Autocrine stimulation of osteoblast activity by Wnt5a in response to TNF-α in human mesenchymal stem cells.

Although anti-tumor necrosis factor (TNF)-α treatments efficiently block inflammation in ankylosing spondylitis (AS), they are inefficient to prevent excessive bone formation. In AS, ossification seems more prone to develop in sites where inflammation has resolved following anti-TNF therapy, suggesting that TNF-α indirectly stimulates ossification. In this context, our objectives were to determine and compare the involvement of Wnt proteins, which are potent growth factors of bone formation, in the effects of TNF-α on osteoblast function. In human mesenchymal stem cells (MSCs), TNF-α significantly increased the levels of Wnt10b and Wnt5a. Associated with this effect, TNF-α stimulated tissue-non specific alkaline phosphatase (TNAP) and mineralization. This effect was mimicked by activation of the canonical ß-catenin pathway with either anti-Dkk1 antibodies, lithium chloride (LiCl) or SB216763. TNF-α reduced, and activation of ß-catenin had little effect on expression of osteocalcin, a late marker of osteoblast differentiation. Surprisingly, TNF-α failed to stabilize ß-catenin and Dkk1 did not inhibit TNF-α effects. In fact, Dkk1 expression was also enhanced in response to TNF-α, perhaps explaining why canonical signaling by Wnt10b was not activated by TNF-α. However, we found that Wnt5a also stimulated TNAP in MSCs cultured in osteogenic conditions, and increased the levels of inflammatory markers such as COX-2. Interestingly, treatment with anti-Wnt5a antibodies reduced endogenous TNAP expression and activity. Collectively, these data suggest that increased levels of Dkk1 may blunt the autocrine effects of Wnt10b, but not that of Wnt5a, acting through non-canonical signaling. Thus, Wnt5a may be potentially involved in the effects of inflammation on bone formation.

Evidences for a role of p38 MAP kinase in the stimulation of alkaline phosphatase and matrix mineralization induced by parathyroid hormone in osteoblastic cells.

Increased bone formation by PTH mainly results from activation of osteoblasts, an effect largely mediated by the cAMP-PKA pathway. Other pathways, however, are likely to be involved in this process. In this study we investigated whether PTH can activate p38 MAPK and the role of this kinase in osteoblastic cells. Bovine PTH(1-34) and forskolin markedly increased alkaline phosphatase (ALP) activity and doubled osteocalcin (Oc) expression in early differentiating MC3T3-E1 cells. These effects were associated with increase in cellular cAMP and activation of the MAP kinases ERK and p38. Activation of these MAP kinases was detectable after 1 h incubation with 10(-7) M PTH and lasted 1-2 h. Activation of p38 was mimicked by 10 microM forskolin and prevented by H89 suggesting a cAMP-PKA-dependent mechanism of p38 activation. Interestingly, PTH-induced ALP stimulation was dose-dependently inhibited by a specific p38 inhibitor with no change in the generation of cAMP and the production of osteocalcin. Similar inhibitory effect was obtained in cells stably expressing a dominant-negative p38 molecule. Finally, treatment of MC3T3-E1 cells with PTH for 3 weeks significantly enhanced matrix mineralization and this effect was markedly reduced by a selective p38 but not a specific MEK inhibitor. In conclusion, data presented in this study indicate that PTH can activate p38 in early differentiating osteoblastic cells. Activation of p38 is cAMP-PKA-dependent and mediates PTH-induced stimulation of ALP which plays a critical role for the calcification of the bone matrix.

Activation of p38 mitogen-activated protein kinase and c-Jun-NH2-terminal kinase by BMP-2 and their implication in the stimulation of osteoblastic cell differentiation.

