In vitro and in vivo induction of bone formation using a recombinant adenoviral vector carrying the human BMP-2 gene.

It has been well established that bone morphogenetic protein-2 (BMP-2) can induce bone formation both in vivo and in vitro, although high concentrations (up to milligrams) of BMP-2 have been required to achieve this effect in vivo. Further, clinical applications are usually limited to a single dose at the time of implantation. In an attempt to prolong the transforming effect of BMP-2 we used a recombinant adenoviral vector carrying the human BMP-2 gene (Adv-BMP2) to transduce marrow-derived mesenchymal stem cells (MSC) of skeletally mature male New Zealand white rabbits. The pluripotential MSC were incubated with Adv-BMP2 overnight followed by culture in growth medium for 1 week. Assays on tissue cultures demonstrated that these Adv-BMP2 transduced MSC produced BMP-2 protein, differentiated into an osteoprogenitor line, and induced bone formation in vitro. These MSC had increased alkaline phosphatase activity, increased expression of type I collagen, osteopontin, and osteocalcin mRNA, and induced matrix mineralization compared with both non-transduced cells and cells transduced with a control adenoviral construct. To analyze the osteogenic potential in vivo, Adv-BMP2-transduced MSC were autologously implanted into the intertransverse process space between L5 and L6 of the donor rabbits. The production of new bone was demonstrated by radiographic examination 4 weeks later in areas implanted with cells transduced with Adv-BMP2, whereas no bone was evident at sites implanted with cells transduced with the control adenoviral construct. Histological examination further confirmed the presence of new bone formation. These accumulated data indicate that it is possible to successfully transduce mesenchymal stem cells with a recombinant adenoviral vector carrying the gene for BMP-2 such that these cells will produce BMP-2, differentiate into an osteoprogenitor line, and induce bone formation both in vitro and in vivo. Moreover, incubation of the Adv-BMP2-transduced cells for an additional 7 days in culture before transplantation enhances the success rate in bone formation (three out of three) as compared with our previous report (one out of five, Calcif Tissue Int 63:357-360, 1998).

Erk is essential for growth, differentiation, integrin expression, and cell function in human osteoblastic cells.

Extracellular signal-regulated kinases (Erks), members of the mitogen-activated protein kinase superfamily, play an important role in cell proliferation and differentiation. In this study we employed a dominant negative approach to determine the role of Erks in the regulation of human osteoblastic cell function. Human osteoblastic cells were transduced with a pseudotyped retrovirus encoding either a mutated Erk1 protein with a dominant negative action against both Erk1 and Erk2 (Erk1DN cells) or the LacZ protein (LacZ cells) as a control. Both basal and growth factor-stimulated MAPK activity and cell proliferation were inhibited in Erk1DN cells. Expression of Erk1DN protein suppressed both osteoblast differentiation and matrix mineralization by decreasing alkaline phosphatase activity and the deposition of bone matrix proteins. Cell adhesion to collagen, osteopontin, and vitronectin was decreased in Erk1DN cells as compared with LacZ cells. Cell spreading and migration on these matrices were also inhibited. In Erk1DN cells, expression of alphabeta(1), alpha(v)beta(3), and alpha(v)beta(5) integrins on the surface was decreased. Metabolic labeling indicated that the synthesis of these integrins was inhibited in Erk1DN cells. These data suggest that Erks are not only essential for the growth and differentiation of osteoblasts but also are important for osteoblast adhesion, spreading, migration, and integrin expression.

Bone mineralization and osteoblast differentiation are negatively modulated by integrin alpha(v)beta3.

