Differential Effects of Two Protein Tyrosine Kinase Inhibitors, Tyrphostin and Genistein, on Human Bone Cell Proliferation as Compared with Differentiation

Protein tyrosyl phosphorylation is a key determinant of cell proliferation and differentiation. The aim of this study was to test the hypothesis that the signal transduction pathway(s) responsible for human bone cell proliferation may involve different groups of protein tyrosine kinase (PTKs) as compared with that for differentiation. To achieve this, we investigated the effects of two structurally different PTK inhibitors, viz, tyrphostin A51 and genistein, on the proliferation ([³H]thymidine incorporation) and differentiation [alkaline phosphatase (ALP) specific activity and collagen synthesis] of two normal human bone cell types: mandible-derived and vertebra-derived bone cells. Tyrphostin A51 and genistein each markedly reduced cellular tyrosyl phosphorylation level (assessed by Western analysis using a commercial anti-phosphotyrosine antibody and the enhanced chemiluminescence detection assay), confirming that these two effectors are potent PTK inhibitors in human bone cells. Regarding bone cell proliferation, tyrphostin A51 (5–30 μM) caused, a dose-dependent inhibition of basal [³H]thymidine incorporation of both human bone cell types. In contrast, genistein (5–20 μM), not only did not inhibit, but significantly stimulated [³H]thymidine incorporation of these same cell types in a dose-dependent, biphasic manner, with the optimal stimulatory dose between 10 and 20 μM. These effects on cell proliferation were confirmed by cell number counting. In addition, whereas the mitogenic activity of 10 ng/ml epidermal growth factor (EGF) on human mandible-derived bone cells was completely abolished by 5–30 μM tyrphostin A51, genistein at 5–30 μM enhanced the EGF-induced bone cell proliferation in an additive manner. With respect to bone cell differentiation, tyrphostin A51 and genistein each significantly increased basal ALP specific activity and collagen synthesis in human bone cells. In summary, (1) PTKs are involved in human bone cell proliferation and differentiation; (2) tyrphostin A51 inhibited both basal and EGF-induced cell proliferation, thus tyrphostin-sensitive PTKs are involved in basal and EGF-induced human bone cell proliferation; (3) genistein stimulated basal proliferation and enhanced EGF-mediated cell proliferation, suggesting that genistein-sensitive PTKs may play an inhibitory role in human bone cell proliferation; and (4) these differential effects of PTK inhibitors on human bone cell proliferation and differentiation are independent of basal differentiation status of the cells. Received: 30 July 1997 / Accepted: 20 February 1998     

Glucocorticoid-induced osteoporosis in the rat is prevented by the tyrosine phosphatase inhibitor,sodium orthovanadate

Glucocorticoid-induced osteoporosis is characterized by decreased osteoblast numbers and a marked impairment of new bone formation. We found that, in vitro, dexamethasone inhibits both preosteoblast proliferation and mitogenic kinase activity in response to mitogens, and that inhibition of protein tyrosine phosphatases (PTPs) using sodium orthovanadate prevents this. Therefore, dexamethasone may act by either upregulating antiproliferative PTPs or downregulating promitogenic tyrosine-phosphorylated substrates. In this study, osteoporosis was induced in 3.5-month-old rats by subcutaneous injection with methylprednisolone 3.5 mg/kg per day for 9 weeks. Rats were treated with steroid alone or in combination with 0.5 mg/mL sodium orthovanadate, administered continuously in drinking water. Steroid-treated bones were significantly (p < 0.005) osteopenic (according to dual-energy X-ray absorptiometry) and physically weaker (p < 0.05) than controls. Quantitative bone histology confirmed a significant decrease in osteoid surfaces (p < 0.001), osteoblast numbers (p < 0.05), and rate of bone formation (p < 0.001). Concomitant treatment with vanadate largely prevented the densitometric, histologic, and physical abnormalities induced by prednisolone. This study supports our finding that PTPs are central to the negative regulation of osteoblast proliferation by glucocorticoids and, furthermore, suggests that PTP inhibitors such as sodium orthovanadate should be considered as novel anabolic agents for the treatment of steroid-induced osteoporosis.     