UNLABELLED: Signaling involved in osteoblastic cell differentiation remains largely unknown. This study further investigates mechanisms involved in BMP-2-induced osteoblastic cell differentiation. We report that BMP-2 can activate JNK and p38 in osteoblastic cells and provide evidences that these MAP kinases have distinct roles in regulating alkaline phosphatase and osteocalcin expression. INTRODUCTION: Bone morphogenetic protein (BMP)-2 exerts many of its biological effects through activation of the Smad pathway. Cooperative interactions between the Smads and the stress-activated protein kinase (SAPK) p38 and c-Jun-NH2-terminal kinase (JNK) pathways have recently been observed in TGF-beta signaling. MATERIALS AND METHODS: Activation of mitogen-activated protein (MAP) kinases by BMP-2 and the role of these signaling pathways for cell differentiation induced by BMP-2 was investigated in mouse MC3T3-E1 and primary cultured calvaria-derived osteoblastic cells using immunoprecipitation, in vitro kinase assay and Western blot analysis, as well as specific MAP kinase inhibitors. RESULTS: Associated with the rapid activation of Smads, BMP-2 barely affected extracellular-signal regulated kinase (ERK) activity, whereas it induced a transient activation of p38 and JNK. The role of p38 and JNK in mediating BMP-2-induced stimulation of osteoblastic cell differentiation was evaluated using the respective specific inhibitors SB203580 and SP600125. Inhibition of p38 by SB203580 was mainly associated with decreased alkaline phosphatase (ALP) activity, whereas inhibition of JNK by SP600125 was associated with a marked reduction in osteocalcin (OC) production induced by BMP-2. Corresponding alterations in ALP and OC mRNA levels were found in cells treated with BMP-2 and inhibitors, suggesting an implication of p38 and JNK pathways in BMP-2-induced osteoblastic cell differentiation at a transcriptional level. CONCLUSION: Data presented in this study describe p38 and JNK as new signaling pathways involved in BMP-2-induced osteoblastic cell differentiation with evidences for a distinct role of each MAP kinase in the control of alkaline phosphatase and osteocalcin expression.

Phosphate is a specific signal for ATDC5 chondrocyte maturation and apoptosis-associated mineralization: possible implication of apoptosis in the regulation of endochondral ossification.

UNLABELLED: Involvement of Pi and Ca in chondrocyte maturation was studied because their levels increase in cartilage growth plate. In vitro results showed that Pi increases type X collagen expression, and together with Ca, induces apoptosis-associated mineralization, which is similar to that analyzed in vivo, thus suggesting a role for both ions and apoptosis during endochondral ossification. INTRODUCTION: During endochondral ossification, regulation of chondrocyte maturation governs the growth of the cartilage plate. The role of inorganic phosphate (Pi), whose levels strongly increase in the hypertrophic zone of the growth plate both in intra- and extracellular compartments, on chondrocyte maturation and mineralization of the extracellular matrix has not yet been deciphered. MATERIALS AND METHODS: The murine chondrogenic cell line ATDC5 was used. Various Pi and calcium concentrations were obtained by adding NaH2PO4/Na2HPO4 and CaCl2, respectively. Mineralization was investigated by measuring calcium content in cell layer by atomic absorption spectroscopy and by analyzing crystals with transmission electron microscopy and Fourier transform infrared microspectroscopy. Cell differentiation was investigated at the mRNA level (reverse transcriptase-polymerase chain reaction [RT-PCR] analysis). Cell viability was assessed by methyl tetrazolium salt (MTS) assay and staining with cell tracker green (CTG) and ethidium homodimer-(EthD-1). Apoptosis was evidenced by DNA fragmentation and caspase activation observed in confocal microscopy, as well as Bcl-2/Bax mRNA ratio (RT-PCR analysis). RESULTS: We showed that Pi increases expression of the hypertrophic marker, type X collagen. When calcium concentration is slightly increased (like in cartilage growth plate), Pi also induces matrix mineralization that seems identical to that observed in murine growth plate cartilage and stimulates apoptosis of differentiated ATDC5 cells, with a decrease in Bcl-2/Bax mRNA ratio, DNA fragmentation, characteristic morphological features, and caspase-3 activation. In addition, the use of a competitive inhibitor of phosphate transport showed that these effects are likely dependent on Pi entry into cells through phosphate transporters. Finally, inhibition of apoptosis with ZVAD-fmk reduces pi-induced mineralization. CONCLUSIONS: These findings suggest that Pi regulates chondrocyte maturation and apoptosis-associated mineralization, highlighting a possible role for Pi in the control of skeletal development.

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.

Species-specific mechanisms control the activity of the Pit1/PIT1 phosphate transporter gene promoter in mouse and human.

