Integrin‐Mediated Expression of Bone Formation‐Related Genes in Osteoblast‐Like Cells in Response to Fluid Shear Stress: Roles of Extracellular Matrix,Shc, and Mitogen‐Activated Protein Kinase

Integrins play significant roles in mechanical responses of cells on extracellular matrix (ECM). We studied the roles of integrins and ECM proteins (fibronectin [FN], type I collagen [COL1], and laminin [LM]) in shear‐mediated signaling and the expression of bone formation‐related genes (early growth response‐1 [Egr‐1], c‐fos, cyclooxygenase‐2 [Cox‐2], and osteopontin [OPN]) in human osteosarcoma MG63 cells. MG63 cells on FN, COL1, and LM were kept as controls or subjected to shear stress (12 dynes/cm²), and the association of α_vβ₃ and β₁ integrins with Shc, phosphorylation of mitogen‐activated protein kinases (MAPKs, i.e., extracellular signal‐regulated kinase [ERK], c‐jun‐NH₂‐terminal kinase [JNK], and p38), and expressions of Egr‐1, c‐fos, Cox‐2, and OPN were determined. In MG63 cells, shear stress induces sustained associations of α_vβ₃ and β₁ with Shc when seeded on FN, but sustained associations of only β₁ with Shc when seeded on COL1/LM. Shear inductions of MAPKs and bone formation‐related genes were sustained (24 h) in cells on FN, but some of these responses were transient in cells on COL1/LM. The shear activations of ERK, JNK, and p38 were mediated by integrins and Shc, and these pathways differentially modulated the downstream bone formation‐related gene expression. Our findings showed that β₁ integrin plays predominant roles for shear‐induced signaling and gene expression in osteoblast‐like MG63 cells on FN, COL1, and LM and that α_vβ₃ also plays significant roles for such responses in cells on FN. The β₁/Shc association leads to the activation of ERK, which is critical for shear induction of bone formation‐related genes in osteoblast‐like cells.     

Estrogen augments shear stress–induced signaling and gene expression in osteoblast‐like cells via estrogen receptor–mediated expression of β1‐integrin

Estrogen and mechanical forces are positive regulators for osteoblast proliferation and bone formation. We investigated the synergistic effect of estrogen and flow‐induced shear stress on signal transduction and gene expression in human osetoblast‐like MG63 cells and primary osteoblasts (HOBs) using activations of extracellular signal‐regulated kinase (ERK) and p38 mitogen‐activated protein kinase (MAPK) and expressions of c‐fos and cyclooxygenase‐2 (I) as readouts. Estrogen (17β‐estradiol, 10 nM) and shear stress (12 dyn/cm²) alone induced transient phosphorylations of ERK and p38 MAPK in MG63 cells. Pretreating MG63 cells with 17β‐estradiol for 6 hours before shearing augmented these shear‐induced MAPK phosphorylations. Western blot and flow cytometric analyses showed that treating MG63 cells with 17β‐estradiol for 6 hrs induced their β₁‐integrin expression. This estrogen‐induction of β₁‐integrin was inhibited by pretreating the cells with a specific antagonist of estrogen receptor ICI 182,780. Both 17β‐estradiol and shear stress alone induced c‐fos and Cox‐2 gene expressions in MG63 cells. Pretreating MG63 cells with 17β‐estradiol for 6 hrs augmented the shear‐induced c‐fos and Cox‐2 expressions. The augmented effects of 17β‐estradiol on shear‐induced MAPK phosphorylations and c‐fos and Cox‐2 expressions were inhibited by pretreating the cells with ICI 182,780 or transfecting the cells with β₁‐specific small interfering RNA. Similar results on the augmented effect of estrogen on shear‐induced signaling and gene expression were obtained with HOBs. Our findings provide insights into the mechanism by which estrogen augments shear stress responsiveness of signal transduction and gene expression in bone cells via estrogen receptor–mediated increases in β₁‐integrin expression. © 2010 American Society for Bone and Mineral Research     

Mst2 Controls Bone Homeostasis by Regulating Osteoclast and Osteoblast Differentiation

Mammalian sterile 20‐like kinase 2 (Mst2) plays a central role in the Hippo pathway, controlling cell proliferation, differentiation, and apoptosis during development. However, the roles of Mst2 in osteoclast and osteoblast development are largely unknown. Here, we demonstrate that mice deficient in Mst2 exhibit osteoporotic phenotypes with increased numbers of osteoclasts and decreased numbers of osteoblasts as shown by micro–computed tomography (µCT) and histomorphometric analyses. Osteoclast precursors lacking Mst2 exhibit increased osteoclastogenesis and Nfatc1, Acp5, and Oscar expression in response to receptor activator of NF‐κB ligand (RANKL) exposure. Conversely, Mst2 overexpression in osteoclast precursors leads to the inhibition of RANKL‐induced osteoclast differentiation. Osteoblast precursors deficient in Mst2 exhibit attenuated osteoblast differentiation and function by downregulating the expression of Runx2, Alpl, Ibsp, and Bglap. Conversely, ectopic expression of Mst2 in osteoblast precursors increases osteoblastogenesis. Finally, we demonstrate that the NF‐κB pathway is activated by Mst2 deficiency during osteoclast and osteoblast development. Our findings suggest that Mst2 is involved in bone homeostasis, functioning as a reciprocal regulator of osteoclast and osteoblast differentiation through the NF‐κB pathway. © 2015 American Society for Bone and Mineral Research.     

