Primary Mouse Osteoblasts Research Articles

Effects of water extract of Cajanus cajan leaves on the osteogenic and adipogenic differentiation of mouse primary bone marrow stromal cells and the adipocytic trans-differentiation of mouse primary osteoblasts

The effects of water extract of Cajanus cajan (Linn.) Millsp. (Leguminosae) leaves (WECML) on the osteogenic and adipogenic differentiation of mouse primary bone marrow stromal cells (BMSCs) and the adipocytic trans-differentiation of mouse primary osteoblasts (OBs) were studied. The results indicated that WECML promoted the proliferation of BMSCs and OBs at most concentrations. WECML promoted the osteogenic differentiation and formation of mineralized matrix nodules of BMSCs at concentrations of 0.1, 1, and 10 μg/mL, but inhibited the osteogenic differentiation and formation of mineralized matrix nodules of BMSCs at concentration of 0.01 μg/mL. WECML inhibited the adipogenic differentiation of BMSCs and adipocytic trans-differentiation of OBs at concentrations of 0.001, 0.1, 1, 10, and 100 μg/mL, but had no effects at concentration of 0.01 μg/mL. The results suggest that WECML has protective effects on bone and these protective effects may be mediated by decreasing adipocytic cell formation from BMSCs, which may promote the proliferation, differentiation, and mineralization function of OBs. The defined active ingredients in the WECML and the active mechanism need to be further studied.

Effects of LIMK2 RNA interference on the mechanosensitivity of c-fos gene in osteoblast

To study the effects of cytoskeleton reorganization inhibition with LIMK2 RNAi upon the mechanosensitivity of c-fos gene in osteoblast. Mouse primary osteoblast was treated with LIMK2 specific siRNA (RNAi Group), negative control siRNA (NC Group), and then were loaded or unloaded by fluid shear stress. Real-time PCR and immunofluorescence were used to detect the c-fos expression levels and statistics analysis was performed. When the cytoskeleton reorganization was inhibited with RNAi only, the c-fos mRNA (0.0108 +/- 0.0074 and 0.0042 +/- 0.0018, t = -1.86, P > 0.05) and protein (121 +/- 7 and 119 +/- 6, t = -1.272, P > 0.05) expression levels of each unloaded group had no significant difference; Fluid shear stress could up-regulate the c-fos mRNA (0.2203 +/- 0.1532 vs 0.0042 +/- 0.0018, t = -707.35, P < 0.05)and protein (178 +/- 12 vs 119 +/- 6, t = -30.761, P < 0.05) expression; After the cytoskeleton reorganization was inhibited with RNAi, the c-fos mRNA (0.5280 +/- 0.0879 vs 0.2203 +/- 0.1532, t = -1007.00, P < 0.05) and protein (224 +/- 46 vs 178 +/- 12, t = -6.853, P < 0.05) expression induced by fluid shear stress had significant difference. Cytoskeleton reorganization inhibition with RNAi had synergistic effect upon the expression of c-fos mRNA (F = 84.388, P < 0.05) and protein (F = 42.409, P < 0.05) induced by fluid shear stress. Using RNAi against LIMK2 to inhibit the cytoskeleton reorganization can promote the expression of c-fos gene and thus enhance the mechanosensitivity of c-fos gene in osteoblast.

A novel microRNA targeting HDAC5 regulates osteoblast differentiation in mice and contributes to primary osteoporosis in humans

MicroRNAs (miRNAs) interfere with translation of specific target mRNAs and are thought to thereby regulate many cellular processes. Recent studies have suggested that miRNAs might play a role in osteoblast differentiation and bone formation. Here, we identify a new miRNA (miR-2861) in primary mouse osteoblasts that promotes osteoblast differentiation by repressing histone deacetylase 5 (HDAC5) expression at the post-transcriptional level. miR-2861 was found to be transcribed in ST2 stromal cells during bone morphogenetic protein 2-induced (BMP2-induced) osteogenesis, and overexpression of miR-2861 enhanced BMP2-induced osteoblastogenesis, whereas inhibition of miR-2861 expression attenuated it. HDAC5, an enhancer of runt-related transcription factor 2 (Runx2) degradation, was confirmed to be a target of miR-2861. In vivo silencing of miR-2861 in mice reduced Runx2 protein expression, inhibited bone formation, and decreased bone mass. Importantly, miR-2861 was found to be conserved in humans, and a homozygous mutation in pre-miR-2861 that blocked expression of miR-2861 was shown to cause primary osteoporosis in 2 related adolescents. Consistent with the mouse data, HDAC5 levels were increased and Runx2 levels decreased in bone samples from the 2 affected individuals. Thus, our studies show that miR-2861 plays an important physiological role in osteoblast differentiation and contributes to osteoporosis via its effect on osteoblasts.

