c PGC-1␣ is a transcriptional coactivator promoting oxidative metabolism in many tissues. Its expression in skeletal muscle (SKM) is induced by hypoxia and reactive oxidative species (ROS) generated during exercise, suggesting that PGC-1␣ might mediate the cross talk between oxidative metabolism and cellular responses to hypoxia and ROS. Here we found that PGC-1␣ directly interacted with Bhlhe40, a basic helix-loop-helix (bHLH) transcriptional repressor induced by hypoxia, and protects SKM from ROS damage, and they cooccupied PGC-1␣-targeted gene promoters/enhancers, which in turn repressed PGC-1␣ transactivational activity. Bhlhe40 repressed PGC-1␣ activity through recruiting histone deacetylases (HDACs) and preventing the relief of PGC-1␣ intramolecular repression caused by its own intrinsic suppressor domain. Knockdown of Bhlhe40 mRNA increased levels of ROS, fatty acid oxidation, mitochondrial DNA, and expression of PGC-1␣ target genes. Similar effects were also observed when the Bhlhe40-mediated repression was rescued by a dominantly active form of the PGC-1␣-interacting domain (PID) from Bhlhe40. We further found that Bhlhe40-mediated repression can be largely relieved by exercise, in which its recruitment to PGC-1␣-targeted cis elements was significantly reduced. These observations suggest that Bhlhe40 is a novel regulator of PGC-1␣ activity repressing oxidative metabolism gene expression and mitochondrion biogenesis in sedentary SKM. Skeletal muscle (SKM) is one of the major metabolic organs, and it has the ability to adapt to various physiological conditions, such as cold, exercise, and sedentary life, by adjusting the balance between glycolytic and oxidative metabolism to meet the energy requirement under these conditions (1). Recent studies suggest that this adaptation relies much on the expression and activity of PGC-1␣, a transcriptional coactivator highly expressed in tissues with high energy metabolism (1, 2). PGC-1␣ enhances the activity of many nuclear receptors (NR), including peroxisome proliferator-activated receptor ␥ (PPAR␥), estrogen receptor (ER), and glucocorticoid receptor (GR), and non-NR transcription factors, such as MEF2, Sox9, FoxO1, and SREBP1 (reviewed in reference 3 and references therein), to promote oxidative metabolism, metabolic thermogenesis adaptation, biogenesis of mitochondria, gluconeogenesis, and fatty acid oxidation in various tissues (4-6). Overexpression of PGC-1␣ can promote metabolic switch from anaerobic glycolysis to oxidative metabolism (7,8). Current studies also show that PGC-1␣ plays critical roles in orchestrating the cellular response to hypoxia (9).In SKM, PGC-1␣ is preferentially expressed in oxidative metabolism-dependent slow-twitch fibers (10), and its overexpression can convert putative fast-twitch fibers into slow-twitch fibers (10). The activation of oxidative and slow-twitch muscle-specific genes by PGC-1␣ is mediated through its coactivation of Mef2 and PPAR binding to the upstream regulatory sites of these target genes (11). PGC-1␣ null mice sho...

A series of novel tylophorine-derived dibenzoquinolines has been synthesized and their biological activity evaluated. Three assays were conducted: inhibition of cancer cell proliferation, inhibition of TGEV replication for anticoronavirus activity, and suppression of nitric oxide production in RAW264.7 cells (a measure of anti-inflammation). The most potent compound from these assays, dibenzoquinoline 33b, showed improved solubility compared to tylophorine 9a, in vivo efficacies in a lung A549 xenografted tumor mouse model and a murine paw edema model, good bioavailability, and no significant neurotoxicity (as tested by a rota-rod test for motor coordination). This is the first study to explore in detail the role of the tylophorine E ring on biological activity and very strongly suggests that tylophorine-derived dibenzoquinolines merit further development into orally active agents.

Previously, we found that MRFs (myogenic regulatory factors) regulated the expression of PGC-1alpha (peroxisome-proliferator-activated receptor gamma co-activator 1alpha) by targeting a short region, from nt -49 to +2 adjacent to the transcription initiation site, that contained two E-boxes. However, only the E2-box had significant affinity for MRFs, and the E1-box was predicted to be the target of Bhlhe40 (basic helix-loop-helix family, member e40, also known as Stra13, Bhlhb2, DEC1 and Sharp2), a transcriptional repressor implicated in the regulation of several physiological processes. In the present study, by using EMSA (electrophoresis mobility-shift assay), we confirmed that Bhlhe40 targeted the E1-box and formed a complex with the basic helix-loop-helix transcription factor MyoD (myogenic differentiation factor D) on the PGC-1alpha core promoter. We demonstrate that Bhlhe40 binds to the promoters of PGC-1alpha and myogenic genes in vivo and that Bhlhe40 represses the MyoD-mediated transactivation of these promoters. Furthermore, we found that this repression could be relieved by P/CAF (p300/CBP-associated factor) in a dose-dependent manner, but not by CBP [CREB (cAMP-response-element-binding protein)-binding protein]. Bhlhe40 interacted with P/CAF and this interaction disrupted the interaction between P/CAF and MyoD. These results suggest that Bhlhe40 functions as a repressor of MyoD by binding to adjacent E-boxes and sequestering P/CAF from MyoD.

Cardenolides are plant-derived toxic substances. Their cytotoxicity and the underlying mechanistic signaling axes have been extensively documented, but only a few anti-viral activities of cardenolides and the associated signaling pathways have been reported. Previously, we reported that a variety of cardenolides impart anti-transmissible gastroenteritis coronavirus (TGEV) activity in swine testicular (ST) cells, through targeting of the cell membrane sodium/potassium pump, Na/K-ATPase. Herein, we further explore the potential signaling cascades associated with this anti-TGEV activity in ST cells. Ouabain, a representative cardenolide, was found to potently diminish TGEV titers and inhibit the TGEV-induced production of IL-6 in a dose dependent manner, with 50% inhibitory concentrations of 37 nM and 23 nM respectively. By pharmacological inhibition and gene silencing, we demonstrated that PI3K_PDK1_RSK2 signaling was induced in TGEV-infected ST cells, and ouabain imparted a degree of anti-TGEV activity via further augmentation of this existing PI3K_PDK1 axis signaling, in a manner dependent upon its association with the Na/K-ATPase. Finally, inhibition of PI3K by LY294002 or PDK1 by BX795 antagonized the anti-viral activity of ouabain and restored the TGEV virus titer and yields. This finding is the first report of a PI3K_PDK1 signaling axis further induced by ouabain and implicated in the suppression of TGEV activity and replication; greatly illuminates the underlying mechanism of cardenolide toxicity; and is expected to result in one or more anti-viral applications for the cardenolides in the future.

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