Transcriptional Regulation of Osteopontin Production in Rat Osteoblast-like Cells by Parathyroid Hormone

Osteopontin (OP) or bone sialoprotein is a recently characterized extracellular matrix protein which is abundant in bone and is produced by osteoblasts. Parathyroid hormone (PTH) is a potent calcitropic hormone which regulates osteoblastic function including the synthesis of extracellular matrix proteins. This study examines the effect of human PTH (hPTH[1-34]) on the expression of this novel protein in rat osteoblast-like cells, hPTH(1-34) significantly decreased the amount of OP in culture media of the rat osteoblastic osteosarcoma cell line, ROS 17/2.8, detected by Western immunoblot analysis, hPTH(1-34) also suppressed the steady-state level of OP mRNA twoto threefold with an EDso of ~ 3 x 10 -'° M. This inhibition was detectable at 24 h, reached its nadir at 48 h, and lasted at least up to 96 h. The hPTH(1-34) effects were mimicked by isobutylmethylxanthine, cholera toxin, 8-bromo-cAMP, forskolin, and isoproterenol, hPTH(I-34) suppressed by twoto threefold the rate of OP gene transcription, estimated by nuclear run-on assays. The suppression of OP mRNA levels by hPTH(1-34) was also seen when basal levels were increased by transforming growth factor type/3, or 1,25-dihydroxyvitamin D3, or were decreased by dexamethasone. A similar decrease in the steady-state level of OP mRNA by hPTH(1-34) was also observed in primary cultures of osteoblast-enriched cells from fetal rat calvaria. These findings indicate that hPTH(1-34) suppresses the production of the novel extracellular matrix protein, OP, in osteoblasts at least in part through transcriptional control. STEOPONTIN (OP)' is a 44-kD glycoprotein which is rich in aspartic acid and glutamic acid and contains one phosphoserine and 12 phosphothreonine residues (Prince et al., 1987). OP is an abundant noncollagenous protein in bone (Franzen and Heinegard, 1985) and was immunohistochemically detected in bone matrix, osteoid, osteoblasts, osteocytes, preosteoblast-like cells, neurons, neurosensory cells, and the proximal convoluted tubules of the kidney (Mark et al., 1987a,b, 1988). OP messenger RNA was found in bone and kidney and at much lower levels in brain and lung (Yoon et al., 1987). In embryonic rat calvaria, OP mRNA expression was shown to increase between 15 and 21 d of gestation during development (Yoon et al., 1987). The amino acid sequence of OP predicted from a cDNA cloned from a rat osteosarcoma (ROS 17/2.8) cell library (Oldberg et al., 1986) was shown to be >92 % identical to that of2ar (Nomura et al., 1988). The 2ar gene was originally isolated on the basis of its enhanced expression in the mouse JB4 epidermal cell line in response to 12-O-tetradecanoyl-phorbol-13-acetate (TPA) (Smith and Denhardt, 1987). Since 2ar appears to be the product of a single copy 1. Abbreviations used in this paper: hPTH(1-34), human parathyroid hormone (1-34); OP, osteopontin; PTH, parathyroid hormone; TGF-/3, transforming growth factor type 8; TPA, 12-O-tetradecanoyl-phorbol-13-acetate. gene, it is probably the mouse homologue of rat OP (Nomura et al., 1988). In fact, 2ar mRNA, similar to rat OP, was expressed in mouse calvaria, vertebrae, and kidney (Nomura et al., 1988). In addition, 2ar mRNA was also expressed in placenta and decidua (Nomura et al., 1988). Although OP/ 2ar mRNA was expressed in cultured fibroblasts in response to TPA, neither the mRNA nor the antigen was detected in situ in rat or mouse skin (Mark et al., 1987b, 1988; Yoon et al., 1987; Nomura et al., 1988). The cDNA-predicted amino acid sequence of OP contains the Arg-Gly-Asp-Ser(RGDS) motif (Oldberg et al., 1986), which is found in fibronectin and other cell-adhesion proteins, and was shown to interact with cell surface receptors (Pytela et al., 1986; Tamkun et al., 1986). OP also has high affinity to hydroxyapatite, possibly due to its nine consecutive aspartic acid residues (Oldberg et al., 1986; Prince et al., 1987). Based on these features, OP was proposed to participate in cell attachment to mineralized matrix (Oldberg et al., 1986). Indeed, OP promoted the attachment of rat osteosarcoma cells and human gingiva fibroblasts to plastic dishes, and this effect was inhibited by RGD-containing peptides (Oldberg et al., 1986; Somerman et al., 1987). The physiological function of OP is not yet known. The temporal and tissue-specific OP/2ar expression seen in vivo (Yoon et al., 1987; Nomura et al., 1988) indicates that this protein is © The Rockefeller University Press, 0021-9525/89/02/713/6 $2.00 The Journal of Cell Biology, Volume 108, February 1989 713-718 713 on July 1, 2017 jcb.rress.org D ow nladed fom restricted to bone, kidney, neural tissue, and placenta and suggests a functional role in these tissues. The steady-state level of OP mRNA or OP in osteoblastlike cells is increased by 1,25-dihydroxyvitamin I)3 (Yoon et al., 1987; Prince and Butler, 1987) and is reduced by glucocorticoids (Yoon et al., 1987). 2ar mRNA in epidermal cells and fibroblasts is induced by embryonic carcinomaderived growth factor and basic fibroblast growth factor (Nomura et al., 1988) in addition to TPA (Smith and Denhardt, 1987). We have recently shown that transforming growth factor type fl (TGF-/5) enhances OP gene expression through transcriptional control in the rat osteoblast-like osteosarcoma cells, ROS 17/2.8, and in newborn mouse calvaria-derived osteoblast-like cells, MC3T3E1 (Noda et al., 1988a). These observations suggest that OP/2ar expression is regulated by factors which control growth and/or differentiation. Parathyroid hormone (PTH) has pronounced effects on the production of many extracellular matrix proteins in osteoblastic cells (Luben et al., 1976; Wong et al., 1977; Kream et al., 1980, 1986; Majeska and Rodan, 1982; Hamilton et al., 1985; Noda et al., 1988b). Moreover, target cells of PTH correspond to OP-producing cells which include proximal tubule, distal tubule, and ascending loop of Henle cells (Klahr and Hruska, 1984), in addition to osteoblasts (Rodan and Rodan, 1984). This study examined the effects of PTH on OP gene expression in the osteoblastic osteosarcoma ROS 17/2.8 cells and in primary cultures of fetal rat calvaria cells. We found that PTH inhibited OP gene expression at least in part through transcriptional control. Materials and Methods Human PTH (1-34) (hPTH[1-34]) was purchased from Bachem Biochemical (Torrance, CA). Porcine TGF-BI was obtained from R&D Systems, Inc. (Minneapolis, MN). 1,25-dihydroxyvitamin D3 was a kind gift from Dr. Uskokovic at Hoffman-LaRoche (Nutley, N J). Forskolin was purchased from Calbiochem-Behring Corp. (La Jolla, CA). Cholera toxin, isobutylmethylxanthine, (-)isoproterenol, and 8-bromo-cAMP were purchased from Sigma Chemical Co. (St. Louis, MO). Proteinase K and vanadyl ribonucleoside complex were obtained from Bethesda Research Laboratories (Gaithersburg, MD).