IGFBP‐2 Directly Stimulates Osteoblast Differentiation

Insulin‐like growth factor binding protein 2 (IGFBP‐2) is important for acquisition of normal bone mass in mice; however, the mechanism by which IGFBP‐2 functions is not defined. These studies investigated the role of IGFBP‐2 in stimulating osteoblast differentiation. MC‐3T3 preosteoblasts expressed IGFBP‐2, and IGFBP‐2 knockdown resulted in a substantial delay in osteoblast differentiation, reduced osteocalcin expression and Alizarin red staining. These findings were replicated in primary calvarial osteoblasts obtained from IGFBP‐2^?/? mice, and addition of IGFBP‐2 rescued the differentiation program. In contrast, overexpression of IGFBP‐2 accelerated the time course of differentiation as well as increasing the total number of differentiating cells. By day 6, IGFBP‐2–overexpressing cells expressed twice as much osteocalcin as control cultures and this difference persisted. To determine the mechanism by which IGFBP‐2 functions, the interaction between IGFBP‐2 and receptor tyrosine phosphatase β (RPTPβ) was examined. Disruption of this interaction inhibited the ability of IGFBP‐2 to stimulate AKT activation and osteoblast differentiation. Knockdown of RPTPβ enhanced osteoblast differentiation, whereas overexpression of RPTPβ was inhibitory. Adding back IGFBP‐2 to RPTPβ‐overexpressing cells was able to rescue cell differentiation via enhancement of AKT activation. To determine the region of IGFBP‐2 that mediated this effect, an IGFBP‐2 mutant that contained substitutions of key amino acids in the heparin‐binding domain‐1 (HBD‐1) was prepared. This mutant had a major reduction in its ability to stimulate differentiation of calvarial osteoblasts from IGFBP‐2^?/? mice. Addition of a synthetic peptide that contained the HBD‐1 sequence to calvarial osteoblasts from IGFBP‐2^?/? mice rescued differentiation and osteocalcin expression. In summary, the results clearly demonstrate that IGFBP‐2 stimulates osteoblast differentiation and that this effect is mediated through its heparin‐binding domain‐1 interacting with RPTPβ. The results suggest that stimulation of differentiation is an important mechanism by which IGFBP‐2 regulates the acquisition of normal bone mass in mice. © 2014 American Society for Bone and Mineral Research.     

Positive Regulation of Adult Bone Formation by Osteoblast‐Specific Transcription Factor Osterix

Osterix (Osx) is essential for osteoblast differentiation and bone formation, because mice lacking Osx die within 1 h of birth with a complete absence of intramembranous and endochondral bone formation. Perinatal lethality caused by the disruption of the Osx gene prevents studies of the role of Osx in bones that are growing or already formed. Here, the function of Osx was examined in adult bones using the time‐ and site‐specific Cre/loxP system. Osx was inactivated in all osteoblasts by Col1a1‐Cre with the activity of Cre recombinase under the control of the 2.3‐kb collagen promoter. Even though no bone defects were observed in newborn mice, Osx inactivation with 2.3‐kb Col1a1‐Cre exhibited osteopenia phenotypes in growing mice. BMD and bone‐forming rate were decreased in lumbar vertebra, and the cortical bone of the long bones was thinner and more porous with reduced bone length. The trabecular bones were increased, but they were immature or premature. The expression of early marker genes for osteoblast differentiation such as Runx2, osteopontin, and alkaline phosphatase was markedly increased, but the late marker gene, osteocalcin, was decreased. However, no functional defects were found in osteoclasts. In summary, Osx inactivation in growing bones delayed osteoblast maturation, causing an accumulation of immature osteoblasts and reducing osteoblast function for bone formation, without apparent defects in bone resorption. These findings suggest a significant role of Osx in positively regulating osteoblast differentiation and bone formation in adult bone.     