The Pit1 phosphate transporter is involved in regulated phosphate handling in bone forming cells. In this study, we compared the structure of the murine and human Pit1/PIT1 promoters and characterized cis-acting elements controlling Pit1/PIT1 expression. The Pit1/PIT1 promoter sequence and its location relative to the first transcribed exon are conserved and similar transcription factor binding sites are found at identical positions in mouse and human. Luciferase reporter gene assays in transiently transfected mouse ATDC5 chondrocytes and human SaOS-2 osteoblasts indicated that the activity of the mouse Pit1 promoter depends on several cis-acting elements, including ATF/CREB, Sp1 and AP-1 sites, an E-box and a TATA box. In contrast, the activity of the human promoter essentially requires a TATA-like sequence and one single Sp1 site. This Sp1 site binds Sp1, Sp3, as well as unidentified proteins present in SaOS-2 nuclear extracts and co-transfection experiments in SL2 cells indicate that Sp1 and Sp3 activate transcription from the human PIT1 promoter. These data suggest that, despite similarities in promoter structure, changes in the relative importance of conserved transcription factor binding sites cause species-dependent differences in Pit1 promoter function, which allow Sp1-related proteins to play a particularly important role in human.

Stimulation of sodium-dependent inorganic phosphate transport by activation of Gi/o-protein-coupled receptors by epinephrine in MC3T3-E1 osteoblast-like cells.

Recent data have shown that activation of Gi-protein-coupled receptors in osteoblast-like cells enhances the proliferation and differentiation of these cells. In the present study, we investigated the effect of epinephrine, an agonist of Gi-protein-coupled receptors in MC3T3-E1 cells, on Pi transport, type III Pi transporter expression, and the signaling mechanism(s) involved in this response. Epinephrine time- and dose-dependently stimulated sodium-dependent Pi transport and this effect was dependent on RNA and protein synthesis. This effect was associated with a related time-dependent increase in Pit-1 mRNA expression. Kinetic analysis indicated that the change in Pi transport activity induced by epinephrine was due to alteration in the maximal rate of Pi transport. Investigation of Pi transport stimulation by several adrenergic agonists and its inhibition by spiperone suggest that the effect of epinephrine on Pi transport was mediated by alpha1-adrenergic receptors. Pertussis toxin, which inactivates Gi/o proteins, significantly blunted the stimulatory effect of epinephrine on Pi transport. Analysis of the signaling pathways involved in this response has suggested a major role of protein kinase C and a small contribution from the mitogen-activated protein kinase Erk (MAPK(erk)). The results show that, in MC3T3-E1 osteoblast-like cells, activation of Gi/o-protein-coupled receptors induces stimulation of Pi transport. This effect is mediated by activation of protein kinase C and the MAPK(erk) pathway and probably involves the synthesis of Pit-1 transporters.

Role of N-cadherin and protein kinase C in osteoblast gene activation induced by the S252W fibroblast growth factor receptor 2 mutation in Apert craniosynostosis.

Apert (Ap) syndrome is characterized by premature cranial suture ossification caused by fibroblast growth factor receptor 2 (FGFR-2) mutations. We studied the role of cadherins and signaling events in the phenotypic alterations induced by the Ap FGFR-2 S252W mutation in mutant immortalized fetal human calvaria osteoblasts. The FGFR-2 mutation caused increased expression of the osteoblast markers alkaline phosphatase (ALP), type 1 collagen (COLIA1), and osteocalcin (OC) in long-term culture. The mutation also increased cell-cell aggregation, which was suppressed by specific neutralizing anti-N- and anti-E-cadherin antibodies. Mutant osteoblasts showed increased N- and E-cadherin, but not N-cell adhesion molecule (N-CAM) messenger RNA (mRNA) and protein levels. This was confirmed in vivo by the abundant immunoreactive N- and E-cadherins in preosteoblasts in the Ap suture whereas N-CAM and alpha- and beta-catenins were unaffected. Neutralizing anti-N-cadherin antibody or N-cadherin antisense (AS) oligonucleotides but not anti-E-cadherin antibody or AS reduced ALP activity as well as ALP, COLIA1, and OC mRNA overexpression in mutant osteoblasts. Analysis of signal transduction revealed increased phospholipase Cgamma (PLCgamma) and protein kinase Calpha (PKCalpha) phosphorylation and increased PKC activity in mutant cells in basal conditions. Inhibition of PKC by calphostin C or the PKCalpha-specific inhibitor Gö6976 suppressed the increased N-cadherin mRNA and protein levels as well as the overexpression of ALP, COLIA1, and OC mRNA in mutant cells. Thus, N-cadherin plays a role in the activation of osteoblast differentiation marker genes in mutant osteoblasts and PKCalpha signaling appears to be involved in the increased N-cadherin and osteoblast gene expression induced by the S252W FGFR-2 mutation in human osteoblasts.

