RANKL is a downstream mediator for insulin-induced osteoblastic differentiation of vascular smooth muscle cells

Several reports have shown that circulating insulin level is positively correlated with arterial calcification; however, the relationship between insulin and arterial calcification remains controversial and the mechanism involved is still unclear. We used calcifying vascular smooth muscle cells (CVSMCs), a specific subpopulation of vascular smooth muscle cells that could spontaneously express osteoblastic phenotype genes and form calcification nodules, to investigate the effect of insulin on osteoblastic differentiation of CVSMCs and the cell signals involved. Our experiments demonstrated that insulin could promote alkaline phosphatase (ALP) activity, osteocalcin expression and the formation of mineralized nodules in CVSMCs. Suppression of receptor activator of nuclear factor κB ligand (RANKL) with small interfering RNA (siRNA) abolished the insulin-induced ALP activity. Insulin induced the activation of extracellular signal-regulated kinase (ERK)1/2, mitogen-activated protein kinase (MAPK) and RAC-alpha serine/threonine-protein kinase (Akt). Furthermore, pretreatment of human osteoblasts with the ERK1/2 inhibitor PD98059, but not the phosphoinositide 3-kinase (PI3K) inhibitor, LY294002, or the Akt inhibitor, 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate (HIMO), abolished the insulin-induced RANKL secretion and blocked the promoting effect of insulin on ALP activities of CVSMCs. Recombinant RANKL protein recovered the ALP activities decreased by RANKL siRNA in insulin-stimulated CVSMCs. These data demonstrated that insulin could promote osteoblastic differentiation of CVSMCs by increased RANKL expression through ERK1/2 activation, but not PI3K/Akt activation.     

Advanced glycation end products suppress osteoblastic differentiation of stromal cells by activating endoplasmic reticulum stress

Advanced glycation end products (AGEs) are involved in bone quality deterioration in diabetes mellitus. We previously showed that AGE2 or AGE3 inhibited osteoblastic differentiation and mineralization of mouse stromal ST2 cells, and also induced apoptosis and decreased cell growth. Although quality management for synthesized proteins in endoplasmic reticulum (ER) is crucial for the maturation of osteoblasts, the effects of AGEs on ER stress in osteoblast lineage are unknown. We thus examined roles of ER stress in AGE2- or AGE3-induced suppression of osteoblastogenesis of ST2 cells. An ER stress inducer, thapsigargin (TG), induced osteoblastic differentiation of ST2 cells by increasing the levels of Osterix, type 1 collagen (Col1), alkaline phosphatase (ALP) and osteocalcin (OCN) mRNA. AGE2 or AGE3 suppressed the levels of ER stress sensors such as IRE1α, ATF6 and OASIS, while they increased the levels of PERK and its downstream molecules, ATF4. A reduction in PERK level by siRNA did not affect the AGEs-induced suppression of the levels of Osterix, Col1 and OCN mRNA. In conclusion, AGEs inhibited the osteoblastic differentiation of stromal cells by suppressing ER stress sensors and accumulating abnormal proteins in the cells. This process might accelerate AGEs-induced suppression of bone formation found in diabetes mellitus.     

Serpinb1a suppresses osteoclast formation

Serpinb1a, a serine protease inhibitor family protein, has been implicated in immunoregulation and several metabolic disorders, such as diabetes and obesity; however, its roles in bone remain unknown. Therefore, we herein investigated the physiological functions of Serpinb1a in osteoclastic and osteoblastic differentiation using mouse cell lines. Serpinb1a overexpression markedly reduced the number of tartrate-resistant acid phosphatase (TRAP)- and calcitonin receptor-positive multinucleated cells increased by receptor activator nuclear factor κB ligand (RANKL) in mouse preosteoclastic RAW 264.7 cells. Moreover, it significantly decreased the mRNA levels of nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), TRAP and cathepsin K in these cells. Regarding osteoblasts, Serpinb1a overexpression significantly reduced the mRNA levels of alkaline phosphatase (ALP) and osteocalcin as well as ALP activity induced by bone morphogenetic protein-2 (BMP-2) in mouse mesenchymal ST2 cells, although it did not alter osteoblast differentiation in mouse osteoblastic MC3T3-E1 cells. Concerning the pathophysiological relevance of Serpinb1a, Serpinb1a mRNA levels were decreased in the soleus and gastrocnemius muscles of mice 4 weeks after bilateral sciatic nerve resection. In conclusion, we herein revealed for the first time that Serpinb1a inhibited osteoclast formation induced by RANKL in RAW 264.7 cells and suppressed BMP-2-induced ALP activity in ST2 cells.     

