Charu Dogra

TNF-related weak inducer of apoptosis (TWEAK) is a potent skeletal muscle-wasting cytokine

TWEAK cytokine has been implicated in several biological responses including inflammation, angiogenesis, and osteoclastogenesis. We have investigated the role of TWEAK in regulating skeletal muscle mass. Addition of soluble TWEAK protein to cultured myotubes reduced the mean myotube diameter and enhanced the degradation of specific muscle proteins such as CK and MyHCf. The effect of TWEAK on degradation of MyHCf was stronger than its structural homologue, TNF‐α. TWEAK increased the ubiquitination of MyHCf and the transcript levels of atrogin‐1 and MuRF1 ubiquitin ligases. TWEAK inhibited phosphor‐ylation of Akt kinase and its downstream targets GSK‐3β, FOXO1, mTOR, and p70S6K Furthermore, TWEAK increased the activation of NF‐κB transcription factor in myotubes. Adenoviral‐mediated overexpression of IκBαΔN (a degradation‐resistant mutant of NF‐κB inhibitory protein ΙκBα) in myotubes blocked the TWEAK‐induced degradation of MyHCf. Chronic administration of TWEAK in mice resulted in reduced body and skeletal muscle weight with an associated increase in the activity of ubiquitin‐proteasome system and NF‐κB. Finally, muscle‐specific transgenic overexpression of TWEAK decreased the body and skeletal muscle weight in mice. Collectively, our data suggest that TWEAK induces skeletal muscle atrophy through inhibition of the PI3K/Akt signaling pathway and activation of the ubiquitin‐proteasome and NF‐kB systems.—Dogra, C., Changotra, H., Wedhas, N., Qin, X., Wergedal, J. E., Kumar, A. TNF‐related weak inducer of apoptosis (TWEAK) is a potent skeletal muscle‐wasting cytokine. FASEB J. 21, 1857–1869 (2007)

Tumor Necrosis Factor-like Weak Inducer of Apoptosis Inhibits Skeletal Myogenesis through Sustained Activation of Nuclear Factor-κB and Degradation of MyoD Protein

In this study we have investigated the effect and the mechanisms by which tumor necrosis factor-like weak inducer of apoptosis (TWEAK) modulates myogenic differentiation. Treatment of C2C12 myoblasts with TWEAK inhibited their differentiation evident by a decrease in the expression of creatine kinase, myosin heavy chain-fast twitch, myogenin, and the formation of multinucleated myotubes. TWEAK also inhibited the differentiation of mouse primary myoblasts. Conversely, the proliferation of C2C12 myoblasts and the expression of a cell-cycle regulator cyclin D1 were increased in response to TWEAK treatment. Inhibition of cellular proliferation using hydroxyurea only partially reversed the inhibitory effect of TWEAK on myogenic differentiation. Treatment of C2C12 myoblasts with TWEAK resulted in the activation of nuclear factor-κB (NF-κB), the (IkappaB) IκB kinase (IKK) complex, and the phosphorylation and degradation of IκBα protein. Inhibition of NF-κB activity by overexpression of a dominant negative mutant of IκBα (IκBαΔN) significantly increased the myogenic differentiation in TWEAK-treated C2C12 cultures. Furthermore, overexpression of a dominant negative mutant of IKKβ (IKKβK44A) but not IKKα (IKKαK44M) reversed the inhibitory effect of TWEAK on myogenesis. TWEAK inhibited the expression of myogenic regulatory factors MyoD and myogenin and also induced the degradation of MyoD protein. Finally, inhibition of NF-κB activation through overexpression of IKKβK44A prevented the degradation of MyoD protein. Overall, our data suggest that TWEAK inhibits myogenesis through the activation of NF-κB signaling pathway and degradation of MyoD protein.

Regulation of phosphatidylinositol 3-kinase (PI3K)/Akt and nuclear factor-kappa B signaling pathways in dystrophin-deficient skeletal muscle in response to mechanical stretch

Phosphatidylinositol 3‐kinase (PI3K)/Akt and nuclear factor‐kappa B (NF‐κB) signaling pathways play a critical role in mediating survival signals. In this study we have investigated how loss of dystrophin (the primary cause of Duchenne muscular dystrophy) modulates the activation of PI3K/Akt and NF‐κB signaling pathways in skeletal muscle in response to mechanical stimulation. Activation of Akt was significantly higher in diaphragm muscle from dystrophin‐deficient mdx mice compared to normal mice at both prenecrotic and necrotic states. Higher activation of Akt was also observed in cultured dystrophin‐deficient primary myotubes differentiated in vitro. Application of passive mechanical stretch ex vivo synergistically increased the activation of Akt in diaphragm of mdx mice. Stretch‐induced activation of PDK‐1 and PI3K were also higher in diaphragm of mdx mice compared to normal mice. Pretreatment of diaphragm with PI3K inhibitor LY294002 blocked the activation of Akt in normal and mdx mice. Higher activation of Akt was associated with increased phosphorylation of its downstream targets glycogen synthase kinase 3β (GSK3β), FKHR, and mammalian target of rapamycin (mTOR). Treatment of diaphragm muscle with LY294002 inhibited the stretch‐induced activation of IkappaB (IκB) kinase (IKK) and NF‐κB transcription factor in normal and mdx mice. Mechanical stretch also reduced the interaction of HDAC1 with RelA subunit of NF‐κB in diaphragm muscle. Finally, cellular levels of Bcl‐2, cIAP1, and integrin β1 and activation of integrin linked kinase were higher in diaphragm muscle of mdx mice compared to normal mice. Taken together, our data suggest that loss of dystrophin and/or mechanical stretch results in the up‐regulation of PI3K/Akt and NF‐κB signaling pathways in skeletal muscle. J. Cell. Physiol. 208: 575–585, 2006.

