The Transcriptional Regulation of the Tissue Inhibitor of Metalloproteinases-1 (Timp-1) Gene in Hepatic Stellate Cells

This work has shown in vitro that the AP-1 site at the 5'end of the TIMP-1 minimal promoter is crucial for transcriptional activity in the HSC. Furthermore this study has established that the Jun D homodimer is the main positive regulator of AP-1 mediated TIMP-1 gene transcription. The other endogenous AP-1 proteins, Fos B and Fra 2, were shown to function as negative regulators through their formation of inhibitory heterodimers. In contrast, investigations of the IL-6 promoter indicated that it was differentially regulated from TIMP-1, although both promoters are up regulated in the activated HSC and dependent on Jun D for transcription. The IL-6 promoter activity however, was shown to be dependent on the binding of a Jun D containing dimer but not a homodimer and it was suggested that the positive transcriptional effects were mediated via another as yet undetermined site. Work in N1H3T3 fibroblasts confirmed the classical c-Jun/c-Fos regulation of TIMP-1 transcription in the fibroblast line, suggesting the Jun D mediated mechanism of TIMP-1 regulation may be HSC specific.Investigations in vivo also confirmed the results of the in vitro studies, indicating that HSC activation was accompanied by an increase in Jun D expression and a change in its molecular weight. The change in Jun D size may further regulate gene expression, as the short form of Jun D is believed to be inactive, possessing no transactivation domain.Through the use of a mouse model it was possible to demonstrate that Jun D-/- knockout mice developed significantly less fibrosis than the wild type controls following an 8week CCI4 liver injury. Using Taqman quantitative RT PCR it was possible to demonstrate a biological mechanism for the reduced fibrosis seen in the Jun D-/- knockouts, as their livers contained significantly less TIMP-1 and pro-collagen mRNA than the controls.Other investigations identified that both the SP-1 and LBP-1 sites in the TIMP-1 promoter were crucial for high level transcription. Protein and supershift analysis suggested that the SP-1 binding complexes differ between the HSC and fibroblasts, implicating the interactions of SP-1 & SP-4 and SP-1 & SP-3 respectively. A single strand binding complex was also identified, binding to the first LBP-1 site of the TIMP-1 promoter, however the content and function of this complex is at present unknown.