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)     

Fluoride and bovine bone extract influence cell proliferation and phosphatase activities in human bone cell cultures

The effects of fluoride (20 mumol/L) and bovine bone extract (17 micrograms/ml) were determined on cultures of human bone cells, embryonic chick bone cells, and human skin fibroblasts. The incorporation of [3H]thymidine into DNA was measured 16 hours after the addition of factors. After three to five days treatment, Triton X-100 extracts of the cells were assayed for acid phosphatase activity, in the presence and absence of tartrate, and for alkaline phosphatase activity. Fluoride stimulated [3H]thymidine incorporation and specific activity of alkaline phosphatase in human bone cells and chick bone cells but not in human skin cells. Fluoride also stimulated the cell population doubling rate of the human bone cells with an optimum of approximately 20 mumol/L. Bovine bone extract stimulated thymidine uptake into DNA several-fold and decreased alkaline phosphatase activity in all three types of cultured cells. The specific activity of tartrate-resistant acid phosphatase was increased in bone cells but not in skin fibroblasts. These results suggest that fluoride specifically stimulates the proliferation and differentiation of osteoblasts, while the growth factors in bovine bone extract primarily stimulate proliferation of bone cells. Cultures of human bone cells respond similarly to chick calvarial cells when treated with fluoride or bovine bone extract.     

睾酮对离体胎鼠头盖骨成骨细胞影响的研究

目的 探讨雄激素对成骨细胞代谢的影响。方法 胎鼠头盖骨成骨细胞培养于含不同浓度睾酮的培养基中．观察细胞胸腺嘧啶核苷掺入、碱性磷酸薛(ALP)活性、骨钙素基因表达及骨钙素合成等细胞增殖及分化等指标的变化情况。结果 胎鼠头盖骨成骨细胞的增殖不受睾酮影响,但ALP活性、骨钙素基因表达及其合成在不同程度上受睾酮的调节。结论 雄激素对成骨细胞的分化具有促进作用,但对其增殖无影响。     

The protein tyrosine phosphatase Rptpzeta is expressed in differentiated osteoblasts and affects bone formation in mice

Tyrosine phosphorylation of intracellular substrates is one mechanism to regulate cellular proliferation and differentiation. Protein tyrosine phosphatases (PTPs) act by dephosphorylation of substrates and thereby counteract the activity of tyrosine kinases. Few PTPs have been suggested to play a role in bone remodeling, one of them being Rptpzeta, since it has been shown to be suppressed by pleiotrophin, a heparin-binding molecule affecting bone formation, when over-expressed in transgenic mice. In a genome-wide expression analysis approach we found that Ptprz1, the gene encoding Rptpzeta, is strongly induced upon terminal differentiation of murine primary calvarial osteoblasts. Using RT-PCR and Western Blotting we further demonstrated that differentiated osteoblasts, in contrast to neuronal cells, specifically express the short transmembrane isoform of Rptpzeta. To uncover a potential role of Rptpzeta in bone remodeling we next analyzed the skeletal phenotype of a Rptpzeta-deficient mouse model using non-decalcified histology and histomorphometry. Compared to wildtype littermates, the Rptpzeta-deficient mice display a decreased trabecular bone volume at the age of 50 weeks, caused by a reduced bone formation rate. Likewise, Rptpzeta-deficient calvarial osteoblasts analyzed ex vivo display decreased expression of osteoblast markers, indicating a cell-autonomous defect. This was confirmed by the finding that Rptpzeta-deficient osteoblasts had a diminished potential to form osteocyte-like cellular extensions on Matrigel-coated surfaces. Taken together, these data provide the first evidence for a physiological role of Rptpzeta in bone remodeling, and thus identify Rptpzeta as the first PTP regulating bone formation in vivo.     

