Sclerostin blockade inhibits bone resorption through PDGF receptor signaling in osteoblast lineage cells.

While sclerostin-neutralizing antibodies (Scl-Abs) transiently stimulate bone formation by activating Wnt signaling in osteoblast lineage cells, they exert sustained inhibition of bone resorption, suggesting an alternate signaling pathway by which Scl-Abs control osteoclast activity. Since sclerostin can activate platelet-derived growth factor receptors (PDGFRs) in osteoblast lineage cells in vitro and PDGFR signaling in these cells induces bone resorption through M-CSF secretion, we hypothesized that the prolonged anticatabolic effect of Scl-Abs could result from PDGFR inhibition. We show here that inhibition of PDGFR signaling in osteoblast lineage cells is sufficient and necessary to mediate prolonged Scl-Ab effects on M-CSF secretion and osteoclast activity in mice. Indeed, sclerostin coactivates PDGFRs independently of Wnt/ß-catenin signaling inhibition, by forming a ternary complex with LRP6 and PDGFRs in preosteoblasts. In turn, Scl-Ab prevents sclerostin-mediated coactivation of PDGFR signaling and consequent M-CSF upregulation in preosteoblast cultures, thereby inhibiting osteoclast activity in preosteoblast/osteoclast coculture assays. These results provide a potential mechanism explaining the dissociation between anabolic and antiresorptive effects of long-term Scl-Ab.

PDGF Receptor Signaling in Osteoblast Lineage Cells Controls Bone Resorption Through Upregulation of Csf1 Expression.

The physiological functions of platelet-derived growth factor receptors (PDGFRs) α and ß in osteoblast biology and bone metabolism remain to be established. Here, we show that PDGFRA and PDGFRB genes are expressed by osteoblast-lineage canopy and reversal cells in close proximity to PDGFB-expressing osteoclasts within human trabecular bone remodeling units. We also report that, although removal of only one of the two PDGFRs in Osterix-positive cells does not affect bone phenotype, suppression of both PDGFRs in those osteoblast lineage cells increases trabecular bone volume in male mice as well as in female gonad-intact and ovariectomized mice. Furthermore, osteoblast lineage-specific suppression of PDGFRs reduces Csf1 expression, bone marrow level of macrophage colony-stimulating factor (M-CSF), number of osteoclasts, and, therefore, bone resorption, but does not change bone formation. Finally, abrogation of PDGFR signaling in osteoblasts blocks PDGF-induced ERK1/2-mediated Csf1 expression and M-CSF secretion in osteoblast cultures and calcitriol-mediated osteoclastogenesis in co-cultures. In conclusion, our results indicate that PDGFR signaling in osteoblast lineage cells controls bone resorption through ERK1/2-mediated Csf1 expression. © 2020 American Society for Bone and Mineral Research (ASBMR).

Selective inhibition of Src family kinases by SU6656 increases bone mass by uncoupling bone formation from resorption in mice.

Mice deficient in the non-receptor tyrosine kinase Src exhibit high bone mass due to impaired bone resorption and increased bone formation. Although several Src family kinase inhibitors inhibit bone resorption in vivo, they display variable effects on bone formation. SU6656 is a selective Src family kinase inhibitor with weaker activity towards the non-receptor tyrosine kinase Abl and receptor tyrosine kinases which are required for appropriate osteoblast proliferation, differentiation and function. Therefore, we sought to determine whether SU6656 could increase bone mass by inhibiting bone resorption and by stimulating bone formation, and to explore its mechanisms of action. Four-month-old female C57Bl/6J mice received intraperitoneal injections of either 25 mg/kg SU6656 or its vehicle every other day for 12 weeks. SU6656-treated mice exhibited increased bone mineral density, cortical thickness, cancellous bone volume and trabecular thickness. SU6656 inhibited bone resorption in mice as shown by reduced osteoclast number, and diminished expressions of Oscar, Trap5b and CtsK. SU6656 did not affect Rankl or Opg expressions. However, it blocked c-fms signaling, osteoclastogenesis and matrix resorption, and induced osteoclast apoptosis in vitro. In addition, SU6656 stimulated bone formation rates at trabecular, endosteal and periosteal bone envelopes, and increased osteoblast number in trabecular bone. SU6656 did not affect expressions of clastokines favoring bone formation in mice. However, it stimulated osteoblast differentiation and matrix mineralization by specifically facilitating BMP-SMAD signaling pathway in vitro. Knockdown of Src and Yes mimicked the stimulatory effect of SU6656 on osteoblast differentiation. In conclusion, SU6656 uncouples bone formation from resorption by inhibiting osteoclast development, function and survival, and by enhancing BMP-mediated osteoblast differentiation.

