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J Gebken, A Feydt, J Brinckmann, H Notbohm, PK Muller and B Batge

High concentrations of transforming growth factor b (TGF-beta) are found in the bone matrix, reflecting a pivotal role of this growth factor in the coupling of bone resorption and formation. TGF-beta strongly stimulates the synthesis of extracellular matrix proteins, but in vitro studies show an inhibitory effect on the final mineralization process, which in vivo occurs despite high concentrations of TGF-beta. Little is known about how bone-forming cells respond to different concentrations of TGF-beta and if they can transiently adapt receptor numbers in order to modulate cellular activity. Against this background, we studied the cell-surface expression of TGF-beta receptors (TbetaR) I, II and III (betaglycan) on human osteoblast-like cells from adult donors, and examined the TbetaR presentation on these cells after a preceding exposure to TGF-beta1. Affinity crosslinking studies with disuccinimidylsuberate showed the presence of all three receptor types. Preincubation with TGF-beta1 markedly reduced 125I-TGF-beta1 binding in a time-dependent and dose-dependent manner and revealed a 95% reduction after an 18-h preincubation with 200 pM TGF-beta1. In parallel, Scatchard analysis showed that the binding affinity did not change as a consequence of TGF-beta1 preincubation. Immunoblotting analyses revealed an almost complete disappearance of immunoreactive TbetaR-II and TbetaR-III proteins after a 24-h preincubation with TGF-beta1. Using semi-quantitative reverse transcription PCR, no effect of TGF-beta1 on the expression of TbetaR-II mRNA was observed. These studies demonstrate a ligand-induced downregulation of TbetaRs-II and -III on human osteoblast-like cells, without any evidence for recovery within the first 24 h, both in the presence and after the removal of the ligand. The underlying mechanism appears to be based on post-transcriptional events. The results suggest that high concentrations of active TGF-beta1 decrease the responsiveness of osteoblasts towards this growth factor.

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M C Slootweg, J P Salles, C Ohlsson, C P de Vries, M J E Engelbregt and J C Netelenbos


Growth hormone (GH) exerts direct differentiative and proliferative effects on osteoblasts. We studied 125I-labeled human (h) GH binding to primary mouse osteoblasts derived from collagenase-treated 18-day fetal mouse calvaria. Scatchard analysis of the data revealed a single class of high affinity GH receptors (apparent K a= 5·74 × 109 m −1) with 2200 sites per cell. Affinity cross-linking and SDS-PAGE electrophoresis showed two bands with apparent molecular masses of 120 and 70 kDa. Mouse osteoblasts express GH receptor mRNA with gene transcripts of 4·2 and 1·2 kb, at levels which reach approximately 1/6 of those in mouse liver and 1/3 of those in mouse muscle. Two populations of undifferentiated and diffentiated osteoblasts, obtained by sequential collagenase digestion of mouse calvaria, were used to study the relationship between osteoblastic phenotype and GH receptor expression. Although the affinity of the receptors in undifferentiated and differentiated cells was the same, the capacity was significantly higher (1·45 ± 1·0% vs 2·39 ± 0·9%, P=0·03) in differentiated cells. This stresses the specific importance of the osteoblast as a target cell for GH. The differentiating potential of the vitamin A derivative retinoic acid was subsequently used experimentally to induce differentiation in the cells. Retinoic acid increased 125I-hGH binding to preosteoblasts (153%, P=0·02). Together, these data demonstrate the presence of a high affinity GH receptor in mouse osteoblasts which is related to differentiation.

Journal of Endocrinology (1996) 150, 465–472

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MC Slootweg, D Swolin, JC Netelenbos, OG Isaksson and C Ohlsson

Postmenopausal bone loss is primarily due to estrogen deficiency. Recent clinical observation demonstrate that GH increases bone mass in GH deficient patients. The present study investigates whether estrogen regulates GH action and GH receptor expression in osteoblasts. 17 beta-estradiol or GH added to the culture medium as single substances did not influence rat osteosarcoma cell proliferation nor human osteoblast-like (hOB) cell proliferation. However, together they synergistically induced osteoblast proliferation (rat osteosarcoma cells 160.1 +/- 15.5% of control cells; human osteoblast-like cells 159.6 +/- 5.1% of control cells). 17 beta-estradiol stimulated 125I-GH binding and GH receptor (GHR) mRNA levels in rat osteosarcoma cells. The stimulatory effect of estradiol was time dependent, reaching a peak after 8 h of incubation with 17 beta-estradiol (binding 216.9 +/- 27.8% and mRNA 374.6 +/- 30.8% of control). The finding that estradiol stimulated 125I-GH binding was confirmed in human osteoblast-like cells. In these cells, 17 beta-estradiol (10(-12) M) increased 125I-GH binding to 203.8 +/- 3.6% of control levels. We conclude that estrogen stimulates GH activity as well as GH binding and GHR mRNA levels in osteoblasts. These findings indicate that estrogen potentiates the effect of GH at the receptor level.