Fluid flow shear stress stimulates human osteoblast proliferation and differentiation through multiple interacting and competing signal transduction pathways

This study sought to assess the role of several signaling pathways in the fluid flow shear stress-induced proliferation and differentiation of normal human osteoblasts. We evaluated the effects of an effective dose of selective inhibitors of the extracellular signal-regulated kinases (ERK) pathway (PD98059 and U0126), the nitric oxide synthase pathway (N(omega)-nitro-L-arginine methyl ester), the cyclo-oxygenase pathway (indomethacin), or the Gi/o pathway (pertussis toxin [PTX]) on the flow-mediated effects. A 30-min steady flow shear stress at 20 dynes/cm(2) increased significantly [(3)H]thymidine incorporation (an indicator of proliferation), alkaline phosphatase activity (an index of osteoblast differentiation), phosphorylation of ERK, and expression of integrin beta1. PD98059, U0126, and N(omega)-nitro-L-arginine methyl ester completely blocked the shear stress-induced increases in ERK phosphorylation, [(3)H]thymidine incorporation, and alkaline phosphatase, but without an effect on integrin beta1 expression, indicating that the ERK and nitric oxide synthase pathways are essential for the shear stress-induced proliferation and differentiation of normal human osteoblasts and that each involves ERK activation but not integrin beta1 upregulation. Indomethacin blocked the shear stress-induced osteoblast proliferation and differentiation and integrin beta1 upregulation but not ERK activation, suggesting that the cyclo-oxygenase pathway (i.e., prostacyclin and/or prostaglandin E(2)) mediates the shear stress-induced osteoblast proliferation in an ERK-independent manner. In contrast, PTX completely blocked the flow-induced increase in integrin beta1 expression but had no effect on the increase in the ERK phosphorylation or [(3)H]thymidine incorporation. PTX not only did not inhibit but also significantly enhanced the stimulatory effect of shear stress on alkaline phosphatase activity, suggesting that a PTX-sensitive signaling pathway may have an inhibitory role in osteoblast differentiation. In summary, this study shows, for the first time, that the signal transduction mechanism of shear stress in osteoblasts is complex and involves multiple ERK-dependent and independent pathways, and provides circumstantial evidence that there may be a PTX-sensitive pathway that has completing effects with an unknown pathway on the differentiation of normal human osteoblasts.     

Infantile hypophosphatasia fibroblasts proliferate normally in culture: Evidence against a role for alkaline phosphatase (tissue nonspecific isoenzyme) in the regulation of cell growth and differentiation

Summary To explore the hypothesis that alkaline phosphatase (ALP) functions in the regulation of cell growth and differentiation, we examined in tissue culture several parameters of cell proliferation demonstrated by dermal fibroblasts from patients with infantile hypophosphatasia—a heritable form of rickets/osteomalacia which is characterized biochemically by marked deficiency of activity of the “tissue nonspecific” (bone/liver/kidney) ALP isoenzyme. Methylumbelliferyl phosphate was used as the fluorogenic substrate to assay ALP activity in cell homogenates from patients and age-, sex-, and passage-matched control cells. The nature of the enzymatic defect in the patient fibroblasts involved their failure to increase the specific activity of ALP during growth to confluency. Patient cell monolayers contained, on average, about 2–3% of control ALP activity (several lines consistently demonstrated less than 1%). Nevertheless, patient cells grew normally both in early and late passage, and in either serum-containing or defined medium. Contact inhibition appeared to be intact in the patient fibroblasts, since the protein and DNA content of their culture dishes were similar to controls just as the cells became visually confluent, and when examined 1 week after they reached monolayer. Light and phase-contrast microscopy revealed no abnormalities in the appearance of patient cells during growth or at confluency. The mean volume of proliferating patient cells was also normal. Despite profound deficiency of constitutive ALP activity, dermal fibroblasts from infants with hypophosphatasia exhibit normal growth parameters in cell culture. Although the precise biochemical defect(s) in hypophosphatasia is unknown, these findings fail to support a role for tissue nonspecific ALP in the regulation of cell growth and differentiation.     

