Biomedical subjects

P J Marie

Publications and source records attributed to P J Marie.

At least 19 recordsLinked to original sources

PubMed2006

Skeletal unloading induces osteoblast apoptosis and targets alpha5beta1-PI3K-Bcl-2 signaling in rat bone.

The mechanisms underlying the altered osteoblastogenesis and bone loss in response to disuse are incompletely understood. Using the rat tail suspension model, we studied the effect of skeletal unloading on osteoblast and osteocyte apoptosis. Tail suspension for 2 to 7 days decreased tibial bone mass and induced early apoptotic loss of osteoblasts and delayed apoptotic loss of osteocytes. Surrenal gland weight and plasma corticosterone levels did not differ in loaded and unloaded rats at any time point, indicating that osteoblast/osteocyte apoptosis occurred independently of endogenous glucocorticoids. The mechanistic basis for the disuse-induced osteoblast/osteocyte apoptosis was examined. We found that alpha5beta1 integrin and phosphorylated phosphatidyl-inositol-3 kinase (p-PI3K) protein levels were transiently decreased in unloaded metaphyseal long bone compared to loaded bones. In contrast, p-FAK and p-ERK p42/44 levels were not significantly altered. Interestingly, the reduced p-PI3K levels in unloaded long bone was associated with decreased levels of the survival protein Bcl-2 with unaltered Bax levels, causing increased Bax/Bcl-2 levels. The results indicate that skeletal unloading in rats induces a glucocorticoid-independent, immediate increase in osteoblast apoptosis associated with decreased alpha5beta1-PI3K-Bcl-2 survival pathway in rat bone, which may contribute to the altered osteoblastogenesis and osteopenia induced by unloading.

Adrenal Glands↗

PubMed2006

RhoA GTPase inactivation by statins induces osteosarcoma cell apoptosis by inhibiting p42/p44-MAPKs-Bcl-2 signaling independently of BMP-2 and cell differentiation.

Osteosarcoma is the most common primary bone tumour in young adults. Despite improved prognosis, resistance to chemotherapy remains responsible for failure of osteosarcoma treatment. The identification of signals that promote apoptosis may provide clues to develop new therapeutic strategies for chemoresistant osteosarcoma. Here, we show that lipophilic statins (atorvastatin, simvastatin, cerivastatin) markedly induce caspases-dependent apoptosis in various human osteosarcoma cells, independently of bone morphogenetic protein (BMP)-2 signaling and cell differentiation. Although statins increased BMP-2 expression, the proapoptotic effect of statins was not prevented by the BMP antagonist noggin, and was abolished by mevalonate and geranylgeranylpyrophosphate, suggesting the involvement of defective protein geranylgeranylation. Consistently, lipophilic statins induced membrane RhoA relocalization to the cytosol and inhibited RhoA activity, which resulted in decreased phospho-p42/p44- mitogen-activated protein kinases (MAPKs) and Bcl-2 levels. Constitutively active RhoA rescued phospho-p42/p44-MAPKs and Bcl-2 and abolished statin-induced apoptosis. Thus, lipophilic statins induce caspase-dependent osteosarcoma cell apoptosis by a RhoA-p42/p44 MAPKs-Bcl-2-mediated mechanism, independently of BMP-2 signaling and cell differentiation.

Anticholesteremic Agents↗

PubMed2006

Strontium ranelate: a physiological approach for optimizing bone formation and resorption.

Osteoporosis associated with estrogen deficiency results from an imbalance between bone resorption and formation, causing deterioration of bone architecture and decreased bone mass. Anti-osteoporotic therapies that have been developed so far include either anticatabolic or anabolic drugs. Strontium ranelate is a newly developed drug that induces opposite effects on bone resorption and formation. This dual original mode of action was demonstrated in experimental studies on bone cells and pharmacological studies in animals. In vitro, strontium ranelate was shown to decrease bone resorption. This effect resulted from a decreased differentiation and resorbing activity of osteoclasts and increased osteoclast apoptosis. In contrast, strontium ranelate was shown to enhance preosteoblastic cell replication and collagen synthesis in culture without affecting bone mineralization. In vivo, strontium ranelate promoted bone formation and reduced bone resorption in intact mice, an effect which resulted in increased vertebral bone mass. Additionally, strontium ranelate was found to reduce resorption and long bone loss induced by hind limb immobilization in rats. Finally, strontium ranelate administration decreased bone resorption and maintained bone formation in adult ovariectomized rats, which resulted in prevention of bone loss. In clinical trials (Spinal Osteoporosis Therapeutic Intervention [SOTI]), bone alkaline phosphatase levels increased, whereas C cross-linking telopeptide of type I collagen (CTX) levels decreased in patients treated with strontium ranelate compared with placebo at all time points. These pharmacological and clinical studies suggest that strontium ranelate acts by increasing bone formation and decreasing bone resorption and that these effects result in improved bone mass in vivo.

