Effects of insulin and insulin-like growth factor 1 on osteoblast proliferation and differentiation: differential signalling via Akt and ERK

Insulin and insulin-like growth factor 1 (IGF-1) are evolutionarily conserved hormonal signalling molecules, which influence a wide array of physiological functions including metabolism, growth and development. Using genetic mouse studies, both insulin and IGF-1 have been shown to be anabolic agents in osteoblasts and bone development primarily through the activation of Akt and ERK signalling pathways. In this study, we examined the temporal signalling actions of insulin and IGF-1 on primary calvarial osteoblast growth and differentiation. First, we observed that the IGF-1 receptor expression decreases whereas insulin receptor expression increases during osteoblast differentiation. Subsequently, we show that although both insulin and IGF-1 promote osteoblast differentiation and mineralization in vitro, IGF-1, but not insulin, can induce osteoblast proliferation. The IGF-1-induced osteoblast proliferation was mediated via both MAPK and Akt pathways because the IGF-1-mediated cell proliferation was blocked by U0126, an MEK/MAPK inhibitor, or LY294002, a PI3-kinase inhibitor. Osteocalcin, an osteoblast-specific protein whose expression corresponds with osteoblast differentiation, was increased in a dose- and time-dependent manner after insulin treatment, whereas it was decreased with IGF-1 treatment. Moreover, insulin treatment dramatically induced osteocalcin promoter activity, whereas IGF-1 treatment significantly inhibited it, indicating direct effect of insulin on osteocalcin synthesis.     

Effects of insulin and insulin‐like growth factor 1 on osteoblast proliferation and differentiation: differential signalling via Akt and ERK

Insulin and insulin‐like growth factor 1 (IGF‐1) are evolutionarily conserved hormonal signalling molecules, which influence a wide array of physiological functions including metabolism, growth and development. Using genetic mouse studies, both insulin and IGF‐1 have been shown to be anabolic agents in osteoblasts and bone development primarily through the activation of Akt and ERK signalling pathways. In this study, we examined the temporal signalling actions of insulin and IGF‐1 on primary calvarial osteoblast growth and differentiation. First, we observed that the IGF‐1 receptor expression decreases whereas insulin receptor expression increases during osteoblast differentiation. Subsequently, we show that although both insulin and IGF‐1 promote osteoblast differentiation and mineralization in vitro, IGF‐1, but not insulin, can induce osteoblast proliferation. The IGF‐1‐induced osteoblast proliferation was mediated via both MAPK and Akt pathways because the IGF‐1‐mediated cell proliferation was blocked by U0126, an MEK/MAPK inhibitor, or LY294002, a PI3‐kinase inhibitor. Osteocalcin, an osteoblast‐specific protein whose expression corresponds with osteoblast differentiation, was increased in a dose‐ and time‐dependent manner after insulin treatment, whereas it was decreased with IGF‐1 treatment. Moreover, insulin treatment dramatically induced osteocalcin promoter activity, whereas IGF‐1 treatment significantly inhibited it, indicating direct effect of insulin on osteocalcin synthesis. Copyright © 2012 John Wiley & Sons, Ltd.     

Rapamycin inhibits osteoblast proliferation and differentiation in MC3T3-E1 cells and primary mouse bone marrow stromal cells

While the roles of the mammalian target of rapamycin (mTOR) signaling in regulation of cell growth, proliferation, and survival have been well documented in various cell types, its actions in osteoblasts are poorly understood. In this study, we determined the effects of rapamycin, a specific inhibitor of mTOR, on osteoblast proliferation and differentiation using MC3T3-E1 preosteoblastic cells (MC-4) and primary mouse bone marrow stromal cells (BMSCs). Rapamycin significantly inhibited proliferation in both MC-4 cells and BMSCs at a concentration as low as 0.1 nM. Western blot analysis shows that rapamycin treatment markedly reduced levels of cyclin A and D1 protein in both cell types. In differentiating osteoblasts, rapamycin dramatically reduced osteoblast-specific osteocalcin (Ocn), bone sialoprotein (Bsp), and osterix (Osx) mRNA expression, ALP activity, and mineralization capacity. However, the drug treatment had no effect on osteoblast differentiation parameters when the cells were completely differentiated. Importantly, rapamycin markedly reduced levels of Runx2 protein in both proliferating and differentiating but not differentiated osteoblasts. Finally, overexpression of S6K in COS-7 cells significantly increased levels of Runx2 protein and Runx2 activity. Taken together, our studies demonstrate that mTOR signaling affects osteoblast functions by targeting osteoblast proliferation and the early stage of osteoblast differentiation.     