Numerous bone matrix proteins can interact with alpha(v)-containing integrins including alpha(v)beta3. To elucidate the net effects of the interaction between these proteins and alpha(v)beta3 on osteoblast function, we developed a murine osteoblastic cell line that overexpressed human alpha(v)beta3. Human alpha(v)beta3-integrin was expressed on cell membrane, in which its presence did not alter the surface level of endogenous mouse alpha(v)beta3. The expressed human alpha(v)beta3 was functional because cell adhesion to osteopontin was increased and this increment was abolished by antibody against human alpha(v)beta3. The proliferation rate of cells overexpressing alpha(v)beta3 (alpha(v)beta3-cells) was increased whereas matrix mineralization was decreased. To elucidate the mechanisms leading to inhibition of matrix mineralization, the expression of proteins important for mineralization was analyzed. Alkaline phosphatase activity and the expression of osteocalcin, type I collagen, and bone sialoprotein (BSP) were decreased whereas osteopontin was stimulated in alpha(v)beta3-cells. The regulation of osteopontin, osteocalcin, and BSP expression was mediated via transcriptional mechanism because their promoter activities were altered. Examination of molecules involved in integrin signaling indicated that activator protein-1 (AP-1) and extracellular signal-regulated kinase (Erk) activities were enhanced whereas c-jun N-terminal kinase (JNK) activity was decreased in alpha(v)beta3-cells. The activity of p38 and the levels of focal adhesion kinase (FAK) and vinculin were not altered. Moreover, the adhesions of alpha(v)beta3-cells to type I collagen and fibronectin were inhibited, which was attributed to decreased beta1-integrin levels on cell surface. In conclusion, overexpressing alpha(v)beta3-integrin in osteoblasts stimulated cell proliferation but retarded differentiation, which were derived via altered integrin-matrix interactions, signal transduction, and matrix protein expression.

In Vitro andIn Vivo induction of bone formation using a recombinant adenoviral vector carrying the human BMP-2 gene.

It has been well established that bone morphogenetic protein-2 (BMP-2) can induce bone formation bothin vivo andin vitro, although high concentrations (up to milligrams) of BMP-2 have been required to achieve this effectin vivo. Further, clinical applications are usually limited to a single dose at the time of implantation. In an attempt to prolong the transforming effect of BMP-2 we used a recombinant adenoviral vector carrying the human BMP-2 gene (Adv-BMP2) to transduce marrow-derived mesenchymal stem cells (MSC) of skeletally mature male New Zealand white rabbits. The pluripotential MSC were incubated with Adv-BMP2 overnight followed by culture in growth medium for 1 week. Assays on tissue cultures demonstrated that these Adv-BMP2 transduced MSC produced BMP-2 protein, differentiated into an osteoprogenitor line, and induced bone formationin vitro. These MSC had increased alkaline phosphatase activity, increased expression of type I collagen, osteopontin, and osteocalcin mRNA, and induced matrix mineralization compared with both nontransduced cells and cells transduced with a control adenoviral construct. To analyze the osteogenic potentialin vivo, Adv-BMP2-transduced MSC were autologously implanted into the intertransverse process space between L5 and L6 of the donor rabbits. The production of new bone was demonstrated by radiographic examination 4 weeks later in areas implanted with cells transduced with Adv-BMP2, whereas no bone was evident at sites implanted with cells transduced with the control adenoviral construct. Histological examination further confirmed the presence of new bone formation. These accumulated data indicate that it is possible to successfully transduce mesenchymal stem cells with a recombinant adenoviral vector carrying the gene for BMP-2 such that these cells will produce BMP-2, differentiate into an osteoprogenitor line, and induce bone formation bothin vitro andin vivo. Moreover, incubation of the Adv-BMP2-transduced cells for an additional 7 days in culture before transplantation enhances the success rate in bone formation (three out of three) as compared with our previous report (one out of five, Calcif Tissue Int 63:357-360, 1998).

Transforming growth factor-beta up-regulates the beta 5 integrin subunit expression via Sp1 and Smad signaling.

