Inductive effects of prostaglandins on alkaline phosphatase in osteoblastic cells, clone MC3T3-E1

The effects of prostaglandins (PGs) on the induction of alkaline phosphatase (ALP) were investigated in osteoblastic clone MC3T3-E1 cells cultured in serum-free medium. Prostaglandin E2 (PGE2) stimulated ALP activity in the cells in a dose-dependent fashion with a maximal effect which was about twice that in the control cells at concentrations of 100-500 ng/ml. Actinomycin D and cycloheximide inhibited the stimulative effect of PGE2 on ALP activity in the cells. PGE2-induced and native ALPs in the cells were of the same type as that in adult mouse calvaria, being heat-labile, L-homoarginine- and levamisole-sensitive, and L-phenylalanine-insensitive. Isobutyl methylxanthine (IBMX), a cAMP phosphodiesterase inhibitor, stimulated the inductive effect of PGE2 on ALP activity at 0.1 mM, at which concentration IBMX alone had little effect on the activity. PGE2 also increased the intracellular cAMP content in a dose-dependent fashion with a maximal effect at 100 ng/ml. PGE1, PGF1 alpha, and PGF2 alpha (primary PGs like PGE2) increased the activity. Our present results suggest that PGs stimulate the differentiation of osteoblasts and are involved in bone formation in vivo, as well as in bone resorption.     

Bone morphogenetic protein-2 stimulates alkaline phosphatase activity and collagen synthesis in cultured osteoblastic cells, MC3T3-E1

The activities of three bone morphogenetic proteins (BMPs), BMP-1, BMP-2 and BMP-3, on alkaline phosphatase activity, collagen synthesis and DNA synthesis were studied in cultured osteoblastic cells, MC3T3-E1. Treatment of cells with BMP-2 for 48 h induces an increase in cellular alkaline phosphatase activity. This stimulatory effect is evident at a concentration as low as 20 ng/ml of BMP-2 and becomes greater with increasing doses of BMP-2. The BMP-2-induced increase in alkaline phosphatase activity is enhanced by the presence of beta-estradiol, dexamethasone or 1 alpha, 25(OH)2D3. BMP-2 and BMP-3 slightly but significantly stimulate collagen synthesis. None of the BMPs stimulates DNA synthesis in MC3T3-E1 cells at doses tested. These results indicate that BMPs act directly on osteoblastic cells and stimulate the expression of the osteoblastic phenotypes.     

Tumor necrosis factor type alpha (cachectin) stimulates mouse osteoblast-like cells (MC3T3-E1) to produce macrophage-colony stimulating activity and prostaglandin E2

To elucidate the mechanism of tumor necrosis factor alpha (TNF-alpha)-induced bone resorption, the effects of recombinant human TNF-alpha on mouse osteoblast-like cells (MC3T3-E1) were studied. TNF-alpha stimulated MC3T3-E1 cells to produce prostaglandin E2 (PGE2) and macrophage colony stimulating activity (M-CSA) in a dose-dependent manner. TNF decreased alkaline phosphatase (AL-P) activity of MC3T3-E1 cells. These TNF effects were observed at 1 ng/ml (approximately 6 X 10(-11)M). The inhibitory effect on AL-P activity was reversible and the cell growth of MC3T3-E1 cells was only slightly affected by TNF. These findings suggest that both PGE2 and M-CSA stimulated by TNF-alpha are possibly involved in osteoblast-mediated osteoclastic bone resorption, whereas inhibition of AL-P activity may lead to a decrease in bone formation.     

Recombinant human interleukin 1 alpha and beta stimulate mouse osteoblast-like cells (MC3T3-E1) to produce macrophage-colony stimulating activity and prostaglandin E2

The effects of interleukin 1 (IL-1) on MC3T3-E1 cells (clonal osteoblast-like cells established from mouse calvaria) were studied to elucidate the mechanism of IL-1-induced bone resorption. Recombinant human interleukin 1 alpha (rhIL-1 alpha) and beta (rhIL-1 beta) stimulated PGE2 production in MC3T3-E1 cells in a dose dependent manner. rhIL-1 alpha and 1 beta also stimulated MC3T3-E1 cells to produce macrophage-colony stimulating activity (M-CSA) in a dose-dependent manner. Indomethacin completely abolished PGE2 production but did not affect CSA. These results suggest that bone resorption induced by IL-1s is at least in part mediated by PGE2 produced by osteoblasts, and that M-CSA produced by osteoblasts may synergistically potentiate bone resorption by recruiting osteoclast precursors.     

