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-alpha. TWEAK increased the ubiquitination of MyHCf and the transcript levels of atrogin-1 and MuRF1 ubiquitin ligases. TWEAK inhibited phosphorylation of Akt kinase and its downstream targets GSK-3beta, FOXO1, mTOR, and p70S6K. Furthermore, TWEAK increased the activation of NF-kappaB transcription factor in myotubes. Adenoviral-mediated overexpression of IkappaB alpha deltaN (a degradation-resistant mutant of NF-kappaB inhibitory protein IkappaB alpha) 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-kappaB. 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-kappaB systems.

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 ␣-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.

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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 alpha-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.

ObjectiveCerebral arteries in the fetus differ functionally from adult. We tested the hypothesis that developmental differences are a consequence of differential activities of α1‐adrenergic receptor (α1‐AR) subtypes.MethodsIn middle cerebral arteries from fetal and adult sheep, we measured responses of Ins(1,4,5)P3, vascular tension, and [Ca2+]i to the α1‐AR agonist phenylephrine (PHE, 10−5 M), in presence of blockers.ResultsIn fetal cerebral arteries, expression levels of each α1‐AR subtype was only ~20% that of adult. In fetus, each subtype specific blocker, 5‐MU (α1A), CEC (α1B) and BMY‐7378 (α1D) inhibited to a degree greater degree PHE‐induced tension and [Ca2+]i, compared to adult. Although in arteries of both age groups, 5‐MU and CEC reduced Ins(1,4,5)P3 responses to PHE ~35%, BMY‐7378 showed no significant decrease in Ins(1,4,5)P3 responses. Also in fetal arteries, the PHE‐induced increase in activated ERKs was blocked by CEC and BMY‐7378, but not by 5‐MU or WB‐4101.ConclusionsIn both age groups α1A‐ and α1B‐AR appear to mediate α1‐adrenergic‐Ins(1,4,5)P3 responses. In the fetus in contrast, α1D‐AR appear to activate the MAPK/ERK1/2 cascade by non‐Ins(1,4,5)P3‐dependent mechanisms. In the immature organism, relative deficiency of specific α1‐AR subtypes, and their differing signaling pathways, may be critical factors associated with dysregulation of cerebral blood flow. (USPHS HD 03708)

ObjectiveHigh altitude, long‐term hypoxia (LTH) is associated with a number of changes in pre‐ and post‐junctional signaling in both fetal and adult cerebrovasculature. Because PKC and downstream pathways play a key role in Ca2+ sensitization of vascular contraction, and are less abundant in fetus, we tested the hypothesis that LTH alters PKC activation pathways differently in the fetus, as compared to the adult.MethodsIn cerebral arteries we quantified both total and activated (phosphorylated) PKC, ERK1/2, caldesmon, Rho A, CPI‐17, and myosin light chain20 (MLC20), and PDBu‐ (PKC agonist) induced contractions and their responses to blockade of ERKs (U‐0126) or Rho kinase (Y‐27632).ResultsIn adult cerebral arteries, LTH acclimatization was associated with significant decreases in total and activated PKC, with increases in activated ERK1/2, CPI‐17, and MLC20 and its kinase. In contrast, in fetal arteries LTH increased PKC expression and increased ERK1/2 activation, with no change in caldesmon, Rho A, CPI‐17, or MLC20.ConclusionsIn adult cerebral arteries, acclimatization to LTH is associated with significant up‐ or down‐regulation of a number of elements of the PKC‐dependent Ca2+ sensitization pathway. In contrast, acclimatization to LTH in the fetus is associated with relatively minor changes in the Ca2+ sensitization pathway. (Supported by USPHS HD 31226)

ObjectiveWe tested the hypotheses that developmental maturation is associated with significant alterations in PKC mechanisms in Ca2+‐independent vascular contraction. In fetal and adult ovine cerebral arteries, in response to PKC stimulation, we quantified both total and phosphorylated levels of several proteins and tension and intracellular [Ca2+]i.ResultsIn both adult and fetus, PDBu‐induced contractions were 65% and 25% of Kmax, respectively, and ERK1/2 inhibition by U‐0126 decreased these contractions 40%. The ROCK inhibitor Y‐27632, decreased the response 50% in fetus but not adult. In adult cerebral arteries, PDBu increased phosphorylation of ERK1/2, ERK1/2‐dependent CaDSer789, CPI‐17Thr38, but not Rho A. In contrast, in fetal cerebral arteries activated Rho A, but not CaDSer789 or CPI‐17Thr38. By co‐immunoprecipatation, in adult, but not fetal, cerebral arteries PDBu‐simulated interaction between PKCα andCPI‐17, and PKCα and caldesmon.ConclusionsIn cerebral arteries, maturation with increasing myofilament Ca2+ sensitivity by MLCP regulation is associated with a transition from a PKC‐ERK1/2, Rho A‐dependent pathway in the fetus to one of CaDSer789‐ and CPI‐17Thr38‐dependent phosphorylation and increased myofilament Ca2+ sensitivity in the adult. In the immature organism, these differing signaling pathways may be associated with dysregulation of cerebral blood flow. (USPHS HD 03708)