal myoblast differentiation

Apoptosis rather than differentiation is a physiological process during myogenesis and muscle regeneration. When cultured myoblasts were induced to differentiate, we detected an increase in caspase 8 activity. Pharmacological inhibition of caspase 8 activity decreased apoptosis. Expression of a dominant-negative mutant of the adapter protein FADD also abrogated apoptosis, implicating a death ligand pathway. Treatment with TRAIL, but not Fas, induced apoptosis in these myoblasts. Accordingly, treatment with a soluble TRAIL decoy receptor or expression of a dominant-negative mutant of the TRAIL receptor DR5 abrogated apoptosis. While TRAIL expression levels remained unaltered in apoptotic myoblasts, DR5 expression levels increased. Finally, we also detected a reduction in FLIP, a death-receptor effector protein and caspase 8 competitive inhibitor, to undetectable levels in apoptotic myoblasts. Thus, our data demonstrate an important role for the TRAIL/DR5/FADD/caspase 8 pathway in the apoptosis associated with skeletal myoblast differentiation. Identifying the functional apoptotic pathways in skeletal myoblasts may prove useful in minimizing the myoblast apoptosis that contributes pathologically to a variety of diseases and in minimizing the apoptosis of transplanted myoblasts to treat these and other disease states.

IFN-γ Induces Apoptosis in HL-60 Cells Through Decreased Bcl-2 and Increased Bak Expression

Interferons (IFNs) are pleiotropic cytokines responsible for inducing innate and adaptive immunities against a wide range of viruses and other microbial pathogens. In addition, IFNs also exert antitumor activities due to their antiproliferative, immunomodulatory, proapoptotic functions. In the last decades, the successful clinical application of IFNs for treatment of cancer, particularly leukemia, has improved the quality and longevity of life for many patients. The induction of tumor cell apoptosis by IFNs is believed to contribute, at least in part, to the beneficial effects. IFN subtypes, such as IFN-alpha, IFN-beta, and IFN-gamma, induce apoptosis through cell type-specific signaling pathways, and several putative IFN-stimulated genes (ISGs) with proapoptotic functions have been identified. Here, we analyzed the ability of IFN-alpha, IFN-beta, or IFN-gamma to induce apoptosis in several malignant hematologic cell lines. We found that treatment with IFN-gamma, but not IFN-alpha, or IFN-beta, specifically induces HL-60 leukemia cells to undergo apoptosis. Roughly 30% of HL-60 cells treated for 48 h with IFN-gamma, but not IFN-gamma, or IFN-beta, underwent apoptosis as monitored by annexin V labeling to determine changes in phosphatidylserine (PS) asymmetry and TUNEL assay to detect DNA fragmentation. Consistent with these results, treatment with IFN-gamma, but not IFN-alpha or IFN-beta, induced the release of cytochrome c, activation of caspase-3, and cleavage of poly (ADP-ribose) polymerase (PARP), a well-characterized caspase-3 substrate. Further investigation into the potential mechanism responsible for mitochondrial disruption revealed that treatment with IFN-gamma caused decreased levels of Bcl-2 and increased levels of Bak. This study thus provides the basis for additional research to uncover the molecular mechanism by which IFN-gamma regulates the expression of Bcl-2 family members in various cell types.

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 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.

Non-canonical role for the TRAIL receptor DR5/FADD/caspase pathway in the regulation of MyoD expression and skeletal myoblast differentiation

We report herein that the TRAIL receptor DR5/FADD/caspase pathway plays a role in skeletal myoblast differentiation through modulation of the expression of the muscle regulatory transcription factor MyoD. Specifically, treatment with the selective caspase 3 inhibitor DEVD-fmk or the selective caspase 8 inhibitor IETD-fmk in growth media (GM), prior to culture in differentiation media (DM), inhibited differentiation. Further, this treatment resulted in decreased levels of MyoD message and protein. We next explored a role for the TRAIL receptor DR5/FADD pathway. We found that expression of either dominant negative (dn) FADD or dominant negative (dn) DR5 also resulted in decreased levels of MyoD mRNA and protein and blocked differentiation. This decreased level of MyoD mRNA was not a consequence of altered stability. Treatment with TSA, an inhibitor of histone deacetylases (HDACs), allowed MyoD expression in myoblasts expressing dnDR5. Finally, acetylation of histones associated with the distal regulatory region (DRR) enhancer of MyoD was decreased in myoblasts expressing dnDR5. Thus, our data suggests a non-canonical role for the TRAIL receptor/FADD pathway in the regulation of MyoD expression and skeletal myoblast differentiation.

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.

Differential signaling through NFκB does not ameliorate skeletal myoblast apoptosis during differentiation

During 23A2 skeletal myoblast differentiation, roughly 30% of the population undergoes apoptosis. Further, constitutive signaling by G12V:H‐Ras or Raf:CAAX abrogates this apoptosis. In this study, we demonstrate an increase in NFκB activity in myoblasts that have survived and are expressing muscle‐specific genes. NFκB activity is also elevated in myoblasts expressing constitutively active G12V:H‐Ras but not Raf:CAAX. Expression of a dominant negative IκB (IκB‐SR) sufficient to eliminate this elevated level of NFκB activity, in either the 23A2 myoblasts or their G12V:H‐Ras‐expressing counterparts, however, does not affect survival. Furthermore, expression of a constitutively active IκB kinase in 23A2 myoblasts does not protect these cells from the apoptosis associated with differentiation. Since signaling by IκB kinase can abrogate differentiation, this result demonstrates that abrogated differentiation and abrogated apoptosis are separable phenotypes.