Endogenous FGF‐2 is critically important in PTH anabolic effects on bone

Parathyroid hormone (PTH) increases fibroblast growth factor receptor‐1 (FGFR1) and fibroblast growth factor‐2 (FGF‐2) expression in osteoblasts and the anabolic response to PTH is reduced in Fgf2?/? mice. This study examined whether candidate factors implicated in the anabolic response to PTH were modulated in Fgf2?/? osteoblasts. PTH increased Runx‐2 protein expression in Fgf2+/+ but not Fgf2?/? osteoblasts. By immunocytochemistry, PTH treatment induced nuclear accumulation of Runx‐2 only in Fgf2+/+ osteoblasts. PTH and FGF‐2 regulate Runx‐2 via activation of the cAMP response element binding proteins (CREBs). Western blot time course studies showed that PTH increased phospho‐CREB within 15 min that was sustained for 24 h in Fgf2+/+ but had no effect in Fgf2?/? osteoblasts. Silencing of FGF‐2 in Fgf2+/+ osteoblasts blocked the stimulatory effect of PTH on Runx‐2 and CREBs phosphorylation. Studies of the effects of PTH on proteins involved in osteoblast precursor proliferation and apoptosis showed that PTH increased cyclinD1‐cdk4/6 protein in Fgf2+/+ but not Fgf2?/? osteoblasts. Interestingly, PTH increased the cell cycle inhibitor p21/waf1 in Fgf2?/? osteoblasts. PTH increased Bcl‐2/Bax protein ratio in Fgf2+/+ but not Fgf2?/? osteoblasts. In addition PTH increased cell viability in Fgf2+/+ but not Fgf2?/? osteoblasts. These data suggest that endogenous FGF‐2 is important in PTH effects on osteoblast proliferation, differentiation, and apoptosis. Reduced expression of these factors may contribute to the reduced anabolic response to PTH in the Fgf2?/? mice. Our results strongly indicate that the anabolic PTH effect is dependent in part on FGF‐2 expression. J. Cell. Physiol. 219: 143–151, 2009. © 2008 Wiley‐Liss, Inc.     

Zoledronate promotes osteoblast differentiation in high-glucose conditions via the p38MAPK pathway

Zoledronate (ZOL) were found to inhibit bone resorption in an animal model of diabetes, high glucose concentrations have been shown to decreased the osteogenesis-related gene expression. But the molecular mechanism by which high glucose levels affect osteoblasts and the effects of ZOL on osteoblast differentiation in a high-glucose environment remain unclear. Therefore, we aimed to investigate the effect of ZOL on osteoblast differentiation in a high-glucose environment and determine the responsible mechanism. Cell proliferation was detected by MTT assay, and cell differentiation was evaluated by immunofluorescence staining for alkaline phosphatase expression, alizarin red staining, cytoskeletal arrangement, and actin fiber formation. Real-time PCR and western blot analyses were performed to detect the mRNA and protein expression of p38MAPK, phosphorylated (p)-p38MAPK, CREB, p-CREB, collagen (COL) I, osteoprotegerin (OPG), and RANKL. The results showed that cell proliferation activity did not differ among the groups. But high glucose inhibited osteoblast differentiation; actin fiber formation; and p38MAPK, p-p38MAPK, CREB, p-CREB, COL I, and OPG expression, while promoting RANKL expression. However, we found that treatment with ZOL reversed these effects of high glucose. And further addition of a p38MAPK inhibitor led to inhibition of osteoblast differentiation and actin fiber formation, and lower p38MAPK, p-p38MAPK, CREB, p-CREB, COL I, and OPG expression than in the high glucose +ZOL group with higher RANKL expression than in the high glucose +ZOL group. Collectively, this study demonstrates that high glucose inhibits the differentiation of osteoblasts, and ZOL could partly overcome these effects by regulating p38MAPK pathway activity.     

