Apoptosis and survival of osteoblast-like cells are regulated by surface attachment

We tested the hypothesis that RGDS peptides regulate osteoblast survival in culture. Osteoblast-like MC3T3-E1 cells were allowed to attach to RGDS peptides that had been tethered to a silicone surface utilizing a previously described grafting technique. The RGDS-modified surface caused up-regulation of alpha(v)beta(3) integrin. We noted that there was an increase in expression of activated focal adhesion kinase and activated Akt. There was no change in the expression level of the anti-apoptotic protein Bcl-2, the pro-apoptotic protein Bad, or the inactivated form of Bad, pBad. Attachment to the RGDS-treated membrane completely abolished apoptosis induced by staurosporine, the Ca(2+).P(i) ion pair, and sodium nitroprusside. However, the surface modification did not interfere with apoptosis mediated by the free RGDS peptide or serum-free medium. When the activity of the phosphatidylinositol 3-kinase pathway was inhibited, RGDS-dependent resistance to apoptosis was eliminated. These results indicated that the binding of cells to RGDS abrogated apoptosis via the mitochondrial pathway and that the suppression of apoptosis was dependent on the activity of phosphatidylinositol 3-kinase.     

A novel human insulin-like growth factor binding protein secreted by osteoblast-like cells.

Insulin-like growth factor binding proteins (IGFBPs) modulate the cellular action of the insulin-like growth factors. Inhibition or enhancement of IGF effects by these cell-secreted binding proteins have been described. We have purified two IGFBPs (23 and 29 kDa) from media conditioned by U-2 human osteosarcoma cells using ligand-affinity chromatography and reversed phase HPLC. N-terminal amino acid analysis of the 23 kDa protein revealed a unique sequence with variable homology to IGFBPs 1-4. The 29 kDa IGFBP was found to be nearly identical to a recently reported IGFBP. Because the affinity purified U-2 IGFBPs enhanced IGF-I-stimulated osteoblast mitogenesis, we suggest that one or both of these binding proteins enhance IGF action in bone.     

Adaptation to phosphate deprivation in osteoblast-like cells

The rat osteosarcoma cell line UMR-106–01 has an osteoblast-like phenotype. When grown in monolyer culture these cells transport inroganic phosphate and L-alanine via Na⁺-dependent transport systems. Exposure of these cells to a low phosphate medium for 4 h produced a 60–70 per cent increase in Na⁺-dependent phosphate uptake compared to control cells maintained in medium with a normal phosphate concentration. In contrast, Na⁺-dependent alanine uptake and Na⁺-independent phosphate uptake were not changed during phosphate deprivation. The increased phosphate uptake was due, in part, to an increased V_max and was blocked completely by pretreatment with cycloheximide (70 μM). In these cells recovery of intracellular pH after acidification with NH₄Cl is due primarily to the Na⁺/H⁺ exchange system. The rate of this recovery process, monitored with a pH sensitive indicator (BCECF), was decreased by more than 50 per cent in phosphate-deprived cells compared to controls indicating that Na⁺/H⁺ exchange was inhibited during phosphate deprivation.     

Involvement of prostaglandin E2 in the inhibition of osteocalcin synthesis by human osteoblast-like cells in response to cytokines and systemic hormones

