Type beta transforming growth factor (TGF beta) regulation of alkaline phosphatase expression and other phenotype-related mRNAs in osteoblastic rat osteosarcoma cells

TGF beta 1 from porcine platelets increased alkaline phosphatase (AP) activity in the rat osteoblastic cell line ROS 17/2.8 about three-fold. This effect was dose-dependent with an ED50 of about approximately 0.2 ng/ml and was larger during logarithmic growth than at confluence. TGF beta 1 inhibited cell growth by about 30% with similar dose dependence. Thirty min exposure to TGF beta 1 was sufficient to increase AP activity 3 days later by about two-fold but did not affect cell growth, suggesting dissociation between effects on proliferation and differentiation. The rise in AP activity started 6 h after TGF beta 1 addition and was blocked by cycloheximide and actinomycin D. TGF beta 1 also increased AP mRNA by two- to three-fold and this effect was not blocked by cycloheximide. The half-life of AP mRNA, estimated following the addition of 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole was about ten h in both control and TGF beta 1-treated cells. The mRNAs for type I procollagen and osteonectin were also increased by TGF beta 1 but fibronectin mRNA was decreased. TGF beta 2 effects on AP and cell growth were similar to those of TGF beta 1, except for lack of activity following transient exposure. At saturating concentrations, TGF beta 2 (2 ng/ml) or dexamethasone (10(-7) M), which has similar effects on these cells, did not further augment the effects of TGF beta 1 (at 2 ng/ml). Above findings suggest that TGF beta promotes osteoblastic differentiation in rat osteosarcoma cells at least in part by acting at the pretranslational level.     

Stimulation of plasma membrane and matrix vesicle enzyme activity by transforming growth factor-beta in osteosarcoma cell cultures

Transforming growth factor-beta (TGF beta) serves an important role in extracellular matrix formation by stimulating the production of numerous extracellular matrix proteins by connective tissue cells and by osteoblasts or bone-forming cells. TGF beta has been shown to stimulate alkaline phosphatase (ALPase) activity in the rat osteoblast-like osteosarcoma cell line ROS 17/2.8. Previous studies have shown that this enzyme is elevated during calcification of bone and that it is enriched in matrix vesicles, an extracellular organelle associated with initial hydroxyapatite formation. To test the hypothesis that TGF beta plays a role in regulating mineral deposition in the matrix, the effects of TGF beta on ALPase and phospholipase A2, two enzymes associated with mineralization, were examined. ROS 17/2.8 cells were cultured at high and low density with recombinant human TGF beta (0.1-10 ng/ml) to examine the influence of cell maturation on response to TGF beta. Maximal stimulation of ALPase activity in the low density cultures was seen at 5 ng/ml; in high-density cultures, there was further stimulation at 10 ng/ml. There was a dose-dependent increase in ALPase activity seen in the matrix vesicles and plasma membranes in both types of cultures. Matrix vesicle ALPase exhibited a greater response to factor than did the plasma membrane enzyme. However, in low-density cultures, the two membrane fractions exhibited a parallel response with greatest activity consistently in the matrix vesicles. There was a dose-dependent increase in phospholipase A2-specific activity in the plasma membranes and matrix vesicles of both high- and low-density cultures. In agreement with previous studies, TGF beta inhibited cellular proliferation 50%. The results show that addition of TGF beta stimulates the activity of enzymes associated with calcification. The effect of TGF beta is dependent on the stage of maturation of the cell. This study indicates that TGF beta may play an important role in induced bone formation, calcification, and fracture repair in addition to its role in promoting chondrogenesis.     

Effects of GDF7/BMP12 on proliferation and alkaline phosphatase expression in rat osteoblastic osteosarcoma ROS 17/2.8 cells

Growth and differentiation factor 7(GDF7), also later called as bone morphogenetic protein (BMP)12, is a new member of the BMP superfamily, which induces formation of tendon-like tissue formation in the ectopic implantation experiments. We examined the effect of BMP12 on proliferation and expression of phenotype-related genes in rat osteoblastic osteosarcoma ROS17/2.8 cells. BMP12 treatment enhanced proliferation of ROS17/2.8 cells within 3 days and this effect was observed at least up to day 6 of the treatment. The cell number was increased by about 50% on day 3 and about two-fold by day 6. These effects were observed at the dose range between 40 and 1,000 ng/ml. Treatment with BMP12 also enhanced alkaline phosphatase activity by about 50% in ROS17/2.8 cells within 24 h of the treatment. The effect peaked at 48 h and was still observed at 72 h. The enhancing effect of BMP12 on alkaline phosphatase was observed similarly at the doses ranging from 40 to 1,000 ng/ml. These data indicate that BMP12 has positive effects on proliferation and phenotypic expression of ROS 17/2.8 cells.     