Fibroblast growth factor-2 (FGF-2) increases N-cadherin expression through protein kinase C and Src-kinase pathways in human calvaria osteoblasts.

Fibroblast growth factors (FGFs) are important factors regulating osteogenesis. However, the early mechanisms and signaling pathways involved in FGF actions in osteoblasts are unknown. We investigated the effects of FGF-2 on cell-cell adhesion and cadherin expression and the underlying signaling pathways in immortalized human neonatal calvaria (IHNC) cells. These cells express E- and N-cadherins, as shown by immunocytochemical and Western blot analyses. rhFGF-2 increased cell-cell adhesion at 24-72 h, as measured in a cell aggregation assay, and this effect was blocked by specific neutralizing anti-N-cadherin, but not anti-E-cadherin antibodies. Accordingly, ELISA and Western blot analyses showed that rhFGF-2 (10-100 ng/ml) dose dependently increased N-cadherin but not E-cadherin protein levels. RT-PCR analysis showed that rhFGF-2 transiently increased N-cadherin mRNA levels in IHNC cells. The RNA polymerase II inhibitor 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole prevented the rhFGF-2-induced up-regulation of N-cadherin mRNA, suggesting that transcription is necessary for this effect. Analysis of signaling molecules showed evidence that PLCgamma-PKC, Src, Erk 1/2 and p38 MAPK pathways are activated by rhFGF-2 in IHNC cells. The selective PKC inhibitors calphostin C, Ro-31-8220, Gö6976 and Gö6983 abrogated the stimulatory effect of rhFGF-2 on N-cadherin mRNA levels. The src-family tyrosine kinase inhibitor PP1 also blocked rhFGF-2-promoted N-cadherin expression. In contrast, the p38 MAP kinase inhibitor SB 203580 or the MEK inhibitor PD98059 had no effect on rhFGF-2-induced N-cadherin mRNA levels. Our data indicate that FGF-2 increases N-cadherin expression and function in human calvaria osteoblasts via activation of PKC and src-kinase pathways. This study identifies N-cadherin as a previously unrecognized target gene for FGF-2 signaling pathway that regulates cell-cell adhesion in human osteoblasts.

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.

Transforming growth factor-beta stimulates inorganic phosphate transport and expression of the type III phosphate transporter Glvr-1 in chondrogenic ATDC5 cells.

Members of the transforming growth factor (TGF)-beta family are important regulators of skeletal development. In this study, we investigated the effect of TGF-beta1 on inorganic phosphate (Pi) transport and on expression of the type III Pi carriers Glvr-1 and Ram-1 in murine ATDC5 chondrocytes. TGF-beta1 induced a selective, dose- and time-dependent increase in sodium-dependent Pi transport in ATDC5 cells. This response was dependent on RNA and protein synthesis and reflected a change in the maximal rate of the transport system, suggesting that TGF-beta1 induces the synthesis of new Pi carriers and their insertion into the plasma membrane. Consistently, Northern blotting analysis showed a dose-dependent increase in Glvr-1 messenger RNA expression in response to TGF-beta1, which preceded the maximal stimulation of Pi transport by several hours. Glvr-1 thus likely mediates at least part of the increase in Pi uptake induced by TGF-beta1. Ram-1 messenger RNA expression was not affected by TGF-beta1. TGF-beta1 activated the Smad signaling pathway and the mitogen-activated protein kinases ERK and p38 in ATDC5 cells. Unlike the regulation of Pi transport by receptor tyrosine kinase agonists in osteoblasts, the effect of TGF-beta1 on Pi uptake in ATDC5 cells did not involve protein kinase C or mitogen-activated protein kinases, suggesting that a specific, possibly Smad-dependent, signal mediates this response. In conclusion, TGF-beta1 stimulates Pi transport and Glvr-1 expression in chondrocytes, suggesting that, like proliferation, differentiation, and matrix synthesis, Pi handling is subject to regulation by TGF-beta3 family members in bone-forming cells.