Animals↗

PubMed2005

FGF and FGFR signaling in chondrodysplasias and craniosynostosis.

The first experimental mouse model for FGF2 in bone dysplasia was made serendipitously by overexpression of FGF from a constitutive promoter. The results were not widely accepted, rightfully drew skepticism, and were difficult to publish; because of over 2,000 studies published on FGF-2 at the time (1993), only a few reported a role of FGF-2 in bone growth and differentiation. However, mapping of human dwarfisms to mutations of the FGFRs shortly, thereafter, made the case that bone growth and remodeling was a major physiological function for FGF. Subsequent production of numerous transgenic and targeted null mice for several genes in the bone growth and remodeling pathways have marvelously elucidated the role of FGFs and their interactions with other genes. Indeed, studies of the FGF pathway present one of the best success stories for use of experimental genetics in functionally parsing morphogenetic regulatory pathways. What remains largely unresolved is the pleiotropic nature of FGF-2. How does it accelerate growth in one cell then stimulate apoptosis or retard growth for another cell in the same type of tissue? Some of the answers may come through distinguishing the FGF-2 protein isoforms, made from alternative translation start sites, these appear to have substantially different functions. Although we have made substantial progress, there is still much to be learned regarding FGF-2 as a most complex, enigmatic protein. Studies of genetic models in mice and human FGFR mutations have provided strong evidence that FGFRs are important modulators of osteoblast function during membranous bone formation. However, there is some controversy regarding the effects of FGFR signaling in human and murine genetic models. Although significant progress has been made in our understanding of FGFR signaling, several questions remain concerning the signaling pathways involved in osteoblast regulation by activated FGFR. Additionally, little is known about the specific role of FGFR target genes involved in cranial bone formation. These issues need to be addressed in future in in vitro and in vivo approaches to better understand the molecular mechanisms of action of FGFR signaling in osteoblasts that result in anabolic effects in bone formation.

Animals↗

PubMed2005

Transforming growth factor-beta inhibits CCAAT/enhancer-binding protein expression and PPARgamma activity in unloaded bone marrow stromal cells.

The molecular mechanisms regulating the adipogenic differentiation of bone marrow stromal cells in vivo remain largely unknown. In this study, we investigated the regulatory effects of transforming growth factor beta-2 (TGF-beta2) on transcription factors involved in adipogenic differentiation induced by hind limb suspension in rat bone marrow stromal cells in vivo. Time course real-time quantitative reverse-transcription polymerase chain reaction (RT-PCR) analysis of gene expression showed that skeletal unloading progressively increases the expression of CCAAT/enhancer-binding protein (C/EBP)alpha and C/EBPbeta alpha at 5 days in bone marrow stromal cells resulting in increased peroxisome proliferator-activated receptor gamma (PPARgamma2) transcripts at 7 days. TGF-beta2 administration in unloaded rats corrected the rise in C/EBPalpha and C/EBPbeta transcripts induced by unloading in bone marrow stromal cells. This resulted in inhibition of PPARgamma2 expression that was associated with increased Runx2 expression. Additionally, the inhibition of C/EBPalpha and C/EBPbeta expression by TGF-beta2 was associated with increased PPARgamma serine phosphorylation in bone marrow stromal cells, a mechanism that inhibits PPARgamma transactivating activity. The sequential inhibitory effect of TGF-beta2 on C/EBPalpha, C/EBPbeta, and PPARgamma2 resulted in reduced LPL expression and abolition of bone marrow stromal cell adipogenic differentiation, which contributed to prevent bone loss induced by skeletal unloading. We conclude that TGF-beta2 inhibits the excessive adipogenic differentiation of bone marrow stromal cells induced by skeletal unloading by inhibiting C/EBPalpha, C/EBPbeta, and PPARgamma expression and activity, which provides a sequential mechanism by which TGF-beta2 regulates adipogenic differentiation of bone marrow stromal cells in vivo.

