The Journal of Steroid Biochemistry and Molecular Biology
Regulation of a promoter of the fibroblast growth factor 1 gene in prostate and breast cancer cells
Introduction
The human fibroblast growth factor 1 (FGF-1) is one member of a growing number of proteins that constitute the fibroblast growth factor family1, 2, 3, 4. FGF-1 plays a significant role in several biological processes including cell growth and development and malignant transformation. It is a mitogen for mesoderm- and neuroectoderm-derived cells[1]. When overexpressed in NIH/3T3 cells, FGF-1 causes transformation; when injected into nude mice, it induces tumor formation[5]. FGF-1 has been shown to be a potent angiogenic factor both in vivo and in vitro[6]and as such may play a role in tumor progression by promoting tumor vascularization[7]or in proliferative diabetic retinopathy[8]. FGF-1 was shown to be an inducer of mesoderm development in the Xenopus embryo[9]. Its expression in the smooth muscle cells of the vascular wall has been implicated in atherosclerosis[10].
FGF and FGF receptor (FGFR) gene expressions have been detected in normal, benign, and malignant tissues as well as tumor cell lines. Penaul-Llorca et al.[11]found FGF-1 gene expression in many breast tumor samples tested and concomitant high-level gene expression of FGFR1, 2 and 4 in many of these same samples. Smith et al.[12]found FGF-1 protein levels to be higher in malignant breast tissues compared with normal or benign tissues. By contrast, Bansal et al.[13]and Anandappa et al.[14]found that FGF-1 expression is lower in breast cancer tissues than in normal breast tissues. FGF-1 expression has been reported in various breast and prostate cancer cell lines. For example, FGF-1 mRNA expression has been found in the malignant human mammary epithelial cell lines MDA-MB-231, MCF-7 and BT-2015, 16and the prostate carcinoma cell lines PC-3[17]and LNCaP[18].
FGF-1 mRNA levels are up-regulated by a variety of mitogens including serum19, 20, 21, phorbol esters21, 22and combinations of growth factors[21]. Depending on the cell type, serum either stimulates FGF-1 mRNA expression as in vascular smooth muscle cells19, 21, or decreases its expression as in lens epithelial cells[23]. Steroid hormones appear to regulate the growth of hormone-dependent carcinoma cells indirectly through steroid-induced polypeptide growth factors24, 25. In LNCaP cells, for example, Harris et al.[18]found that the levels of FGF-1 mRNA increased following androgen stimulation.
The human FGF-1 gene spans more than 100 kb and contains three protein-coding exons and at least four 5′ untranslated exons. Multiple FGF-1 transcripts arise from alternative splicing17, 26, multiple promoter usage[27], and alternative transcriptional terminations[28]. Splicing of upstream exons −1A, −1B, −1C, or −1D to the first protein-coding exon results in FGF-1 transcripts 1.A, 1.B, 1.C, and 1.D that are expressed in a tissue- and cell-specific manner17, 19, 26. The biological significance of having alternative 5′ untranslated exons may include transcript stabilization[29]and compartmentalization[30], and translational regulation[31].
To better understand the potential role of FGF-1 in human prostate and breast cancer, we began an analysis of the cis- and trans-acting elements of one of its promoters required for the serum, PMA, and androgen regulation in breast and prostate cancer cell lines. We show that FGF-1.C mRNA expression is increased following serum or PMA treatment of PC-3 cells. Using luciferase reporter analysis, we show that the sequence from −1614 to the FGF-1.C start site is sufficient to stimulate promoter activity following FGF-1 plus androgen treatment of LNCaP cells or serum treatment of MDA-MB-231 cells.
Section snippets
Chemicals
The synthetic androgen, 17β-hydroxyl-17α-methyl-estra-4,9,11-trien-3-one (R1881), was obtained from Roussel-UCLAF, Romainvelle, France. Dihydrotestosterone (DHT), testosterone, and phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma (St. Louis, MO). Epidermal growth factor (EGF) and FGF-1 were purchased from Upstate Biotechnology (Lake Placid, NY).
Cell culture
PC-3 cells (ATCC, passage number 16) were grown in DMEM (Gibco-BRL) supplemented with nonessential amino acids, 10% fetal bovine serum
FGF-1.C mRNA is increased following serum or PMA treatment of PC-3 cells
Since FGF-1 mRNA levels were shown to be up-regulated by serum19, 20, 21and phorbol esters21, 22, we specifically wanted to know which FGF-1 prmoter(s) is activated under the same conditions. PC-3 cells were grown to 80% confluency and then serum starved for 72 h in medium containing 0.5% FBS. Serum-starved cells were then stimulated with either 10% FBS or PMA (100 ng/ml) for 6 h, the time point shown previously to result in the greatest FGF-1.C stimulation in saphenous vein smooth muscle cells[19]
Discussion
The induction of the FGF-1.C mRNA by serum in PC-3 cells and MDA-MB-231 cells as demonstrated in RNase protection analysis (Fig. 1) and transient transfection analysis (Fig. 4, Fig. 7) provide model systems to study the signal transduction pathways involved in the stimulation of the FGF-1.C promoter. In the 5′-flanking sequences of FGF-1.C are consensus or near consensus AP1, AP2, CRE, and Sp1 sites (Fig. 3). Using EMSA, we showed binding of protein from PC-3 nuclear extracts to AP1, AP2, and
Acknowledgements
We thank Yang Liu for her technical help. This work was supported by grants R01CA45611 and R01DK47506 from the National Institutes of Health. I.-M.C. was supported by a Research Career Development award (K04CA01369) and R.A.P. was supported by a National Research Service Award (T32CA09498) from the National Institutes of Health.
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2000, Journal of Biological ChemistryCitation Excerpt :The 1A reporter was constructed by placing the 1A promoter region from −826 to +77 upstream of the luciferase reporter gene (pGL2-Basic vector, Promega Corp, Madison, WI). The FGF1B and -1C luciferase constructs have previously been described (5, 8). The previously described FGF1Dminigene (10) was used as DNA template to polymerase chain reaction amplify the FGF1D promoter region −150 to +40, and including 9 bp of the exon 1 sequence.