Suppression of p38α MAPK Signaling in Osteoblast Lineage Cells Impairs Bone Anabolic Action of Parathyroid Hormone.

Intermittent parathyroid hormone administration (iPTH) increases bone mass and strength by stimulating osteoblast number and activity. PTH exerts its anabolic effects through cAMP/protein kinase A (PKA) signaling pathway in mature osteoblasts and osteocytes. Here, we show that inactivation of the p38α MAPK-encoding gene with the use of an osteocalcin-cre transgene prevents iPTH bone anabolic action. Indeed, iPTH fails to increase insulin-like growth factor 1 expression, osteoblast number and activity, and bone formation in mice lacking p38α in osteoblasts and osteocytes. Moreover, iPTH-induced expression of receptor activator of NF-κB ligand (RANKL) and subsequent increased bone resorption are suppressed in those mice. Finally, we found that PTH activates p38α MAPK downstream of cAMP/PKA signaling pathway in mature osteoblasts. Our findings identify p38α MAPK as a key component of PTH signaling in osteoblast lineage cells and highlight its requirement in iPTH osteoanabolic activity. © 2015 American Society for Bone and Mineral Research.

Sclerostin inhibits osteoblast differentiation without affecting BMP2/SMAD1/5 or Wnt3a/ß-catenin signaling but through activation of platelet-derived growth factor receptor signaling in vitro.

Sclerostin inhibits bone formation mostly by antagonizing LRP5/6, thus inhibiting Wnt signaling. However, experiments with genetically modified mouse models suggest that a significant part of sclerostin-mediated inhibition of bone formation is due to interactions with other binding partners. The objective of the present work was to identify signaling pathways affected by sclerostin in relation with its inhibitory action on osteogenic differentiation of C3H10T1/2 cells, MC3T3-E1 cells and primary osteoblasts. Sclerostin inhibited BMP2-induced osteoblast differentiation without altering SMAD1/5 phosphorylation and transcriptional activity. Moreover, sclerostin prevented Wnt3a-mediated osteoblastogenesis without affecting LRP5/6 phosphorylation or ß-catenin transcriptional activity. In addition, sclerostin inhibited mineralization promoted by GSK3 inhibition, which mimics canonical Wnt signaling without activation of LRP5/6, suggesting that sclerostin can prevent osteoblast differentiation without antagonizing LRP5/6. Finally, we found that sclerostin could activate platelet-derived growth factor receptor (PDGFR) and its downstream signaling pathways PLCγ, PKC, Akt and ERK1/2. PDGFR inhibition could reverse sclerostin-mediated inhibitory activity on BMP2-induced osteoblast differentiation. Therefore, our data suggest that sclerostin can activate PDGFR signaling by itself, and this functional interaction may be involved in the negative effect of sclerostin on osteoblast differentiation.

Ablation of p38α MAPK Signaling in Osteoblast Lineage Cells Protects Mice From Bone Loss Induced by Estrogen Deficiency.

Estrogen deficiency causes bone loss by increasing the number of bone-resorbing osteoclasts. Selective p38α MAPK inhibitors prevent bone-wasting effects of estrogen withdrawal but implicated mechanisms remain to be identified. Here, we show that inactivation of the p38α-encoding gene in osteoblast lineage cells with the use of an osteocalcin-cre transgene protects mice from ovariectomy-induced bone loss (a murine model of postmenopausal osteoporosis). Ovariectomy fails to induce bone loss, increase bone resorption, and stimulate receptor activator of nuclear factor κB ligand and IL-6 expression in mice lacking p38α in osteoblasts and osteocytes. Finally, TNFα or IL-1, which are osteoclastogenic cytokines overproduced in the bone marrow under estrogen deficiency, can activate p38α signaling in osteoblasts, but those cytokines cannot enhance Rankl and Il6 expressions or increase osteoclast formation in p38a-deficient osteoblast cultures. These findings demonstrate that p38α MAPK signaling in osteoblast lineage cells mediates ovariectomy-induced bone loss by up-regulating receptor activator of nuclear factor κB ligand and IL-6 production.

