Estrogen activates cyclin-dependent kinases 4 and 6 through induction of cyclin D in rat primary osteoblasts

Estrogen plays important roles in maintaining bone density and protecting against osteoporosis, but the underlying mechanisms of estrogen action via estrogen receptors (ERs) in bone remain to be clarified. In the present study, we isolated primary osteoblasts derived from transgenic rats harboring a dominant negative ER mutant, rat ERalpha (1-535) cDNA, and from their wild-type littermates. We observed that the rate of cell growth of osteoblasts from the transgenic rats was reduced compared to that of wild-type osteoblasts. Utilizing cDNA microarray analysis, we found that mRNA level of cyclin D2 was lower in the osteoblasts from the transgenic rats. D-type cyclins including cyclin D1, cyclin D2, and cyclin D3 are cell cycle regulators that promote progression through the early-to-mid G1 phase of the cell cycle. The protein levels of D-type cyclins including cyclin D2 and cyclin D3 but not cyclin D1 were elevated in wild-type osteoblasts with 17beta-estradiol treatment, resulting in the activation of cyclin-dependent kinases 4 and 6 (Cdk4/6) activities and the promotion of cell growth. Moreover, an anti-estrogen ICI 182,780 abolished the induction of the expression of D-type cyclins by 17beta-estradiol. Our findings indicate that estrogen and its receptors enhance Cdk4/6 activities through the induction of D-type cyclins, leading to the growth promotion of osteoblasts.     

Effects of targeted overexpression of pleiotrophin on postnatal bone development

Pleiotrophin (PTN) is an extracellular matrix-associated growth/differentiation factor that, in post-natal life, is found mainly in bone and brain. Bone development was investigated in ptn-overexpressing mice between 1 and 30 weeks. In transgenics and controls, PTN (and its receptor syndecan-3) was synthesized by osteoblasts and was present in striated muscle. ptn over-expression enhanced intramembranous bone formation and had multiple effects on long-term bone growth. The pubertal growth spurt did not take place in transgenic mice, in which the growth trajectory was steady and continuous until 25 weeks. By 30 weeks, transgenic and control mice were of the same size, but the calcium content/mg bone was approximately 10% higher in the transgenics. PTN was also localized in growth plate and articular chondrocytes, but only in transgenic mice. In these, synthesis of type I collagen by articular chondrocytes was observed, as well as an encroachment of subchondral bone into the articular cartilage. The results suggest that PTN has multiple roles during in vivo bone formation and remodeling, probably acting as a co-factor or accessory protein that modulates the effects of primary signaling molecules.     

HtrA1 is upregulated during RANKL-induced osteoclastogenesis,and negatively regulates osteoblast differentiation and BMP2-induced Smad1/5/8, ERK and p38 phosphorylation

Bone remodeling is regulated by secreted factors in the bone microenvironment. However, data regarding osteoclast-derived factors that influence osteoblast differentiation are lacking. Here, we show that HtrA1 is produced as a secreted protein during osteoclastogenesis, and negatively regulates osteoblast differentiation. Exogenous addition of recombinant HtrA1 attenuates osteoblast differentiation and BMP2-induced Smad1/5/8, ERK1/2 and p38 phosphorylation in pre-osteoblasts. Our studies imply a unique mode of crosstalk in which HtrA1 is produced by both osteoclasts and osteoblasts and negatively regulates osteoblast differentiation, suggesting that HtrA1 may mediate the fine tuning of paracrine and autocrine regulations during bone remodeling processes.     

Galectin-9 induces osteoblast differentiation through the CD44/Smad signaling pathway

Galectin-9 is a β-galactoside-binding lectin expressed in various tissues. It binds various glycoconjugates and modulates a variety of biological functions in various cell types. Although galectin-9 is expressed in bone, its function in human osteoblasts remains unclear. We demonstrate that galectin-9 induces osteoblast differentiation through the CD44/Smad signaling pathway in the absence of bone morphogenetic proteins (BMPs). Galectin-9 increases alkaline phosphatase activities in human osteoblasts and induces the phosphorylation of Smad1/5/8 and translocation of Smad4 to the nucleus in the absence of BMPs. Galectin-9 also induces binding of Smad4 to the Id1 promoter and increases its activity. Anti-CD44 antibody inhibits Smad1/5/8 phosphorylation by galectin-9. Galectin-9 binds to CD44 and induces the formation of a CD44/BMP receptor complex. Because Smad1 is phosphorylated by BMP receptors, we propose that formation of the CD44/BMP receptor complex induced by galectin-9 may provide a trigger for the activation of Smads.     