Effect of cytoskeleton integrity on the expression of c-fos in osteoblasts induced by fluid shear stress

To investigate the effect of cytoskeleton integrity on the expression of c-fos gene in osteoblasts induced by fluid shear stress. BALB/c mouse primary osteoblasts were divided into four groups (according to fluid shear stress loaded or not and cytochalasin D used or not). The Tagman probe real-time PCR and immunofluorescence were performed to detect the expression levels of c-fos mRNA, c-fos protein and cytoskeleton, respectively. The data were analysed using two-way ANOVA. In control group and cytochalasin D group, fluid shear stress could significantly increase the expression levels of c-fos mRNA (0.1637 +/- 0.0303 and 0.0104 +/- 0.0070, respectively) and protein (177.14 +/- 9.37 and 150.95 +/- 6.17, respectively) in osteoblasts, compared with the unloaded osteoblasts of the control group and the cytochalasin D group (0.0057 +/- 0.0021 and 0.0032 +/- 0.0014, respectively for c-fos mRNA, and 117.96 +/- 4.11 and 119.77 +/- 5.19, respectively for protein, P < 0.05). Induced by the fluid shear stress, the expression levels of c-fos mRNA and protein in cytochalasin D group were lower than control group, and the difference had statistical significance (P < 0.05). The cytoskeleton integrity in osteoblasts was essential to the expression of c-fos gene induced by fluid shear stress.

BMP-4 Induction of Arrest and Differentiation of Osteoblast-Like Cells via p21CIP1and p27KIP1Regulation

Cell cycle regulation by differentiation signals is critical for eukaryote development. We investigated the roles of bone morphogenetic protein (BMP)-4, an important stimulator of osteoblast differentiation and bone formation, in regulating cell cycle distribution in four osteoblast-like cell lines and mouse primary osteoblasts, and the underlying mechanisms. In all cells used, BMP-4 induced G(0)/G(1) arrest. The molecular basis of the BMP-4 effect was analyzed, and the presentation on molecular mechanism is focused on human MG63 cells. BMP-4 induced p21(CIP1) and p27(KIP1) expressions and hence cell differentiation but had no effects on the expressions of cyclins A, B1, D1, and E, cyclin-dependent protein kinase-2, -4, and -6. Using specific small interfering RNA (siRNA), we found that BMP-4-induced G(0)/G(1) arrest, and p21(CIP1) and p27(KIP1) expressions were mediated by BMP receptor type IA (BMPRIA)-specific Sma- and Mad-related protein (Smad)1/5. BMP-4 induced transient phosphorylations of ERK; transfection of MG63 cells with ERK2, but not ERK1, -specific siRNA inhibited the BMP-4-induced responses in MG63 cells. Pretreatment of MG63 cells with Arg-Gly-Asp-Ser, which blocks the cell-extracellular matrix interaction, or transfection with beta(3) integrin-specific siRNA inhibited BMP-4-induced ERK and Smad1/5 phosphorylations. BMP-4 induced transient increases in associations of beta(3)-integrin with focal adhesion kinase and Shc, the dominant-negative mutants of which inhibited BMP-4-induced ERK and Smad1/5 phosphorylations. Our results indicate that BMP-4 induces G(0)/G(1) arrest and hence differentiation in osteoblast-like cells through increased expressions of p21(CIP1) and p27(KIP1), which are mediated by BMPRIA-specific Smad1/5. The extracellular matrix/beta(3) integrin/ focal adhesion kinase/Shc/ERK2 signaling pathway is involved in these BMP-4-induced responses in osteoblast-like cells.