Green tea catechin enhances osteogenesis in a bone marrow mesenchymal stem cell line

Green tea has been reported to possess antioxidant, antitumorigenic, and antibacterial qualities that regulate the endocrine system. Previous epidemiological studies found that the bone mineral density (BMD) of postmenopausal women with a habit of tea drinking was higher than that of women without habitual tea consumption. However, the effects of green tea catechins on osteogenic function have rarely been investigated. In this study, we tested (-)-epigallocatechin-3-gallate (EGCG), one of the green tea catechins, on cell proliferation, the mRNA expressions of relevant osteogenic markers, alkaline phosphatase (ALP) activity and mineralization. In a murine bone marrow mesenchymal stem cell line, D1, the mRNA expressions of core binding factors a1 (Cbfa1/Runx2), osterix, osteocalcin, ALP increased after 48 h of EGCG treatment. ALP activity was also significantly augmented upon EGCG treatment for 4 days, 7 days and 14 days. Furthermore, mineralizations assayed by Alizarin Red S and von Kossa stain were enhanced after EGCG treatment for 2–4 weeks in D1 cell cultures. However, a 24-h treatment of EGCG inhibited thymidine incorporation of D1 cells. These results demonstrated that long-term treatment of EGCG increases the expressions of osteogenic genes, elevates ALP activity and eventually stimulates mineralization, in spite of its inhibitory effect on proliferation. This finding suggests that the stimulatory effects of EGCG on osteogenesis of mesenchymal stem cells may be one of the mechanisms that allow tea drinkers to possess higher BMD.     

The effect of phospholipase A2 inhibitors on proliferation and apoptosis of murine intestinal cells.

BACKGROUND: Group II phospholipase A2 (PLA2) enzymes, the rate controlling enzymes in arachidonic acid metabolism, have been well characterized and subdivided into secretory 14-kDa PLA2 (sPLA2) and cytoplasmic 85-kDa PLA2 (cPLA2). Previous research has demonstrated increased PLA2 in colorectal tumors. The present study was performed to determine the effect of specific PLA2 inhibitors on the proliferation and induction of apoptosis of intestinal epithelial cells. METHODS: A continuously proliferating rat small intestinal cell line (IEC-18) and a mouse colon cancer cell line (WB-2054-M4) were utilized for these experiments. The cells were placed in microwells with serum-free or serum-supplemented media. The effects of serum on proliferation were then evaluated in the presence of the cPLA2 inhibitor, methylarachidonyl fluorophosphate (MAFP), or the sPLA2 inhibitor p-bromophenacyl bromide (BPB). The sPLA2 and cPLA2 protein was estimated by Western blotting. Proliferation of intestinal cells was quantitated by incorporation of [3H]thymidine into DNA and PLA2 activity was evaluated by quantitating arachidonic acid formation and prostaglandin E2 production. RESULTS: Western blotting of IEC-18 and WB-2054 cell protein demonstrated sPLA2 and cPLA2 enzyme in cells incubated in media containing 10% serum. Spontaneous DNA synthesis was present in both cell lines and serum consistently increased proliferation. In IEC-18 cells [3H]thymidine incorporation stimulated by serum was inhibited by MAFP and BPB, while in the malignant cell line, proliferation was inhibited only by BPB. BPB, but not MAFP, produced a dose-dependent increase in apoptotic ratios in both cell lines. Arachidonic acid and PGE2 formation, stimulated by serum, was inhibited by MAFP and BPB. CONCLUSIONS: A differential effect on intestinal cell mitogenesis was seen with different PLA2 inhibitors. The sPLA2 inhibitor, but not the cPLA2 inhibitor, significantly inhibited [3H]thymidine incorporation in the malignant cell line. This occurred with an induction of apoptosis. sPLA2 inhibitors may be specific inhibitors of growth of malignant cells. The inhibition of arachidonic acid and PGE2 production did not correlate with the inhibition of proliferation, suggesting that the two processes may be unrelated.     