Animals↗

PubMed2005

Sp1/Sp3 and the myeloid zinc finger gene MZF1 regulate the human N-cadherin promoter in osteoblasts.

To determine the molecular mechanisms by which N-cadherin transcription is regulated, we cloned and sequenced a 3681-bp of the 5'-flanking region of the human N-cadherin gene. Deletion analysis of the proximal region identified a minimal 318-bp region with strong promoter activity in human osteoblasts. The cryptic promoter is characterized by high GC content and a GA-rich binding core that may bind zing finger transcription factors. Electrophoretic mobility shift assays (EMSA), competition and supershift EMSA revealed that an Sp1/Sp3 binding site acts as a basal regulatory element of the promoter in osteoblasts. Incubation of osteoblast nuclear extracts with -163/-131 wild-type probe containing the GA-rich binding core revealed another specific complex, which was not formed with a -163/-131 probe mutated in the GA repeat. EMSA identified the nuclear factor involved as myeloid zinc finger-1 (MZF1). Mutation analysis showed that Sp1/Sp3 and MZF1 binding sites contribute to basal promoter activity. Cotransfection analyses showed that Sp1 and MZF1 overexpression increases whereas Sp3 antagonizes Sp1-induced N-cadherin promoter activity in osteoblasts. RT-PCR analysis showed that human osteoblastic cells express MZF1 and that Sp1/MZF1 overexpression increased N-cadherin expression. These results indicate that Sp1/Sp3 and MZF1 are important transcription factors regulating N-cadherin promoter activity and expression in osteoblasts.

5' Flanking Region↗

PubMed2005

Strontium ranelate: a novel mode of action optimizing bone formation and resorption.

Strontium ranelate has been shown to decrease the risk of fractures in postmenopausal women. Its efficacy in clinical studies results from its unique mode of action, on both bone resorption and bone formation. Pharmacological studies in animals have shown that strontium ranelate decreases bone resorption and increases bone formation, resulting in increased bone mass. In ovariectomized rats, strontium ranelate prevented the reduction in bone mineral content and the decrease in trabecular bone volume induced by estrogen deficiency. In this model, strontium ranelate decreased bone resorption, whereas bone formation was maintained at a high level as documented by plasma biochemical markers and histomorphometric indices of bone formation. In the model of osteopenia induced by hind-limb immobilization in rats, strontium ranelate reduced histomorphometric parameters of bone resorption and partially prevented long-bone loss, as assessed by bone mineral content, bone volume, and biochemical indices of bone resorption. In intact mice, strontium ranelate increased bone formation and vertebral bone mass. In intact growing rats, strontium ranelate increased the bone trabecular volume without alteration of mineralization. The unique mode of action of strontium ranelate on bone formation and resorption was supported by in vitro studies. In rat calvaria culture systems and rat osteoblastic cell cultures, strontium ranelate enhanced preosteoblastic cell replication and increased collagen synthesis by osteoblasts. Moreover, strontium ranelate decreased bone resorption in organ cultures and decreased the resorbing activity of isolated mouse osteoclasts. The assessment of bone markers in a clinical trial [Spinal Osteoporosis Therapeutic Intervention (SOTI)] supports the mode of action of strontium ranelate: bone alkaline phosphatase levels increased and C-telopeptide of type I collagen levels decreased in treated patients compared with the placebo group at all time points. Thus, pharmacological and clinical studies suggest that strontium ranelate optimizes bone resorption and bone formation, resulting in increased bone mass, which may be of great value in the treatment of osteoporosis.

Alkaline Phosphatase↗

PubMed2004

Fibroblast growth factor-2 induces osteoblast survival through a phosphatidylinositol 3-kinase-dependent, -beta-catenin-independent signaling pathway.