Protein Kinase C-δ Transactivates Platelet-derived Growth Factor Receptor-α in Mechanical Strain-induced Collagenase 3 (Matrix Metalloproteinase-13) Expression by Osteoblast-like Cells

Matrix metalloproteinase-13 (MMP-13, or collagenase 3) has been shown to degrade intact collagen and to participate in situations where rapid and effective remodeling of collagenous ECM is required. Mechanical strain induction of MMP-13 is an example of how osteoblasts respond to high mechanical forces and participate in the bone-remodeling mechanism. Using MC3T3-E1 osteoblast-like cells, we dissected the signaling molecules involved in MMP-13 induction by mechanical strain. Reverse transcription-PCR and zymogram analysis showed that platelet-derived growth factor receptor (PDGFR) inhibitor, AG1296, inhibited the mechanical strain-induced MMP-13 gene and activity. However, the induction was not affected by anti-PDGF-AA serum. Immunoblot analysis revealed time-dependent phosphorylation of PDGFR-alpha up to 2.7-fold increases within 3 min under strain. Transfection with shPDGFR-alpha (at 4 and 8 microg/ml) abolished PDGFR-alpha and reduced MMP-13 expression. Moreover, time-dependent recruitments of phosphoinositide 3-kinase (PI3K) by PDGFR-alpha were detected by immunoprecipitation with anti-PDGFR-alpha serum followed by immunoblot with anti-PI3K serum. AG1296 inhibited PDGFR-alpha/PI3K aggregation and Akt phosphorylation. Interestingly, protein kinase C-delta (PKC-delta) inhibitor, rottlerin, inhibited not only PDGFR-alpha/PI3K aggregation but PDGFR-alpha phosphorylation. The sequential activations were further confirmed by mutants DeltaPKC-delta, DeltaAkt, and DeltaERK1. Consistently, the primary mouse osteoblast cells used the same identified signaling molecules to express MMP-13 under mechanical strain. These results demonstrate that, in osteoblast-like cells, the MMP-13 induction by mechanical strain requires the transactivation of PDGFR-alpha by PKC-delta and the cross-talk between PDGFR-alpha/PI3K/Akt and MEK/ERK pathways.

Thyroid hormone non-genomically suppresses Src thereby stimulating osteocalcin expression in primary mouse calvarial osteoblasts

To provide further insights into non-genomic action of thyroid hormone (T3), we investigated whether Src is under control of T3 in primary calvarial osteoblasts prepared from neonatal mice. Treatment of the cells with T3 rapidly decreased Src Y416 autophosphorylation, followed by the decrease of phosphorylated extracellular signal-regulated kinases, suggesting that T3 non-genomically suppresses Src activity. Furthermore, this T3 effect was rapid and persistent, and was associated with the increased expression of osteocalcin (OC). To confirm the contribution of Src to the effect of T3 on OC expression, a constitutively active Src (Y527F) was overexpressed in osteoblasts. In such cells, Y416 phosphorylation was markedly increased even in the presence of T3, and T3-dependent expression of OC was markedly attenuated. The present study demonstrates a novel, non-genomic action of T3 in primary mouse osteoblasts, by which T3 suppresses Src thereby stimulating OC expression.

NFATc1 mediates HDAC-dependent transcriptional repression of osteocalcin expression during osteoblast differentiation

