Osteogenic protein-1 (OP-1) is a member of the bone morphogenetic protein family and has been shown to induce new bone formation in vivo. In the present study, we determined whether the expression of the IGF system, a significant growth factor system in bone, was altered by OP-1 in primary cultures of fetal rat calvarial cells. Levels of messenger RNA (mRNA) encoding insulin-like growth factor I (IGF-I), IGF-II, IGF-I receptor, and IGF-binding proteins (IGFBP-1 to -6) were determined after OP-1 treatment. The level of total IGF-I mRNA was elevated in an OP-1 concentration (0-1000 ng/ml)-dependent manner, with maximal stimulation of IGF-I mRNA of 2- to 3-fold apparent 24 h after treatment. The increase in the IGF-I mRNA level involved a preferential stimulation of transcripts initiated at start site 2 in the exon 1 promoter. The level of IGF-II mRNA also increased by approximately 2-fold in OP-1 treated cells in a concentration-dependent manner. The level of IGF-I receptor mRNA was not altered by treatment. Whereas IGFBP-1 mRNA was not detected in these cells, IGFBP-2 mRNA was expressed, but the expression was not changed after treatment for 48 h in the concentration range (0-1000 ng/ml) tested. The IGFBP-3 mRNA level was increased slightly 48 h after OP-1 treatment in a concentration-dependent manner. The IGFBP-4, -5, and -6 mRNA levels decreased dramatically in an OP-1 concentration-dependent manner. In addition, coincubation of antisense oligonucleotides corresponding to IGF-I or -II mRNA sequence with OP-1 reduced the OP-1 induced elevation in alkaline phosphatase activity. The present results suggest that the differentiation of rat osteoblastic cells in response to OP-1 is mediated in part by increased IGF-I -II gene expression and alterations in the gene expression of different IGFBPs.
Osteogenic protein-1 (OP-1 or BMP-7) is a multifunctional cytokine that regulates the development of several tissues during embryogenesis, including the skeleton, eye, and kidney. In postnatal life, OP-1 expression is most abundant in the kidney, although the cellular localization of this expression has not been described. In this study, we utilized a cell culture approach to localize OP-1 mRNA expression in various renal cell types and to determine potential target cells for OP-1 effects. OP-1 mRNA expression was demonstrated in several glomerular cell types, such as mesangial, epithelial, and endothelial cells. Distal tubule MDCK cells also expressed OP-1 mRNA but human proximal tubule HK-2 cells did not. Multiple OP-1 transcripts, which ranged in size from 1.6 to 3.8 kb, were observed in both glomerular and tubule cells. Interestingly, the pattern of expression varied among the different cell types, suggesting cell-specific expression of OP-1 mRNA. Analysis of OP-1 receptor expression revealed transcripts for BMP receptors type IA and IB in HK-2 cells and transcripts for BMPR-IA and ALK-2 in mesangial cells. Treatment of HK-2 cells with OP-1 (300 ng/ml) for 24-48 hr increased cellular proliferation whereas treatment of cells with transforming growth factor-beta had no effect. Mesangial cell proliferation was not affected by OP-1. The results suggest that OP-1 is produced in the renal glomerulus and then travels to the proximal tubule to regulate the proliferation of cells in this region of the nephron.
Endothelin-1 (ET-1) is a vasoactive peptide that modulates bone metabolism via regulatory effects on osteoblasts, chondrocytes, and osteoclasts. While ET-1 may circulate in the blood stream, tissue-specific expression of this peptide is more physiologically relevant. In the present study we measured ET-1 synthesis in sections of fetal rat calvaria (FRC) and in cultured FRC osteoblasts. Regulation of ET-1 synthesis in FRC osteoblasts by bone morphogenetic protein-7 (BMP-7) and transforming growth factor-beta1 (TGF-beta1) also was examined. Immunohistochemical analysis revealed ET-1 staining in calvarial osteoblasts, endothelial cells, and osteocytes. ET-1 mRNA expression was detected in cultured FRC cells and ET-1 peptide was present in conditioned media. During long-term culture of FRC cells (26 days) ET-1 peptide production rose sharply and peaked during the time of cellular proliferation (Days 0-3) then returned to baseline levels by Day 18, when mineralized nodules were forming. Treatment of FRC cells with BMP-7 enhanced ET-1 levels by three-fold on Day 3 and enhanced nodule formation by 15-fold on Day 26. To determine whether ET-1 was involved in an autocrine manner in BMP-7-induced nodule formation, cells were cultured in the presence of BMP-7 and BQ-123, an ET(A) receptor antagonist. BQ-123 had no effect on nodule formation in control or BMP-7-treated cells, indicating that osteoblast-derived ET-1 regulates other cell types in vivo during the bone formation process.
The temporal pattern with which phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC), modulates parathyroid hormone (PTH)-responsive adenylyl cyclase (AC) was evaluated in a clonal osteoblast-like cell line (UMR-106). Brief (< or = 1 h) exposure of UMR-106 cells to PMA enhanced PTH stimulation of AC, whereas more prolonged PMA treatment decreased the PTH response, with maximum inhibition occurring at < or = 6 h. PMA treatment also resulted in initial activation followed by downregulation of PKC. Exposure of cells to 1,2-dioctanoyl-sn-glycerol, which activated but did not downregulate PKC, resulted in bidirectional modulation of PTH-responsive AC identical to that produced by PMA. Prolonged PMA exposure decreased PTH receptor number, as determined by radioligand binding studies, and reduced PTH receptor mRNA levels, assessed by Northern blot analysis. Forskolin activation of the catalytic subunit of AC was also decreased after prolonged PMA treatment. The results suggest that activation of PKC sequentially stimulates and then inhibits PTH responsiveness. Inhibition of the PTH response occurs by PKC actions exerted on the PTH receptor and the AC catalytic subunit.
Osteogenic protein-1 (OP-1) stimulates bone morphogenesis in vivo and modulates osteoblast growth and differentiation in vitro. Treatment of ROS 17/2.8 cells with OP-1 resulted in a time- and concentration-dependent inhibition of [3H]thymidine incorporation. In contrast, OP-1 treatment stimulated phenotypic differentiation in ROS 17/2.8 cells, as indicated by enhanced 1) alkaline phosphatase activity (4-fold); 2) alkaline phosphatase mRNA (5-fold); 3) parathyroid hormone receptor mRNA (2-fold), and 4) parathyroid hormone-stimulated adenosine 3',5'-cyclic monophosphate accumulation (2-fold). OP-1-induced changes in cell growth and gene expression were sensitive to cycloheximide and actinomycin D. Measurement of [3H]thymidine incorporation and alkaline phosphatase activity in situ revealed heterogeneity in the cellular responses to OP-1. Proliferating cells exhibited less alkaline phosphatase activity than nonproliferating cells, whereas cells expressing high levels of alkaline phosphatase incorporated little [3H]thymidine. Our data delineating the responses of mature differentiated osteoblasts to OP-1 suggest that potentiation of osteoblast differentiated function is an important component of bone morphogenesis in vivo.
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