Levels Are Negatively Regulated by Serum Growth Factors 1

p202 is an IFN-inducible phosphoprotein (Mr 52,000) whose expression in transfected cells retards proliferation. Interestingly, the reduced levels of p202 in fibroblasts (in consequence of the expression of antisense to 202 RNA), under reduced serum conditions, increase the susceptibility of cells to apoptosis. To identify the functional role of p202 in cell growth regulation, we tested whether serum growth factor levels in the culture medium affect p202 levels. Here we report that, under reduced serum conditions, the p202 levels were increased in fibroblasts, and the increase was seen at both the mRNA and protein levels. Moreover, an increase in p202 levels was correlated with cell growth arrest in the G1 phase of the cell cycle. Interestingly, the presence of plateletderived growth factor AB, basic fibroblast growth factor, or transforming growth factor b1 in the culture medium abrogated the increase in p202 levels seen under reduced serum conditions. We found that the increase in p202 levels was accompanied by an increase in JunD/activation protein 1 (AP-1) levels, and transfection of a JunD-encoding plasmid along with a reporter plasmid in which transcription of the reporter gene (luciferase) was driven by the 5*-regulatory region of the 202 gene resulted in an increase in the activity of luciferase. Additionally, stable overexpression of JunD in cells, under reduced serum conditions, also resulted in an increase in p202 levels. Interestingly, one of the AP-1-like DNA-binding sequences present in the 5*-regulatory region of the 202 gene could selectively bind to the JunD/AP-1 transcription factor. Taken together, our observations reported herein suggest that in fibroblasts, under reduced serum conditions, the increased levels of JunD/AP-1 contribute to the transcriptional up-regulation of p202 levels, which may be important for the regulation of apoptosis. Introduction p202 is an IFN-inducible phosphoprotein (Mr 52,000) whose ectopic expression in fibroblasts retards proliferation (1–7). p202 has the ability to bind retinoblastoma protein (pRb) and other members of the “pocket” protein family (4, 8). Furthermore, p202 modulates the transcriptional activity of several factors including p53 (9), AP-1 (c-Fos and c-Jun; Ref. 6), and E2F (E2F-1/DP-1 and E2F-4/DP-1) (5, 8). Whereas the ability of p202 to inhibit the transcriptional activity of these factors provides a potential mechanism to regulate cell proliferation and apoptosis, it remains unclear how p202 negatively regulates cell proliferation. Additionally, it remains to be seen whether growth-regulatory molecules other than IFNs regulate the expression of p202. The Jun family transcription-regulatory proteins are components of the AP-1 transcription factor, which includes three members: (a) c-Jun; (b) JunB; and (c) JunD (10, 11). These proteins share a high degree of homology at the primary sequence level, and the genes encoding them undergo similar but not identical regulation. Both c-jun and junB are considered “classical” immediate early response genes based on their rapid, large, and transient increase in transcription after stimulation of cells with mitogenic agents. By contrast, the junD gene is relatively refractory to such growth-stimulatory signals (12). Furthermore, JunD gene is constitutively expressed in quiescent fibroblasts (13, 14). The JunD protein has been implicated as a negative regulator of cell proliferation, and serum growth factors are shown to modulate JunD activity in a variety of apparently independent ways to overcome its negative regulatory effect in controlling cell proliferation (12, 14, 15). These include the induction of a transient increase in JunD phosphorylation, repression of JunD/AP-1 DNA binding activity, and downregulation of JunD expression (11, 12, 16). When NIH 3T3 fibroblasts become quiescent, the levels of c-Jun decrease, whereas the levels of JunD increase (12). Furthermore, overexpression of JunD results in slower growth and an increase in the percentage of cells in G0-G1 (12, 17). Together, these observations suggest that an important function of JunD is to negatively regulate the proliferation of fibroblasts. Although JunD has been reported to activate transcription of several genes (18), it is not known how JunD retards cell proliferation. Our previous studies have revealed that a decrease in p202 levels in murine fibroblasts, under reduced serum conditions, increases susceptibility to apoptosis (19). Therefore, to elucidate the functional role of p202 in the regulation of cell Received 2/28/00; revised 6/15/00; accepted 7/25/00. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by NIH Grant CA69031. 2 To whom requests for reprints should be addressed. Present address: Department of Radiation Oncology, Loyola University Medical CenterHines, Fifth Avenue and Roosevelt Road, Building 1, Room F-317, Hines, IL 60141. Phone: (708) 202-8387. 3 The abbreviations used are: AP-1, activation protein 1; PDGF, plateletderived growth factor; bFGF, basic fibroblast growth factor; TGF, transforming growth factor; FBS, fetal bovine serum. 475 Vol. 11, 475–483, September 2000 Cell Growth & Differentiation