Stimulation by 1,25-dihydroxyvitamin D3 of in vitro mineralization induced by osteoblast-like MC3T3-E1 cells

Although vitamin D is essential for mineralization of bone, it is as yet unclear whether vitamin D has a direct stimulatory effect on the bone mineralization process. In the present study, the effect of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on in vitro mineralization mediated by osteoblast-like MC3T3-E1 cells was examined. MC3T3-E1 cells continued to grow after they reached confluency, and DNA content and alkaline phosphatase activity increased linearly until about 16 days of culture, whereas 45Ca accumulation into cell and matrix layer remained low. After this period, DNA content plateaued, and 45Ca accumulation increased sharply. Histological examination by von Kossa staining revealed that calcium was accumulated into extracellular matrix. In addition, needle-shaped mineral crystals similar to hydroxyapatite crystals could be demonstrated in between collagen fibrils by electron microscopy. Thus, MC3T3-E1 cells differentiate in vitro into cells with osteoblastic phenotype and exhibit mineralization. When MC3T3-E1 cells were treated with 1,25(OH)2D3 at this stage of culture, there was a dose-dependent stimulation of 45Ca accumulation by 1,25(OH)2D3, and a significant stimulation of 45Ca accumulation was observed with 3 x 10(-10) M 1,25(OH)2D3. Although 1,25(OH)2D3 enhanced alkaline phosphatase activity and collagen synthesis at the early phase of culture, it did not affect any of these parameters at the late phase when 1,25(OH)2D3 stimulated mineralization. Neither 24,25-dihydroxyvitamin D3 nor human PTH(1-34) affected mineralization in the presence or absence of 1,25(OH)2D3. These results demonstrate that 1,25(OH)2D3 stimulates matrix mineralization induced by osteoblastic MC3T3-E1 cells, and are consistent with the possibility that 1,25(OH)2D3 has a direct stimulatory effect on bone mineralization process.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential regulation of prostaglandin E2 synthesis and phospholipase A2 activity by 1,25-(OH)2D3 in three osteoblast-like cell lines (MC-3T3-E1, ROS 17/2.8, and MG-63).

Both 1,25-(OH)2D3 and prostaglandin E2 (PGE2) stimulate alkaline phosphatase activity in MC-3T3-E1 cells. Previous studies, demonstrating a correlation between 1,25-(OH)2D3-dependent alkaline phosphatase and phospholipase A2 activities in matrix vesicles isolated from growth cartilage chondrocyte cultures, suggest that one mechanism of vitamin D action may be via autocrine or paracrine action of PGE2. Since most PGE2 is derived from arachidonic acid released by the action of phospholipase A2, we examined whether 1,25-(OH)2D3 stimulates phospholipase A2 activity in three osteoblastic cell lines: ROS 17/2.8 cells, MC-3T3-E1 cells, and MG-63 cells. 1,25-(OH)2D3-dependent alkaline phosphatase and phospholipase A2 activity were correlated with production of PGE2 and PGE1 in the MC-3T3-E1 cells. Alkaline phosphatase specific activity was enriched in the matrix vesicles produced by all three cell types and was stimulated by 1,25-(OH)2D3 at 10(-8) to 10(-7) M. Although phospholipase A2 specific activity was enriched in the matrix vesicles produced only by the ROS 17/2.8 cell cultures, stimulation of this enzyme activity was observed only in the MC-3T3-E1 cell cultures. The effects of 1,25-(OH)2D3 on phospholipase A2 were dose-dependent and were significant at 10(-8) to 10(-7) M. There was a significant increase in PGE2 production in the MC-3T3-E1 cell cultures only. Indomethacin reduced PGE2 production to base line values. Even at baseline, MC-3T3-E1 cells produced ten times more PGE2 than did the ROS 17/2.8 or MG-63 cell cultures. The effects of 1,25-(OH)2D3 on PGE1 were comparable to those on PGE2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibitory Effects of 1,25(OH)2D3 on Membrane-Associated and Cytosolic Casein Kinase Activity in MC3T3-E1 Osteoblast-like Cells