We previously reported that the in vivo and in vitro suppression of Nuclear Factor of Activated T Cells (NFAT) signaling increases osteoblast differentiation and bone formation. To investigate the mechanism by which NFATc1 regulates osteoblast differentiation, we established an osteoblast cell line that overexpresses a constitutively active NFATc1 (ca-NFATc1). The activation of NFATc1 significantly inhibits osteoblast differentiation and function, demonstrated by inhibition of alkaline phosphatase activity and mineralization as well as a decrease in gene expression of early and late markers of osteoblast differentiation such as osterix and osteocalcin, respectively. By focusing on the specific role of NFATc1 during late differentiation, we discovered that the inhibition of osteocalcin gene expression by NFATc1 was associated with a repression of the osteocalcin promoter activity, and a decrease in TCF/LEF transactivation. Also, overexpression of NFATc1 completely blocked the decrease in total histone deacetylase (HDAC) activity during osteoblast differentiation and prevented the hyperacetylation of histones H3 and H4. Mechanistically, we show by Chromatin Immunoprecipitation (ChIP) assay that the overexpression of NFATc1 sustains the binding of HDAC3 on the proximal region of the osteocalcin promoter, resulting in complete hypoacetylation of histones H3 and H4 when compared to GFP-expressing osteoblasts. In contrast, the inhibition of NFATc1 nuclear translocation either by cyclosporin or by using primary mouse osteoblasts with deleted calcineurin b1 prevents HDAC3 from associating with the proximal regulatory site of the osteocalcin promoter. These preliminary results suggest that NFATc1 acts as a transcriptional co-repressor of osteocalcin promoter, possibly in an HDAC-dependent manner.

Identification of LRRc17 as a Negative Regulator of Receptor Activator of NF-κB Ligand (RANKL)-induced Osteoclast Differentiation

Osteoblasts are the primary cells responsible for bone formation. They also support osteoclast formation from bone marrow precursors in response to osteotropic factors by inducing receptor activator of NF-kappaB ligand (RANKL) expression and down-regulating osteoprotegerin (OPG) production. In addition to the RANKL-RANK-OPG signaling axis, other factors produced by osteoblasts/stromal cells are involved in osteoclastogenesis. Here, we describe the identification and characterization of leucine-rich repeat-containing 17 (LRRc17), a member of the LRR superfamily that acts as a negative regulator of RANKL-induced osteoclast differentiation. Osteoblasts showed high levels of LRRc17 expression, which was down-regulated in response to the pro-osteoclastogenic factor 1,25-dihydroxyvitamin D(3). Recombinant LRRc17 protein inhibited RANKL-induced osteoclast differentiation from bone marrow precursors, whereas it did not affect the differentiation or activation of macrophages and dendritic cells. These results suggest that among the cell types derived from common myeloid precursors, LRRc17 specifically regulates osteoclasts. Further analysis revealed that LRRc17 attenuated RANKL-induced expression of NFATc1 by blocking phospholipase C-gamma signaling, which, in turn, inhibited RANKL-mediated osteoclast differentiation. Taken together, our results demonstrated a novel inhibitory activity of LRRc17 in RANKL-induced osteoclastogenesis.

Involvement of p53 in phthalate effects on mouse and rat osteoblasts

The role of two estrogen-mimicking compounds in regulating osteoblast activities were examined. Previously, our attention was focused on benzyl butyl phthalate (BBP) and di-n-butyl phthalate (DBP) since previous works showed that they enter the cytoplasm, bioaccumulate, modify actin cytoarchitecture and exert mitogenic effects involving microfilament disruption, and nuclear actin and lamin A regulation in Py1a rat osteoblasts. In this study we showed that BBP and DBP cause DNA base lesions both in MT3T3-E1 osteoblasts and in mouse primary calvarial osteoblasts (COBs). In addition, treatment with the above effectors caused an increase of p53 and phospho-p53 (ser-15 and ser-20) as well as an increase of apoptotic proteins with consequent decrease of cell viability. Moreover, treatment with phthalates did not modified p53 and phospho-p53 expression in Py1a rat osteoblasts. It is of relevance that in p53 knockdown mouse osteoblasts a proliferative effect of phthalates, similar to that observed in rat Py1a osteoblasts, was found. In conclusion, our data demonstrated that phthalates induce osteoblast apoptosis, which is, at least in part, mediated by p53 activation, suggesting that the proliferative effects could be due to p53 missing activation or p53 mutation.