Activation of PERK in ET-1- and thrombin-induced pulmonary fibroblast differentiation: Inhibitory effects of curcumin

In the present study, we investigated the role of PKR-like endoplasmic reticular kinase (PERK), an endoplasmic reticulum (ER) stress kinase, in endothelin 1 (ET-1)- and thrombin-induced pulmonary fibrosis (PF), and the preventive effects of curcumin (CUR). Using the human embryonic WI-38 lung fibroblast cell line, ET-1 and thrombin induced the expression of ER stress-related proteins (CCAAT-enhancer-binding protein homologous protein, PERK, and binding immunoglobulin protein), a profibrogenic factor (cellular communication network factor 2 [CCN2]), and differentiation markers including α-smooth muscle actin (α-SMA), collagen I (Col I), and Col IV. Knockdown of PERK expression via small interfering RNA (siRNA) significantly reduced the increases in CCN2, α-SMA, Col I, and Col IV proteins in WI-38 cells according to western blot analysis and immunohistochemistry (IHC). Activation of c-Jun N-terminal kinase (JNK) was observed in ET-1- and thrombin-treated WI-38 cells, and the addition of a JNK inhibitor (SP) suppressed the induction of the indicated proteins by ET-1 and thrombin. Thapsigargin (TG), an ER stress inducer, elevated expressions of PERK and ER stress-related proteins with increased differentiation of WI-38 cells. Knockdown of PERK by siRNA or the PERK inhibitor glycogen synthesis kinase reduced expressions of the differentiation markers, α-SMA and Col IV, in WI-38 cells. CUR concentration-dependently inhibited ET-1- or thrombin-induced CCN2, α-SMA, and vimentin proteins with decreased levels of phosphorylated mitogen-activated protein kinase and PERK in WI-38 cells. An in vivo bleomycin-induced PF study showed that an intraperitoneal injection of CUR (30 mg/kg) reduced expressions of α-SMA, CCN2, Col IV, and vimentin in lung tissues via IHC staining using specific antibodies. This study is the first to demonstrate that PERK activation contributes to pulmonary fibroblast differentiation elicited by ET-1 or thrombin, and the inhibitory activity of CUR against PF is demonstrated herein.     

8-Isoprostaglandin E2 enhances receptor-activated NFkappa B ligand (RANKL)-dependent osteoclastic potential of marrow hematopoietic precursors via the cAMP pathway

Lipid oxidation products promote atherosclerosis and may also affect osteoporosis. We showed previously that oxidized lipids including 8-isoprostaglandin E2 (isoPGE2) inhibit osteoblastic differentiation of preosteoblasts. Since osteoporosis is mediated both by decreased osteoblastic bone formation and by increased osteoclastic bone resorption, we assessed whether oxidized lipids regulate the osteoclastic potential of marrow hematopoietic cells. Treatment of marrow-derived preosteoclasts with isoPGE2 enhanced osteoclastic differentiation as evidenced by increased tartrate-resistant acid phosphatase (TRAP) activity and multinucleation, which were inhibited by calcitonin, and increased numbers of resorption pits. The enhanced osteoclastic differentiation by isoPGE2 was observed whether preosteoclasts were in coculture with stromal cells or in monoculture in the presence of receptor-activated NFkappaB ligand (RANKL) and macrophage colony-stimulating factor. Receptor antagonist studies suggest that isoPGE2 effects were mediated by prostaglandin receptor subtypes EP2/DP on preosteoclasts and subtype EP1 and thromboxane receptors on stromal/osteoblast cells. The enhanced TRAP activity was also inhibited by cAMP-dependent protein kinase inhibitors, and isoPGE2 elevated intracellular cAMP levels of preosteoclast monocultures. Other oxidized lipids also enhanced the TRAP activity of preosteoclast monocultures. These data suggest that isoPGE2 enhances osteoclastic differentiation of marrow preosteoclasts and that this regulation occurs via the cAMP-dependent protein kinase pathway.     