IL-6 is produced by osteoblasts and induces bone resorption

To examine the possible involvement of IL-6 in bone metabolism, a mouse osteoblastic cell line (MC3T3-E1) and primary osteoblast-like cells from fetal mouse calvaria were cultured with several systemic and local bone-resorbing agents and their expression of IL-6 mRNA was determined. Local bone-resorbing agents such as IL-1 alpha, IL-1 beta, TNF-alpha, and LPS greatly induced IL-6 mRNA expression in both MC3T3-E1 cells and primary osteoblast-like cells. Parathyroid hormone slightly increased expression of IL-6 mRNA in primary osteoblast-like cells but not in MC3T3-E1 cells. Neither IL-6 nor 1 alpha,25-dihydroxyvitamin D3 increased expression of IL-6 mRNA in either of the osteoblast-like cells. In agreement with the expression of IL-6 mRNA, biologically active IL-6 was produced in response to the treatment with IL-1 alpha, TNF-alpha, and LPS in MC3T3-E1 cells. Adding IL-6 dose dependently stimulated the release of 45Ca from prelabeled fetal mouse calvaria. Simultaneously adding suboptimal concentrations of IL-6 and IL-1 alpha induced bone resorption cooperatively. In accord with the increase in the release of 45Ca by IL-6, there were three times as many osteoclasts in the bone sections of calvaria cultured with IL-6 for 5 days as in the controls. IL-6 slightly suppressed alkaline phosphatase activity and collagen synthesis in MC3T3-E1 cells. These results indicate that IL-6 is also produced by osteoblasts, preferentially in response to local bone-resorbing agents, and it induces bone resorption both alone and in concert with other bone-resorbing agents.     

Serial passage of MC3T3-E1 cells alters osteoblastic function and responsiveness to transforming growth factor-beta1 and bone morphogenetic protein-2.

The murine-derived clonal MC3T3-E1 cell is a well-studied osteoblast-like cell line. To understand the effects of serial passages on its cellular function, we examined changes in cell morphology, gap junctional intercellular communication (GJIC), proliferation, and osteoblastic function between early passage (<20) and late passage (>65) cells. MC3T3-E1 cells developed an elongated, spindle shape after multiple passages. Intercellular communication decreased significantly (33%) in late vs. early passage cells. Transforming growth factor-beta1 (TGF-beta1) stimulated cell proliferation in early passage cells and induced c-fos expression, while it inhibited proliferation in late passage cells. Using alkaline phosphatase (ALP) activity and osteocalcin (OC) secretion as markers for osteoblastic function and differentiation, we demonstrated that both markers were significantly reduced after multiple cell passages. Bone morphogenetic protein-2 (BMP-2) significantly enhanced ALP activity and OC secretion in early passage cells while TGF-beta1 exerted an opposite effect. Both BMP-2 and TGF-beta1 had minimal effects on late passage cells. We conclude that serial passage alters MC3T3-E1 cell morphology, and significantly diminishes GJIC, osteoblastic function, TGF-beta1-mediated cell proliferation, and responsiveness to TGF-beta1 and BMP-2. Cell passage numbers should be clearly defined in functional studies involving MC3T3-E1 cells.     

Stimulation of prostaglandin E2 synthesis in cloned osteoblastic cells of mouse (MC3T3-E1) by epidermal growth factor

Prostaglandin (PG) E2, known as a bone-resorption factor, was released as a predominant arachidonate metabolite in the culture medium of an osteoblastic cell line cloned from mouse calvaria (MC3T3-E1). Epidermal growth factor (EGF) (10 ng/ml) prominently enhanced endogenous PGE2 synthesis, requiring the simultaneous presence of unidentified factor(s) contained in bovine serum. PGE2 synthesis increased after a lag phase for 1-2 h and reached a maximum level at about 3 h after EGF addition. EGF-stimulated PGE2 synthesis was almost completely blocked by 10 microM cycloheximide or 1 microM actinomycin D. Furthermore, when the cells were pretreated with EGF, the microsomes exhibited an increased activity of fatty acid cyclooxygenase (arachidonic acid----PGH2), whereas the activity of PGE synthase (PGH2----PGE2) remained unchanged. These results suggested an EGF-mediated induction of cyclooxygenase. Following increased PGE2 synthesis, DNA synthesis increased and alkaline phosphatase activity decreased in a slower response to EGF. PGE2 (above 0.1 microM) added to the cells could replace EGF. However, such effects of EGF on the osteoblasts could not be attributed totally to an autocrine function of PGE2 produced by stimulation with EGF because these effects of EGF were not abolished by indomethacin, which blocked the PGE2 synthesis.     