Focus on the p38 MAPK signaling pathway in bone development and maintenance.

The p38 mitogen-activated protein kinase (MAPK) signaling pathway can be activated in response to a wide range of extracellular signals. As a consequence, it can generate many different biological effects that depend on the stimulus and on the activated cell type. Therefore, this pathway has been found to regulate many aspects of tissue development and homeostasis. Recent work with the aid of genetically modified mice has highlighted the physiological functions of this pathway in skeletogenesis and postnatal bone maintenance. In this review, emphasis is given to the roles of the p38 MAPK pathway in chondrocyte, osteoblast and osteoclast biology. In particular, we describe the molecular mechanisms of p38 MAPK activation and downstream targets. The requirement of this pathway in physiological bone development and homeostasis is demonstrated by the ability of p38 MAPK to regulate master transcription factors controlling geneses and functions of chondrocytes, osteoblasts and osteoclasts.

Crosstalk between tyrosine kinase receptors, GSK3 and BMP2 signaling during osteoblastic differentiation of human mesenchymal stem cells.

Bone morphogenic proteins (BMPs) promote mesenchymal stem cell (MSC) osteogenic differentiation, whereas platelet derived growth factor (PDGF) and fibroblast growth factor (FGF) activate their proliferation through receptors tyrosine kinase (RTK). The effects of PDGF or FGF receptor signaling pathway on BMP2-induced osteoblastic differentiation was investigated in human MSC (HMSC). Inhibition of PDGF or/and FGF receptors enhanced BMP2-induced alkaline phosphatase (ALP) activity, expression of Osterix, ALP and Bone sialoprotein, and matrix calcification. These effects were associated with increased Smad-1 activity, indicating that mitogenic factors interfere with Smad signaling in HMSC differentiation. RTK activate MAPK and inhibit GSK3 through the PI3K/Akt pathway. Biochemical analysis indicated that MAPK JNK and GSK3 especially are potential signaling molecules regulating BMP-induced osteoblastic HMSC differentiation. These observations highlight that the osteogenic effects of BMP2 are modulated by mitogenic factors acting through RTK.

Predominant role of PDGF receptor transactivation in Wnt3a-induced osteoblastic cell proliferation.

Previous studies have shown that Wnt3a enhances the proliferation and inhibits the osteogenic differentiation of human mesenchymal stem cells (hMSCs). In this study, we investigated the signaling pathways involved in Wnt3a-induced osteoblastic cell proliferation. Experiments with DKK1, a natural antagonist of Lrp5/6, indicated that Wnt/ß-catenin did not play a major role in Wnt3a-induced osteoblastic cell proliferation. The use of selective inhibitors of known mitogenic pathways implicates Src family kinases (SFKs) and a protein kinase C (PKC) in this cellular response. Time-dependent analysis of signaling molecules activated by Wnt3a in MC3T3-E1 cells revealed parallel activation of the canonical pathway and of several tyrosine kinases, including SFKs and PDGF receptors (PDGF-Rs). Functional analysis with specific inhibitors suggested a major role of PDGF-Rs in mediating Wnt3a-induced cell proliferation. Further investigation with an si-RNA approach confirmed a predominant role of this receptor in this cellular response. The use of soluble decoy PDGF-Rs that can sequester extracellular PDGFs excluding that part of the increased PDGF receptor phosphorylation by Wnt3a was the result of autocrine production of PDGFs. A selective SFK inhibitor blunted the enhanced PDGF-R phosphorylation and cell proliferation induced by Wnt3a. Studies of initial events involved in the regulation of this pathway suggest a role of dishevelled. In conclusion, data presented in this study indicate that cell proliferation induced by Wnt3a in osteoblastic cells is mediated by a dishevelled-dependent and ß-catenin-independent pathway, which involves the transactivation of PDGF receptors.