Sodium hydrogen sulfide inhibits nicotine and lipopolysaccharide‐induced osteoclastic differentiation and reversed osteoblastic differentiation in human periodontal ligament cells

Although previous studies have demonstrated that hydrogen sulfide (H₂S) stimulated or inhibited osteoclastic differentiation, little is known about the effects of H₂S on the differentiation of osteoblasts and osteoclasts. To determine the possible bioactivities of H₂S on bone metabolism, we investigated the in vitro effects of H₂S on cytotoxicity, osteoblastic, and osteoclastic differentiation as well as the underlying mechanism in lipopolysaccharide (LPS) and nicotine‐stimulated human periodontal ligament cells (hPDLCs). The H₂S donor, NaHS, protected hPDLCs from nicotine and LPS‐induced cytotoxicity and recovered nicotine‐ and LPS‐downregulated osteoblastic differentiation, such as alkaline phosphatase (ALP) activity, mRNA expression of osteoblasts, including ALP, osteopontin (OPN), and osteocalcin (OCN), and mineralized nodule formation. Concomitantly, NaHS inhibited the differentiation of tartrate‐resistant acid phosphatase (TRAP)‐positive osteoclasts in mouse bone marrow cells and blocked nicotine‐ and LPS‐induced osteoclastogenesis regulatory molecules, such as RANKL, OPG, M‐CSF, MMP‐9, TRAP, and cathepsin K mRNA. NaHS blocked nicotine and LPS‐induced activation of p38, ERK, MKP‐1, PI3K, PKC, and PKC isoenzymes, and NF‐κB. The effects of H₂S on nicotine‐ and LPS‐induced osteoblastic and osteoclastic differentiation were remarkably reversed by MKP‐1 enzyme inhibitor (vanadate) and expression inhibitor (triptolide). Taken together, we report for the first time that H₂S inhibited cytotoxicity and osteoclastic differentiation and recovered osteoblastic differentiation in a nicotine‐ and periodontopathogen‐stimulated hPDLCs model, which has potential therapeutic value for treatment of periodontal and inflammatory bone diseases. J. Cell. Biochem. 114: 1183–1193, 2013. © 2012 Wiley Periodicals, Inc.     

Inhibition of insulin-like growth factor-1 (IGF-1) expression by prolonged transforming growth factor-β1 (TGF-β1) administration suppresses osteoblast differentiation

TGF-β1 can regulate osteoblast differentiation not only positively but also negatively. However, the mechanisms of negative regulation are not well understood. We previously established the reproducible model for studying the suppression of osteoblast differentiation by repeated or high dose treatment with TGF-β1, although single low dose TGF-β1 strongly induced osteoblast differentiation. The mRNA expression and protein level of insulin-like growth factor-1 (IGF-1) were remarkably decreased by repeated TGF-β1 administration in human periodontal ligament cells, human mesenchymal stem cells, and murine preosteoblast MC3T3-E1 cells. Repeated TGF-β1 administration subsequently decreased alkaline phosphatase (ALP) activity and mRNA expression of osteoblast differentiation marker genes, such as RUNX2, ALP, and bone sialoprotein (BSP). Additionally, repeated administration significantly reduced the downstream signaling pathway of IGF-1, such as Akt phosphorylation in these cells. Surprisingly, exogenous and overexpressed IGF-1 recovered ALP activity and mRNA expression of osteoblast differentiation marker genes even with repeated TGF-β1 administration. These facts indicate that the key mechanism of inhibition of osteoblast differentiation induced by repeated TGF-β1 treatment is simply due to the down-regulation of IGF-1 expression. Inhibition of IGF-1 signaling using small interfering RNA (siRNA) against insulin receptor substrate-1 (IRS-1) suppressed mRNA expression of RUNX2, ALP, BSP, and IGF-1 even with single TGF-β1 administration. This study showed that persistence of TGF-β1 inhibited osteoblast differentiation via suppression of IGF-1 expression and subsequent down-regulation of the PI3K/Akt pathway. We think this fact could open the way to use IGF-1 as a treatment tool for bone regeneration in prolonged inflammatory disease.     