A novel in vitro culture system for analysis of functional role of phosphate transport in endochondral ossification.

In vivo expression of the type III sodium-dependent phosphate transporter (NaPiT) Glvr-1 during endochondral ossification, suggests a functional role for inorganic phosphate (Pi) transport in cartilage calcification. For further analysis of this relationship, an in vitro model of endochondral ossification is required. In this context, we investigated the characteristics of Pi transport in the new chondrogenic cell line ATDC5 in relation to extracellular matrix (ECM) formation and mineralization. Pi uptake in ATDC-5 cells and in isolated matrix vesicles (MVs) is mediated by an Na-dependent Pi transporter with a pH dependency characteristic of a type III Pi carrier (lower activity at alkaline pH). Northern blot analysis indicated that ATDC-5 cells express Glvr-1 transcripts during the various stages of their maturation with a maximal level during the proliferating stage. In isolated MVs, Pi transport activity was maximal at day 21, concomitant with the beginning of type X collagen messenger RNA expression. These events preceded the initiation of matrix mineralization, which was apparent at day 25, and then gradually increased until day 47. This temporal relationship between maximal Pi transport activity in MVs and the expression of a marker of mineralizing chondrocytes is compatible with the possible involvement of Pi transport in the ECM calcification observed in ATDC-5 cell cultures. In conclusion, these observations suggest that ATCD-5 cells in culture represent a promising model for the analysis of a functional role of Pi transport in the initial events of endochondral ossification.

Structure of the murine Pit1 phosphate transporter/retrovirus receptor gene and functional characterization of its promoter region.

The Pit1 phosphate transporter (formerly also called Glvr-1) probably plays an important role in regulated phosphate handling in bone-forming cells. In this study, we describe the structure of the mouse Pit1 gene, as well as some functional characteristics of its promoter region in murine bone cells. Screening of a genomic library led to the isolation of two overlapping lambda clones containing 7kb of 5' flanking region, as well as the 10 exons of the mouse Pit1 gene corresponding to the published cDNA. The translation start site is located within exon I and the stop codon within exon X. The overall structure of the mouse gene is very similar to that of its human homolog, except for the presence of an additional 5' untranslated exon in human. The structure of the 5' untranslated region of the mouse gene was thus further investigated using rapid amplification of cDNA ends in murine ATDC5, MC3T3-E1 and Swiss 3T3 cells. The results indicate that, compared to the published cDNA, the mouse Pit1 gene contains in fact one additional 5' exon, which we named exon IA. Reporter gene assays demonstrate the presence of a functional TATA box containing promoter upstream of exon IA. This description of the murine Pit1 gene and of its promoter region paves the way to more detailed analyses concerning the regulation of Pit1 transcription in mouse cells. Furthermore, a comparison of mouse and human promoters will hopefully allow a better understanding of general mechanisms regulating Pit1 expression in different species.

Stimulation of sodium-dependent phosphate transport and signaling mechanisms induced by basic fibroblast growth factor in MC3T3-E1 osteoblast-like cells.