Integrin-mediated cell-matrix interactions play important roles in regulating cell function. Since transforming growth factor-beta (TGF-beta) modulates many osteoblast activities, we hypothesized that the growth factor acts in part by modulating integrin expression. TGF-beta increased cell adhesion to vitronectin and up-regulated the surface level of alpha(v)beta(5) via increasing beta(5) protein synthesis by a transcriptional mechanism. Promoter activity analysis demonstrated that a TGF-beta-responsive element resides between nucleotides -63 and -44. Electrophoretic mobility shift assay and immunoprecipitation/Western studies indicated that the nuclear complex formed using the -66/-42 oligonucleotide contained both Sp1/Sp3 and Smad proteins. Since nuclear Sp1/Sp3 levels were not altered, whereas Smad levels were increased by TGF-beta, we investigated the roles of Smad proteins in the up-regulation of beta(5) gene activation. Co-transfection of cells with beta(5) promoter reporter construct and expression vectors for Smad3, Smad4, and Sp1 increased the stimulatory effect of TGF-beta. Furthermore, expression of dominant negative Smad3 or Smad4 in cells decreased or abolished the stimulation of beta(5) promoter activity by TGF-beta. Smad4 mutant also inhibited the up-regulation of surface beta(5) level by TGF-beta. Thus, TGF-beta increases expression of the integrin beta(5) gene by mechanisms involving Sp1/Sp3 and Smad transcription factors.

Differential regulation of cadherins by dexamethasone in human osteoblastic cells.

Human osteoblasts express a repertoire of cadherins, including N-cadherin (N-cad), cadherin-11 (C11), and cadherin-4 (C4). We have previously shown that direct cell-cell adhesion via cadherins is critical for BMP-2-induced osteoblast differentiation. In this study, we have analyzed the regulation of cadherin expression in normal human trabecular bone osteoblasts (HOB), and osteoprogenitor marrow stromal cells (BMC), during exposure to dexamethasone, another inducer of human bone cell differentiation. Dexamethasone inhibited the expression of both C11 and N-cad mRNA in both BMC and HOB, although the effect was much more pronounced on N-cad than on C11. This action of the steroid was dose dependent, was maximal at 10(-7) M concentration, and occurred as early as after 1 day of incubation. By contrast, expression of C4 mRNA and protein was strongly induced by dexamethasone in BMC and was stimulated in HOB. This stimulatory effect lasted for at least 2 weeks of incubation. A cadherin inhibitor, HAV-containing decapeptide only partially ( approximately 50%) prevented dexamethasone-induced stimulation of alkaline phosphatase activity by BMC, which instead was not altered by incubation with a neutralizing antibody against C4. Therefore, the pattern of cadherin regulation by dexamethasone radically differs form that observed with BMP-2. Dexamethasone effects on certain osteoblast differentiated features, such as induction of alkaline phosphatase activity are not strictly dependent on cadherin function.

Sp1/Sp3 and PU.1 differentially regulate beta(5) integrin gene expression in macrophages and osteoblasts.

Murine osteoclast precursors and osteoblasts express the integrin alpha(v)beta(5), the appearance of which on the cell surface is controlled by the beta(5), and not the alpha(v), subunit. Here, we show that a 173-base pair proximal region of the beta(5) promoter mediates beta(5) basal transcription in macrophage (osteoclast precursor)-like and osteoblast-like cells. DNase I footprinting reveal four regions (FP1-FP4) within the 173-base pair region, protected by macrophage nuclear extracts. In contrast, osteoblast nuclear extracts protect only FP1, FP2, and FP3. FP1, FP2, and FP3 bind Sp1 and Sp3 from both macrophage and osteoblast nuclear extracts. FP4 does not bind osteoblast proteins but binds PU.1 from macrophages. Transfection studies show that FP1 and FP2 Sp1/Sp3 sites act as enhancers in both MC3T3-E1 (osteoblast-like) and J774 (macrophage-like) cell lines, whereas the FP3 Sp1/Sp3 site serves as a silencer. Mutation of the FP2 Sp1/Sp3 site totally abolishes promoter activity in J774 cells, with only partial reduction in MC3T3-E1 cells. Finally, we demonstrate that PU.1 acts as a beta(5) silencer in J774 cells but plays no role in MC3T3-E1 cells. Thus, three Sp1/Sp3 sites regulate beta(5) gene expression in macrophages and osteoblast-like cells, with each element exhibiting cell-type and/or activation-suppression specificity.

