L6 myoblast differentiation is modulated by Cdk5 via the PI3K-AKT-p70S6K signaling pathway.

Cdk5 regulates myogenesis but the signaling cascade through which Cdk5 modulates this process remains to be characterized. Here, we investigated whether PI3K, Akt, p70S6K, p38 MAPK, p44/42 MAPK, and Egr-1 serve as upstream regulators of Cdk5 during L6 myoblast differentiation. Upon serum reduction, we found that besides elevated expression of Cdk5 and its activator, p35, and increased Cdk5/p35 activity, Egr-1, Akt, p70S6K, and p38 MAPK activity were upregulated in differentiating L6 cells. However, p44/42 MAPK was downregulated and SAPK/JNK was unaffected. LY294002, a PI3K inhibitor, blocked the activation of Akt and p70S6K, indicating that Akt and p70S6K activation is linked to PI3K activation. The lack of LY294002 effect on p38 MAPK suggests that p38 MAPK activation is not associated with PI3K activation. Rapamycin, a specific inhibitor of FRAP/mTOR (the upstream kinase of p70S6K), also blocked p70S6K activation, indicating the involvement of FRAP/mTOR activation. LY294002 and rapamycin also blocked the enhancement of Egr-1 level, Cdk5 activity, and myogenin expression, suggesting that upregulation of these factors is coupled to PI3K-p70S6K activation. Overexpression of dominant-negative-Akt also reduced Cdk5/p35 activity and myogenin expression, indicating that the PI3K-p70S6K-Egr-1-Cdk5 signaling cascade is linked to Akt activation. SB2023580, a p38 MAPK inhibitor, had no effect on p70S6K, Egr-1, or Cdk5 activity, suggesting that p38 MAPK activation lies in a pathway distinct from the PI3K-Akt-p70S6K-Egr-1 pathway that we identify as the upstream modulator of Cdk5 activity during L6 myoblast differentiation.

Identification of a novel function for the chromatin remodeling protein ING2 in muscle differentiation.

The inhibitor of growth (ING) family of zinc-finger plant homeodomain (PHD)-containing chromatin remodeling protein controls gene expression and has been implicated in the regulation of cell proliferation and death. However, the role of ING proteins in cell differentiation remains largely unexplored. Here, we identify an essential function for ING2 in muscle differentiation. We find that knockdown of ING2 by RNA interference (RNAi) blocks the differentiation of C2C12 cells into myotubes, suggesting that ING2 regulates the myogenic differentiation program. We also characterize a mechanism by which ING2 drives muscle differentiation. In structure-function analyses, we find that the leucine zipper motif of ING2 contributes to ING2-dependent muscle differentiation. By contrast, the PHD domain, which recognizes the histone H3K4me3 epigenetic mark, inhibits the ability of ING2 to induce muscle differentiation. We also find that the Sin3A-HDAC1 chromatin remodeling complex, which interacts with ING2, plays a critical role in ING2-dependent muscle differentiation. These findings define a novel function for ING2 in muscle differentiation and bear significant implications for our understanding of the role of the ING protein family in cell differentiation and tumor suppression.

ING2 as a novel mediator of transforming growth factor-beta-dependent responses in epithelial cells.

Members of the ING (inhibitor of growth) family of chromatin modifying proteins (ING1-ING5) have emerged as critical regulators of gene expression and cellular responses, suggesting that the ING proteins may impinge on specific signal transduction pathways and their mediated effects. Here, we demonstrate a role for the protein ING2 in mediating responses by the transforming growth factor (TGF)-beta-Smad signaling pathway. We show that ING2 promotes TGF-beta-induced transcription. Both gain-of-function and RNA interference-mediated knockdown of endogenous ING2 reveal that ING2 couples TGF-beta signals to the induction of transcription and cell cycle arrest. We also find that the Smad-interacting transcriptional modulator SnoN interacts with ING2 and promotes the assembly of a protein complex containing SnoN, ING2, and Smad2. Knockdown of endogenous SnoN blocks the ability of ING2 to promote TGF-beta-dependent transcription, and conversely expression of SnoN augments ING2 enhancement of the TGF-beta response. Collectively, our data suggest that ING2 collaborates with SnoN to mediate TGF-beta-induced Smad-dependent transcription and cellular responses.

Sumoylated SnoN represses transcription in a promoter-specific manner.

The transcriptional modulator SnoN controls a diverse set of biological processes, including cell proliferation and differentiation. The mechanisms by which SnoN regulates these processes remain incompletely understood. Recent studies have shown that SnoN exerts positive or negative regulatory effects on transcription. Because post-translational modification of proteins by small ubiquitin-like modifier (SUMO) represents an important mechanism in the control of the activity of transcriptional regulators, we asked if this modification regulates SnoN function. Here, we show that SnoN is sumoylated. Our data demonstrate that the SUMO-conjugating E2 enzyme Ubc9 is critical for SnoN sumoylation and that the SUMO E3 ligase PIAS1 selectively interacts with and enhances the sumoylation of SnoN. We identify lysine residues 50 and 383 as the SUMO acceptor sites in SnoN. Analyses of SUMO "loss-of-function" and "gain-of-function" SnoN mutants in transcriptional reporter assays reveal that sumoylation of SnoN contributes to the ability of SnoN to repress gene expression in a promoter-specific manner. Although this modification has little effect on SnoN repression of the plasminogen activator inhibitor-1 promoter and only modestly potentiates SnoN repression of the p21 promoter, SnoN sumoylation robustly augments the ability of SnoN to suppress transcription of the myogenesis master regulatory gene myogenin. In addition, we show that the SnoN SUMO E3 ligase, PIAS1, at its endogenous levels, suppresses myogenin transcription. Collectively, our findings suggest that SnoN is directly regulated by sumoylation leading to the enhancement of the ability of SnoN to repress transcription in a promoter-specific manner. Our study also points to a physiological role for SnoN sumoylation in the control of myogenin expression in differentiating muscle cells.