Human purified interleukin-1 inhibits DNA synthesis and cell growth of osteoblastic cell line (MC3T3-E1), but enhances alkaline phosphatase activity in the cells

We examined the effects of human purified interleukin-1 (IL-1) on DNA synthesis, cell growth, and alkaline phosphatase activity in the osteoblastic cell line MC3T3-E1 under both preconfluent and confluent culture conditions. Addition of IL-1 to the cells markedly inhibited their DNA synthesis and growth over the range 1-10 U/ml. Such significant inhibitory effects were observed in cells cultivated in 1 or 5% fetal calf serum (FCS)-containing alpha modification Eagle's medium (alpha-MEM), but not in alpha-MEM containing 10% FCS. In contrast, alkaline phosphatase activity was enhanced significantly by IL-1 in the cell line cultivated in 1% FCS-containing alpha-MEM. These results demonstrate that human purified IL-1 is effective in inducing the differentiation of osteoblastic cell MC3T3-E1.     

The existence of distinct classes of prostaglandin E2 receptors mediating adenylate cyclase and phospholipase C pathways in osteoblastic clone MC3T3-E1.

We have reported previously that PGE2 evoked an increase in intracellular calcium level ([Ca2+]i) in mouse osteoblastic cells (1). Here, we investigated the effects of PGE1 and PGF2 alpha on cAMP production and [Ca2+]i in comparison with those of PGE2. In osteoblastic clone, MC3T3-E1 cells, PGE1 stimulated cAMP production, but had no effect on [Ca2+]i, whereas PGF2 alpha evoked only [Ca2+]i increase. In contrast, PGE2 not only stimulated cAMP production, but also increased [Ca2+]i. From the Scatchard plot analysis of PGE2 it was confirmed that there were two classes of PGE2 binding sites (Kd value, 9.2 nM; binding site, 29 fmole/mg protein, and Kd value, 134 nM; binding site, 148 fmole/mg protein). As the increase in [Ca2+]i was caused by PGF2 alpha and PGE2, but not by PGE1, we investigated the displacement of [3H]-PGF2 alpha binding. The displacement capacity of unlabeled PGE2 was about 110 of that of PGF2 alpha, while that of PGE1 was very low even at 500-fold excess. These data indicate the possibility that the dual action of PGE2 is mediated by distinct receptor systems.     

Differential stimulation by PGE(2) and calcemic hormones of IL-6 in stromal/osteoblastic cells

Formation of osteoclast-like cells in mouse bone marrow cultures induced by either 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)), parathyroid hormone (PTH) or prostaglandin E(2) (PGE(2)), respectively, shows partial dependence on interleukin-6 receptor (IL-6R) activation. This suggests that locally produced IL-6 could be relevant for osteoclast formation. Therefore, we evaluated the effects of 1,25-(OH)(2)D(3), PTH, and PGE(2) on IL-6 production in stromal/osteoblastic cell lines. It appeared that these bone resorptive factors differed widely in their ability to modulate IL-6 mRNA expression and, consequently, protein synthesis in each of the cell lines studied. While 1,25-(OH)(2)D(3) was marginally effective only in ST2 cells, and PTH caused a 2- to 20-fold increase in IL-6 levels MC3T3-E1 and UMR-106 cells, PGE(2) enhanced IL-6 production in the ST2 and MC3T3-E1 cell line by two to three orders of magnitude, respectively, and also induced IL-6 in fibroblastic L929 cells. PGE(2)-stimulated IL-6 release from mesenchymal cells seems to be important for autocrine/paracrine control of osteoclast formation in health and disease.     