Fibroblast growth factor-2 (FGF-2) is an important molecule that controls bone formation through activation of osteoblastic cell replication and differentiation. The role of FGF-2 on human osteoblast survival and the signaling pathway that mediates its effect are not known. We studied the effect of FGF-2 on apoptosis induced by low serum concentration and the signal transduction pathway involved in this effect in human primary calvaria osteoblasts and immortalized osteoblastic cells. Treatment with FGF-2 for 24-48 h protected against osteoblast apoptosis induced by low serum concentration, through specific inhibition of caspase-2 and caspase-3 activity. Pharmacological inhibition of MEK-1 and p38 MAPK had no effect on the inhibition of caspases-2 and -3 induced by FGF-2. In contrast, inhibition of PI3K with LY294002 abolished the FGF-2-induced inhibition of caspases-2 and -3. FGF-2 increased PI3K activity but did not induce phosphorylation of Akt or the downstream effector p70 S6 kinase. FGF-2 also induced GSK-3alpha and beta phosphorylation in osteoblastic cells, which however did not result in beta-catenin accumulation or Lef/Tcf transcriptional activity. In contrast, lithium induced beta-catenin accumulation, Lef/Tcf transcriptional activation and increased caspase-2 and -3 activity. The results indicate that the immediate protective effect of FGF-2 on human osteoblastic cell apoptosis involves PI3K and inhibition of downstream caspases, independently of GSK-3 and beta-catenin-Lef/Tcf-mediated transcription.

Apoptosis↗

PubMed2004

Normal matrix mineralization induced by strontium ranelate in MC3T3-E1 osteogenic cells.

There is growing evidence that strontium ranelate (SR; S12911-2, PROTELOS; Institut de Recherches Internationales Servier, Courbevoie, France), a compound containing 2 atoms of stable strontium (Sr), influences bone cells and bone metabolism in vitro and in vivo. We previously reported that SR increases bone mass in rats and mice by stimulating bone formation and inhibiting bone resorption. We also showed that short-term treatment with SR enhances osteoblastic cell recruitment and function in short-term rat calvaria cultures. Because Sr incorporates into the bone matrix, it was of interest to determine whether SR may affect matrix mineralization in long-term culture. To this goal, osteogenic mouse calvaria-derived MC3T3-E1 osteoblastic cells were cultured for up to 14 days in the presence of ascorbic acid and phosphate to induce matrix formation and mineralization. Matrix formation was determined by incorporation of tritiated proline during collagen synthesis. Matrix mineralization was quantified by measuring the number and surface of mineralized nodules using a digital image analyzer. In this model, 1,25(OH)2 vitamin D (1 nmol/L) used as internal control, increased alkaline phosphatase (ALP) activity, an early osteoblast marker, on days 4, 10, and 14 of culture. Treatment with SR (1 mmol/L Sr(2+)) increased ALP activity at days 4 and 14 of culture. SR also increased collagen synthesis at days 4 and 10 of culture. In contrast, 1,25(OH)2 vitamin D (1 nmol/L) inhibited collagen synthesis at 4 to 14 days of culture. Long-term treatment with SR (0.1 to 1 mmol/L Sr(2+)) dose dependently increased Sr concentration into the calcified nodules, but did not alter matrix mineralization in long-term culture, as shown by the ratio of the surface of mineralized nodules to the number of mineralized nodules on day 14 of culture. These results show that long-term treatment with SR increases collagenous matrix formation by MC3T3-E1 osteoblasts without inducing deleterious effect on matrix mineralization.

Alkaline Phosphatase↗

PubMed2004

Plasticity and regulation of human bone marrow stromal osteoprogenitor cells: potential implication in the treatment of age-related bone loss.

Human bone marrow stroma contains pluripotent mesenchymal progenitor cells that can give rise to many mesenchymal lineages, including chondroblasts, adipocytes or osteoblasts. The differentiation of these cells towards a specific lineage is dependent on hormonal and local factors activating specific transcription factors. Attempts have been recently made to identify osteoprogenitor cells in the human bone marrow and to identify the molecular mechanisms responsible for lineage-specific differentiation of human bone marrow stromal cells. Using a clonal pluripotent human bone marrow stromal cell line with tri-potential characteristics, we have provided evidence for a controlled reciprocal regulation of osteoblast/chondroblast and osteoblast/adipocyte differentiation of human bone marrow stromal cells. We have also shown that administration of TGFss that regulates the expression of specific osteoblast and adipocyte transcription factors can promote osteoblast differentiation and inhibit adipocyte conversion of rat marrow stromal cells in vivo. This indicates that the reciprocal relationship between osteoblastogenesis and adipogenesis can be manipulated in vivo in order to improve bone formation. Future studies will have to identify key signals for lineage-specific differentiation of human marrow stromal cells. This may result in the development of therapeutic strategies to promote the differentiation of these cells towards the osteoblast lineage and to inhibit excessive bone marrow adipogenesis associated with aging.