The "bone morphogenic proteins" pathways in bone and joint diseases: translational perspectives from physiopathology to therapeutic targets.

A large body of evidence supports an important role of bone morphogenic proteins (BMPs) pathways in skeletal development in the embryo. BMPs are also involved in skeletal homeostasis and diseases in the adult. They were first identified as major bone anabolic agents and recent advances indicate that they also regulate osteoclastogenesis and joint components via multiple cross-talks with other signaling pathways. This review attempts to integrate these data in the pathogenesis of bone and joints diseases, such as osteoporosis, fracture healing, osteoarthritis, inflammatory arthritis, or bone metastasis. The use of recombinant BMPs in bone tissue engineering and in the treatment of skeletal diseases, or future therapeutic strategies targeting BMPs signal and its regulators, will be discussed based on these considerations.

Fibroblast growth factor 2 inhibits up-regulation of bone morphogenic proteins and their receptors during osteoblastic differentiation of human mesenchymal stem cells.

Understanding the interactions between growth factors and bone morphogenic proteins (BMPs) signaling remains a crucial issue to optimize the use of human mesenchymal stem cells (HMSCs) and BMPs in therapeutic perspectives and bone tissue engineering. BMPs are potent inducers of osteoblastic differentiation. They exert their actions via BMP receptors (BMPR), including BMPR1A, BMPR1B and BMPR2. Fibroblast growth factor 2 (FGF2) is expressed by cells of the osteoblastic lineage, increases their proliferation and is secreted during the healing process of fractures or in surgery bone sites. We hypothesized that FGF2 might influence HMSC osteoblastic differentiation by modulating expressions of BMPs and their receptors. BMP2, BMP4, BMPR1A and mainly BMPR1B expressions were up-regulated during this differentiation. FGF2 inhibited HMSCs osteoblastic differentiation and the up-regulation of BMPs and BMPR. This effect was prevented by inhibiting the ERK or JNK mitogen-activated protein kinases which are known to be activated by FGF2. These data provide a mechanism explaining the inhibitory effect of FGF2 on osteoblastic differentiation of HMSCs. These crosstalks between growth and osteogenic factors should be considered in the use of recombinant BMPs in therapeutic purpose of fracture repair or skeletal bioengineering.

The p38α MAPK positively regulates osteoblast function and postnatal bone acquisition.

Bone continuously remodels throughout life by coordinated actions of osteoclasts and osteoblasts. Abnormalities in either osteoclast or osteoblast functions lead to bone disorders. The p38 MAPK pathway has been shown to be essential in controlling osteoblast differentiation and skeletogenesis. Although p38α is the most abundant p38 member in osteoblasts, its specific individual contribution in regulating postnatal osteoblast activity and bone metabolism is unknown. To elucidate the specific role of p38α in regulating osteoblast function and bone homeostasis, we generated mice lacking p38α in differentiated osteoblasts. Osteoblast-specific p38a knockout mice were of normal weight and size. Despite non-significant bone alterations until 5 weeks of age, mutant mice demonstrated significant and progressive decrease in bone mineral density from that age. Adult mice deficient in p38a in osteoblasts displayed a striking reduction in cancellous bone volume at both axial and appendicular skeletal sites. At 6 months of age, trabecular bone volume was reduced by 62% in those mice. Mutant mice also exhibited progressive decrease in cortical thickness of long bones. These abnormalities correlated with decreased endocortical and trabecular bone formation rate and reduced expressions of type 1 collagen, alkaline phosphatase, osteopontin and osteocalcin whereas bone resorption and osteoclasts remained unaffected. Finally, osteoblasts lacking p38α showed impaired marker gene expressions and defective mineralization in vitro. These findings indicate that p38α is an essential positive regulator of osteoblast function and postnatal bone formation in vivo.

Activation of FGF receptors is a new mechanism by which strontium ranelate induces osteoblastic cell growth.