Acerogenin A, a natural compound isolated from Acer nikoense Maxim, stimulates osteoblast differentiation through bone morphogenetic protein action

We investigated the effects of acerogenin A, a natural compound isolated from Acer nikoense Maxim, on osteoblast differentiation by using osteoblastic cells. Acerogenin A stimulated the cell proliferation of MC3T3-E1 osteoblastic cells and RD-C6 osteoblastic cells (Runx2-deficient cell line). It also increased alkaline phosphatase activity in MC3T3-E1 and RD-C6 cells and calvarial osteoblastic cells isolated from the calvariae of newborn mice. Acerogenin A also increased the expression of mRNAs related to osteoblast differentiation, including Osteocalcin, Osterix and Runx2 in MC3T3-E1 cells and primary osteoblasts: it also stimulated Osteocalcin and Osterix mRNA expression in RD-C6 cells. The acerogenin A treatment for 3 days increased Bmp-2, Bmp-4, and Bmp-7 mRNA expression levels in MC3T3-E1 cells. Adding noggin, a BMP specific-antagonist, inhibited the acerogenin A-induced increase in the Osteocalcin, Osterix and Runx2 mRNA expression levels. These results indicated that acerogenin A stimulates osteoblast differentiation through BMP action, which is mediated by Runx2-dependent and Runx2-independent pathways.     

Apelin stimulates proliferation and suppresses apoptosis of mouse osteoblastic cell line MC3T3-E1 via JNK and PI3-K/Akt signaling pathways

The aim of this study was to investigate the effects of apelin on proliferation and apoptosis of mouse osteoblastic MC3T3-E1 cells. APJ was expressed in MC3T3-E1 cells. Apelin did not affect Runx2 expression, alkaline phosphatase (ALP) activity, osteocalcin and type I collagen secretion, suggesting that it has no effect on osteoblastic differentiation of MC3T3-E1 cells. However, apelin stimulated MC3T3-E1 cell proliferation and inhibited cell apoptosis induced by serum deprivation. Our study also shows that apelin decreased cytochrome c release and caspase-3, capase-8 and caspase-9 activation in serum-deprived MC3T3-E1 cells. Apelin activated c-Jun N-terminal kinase (JNK) and Akt (phosphatidylinositol 3-kinase downstream effector), and the JNK inhibitor SP600125, the phosphatidylinositol 3-kinase (PI3-K) inhibitor LY294002 or the Akt inhibitor 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate (HIMO) inhibited its effects on proliferation and serum deprivation-induced apoptosis. Furthermore, apelin protected against apoptosis induced by the glucocorticoid dexamethasone or TNF-alpha. Apelin stimulates proliferation and suppresses serum deprivation-induced apoptosis of MC3T3-E1 cells and these actions are mediated via JNK and PI3-K/Akt signaling pathways.     

Interleukin (IL)-17 enhances prostaglandin F(2 alpha)-stimulated IL-6 synthesis in osteoblasts

We previously showed that prostaglandin F(2alpha) (PGF(2alpha)) and endothelin-1 (ET-1) induce interleukin (IL)-6 through the activation of protein kinase C-dependent p44/p42 mitogen-activated protein (MAP) kinase in osteoblast-like MC3T3-E1 cells. It has recently been reported that tumor necrosis factor-alpha-induced IL-6 synthesis is amplified by IL-17 in these cells. In the present study, we investigated the effect of IL-17 on the IL-6 synthesis stimulated by PGF(2alpha) in MC3T3-E1 cells. IL-17 significantly enhanced the PGF(2alpha)-induced IL-6 synthesis in a dose-dependent manner in the range between 0.1 and 10 ng/ml. IL-17 also enhanced the IL-6 synthesis stimulated by 12- O -tetradecanoylphorbol-13-acetate, a direct activator of protein kinase C. In addition, IL-17 amplified the IL-6 synthesis induced by ET-1. However, IL-17 hardly affected the phosphorylation of p44/p42 MAP kinase induced by PGF(2alpha) or ET-1. These results strongly suggest that IL-17 enhances the IL-6 synthesis stimulated by PGF(2alpha) as well as ET-1 in osteoblasts, and that the effect is exerted at a point downstream from p44/p42 MAP kinase.     

Transforming growth factor-beta-induced gene expression of monocyte chemoattractant JE in mouse osteoblastic cells, MC3T3-E1

A recent study demonstrated that PDGF-inducible JE is an inflammatory cytokine that directs chemotactic activity of monocytes. Accumulation of monocyte/macrophage lineage cells at site of bone tissue sites is very important for formation of multinucleate osteoclasts, which mediate bone resorption. Since transforming growth factor-beta (TGF-beta) is a potent regulator in bone remodeling, we examined whether TGF-beta induced JE gene expression in mouse osteoblastic cells, MC3T3-E1. TGF-beta induced a maximum JE mRNA expression at 3 hr after initiation of the cytokine treatment. This maximal expression was observed in when TGF-beta was used at a concentration of 1 ng/ml. The chemotactic activity for human monocytes was detected in conditioned medium of TGF-beta-treated cells, and the chemotactic activity was neutralized by anti-JE serum treatment.     