The secretion of phosphorylated matrix proteins is high in osteoblasts. Phosphorylation of these proteins may be catalyzed by casein kinases (CK), and CK may play an important role in the site of bone mineralization. In this study, we examined the effects of 1,25(OH)₂D₃ on CK activities in MC3T3-E1 osteoblast-like cells. Different concentrations (ranging from 10⁻⁷ to 10⁻¹¹M) of 1,25(OH)₂D₃ were included in a culture medium. After incubation for various lengths of time, MC3T3-E1 cells were homogenized and segregated into cytosolic (c) and microsomal (m) fractions. To measure CK activity, each fraction was used as an enzyme source to phosphorylate casein. MC3T3-E1 cells showed the highest cCK activity after incubation for 21 days, and showed the highest mCK activity after incubation for 14 days. 1,25(OH)₂D₃ inhibited mCK activity at the early stage of culture, but inhibited cCK activity at the late stage of culture. In contrast, 1,25(OH)₂D₃ had a slight stimulatory effect on CK activity in the culture medium of MC3T3-E1 cells. Our data suggest that cCK and mCK may play different roles in the function of osteoblasts, and 1,25(OH)₂D₃ regulates intracellular and extracellular casein kinase activities related to the function of osteoblasts. Received: 26 June 1997 / Accepted: 23 March 1998     

Analysis of vitamin D-regulated gene expression in LNCaP human prostate cancer cells using cDNA microarrays

BACKGROUND: 1,25-dihydroxyvitamin D(3) [1,25(OH)2D3] exerts growth inhibitory, pro-differentiating, and pro-apoptotic effects on prostate cells. To better understand the molecular mechanisms underlying these actions, we employed cDNA microarrays to study 1,25(OH)2D3-regulated gene expression in the LNCaP human prostate cancer cells. METHODS: mRNA isolated from LNCaP cells treated with vehicle or 50 nM 1,25(OH)2D3 for various lengths of time were hybridized to microarrays carrying approximately 23,000 genes. Some of the putative target genes revealed by the microarray analysis were verified by real-time PCR assays. RESULTS: 1,25(OH)2D3 most substantially increased the expression of the insulin-like growth factor binding protein-3 (IGFBP-3) gene. Our analysis also revealed several novel 1,25(OH)2D3-responsive genes. Interestingly, some of the key genes regulated by 1,25(OH)2D3 are also androgen-responsive genes. 1,25(OH)2D3 also down-regulated genes that mediate androgen catabolism. CONCLUSIONS: The putative 1,25(OH)2D3 target genes appear to be involved in a variety of cellular functions including growth regulation, differentiation, membrane transport, cell-cell and cell-matrix interactions, DNA repair, and inhibition of metastasis. The up-regulation of IGFBP-3 gene has been shown to be crucial in 1,25(OH)2D3-mediated inhibition of LNCaP cell growth. 1,25(OH)2D3 regulation of androgen-responsive genes as well as genes involved in androgen catabolism suggests that there are interactions between 1,25(OH)2D3 and androgen signaling pathways in LNCaP cells. Further studies on the role of these genes and others in mediating the anti-cancer effects of 1,25(OH)2D3 may lead to better approaches to the prevention and treatment of prostate cancer.     

Osteotropic agents induce the differential secretion of granulocyte-macrophage colony-stimulating factor by the osteoblast cell line MC3T3-E1

Osteoblasts play a central role in the regulation of bone remodeling. Not only are they responsible for the formation of new bone, but they also regulate bone resorption. These cells also exert regulatory influences outside the bone in that they are able to regulate hematopoiesis. However, obtaining pure populations of osteoblasts devoid of contaminating cell types remains problematic. One approach to this problem is the use of cloned osteoblastic cell lines. To this end we have used MC3T3-E1, a cloned murine osteoblast cell line of C57BL/6 origin. We report that MC3T3-E1 cells respond to lipopolysaccharide (LPS) and, to a lesser extent, parathyroid hormone (PTH) by the secretion of granulocyte-macrophage colony-stimulating factor (GM-CSF). However, 1,25-(OH)2D3, a potent activator of osteoblasts, fails to induce these cells to secrete GM-CSF. These results suggest that MC3T3-E1 cells respond to osteotropic agents in a hierarchical fashion. Secretion of GM-CSF is not constitutive but rather requires active induction of the cells. MC3T3 cells fail to secrete detectable levels of interleukin-2 (IL-2), IL-3, or IL-4, regardless of whether or not the cells are activated. The data indicate that MC3T3-E1 cells secrete cytokines in response to osteotropic agents in a way similar to that of normal primary osteoblasts. Therefore, MC3T3-E1 cells may serve as a good in vitro model for primary osteoblasts.     