BACKGROUND/AIMS: Strontium ranelate (SrRan) is an anti-osteoporotic treatment that reduces the risk of vertebral and hip fractures. Recent in vitro studies suggest that the effect of strontium ranelate on osteoblastic cell growth likely involves two processes including activation of the calcium sensing receptor (CaSR) and a yet undefined mechanism. In the present study, we investigated the CaSR-independent molecular mechanism by which SrRan stimulates osteoblast growth. METHODS: MC3T3-E1 and primary osteoblastic cells, specific inhibitors of receptor tyrosine kinases (RTK) and western blot analysis were used to characterize the CaSR-independent mechanism in osteoblastic cells. RESULTS: A selective inhibitor of FGF receptor but not other RTK inhibitors markedly blunted cell growth induced by SrRan in osteoblastic cells. Associated with this observation, SrRan induced rapid activation of FGFR signaling pathways such as PLCγ, FRS2, Akt, ERK1,2 and p38. FGFR-dependent stimulation of osteogenic cell growth was also observed with other cations but not with neomycin, a selective CaSR agonist. Also, in cultured conditions used in this study, MC3T3-E1 cells and primary osteoblasts did not express the CaSR. CONCLUSION: data presented in this study suggest that activation of FGFRs is a new potential mechanism by which strontium can stimulate osteoblastic cell growth. Activation of FGFR-dependent cell growth is also observed in response to other cations suggesting that activation of FGF receptors is a new cation sensing mechanism in osteoblasts.

Regulation of proliferation and differentiation of human fetal bone cells.

During the last decade, extensive research has been performed in the field of orthopedic medicine to develop cell-based therapies for the restoration of injured bone tissue. We previously demonstrated that human primary fetal bone cells (HFBCs) associated with porous scaffolds induced a bone formation in critical calvaria defect; however, the environmental factors regulating their behavior in culture have not been identified. HFBCs (human fetal femur,12 week development) were compared to marrow-derived human mesenchymal stem cells (HMSCs) for their capacity to proliferate and differentiate into osteoblasts under various culture conditions. When cultured in standard alphaMEM medium, PDGF and FGF-2 increased cell proliferation of both cell types. Investigation of the differentiating capacity of HFBCs and HMSCs in a normal culture medium indicated that HFBCs expressed higher expression levels of RUNX2, OSX, and osteogenic markers compared with HMSCs, while SOX9 was expressed at very low levels in both cells types. However, HMSCs, but not HFBCs enhanced osteoblastic markers in response to osteogenic factors. Surprisingly, BMP-2 with osteogenic factors increased cell numbers and reduced osteoblastic differentiation in HFBCs with the opposite effect seen in HMSCs. Associated with a higher expression of osteoblastic markers, HFBCs produced a higher calcified extra cellular matrix compared with HMSCs. Taken together, data presented in this study suggest that HFBCs have characteristics of osteoprecursor cells that are more advanced in their osteogenesis development compared with mesenchymal stem cells, making fetal cells an interesting biological tool for treatment of skeletal defects and diseases.

Effects of transgenic Pit-1 overexpression on calcium phosphate and bone metabolism.

The type III inorganic phosphate (Pi) transporter Pit-1 was previously found to be preferentially expressed in developing long bones. Several studies also described a regulation of its expression in cultured bone cells by osteotropic factors, suggesting a role of this transporter in bone metabolism. In the present study, we investigated the effects of the transgenic overexpression of Pit-1 in Wistar male rats on calcium phosphate and bone metabolism. A threefold increase and doubling of Pi transport activity were recorded in primary cultured osteoblastic cells derived from calvaria of two transgenic (Tg) lines compared with wild-type littermates (WT), respectively. Skeletal development was not affected by the transgene, and bone mass, analyzed by DXA, was slightly decreased in Tg compared with WT. Enhanced Pi uptake in calvaria-derived osteoblasts from Pit-1 Tg was associated with a significantly decreased expression of alkaline phosphatase activity and a normal deposition and calcification of the collagenous matrix. In 4-month-old adult Tg rats, serum Pi and renal Pi transport were increased compared with WT. The decrease of serum Ca concentration was associated with increased serum parathyroid hormone levels. Variations in serum Pi in Pit-1 Tg rats were negatively correlated with serum fibroblast growth factor-23, whereas 1,25-dihydroxyvitamin D(3) was not affected by Pit-1 overexpression. In conclusion, transgenic Pit-1 overexpression in rats affected bone and calcium phosphate metabolism. It also decreased alkaline phosphatase activity in osteoblasts without influencing bone matrix mineralization as well as skeletal development.