Wnt inhibitory factor 1 is epigenetically silenced in human osteosarcoma, and targeted disruption accelerates osteosarcomagenesis in mice

Wnt signaling increases bone mass by stimulating osteoblast lineage commitment and expansion and forms the basis for novel anabolic therapeutic strategies being developed for osteoporosis. These strategies include derepression of Wnt signaling by targeting secreted Wnt pathway antagonists, such as sclerostin. However, such therapies are associated with safety concerns regarding an increased risk of osteosarcoma, the most common primary malignancy of bone. Here, we analyzed 5 human osteosarcoma cell lines in a high-throughput screen for epigenetically silenced tumor suppressor genes and identified Wnt inhibitory factor 1 (WIF1), which encodes an endogenous secreted Wnt pathway antagonist, as a candidate tumor suppressor gene. In vitro, WIF1 suppressed beta-catenin levels in human osteosarcoma cell lines, induced differentiation of human and mouse primary osteoblasts, and suppressed the growth of mouse and human osteosarcoma cell lines. Wif1 was highly expressed in the developing and mature mouse skeleton, and, although it was dispensable for normal development, targeted deletion of mouse Wif1 accelerated development of radiation-induced osteosarcomas in vivo. In primary human osteosarcomas, silencing of WIF1 by promoter hypermethylation was associated with loss of differentiation, increased beta-catenin levels, and increased proliferation. These data lead us to suggest that derepression of Wnt signaling by targeting secreted Wnt antagonists in osteoblasts may increase susceptibility to osteosarcoma.

PIAS3 negatively regulates RANKL-mediated osteoclastogenesis directly in osteoclast precursors and indirectly via osteoblasts

AbstractCytokine signaling via various transcription factors regulates receptor activator of nuclear factor (NF)–κB ligand (RANKL)–mediated osteoclast differentiation from monocyte/macrophage lineage cells involved in propagation and resolution of inflammatory bone destruction. Protein inhibitor of activated STAT3 (PIAS3) was initially identified as a molecule that inhibits DNA binding of STAT3 and regulates many transcription factors through distinct mechanisms. To analyze PIAS3 function in osteoclasts in vivo, we have generated transgenic mice in which PIAS3 is specifically expressed in the osteoclast lineage using the tartrate-resistant acid phosphatase (TRAP) gene promoter. PIAS3 transgenic mice showed an osteopetrotic phenotype due to impairment of osteoclast differentiation. Overexpression of PIAS3 in RAW264.7 cells suppressed RANKL-induced osteoclastogenesis by inhibiting the expression of c-Fos and NFATc1. Interestingly, PIAS3 inhibits the transcriptional activity of microphthalmiaassociated transcription factor (MITF) independent of sumoylation. Down-regulation of PIAS3 markedly enhances RANKL-mediated osteoclastogenesis in RAW264.7 cells. Furthermore, overexpression of PIAS3 in mouse primary osteoblast (POB), down-regulates RANKL expression induced by interleukin-6 (IL-6) cytokine family, and inhibits osteoclast formation from bone marrow macrophages (BMMs) in vitro coculture system. Down-regulation of PIAS3 leads to the accelerated expression of RANKL in POB stimulated with IL-6 and soluble IL-6 receptor (sIL-6R). Taken together, our results clearly indicate that PIAS3 negatively regulates RANKL-mediated osteoclastogenesis directly in osteoclast precursors and indirectly via osteoblasts.

P53 Deficiency Leads to Compensatory Up-Regulation of p16INK4a

p53-p21-cyclin-dependent kinase and p16(INK4a)-cyclin-dependent kinase pathways have parallel functions in preventing tumorigenesis. In cancer patients, tumor suppressor p53 is frequently inactivated through mutations, whereas p16(INK4a) is silenced through promoter methylation. However, the interaction between these two pathways is less well understood. Here, we report that p53 controls p16(INK4a) expression in a unique way. p53 deficiency led to up-regulation of p16(INK4a) in primary mouse embryonic fibroblasts, osteoblasts, and various mouse organs, and an increase in the p16(INK4a) promoter activity, without affecting the half-life of p16(INK4a). Reconstitution of p53, but not mutant p53, restored the proper expression of p16(INK4a). These results indicate that p53 is necessary in repressing p16(INK4a) expression. However, up-regulation of p53 in response to genotoxic stress or nutlin-3 treatment did not down-regulate p16(INK4a). p53 did not repress the p16(INK4a) promoter activity either. These findings suggest that p53 has a necessary but not sufficient role in repressing p16(INK4a) expression. p16(INK4a) elevation in p53(-/-) cells is, at least partially, mediated by Ets1, a known positive regulator of p16(INK4a), as p53 deficiency up-regulated Ets1 through protein stabilization and knockdown of Ets1 down-regulated p16(INK4a) expression in p53(-/-) mouse embryonic fibroblasts. These studies uncover a compensatory mechanism for the loss of p53 and provide a basis for targeting both p53 and p16(INK4a) in cancer therapy.