Regulation of alphaVbeta3 and alphaVbeta5 integrins by dexamethasone in normal human osteoblastic cells.

Long-term administration of pharmacological doses of glucocorticoids inhibits bone formation and results in osteoporosis. Since integrin-mediated cell-matrix interactions are essential for osteoblast function, we hypothesized that the detrimental effect of glucocorticoids on bone derived, at least in part, from decreased integrin-matrix interactions. Because alphavbeta3 and alphavbeta5 integrins can interact with several bone matrix proteins, we analyzed the effects of dexamethasone (Dex) on the expression of these integrins in normal human osteoblastic cells. We found adhesion of these cells to osteopontin and vitronectin to be dependent on alphavbeta3 and alphavbeta5, respectively; this ligand specificity was not altered by Dex. The effects of Dex on the adhesion of human osteoblastic cells to osteopontin and vitronectin were biphasic with an increase after 2 days, followed by a decrease after 8 days of treatment. Consistently, surface alphavbeta3 and alphavbeta5 integrins, which were increased after 2 days of Dex treatment, were decreased after 8 days. Similarly, total cellular alphav, beta3, and beta5 proteins, which were increased by Dex early in the culture, were diminished after 8 days. Metabolic labeling studies indicated that Dex exhibited biphasic regulation on the biosynthesis of alphavbeta5, with stimulation observed during the second day of treatment, followed by inhibition during the 8th day of exposure. By contrast, the biosynthesis of alphavbeta3 was inhibited by Dex on day 1 and remained inhibited on day 8. Analysis of the mRNA indicated that alphav and beta5 levels were increased by Dex during early exposure (1-3 days), followed by inhibition after prolonged exposure (>/=7 days). By contrast, Dex decreased beta3 mRNA level at all the time points analyzed. Consistently, Dex decreased beta3 promoter activity after 1 day and persisted over 8-day period. By contrast, Dex stimulated beta5 promoter activity after 1 or 2 days but had no effect after 8 days. To further evaluate mechanism(s) leading to the decreased integrin expression after prolonged Dex treatment, mRNA stability was analyzed. Dex was found to accelerate the degradation of alphav, beta3 and beta5 mRNA after an 8-day treatment. Thus, the regulation of alphavbeta3 was dependent on transcription and posttranscriptional events whereas the expression of alphavbeta5 was dependent mainly on posttranscriptional events after prolonged Dex treatment. In conclusion, Dex exhibited time-dependent regulation on the expression of alphavbeta3 and alphavbeta5 integrins in normal human osteoblastic cells. Short-term exposure to Dex increased the levels of alphavbeta3 and alphavbeta5 on the surface and cell adhesion to osteopontin and vitronectin whereas long-term exposure to Dex decreased the expression of both integrins and inhibited the cell adhesion to matrix proteins.

Extracellular-signal regulated kinase signaling pathway mediates downregulation of type I procollagen gene expression by FGF-2, PDGF-BB, and okadaic acid in osteoblastic cells.

Although basic fibroblast growth factor (FGF-2) had been shown to inhibit type I collagen gene expression in osteoblast, its inhibitory mechanism is unknown. In the present study, we investigated the underlying mechanisms by which growth factors downregulate type I collagen gene expression. Treatment of mouse osteoblastic MC3T3-E1 cells with okadaic acid (40 ng/ml), an inhibitor of phosphoserine/threonine-specific protein phosphatase and activator of ERK1/2, for 24 h and 48 h completely inhibited steady-state mRNA levels of type I collagen. FGF-2 (30 ng/ml), platelet-derived growth factor-BB (PDGF-BB), 30 ng/ml, and serum, which activate ERK mitogen-activated protein kinase (MAPK) pathway also inhibited collagen type I gene expression, suggesting that the activation of ERK pathway mediates inhibition of type I collagen mRNA. This observation was further confirmed by experiments using inhibitors of the ERK pathway (i.e., PD and U0126), which increased type I collagen mRNA in MC3T3-E1 cells, indicating that the inhibition of ERK pathway upregulates type I collagen gene expression. Low serum (0.3%) markedly increased type I collagen mRNA. MEK inhibitor PD inhibited c-fos induction by FGF-2 and PDGF-BB, suggesting that c-fos is the downstream target of ERK pathway. Our data have clearly demonstrated for the first time that the ERK MAPK pathway play an important role in the regulation of type I collagen gene expression in osteoblastic cells. Results also showed that one of the mechanisms by which FGF-2 and PDGF-BB downregulate type I collagen gene expression in the osteoblast is through the activation of ERK signaling pathway.