Dexamethasone inhibits and thyroid hormone promotes differentiation of mouse chondrogenic ATDC5 cells

The effects of glucocorticoid (GC) excess, thyrotoxicosis, and hypothyroidism on linear growth indicate that growth plate chondrocytes are exquisitely sensitive to GC and thyroid hormone (T(3)). Murine ATDC5 cells undergo chondrogenesis in vitro and were used to evaluate the effects of dexamethasone (Dex) and T(3) on cell proliferation and differentiation. Immature and differentiated ATDC5 cells expressed glucocorticoid and T(3)-receptor mRNAs. Cells proliferated and organized into cartilage-like nodules after 7 days. Chondrocyte maturation progressed over 9-40 days, with increasing alkaline phosphatase (ALP) activity, secretion of an Alcian blue-positive matrix, and mineralization of cartilage-like nodules. Dex reduced cell number over the 40 day period, causing inhibition of ALP activity and matrix production with failure of mineralization. Following withdrawal of Dex, chondrocytes proliferated and re-entered the differentiation and mineralization program, indicating that GC inhibition of chondrogenesis is reversible. In contrast, T(3) reduced cell proliferation, but induced ALP activity and increased matrix secretion earlier than in control cultures. Thus, GCs and T(3) regulate growth plate chondrocyte differentiation by distinct mechanisms. GCs arrest cell proliferation, differentiation, and cartilage mineralization and maintain chondrocyte precursors in a state of quiescence with the capacity to re-enter chondrogenesis. T(3) inhibits cell proliferation but accelerates differentiation to stimulate chondrogenesis.     

Biphasic effects of transforming growth factor β on bone morphogenetic protein–induced osteoblast differentiation

Bone morphogenetic proteins (BMPs) exert an important role in skeletal development, adult bone homeostasis, and fracture healing and have demonstrated clinical utility for bone regeneration. However, BMPs fall short as regenerative agents because high doses need to be used to obtain therapeutic effects. Determining the molecular mechanisms controlling BMP‐induced bone formation may lead to the development of more effective BMP‐based therapies. To identify kinases mediating BMP‐induced osteoblast differentiation, we performed an siRNA screen to find kinases modulating BMP‐6‐induced alkaline phosphatase (ALP) activity. Surprisingly, although transforming growth factor β (TGF‐β) generally is considered to antagonize BMP‐induced osteoblast differentiation, C2C12 cells transfected with siRNAs targeting TGF‐β receptors displayed reduced BMP‐6‐induced ALP activity. Furthermore, pharmacologic inhibitors blocking the TGF‐β type I receptor impaired BMP‐induced ALP activity in KS483 and C2C12 cells and mineralization of KS483 cells. Consistently, costimulation with BMPs and TGF‐β further increased expression of osteoblast‐specific genes, ALP activity, and mineralization of KS483 cells and primary mesenchymal stem cells compared with BMPs alone. The stimulatory and inhibitory effects of TGF‐β were found to depend on timing and duration of the costimulation. TGF‐β inhibited BMP‐induced activation of a BMP‐Smad‐dependent luciferase reporter, suggesting that the stimulatory effect of TGF‐β is not due to increased BMP‐Smad activity. TGF‐β also inhibited the BMP‐induced expression of the BMP antagonist noggin and prolonged BMP activity. In conclusion, TGF‐β, besides acting as an inhibitor, also can, by dampening the noggin‐mediated negative‐feedback loop, enhance BMP‐induced osteoblast differentiation, which might be beneficial in fracture healing. © 2011 American Society for Bone and Mineral Research.     

Evidence for a role of p38 MAP kinase in expression of alkaline phosphatase during osteoblastic cell differentiation.