Thrombin-stimulated growth factor and cytokine expression in osteoblasts is mediated by protease-activated receptor-1 and prostanoids

Thrombin exerts multiple effects upon osteoblasts including stimulating proliferation, and inhibiting osteoblast differentiation and apoptosis. Some of these effects are believed to be mediated by the synthesis and secretion of autocrine factors such as growth factors and cytokines. Many but not all cellular responses to thrombin are mediated by members of the protease-activated receptor (PAR) family of G protein-coupled receptors. The current study was undertaken to investigate the nature of thrombin's induction of autocrine factors by analysing the expression of twelve candidate genes in thrombin-stimulated primary mouse osteoblasts. Analysis by quantitative reverse transcription polymerase chain reaction (qRT-PCR) demonstrated that thrombin induced transforming growth factor β, cyclooxygenase-2, tenascin C, fibroblast growth factor-1 and -2, connective tissue growth factor and interleukin-6 expression in wild type osteoblasts, but not PAR-1 null mouse osteoblasts. Induction of all the thrombin-responsive genes was blocked by the presence of the non-selective cyclooxygenase inhibitor indomethacin. Further studies were conducted on interleukin-6, which was the gene that showed the greatest increase in expression following stimulation of osteoblast-like cells with thrombin. A PAR-1-specific activating peptide, but neither a PAR-4-activating peptide nor catalytically inactive thrombin induced release of interleukin-6 by osteoblasts. Furthermore, in the presence of the selective cyclooxygenase-1 and -2 inhibitors SC-560 and NS-398 thrombin-induced interleukin-6 release was prevented. Levels of both prostaglandin E2 and interleukin-6 in medium conditioned by thrombin-stimulated osteoblast-like cells were found to be significantly increased compared to medium conditioned by non-stimulated cells, however release of prostaglandin E2 was found to precede release of interleukin-6. Treatment of isolated osteoblast-like cells with a number of synthetic prostanoids stimulated secretion of interleukin-6 with differing potencies. These studies suggest that activation of PAR-1 on osteoblasts by thrombin induces cyclooxygenase activity, which in turn results in the increased expression of multiple secreted factors. The induction of these secreted factors may act in an autocrine fashion to alter osteoblast function, allowing these cells to participate in the earliest stages of bone healing by both autocrine and paracrine mechanisms.

Effect of Dy3+on osteogenic and adipogenic differentiation of mouse primary bone marrow stromal cells and adipocytic trans-differentiation of mouse primary osteoblasts

A series of experimental methods including 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) test, alkaline phosphatase (ALP) activity measurement, mineralized function, Oil Red O stain and measurement were employed to assess the effect of Dy3+ on the osteogenic and adipogenic differentiation of mouse primary bone marrow stromal cells (BMSCs) and the adipogenic trans-differentiation of mouse primary osteoblasts (OBs). The results showed that Dy3+ had no effect on BMSC proliferation at concentrations of 1×10−8 and 1×10−5 mol/L, but inhibited BMSC proliferation at other concentrations. Dy3+ had no effect on OB proliferation at concentrations of 1×10−10 and 1×10−9 mol/L, but inhibited OB proliferation at other concentrations. Dy3+ had no effect on the osteogenic differentiation of BMSCs at concentrations of 1×10−9 and 1×10−7 mol/L, and promoted osteogenic differentiation of BMSCs at other concentrations at the 7th day. The osteogenic differentiation of BMSCs was inhibited by Dy3+ at concentration of 1×10−5 mol/L at the 14th day, but promoted osteogenic differentiation of BMSCs at concentrations of 1×10−9, 1×10−8, 1×10−7 and 1×10−6 mol/L with the maximal effect at concentration of 10−6 mol/L. Dy3+ promoted mineralized function of BMSCs at any concentration. Dy3+ had no effect on adipogenic differentiation of BMSCs at concentration of 1×10−7 mol/L, but inhibited adipogenic differentiation of BMSCs at other concentrations. Dy3+ inhibited adipocytic trans-differentiation of OBs at any concentration, suggesting that Dy3+ had protective effect on bone and the protective effect on bone may be mediated by modulating differentiation of BMSCs away from the adipocyte and inhibiting adipocytic trans-differentiation of OBs which may promote differentiation and mineralization of OBs. These results may be valuable for better understanding the mechanism of the effect of Dy3+ on pathogenesis of osteoporosis.