MLO-Y4 osteocyte-like cells support osteoclast formation and activation.

Osteocytes are terminally differentiated cells of the osteoblast lineage that have become embedded in mineralized matrix and may send signals that regulate bone modeling and remodeling. The hypothesis to be tested in this study is that osteocytes can stimulate and support osteoclast formation and activation. To test this hypothesis, an osteocyte-like cell line called MLO-Y4 and primary murine osteocytes were used in coculture with spleen or marrow cells. MLO-Y4 cells support osteoclast formation in the absence of 1,25-dihydroxyvitamin D3 [1,25(OD)2D3] or any other exogenous osteotropic factor. These cells alone stimulate osteoclast formation to the same extent or greater than adding 1,25(OH)2D3. Coaddition of 1,25(OH)2D3 with MLO-Y4 cells synergistically increased osteoclast formation. Optimal osteoclast formation and pit formation on dentine was observed with 200-1,000 MLO-Y4 cells per 0.75-cm2 well. No osteoclast formation was observed with 2T3, OCT-1, or MC3T3-E1 osteoblast cells (1,000 cells/well). Conditioned media from the MLO-Y4 cells had no effect on osteoclast formation, indicating that cell contact is necessary. Serial digestions of 2-week-old mouse calvaria yielded populations of cells that support osteoclast formation when cocultured with 1,25(OH)2D3 and marrow, but the population that remained in the bone particles supported the greatest number of osteoclasts with or without 1,25(OH)2D3. To examine the mechanism whereby these cells support osteoclast formation, the MLO-Y4 cells were compared with a series of osteoblast and stromal cells for expression of macrophage colony-stimulating factor (M-CSF), RANKL, and osteoprotegerin (OPG). MLO-Y4 cells express and secrete large amounts of M-CSF. MLO-Y4 cells express RANKL on their surface and their dendritic processes. The ratio of RANKL to OPG mRNA is greatest in the MLO-Y4 cells compared with the other cell types. RANK-Fc and OPG-Fc blocked the formation of osteoclasts by MLO-Y4 cells. These studies suggest that both RANKL and OPG may play a role in osteocyte signaling, OPG and M-CSF as soluble factors and RANKL as a surface molecule that is functional in osteocytes or along their exposed dendritic processes.     

健骨胶囊对成骨样细胞MC3T3-E1增殖及分化作用机制

目的 探讨国医大师刘柏龄“健骨胶囊”对MC3T3-E1成骨细胞分化及增殖的影响。方法 制备健骨胶囊水提物,采用CCK-8法和细胞迁移实验检测健骨胶囊提取物对MC3T3-E1细胞增殖和细胞迁移的影响;茜素红染色检测MC3T3-E1细胞的矿化能力;实时荧光定量PCR检测成骨分化基因Runx2、OCN、OPN、Col1a1、ALP、Bcl2、RASSF1A等mRNA表达水平;蛋白质印迹法Western blot检测Col1a1、Bcl2的蛋白表达水平。结果 通过实验结果比对得出,健骨胶囊提取物能促进MC3T3-E1细胞增殖、使细胞迁移率提高;同时健骨胶囊提取物组能明显提高MC3T3-E1细胞钙化能力(P＜0.01),促进Runx2、OCN、OPN、Col1a1、ALP、Bcl2的mRNA表达(P＜0.05),上调Col1a1、Bcl2蛋白量的表达。结论 健骨胶囊能促进成骨细胞MC3T3-E1的增殖及细胞迁移能力,并通过上调成骨基因的表达水平如Runx2、OCN、OPN、Col1a1、ALP、Bcl2等,提高MC3T3-E1细胞的成骨能力。     

Cyclooxygenase inhibitors enhance cell growth in an osteoblastic cell line, MC3T3-E1

