The liver performs a wider range of metabolic functions than most organs, yet also undergoes orderly compensatory growth and is, therefore, an approachable model to study broad questions in regulatory cell and molecular biology both in vivo and in vitro. The heparin-binding (fibroblast) growth factor (HBGF) family of polypeptide regulators and its receptors has been implicated in normal and hepatoma cell growth and function.
The specific aims are to (1) identify and structurally characterize a potentially unique HBGF-like activity from human hepatoma cells; (2) characterize expression of HBGF receptor subtypes in hepatoma cells and parenchymal and non-parenchymal cells of regenerating liver; (3) characterize role of EGF/TGF-alpha, TGF-beta and hepatocyte growth factor (HGF) on HBGF receptor subtypes; (4) test for a differential role of HBGF receptor subtypes in liver cell growth and function; and (5) utilize define polypeptide growth factors to select and identify specific liver cell subtypes in cell culture. The goals will be achieved by application of combined methods in molecular and cell biology including cell culture. The hepatoma cell-derived HBGF-like factor will be cloned and sequenced by using low stringency cDNA probes for known HBGF family members. The comparative temporal expression of HBGF receptor subtypes and effect of EGF/TGF-alpha, TGF-beta, and HGF in liver cells will be quantified by analysis of specific mRNA expression and immunoassay with receptor subtype- specific antibodies. Sense- and anti-sense constructions transfected into liver and non-liver host cells and immunochemical blocking experiments will be utilized to determine which HBGF receptor subtype contributes to high- and low-affinity HBGF binding and which contributes to growth, growth inhibition or expression of secretory function. Experiments will be performed to determine if specific receptor subtypes activate specific growth-related (c-fos, c-jun, c-myc) or function-related (alpha1 antitrypsin, alpha1 acidglycoprotein) gene expression. Lastly, specific media conditions will be used to select for and synchronize proliferative cell types from normal adult liver that are candidate """"""""facultative stem cells"""""""". These cell types will be characterized by specific growth factor gene expression and receptor subtype and accepted markers of candidate """"""""facultative stem cells"""""""". The combined results will constitute a global test of the autocrine/paracrine hypothesis of sequential and ordered liver cell regeneration and possibly reveal whether specific liver cell subtypes are involved. The results will pave the way for controlled reconstitution of the regenerative process for cellular and molecular studies.
Showing the most recent 10 out of 69 publications
