No abstract

Hepatic stellate cells (HSCs) are responsible for type I collagen deposition in liver fibrosis that leads to cirrhosis. The purpose of this study was to examine potential molecular signals that lead to increased alpha(2)(I) collagen gene expression by acetaldehyde, the primary metabolite of alcohol and malondialdehyde (MDA), a lipid peroxidation product known to be associated with chronic liver injury. MDA and the combination of MDA and acetaldehyde were employed to determine the effect on alpha(2)(I) collagen gene expression as assessed by transient transfection analysis and reverse transcriptase polymerase chain reaction (RT-PCR). Immunoblot and subsequent immunoprecipitation analysis examined stress-activated protein kinase (SAPK) activity. Cotransfection with a dominant negative mutant for c-jun nuclear kinase (dnJNK1) was also employed with the alpha(2)(I) collagen promoter. MDA increased alpha(2)(I) collagen gene expression nearly 2.5- to 3-fold, however there was no synergistic effect of the combination of acetaldehyde and MDA on alpha(2)(I) collagen gene activation and expression. Acetaldehyde, MDA, or both significantly increased JNK activity when compared to untreated stellate cells. The dnJNK1 expression vector abrogated alpha(2)(I) collagen transgene activity. In conclusion, JNK activation appears to be critical in the signaling cascade of oxidative metabolites of chronic alcohol-related liver injury and collagen gene activation.

Protein kinase C (PKC) inhibitors decrease alpha1(I) collagen mRNA in stellate cells exposed to 200 micromol/liter of acetaldehyde. The purpose of these studies was to determine whether PKC activation plays a role in transcriptional activation of the alpha2(I) collagen gene. Cultured stellate cells were exposed to 200 micromol/liter of acetaldehyde. PKC, inositol triphosphate, diacylglycerol (DAG), and intracellular free calcium (Ca2+i) were measured. Alpha1(I) and alpha2(I) collagen messages were determined by reverse transcriptase-polymerase chain reaction. Activation of the alpha2(I) collagen promoter was determined in transiently transfected stellate cells. Acetaldehyde exposure enhanced PKC activity translocation to the particulate fraction at 20 min. Acetaldehyde did not increase Ca2+i, or inositol triphosphate but increased DAG levels at 20 min and 3 hr. Acetaldehyde increased both the alpha1(I) and alpha2(I) collagen messages in stellate cells. Calphostin C, a specific PKC inhibitor, which blocks DAG binding, eliminated both activation of the alpha2(I) collagen promoter by acetaldehyde and mRNA production by reverse transcriptase-polymerase chain reaction analysis. Similarly, D609, an inhibitor of DAG production, also inhibited alpha2(I) collagen gene expression. This study shows that collagen production by acetaldehyde is mediated by a calcium-independent PKC mechanism.

Protein kinase C (PKC) inhibitors decrease alpha1(I) collagen mRNA in stellate cells exposed to 200 micromol/liter of acetaldehyde. The purpose of these studies was to determine whether PKC activation plays a role in transcriptional activation of the alpha2(I) collagen gene. Cultured stellate cells were exposed to 200 micromol/liter of acetaldehyde. PKC, inositol triphosphate, diacylglycerol (DAG), and intracellular free calcium (Ca2+i) were measured. Alpha1(I) and alpha2(I) collagen messages were determined by reverse transcriptase-polymerase chain reaction. Activation of the alpha2(I) collagen promoter was determined in transiently transfected stellate cells. Acetaldehyde exposure enhanced PKC activity translocation to the particulate fraction at 20 min. Acetaldehyde did not increase Ca2+i, or inositol triphosphate but increased DAG levels at 20 min and 3 hr. Acetaldehyde increased both the alpha1(I) and alpha2(I) collagen messages in stellate cells. Calphostin C, a specific PKC inhibitor, which blocks DAG binding, eliminated both activation of the alpha2(I) collagen promoter by acetaldehyde and mRNA production by reverse transcriptase-polymerase chain reaction analysis. Similarly, D609, an inhibitor of DAG production, also inhibited alpha2(I) collagen gene expression. This study shows that collagen production by acetaldehyde is mediated by a calcium-independent PKC mechanism.