p85alpha regulates osteoblast differentiation by cross-talking with the MAPK pathway

Class IA phosphoinositide 3-kinase (PI3K) is involved in regulating many cellular functions including cell growth, proliferation, cell survival, and differentiation. The p85 regulatory subunit is a critical component of the PI3K signaling pathway. Mesenchymal stem cells (MSC) are multipotent cells that can be differentiated into osteoblasts (OBs), adipocytes, and chondrocytes under defined culture conditions. To determine whether p85α subunit of PI3K affects biological functions of MSCs, bone marrow-derived wild type (WT) and p85α-deficient (p85α(-/-)) cells were employed in this study. Increased cell growth, higher proliferation rate and reduced number of senescent cells were observed in MSCs lacking p85α compare with WT MSCs as evaluated by CFU-F assay, thymidine incorporation assay, and β-galactosidase staining, respectively. These functional changes are associated with the increased cell cycle, increased expression of cyclin D, cyclin E, and reduced expression of p16 and p19 in p85α(-/-) MSCs. In addition, a time-dependent reduction in alkaline phosphatase (ALP) activity and osteocalcin mRNA expression was observed in p85α(-/-) MSCs compared with WT MSCs, suggesting impaired osteoblast differentiation due to p85α deficiency in MSCs. The impaired p85α(-/-) osteoblast differentiation was associated with increased activation of Akt and MAPK. Importantly, bone morphogenic protein 2 (BMP2) was able to intensify the differentiation of osteoblasts derived from WT MSCs, whereas this process was significantly impaired as a result of p85α deficiency. Addition of LY294002, a PI3K inhibitor, did not alter the differentiation of osteoblasts in either genotype. However, application of PD98059, a Mek/MAPK inhibitor, significantly enhanced osteoblast differentiation in WT and p85α(-/-) MSCs. These results suggest that p85α plays an essential role in osteoblast differentiation from MSCs by repressing the activation of MAPK pathway.     

Lentiviral-mediated RNAi targeting p38MAPK ameliorates high glucose-induced apoptosis in osteoblast MC3T3-E1 cell line

The p38 mitogen activated protein kinase (p38MAPK) pathway is an important signaling cascade involved in cell growth, differentiation and apoptosis. High glucose activates p38MAPK pathway in different cells, including osteoblasts. In the present study, role of p38MAPK in high glucose induced osteoblast apoptosis and potential of RNA interference (RNAi) targeting p38MAPK as a therapy strategy have been reported. Lentiviral-mediated RNAi effectively reduced p38MAPK and p-p38MAPK expressions in osteoblastic cell line (MC3T3-E1) following high glucose (22 mM) induction. Inhibition of p38MAPK activity significantly suppressed high glucose induced apoptosis of MC3T3-E1 cell and was confirmed by flow cytometry and ultra-structural examination by transmission electronic microscope. Inhibition of p38MAPK also significantly attenuates caspase-3 and bax protein expressions, but increased significantly bcl-2 expression as determined by Western blot analysis. The results suggested that p38MAPK mediates high glucose induced osteoblast apoptosis, partly through modulating the expressions of caspase-3, bax and bcl-2. Inhibition of p38MAPK with lentiviral-mediated RNAi or its specific inhibitor provides a new strategy to treat high glucose induced osteoblast apoptosis.     

PKR-mediated degradation of STAT1 regulates osteoblast differentiation

The double-stranded RNA-dependent protein kinase (PKR) plays a critical role in various biological responses including antiviral defense, cell differentiation, apoptosis, and tumorigenesis. In this study, we investigated whether PKR could affect the post-translational modifications of STAT1 protein and whether these modifications regulate osteoblast differentiation. We demonstrated that PKR was necessary for the ubiquitination of STAT1 protein. The expressions of bone-related genes such as type I collagen, integrin binding sialoprotein, osteopontin, and osterix were suppressed in osteoblasts lacking PKR activity. In contrast, the expressions of interleukin-6 and matrix metalloproteinases 8 and 13 increased in PKR-mutated osteoblasts. The expression and degradation of STAT1 protein were regulated by PKR in a SLIM-dependent pathway. Inhibition of SLIM by RNA interference resulted in the decreased activity of Runx2 in osteoblasts. Stimulation of interleukin-6 expression and suppression of alkaline phosphatase activity were regulated through by SLIM-dependent pathway. However, expressions of bone-related genes and MMPs were regulated by SLIM-independent pathway. Our present results suggest that the aberrant accumulation of STAT1 protein induced by loss of PKR regulate osteoblast differentiation through both SLIM/STAT1-dependent and -independent pathways.     