Reduction of vertebral fracture risk in postmenopausal women with osteoporosis treated with raloxifene: results from a 3-year randomized clinical trial. Multiple Outcomes of Raloxifene Evaluation (MORE) Investigators.

CONTEXT: Raloxifene hydrochloride, a selective estrogen receptor modulator, prevents bone loss in postmenopausal women, but whether it reduces fracture risk in these women is not known. OBJECTIVE: To determine the effect of raloxifene therapy on risk of vertebral and nonvertebral fractures. DESIGN: The Multiple Outcomes of Raloxifene Evaluation (MORE) study, a multicenter, randomized, blinded, placebo-controlled trial. SETTING AND PARTICIPANTS: A total of 7705 women aged 31 to 80 years in 25 countries who had been postmenopausal for at least 2 years and who met World Health Organization criteria for having osteoporosis. The study began in 1994 and had up to 36 months of follow-up for primary efficacy measurements and nonserious adverse events and up to 40 months of follow-up for serious adverse events. INTERVENTIONS: Participants were randomized to 60 mg/d or 120 mg/d of raloxifene or to identically appearing placebo pills; in addition, all women received supplemental calcium and cholecalciferol. MAIN OUTCOME MEASURES: Incident vertebral fracture was determined radiographically at baseline and at scheduled 24- and 36-month visits. Nonvertebral fracture was ascertained by interview at 6-month-interim visits. Bone mineral density was determined annually by dual-energy x-ray absorptiometry. RESULTS: At 36 months of the evaluable radiographs in 6828 women, 503 (7.4%) had at least 1 new vertebral fracture, including 10.1% of women receiving placebo, 6.6% of those receiving 60 mg/d of raloxifene, and 5.4% of those receiving 120 mg/d of raloxifene. Risk of vertebral fracture was reduced in both study groups receiving raloxifene (for 60-mg/d group: relative risk [RR], 0.7; 95% confidence interval [CI], 0.5-0.8; for 120-mg/d group: RR, 0.5; 95% CI, 0.4-0.7). Frequency of vertebral fracture was reduced both in women who did and did not have prevalent fracture. Risk of nonvertebral fracture for raloxifene vs placebo did not differ significantly (RR, 0.9; 95% CI, 0.8-1.1 for both raloxifene groups combined). Compared with placebo, raloxifene increased bone mineral density in the femoral neck by 2.1 % (60 mg) and 2.4% (120 mg) and in the spine by 2.6% (60 mg) and 2.7% (120 mg) P<0.001 for all comparisons). Women receiving raloxifene had increased risk of venous thromboembolus vs placebo (RR, 3.1; 95% CI, 1.5-6.2). Raloxifene did not cause vaginal bleeding or breast pain and was associated with a lower incidence of breast cancer. CONCLUSIONS: In postmenopausal women with osteoporosis, raloxifene increases bone mineral density in the spine and femoral neck and reduces risk of vertebral fracture.