To elucidate the significance of endogenous prostaglandin E2 (PGE2) in osteoblastic cell function, we studied the effects of cyclooxygenase inhibitors on cell growth and alkaline phosphatase (ALP) activity in MC3T3-E1 cells. UMR-106 cells were also used as references in our experiments. MC3T3-E1 cells, cultured in alpha-minimal essential medium containing 10% fetal bovine serum, were shown to produce PGE2, which was markedly suppressed in the presence of indomethacin. Addition of indomethacin resulted in an increase in DNA content and [3H]thymidine incorporation. A similar growth stimulatory effect was observed when structurally different cyclooxygenase inhibitors, that is, acetyl salicylic acid (ASA), flurbiprofen, and piroxicam, were added. These cyclooxygenase inhibitors, however, differed in their effects on ALP activity. Indomethacin and ASA enhanced ALP activity, whereas flurbiprofen and piroxicam suppressed it. We then examined the effects of exogenous addition of PGE2. Although exogenous PGE2 at 6 x 10(-6) M slightly stimulated cell growth, it inhibited cell growth at 6 x 10(-8) M and 6 x 10(-7) M. ALP activity was reduced in a dose-dependent fashion by exogenous PGE2. These results suggest that PGE2 produced by MC3T3-E1 may be suppressing cell proliferation and that cyclooxygenase inhibitors, per se, may stimulate cell growth by inhibiting endogenous PGE2 production in MC3T3-E1 cells. UMR-106 cells also produced PGE2, although less than MC3T3-E1 cells. In UMR-106 cells, the cyclooxygenase inhibitors did not influence DNA content or ALP activity as distinctly as in MC3T3-E1 cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vitamin D analogs with low affinity for the vitamin D binding protein: enhanced in vitro and decreased in vivo activity.

The affinity of 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25-(OH)2D3] and analogs with side-chain modifications [MC 903 or calcipotriol, MC 1147 or 24,24-dihomo-1 alpha,25-(OH)2D3 and 1,25-(OH)2-16ene-23yne-D3] for the vitamin D receptor and the serum vitamin D binding protein (DBP) were compared. The affinity of MC 903 for the receptor from chick and rat duodenum or from human peripheral blood mononuclear cells or HL-60 cells varied between 60 and 100% relative to the affinity of 1,25-(OH)2D3. The relative affinity of 1,25-(OH)2-16ene-23yne-D3 and MC 1147 varied for the same receptors between 45-70 and 3.5-25%, respectively. The relative affinity of MC 903 for human DBP was 30-fold decreased, whereas the two other analogs did not bind to DBP at all even in more than 1000-fold excess. The in vitro biologic activity of 1 alpha,25-(OH)2D3 on phytohemagglutinin-stimulated normal human lymphocyte proliferation was markedly inhibited by the addition of physiologic amounts of DBP to the cell culture medium. No such inhibition was observed when MC 903 or 1147 was evaluated similarly. DBP therefore reversed the rank order of the in vitro potency of these analogs. Intramuscular injections for 10 consecutive days to vitamin D-deficient chicks demonstrated a greater than or equal to 100-fold lower biologic activity of MC 903, MC 1147, and 1,25-(OH)2-16ene-23yne-D3 compared to that of 1 alpha,25-(OH)2D3 as evaluated by serum calcium and osteocalcin concentrations, as well as by duodenal calbindin D28K and bone calcium content.(ABSTRACT TRUNCATED AT 250 WORDS)     

Insulin-like growth factor binding protein-3 mediates 1 alpha,25-dihydroxyvitamin d(3) growth inhibition in the LNCaP prostate cancer cell line through p21/WAF1

