The metabolic regulator PGC-1α links hepatitis C virus infection to hepatic insulin resistance.

BACKGROUND & AIMS: Chronic hepatitis C virus (HCV) infection is strongly associated with insulin resistance and diabetes mellitus. Peroxisome proliferator-activated receptor-gamma co-activator 1α (PGC-1α) is a transcriptional co-activator involved in the initiation of gluconeogenesis in the liver. Increased hepatic expression of PGC-1α has been implicated in insulin resistance. We investigated whether modulation of PGC-1α levels following HCV infection underlies HCV-associated hepatic insulin resistance. METHODS: HCV genomes were expressed in hepatoma cells followed by analysis of PGC-1α and gluconeogenesis levels. RESULTS: PGC-1α was robustly induced in HCV infected cells. PGC-1α induction was accompanied by an elevated expression of the gluconeogenic gene glucose-6 phosphatase (G6Pase) and increased glucose production. The induction of gluconeogenesis is HCV dependent, since interferon treatment abolishes PGC-1α and G6Pase elevation and decreases glucose output. Moreover, PGC-1α knockdown resulted in a significant reduction of G6Pase levels in HCV full length replicon cells, emphasizing the central role of PGC-1α in the exaggerated gluconeogenic response observed in HCV patients. Treatment of HCV replicon cells with the antioxidant N-acetylcysteine resulted in reduction of PGC-1α levels, suggesting that HCV-induced oxidative stress promoted PGC-1α upregulation. Finally, both PGC-1α and G6Pase RNA levels were significantly elevated in liver samples of HCV infected patients, highlighting the clinical relevance of these results. CONCLUSIONS: PGC-1α is robustly induced following HCV infection, resulting in an upregulated gluconeogenic response, thereby linking HCV infection to hepatic insulin resistance. Our results suggest that PGC-1α is a potential molecular target for the treatment of HCV-associated insulin resistance.

Curcumin inhibits hepatitis B virus via down-regulation of the metabolic coactivator PGC-1alpha.

Hepatitis B virus (HBV) infects the liver and uses its cell host for gene expression and propagation. Therefore, targeting host factors essential for HBV gene expression is a potential anti-viral strategy. Here we show that treating HBV expressing cells with the natural phenolic compound curcumin inhibits HBV gene expression and replication. This inhibition is mediated via down-regulation of PGC-1alpha, a starvation-induced protein that initiates the gluconeogenesis cascade and that has been shown to robustly coactivate HBV transcription. We suggest curcumin as a host targeted therapy for HBV infection that may complement current virus-specific therapies.

Different domains regulate homomeric and heteromeric complex formation among type I and type II transforming growth factor-beta receptors.

Transforming growth factor-beta (TGF-beta) binds to and signals via two serine-threonine kinase receptors, type I (TbetaRI) and type II (TbetaRII). The oligomerization of TGF-beta receptors modulates ligand binding and receptor trafficking and may contribute to signal diversification. However, numerous features of the molecular domains that determine the homo- and hetero-oligomerization of full-length receptors at the cell surface and the mode of these interactions remain unclear. Here, we address these questions through computerized immunofluorescence co-patching and patch/fluorescence recovery after photobleaching measurements of different combinations of epitope-tagged receptors and their mutants in live cells. We show that TbetaRI and TbetaRII are present on the plasma membrane both as monomers and homo- and hetero-oligomers. The homodimerization of TbetaRII depends on a cytoplasmic juxtamembrane region (amino acid residues 200-220). In contrast, the cytoplasmic domain of TbetaRI is dispensable for its homodimerization. TbetaRI.TbetaRII hetero-oligomerization depends on the cytoplasmic domain of TbetaRI and on a C-terminal region of TbetaRII (residues 419-565). TGF-beta1 elevates TbetaRII homodimerization to some degree and strongly enhances TbetaRI.TbetaRII heteromeric complex formation. Both ligand-induced effects depend on the region encompassed between residues 200-242 of TbetaRII. Furthermore, the kinase activity of TbetaRI is also necessary for the latter effect. All forms of the homo- and hetero-oligomers, whether constitutively present on the membrane or formed upon TGF-beta1 stimulation, were stable in the time-scale of our patch/FRAP measurements. We suggest that the different forms of receptor oligomerization may serve as a basis for the heterogeneity of TGF-beta signaling responses.

Different routes of bone morphogenic protein (BMP) receptor endocytosis influence BMP signaling.

Endocytosis is important for a variety of functions in eukaryotic cells, including the regulation of signaling cascades via transmembrane receptors. The internalization of bone morphogenetic protein (BMP) receptor type I (BRI) and type II (BRII) and its relation to signaling were largely unexplored. Here, we demonstrate that both receptor types undergo constitutive endocytosis via clathrin-coated pits (CCPs) but that only BRII undergoes also caveola-like internalization. Using several complementary approaches, we could show that (i) BMP-2-mediated Smad1/5 phosphorylation occurs at the plasma membrane in nonraft regions, (ii) continuation of Smad signaling resulting in a transcriptional response requires endocytosis via the clathrin-mediated route, and (iii) BMP signaling leading to alkaline phosphatase induction initiates from receptors that fractionate into cholesterol-enriched, detergent-resistant membranes. Furthermore, we show that BRII interacts with Eps15R, a constitutive component of CCPs, and with caveolin-1, the marker protein of caveolae. Taken together, the localization of BMP receptors in distinct membrane domains is prerequisite to their taking different endocytosis routes with specific impacts on Smad-dependent and Smad-independent signaling cascades.

Monomeric and dimeric GDF-5 show equal type I receptor binding and oligomerization capability and have the same biological activity.

Growth and differentiation factor 5 (GDF-5) is a homodimeric protein stabilized by a single disulfide bridge between cysteine 465 in the respective monomers, as well as by three intramolecular cysteine bridges within each subunit. A mature recombinant human GDF-5 variant with cysteine 465 replaced by alanine (rhGDF-5 C465A) was expressed in E. coli, purified to homogeneity, and chemically renatured. Biochemical analysis showed that this procedure eliminated the sole interchain disulfide bond. Surprisingly, the monomeric variant of rhGDF-5 is as potent in vitro as the dimeric form. This could be confirmed by alkaline phosphatase assays and Smad reporter gene activation. Furthermore, dimeric and monomeric rhGDF-5 show comparable binding to their specific type I receptor, BRIb. Studies on living cells showed that both the dimeric and monomeric rhGDF-5 induce homomeric BRIb and heteromeric BRIb/BRII oligomers. Our results suggest that rhGDF-5 C465A has the same biological activity as rhGDF-5 with respect to binding to, oligomerization of and signaling through the BMP receptor type Ib.