Effect of LIMK2 RNAi on reorganization of the actin cytoskeleton in osteoblasts induced by fluid shear stress

The biomechanical characteristics of bone tissue and its cells under mechanical stress are significant for bone biomechanics research, but the mechanism of mechanotransduction is still unknown. It has been established that the actin cytoskeleton of osteoblasts plays an important role in this process. However, the structure of the actin cytoskeleton is reorganized when loaded with mechanical stress, which results in changes in cell stiffness. These phenomena suggest that an actin-cytoskeleton-induced feedback regulation mechanism may be involved in the mechanotransduction of osteoblasts, but this has not yet been proven. The aim of this study was to explore the role of LIMK2 in the reorganization of the actin cytoskeleton induced by fluid shear stress in osteoblasts by using RNA interference. Balb/c mouse primary osteoblasts were divided into four groups. Cells in Groups 1 and 3 were transfected with negative control RNA, while cells in Groups 2 and 4 were transfected with a specific siRNA designed to silence the LIMK2 gene. Twenty-four hours after transfection, cells in Groups 1 and 2 were loaded with fluid shear stress at 12 dyne/cm 2 while cells in Groups 3 and 4 were not. Compared with Group 1, the mean fluorescence density of the actin cytoskeleton in the other three groups was 28.9%, 45.7%, and 33.0%, respectively. These results indicate that LIMK2 plays an important role in the reorganization of the actin cytoskeleton induced by fluid shear stress.

Activating transcription factor 4 is critical for proliferation and survival in primary bone marrow stromal cells and calvarial osteoblasts

Activating transcription factor 4 (ATF4) is essential for bone formation. However, the mechanism of its actions in bone is poorly understood. The present study examined the role for ATF4 in the regulation of proliferation and survival of primary mouse bone marrow stromal cells (BMSCs) and osteoblasts. Results showed that Atf4(-/-) cells display a severe proliferative defect as measured by multiple cell proliferation assays. Cell cycle progression of Atf4(-/-) BMSCs was largely delayed with significant G1 arrest. Expression of cyclin D1 was decreased both at the mRNA and protein level. A similar proliferation defect was observed in Atf4(-/-) calvarial periosteal osteoblasts when compared with wt control. Knocking down Atf4 mRNA by small interfering RNA in MC3T3-E1 subclone 4 preosteoblasts markedly reduced expression of cyclin D1 and cell proliferation. In contrast, overexpression of ATF4 increased cyclin D1 expression as well as cell proliferation in Atf4(-/-) BMSCs. In addition, apoptosis was significantly increased in Atf4(-/-) BMSCs and calvarial periosteal osteoblasts relative to wt controls. Taken together, these results for the first time demonstrate that ATF4 is a critical regulator of proliferation and survival in BMSCs and osteoblasts in vitro and in vivo.

