Regulation of mitogen regulated protein/proliferin gene expression in 3T3 cells by basic fibroblast growth factor

Mohideen, Manzoor
Major Professor
Marit Nilsen-Hamiliton
Committee Member
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Biochemistry, Biophysics and Molecular Biology

Mitogen regulated protein/proliferin (MRP/PLF) is a murine uterine growth factor belonging to the prolactin/growth hormone gene superfamily. Basic fibroblast growth factor (bFGF) regulates its expression in 3T3 cells and is speculated to be among its regulators in vivo. In order to gain insights into its regulation, experiments were performed to elucidate the mechanisms by which bFGF regulates mrp/plf gene expression in 3T3 cells. RT-PCR analyses demonstrated that of the three different forms of MRP/PLF proteins only one, PLF1, is expressed in these cells in response to bFGF. PLF1 could be the product of either plf42 or plf149 genes. bFGF stimulation results in an 8-fold increase in PLF1 mRNA. This increase is not due to transcriptional activation of the plf42 or plf149 genes as evidenced by nuclear run-on in vitro transcription assays as well as by stable transfection analyses of promoters attached to the reporter gene, chloramphenicol acetyl transferase. Northern analyses following treatment of cells with cordycepin, an inhibitor of transcription, and pulse chase analyses of mRNA that was metabolically radiolabeled with 32P-PO4, indicated that PLF1 expression is not regulated at turnover as well. However, radiolabeled pulse chase assays of nuclear RNA demonstrated that bFGF specifically enhances the rate of processing of PLF1 heterogenous nuclear RNA (hnRNA), which accounts for the increase in the PLF1 mRNA in the cytoplasm;One of the three mrp/plf genes, mrp/plf3, was transcriptionally activated by bFGF in stable transfection assays. By performing systematic deletion analyses of the three promoters, we have identified a 10 bp long bFGF response element (FRE) in the mrp/plf3 promoter. DNA gel mobility shift assays revealed that the FRE binds nuclear factors specifically from bFGF-stimulated cells. The corresponding sequence from plf42 and plf149 gene promoters do not exhibit such binding. Nuclear factors recognized by the FRE do not include the AP1 complex. The FRE is active in living cells as evidenced by in vivo competition analyses. Nuclear factors from cells stimulated by other mitogens such as epidermal growth factor, platelet-derived growth factor-aa and phorbol myristate acetate are also recognized by the FRE suggesting that this element may be a general mitogen response element.