Transforming Growth Factor β Acts as a Regulatory Molecule for Lipogenic Pathways among Hepatitis C Virus Genotype-Specific Infections

Abstract

Hepatitis C virus (HCV) infection promotes metabolic disorders, and the severity of lipogenic disease depends upon the infecting virus genotype. Here, we have examined HCV genotype 1-, 2-, or 3-specific regulation of lipid metabolism, involving transforming growth factor β (TGF-β)-regulated phospho-Akt (p-Akt) and peroxisome proliferator-activated receptor alpha (PPARα) axes. Since HCV core protein is one of the key players in metabolic regulation, we also examined its contribution in lipid metabolic pathways. The expression of regulatory molecules, TGF-β1/2, phospho-Akt (Ser473), PPARα, sterol regulatory element-binding protein 1 (SREBP-1), fatty acid synthase (FASN), hormone-sensitive lipase (HSL), and acyl dehydrogenases was analyzed in virus-infected hepatocytes. Interestingly, HCV genotype 3a exhibited much higher activation of TGF-β and p-Akt, with a concurrent decrease in PPARα expression and fatty acid oxidation. A significant and similar decrease in HSL, unlike in HCV genotype 1a, was observed with both genotypes 2a and 3a. Similar observations were made from ectopic expression of the core genomic region from each genotype. The key role of TGF-β was further verified using specific small interfering RNA (siRNA). Together, our results highlight a significant difference in TGF-β-induced activity for the HCV genotype 2a- or 3a-induced lipogenic pathway, exhibiting higher triglyceride synthesis and a decreased lipolytic mechanism. These results may help in therapeutic modalities for early treatment of HCV genotype-associated lipid metabolic disorders.IMPORTANCE Hepatic steatosis is a frequent complication associated with chronic hepatitis C virus (HCV) infection and is a key prognostic indicator for progression to fibrosis and cirrhosis. Several mechanisms are proposed for the development of steatosis, especially with HCV genotype 3a. Our observations suggest that transforming growth factor β (TGF-β) and peroxisome proliferator-activated receptor alpha (PPARα)-associated mechanistic pathways in hepatocytes infected with HCV genotype 2a and 3a differ from those in cells infected with genotype 1a. The results suggest that a targeted therapeutic approach for enhanced PPARα and lipolysis may reduce HCV genotype-associated lipid metabolic disorder in liver disease.

Keywords: TGF-beta; core protein; hepatitis C virus; metabolic regulation.

FIG 1

HCV genotypes differentially activate TGF-β and p-Akt and express PPARα expression in infected cells. (A to D) Cell culture-grown HCV genotype 1a-, 2a-, or 3a-infected immortalized human hepatocytes (IHH) were analyzed for expression of TGF-β1, TGF-β2, phosphorylated Akt (S473), and PPARα by Western blotting and normalized with the level of actin expression in each lane. Results from densitometric scanning of the blot are shown on the right. Statistical significance was examined using the two-tailed Student’s t test. *, P < 0.05; **, P < 0.01.

FIG 2

HCV genotypes 2a and 3a enhance SREBP-1 and FASN expression. (A) Cell culture-grown HCV genotype 1a-, 2a-, or 3a-infected cell lysates were analyzed for expression of SREBP-1 by Western blotting and normalized with actin. (B) Subcellular localization of SREBP-1 (green) and HCV NS5A protein (red) in HCV genotype 3a-infected IHH were analyzed by confocal microscopy. Mock-infected IHH were used as control for comparison. Cells were fixed, permeabilized for staining with DAPI or respective antibodies, and are shown at a magnification of ×60. (C) HCV genotype 1a-, 2a-, or 3a-infected cell lysates were analyzed for expression of FASN by Western blotting and normalized with actin. Densitometric scanning results of the protein bands are shown on the right. Statistical significance was examined using the two-tailed Student’s t test. *, P < 0.05; **, P < 0.01.

FIG 3

HCV genotype 1a infection enhances lipolytic enzymes expression. IHH were infected with HCV genotype 1a, 2a, or 3a. (A to C) Infected cells were analyzed for expression of MCAD, SCAD, and phosphorylated HSL (S563) by Western blot and normalized with actin level. Densitometric scanning results of the protein bands are shown on the right of each panel. Statistical significance was analyzed using the two-tailed Student’s t test. *P < 0.05, **P < 0.01.

FIG 4

Differential expression of TGF-β and p-Akt regulation by core protein from HCV genotypes. (A) TGF-β2 expression was examined by Western blotting of HepG2 cells transfected with HCV core from genotypes 1a, 2a, and 3a and compared with mock-transfected control. (B) The role of core protein from genotype 1a and 3a upon TGF-β2 expression was separately verified using specific siRNA in transfected cells. Expression level of actin in each lane is shown as a loading control for comparison with expression of other proteins. (C and D) Densitometric scanning results of the protein bands are shown at the bottom of each panel. Phosphorylated Akt (S473) level was analyzed similarly. (E) Effect of exogenous TGF-β on Akt activation (S473) in naive IHH and HepG2 cells was also analyzed. Statistical significance using the two-tailed Student’s t test is shown. *, P < 0.05; **, P < 0.01.

FIG 5

SREBP-1 and FASN are elevated in HepG2 cells transfected with HCV genotype 2a and 3a core proteins. (A) SREBP-1 expression was examined in HepG2 cells transfected with HCV core from genotypes 1a, 2a, and 3a and compared with mock-transfected control by Western blotting. Expression level of actin is shown as a loading control for comparison of SREBP-1 protein expression. Densitometric scanning results of the protein bands are shown at the bottom. (B) FASN level in core transfected cell lysates were separately analyzed by ELISA. Statistical significance using the two-tailed Student's t test is shown. *, P < 0.05; **, P < 0.01.

FIG 6

Differential expression of lipolytic pathway-related key molecules by HCV core protein from distinct genotypes. (A) PPARα expression was examined by Western blot in HCV core from genotype 1a-, 2a-, and 3a-transfected HepG2 cells and compared with mock-transfected controls. Expression level of actin in each lane is shown as a loading control for comparison with expression of other proteins. Densitometric scanning results of the protein bands are shown at the bottom. (B) The role of core protein from genotype 1a and 3a on TGF-β expression and its consequence on PPARα were separately verified using specific siRNAs in transfected cells. (C) Effect of exogenous TGF-β on PPARα expression in naive IHH and HepG2 cells was also analyzed. (D) The roles of HCV core protein from genotypes 1a, 2a, and 3a on phosphorylation and activation of HSL (S563) in transfected HepG2 cells were compared with that in mock-transfected controls by Western blotting. (E) HCV core transfected cells were analyzed for total triglyceride level by ELISA and compared with mock-transfected control cells. Statistical significance was analyzed using the two-tailed Student's t test. *, P < 0.05; **, P < 0.01.

FIG 7

Schematic diagram showing mechanism of HCV-associated lipid metabolic regulation. HCV genotype 1a significantly differs from genotypes 2a and 3a in the regulation of TGF-β and PPARα axes for dampening lipid catabolism.