Opposing effects of fibroblast growth factor and pertussis toxin on alkaline phosphatase, osteopontin, osteocalcin, and type I collagen mRNA levels in ROS 17/2.8 cells

In rat osteosarcoma (ROS 17/2.8) cells, which express osteoblastic features in culture, basic fibroblast growth factor (bFGF) reduces the level of alkaline phosphatase, type I collagen, and osteocalcin mRNA and increases osteopontin mRNA, independent of growth stimulation. The fibroblast growth factor (FGF) effects are dose dependent (EC50 about 6 pM) and are detected 24 h after addition of the growth factor. bFGF also reduces parathyroid hormone-stimulatable adenylate cyclase and alkaline phosphatase activity in these cells. Concomitant treatment with pertussis toxin (20 ng/ml) opposes the FGF effects. Although cyclic AMP elevating agents mimic pertussis toxin action on some parameters, they produce opposite effects on others, indicating that antagonism between pertussis toxin and bFGF is not mediated by cyclic AMP. bFGF caused a small reduction in steady state NAD-dependent ADP-ribosylation and had no detectable effects on the steady-state levels of the Gi alpha (alpha subunit of the inhibitory G protein) 1, 2, and 3, visualized with specific antibodies in these cells. Although the site of interaction of pertussis toxin and FGF remains to be determined, the findings presented here suggest separate control of growth and differentiation by bFGF and show that pertussis toxin treatment can modulate differentiation in these cells, presumably via Gi proteins.     

Transforming growth factor-beta (TGF beta) inhibits TGF alpha expression in bovine anterior pituitary-derived cells.

Transforming growth factor-beta 1 (TGF beta 1) is a multifunctional regulator of cell growth and differentiation. We report here that TGF beta 1 decreased the proliferation of nontransformed bovine anterior pituitary-derived cells grown in culture. We have previously demonstrated that these cells express both TGF alpha and its receptor [the epidermal growth factor (EGF) receptor] and that expression can be stimulated by phorbol ester (TPA) and EGF. TGF beta 1 treatment over a 2-day period decreased the proliferation of pituitary cells. This decreased growth rate was accompanied by a decrease in the TGF alpha mRNA level. The effect of TGF beta 1 on TGF alpha mRNA down-regulation was both dose dependent (maximal effect observed at 1.0 ng/ml TGF beta 1) and time dependent (minimum of 2-day treatment with TGF beta 1 was required before a decrease in TGF alpha mRNA was observed). Studies on TGF alpha mRNA stability indicated that TGF beta 1 did not alter the TGF alpha mRNA half-life. Treatment of the TGF beta 1 down-regulated cells with EGF resulted in the stimulation of TGF alpha mRNA levels; thus, the TGF beta 1-treated cells remained responsive to EGF. The decreased proliferation in response to TGF beta 1 could be only partially reversed by simultaneous treatment of the cells with EGF (10(-9)M) and TGF beta 1 (3.0 ng/ml). Qualitatively, the TGF beta 1-induced reduction of TGF alpha mRNA content was independent of cell density. TGF beta 1 treatment of the anterior pituitary-derived cells also reduced the levels of c-myc and EGF receptor mRNA. These results represent the first demonstration of the down-regulation of TGF alpha synthesis by a polypeptide growth factor and suggest that TGF beta 1 may be a physiological regulator of TGF alpha production in vivo.     

Inhibition of osteoblastic cell differentiation by conditioned medium derived from the human prostatic cancer cell line PC-3 in vitro

Human prostatic carcinoma frequently metastasizes to bone tissue and activates bone metabolism, especially bone formation, at the site of metastasis. It has been reported that an extract of prostatic carcinoma and conditioned medium (CM) of a human prostatic carcinoma cell line, PC-3, established from a bone metastastic lesion, stimulate osteoblastic cell proliferation. However, there is little information about the effect of PC-3 CM on the differentiation of osteoblastic cells. In this study, we investigated the effect of PC-3 CM on the differentiation of two types of osteoblastic cells, primary fetal rat calvaria (RC) cells containing many undifferentiated osteoprogenitor cells, and ROS 17/2.8, a well-differentiated rat osteosarcoma cell line. PC-3 CM inhibited bone nodule formation and the activity of alkaline phosphatase (ALPase), an osteoblastic marker enzyme, on days 7, 14, and 21 (RC cells) or 3, 6, and 9 (ROS 17/2.8 cells) in a dose-dependent manner (5–30% CM). However, the CM did not affect cell proliferation or cell viability. PC-3 CM was found to markedly block the gene expression of ALPase and osteocalcin (OCN) mRNAs but had no effect on the mRNA expression of osteopontin (OPN), the latter two being noncollagenous proteins related to bone matrix mineralization. These findings suggest that PC-3 CM contains a factor that inhibits osteoblastic cell differentiation and that this factor may be involved in the process of bone metastasis from prostatic carcinoma. J. Cell. Biochem. 67:248–256, 1997. © 1997 Wiley-Liss, Inc.