A novel T cell cytokine stimulates interleukin-6 in human osteoblastic cells.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by a heavy lymphocytic infiltration into the synovial cavity, resulting in the secretion of a variety of cytokines which ultimately leads to destruction of joint tissue. Among the infiltrating cells are activated T cells which produce specific cytokines capable of osteoclast progenitor cell expansion, fusion, and activation. Cultures of activated human T cells and human osteoblasts (hOBs) were used to study the possibility that lymphokines may act on osteoblasts to produce the osteoclastogenic factor interleukin-6 (IL-6). Purified T cells were activated with a combination of anti-CD3 and anti-CD28 antibodies, cocultured with hOBs in direct physical contact or separated by a transwell system, and conditioned media (CM) were assayed for IL-6 production. After a 72 h incubation period, activated T cell-hOB interaction resulted in a 100-fold increase of IL-6 production over basal levels. The immunosuppressant cyclosporine A (CsA) inhibited T cell tumor necrosis factor alpha and IL-6 production but did not inhibit the T cell induction of IL-6 from hOB. Assay of activated T-cell CM on hOB revealed that a soluble factor, not cell-cell contact, was the major inducer of IL-6. The induction of IL-6 mRNA by both activated T cell CM and CsA-treated activated T cell CM was confirmed by Northern blot analysis. Neutralizing antibodies to IL-13 and IL-17 did not affect IL-6 production. These findings suggest that activated T cells produce a novel, potent, IL-6 inducing factor that may be responsible for the bone loss observed in RA patients.

Regulation of insulin-like growth factors I and II and their binding proteins in human bone marrow stromal cells by dexamethasone.

Glucocorticoids inhibit the proliferation, but induce the differentiation, of bone marrow stromal cells into osteoblast-like cells. The mechanisms, however, are still conjectural. Since insulin-like growth factors (IGFs) have profound effects on osteoblast growth and differentiation, it is possible that glucocorticoids exert their effects on bone marrow stromal cells in part via regulation of IGFs. Therefore, we analyzed the effects of dexamethasone (Dex) on the expression of IGF I and IGF II in cultured preosteoblastic normal human bone marrow stromal cells (HBMSC). Whereas Dex decreased the concentration of IGF I in the conditioned medium since early in the treatment, the concentration of IGF II was increased progressively as culture period lengthened. As the activities of IGF I and IGF II are regulated by the IGF binding proteins (IGFBPs), we analyzed the effects of Dex on the expression of IGFBPs. Dex increased IGFBP-2 in a time-dependent manner. The increase in IGFBP-2, however, was only to the same extent as that of IGF II at most, depending on the length of treatment. Therefore, the increase in IGFBP-2 would dampen, but not eliminate, the increased IGF II activities. By contrast, Dex decreased IGFBP-3 levels, the latter increasing the bioavailability of IGF II. Although IGFBP-4 mRNA levels were stimulated by Dex, IGFBP-4 concentration in the conditioned medium was unchanged as measured by RIA. IGFBP-5 and IGFBP-6 mRNA levels were decreased by Dex in a time-dependent fashion. IGFBP-5 protein level was also decreased 1-4 days after Dex treatment. IGFBP-1 mRNA was not detectable in HBMSC. These accumulated data indicate that Dex regulates IGF I and IGF II and their binding proteins differentially in normal human bone marrow stromal cells. The progressive increase in IGF II may contribute to Dex-induced cell differentiation.

Induction of bone formation using a recombinant adenoviral vector carrying the human BMP-2 gene in a rabbit spinal fusion model.

Bone marrow-derived mesenchymal stem cells are pluripotential cells that have the capacity to differentiate into an osteoprogenitor line. It has been demonstrated that BMP-2 can enhance this differentiation process. In an attempt to prolong the transforming effect of BMP-2, we used an adenoviral vector carrying the human BMP-2 gene to transduce marrow-derived mesenchymal stem cells of New Zealand white rabbits. Assays on tissue culture demonstrated that these cells indeed produced the BMP-2 protein. These transduced stem cells were then autologously reimplanted into the donor rabbits. The cells were placed in the intertransverse process area of five rabbits. In one out of the five rabbits, this resulted in the production of new bone which was demonstrable on both radiographic and histologic examination. We conclude that it is possible to successfully transduce mesenchymal stem cells with the gene for BMP-2 such that these cells will produce the BMP-2 protein in vitro. Further, the transduction results in transformation of these cells into an osteoprogenitor line capable of producing bone in vivo. These data suggest the feasibility of employing gene therapy using recombinant adenoviral vectors as a tool for enhancing spine fusion. Further work to improve the fidelity and longevity of the gene transfer is warranted.