IGF-I secreted by osteoblasts acts as a potent chemotactic factor for osteoblasts

Osteoblast recruitment to the site of future bone formation is essential for skeletal development, bone remodeling and fracture healing. A number of factors associated with bone tissue have been reported to induce directional migration of osteoblasts but the mechanism remains to be clarified. In this study, to explore a major chemotactic factor(s) for osteoblasts, we examined the serum-free medium conditioned by MC3T3-E1 osteoblast-like cells for its ability to induce osteoblast migration. Employing sequential chromatography and tandem mass spectrometry analysis, we purified and identified IGF-I as a potent chemotactic factor from the conditioned medium. IGF-I induced cell migration of both MC3T3-E1 cells and primary mouse osteoblasts, and checkerboard analysis revealed that IGF-I markedly induced directional migration (chemotaxis) of osteoblasts. Neutralization of mouse IGF-I with monoclonal antibodies resulted in delayed osteoblast monolayer wound healing and cellular polarization but addition of human IGF-I reversed these effects. IGF-I also promoted cell spreading on fibronectin in an integrin β1-dependent manner. IGF-I induced Akt and Rac activation and localized accumulation of phosphatidylinositol 3,4,5-triphosphate (PtdIns (3,4,5)P 3) at the membrane in osteoblasts. The phosphatidyl inositol 3 kinase (PI3K) inhibitor LY294002 inhibited IGF-I-induced cell migration and wound healing. Together, the results suggest that IGF-I secreted from osteoblasts in the bone tissue is a potent chemotactic factor that may play a major role in recruitment of osteoblasts during bone formation.

Saturated Fatty Acids Modulate Cell Response to DNA Damage: Implication for Their Role in Tumorigenesis

DNA damage triggers a network of signaling events that leads to cell cycle arrest or apoptosis. This DNA damage response acts as a mechanism to prevent cancer development. It has been reported that fatty acids (FAs) synthesis is increased in many human tumors while inhibition of fatty acid synthase (FASN) could suppress tumor growth. Here we report that saturated fatty acids (SFAs) play a negative role in DNA damage response. Palmitic acid, as well as stearic acid and myristic acid, compromised the induction of p21 and Bax expression in response to double stranded breaks and ssDNA, while inhibition or knockdown of FASN enhanced these cellular events. SFAs appeared to regulate p21 and Bax expression via Atr-p53 dependent and independent pathways. These effects were only observed in primary mouse embryonic fibroblasts and osteoblasts, but not in immortalized murine NIH3T3, or transformed HCT116 and MCF-7 cell lines. Accordingly, SFAs showed some positive effects on proliferation of MEFs in response to DNA damage. These results suggest that SFAs, by negatively regulating the DNA damage response pathway, might promote cell transformation, and that increased synthesis of SFAs in precancer/cancer cells might contribute to tumor progression and drug resistance.

ODDD-Linked Cx43 Mutants Reduce Endogenous Cx43 Expression and Function in Osteoblasts and Inhibit Late Stage Differentiation

Bone development and modeling requires precise gap junctional intercellular communication (GJIC). Oculodentodigital dysplasia (ODDD) is an autosomal dominant human disease caused by mutations in the gene (GJA1) encoding the gap junction protein, connexin43 (Cx43). The disease is characterized by craniofacial bone deformities and limb abnormalities. It is our hypothesis that Cx43 mutation causes osteoblast dysfunction, which may contribute to the bone phenotype of ODDD. We expressed human and mouse ODDD-linked Cx43 mutants in MC3T3-E1 cells and primary mouse osteoblasts by retroviral infection and evaluated their in vitro differentiation as an index of osteoblast function. We compared these findings to the differentiation of osteoblasts isolated from a mouse model of ODDD that harbors a germ line Cx43 mutation and exhibits craniofacial and limb defects mimicking human ODDD. We determined the differentiation status of osteoblasts by analyzing alkaline phosphatase activity and the expression levels of osteoblast markers including bone sialoprotein and osteocalcin. We showed that ODDD-linked Cx43 mutants are loss-of-function and dominant-negative to co-expressed Cx43 and, furthermore, greatly inhibit functional GJIC in osteoblasts. Surprisingly, the mutants had only a minor effect on osteoblast differentiation when introduced into lineage committed cells. In contrast, osteoblasts isolated from the ODDD mouse model exhibited impaired late stage differentiation. Expression of human and mouse ODDD-linked Cx43 mutants failed to significantly impair differentiation in cells predisposed to the osteoblast lineage; however, germ line reduction of Cx43-based GJIC leads to impaired osteoblast differentiation, which may account for the bone phenotypes observed in ODDD patients.