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A Suzuki, J Shinoda, Y Oiso and O Kozawa


We have previously reported that extracellular ATP stimulates Ca2+ influx from extracellular space, resulting in the production of prostaglandin E2 which mediates, at least in part, its proliferative effect on osteoblast-like MC3T3-E1 cells, and that the activation of protein kinase C (PKC) stimulates phospholipase D in these cells. In the present study, we examined the effect of extracellular ATP on phosphatidylcholine-hydrolysing phospholipase D activity in MC3T3-E1 cells. ATP stimulated the formation of both choline and inositol phosphates dose-dependently in the range between 0·1 and 1 mm. The formation of choline by a combination of ATP and NaF, an activator of GTP-binding protein, was synergistic, whereas that of inositol phosphates was not. A combination of ATP and 12-O-tetradecanoylphorbol-13-acetate, a PKC activating phorbol ester, additively stimulated the formation of choline. Staurosporine, an inhibitor of PKC, had little effect on ATP-stimulated formation of choline. Choline formation was significantly reduced by chelating extracellular Ca2+ with EGTA, while being inhibited by W-7, an antagonist of calmodulin. These results suggest that extracellular ATP stimulates phospholipase D in a Ca2+/calmodulin-dependent manner in osteoblast-like cells, and that neither PKC activation nor GTP-binding protein is involved in this mechanism.

Journal of Endocrinology (1995) 145, 81–86

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E Zoidis, C Ghirlanda-Keller, M Gosteli-Peter, J Zapf and C Schmid

In osteoblasts only the type III Na(+)-dependent phosphate (NaPi) transporter isoforms Pit-1 and Pit-2 have been identified. We tested the effects of extracellular Pi, Ca(2+) and IGF-I on Na(d)Pi transport and Pit-1 or Pit-2 mRNA expression in rat osteoblastic (PyMS) cells. The v(max) of Na(d)Pi transport was higher in cells kept in Pi-free, serum-free medium for 24 h than in controls at 1 mM Pi (2.47+/-0.20 vs 1.83+/-0.17 nmol/mg protein x 10 min). The apparent affinity constant (K(M)) for Pi remained unchanged. Pi withdrawal for 24 h did not impair cell viability whereas increasing the extracellular Pi to 5 mM resulted in cell death. Pit-1 (but not Pit-2) mRNA was upregulated following Pi deprivation, Ca(2+) treatment or after treatment with 1 nM IGF-I, known to stimulate Na(d)Pi transport and cell proliferation. IGF-I also stimulated Na(d)Pi transport and Pit-1 mRNA in primary rat calvarial osteoblasts. Expression of Pit-1 mRNA in vivo and the coordinate regulation of Pit-1 mRNA and Pi transport in osteoblastic cells suggest that Pit-1 is a candidate transporter of physiological relevance in bone.

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D Swolin-Eide, A Nilsson and C Ohlsson

It is well known that high levels of glucocorticoids cause osteoporosis and that physiologic levels of growth hormone (GH) are required for normal bone remodeling. It has been suggested that glucocorticoids regulate GH-responses via the regulation of GH-receptor expression. The aim of the present study was to investigate whether cortisol plays a role in the regulation of GH-receptor expression in cultured human osteoblasts. The effect of serum starvation and cortisol on GH-receptor expression was tested in human osteoblast (hOB)-like cells. Serum starvation for 24 h resulted in an increase in GH-receptor mRNA levels (90 +/- 1% over control culture). Cortisol increased GH-receptor mRNA levels in a dose-dependent manner with a maximal effect at 10(-6)M. The stimulating effect of cortisol on GH-receptor mRNA levels was time-dependent, reaching a peak 12 h after the addition of cortisol (126 +/- 29% over control culture) and remaining up to 12 h later. The increase in GH-receptor mRNA levels was accompanied by an increase in 125I-GH binding which reached a maximum at 24 h (196 +/- 87% over control culture). In conclusion, glucocorticoids increase GH-receptor expression in hOB-like cells. Further studies are needed to clarify whether glucocorticoid-induced regulation of the GH-receptor is important in human bone physiology.

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R S Birnbaum, R R Bowsher and K M Wiren


IGF-I and -II have potent effects on proliferation and differentiation of osteoblasts in vitro. These cells secrete both IGFs and expression of these peptides is regulated by several of the hormones and growth factors that promote bone resorption and/or formation. However, the physiological role(s) of IGFs in the remodelling process of adult bone is still unclear. Some confusion may arise from results influenced, in part, by differences in the state of osteoblast development of in vitro cultures. Several laboratories have demonstrated that murine osteoblast cultures progress from proliferating preosteoblasts, to mature differentiated osteoblasts that form an extracellular matrix, to cultures that form a mineralized matrix. We have recently documented changes in IGF-binding protein expression and secretion in these cultures. To complement and extend this work, we have examined IGF-I expression and secretion and IGF-II expression during in vitro osteoblast development.