The role of calcitonin in the prevention of osteoporosis.

This article discusses calcitonin as an alternative for patients with established osteoporosis who cannot, will not, or should not proceed with estrogen replacement therapy. Clinical trial data for calcitonin in both injectable and nasal spray formulations and for both lower-dose and higher-dose regimens are presented.

Identification and activation of mitogen-activated protein (MAP) kinase in normal human osteoblastic and bone marrow stromal cells: attenuation of MAP kinase activation by cAMP, parathyroid hormone and forskolin.

The mitogen-activated protein (MAP) kinases (p44mapk and p42mapk), also known as extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2), are activated in response to a variety of extracellular signals, including growth factors, hormones and, neurotransmitters. We have investigated MAP kinase signal transduction pathways in normal human osteoblastic cells. Normal human bone marrow stromal (HBMS), osteoblastic (HOB), and human (TE85, MG-63, SaOS-2), rat (ROS 17/2.8, UMR-106) and mouse (MC3T3-E1) osteoblastic cell lines contained immunodetectable p44mapk/ERK1 and p42mapk/ERK2. MAP kinase activity was measured by 'in-gel' assay using myelin basic protein as the substrate. Mainly ERK2 was rapidly activated (within 10 min) by bFGF, IGF-I and PDGF-BB in normal HOB, HBMS and human osteosarcoma cells, whereas both ERK1 and ERK2 were activated by growth factors in rat osteoblast-like cell lines, ROS 17/2.8 and UMR-106. The ERK1 activation was greater than the ERK2 in ROS 17/2.8 cells. Furthermore, ERK2 was also activated by bFGF and PDGF-BB in the mouse osteoblastic cell line, MC3T3-E1. This is the first demonstration of inter-species differences in the activation of MAP kinases in osteoblastic cells. Cyclic AMP derivatives or cAMP generating agents such as PTH and forskolin inhibited ERK2 activation by bFGF and PDGF-BB suggesting a 'cross-talk' between the two different signalling pathways activated by receptor tyrosine kinases and cAMP-dependent protein kinase. The accumulated results also suggest that the MAP kinases may be involved in mediating mitogenic and other biological actions of bFGF, IGF-I and PDGF-BB in normal human osteoblastic and bone marrow stromal cells.

Activation of c-Jun NH2-terminal kinases by interleukin-1 beta in normal human osteoblastic and rat UMR-106 cells.

We recently demonstrated the activation of extracellular signal- regulated protein kinase 1 and 2 (ERK1 and ERK2) by IGF-1, FGF-2, and PDGF-BB in normal human osteoblastic (HOB) cells as well as in rat and mouse osteoblastic cells. In this report, we have examined whether c-Jun NH2-Terminal Kinase (JNK) pathway is activated by growth factors and interleukin-1 beta (IL-1 beta) in normal HOB and rat UMR-106 cells using immune-complex kinase assay and anti-active JNK antibody, which recognizes activated forms of both JNK1 and JNK2. Results have demonstrated the presence of JNK1 and JNK2 proteins in normal HOB and UMR-106 cells. Both JNK1 and JNK2 were activated by IL-1 beta. IL-1 beta preferentially activated JNK pathway in a dose- and time-dependent manner and had little effect on ERK pathway. On the other hand, FGF-2 did not activate JNK but most strongly activated ERK pathway. The activation of JNK was maximal at 20 min whereas maximal activation of ERK1 and ERK2 was observed within 10 min. Results have clearly demonstrated that IL-1 beta preferentially activates JNK pathway whereas FGF-2 activates ERK pathway in normal human and rat UMR-106 osteoblastic cells.

Regulation of bone matrix protein expression and induction of differentiation of human osteoblasts and human bone marrow stromal cells by bone morphogenetic protein-2.