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.

Prevention of trabecular bone loss induced by estrogen deficiency by a selective p38alpha inhibitor.

Increased bone remodeling with estrogen deficiency is mediated by the production of cytokines such as TNFalpha and interleukin (IL)-1. Recent data have indicated that the p38 pathway mediates cytokines effects on enhanced bone turnover in postmenopausal osteoporosis. Thus, in this study, we investigated the effect of a selective p38alpha inhibitor, SD-282, on the prevention of bone loss induced by estrogen deficiency in an adult ovariectomized (OVX) rat model. Results indicate that oral administration of SD-282 for 8 wk dose-dependently blunted the increase in the bone resorption marker DPD/Cr induced by OVX in adult rats. Associated with this effect, SD-282 did not reduce but significantly enhanced by 2-fold the rise in the bone formation marker serum osteocalcin observed in OVX animals. In addition, SD-282 completely blocked vertebral bone loss associated with estrogen deficiency. Furthermore, a partial preventive effect was observed in long bones with reduction of trabecular bone loss and enhancement of cross-sectional area of the diaphysis. Prevention of trabecular bone loss and increased in cortical bone area were associated with improvement of biomechanical resistances. In conclusion, chronic administration of a selective p38alpha inhibitor effectively prevented trabecular bone loss and alteration of bone microarchitecture induced by estrogen deficiency. Prevention of bone loss was associated with inhibition of bone resorption with uncoupled changes in bone formation. These data strongly suggest that the p38 pathway is important for regulation of bone resorption induced by estrogen deficiency, and selective inhibitors of this pathway have potential for prevention of bone loss in postmenopausal osteoporosis.

Essential role of Wnt3a-mediated activation of mitogen-activated protein kinase p38 for the stimulation of alkaline phosphatase activity and matrix mineralization in C3H10T1/2 mesenchymal cells.

Signaling pathways involved in the development of osteoprogenitors induced by Wnts remain poorly understood. In this study, we investigated the role of MAPKs in the development of mesenchymal cells into osteoprogenitors. In C3H10T1/2 mesenchymal cells, Wnt3a induced a rapid and transient activation of MAPKs p38 and ERK. Dickkopf 1, a selective antagonist of Wnt proteins binding to low-density lipoprotein-receptor-related protein-5/6 did not influence activation of p38 and ERK induced by Wnt3a. A MAPK kinase-1/2 (MEK1/2) inhibitor blocked, whereas a p38 inhibitor had no effect on, Wnt3a-induced cell proliferation. In contrast, both inhibitors significantly reduced alkaline phosphatase stimulation with a more pronounced effect of the p38 inhibitor. The p38 inhibitor also blunted nodule mineralization induced by Wnt3a. Associated with these effects, beta-catenin transcriptional activity, assessed with the TOPflash system, was dose-dependently decreased by the p38 but not by the ERK inhibitor. Both the reduced alkaline phosphatase stimulation and blunting of beta-catenin transcriptional activity were mimicked by expression of dominant-negative (dn) p38 and dnMEK 3/6. Inhibition of beta-catenin transcriptional activity by the p38 inhibitor as well as by dnp38 and dnMEK 3/6 molecules were not associated with changes in cytosolic and nuclear beta-catenin levels induced by Wnt3a. In conclusion, Wnt3a activates ERK and p38 in mesenchymal C3H10T1/2 cells by a low-density lipoprotein-receptor-related protein-5/6-independent mechanism. Activation of p38 regulates alkaline phosphatase activity and nodule mineralization induced by Wnt3a probably by interacting with beta-catenin transcriptional activity. These observations suggest that MAPKs ERK and p38 are probably essential pathways activated by Wnt proteins for the development of mesenchymal cells into osteoprogenitors.

Proline-rich motifs in the parathyroid hormone (PTH)/PTH-related protein receptor C terminus mediate scaffolding of c-Src with beta-arrestin2 for ERK1/2 activation.