Adipocytes↗

PubMed2003

Fibroblast growth factor signaling controlling osteoblast differentiation.

Fibroblast growth factors (FGFs) play important roles in skeletal development and postnatal osteogenesis. FGF signaling controls bone formation by regulating the expression of various genes involved in osteoprogenitor cell replication, osteoblast differentiation and apoptosis. Recent genetic manipulation of FGF expression in mice and studies of the phenotype induced by gain-of-function mutations in FGF receptors in humans revealed the important role of FGF signaling in osteoblast function and differentiation. Additionally, cell biology studies allowed to identify some signaling pathways that are involved in the control of FGF actions in osteoblasts. This led to a better understanding of the functional role of FGF signaling in the control of gene expression in osteoblasts. The elucidation of molecular mechanisms by which FGF signaling promotes osteoblast gene expression and differentiation may help to find novel molecular targets and develop new therapeutic approaches to promote bone formation in human bone disorders.

Cell Differentiation↗

PubMed2003

Optimizing bone metabolism in osteoporosis: insight into the pharmacologic profile of strontium ranelate.

Strontium ranelate (SR) is currently being developed for the treatment of osteoporosis. Pharmacologic studies in animal models have shown that its efficacy on bone mass is based on its original mode of action on bone formation and bone resorption. In normal mice, SR increased bone formation and vertebral bone mass. In normal rats, SR increased bone mass and the mechanical properties of vertebral, humeral and femoral bones, associated with increased femoral shaft diameter. Vertebral bone mineral density and bone strength were also increased by SR, whereas stiffness was not altered, underlining that the improvement in bone strength occurs without inducing defective bone mineralization. In normal adult monkey alveolar bone, SR decreased bone resorption and increased bone. In ovariectomized (OVX) rats, SR limited the reduction in bone mineral content and the decrease in trabecular bone volume induced by estrogen deficiency, by inhibiting bone resorption while maintaining bone formation. Curative treatment with SR also partially restored bone mass in OVX rats. In the model of hind limb immobilization in rats, SR reduced bone resorption and partially limited long bone loss, as assessed by bone mineral content, bone volume, and histomorphometric and biochemical indices of bone resorption. The unique mode of action of SR on bone formation and resorption is also supported by in vitro studies. In calvaria culture systems and osteoblastic cell cultures, SR enhanced the replication of preosteoblastic cells and consequently increased collagen synthesis. Moreover, SR inhibited the bone-resorbing activity of isolated mouse osteoclasts and devreased osteoclast differentiation markers in chicken bone marrow cultures. Altogether, these pharmacologic results suggest that SR optimizes bone metabolism by decreasing bone resorption and promoting bone formation, which may be of potential value in the treatment of osteoporosis.

Animals↗

PubMed2003

S12911-2 reduces bone loss induced by short-term immobilization in rats.