SnoN is a cell type-specific mediator of transforming growth factor-beta responses.

The transforming growth factor-beta (TGF-beta) family of secreted proteins have pleiotropic functions that are critical to normal development and homeostasis. However, the intracellular mechanisms by which the TGF-beta proteins elicit cellular responses remain incompletely understood. The Smad proteins provide a major means for the propagation of the TGF-beta signal from the cell surface to the nucleus, where the Smad proteins regulate gene expression leading to TGF-beta-dependent cellular responses including the inhibition of cell proliferation. Recent studies have suggested that a nuclear Smad-interacting protein termed SnoN, when overexpressed in cells, suppresses TGF-beta-induced Smad signaling and TGF-beta inhibition of cell proliferation. However, the physiologic function of endogenous SnoN in TGF-beta-mediated biological responses remained to be elucidated. Here, we determined the effect of genetic knock-down of SnoN by RNA interference on TGF-beta responses in mammalian cells. Unexpectedly, we found that SnoN knock-down specifically inhibited TGF-beta-induced transcription in the lung epithelial cell line Mv1Lu but not in HeLa or HaCaT cells. SnoN knock-down was also found to block TGF-beta-dependent cell cycle arrest in Mv1Lu cells. Collectively, these data indicate that rather than suppressing TGF-beta-induced responses, endogenous SnoN acts as a positive mediator of TGF-beta-induced transcription and cell cycle arrest in lung epithelial cells. Our study also shows that SnoN couples the TGF-beta signal to gene expression in a cell-specific manner.

Anti-atherogenic effects of an egg yolk-enriched garlic supplement.

It has been suggested that oxidation of low-density lipoprotein (LDL) plays an important role in the initiation and progression of atherosclerosis. In the present study, we determined the anti-atherogenic effects of egg yolk-enriched garlic powder (EGP), which has been used as a traditional health-promoting food in southern Japan since ancient times, on LDL oxidation and oxidant stress-induced cell injury models. We confirmed that EGP inhibits copper-induced LDL oxidation in a dose-dependent manner. We also observed that pretreatment of EGP significantly suppressed the production of peroxides in HL60 cells and protected endothelial cells from hydrogen peroxide-induced cell injury. These findings might, in part, be ascribed to the biodistribution of garlic compounds and egg yolk interaction, and suggest that EGP might be useful in the prevention of atherosclerosis.

Ebselen inhibits NO-induced apoptosis of differentiated PC12 cells via inhibition of ASK1-p38 MAPK-p53 and JNK signaling and activation of p44/42 MAPK and Bcl-2.

Ebselen, a selenium-containing heterocyclic compound, prevents ischemia-induced cell death. However, the molecular mechanism through which ebselen exerts its cytoprotective effect remains to be elucidated. Using sodium nitroprusside (SNP) as a nitric oxide (NO) donor, we show here that ebselen potently inhibits NO-induced apoptosis of differentiated PC12 cells. This was associated with inhibition of NO-induced phosphatidyl Serine exposure, cytochrome c release, and caspase-3 activation by ebselen. Analysis of key apoptotic regulators during NO-induced apoptosis of differentiated PC12 cells showed that ebselen blocks the activation of the apoptosis signaling-regulating kinase 1 (ASK1), and inhibits phosphorylation of p38 mitogen-activated protein kinase (MAPK) and c-jun N-terminal protein kinase (JNK). Moreover, ebselen inhibits NO-induced p53 phosphorylation at Ser15 and c-Jun phosphorylation at Ser63 and Ser73. It appears that inhibition of p38 MAPK and p53 phosphorylation by ebselen occurs via a thiol-redox-dependent mechanism. Interestingly, ebselen also activates p44/42 MAPK, and inhibits the downregulation of the antiapoptotic protein Bcl-2 in SNP-treated PC12 cells. Together, these findings suggest that ebselen protects neuronal cells from NO cytotoxicity by reciprocally regulating the apoptotic and antiapoptotic signaling cascades.

Outer dense fibers serve as a functional target for Cdk5.p35 in the developing sperm tail.

Cdk5 is ubiquitously expressed in all tissues, but its activators, p35 and p39, are principally found in brain, and Cdk5 activity has mostly been associated with brain development, particularly neuronal differentiation and migration. Here we show that the p35 transcript and protein are also present in the testis, and an active Cdk5.p35 complex exists in this tissue as well. Cdk5 and p35 are prominently observed in elongating spermatid tails, particularly over the tail outer dense fibers (ODF). The appearance of Cdk5.p35 proceeds from the proximal to the distal end of elongating spermatids, coinciding with the proximal to distal assembly of ODF along the length of the tail axoneme. Incidentally, increased Cdk5.p35 activity is observed in isolated elongating spermatids and at a time when elongating spermatids appear in the developing testis, suggesting a role for Cdk5.p35 in spermiogenesis. The presence of Cdk5 and p35 in ODF isolated from rat sperm tails implies a strong association among these proteins. In vitro ODF phosphorylation by Cdk5.p35 and decreased in vivo sperm tail ODF phosphorylation in p35-deficient mice indicate that Cdk5.p35 is an integral component of ODF and that ODF is a functional Cdk5.p35 target in the testis. Our results demonstrate for the first time that Cdk5.p35 may participate in the regulation of sperm tail development via a mechanism involving ODF phosphorylation. Apparently, as in brain development, Cdk5.p35 plays a part in testis development.