The combination of high glucose and advanced glycation end-products (AGEs) inhibits the mineralization of osteoblastic MC3T3-E1 cells through glucose-induced increase in the receptor for AGEs.

Type 1 diabetes mellitus is known to be associated with reduced bone mass and increased bone fractures. This is thought to be due to a decrease in osteoblastic bone formation rather than an increase in osteoclastic bone resorption, but the precise mechanism is unknown. In this study, we examined whether or not high glucose or advanced glycation end-products (AGEs), which play key roles in the pathogenesis and complications of diabetes, affect the differentiation of osteoblastic MC3T3-E1 cells. First, MC3T3-E1 cells were incubated in media containing either 22 mM glucose, 22 mM mannitol, 300 microg/ml AGE2, or 300 microg/ml AGE3. Each of these agents alone did not affect the mineralization of the cells by von Kossa staining and Alizarin red staining. However, high glucose but not mannitol or AGEs markedly increased mRNA expression of AGE receptor (RAGE) by real-time PCR. Next, we examined the combined effects of high glucose and AGEs on the differentiation of MC3T3-E1 cells. The combination of 22 mM glucose and 300 microg/ml AGE2 significantly inhibited the mineralization of MC3T3-E1 cells, and 22 mM glucose in combination with either 300 microg/ml AGE2 or AGE3 apparently decreased osteocalcin mRNA expression. These results suggest that high glucose or AGEs alone might have no effect on osteoblastic differentiation, but their combination could additionally or synergistically inhibit osteoblastic mineralization through glucose-induced increase in RAGE expression.     

Stimulation of prostaglandin E2 (PGE2) production by arachidonic acid, oestrogen and parathyroid hormone in MG-63 and MC3T3-E1 osteoblast-like cells

Polyunsaturated fatty acids (PUFAs) as well as oestrogen (E2) and parathyroid hormone (PTH) affect bone cells. The aim of the study was to determine whether arachidonic acid (AA), E2, and PTH increase prostaglandin E₂ (PGE₂) synthesis in MG-63 and MC3T3-E1 osteoblastic cells and the level of mediation by COX-1 and COX-2. PGE₂ levels were determined in the conditioned culture media of MG-63 and MC3T3-E1 osteoblasts after exposure to AA, PTH and E2. Cells were pre-incubated in some experiments with the unselective COX inhibitor indomethacin or the COX-2 specific blocker NS-398. Indirect immunofluorescence was performed on MG-63 cells to detect the presence and location of the two enzymes involved. AA increased PGE₂ secretion in both cell lines; production by MC3T3-E1 cells, however, was significantly higher than that of MG-63 cells. This could be due to autoamplification via the EP₁ subtype of PGE receptors in mouse MC3T3-E1 osteoblasts. Both COX-1 and COX-2 affected the regulation of PGE₂ synthesis in MG-63 cells. E2 had no effect on PGE₂ secretion in both cell lines, while PTH caused a slight increase in PGE₂ synthesis in the MG-63 cell line.     

Effects of arachidonic acid, docosahexaenoic acid, prostaglandin E(2) and parathyroid hormone on osteoprotegerin and RANKL secretion by MC3T3-E1 osteoblast-like cells