Skeletal immobilization induces trabecular bone loss resulting from increased bone resorption and decreased formation. In this study we determined the effect of S12911-2, a compound containing two atoms of stable strontium, on trabecular bone loss induced by short-term immobilization of hind limbs in rats. Male Sprague-Dawley rats were randomly allocated to six groups (n = 25 per group). At 9 weeks of age, five groups of rats had their right hind limb immobilized for 10 days, using a plaster cast, whereas one control group was not immobilized (CT). Four groups of immobilized rats were treated for 10 days with 50, 200, or 800 mg/kg/day of S12911-2 or the vehicle. One group of immobilized rats was pretreated (PT) for 2 weeks with 200 mg/kg/day of S12911-2 and continued treatment during the immobilization period. Immobilization of the right hind limb induced bone loss as shown by decreased ash weight (-12%) and bone mineral density measured by dual energy x-ray absorptiometry of the femur (-9%), and confirmed by decreased trabecular bone volume measured by histomorphometry of the tibial metaphysis (-25%). This effect was unrelated to alteration in long bone length and was associated with increased urinary hydroxyproline excretion (+12%), increased osteoclast surface and number (+27%), decreased mineral apposition rate (-30%), and tetracycline double labeled surface (-17%) in the immobilized tibia. S12911-2 (800 mg/kg/day) partially reduced bone loss, as shown by increased bone mineral density (+4%) and trabecular bone volume (+19%) compared with untreated immobilized rats. Furthermore, S12911-2 (800 mg/kg/day) increased bone density (+5%) in the contralateral nonimmobilized leg. These effects resulted from inhibition of bone resorption, as shown by normalization of urinary hydroxyproline excretion and histomorphometric indices of bone resorption. This study shows that the bone resorption induced by immobilization in rats can be suppressed by treatment with S12911-2, resulting in partial reduction of the bone loss.

Animals↗

PubMed2002

Long-term treatment with strontium ranelate increases vertebral bone mass without deleterious effect in mice.

It was previously shown that strontium ranelate (SR; S12911-PROTOS, Institut de Recherches Internationales Servier, Courbevoie, France) can modulate bone metabolism in rats and mice. To determine the long-term effects of SR on vertebral bone metabolism in adult mice, the compound or the vehicle was given in the diet to normal male and female mice for 104 weeks at the dose of 200, 600, or 1,800 mg/kg/d corresponding to 0.78, 2.34 or 7.01 mmol Sr(2+)/kg/d. SR dose-dependently increased plasma strontium concentration, as well as exposure to the drug. Histomorphometric analyses of indices of bone volume, bone formation, and resorption were determined in the endosteal vertebral bone. SR significantly increased the trabecular bone volume by 25% and 59% in females treated with SR 600 and 1,800 mg/kg/d, respectively. This was associated with a 27% and 62% increase in mineralized bone volume. Bone volume was also significantly increased by 17% and 38% in male mice treated with SR 200 and 1,800 mg/kg/d, respectively. In parallel, SR increased the osteoblastic surface by 131% in males. In addition to this stimulatory effect on bone formation, a 52% decrease in osteoclastic surface, and a dose-dependent decrease in osteoclastic number (30% to 47%), was observed in female mice. Finally, SR even at the highest dose tested did not alter the osteoid thickness, indicating no deleterious effect on bone mineralization. Altogether, these findings show that SR simultaneously increases bone formation and decreases bone resorption in male or female mice, which results in increased vertebral bone mass in both genders without deleterious effect on bone mineralization.

Administration, Oral↗

PubMed2002

Regulation of human cranial osteoblast phenotype by FGF-2, FGFR-2 and BMP-2 signaling.

The formation of cranial bone requires the differentiation of osteoblasts from undifferentiated mesenchymal cells. The balance between osteoblast recruitment, proliferation, differentiation and apoptosis in sutures between cranial bones is essential for calvarial bone formation. The mechanisms that control human osteoblasts during normal calvarial bone formation and premature suture ossification (craniosynostosis) begin to be understood. Our studies of the human calvaria osteoblast phenotype and calvarial bone formation showed that premature fusion of the sutures in non-syndromic and syndromic (Apert syndrome) craniosynostoses results from precocious osteoblast differentiation. We showed that Fibroblast Growth Factor-2 (FGF-2), FGF receptor-2 (FGFR-2) and Bone Morphogenetic Protein-2 (BMP-2), three essential factors involved in skeletal development, regulate the proliferation, differentiation and apoptosis in human calvaria osteoblasts. Mechanisms that induce the differentiated osteoblast phenotype have also been identified in human calvaria osteoblasts. We demonstrated the implication of molecules (N-cadherin, Il-1) and signaling pathways (src, PKC) by which these local factors modulate human calvaria osteoblast differentiation and apoptosis. The identification of these essential signaling molecules provides new insights into the pathways controlling the differentiated osteoblast phenotype, and leads to a more comprehensive view in the mechanisms that control normal and premature cranial ossification in humans.

