Characterization of Insulin-Like Growth Factor-Binding Protein-Related Protein-1 in Prostate Cells

0021-972X/98/$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1998 by The Endocrine Society Vol. 83, No. 12 Printed in U.S.A. Characterization of Insulin-Like Growth Factor-Binding Protein-Related Protein-1 in Prostate Cells* VIVIAN HWA, CINDY TOMASINI-SPRENGER, ABEL LÓPEZ BERMEJO, RON G. ROSENFELD, AND STEVEN R. PLYMATE Department of Pediatrics, Oregon Health Sciences University (V.H., A.L.B., R.G.R.), Portland, Oregon 97201; and the Geriatric Research, Education, and Clinical Center, Veterans Affairs Health Care System Puget Sound (C.T.-S., S.R.P.), Tacoma, Washington 98493 ABSTRACT Insulin-like growth factor-binding protein-related protein-1 (IGFBP-rP1; also known as Mac25, TAF, and PSF) is a member of the IGFBP superfamily. It is a cysteine-rich protein that shares structural and functional similarities with the conventional IGFBPs. In situ hybridization of prostate tissue sections show intense IGFBP-rP1 messenger ribonucleic acid (mRNA) expression in normal stroma and glandular epithelium. There was a significant loss of detectable IGFBP-rP1 mRNA in metastatic prostate tissue. IGFBP-rP1 mRNA (Northern blots) and protein (immunoblots) were detectable in primary cultures of prostatic stromal and epithelial cells as well as in the T HE PROLIFERATION of human prostatic cells is controlled by the complex regulation of many hormones and growth factors, including insulin-like growth factors (IGFs) (1). IGFs, of which there are two (IGF-I and IGF-II), are peptides that are structurally related to insulin. Both IGF peptides exert growth-stimulating effects on prostate cells (1– 4), mainly through interactions with the type I IGF receptors (IGF-IR) found on cell surfaces. Perturbation of IGF levels and IGF availability have been hypothesized to contribute to malignant prostatic cell growth, a hypothesis supported by recent clinical studies. In one investigation, men with increased serum levels of IGF-I were found to have a 4-fold higher probability of contracting prostate cancer (5). In another study, young males with acromegaly, a disease characterized by abnormally high levels of circulating IGFs, were found to have enlarged prostates (6). In biological fluids, IGFs are normally sequestered by IGFbinding proteins (IGFBPs), of which there are six, designated IGFBP-1 to -6 (7–10). As IGFs have higher binding affinities for IGFBPs than for the IGF-IR, IGFBPs are important in the modulation of IGF biological activity. The effect can be an inhibition (11, 12) or an enhancement of IGF-IGF-IR interactions (13). In addition to these IGF-dependent actions of IGFBPs, IGFBPs have important biological actions indepenReceived June 15, 1998. Revision received September 1, 1998. Accepted September 8, 1998. Address all correspondence and requests for reprints to: Dr. Vivian Hwa, Department of Pediatrics, NRC5, Oregon Health Sciences University, 3181 SW Sam Jackson Park Road, Portland, Oregon 97201. * This work was supported by NIH Grants CA-58110 and DK-51513 (to R.G.R.), NIH Grant DK-52683, the Veterans Affairs Merit Review Program (to S.R.P.), Grant 97/5309 from the Fondo de Investigación Sanitaria, Spain (to A.L.B.), and Lilly S.A., Spain (Eighth Pediatric Endocrinology Research Award; to A.L.B.). immortalized nonmalignant prostatic human epithelial cells, P69, and in the P69 metastatic subline, M12. IGFBP-rP1 expression was not detectable in the prostatic cancer cell lines PC-3, DU145, and LNCaP. IGFBP-rP1 expression was regulated in P69 cells but not in M12 cells. Protein and mRNA expression was up-regulated by IGF-I, transforming growth factor-b, and retinoic acid. The observations that IGFBP-rP1 expression is significantly diminished in prostate tumorigenesis and is regulated in nonmalignant prostate cells suggest IGFBP-rP1 is important in normal prostatic cell growth. (J Clin Endocrinol Metab 83: 4355– 4362, 1998) dent of their abilities to bind IGFs. The IGF-independent effects of IGFBPs can be antiproliferative (14, 15) or growth stimulatory (16). The IGFBP family has recently been expanded to include a group of additional cysteine-rich proteins that are involved in regulating cell growth (17–19). These IGFBP-related proteins (IGFBP-rPs) share structural features with the conventional IGFBPs, IGFBP-1 to -6 (20). For the conventional IGFBPs, a striking shared feature is the conservation of critical cysteines, clustered at the N-terminus third (12 cysteines) and the C-terminus third (6 cysteines) of the proteins (7). It has been hypothesized that the N- and C-termini are independent domains that together are responsible for the high affinity IGF binding characteristic of IGFBPs (21). The IGFBPrPs contain the N-terminal domain of the IGFBPs, but their C-terminal domains have clearly diverged (17, 20, 22, 23). In addition to structural similarities, there are functional similarities between the IGFBP-rPs and IGFBPs. Two of the IGFBP-rPs, IGFBP-rP1, Mac25 (22), and IGFBP-rP2, CTGF (23), are able to bind IGF-I, although with a 20- to 100-fold reduced affinity compared to that of IGFBP-3 (18, 19). The existence of cysteine-rich proteins with conserved N-terminus domains and demonstrable abilities to bind IGFs, albeit with lower affinities than the conventional IGFBPs, has led to the proposal of an IGFBP superfamily, subdivided into high affinity IGF binders (IGFBP-1 to -6) and low affinity IGF binders (IGFBP-rPs) (19). The ability of these low affinity IGF binders to modulate IGF bioactivity in vivo is not known, and only scant data are available on their IGF-independent actions. The conventional IGFBPs in the prostate have been well characterized, whereas virtually nothing is known regarding prostate IGFBP-rPs. IGFBP-2 to -6 have been detected in 4355 4356 HWA ET AL. stromal and epithelial prostate cells as well as in prostatic epithelial cell lines (1, 24 –30). The pattern of detectable IGFBPs is altered in malignant prostatic cells (31, 32). The significance of changes in IGFBP levels in malignancy has yet to be determined, but probably includes altered modulation of IGF bioactivity (4, 33, 34) as well as biological effects independent of IGFs (35–37). Of the IGFBP-rPs, IGFBP-rP1 (Mac25) messenger ribonucleic acid (mRNA) has been detected in the prostate (18). The complementary DNA (cDNA) for Mac25 (22) was originally cloned from leptomeningial cells by differential display. The mac25 cDNA was found to be preferentially expressed in normal leptomeningial and mammary epithelial cells compared to their counterpart tumor cells (22, 38) and to be up-regulated in senescent human mammary epithelial cells (38). These results suggested that mac25 played a role in growth-regulating pathways that are abrogated in meningiomas and breast carcinoma. The same apparent protein and cDNA have been isolated from human bladder carcinoma cells [tumor-derived adhesion factor (TAF)] (39) and from human diploid fibroblast cells [prostacyclin-stimulating factor (PSF)] ( (...truncated)