Beta-catenin is not activated by downregulation of PTEN in osteoblasts

Selective knockdown of phosphatase and tensin homolog (PTEN) has been recently shown to increase life long accumulation of bone and its ability to increase osteoblast lifespan. In order to determine how loss of PTEN function affects osteoblast differentiation, we created cell lines with stable knockdown of PTEN expression using short hairpin RNA vectors and characterized several clones. The effect of deregulated PTEN in osteoblasts was studied in relationship to cell proliferation and differentiation. Downregulation of PTEN initially affected the cell’s attachment and spreading on plastic but cells recovered after a brief period of time. When cell proliferation was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, we noticed a small but significant increase in growth rates with PTEN reduction. The size of individual cells appeared larger when compared to control cells. Differentiation properties of these osteoblasts were increased as evidenced by higher expression of several of the bone markers tested (alkaline phosphatase, osteocalcin, osterix, bone morphogenetic protein 2, Cbfa1, osteoprotegerin, and receptor activator of NF-kappaB ligand) and their mineralization capacity in culture. As stabilization of beta-catenin is known to be responsible for growth deregulation with PTEN loss in other cell types, we investigated the activation of the canonical Wnt pathway in our cell lines. Immunofluorescence staining, protein expression in subcellular fractions for beta-catenin, and assays for activation of the canonical Wnt/beta-catenin signaling were studied in the PTEN downregulated cells. There was an overall decrease in β-catenin expression in cells with PTEN knockdown. The distribution of β-catenin was more diffuse within the cell in the PTEN-reduced clones when compared to controls where they were mostly present in cell borders. Signaling through the canonical pathway was also reduced in the PTEN knockdown cells when compared to control. The results of this study suggest that while decreased PTEN augments cell proliferation and positively affects differentiation, there is a decrease in β-catenin levels and activity in osteoblasts. Therefore, at least in osteoblasts, β-catenin is not responsible for mediating the activation of osteoblast differentiation with reduction in PTEN function.     

Lung epithelial-C/EBPβ contributes to LPS-induced inflammation and its suppression by formoterol

Muscle mass is related to higher bone mass and a reduction in fracture risk. However, the interactions between muscle tissues and bone metabolism are incompletely understood and there might be some humoral factors that are produced in muscle tissues and exhibit bone anabolic activity. We therefore investigated the role of FAM5C in osteoblast differentiation and the interactions between muscle and bone. A reduction of endogenous FAM5C by siRNA reduced the levels of osterix, alkaline phosphatase (ALP) and osteocalcin (OCN) mRNA as well as the levels of type 1 collagen and β-catenin in mouse osteoblastic MC3T3-E1 cells and mouse calvarial osteoblasts, although FAM5C overexpression significantly antagonized the levels of osterix, ALP and OCN mRNA induced by bone morphogenetic protein-2 in C2C12 cells. The conditioned medium from FAM5C-overexpressed and -suppressed C2C12 cells increased and decreased the levels of osterix, ALP and OCN mRNA in MC3T3-E1 cells, respectively. In conclusion, the present study is the first to show that FAM5C enhances osteoblast differentiation in differentiated osteoblasts, and that the effects of the conditioned medium from FAM5C-modulated myoblastic cells were positively correlated with the effects of FAM5C on osteoblast phenotype in osteoblasts. FAM5C might be an important humoral bone anabolic factor produced from muscle cells.     