Physiological and pathological observations indicate that basic fibroblast growth factor (bFGF) is an important regulator of osteoblastic cell differentiation and in particular of cranial ossification. Experimental evidence suggests that inorganic phosphate (Pi) transport could be an important function of bone matrix calcification. In the present study, we address the influence of bFGF on Pi transport activity in MC3T3-E1 osteoblast-like cells derived from mouse calvaria. The results indicate that bFGF is a potent and selective stimulator of sodium-dependent Pi transport in these cells. The change in Pi transport activity induced by bFGF depends on transcription and translation and corresponds to a change in the maximum velocity of the Pi transport system (Vmax). These observations suggest that enhanced Pi transport activity in response to bFGF may result from insertion of newly synthesized Pi transporters into the plasma membrane. A selective inhibitor of fibroblast growth factor receptor (FGFR) tyrosine kinase, SU5402, blunted the stimulation of Pi transport induced by bFGF. It also prevented the increase in protein tyrosine phosphorylation induced by bFGF, including phosphorylation of FGFR-1, FGFR-2, phospholipase C-gamma (PLC-gamma), and Shc as well as the recruitment of the Grb2/Sos signaling complex. In addition, bFGF-induced the activation of the mitogen-activated protein (MAP) kinases extracellular signal-regulated kinase (ERK) and p38, effects that were prevented by SU5402. Both the protein kinase C (PKC) inhibitor calphostin C and PKC down-regulation suppressed the stimulatory effect of bFGF on Pi transport. Selective inhibitors of ERK and p38 MAP kinases slightly reduced this cellular response with a significant effect observed with the highest concentration of the p38 MAP kinase inhibitor. In conclusion, the results of this study indicate that bFGF selectively stimulates Pi transport in calvaria-derived osteoblastic cells. The main signaling mechanism responsible for this effect involves tyrosine phosphorylation of PLC-gamma and activation of PKC, with a possible contribution of the p38 MAP kinase pathway.

Insulin-induced stimulation of Na+,K(+)-ATPase activity in kidney proximal tubule cells depends on phosphorylation of the alpha-subunit at Tyr-10.

Phosphorylation of the alpha-subunit of Na+,K(+)-ATPase plays an important role in the regulation of this pump. Recent studies suggest that insulin, known to increase solute and fluid reabsorption in mammalian proximal convoluted tubule (PCT), is stimulating Na+,K(+)-ATPase activity through the tyrosine phosphorylation process. This study was therefore undertaken to evaluate the role of tyrosine phosphorylation of the Na+,K(+)-ATPase alpha-subunit in the action of insulin. In rat PCT, insulin and orthovanadate (a tyrosine phosphatase inhibitor) increased tyrosine phosphorylation level of the alpha-subunit more than twofold. Their effects were not additive, suggesting a common mechanism of action. Insulin-induced tyrosine phosphorylation was prevented by genistein, a tyrosine kinase inhibitor. The site of tyrosine phosphorylation was identified on Tyr-10 by controlled trypsinolysis in rat PCTs and by site-directed mutagenesis in opossum kidney cells transfected with rat alpha-subunit. The functional relevance of Tyr-10 phosphorylation was assessed by 1) the abolition of insulin-induced stimulation of the ouabain-sensitive (86)Rb uptake in opossum kidney cells expressing mutant rat alpha1-subunits wherein tyrosine was replaced by alanine or glutamine; and 2) the similarity of the time course and dose dependency of the insulin-induced increase in ouabain-sensitive (86)Rb uptake and tyrosine phosphorylation. These findings indicate that phosphorylation of the Na+,K(+)-ATPase alpha-subunit at Tyr-10 likely participates in the physiological control of sodium reabsorption in PCT.

Regulation of alkaline phosphatase activity by p38 MAP kinase in response to activation of Gi protein-coupled receptors by epinephrine in osteoblast-like cells.

The signaling mechanisms responsible for the regulation of alkaline phosphatase (ALP) activity by exogenous factors in osteoblast-like cells remain poorly understood. Among various agents, epinephrine was recently found to increase ALP activity in differentiating MC3T3-E1 cells by stimulating alpha1 adrenergic receptors coupled to Gi proteins. In the present study, we investigated the role of both ERK2 and p38 mitogen-activated protein (MAP) kinases in mediating this response in MC3T3-E1 cells. Our results indicate that both MAP kinases are transiently stimulated by epinephrine in differentiating cells via a pertussis toxin sensitive mechanism. The role of each MAP kinase pathway in mediating the stimulation of ALP activity by epinephrine was investigated using specific inhibitors. The MEK inhibitor PD98059, blocked ERK2 activity induced by epinephrine but had no effect on the stimulation of ALP activity. In contrast, low concentrations of SB203580, a specific inhibitor of the p38 MAP kinase, completely blunted this cellular response. However, this inhibitor had no influence on the stimulation of ALP activity induced by ascorbic acid. In conclusion, the results of this study suggest distinct roles for ERK and p38 MAP kinase pathways in regulating activity of MC3T3-E1 osteoblastic cells. The ERK pathway is likely involved in the control of cell proliferation whereas the p38 MAP kinase pathway regulates ALP activity in response to activation of Gi protein-coupled receptors.