Parathyroid hormone (PTH) stimulates ERK1/2 through both G-protein signaling and beta-arrestin2-mediated internalization. Beta-arrestin may serve as a scaffold for c-Src. However, the molecular mechanisms for ERK1/2 activation by PTH remain unclear. By using a targeted mutagenesis approach, we investigated the PTH/PTH-related protein receptor (PTH1R) structural determinants for ERK1/2 activation and transcriptional activity in HEK-293 cells. First, ERK1/2 activation was inhibited by PTH1R mutations that specifically abrogate G(q)-protein kinase C signaling without a decrease in cAMP-protein kinase A. Second, PTH1R C-terminal mutations and/or deletions that prevent interaction with beta-arrestin inhibited ERK1/2 activation. Similar results were obtained in HEK-293 cells co-expressing wild-type PTH1R and a dominant-negative beta-arrestin2. Third, the c-Src inhibitor PP2 and a kinase-dead c-SrcK295M mutant co-expressed with wild-type PTH1R both inhibited ERK1/2 activation. Furthermore, c-Src co-precipitated with both PTH1R and beta-arrestin2 in response to PTH. Deleting the PTH1R-proximal C terminus abolished these interactions. However, the need for receptor interaction with beta-arrestin to co-precipitate Src and activate ERK1/2 was obviated by expressing a constitutively active c-SrcY527A mutant, suggesting direct binding of activated Src to PTH1R. Subsequently, we identified and mutated to alanine four proline-rich motifs in the PTH1R distal C terminus, which resulted in loss of both c-Src and arrestin co-precipitation and significantly decreased ERK1/2 activation. These data delineate the multiple PTH1R structural determinants for ERK1/2 activation and newly identify a unique mechanism involving proline-rich motifs in the receptor C terminus for reciprocal scaffolding of c-Src and beta-arrestin2 with a class II G-protein-coupled receptor.

Enhanced expression of the inorganic phosphate transporter Pit-1 is involved in BMP-2-induced matrix mineralization in osteoblast-like cells.

UNLABELLED: Pi handling by osteogenic cells is important for bone mineralization. The role of Pi transport in BMP-2-induced matrix calcification was studied. BMP-2 enhances Pit-1 Pi transporters in osteogenic cells. Experimental analysis suggest that this response is required for bone matrix calcification. INTRODUCTION: Bone morphogenetic proteins (BMPs) are produced by osteogenic cells and play an important role in bone formation. Inorganic phosphate (Pi) is a fundamental constituent of hydroxyapatite, and its transport by osteogenic cells is an important function for primary calcification of the bone matrix. In this study, we investigated the role of Pi transport in BMP-2-induced matrix mineralization. MATERIALS AND METHODS: Confluent MC3T3-E1 osteoblast-like cells were exposed to BMP-2 for various time periods. Pi and alanine transport was determined using radiolabeled substrate, Pit-1 and Pit-2 expression by Northern blot analysis, cell differentiation by alkaline phosphatase activity, matrix mineralization by alizarin red staining, and the characteristics of mineral deposited in the matrix by transmission electron microscopy, electron diffraction analysis, and Fourier transformed infrared resolution (FTIR). RESULTS: BMP-2 time- and dose-dependently stimulated Na-dependent Pi transport in MC3T3-E1 cells by increasing the V(max) of the transport system. This effect was preceded by an increase in mRNA encoding Pit-1 but not Pit-2. BMP-2 also dose-dependently enhanced extracellular matrix mineralization, an effect blunted by either phosphonoformic acid or expression of antisense Pit-1. Enhanced Pi transport and matrix mineralization induced by BMP-2 were blunted by a specific inhibitor of the c-Jun-N-terminal kinase (JNK) pathway. CONCLUSIONS: Results presented in this study indicate that, in addition to its well-known effect on several markers of the differentiation of osteoblastic cells, BMP-2 also stimulates Pi transport activity through a selective increase in expression of type III Pi transporters Pit-1. In MC3T3-E1 cells, this effect is mediated by the JNK pathway and plays an essential role in bone matrix calcification induced by BMP-2.