Hepatic fibrogenesis, resulting, in part, from progressive deposition of type I Collagen, is responsible for the irreversible complications of alcoholic cirrhosis, a major cause of morbidity and mortality in the United States. The activated hepatic stellate cell is the principle collagen-producing cell in mammalian liver. In recent investigations, products of oxidative stress, including malondialdehyde (MDA) and 4-hydroxy-2- nonenal (HNE) can augment type I Collagen gene expression in activated stellate cells, however, the potential signal transduction cascade(s) by such lipid peroxidation (LP) products for collagen gene regulation in these cells is not fully understood. Thus, it would be beneficial to elucidate the signal transduction cascade(s) for potential molecular and pharmacologic targets to arrest liver fibrosis. Therefore, the HYPOTHESIS of this proposal is that MDA or HNE-representing a spectrum of aldehyde reactivity-activate Collagen gene expression through activation and phosphorylation by the mitogen activated protein kinase (MAPK), c-jun NH2-terminal kinase (JNK); and, this pathway employs downstream elements of the transforming growth factor beta one (TGFbeta1) transducing pathways-Smads 2/3 and Smad 4. Preliminary data confirm that MDA can augment alpha2(l) Collagen gene promoter activation and increase its transcription; further, the data also suggest JNK is responsible for this effect. These studies will be performed with an immortalized stellate cell line HSC-T6.
AIM #1 will demonstrate that MDA and HNE increase alphal Collagen gene expression and promoter activation by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and transient transfection analysis.
AIM 9 2 will determine which of the three MAPKs and their respective MAPK kinases (MAPPKS) mediate this effect by immunoprecipitation of phosphorylated MAPK substrates and immunoblot analysis of phosphorylated MAPKS. Finally, AIM 43 will determine whether there is signal cross talk in LP associated Collagen gene activation between the MARK pathways and the mammalian homologues of mothers against Drosophilia, or Smads 2/3 and Smad 4.
| Saxena, Neeraj K; Vertino, Paula M; Anania, Frank A et al. (2007) leptin-induced growth stimulation of breast cancer cells involves recruitment of histone acetyltransferases and mediator complex to CYCLIN D1 promoter via activation of Stat3. J Biol Chem 282:13316-25 |