PURPOSE: We determined that insulin-like growth factor binding protein 3 (IGFBP-3) induction by 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) is a necessary component of 1,25-(OH)(2)D(3) mediated growth inhibition of the LNCaP human prostate cancer cell line. In addition, induction of the cyclin dependent kinase inhibitory protein p21/WAF/CIP1 by 1,25-(OH)(2)D(3) is mediated by IGFBP-3. MATERIALS AND METHODS: Induction of IGFBP-3 by 1,25-(OH)(2)D(3) was determined by enzyme-linked immunosorbent assay for IGFBP-3 protein and by Northern blot analysis for IGFBP-3 messenger (m) RNA. Growth assays for LNCaP cells were determined by measuring DNA content. The contribution of IGFBP-3 toward 1,25-(OH)(2)D(3) mediated growth inhibition was determined by adding either antisense oligonucleotides or immuno-neutralizing antibodies to culture media of growth assays. Regulation of p21/WAF/CIP1 was determined by Western blot analysis. RESULTS: Adding 1,25-(OH)(2)D(3) to LNCaP prostate cancer cells demonstrated that 1,25-(OH)(2)D(3) significantly up-regulated IGFBP-3 at the mRNA and protein levels in these cells approximately 3-fold over control levels. Also, adding IGFBP-3 protein to LNCaP cell growth medium inhibited LNCaP cell growth. Interestingly adding IGFBP-3 antisense oligonucleotides or antibodies directed toward IGFBP-3 abolished the growth inhibitory actions of 1,25-(OH)(2)D(3), indicating that this effect is IGFBP-3 dependent. Furthermore, to connect the mechanisms of IGFBP-3 and 1,25-(OH)(2)D(3) mediated growth inhibition we demonstrated that IGFBP-3 up-regulates the expression of p21/WAF1 protein to approximately 2-fold over the control level. Adding an IGFBP-3 immuno-neutralizing antibody completely prevented the 1,25-(OH)(2)D(3) induced up-regulation of p21/WAF1. CONCLUSIONS: 1,25-(OH)(2)D(3) up-regulates IGFBP-3 in the LNCaP cell line at the mRNA and protein levels. The growth inhibitory action of 1,25-(OH)(2)D(3) on LNCaP cells depends on active IGFBP-3, as evidenced by the loss of growth inhibition induced by IGFBP-3 antisense oligonucleotide and immuno-neutralization experiments. A possible connection between IGFBP-3 and 1,25-(OH)(2)D(3) lies in the cyclin dependent kinase inhibitory protein p21/WAF1 since IGFBP-3 and 1,25-(OH)(2)D(3) each up-regulate this protein and both inhibit LNCaP cell growth. Therefore, we hypothesize that the mechanism of action by which IGFBP-3 and 1,25-(OH)(2)D(3) induce growth inhibition is the induction of p21/WAF1 because IGFPB-3 immuno-neutralizing antibodies completely abrogate the 1,25-(OH)(2)D(3) mediated up-regulation of p21/WAF1 and growth inhibition.     

Actions of calcipotriol (MC 903), a novel vitamin D3 analog, on human bone-derived cells: comparison with 1,25-dihydroxyvitamin D3.

The actions of a novel vitamin D3 analog calcipotriol (MC 903), on human bone-derived cells were compared to those of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. Both calcipotriol and 1,25-(OH)2D3 inhibited the proliferation of human osteoblast-like cells in a dose-dependent manner (10(-10)-10(-6) M), an effect observed at different cell densities. Lower concentrations of either agent exerted no marked effect on the growth of the cells compared to untreated cultures. Calcipotriol and 1,25-(OH)2D3 were equipotent in stimulating the activity of alkaline phosphatase and the synthesis of osteocalcin in human osteoblast-like cells. The stimulation of alkaline phosphatase activity and osteocalcin synthesis by both compounds was evident by 24 h and was increased progressively up to 96 h in a dose-dependent manner over the concentration range of 10(-10)-10(-6) M. The increment in both proteins was dependent on cell density and was attenuated at higher cell densities. In contrast to these actions, neither calcipotriol nor 1,25-(OH)2D3 (10(-14)-10(-6) M) affected the synthesis of prostaglandin E2. These studies indicate that calcipotriol and 1,25-(OH)2D3 exhibit a similar spectrum of activity on human osteoblast-like cells in vitro.     

Modulation of 1,25-dihydroxyvitamin D3 receptor binding and action by sodium butyrate in cultured pig kidney cells (LLC-PK1)