We have examined the effects of BMP-2 on the expression of bone matrix proteins in both human bone marrow stromal cells (HBMSC) and human osteoblasts (HOB) and their proliferation and mineralization. Both HBMSC and HOB express BMP-2/-4 type I and type II receptors. Treatment of these two cell types with BMP-2 for 4 weeks in the presence of beta-glycerophosphate and ascorbic acid results in mineralization of their matrix. BMP-2 increases the mRNA level and activities of alkaline phosphatase and elevates the mRNA levels and protein synthesis of osteopontin, bone sialoprotein, osteocalcin, and alpha 1(I) collagen in both cell types. Whereas the mRNA level of decorin is increased, the mRNA concentration of biglycan is not altered by BMP-2. No effect on osteonectin is observed. The effect of BMP-2 on bone matrix protein expression is dose dependent from 25 to 100 ng/ml and is evident after 1-7 days treatment. In the presence of BMP-2, proliferation of HBMSC and HOB is decreased under either serum-free condition or in the presence of serum. Thus, BMP-2 has profound effects on the proliferation, expression of most of the bone matrix proteins and the mineralization of both relatively immature human bone marrow stromal preosteoblasts and mature human osteoblasts.

Activation of extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2) by FGF-2 and PDGF-BB in normal human osteoblastic and bone marrow stromal cells: differences in mobility and in-gel renaturation of ERK1 in human, rat, and mouse osteoblastic cells.

We recently demonstrated that basic fibroblast growth factor (FGF-2) and platelet-derived growth factor-BB (PDGF-BB) mainly activated extracellular signal-regulated kinase 2 (ERK2) in normal human osteoblastic (HOB) and bone marrow stromal (HBMS) cells by an "in-gel" MAP kinase assay, although both ERK1 and ERK2 proteins were present. In the present study, we examined whether ERK1 is also activated by growth factors by using three different MAPK assay procedures, an "in-gel MAP kinase assay," an immune-complex kinase assay, and western blotting with anti-active MAPK antibody which recognizes specifically activated forms of both ERK1 and ERK2. Results have demonstrated that in addition to ERK2, ERK1 is activated by FGF-2 and PDGF-BB in normal HOB and HBMS cells. The human ERK1 moved faster on SDS-polyacrylamide gel compared to rat and mouse, revealing differences in the apparent molecular weight of FRK1 in normal human osteoblastic and bone marrow osteoprogenitor cells, human (TE-85) and rat (ROS 17/2.8 and UMR-106) osteosarcoma, and mouse (MC3T3E1) osteoblastic cells. ERK1 is less stable in the in-gel renaturation process compared to ERK2; thus, in-gel MAP kinase assay does not provide an accurate estimation of ERK1 activity. Results also showed that anti-active MAPK antibody can be used reliably and accurately to measure the activation of ERK1 and ERK2 in osteoblastic cells.

Skeletal casein kinase activity defect in the HYP mouse.

The Hyp mouse, a model for human X-linked hypophosphatemia (XLH), is characterized by phosphate wasting and defective mineralization. Since osteopontin (OPN) is considered pivotal for biological mineralization, we examined the biosynthesis of OPN in osteoblasts of +/Y and Hyp/Y mice. Immunoprecipitation analyses using a specific antibody to OPN revealed that Hyp/Y and +/Y osteoblasts secrete similar levels of OPN as determined by [35S]-methionine biosynthetic labeling, but a reduced phosphorylation was noted after 32P-PO4 biosynthetic labeling. Northern blot hybridization analysis of +/Y and Hyp/Y mice osteoblast mRNAs, using a cDNA probe for mouse OPN, revealed no difference in the steady state levels of osteopontin mRNA. Analysis of casein kinase II activity in +/Y and Hyp/Y mice osteoblast, kidney, heart and liver membrane fractions revealed that casein kinase II activity in the Hyp/Y mice osteoblasts and kidney is only 35%-50%, respectively, of that of the +/Y mice tissues. The accumulated data are consistent with a post-translation defect in the Hyp/Y mouse osteoblast which results in the under-phosphorylation of osteopontin and subsequent under-mineralization of bone matrix.