Fibroblast Growth Factor Inducible 14 (Fn14) Is Required for the Expression of Myogenic Regulatory Factors and Differentiation of Myoblasts into Myotubes EVIDENCE FOR TWEAK-INDEPENDENT FUNCTIONS OF Fn14 DURING MYOGENESIS

Fibroblast growth factor-inducible 14 (Fn14), distantly related to tumor necrosis factor receptor superfamily and a receptor for TWEAK cytokine, has been implicated in several biological responses. In this study, we have investigated the role of Fn14 in skeletal muscle formation in vitro. Flow cytometric and Western blot analysis revealed that Fn14 is highly expressed on myoblastic cell line C2C12 and mouse primary myoblasts. The expression of Fn14 was decreased upon differentiation of myoblasts into myotubes. Suppression of Fn14 expression using RNA interference inhibited the myotube formation in both C2C12 and primary myoblast cultures. Fn14 was required for the transactivation of skeletal α-actin promoter and the expression of specific muscle proteins such as myosin heavy chain fast type and creatine kinase. RNA interference-mediated knockdown of Fn14 receptor in C2C12 myoblasts decreased the levels of myogenic regulatory factors MyoD and myogenin upon induction of differentiation. Conversely, overexpression of MyoD increased differentiation in Fn14-knockdown C2C12 cultures. Suppression of Fn14 expression in C2C12 myoblasts also inhibited the differentiation-associated increase in the activity of serum response factor and RhoA GTPase. In addition, our data suggest that the role of Fn14 during myogenic differentiation could be independent of TWEAK cytokine. Collectively, our study suggests that the Fn14 receptor is required for the expression of myogenic regulatory factors and differentiation of myoblasts into myotubes.

Inhibition of mechanosensitive cation channels inhibits myogenic differentiation by suppressing the expression of myogenic regulatory factors and caspase-3 activity

Mechanosensitive cation channels (MSC) are ubiquitous in eukaryotic cell types. However, the physiological functions of MSC in several tissues remain in question. In this study we have investigated the role of MSC in skeletal myogenesis. Treatment of C2C12 myoblasts with gadolinium ions (MSC blocker) inhibited myotube formation and the myogenic index in differentiation medium (DM). The enzymatic activity of creatine kinase (CK) and the expression of myosin heavy chain‐fast twitch (MyHCf) in C2C12 cultures were also blocked in response to gadolinium. Treatment of C2C12 myoblasts with gadolinium ions did not affect the expression of either cyclin A or cyclin D1 in DM. Other inhibitors of MSC such as streptomycin and GsTMx‐4 also suppressed the expression of CK and MyHCf in C2C12 cultures. The inhibitory effect of gadolinium ions on myogenic differentiation was reversible and independent of myogenic cell type. Real‐time‐polymerase chain reaction analysis revealed that inhibition of MSC decreases the expression of myogenic transcription factors MyoD, myogenin, and Myf‐5. Furthermore, the activity of skeletal α‐actin promoter was suppressed on MSC blockade. Treatment of C2C12 myoblasts with gadolinium ions prevented differentiation‐associated cell death and inhibited the cleavage of poly (ADP‐ribose) polymerase and activation of caspase‐3. On the other hand, delivery of active caspase‐3 protein to C2C12 myoblasts reversed the inhibitory effect of gadolinium ions on myogenesis. Our data suggest that inhibition of MSC suppresses myogenic differentiation by inhibiting the caspase‐3 activity and the expression of myogenic regulatory factors.—Wedhas, N., Klamut, H. J., Dogra, C., Srivastava, A. K., Mohan, S., Kumar, A. Inhibition of mechanosensitive cation channels inhibits myogenic differentiation by suppressing the expression of myogenic regulatory factors and caspase‐3 activity. FASEB J. 19, 1986–1997 (2005)

Protein–DNA array-based identification of transcription factor activities differentially regulated in skeletal muscle of normal and dystrophin-deficient mdx mice

Inactivation of dystrophin gene is the primary cause of Duchenne muscular dystrophy (DMD) in humans and mdx mice. However, the underpinning mechanisms, which govern the pathogenesis of dystrophin-deficient skeletal muscle, remain poorly understood. We have previously reported activation of mitogen-activated protein kinases (MAPK), nuclear factor-kappa B (NF-κB), and phosphatidyl-inositol 3-kinase/Akt (PI3K/Akt) signaling pathways in diaphragm muscle of mdx mice. In this study, using a protein–DNA array-based approach, we have investigated the activation of 345 transcription factors in diaphragm muscle of 6-week old normal and dystrophin-deficient mdx mice. Our data demonstrate increased activation of a number nuclear transcription factors including AP1, HFH-3, PPARα, c.myb BP, ETF, Fra-1/JUN, kBF-A, N-rasBP, lactoferrin BP, Myb(2), EBP40_45, EKLF(1), p53(2), TFEB, Myc-Max; c-Rel; E2, ISRE; NF-kB; Stat1 p84/p91, Antioxidant RE, EVI-1, Stat3, AP3, p53, Stat4, AP4, HFH-1, FAST-1, Pax-5, and Beta-RE in the diaphragm muscle of mdx mice compared to corresponding normal mice. The level of activation for p53 was highest among all the transcription factors studied. Furthermore, higher activation of p53 in diaphragm muscle of mdx mice was associated with its increased phosphorylation and nuclear translocation. Collectively, our data suggest that the primary deficiency of dystrophin leads to the aberrant activation of nuclear transcription factors which might further contribute to muscle pathogenesis in mdx mice.