In the present study, we investigate the implication of the mitogen-activated protein kinases (MAPKs) Erk, p38, and JNK in mediating the effect of fetal calf serum (FCS) on the differentiation of MC3T3-E1 osteoblast-like cells. Erk is stimulated by FCS in proliferating, early-differentiating, as well as in mature cells. Activation of p38 by FCS is not detected in proliferating cells but is observed as the cells differentiate. JNK is activated in response to FCS throughout the entire differentiation process, but a maximal stimulation is observed in early differentiating cells. The roles of Erk and p38 pathways in mediating MC3T3-E1 cell differentiation was determined using specific inhibitors such as U0126 and SB203580, respectively. These experiments confirmed that the Erk pathway is essential for mediating cell proliferation in response to FCS, but indicated that this MAP kinase has little effect in regulating the differentiation of MC3T3-E1 cells. In contrast, p38 only marginally influenced proliferation, but appeared to be critical for the control of alkaline phosphatase (ALP) expression in differentiating cells. Finally, results obtained with high doses of SB203580, which also affected JNK activity, suggest that p38 and/or JNK are probably also involved in the control of type 1 collagen and osteocalcin expression in differentiating cells. The data indicate that MAPKs regulate different stages of MC3T3-E1 cell development in response to FCS. Distinct MAPK pathways seem to independently modulate osteoblastic cell proliferation and differentiation, with Erk playing an essential role in cell replication, whereas p38 is involved in the regulation of ALP expression during osteoblastic cell differentiation. JNK is also probably involved in the regulation of osteoblastic cell differentiation, but its precise role requires further investigation.     

Role of mitogen‐activated protein kinase in osteoblast differentiation

Local control of osteoblast differentiation and bone formation is not well understood. We have previously seen biphasic effects on cell differentiation in response to the short‐ and long‐term exposure to IL‐1β in rat calvarial osteoblasts. To characterize the signaling pathway mechanisms regulating IL‐1β biphasic effects, we examined the contribution of mitogen‐activated protein kinase (MAPK) family. Cells were pretreated with specific inhibitors to extracellular signal‐regulated kinase (ERK, PD98059), p38 (SB203580), and c‐JUN N‐terminal kinase (JNK, SP600125), then co‐cultured with IL‐1β for 2, 4, and 6 days. Cell differentiation was determined by measuring bone nodules after 10 days of culture. These inhibitors did not alter biphasic effects of IL‐1β on cell differentiation. However, PD98059 and U2016, another inhibitor of ERK activation robustly increased osteoblast differentiation compared to vehicle‐treated control in a time‐ and dose‐dependent manner. PD98059 appears to stimulate alkaline phosphatase (ALP) activity to promote cell differentiation, where IL‐1β appears to suppress it. Interestingly, continuous ERK inhibition with PD98059, after 2 and 4 days of IL‐1β treatment, enhanced the IL‐1β anabolic effect by increasing bone nodules formed. These observations provide a potential mechanism involving ERK pathway in osteoblasts differentiation and suggest that MAPK family may not directly regulate IL‐1β biphasic effects. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:204–210, 2011     

Differentiation-inducing factor-1 alters canonical Wnt signaling and suppresses alkaline phosphatase expression in osteoblast-like cell lines.