Role of Endoplasmic Reticulum Stress in Aberrant Activation of Fluoride-Treated Osteoblasts

The aberrant activation of osteoblasts in the early stage is one of the critical steps during the pathogenesis of skeletal fluorosis. The endoplasmic reticulum (ER) stresses and unfolded protein response (UPR) are initiated to alleviate the accumulation of unfolded proteins against cell injury. The previous researches had demonstrated that fluoride induced ER stress in other cells or tissues. In this study, we determined the ER stress and UPR to investigate their roles in aberrant activation of fluoride-treated osteoblasts. The gene expression of bone markers and UPR factors in MC3T3-E1 cells treated with varying doses of fluoride administration was analyzed. Meantime, levels of glutathione and glutathione disulfide were tested by the ultraperformance liquid chromatography–tandem mass spectrometry applications. Our results indicated that a certain dose and period of fluoride administration induced cell proliferation and differentiation, and Runx2 was involved in the regulation of osteoblastic differentiation of MC3T3-E1 cells. Increase trend of Runx2 expression was consistent with change of marker of ER stress. Fluoride caused ER stress and stimulated UPR during the process of osteoblast maturation, while oxidative stress was also active in the occurrence of ER stress. These data indicated that ER stress and UPR were possibly involved in the action of fluoride on osteoblasts.     

Tunicamycin negatively regulates BMP2-induced osteoblast differentiation through CREBH expression in MC3T3E1 cells

Tunicamycin, an endoplasmic reticulum (ER) stress inducer, specifically inhibits N-glycosylation. The cyclic AMP (cAMP) response element-binding protein H (CREBH) was previously shown to be regulated by UPR-dependent proteolytic cleavage in the liver. On the other hand, the role of CREBH in other tissues is unknown. In the present study, tunicamycin increased the level of CREBH activation (cleavage) as well as mRNA expression in osteoblast cells. Adenoviral (Ad) overexpression of CREBH suppressed BMP2-induced expression of alkaline phosphatase (ALP) and osteocalcin (OC). Interestingly, the BMP2-induced OASIS (structurally similar to CREBH, a positive regulator of osteoblast differentiation) expression was also inhibited by CREBH overexpression. In addition, inhibition of CREBH expression using siRNA reversed the tunicamycin-suppressed ALP and OC expression. These results suggest that CREBH inhibited osteoblast differentiation via suppressing BMP2-induced ALP, OC and OASIS expression in mouse calvarial derived osteoblasts.     

FK506 enhanced osteoblastic differentiation in mesenchymal cells.

Bone morphogenetic protein (BMP) is a bone-derived growth factor capable of promoting the differentiation of mesenchymal cells into osteogenic lineage pathways. Recently, immunosuppressants were reported to cause a moderate increase in osteoblastic differentiation in a rat osteoblast-like osteosarcoma cell line. If immunosuppressants can induce osteoblastic differentiation, it will be useful for bone tissue transplantation. We assessed the effect of immunosuppressants with or without BMP-4 on inducing osteoblastic differentiation in osteoblast-like and other mesenchymal cells. FK506, an immunosuppressant often used clinically, induced a dose- and time-dependent increase in alkaline phosphatase (ALP) activity, one of the markers of osteoblast differentiation, in cells derived from mesenchyma. In the presence of BMP-4, ALP activity, mRNA levels of ALP and osteocalcin increased. FK506 was found to not only stimulate osteoblastic differentiation, but also to enhance BMP-4 induced osteoblastic differentiation. These results suggest that FK506 promotes differentiation of osteoblastic cells.     

RNA interference of the BMPR-IB gene blocks BMP-2-induced osteogenic gene expression in human bone cells

We have previously shown that human bone cells express bone morphogenetic protein receptor-IB (BMPR-IB). However, little is known about the precise role of this receptor in the response of osteoblastic genes to the BMP in these cells. To determine BMPR-IB-dependent osteoblastic gene expression, the present study examined the effects of BMPR-IB knockdown on BMP-induced osteoblast-associated genes. BMPR-IB mRNA and protein were markedly suppressed by transfection of cells with BMPR-IB siRNA. Using three different bone cell samples, BMP-2 stimulation of alkaline phosphatase (ALP), osteocalcin (OC), distal-less homeobox-5 (Dlx5) and core binding factor alpha-1 (Cbfa1) was found to be specifically and significantly reduced in the BMPR-IB siRNA-transfected cultures compared with that of control cultures. Our study has provided evidence that BMPR-IB-dependent signaling plays a crucial role in BMP-2 up-regulation of the ALP, OC, Dlx5 and Cbfa1 genes in bone cells, suggesting a pivotal role of this receptor in BMP-2-induced osteoblast differentiation in vitro. These findings thus suggest the possibility that BMPR-IB could be a therapeutic target for enhancing bone regeneration in vivo.