Bone is continuously remodeled through resorption by osteoclasts and the subsequent synthesis of the bone matrix by osteoblasts. Cell-to-cell contact between osteoblasts and osteoclast precursors is required for osteoclast formation. RANKL (receptor activator of nuclear factor-kappaB ligand) expressed on osteoblastic cell membranes stimulates osteoclastogenesis, while osteoprotegerin (OPG) secreted by osteoblasts inhibits osteoclastogenesis. Although polyunsaturated fatty acids (PUFAs) have been implicated in bone homeostasis, the effects thereof on OPG and RANKL secretion have not been investigated. MC3T3-E1 osteoblasts were exposed to the n-6 PUFA arachidonic acid (AA) and the n-3 PUFA docosahexaenoic acid (DHA); furthermore, the bone-active hormone parathyroid hormone (PTH) and the effects thereof were tested on OPG and RANKL secretion. Prostaglandin E(2) (PGE(2)), a product of AA metabolism that was previously implicated in bone homeostasis, was included in the study. AA (5.0-20 microg/ml) inhibited OPG secretion by 25-30%, which was attenuated by pretreatment with the cyclooxygenase blocker indomethacin, suggesting that the inhibitory effect of AA on OPG could possibly be PGE(2)-mediated. MC3T3-E1 cells secreted very low basal levels of RANKL, but AA stimulated RANKL secretion, thereby decreasing the OPG/RANKL ratio. DHA suppressed OPG secretion to a smaller extent than AA. This could, however, be due to endogenous PGE(2) production. No RANKL could be detected after exposing the MC3T3-E1 cells to DHA. PTH did not affect OPG secretion, but stimulated RANKL secretion. This study demonstrates that AA and PTH reduce the OPG/RANKL ratio and may increase osteoclastogenesis. DHA, however, had no significant effect on OPG or RANKL in this model.     

Autocrine/paracrine regulation of apoptosis in epithelial cells by prostaglandin E2

This study was designed to investigate the effect of IL-1alpha-induced up-regulation of cyclooxygenase-2 (COX-2) on prostaglandin E(2) (PGE(2)) secretion and the subsequent phenotypic effects of PGE(2) on epithelial cells. The effect of IL-1alpha on COX-2 expression was investigated in the T24 bladder epithelial cell line following treatment with 0, 0.05, 0.5, 1 or 10 ng/ml IL-1alpha for 1, 2, 4 or 6 h. Quantitative PCR confirmed up-regulation of expression of COX-2 with maximal expression observed following treatment with 0.5 ng/ml IL-1alpha for 1 h. Co-treatment of the cells with 0.5 ng/ml IL-1alpha in the presence or absence of 100 ng/ml IL-1 receptor antagonist (RA) abolished the up-regulation in COX-2 expression confirming that the effect of IL-1alpha is mediated via its membrane-bound receptors. Treatment with 0.5 ng/ml IL-1alpha resulted in a time-dependent increase in PGE(2) secretion with maximal secretion detected at 24 and 48 h after stimulation with IL-1alpha. Co-treatment of the cells with IL-1alpha and IL-1RA or the COX-2 enzyme inhibitor NS398 abolished the IL-1alpha mediated secretion of PGE(2). Treatment of T24 cells with 100 nM PGE(2) resulted in a significant elevation in cAMP generation confirming the expression of functional PGE(2) receptors. Finally, the effect of exogenous treatment with PGE(2) on apoptosis of T24 cells was assessed using cell death detection ELISA. T24 cells were treated with camptothecin to induce apoptosis in the presence or absence of 50 or 100 nM PGE(2) or 10 microM forskolin. Treatment of T24 cells with increasing doses of camptothecin alone resulted in a significant increase in the induction of apoptosis (P<0.01). However, co-treatment of the cells with 50 or 100 nM PGE(2) or 10 microM forskolin resulted in the inhibition of induction of the apoptotic pathway by camptothecin. These data demonstrate that PGE(2) inhibits apoptosis of epithelial cells possibly via cAMP-dependent pathway.     

Mechanical stimulation of osteopontin mRNA expression and synthesis in bone cell cultures

We have shown earlier that mechanical stimulation by intermittent hydrostatic compression (IHC) promotes alkaline phosphatase and procollagen type I gene expression in calvarial bone cells. The bone matrix glycoprotein osteopontin (OPN) is considered to be important in bone matrix metabolism and cell-matrix interactions, but its role is unknown. Here we examined the effects of IHC (13 kPa) on OPN mRNA expression and synthesis in primary calvarial cell cultures and the osteoblast-like cell line MC3T3-E1. OPN mRNA expression declined during control culture of primary calvarial cells, but not MC3T3-E1 cells. IHC upregulated OPN mRNA expression in late released osteoblastic cell cultures, but not in early released osteoprogenitor-like cells. Also, in both proliferating and differentiating MC3T3-E1 cells, OPN mRNA expression and synthesis were enhanced by IHC, differentiating cells being more responsive than proliferating cells. These results suggest a role for OPN in the reaction of bone cells to mechanical stimuli. The severe loss of OPN expression in primary bone cells cultured without mechanical stimulation suggests that disuse conditions down-regulate the differentiated osteoblastic phenotype. J. Cell. Physiol. 170:174–181, 1997. © 1997 Wiley-Liss, Inc.