Characterization of the human Glvr-1 phosphate transporter/retrovirus receptor gene and promoter region.

The cell surface receptor for gibbon ape leukemia virus (Glvr-1) belongs to the type III sodium-dependent phosphate transporter/retrovirus receptor gene family. Several observations have suggested an important role for Glvr-1 in regulated Pi handling in bone forming cells and prompted us to investigate further the molecular mechanisms regulating Glvr-1 gene expression. In addition, the regulation of Glvr-1 gene expression also has potential applications to gene therapy, since retroviral vectors carrying gibbon ape leukemia virus envelope proteins are used for gene delivery into different cell types. The aim of this study was thus to clone the human Glvr-1 gene in order to describe its structure and its promoter region. Our results indicate that the Glvr-1 gene consists of 11 exons and 10 introns spread over 18kb of genomic DNA. The translation initiation site is located within exon II and the translation stop codon within exon XI. Rapid amplification of cDNA ends (5'-RACE) suggests that, in human SaOS-2 osteoblast-like cells, transcription of Glvr-1 is initiated at multiple sites, mostly located between bp 32 and 50 of the published cDNA sequence, which was initially obtained from HL-60 cells. The 5'-flanking region of the gene is characterized by a very high GC content. Reporter gene assays demonstrate the presence of a functional promoter upstream of exon I and indicate that a GC-rich region, containing two potential SP1 binding sites, is required for high promoter activity in transiently transfected SaOS-2 cells. The description of the human Glvr-1 gene structure, as well as the analysis of some structural and functional characteristics of its promoter region, provide a basis for more detailed investigation of the molecular mechanisms controlling expression of the Glvr-1 gene in bone forming cells and in other cell types.

In vivo expression of transcripts encoding the Glvr-1 phosphate transporter/retrovirus receptor during bone development.

In vitro observations suggest that inorganic phosphate (Pi) transport plays an important functional role in osteogenic cells and in their matrix vesicles for the initiation of matrix calcification. Recent studies have shown that the type III sodium-dependent Pi transporters, Glvr-1 and Glvr-2, are expressed in human osteoblast-like cells and have suggested a potential role for type III transporters in regulated Pi handling in osteogenic cells. To address the relevance of these findings in the context of bone formation in vivo and, in particular, in relation to matrix calcification, we investigated expression of the Glvr-1 transporter by in situ hybridization in developing embryonic murine metatarsals, using human Glvr-1 cDNA as a probe. In this model of endochondral ossification, expression of transcripts encoding Glvr-1 could be detected from day 17 of embryonic development. A hybridization signal for Glvr-1 was specifically observed in a subset of hypertrophic chondrocytes and could not be detected in osteoblasts. The expression of Glvr-1 mRNA was compared with that of transcripts encoding extracellular matrix proteins. Glvr-1 mRNA expression was confined to a population of early hypertrophic chondrocytes expressing type X collagen and to slightly more mature cells that express transcripts encoding osteopontin but lack type X collagen mRNA. No Glvr-1 transcripts were detected in fully differentiated hypertrophic chondrocytes. This pattern of Glvr-1 mRNA expression was maintained throughout embryonic development until after birth. In conclusion, the Glvr-1 phosphate transporter is selectively expressed in a subset of hypertrophic chondrocytes during endochondral bone formation, in a region where matrix mineralization proceeds. This observation represents the first in vivo evidence consistent with a potential role for this phosphate transporter in matrix calcification.

Catecholamines stimulate the proliferation and alkaline phosphatase activity of MC3T3-E1 osteoblast-like cells.