We have studied the effects of sodium butyrate (SB) on 1,25(OH)2D3 receptors in the pig kidney cell line (LLC-PK1). In this system, we have shown that 1,25(OH)2D3 induction of 25-hydroxyvitamin D3-24-hydroxylase (24-hydroxylase) activity is dependent on the level of 1,25(OH)2D3 receptors. Treatment of confluent cells with SB (5 mM/48 h) caused an approximately 50% decrease in total receptors, whereas the affinity for 1,25(OH)2D3 was unchanged. At 5 mm, the action of SB on these receptors required more than 24 h to be detected. The effect of the decrease in receptors on the functional response to the hormone was studied by measuring the 1,25(OH)2D3 induction of 24-hydroxylase activity after treatment with SB. The induction of 24-hydroxylase activity at higher doses of hormone paralleled the reduction in receptors and was diminished by 25-50%. At low doses of hormone, the cells appear to be more sensitive to 1,25(OH)2D3 induction, exhibiting an unexplained increase in 24-hydroxylase activity compared to cells not exposed to SB. An additional effect of SB was also noted: SB decreased cell proliferation and inhibited [3H]thymidine incorporation by 75% when added to cells prior to confluence. At confluence, SB caused a less drastic effect on protein and DNA synthesis. Therefore, most binding experiments were conducted at confluence when the SB effect on cell proliferation was less. Other short chain fatty acids in addition to SB were also tested. The action to decrease 1,25(OH)2D3 receptors was more specific upon exposure to SB. We have previously demonstrated up-regulation of 1,25(OH)2D3 receptors in LLC-PK1 cells after treatment with various vitamin D metabolites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Strontium ranelate promotes osteoblastic cell replication through at least two different mechanisms

The cellular and molecular mechanisms involved in osteoblastic cell replication induced by strontium ranelate are presently under investigation. The calcium-sensing receptor is a suggested target but other potential mechanisms have not been investigated. Signaling pathways involved in strontium ranelate-induced replication were investigated in preosteoblastic MC3T3-E1 and pluripotent mesenchymal C3H10T1/2 cells. Strontium ranelate effects were compared with those of calcium chloride as Ca(2+). In MC3T3-E1 cells, strontium ranelate but not CaCl(2) dose-dependently increased cell number whereas similar effects were observed for both cations in C3H10T1/2 cells. Immunoblot analysis indicated that activation of ERK, PKC and PKD by strontium ranelate in MC3T3-E1 cells was delayed compared with CaCl(2). Indeed, onset of signaling by strontium ranelate was detected after one or several hours whereas CaCl(2) had a maximal effect already after 5 min exposure. In C3H10T1/2 cells, strontium ranelate induced two types of signaling, a rapid effect and a delayed response. In addition to activation of ERK, PKC and PKD, strontium ranelate and CaCl(2) also transiently activated p38 in C3H10T1/2 cells. Functional analysis with specific inhibitors indicated that cell replication induced by strontium ranelate involves a PKC/PKD pathway in MC3T3-E1 cells and p38 in C3H10T1/2 cells. In both cell types, inhibition of the ERK pathway decreased basal cell replication but not the strontium ranelate response. In conclusion, strontium ranelate increases the replication of cells of the osteoblastic lineage by two distinct cellular mechanisms. Strontium ranelate may directly interact with the CaSR and trigger mitogenic signals such as p38 in C3H10T1/2 cells. The delayed activation of several signaling pathways in both cell lines, however, suggests the release of an autocrine growth factor by strontium ranelate that represents another potential mechanism for inducing osteoblastic cell replication.     

Evidence for the involvement of two pathways in activation of extracellular signal-regulated kinase (Erk) and cell proliferation by Gi and Gq protein-coupled receptors in osteoblast-like cells.

The mechanisms by which Gi and Gq protein- coupled receptors mediate mitogenic signaling in osteoblast-like cells are unknown and were investigated in MC3T3-E1 cells using specific receptor agonists such as lysophosphatidic acid (LPA) and prostaglandin F2alpha (PGF2alpha). In contrast to their implication in epidermal growth factor (EGF) receptor tyrosine kinase signaling, the adaptor protein Shc, the Grb2/Sos complex, and the small G protein Ras were not involved in the activation of Erk induced by either LPA or PGF2alpha in MC3T3-E1 cells, suggesting that activation of Erk by Gi and Gq protein-coupled receptors is Ras independent in these cells. Using specific kinase inhibitors and kinetic analyses, we provide evidence for two distinct components in the activation of Erk by Gi and Gq protein-coupled receptors in MC3T3-E1 cells including an Src-like kinase-dependent pathway and a protein kinase C (PKC)-dependent mechanism. Functional analyses suggested that these two components are required for optimal DNA synthesis in response to LPA and PGF2alpha. These results suggest the implication of two pathways in the stimulation of Erk and cell replication by growth factors acting through Gi and Gq protein-coupled receptors in bone-forming cells.