Identification of PSD-95 palmitoylating enzymes

Palmitoylation is a lipid modification that plays a critical role in protein trafficking and function throughout the nervous system. Palmitoylation of PSD-95 is essential for its regulation of AMPA receptors and synaptic plasticity. The enzymes that mediate palmitoyl acyl transfer to PSD-95 have not yet been identified; however, proteins containing a DHHC cysteine-rich domain mediate palmitoyl acyl transferase activity in yeast. Here, we isolated 23 mammalian DHHC proteins and found that a subset specifically palmitoylated PSD-95 in vitro and in vivo. These PSD-95 palmitoyl transferases (P-PATs) showed substrate specificity, as they did not all enhance palmitoylation of Lck, SNAP-25b, Galpha(s), or H-Ras in cultured cells. Inhibition of P-PAT activity in neurons reduced palmitoylation and synaptic clustering of PSD-95 and diminished AMPA receptor-mediated neurotransmission. This study suggests that P-PATs regulate synaptic function through PSD-95 palmitoylation.     

Insulin stimulates osteoblast proliferation and differentiation through ERK and PI3K in MG‐63 cells

Insulin has been proposed to be an anabolic agent in bone, but the mechanisms underlying insulin effects on osteoblast differentiation are still not clear. To explore the mechanisms of action of insulin on osteoblast growth and differentiation, human osteoblastic cell line‐MG‐63 was used and stimulated by insulin in the presence or absence of ERK inhibitor PD98059, PI3‐K inhibitor LY294002, or inhibitor PD98059 + LY294002. The results showed that insulin positively regulated the expression of its receptor. Insulin stimulated the proliferation of MG‐63 cells in a time‐ and dose‐dependent manner and blockade of both MAPK and PI3K pathways could inhibit the cell proliferation. In addition, ALP activity, the secretion of type I collagen, OC gene expression, and mineralized nodule formation were increased in the insulin treated group, whereas these indicators were decreased after treatment with blocking agents. However, treatment with PI3‐K inhibitor LY294002 significantly reversed the down‐regulation of Runx2 expression and treatment with ERK inhibitor PD98059 remarkably decreased up‐regulation of Osx and IGF‐1 expression after insulin treatment. Therefore, the data obtained from this study suggested that insulin promoted osteoblast proliferation and differentiation through MAPK and PI3K pathway in MG‐63 cells. Copyright © 2010 John Wiley & Sons, Ltd.     

Distinct roles for mitogen-activated protein kinase phosphatase-1 (MKP-1) and ERK-MAPK in PTH1R signaling during osteoblast proliferation and differentiation

Parathyroid hormone (PTH) and PTH-related protein (PTHrP) activate one single receptor (PTH1R) which mediates catabolic and anabolic actions in the bone. Activation of PTH1R modulates multiple intracellular signaling responses. We previously reported that PTH and PTHrP down-regulate pERK1/2 and cyclin D1 in differentiated osteoblasts. In this study we investigate the role of MAPK phosphatase-1 (MKP-1) in PTHrP regulation of ERK1/2 activity in relation to osteoblast proliferation, differentiation and bone formation. Here we show that PTHrP increases MKP-1 expression in differentiated osteoblastic MC3T3-E1 cells, primary cultures of differentiated bone marrow stromal cells (BMSCs) and calvarial osteoblasts. PTHrP had no effect on MKP-1 expression in proliferating osteoblastic cells. Overexpression of MKP-1 in MC-4 cells inhibited osteoblastic cell proliferation. Cell extracts from differentiated MC-4 cells treated with PTHrP inactivate/dephosphorylate pERK1/2 in vitro; immunodepletion of MKP-1 blocked the ability of the extract to dephosphorylate pERK1/2; these data indicate that MKP-1 is involved in PTHrP-induced pERK1/2 dephosphorylation in the differentiated osteoblastic cells. PTHrP regulation of MKP-1 expression is partially dependent on PKA and PKC pathways. Treatment of nude mice, bearing ectopic ossicles, with intermittent PTH for 3 weeks, up-regulated MKP-1 and osteocalcin, a bone formation marker, with an increase in bone formation. These data indicate that PTH and PTHrP increase MKP-1 expression in differentiated osteoblasts; and that MKP-1 induces growth arrest of osteoblasts, via inactivating pERK1/2 and down-regulating cyclin D1; and identify MKP-1 as a possible mediator of the anabolic actions of PTH1R in mature osteoblasts.