A number of factors have been shown to influence osteoblastic proliferation, including fluoride. Recent observations suggest that heterotrimeric G proteins are probably involved in the mitogenic response induced by this agent, further suggesting a role of guanosine 5'-triphosphate (GTP)-binding protein-coupled receptors (GPCR) in the regulation of osteoblastic cell growth. We therefore explored what mitogenic factors known to activate GPCR can influence the replication of mouse osteoblast-like MC3T3-E1 cells. Among several candidates, epinephrine was found to be a potent mitogen for these cells, and its effect on the growth and differentiation of these cells was further investigated. Deoxyribonucleic acid (DNA) synthesis was dose dependently enhanced by this catecholamine in the concentration range of 1 nmol/L-10 micromol/L. Stimulation of DNA synthesis by catecholamines was in the order of epinephrine > norepinephrine >> isoproterenol, indicating that alpha adrenergic receptors mediated this cellular response. Further analysis with specific adrenergic receptor agonists and antagonists suggested that the mitogenic response induced by epinephrine in MC3T3-E1 cells is mediated by alpha1 adrenergic receptors. In addition to its effect on cell replication, epinephrine also enhanced alkaline phosphatase (ALP) activity in these cells but had little effect on collagen synthesis and osteocalcin production. As for the mitogenic response, the change in ALP activity was found to be mediated by alpha1 adrenergic receptors. Both effects of epinephrine on cell replication and ALP activity were markedly reduced by pretreatment of the cells with pertussis toxin (PTX), suggesting a role of Gi proteins. These effects were also completely blocked by pretreatment of the cells with 50 micromol/L genistein, a nonselective inhibitor of tyrosine kinase. In conclusion, the results indicate that epinephrine enhances replication and ALP activity of MC3T3-E1 osteoblast-like cells via alpha1 adrenergic receptors coupled to Gi proteins. The signaling mechanism probably involves a tyrosine phosphorylation mechanism. These observations suggest that PTX-sensitive G proteins are potent mediators of cell proliferation and ALP activity of osteoblast-like cells in response to factors acting through G protein-coupled receptors.

Arginine increases insulin-like growth factor-I production and collagen synthesis in osteoblast-like cells.

Protein-energy malnutrition, which is common in elderly patients with osteoporotic hip fractures, is associated with reduced plasma levels of insulin-like growth factor-I (IGF-I). IGF-I is an important regulator of bone metabolism, particularly of osteoblastic bone formation both in vivo and in vitro. Pharmacological doses of arginine (Arg) increase growth hormone (GH) and IGF-I serum levels. Whether amino acids, particularly Arg, can directly modulate the production of IGF-I by osteoblasts is not known. We investigated the effects of increasing concentrations of Arg on IGF-I expression and production, alpha1(I) collagen expression and collagen synthesis, and cell proliferation and cell differentiation, as assessed by alkaline phosphatase (ALP) activity and osteocalcin (OC) release, in confluent mouse osteoblast-like MC3T3-E1 cells. The addition of Arg (7.5-7500 micromol/L, equivalent to 0.1- to 100-fold human plasma concentration) for 48 h increased IGF-I production (adjusted for cell number) in a concentration-dependent manner with a maximum of 2.3 +/- 0.3-fold at 7500 micromol/L Arg [x +/- standard error of the mean (SEM), n = 3 experiments, p < 0.01]. Arg (7.5-7500 micromol/L) increased the percentage of de novo collagen synthesis in a concentration-dependent manner (2.1 +/- 0.4-fold with 7500 micromol/L Arg, p < 0.001) and ALP activity with a maximal stimulation of 144% +/- 13% plateauing at 750 micromol/l Arg (p = 0.002). The steady state level of IGF-I messenger ribonucleic acid (mRNA) and alpha1(I) collagen mRNA (both normalized to cyclophilin mRNA) of cells incubated with Arg at high (100-fold) or low (0.1-fold) human plasma concentrations, was 1.4 +/- 0.2, 1.2 +/- 0.2, and 1.1 +/- 0.2 after 24 h for the 7.5, 1.8, and 0.9 kb IGF-I mRNA transcripts, respectively (n = 3 experiments) and 1.5 +/- 0.2 and 3.1 +/- 0.7 after 24 and 48 h, respectively, for the combined analysis of the 5.6 and 4.7 kb alpha1(I) collagen mRNA transcripts (n = 3 experiments). A maximal mitogenic effect (cell number) of +21% +/- 3% (p < 0.01) was obtained with 1000 micromol/L Arg. In contrast, Arg (7.5-7500 micromol/L) induced a reduction of OC production, which reached 30% +/- 3% with 7500 micromol/L Arg (p = 0.02). In conclusion, Arg stimulated IGF-I production and collagen synthesis in osteoblast-like cells. Thus, Arg may influence bone formation by enhancing local IGF-I production.