The stimulation of the production of osteocalcin by human osteoblast-like cells in response to 1,25(OH)2D3 is antagonized by several agents that induce the synthesis of prostaglandin E2 (PGE2) including interleukin 1 (IL-1), tumour necrosis factor (TNF) and parathyroid hormone (PTH). The mechanism whereby these agents inhibit the synthesis of osteocalcin is not known. In this report we show that exogenous PGE2 inhibits this stimulatory action of 1,25(OH)2D3 on human osteoblast-like cells in a dose-dependent manner, suggesting that PGE2 may contribute to the inhibition of osteocalcin synthesis in response to these agents. Assessment of the inhibitory role of endogenous PGE2 synthesis in the action of rhIL-1 alpha, rhIL-1 beta and rhTNF alpha on the production of osteocalcin demonstrated that the inhibition by these agents could be partially overcome by the addition of indomethacin, an inhibitor of PGE2 synthesis. In contrast, the inhibitory action observed with bPTH (1-84) was unaffected by indomethacin. These observations indicate that endogenous PGE2 synthesis mediates, in part, some of the inhibitory actions of the cytokines on the induction of osteocalcin synthesis in response to 1,25(OH)2D3, but not of PTH. Since the antagonism of the synthesis of osteocalcin by rhIL-1 alpha, rhIL-1 beta and rhTNF alpha was not completely abolished following the inhibition of PGE2 synthesis this would indicate that additional PGE2-independent mechanisms also account for the action of these cytokines on osteocalcin production. The nature of these mechanisms is currently not known.     

Nephronectin expression is regulated by SMAD signaling in osteoblast-like MC3T3-E1 cells

Nephronectin (Npnt) is an extracellular matrix protein known to be a ligand for the integrin α8β1. We previously demonstrated that Npnt expression was suppressed by TGF-β through ERK1/2 and JNK in osteoblasts. In this study, we found that inhibition of a TGF-β type I receptor (TGF-β R1, Alk5) by a specific inhibitor {2-[3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl]-1,5-naphthyridine} strongly induced Npnt expression in osteoblast-like MC3T3-E1 cells. The Alk5 inhibitor-induced increase of Npnt expression occurred in both time- and dose-dependent manners, while that expression was also induced by introduction of an siRNA for Smad2, a central intracellular mediator of TGF-β signaling. These results suggest that the expression of Npnt is regulated by the Alk5-SMAD signaling pathway in osteoblasts.     

A novel endothelial tyrosine kinase cDNA homologous to platelet-derived growth factor receptor cDNA.

Degenerate oligonucleotide primers complementary to the highly conserved subdomains III and VIII of subclass III tyrosine kinase receptors (TKr-III) were utilized to amplify rat aortic cDNA by polymerase chain reaction. Most of the cloned DNA products were rat platelet-derived growth factor receptor beta and macrophage-colony stimulating growth factor receptor cDNAs. Screening of the clones with probes coding for the receptor-specific kinase insert domain allowed the identification of a novel putative TKr-III cDNA, which hybridized with a approximately 6.1 kb mRNA with a distinctive tissue distribution. In situ hybridization on rat tissues and Northern analysis of cultured cells indicate that endothelial cells express a novel putative TKr-III mRNA.     

Basal activities and hormone responsiveness of osteoclast-like and osteoblast-like bone cells are regulated by glucocorticoids.

Isolated bone cells demonstrate cell-type specific responses to glucocorticoids. Osteoclast-like (OC) cells exhibit a large decrease in basal hyaluronate synthesis at physiological doses of glucocorticoids and resistance to further inhibition by pharmacological doses up to 10(-4) M. This effect is not accompanied by decreases in protein synthesis. In contrast, osteoblast-like (OB) cell metabolism is not inhibited by physiological doses of glucocorticoids. However, in OB cells both citrate decarboxylation and collagen synthesis are decreased at pharmacological doses of glucocorticoids and these effects are accompanied by a decrease in general protein synthesis. In addition to these effects on basal and general cell activities, physiological doses of glucocorticoids modulate the hormonal sensitivity of OC and OB bone cells such that lower concentrations of bovine parathyroid hormone (PTH) are necessary to elicit measurable biochemical changes. As a result, the presence of glucocorticoids permits significant responses to PTH to be detected at doses as low as 2 x 10(-13) M in OC and OB bone cells.     

Identification of novel monosodium urate crystal regulated mRNAs by transcript profiling of dissected murine air pouch membranes

Introduction  

The murine air pouch is a bursa-like space that resembles the human synovial membrane. Injection of monosodium urate (MSU) crystals into the pouch elicits an acute inflammatory response similar to human gout. We conducted the present study to identify mRNAs that were highly regulated by MSU crystals in the pouch membrane.