Because DIF-1 has been shown to affect Wnt/beta-catenin signaling pathway, the effects of DIF-1 on osteoblast-like cell lines, SaOS-2 and MC3T3-E1, were examined. We found that DIF-1 inhibited this pathway, resulting in the suppression of ALP promoter activity through the TCF/LEF binding site. INTRODUCTION: Differentiation-inducing factor-1 (DIF-1), a morphogen of Dictyostelium, inhibits cell proliferation and induces cell differentiation in several mammalian cells. Previous studies showed that DIF-1 activated glycogen synthase kinase-3beta, suggesting that this chemical could affect the Wnt/beta-catenin signaling pathway. This pathway has been shown to be involved in bone biology. MATERIALS AND METHODS: We studied the effects of DIF-1 on SaOS-2 and MC3T3-E1, osteosarcoma cell lines widely used as a model system for ostoblastic cells and murine osteoblast-like cell line, respectively. Reporter gene assays were also carried out to examine the effect of DIF-1 on the Wnt/beta-catenin signaling pathway. RESULTS: DIF-1 inhibited SaOS-2 proliferation and reduced alkaline phosphatase (ALP) activity in a concentration- and a time-dependent manner. The expression of ALP was markedly suppressed by DIF-1-treatment in protein and mRNA levels. DIF-1 also suppressed the expression of other osteoblast differentiation markers, including core binding factor alpha1, type I collagen, and osteocalcin, in protein and mRNA levels and inhibited osteoblast-mediated mineralization. Subsequently, we examined the effect of DIF-1 on the Wnt/beta-catenin signaling pathway. We found that DIF-1 suppressed the expression of beta-catenin protein and the activity of the reporter gene containing T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) consensus binding sites. We examined the effect of DIF-1 on a reporter gene driven by the human ALP promoter and found that DIF-1 significantly reduced the ALP reporter gene activity through the TCF/LEF binding site (-1023/-1017 bp). Furthermore, the effect of DIF-1 on MC3T3-E1, a murine osteoblast-like cell line, was examined, and it was found that DIF-1 suppressed ALP mRNA expression by the reduction of the ALP reporter gene activity through the TCF/LEF binding site. CONCLUSIONS: Our data suggest that DIF-1 inhibits Wnt/beta-catenin signaling, resulting in the suppression of ALP promoter activity. To our knowledge, this is the first report to analyze the role of the TCF/LEF binding site (-1023/-1017 bp) of the ALP gene promoter in osteoblast-like cell lines.     

Effects of secreted frizzled-related protein 3 on osteoblasts in vitro.

To examine if sFRP3s act as decoy receptors for Wnt, we examined the effects of recombinant sFRP3 on mouse osteoblast proliferation and differentiation. We found that sFRP3 unexpectedly increased osteoblast differentiation, suggesting it may act through other mechanisms besides acting as a decoy receptor for Wnt's. INTRODUCTION: Secreted frizzled-related proteins (sFRPs) are a truncated form of frizzled receptor, missing both the transmembrane and cytosolic domains. Because previous studies have shown that sFRPs bind and act as decoy receptors for Wnt proteins that promote osteoblast differentiation, we postulated that sFRP3 acts as an inhibitor of osteoblast differentiation. MATERIALS AND METHODS: We examined the effects of mouse recombinant sFRP3 and/or Wnt-3A on cell proliferation and differentiation using MC3T3-E1 mouse osteoblasts and primary cultures of mouse bone marrow stromal cells. We evaluated the effects of sFRP3 on beta-catenin levels using Western immunoblot analyses. RESULTS: We found that sFRP3 suppressed osteoblast cell number in a dose-dependent manner that was the result of a decrease in proliferation and not because of an increase in apoptosis. Surprisingly, sFRP3 increased osteoblast differentiation, which could not be explained based on sFRP3 acting as a decoy receptor for stimulatory Wnt's. Furthermore, sFRP3 did not inhibit Wnt3A-induced increase in alkaline phosphatase (ALP) activity. Wnt3A, but not sFRP3 treatment, increased cellular beta-catenin levels, and sFRP3 failed to block Wnt3A-induced increase in cellular beta-catenin levels. Treatment with endostatin, an agent known to degrade beta-catenin, did not inhibit sFRP3-induced increase in ALP activity. sFRP1, like sFRP3, inhibited proliferation and stimulated ALP activity in MC3T3-E1 mouse osteoblasts. CONCLUSIONS: Based on our findings, we conclude that sFRP3 decreased osteoblast proliferation and unexpectedly increased parameters of osteoblast differentiation. Based on our findings, we propose that sFRP3 may stimulate differentiation through a beta-catenin-independent pathway in addition to its previously known function as a decoy receptor for Wnt's.