Apoptosis↗

PubMed2001

Protein kinase C-dependent upregulation of N-cadherin expression by phorbol ester in human calvaria osteoblasts.

Cell-cell adhesion mediated by cadherins is believed to play an essential role in the control of cell differentiation and tissue formation. Our recent studies indicate that N-cadherin is involved in human osteoblast differentiation. However, the signalling molecules that regulate cadherins in osteoblasts are not known. We tested the possibility that N-cadherin expression and function may be regulated by direct activation of protein kinase C (PKC) in human osteoblasts. Treatment of immortalized human neonatal calvaria (IHNC) cells with phorbol 12,13-dibutyrate (100 nM) transiently increased PKC activity. RT-PCR analysis showed that transient treatment with phorbol ester transiently increased N-cadherin mRNA levels at 4-12 h. Western blot analysis showed that N-cadherin protein levels were increased by phorbol ester at 24-48 h, and this was confirmed by immunocytochemical analysis. In contrast, E-cadherin expression was not affected. Transient treatment of IHNC cells with phorbol ester increased cell-cell aggregation, which was suppressed by neutralizing N-cadherin antibody, showing that the increased N-cadherin induced by phorbol ester was functional. Finally, phorbol ester dose-dependently increased alkaline phosphatase activity, an early marker of osteoblast differentiation. This effect was comparable to the promoting effect of BMP-2, a potent activator of osteoblast differentiation. These data show that direct activation of PKC by phorbol ester increases N-cadherin expression and function, and promotes ALP activity in human calvaria osteoblasts, which provides a signaling mechanism by which N-cadherin is regulated and suggests a role for PKC in N-cadherin-mediated control of human osteoblast differentiation.

Alkaline Phosphatase↗

PubMed2001

Bone morphogenetic protein-2 promotes osteoblast apoptosis through a Smad-independent, protein kinase C-dependent signaling pathway.

Bone morphogenetic protein-2 (BMP-2), a member of the transforming growth factor-beta (TGF-beta) family, regulates osteoblast differentiation and bone formation. Here we show a novel function of BMP-2 in human osteoblasts and identify a signaling pathway involved in this function. BMP-2 promotes apoptosis in primary human calvaria osteoblasts and in immortalized human neonatal calvaria osteoblasts, as shown by terminal deoxynucleotidyl transferase-mediated nick end labeling analysis. In contrast, TGF-beta 2 inhibits apoptosis in human osteoblasts. Studies of the mechanisms of action showed that BMP-2 increases the Bax/Bcl-2 ratio, whereas TG beta-2 has a negative effect. Moreover, BMP-2 increases the release of mitochondrial cytochrome c to the cytosol. Consistent with these results, BMP-2 increases caspase-9 and caspase-3, -6, and -7 activity, and an anti-caspase-9 agent suppresses BMP-2-induced apoptosis. Overexpression of dominant-negative Smad1 effectively blocks BMP-2-induced expression of the osteoblast transcription factor Runx2 but not the activation of caspases or apoptosis induced by BMP-2, indicating that the Smad1 signaling pathway is not involved in the BMP-2-induced apoptosis. The proapoptotic effect of BMP-2 is PKC-dependent, because BMP-2 increases PKC activity, and the selective PKC inhibitor calphostin C blocks the BMP-2-induced increased Bax/Bcl-2, caspase activity, and apoptosis. In contrast, the cAMP-dependent protein kinase A inhibitor H89, the p38 MAPK inhibitor SB203580, and the MEK inhibitor PD-98059 have no effect. The results show that BMP-2 uses a Smad-independent, PKC-dependent pathway to promote apoptosis via a Bax/Bcl-2 and cytochrome c-caspase-9-caspase-3, -6, -7 cascade in human osteoblasts.

Apoptosis↗

PubMed2001

Osteobiology, strain, and microgravity. Part II: studies at the tissue level.

Loading microgravity, and/or defective mechanical strain-forces have important effects on bone cells and bone quality and quantity. The complex mechanisms induced by strain and microgravity on bone cells have been reviewed in Part I of this paper. In Part II, we have considered the data on the alterations induced by unloading and microgravity on the skeleton and the mechanisms that are involved at the tissue level in animals and humans.

Animals↗