Steady-state mRNA levels of both IGF-I and -II increased from the earliest time examined, day 5 in culture, to a maximum at day 11 and, thereafter, declined. IGF-I secreted into the medium also changed in a biphasic manner, but IGF-II could not be quantitated due to the sensitivity of our assay. Secretion of IGF-I was lowest between days 8 and 14. IGF-I secretion on day 5 was significantly greater than day 8. Similarly, IGF-I secretion from day 17 to 26 was also greater than observed for days 8 to 14. If differentiation of the cells was inhibited, this late rise in IGF-I secretion was abolished. We conclude that IGF-I is an autocrine mitogen of the proliferating preosteoblasts. Further, we also suggest that the rise in IGF-I secretion, late in osteoblast development, may lead to sequestration of this mitogen in the extracellular matrix for release during a subsequent remodelling cycle.

Journal of Endocrinology (1995) 144, 251–259

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C S Kovacs, C L Chik, B Li, E Karpinski and A K Ho


Pituitary adenylate cyclase-activating peptide (PACAP) and vasoactive intestinal peptide (VIP) share 68% homology and function as neurotransmitters or neuroendocrine factors. Although VIP immunoreactivity has been detected in bone cells, the presence of PACAP or PACAP receptors in bone has not been determined. In this study, we investigated the role of PACAP and VIP in regulating cAMP accumulation in the UMR 106 osteoblast-like tumor cell line.

PACAP 27 (10−9 to 3 × 10−7 m), PACAP 38 (10−9 to 3 × 10−7 m) and VIP (10−8 to 10−6 m) stimulated cAMP accumulation up to eightfold. PACAP 27 was slightly more potent than PACAP 38, and both were tenfold more potent than VIP. Both PACAP- and VIP-stimulated cAMP accumulation were potentiated by 4β-phorbol 12-myristate 13-acetate, an activator of protein kinase C. Two PACAP antagonists, PACAP 6–27 (3 × 10−6 m) and PACAP 6–38 (3 × 10−6 m), blocked PACAP- and VIP-stimulated cAMP accumulation. Two VIP antagonists ([Lys1,Pro2,5,Arg3,4,Tyr6]-VIP, and 4 Cl-d-Phe6,Leu17]-VIP) did not reduce the PACAP-or VIP-stimulated cAMP accumulation. Pretreatment with PACAP 27, PACAP 38 or VIP equally blocked PACAP- and VIP-stimulated cAMP accumulation.

These results suggest that PACAP is a more potent stimulator of cAMP accumulation than VIP in UMR 106 cells. PACAP and VIP may share a role in the paracrine or neuroendocrine regulation of bone metabolism.

Journal of Endocrinology (1996) 149, 287–295

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Jessica L Pierce, Ke-Hong Ding, Jianrui Xu, Anuj K Sharma, Kanglun Yu, Natalia del Mazo Arbona, Zuleika Rodríguez-Santos, Paul J Bernard, Wendy B Bollag, Maribeth H Johnson, Mark W Hamrick, Dana L Begun, Xing-Ming Shi, Carlos M Isales and Meghan E McGee-Lawrence

, including glucocorticoids, that may interfere with osteoblast function and drive marrow adipose tissue expansion ( Devlin et al. 2010 , Cawthorn et al. 2016 ). The enzyme 11β-hydroxysteroid dehydrogenase type 1 (Hsd11b1) (which converts inactive

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E Lambertini, L Penolazzi, V Sollazzo, F Pezzetti, M de Mattei, L del Senno, GC Traina and R Piva

Estrogen receptor (ER) alpha is expressed during osteoblast differentiation; however, both its functional role in bone metabolism and its involvement in osteoporotic pathogenesis caused by estrogen deficiency are not well understood. Loss of ER alpha gene expression could be one of the mechanisms leading to osteoporosis. Therefore, we investigated a possible modulation of ER alpha gene expression in a human osteoblastic cell line and in four primary osteoblast cultures by using a decoy strategy. Double stranded DNA molecules, mimicking a regulatory region of the ER alpha gene promoter (DNA-102) and acting as a 'silencer' in breast cancer cells, were introduced into osteoblasts as 'decoy' cis-elements to bind and functionally inactivate a putative negative transcription factor, and thus to induce ER alpha gene expression. We found that the DNA-102 molecule was able to specifically bind osteoblast nuclear proteins. Before decoy treatment, absence or variable low levels of ER alpha RNAs in the different cultures were detected. When the cells were transfected with the DNA-102 decoy, an increase in expression of ER alpha and osteoblastic markers, such as osteopontin, was observed, indicating a more differentiated osteoblastic phenotype both in the cell line and in primary cultures. These results showed that the DNA-102 sequence competes with endogenous specific negative transcription factors that may be critical for a decrease in or lack of ER alpha gene transcription. Therefore, osteoblastic transfection with the DNA-102 decoy molecule may be considered a tempting model in a putative therapeutic approach for those pathologies, such as osteoporosis, in which the decrease or loss of ER alpha expression plays a critical role in bone function.