Overexpression of Smurf2 Stimulates Endochondral Ossification Through Upregulation of β‐Catenin

Ectopic expression of Smurf2 in chondrocytes and perichondrial cells accelerated endochondral ossification by stimulating chondrocyte maturation and osteoblast development through upregulation of β‐catenin in Col2a1‐Smurf2 embryos. The mechanism underlying Smurf2‐mediated morphological changes during embryonic development may provide new mechanistic insights and potential targets for prevention and treatment of human osteoarthritis. Introduction : Our recent finding that adult Col2a1‐Smurf2 mice have an osteoarthritis‐like phenotype in knee joints prompted us to examine the role of Smurf2 in the regulation of chondrocyte maturation and osteoblast differentiation during embryonic endochondral ossification. Materials and Methods : We analyzed gene expression and morphological changes in developing limbs by immunofluorescence, immunohistochemistry, Western blot, skeletal preparation, and histology. A series of markers for chondrocyte maturation and osteoblast differentiation in developing limbs were examined by in situ hybridization. Results : Ectopic overexpression of Smurf2 driven by the Col2a1 promoter was detected in chondrocytes and in the perichondrium/periosteum of 16.5 dpc transgenic limbs. Ectopic Smurf2 expression in cells of the chondrogenic lineage inhibited chondrocyte differentiation and stimulated maturation; ectopic Smurf2 in cells of the osteoblastic lineage stimulated osteoblast differentiation. Mechanistically, this could be caused by a dramatic increase in the expression of β‐catenin protein levels in the chondrocytes and perichondrial/periosteal cells of the Col2a1‐Smurf2 limbs. Conclusions : Ectopic expression of Smurf2 driven by the Col2a1 promoter accelerated the process of endochondral ossification including chondrocyte maturation and osteoblast differentiation through upregulation of β‐catenin, suggesting a possible mechanism for development of osteoarthritis seen in these mice.     

Deficiency of Sef Is Associated With Increased Postnatal Cortical Bone Mass by Regulating Runx2 Activity

Sef (similar expression to fgf genes) is a feedback inhibitor of fibroblast growth factor (FGF) signaling and functions in part by binding to FGF receptors and inhibiting their activation. Genetic studies in mice and humans indicate an important role for fibroblast growth factor signaling in bone growth and homeostasis. We, therefore, investigated whether Sef had a function role in skeletal acquisition and remodeling. Sef expression is increased during osteoblast differentiation in vitro, and LacZ staining of Sef+/? mice showed high expression of Sef in the periosteum and chondro‐osseous junction of neonatal and adult mice. Mice with a global deletion of Sef showed increased cortical bone thickness, bone volume, and increased periosteal perimeter by micro‐computed tomography (micro‐CT). Histomorphometric analysis of cortical bone revealed a significant increase in osteoblast number. Interestingly, Sef?/? mice showed very little difference in trabecular bone by micro‐CT and histomorphometry compared with wild‐type mice. Bone marrow cells from Sef?/? mice grown in osteogenic medium showed increased proliferation and increased osteoblast differentiation compared with wild‐type bone marrow cells. Bone marrow cells from Sef?/? mice showed enhanced FGF2‐induced activation of the ERK pathway, whereas bone marrow cells from Sef transgenic mice showed decreased FGF2‐induced signaling. FGF2‐induced acetylation and stability of Runx2 was enhanced in Sef?/? bone marrow cells, whereas overexpression of Sef inhibited Runx2‐responsive luciferase reporter activity. Bone marrow from Sef?/? mice showed enhanced hematopoietic lineage‐dependent and osteoblast‐dependent osteoclastogenesis and increased bone resorptive activity relative to wild‐type controls in in vitro assays, whereas overexpression of Sef inhibited osteoclast differentiation. Taken together, these studies indicate that Sef has specific roles in osteoblast and osteoclast lineages and that its absence results in increased osteoblast and osteoclast activity with a net increase in cortical bone mass. © 2014 American Society for Bone and Mineral Research.     

Connexin 43 and ERK regulate tension‐induced signal transduction in human periodontal ligament fibroblasts

Periodontal ligament (PDL) fibroblasts play an important role in preserving periodontal homeostasis and transmitting mechanical signals to alveolar bone. Connexin 43 (Cx43), a gap junction protein, is essential for bone homeostasis and regulates bone remodeling. However, the function of Cx43 in human PDL fibroblast‐regulated bone remodeling has not yet been elucidated. In this study, human PDL fibroblasts were exposed to cyclic mechanical tension with a maximum 5% elongation for different durations. We then examined the expression of signaling molecules related to osteogenesis and osteoclastogenesis at both the mRNA and protein levels as well as the activity of extracellular signal‐regulated kinase (ERK) in human PDL fibroblasts after loading. We found that mechanical tension increased Cx43, which further upregulated osteogenic (e.g., RUNX2, Osterix, and OPG) and down‐regulated osteoclastogenic (e.g., RANKL) signaling molecules. Suppressing Cx43 gene (Gja1) by siRNA inhibited the increase in osteogenesis‐related molecules but enhanced RANKL expression. Similar to Cx43, activated ERK1/2 was also enhanced by mechanical tension and suppressed by Cx43 siRNA. Inhibition of ERK1/2 signaling using PD98059 reduced the tension‐regulated increase in osteogenesis‐related molecules but enhanced that of osteoclastogenesis‐related ones. These findings suggest that cyclic tension may involve into the osteogenic or osteoclastogenetic differentiation potential of human PDL fibroblasts via the Cx43‐ERK1/2 signaling pathway. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:1008–1014, 2015.