Evidence for a role for the p110-α isoform of PI3K in skeletal function

Signaling through phosphatidylinositol-3 kinases (PI3K) regulates fundamental cellular processes such as survival and growth, and these lipid kinases are currently being investigated as therapeutic targets in several contexts. In skeletal tissue, experiments using pan-specific PI3K inhibitors have suggested that PI3K signaling influences both osteoclast and osteoblast function, but the contributions of specific PI3K isoforms to these effects have not been examined. In the current work, we assessed the effects of pharmacological inhibitors of the class Ia PI3Ks, α, β, and δ, on bone cell growth, differentiation and function in vitro. Each of the class Ia PI3K isoforms is expressed and functionally active in bone cells. No consistent effects of inhibitors of p110-β or p110-δ on bone cells were observed. Inhibitors of p110-α decreased osteoclastogenesis by 60-80% (p < 0.001 vs control) by direct actions on osteoclast precursors, and decreased the resorptive activity of mature osteoclasts by 60% (p < 0.01 vs control). The p110-α inhibitors also decreased the growth of osteoblastic and stromal cells (p < 0.001 vs control), and decreased differentiated osteoblast function by 30% (p < 0.05 vs control). These data suggest that signaling through the p110-α isoform of class Ia PI3Ks positively regulates the development and function of both osteoblasts and osteoclasts. Therapeutic agents that target this enzyme have the potential to significantly affect bone homeostasis, and evaluation of skeletal endpoints in clinical trials of such agents is warranted.     

Macrophage inflammatory protein-1α (MIP-1α) enhances a receptor activator of nuclear factor κB ligand (RANKL) expression in mouse bone marrow stromal cells and osteoblasts through MAPK and PI3K/Akt pathways

Osteolytic lesions are rapidly progressive during the terminal stages of myeloma, and the bone pain or bone fracture that occurs at these lesions decreases the patients’ quality of life to a notable degree. In relation to the etiology of this bone destruction, it has been reported recently that MIP-1α, produced in large amounts in myeloma patients, acts indirectly on osteoclastic precursor cells, and activates osteoclasts by way of bone-marrow stromal cells or osteoblasts, although the details of this process remain obscure. In the present study, our group investigated the mechanism by which RANKL expression is induced by MIP-1α and the effects of MIP-1α on the activation of osteoclasts. RANKL mRNA and RANKL protein expressions increased in both ST2 cells and MC3T3–E1 cells in a MIP-1α concentration-dependent manner. RANKL mRNA expression began to increase at 1 h after the addition of MIP-1α; the increase became remarkable at 2 h, and continuous expression was observed subsequently. Both ST2 and MC3T3-E1 cells showed similar levels of increased RANKL protein expression at 1, 2, and 3 days after the addition of MIP-1α. After the addition of MIP-1α, the amount of phosphorylated ERK1/2 and Akt protein expressions showed an increase, as compared to the corresponding amount in the control group. On the other hand, the amount of phosphorylated p38MAPK protein expression showed a decrease from the amount in the control group after the addition of MIP-1α. U0126 (a MEK1/2 inhibitor) or LY294002 (a PI3K inhibitor) was added to ST2 and MC3T3-E1 cells, and was found to inhibit RANKL mRNA and RANKL protein expression in these cells. When SB203580, a p38MAPK inhibitor, was added, RANKL mRNA and RANKL protein expression were increased in these cells. MIP-1α was found to promote osteoclastic differentiation of C7 cells, an osteoclastic precursor cell line, in a MIP-1α concentration-dependent manner. MIP-1α promoted differentiation into osteoclasts more extensively in C7 cells incubated together with ST2 and MC3T3-E1 cells than in C7 cells incubated alone. These results suggested that MIP-1α directly acts on the osteoclastic precursor cells and induces osteoclastic differentiation. This substance also indirectly induces osteoclastic differentiation through the promotion of RANKL expression in bone-marrow stromal cells and osteoblasts. The findings of this investigation suggested that activation of the MEK/ERK and the PI3K/Akt pathways and inhibition of p38MAPK pathway were involved in RANKL expression induced by MIP-1α in bone-marrow stromal cells and osteoblasts. This finding may be useful in the development of an osteoclastic inhibitor that targets intracellular signaling factors.     

A simple cell motility assay demonstrates differential motility of human periodontal ligament fibroblasts, gingival fibroblasts, and pre-osteoblasts

During periodontal regeneration, multiple cell types can invade the wound site, thereby leading to repair. Cell motility requires interactions mediated by integrin receptors for the extracellular matrix (ECM), which might be useful in guiding specific cell populations into the periodontal defect. Our data demonstrate that fibroblasts exhibit differential motility when grown on ECM proteins. Specifically, gingival fibroblasts are twice as motile as periodontal ligament fibroblasts, whereas osteoblasts are essentially non-motile. Collagens promote the greatest motility of gingival fibroblasts in the following order: collagen III>collagen V>collagen I. Differences in motility do not correlate with cell proliferation or integrin expression. Osteoblasts display greater attachment to collagens than does either fibroblast population, but lower motility. Gingival fibroblast motility on collagen I is generally mediated by α2 integrins, whereas motility on collagen III involves α1 integrins. Other integrins (α10 or α11) may also contribute to gingival fibroblast motility. Thus, ECM proteins do indeed differentially promote the cell motility of periodontal cells. Because of their greater motility, gingival fibroblasts have more of a potential to invade periodontal wound sites and to contribute to regeneration. This finding may explain the formation of disorganized connective tissue masses rather than the occurrence of the true regeneration of the periodontium. This research was supported by the Louisiana Board of Regents through the Millennium Trust Health Excellence Fund, HEF-(2000-05)-04.