Rita Gerard-O'Riley

Focal adhesion kinase is important for fluid shear stress-induced mechanotransduction in osteoblasts.

Mechanical loading of bone is important for maintenance of bone mass and structural stability of the skeleton. When bone is mechanically loaded, movement of fluid within the spaces surrounding bone cells generates fluid shear stress (FSS) that stimulates osteoblasts, resulting in enhanced anabolic activity. The mechanisms by which osteoblasts convert the external stimulation of FSS into biochemical changes, a process known as mechanotransduction, remain poorly understood. Focal adhesions are prime candidates for transducing external stimuli. Focal adhesion kinase (FAK), a nonreceptor tyrosine kinase found in focal adhesions, may play a key role in mechanotransduction, although its function has not been directly examined in osteoblasts. We examined the role of FAK in osteoblast mechanotransduction using short interfering RNA (siRNA), overexpression of a dominant negative FAK, and FAK−/− osteoblasts to disrupt FAK function in calvarial osteoblasts. Osteoblasts were subjected to varying periods oscillatory fluid flow (OFF) from 5 min to 4 h, and several physiologically important readouts of mechanotransduction were analyzed including: extracellular signal‐related kinase 1/2 phosphorylation, upregulation of c‐fos, cyclooxygenase‐2, and osteopontin, and release of prostaglandin E2. Osteoblasts with disrupted FAK signaling exhibited severely impaired mechanical responses in all endpoints examined. These data indicate the importance of FAK for both short and long periods of FSS‐induced mechanotransduction in osteoblasts.

Hypoxia-inducible factor-1 (HIF-1) but not HIF-2 is essential for hypoxic induction of collagen prolyl 4-hydroxylases in primary newborn mouse epiphyseal growth plate chondrocytes

Background: The hypoxic cartilaginous growth plate is rich in extracellular matrix (ECM). Results: Expression of the key enzymes in ECM synthesis, the collagen prolyl 4-hydroxylases (C-P4Hs), is induced specifically by hypoxia-inducible factor 1. Conclusion: Hypoxia inducibility of C-P4Hs ensures sufficient C-P4H activity in hypoxic chondrocytes. Significance: Quantitative regulation of C-P4H may be a key modality by which hypoxia influences early chondrocyte survival and differentiation. Hypoxia-inducible factors (HIFs) are the master regulators of hypoxia-responsive genes. They play a critical role in the survival, development, and differentiation of chondrocytes in the avascular hypoxic fetal growth plate, which is rich in extracellular matrix (ECM) and in its main component, collagens. Several genes involved in the synthesis, maintenance, and degradation of ECM are regulated by HIFs. Collagen prolyl 4-hydroxylases (C-P4Hs) are key enzymes in collagen synthesis because the resulting 4-hydroxyprolines are necessary for the stability of all collagen molecules. The vertebrate C-P4Hs are α2β2 tetramers with three isoforms of the catalytic α subunit, yielding C-P4Hs of types I–III. C-P4H-I is the main form in most cells, but C-P4H-II is the major form in chondrocytes. We postulated here that post-translational modification of collagens, particularly 4-hydroxylation of proline residues, could be one of the modalities by which HIF regulates the adaptive responses of chondrocytes in fetal growth plates. To address this hypothesis, we used primary epiphyseal growth plate chondrocytes isolated from newborn mice with conditionally inactivated genes for HIF-1α, HIF-2α, or the von Hippel-Lindau protein. The data obtained showed that C-P4H α(I) and α(II) mRNA levels were increased in hypoxic chondrocytes in a manner dependent on HIF-1 but not on HIF-2. Furthermore, the increases in the C-P4H mRNA levels were associated with both increased amounts of the C-P4H tetramers and augmented C-P4H activity in hypoxia. The hypoxia inducibility of the C-P4H isoenzymes is thus likely to ensure sufficient C-P4H activity for collagen synthesis occurring in chondrocytes in a hypoxic environment.

Activation of NF-κB by fluid shear stress, but not TNF-α, requires focal adhesion kinase in osteoblasts

When bone is mechanically loaded fluid shear stress (FSS) is generated as a result of the movement of interstitial fluid across the membranes of osteoblasts and osteocytes. This external mechanical loading stimulates changes in the activity of cytoplasmic signaling molecules and alters gene expression in bone cells. This process, referred to as mechanotransduction, is vital for maintaining bone health in vivo by regulating the balance between bone formation and bone resorption. This current study focuses on the role of focal adhesions, sites of integrin-mediated cellular attachment to the extracellular matrix, and their proposed function as mechanosensors in bone cells. We examined the role of a key component of focal adhesions and of mechanotransduction, focal adhesion kinase (FAK) in regulation of FSS- and tumor necrosis factor-alpha (TNF-alpha)-induced activation of nuclear factor-kappa B (NF-kappaB) signaling in osteoblasts. Immortalized FAK(+/+) and FAK(-)(/)(-) osteoblasts were exposed to periods of oscillatory fluid shear stress (OFF) and NF-kappaB activation was analyzed. We determined that FAK is required for OFF-induced nuclear translocation and activation of NF-kappaB in osteoblasts. In addition we found that OFF-induced phosphorylation of the IkappaB kinases (IKKalpha/beta) in both FAK(+/+) and FAK(-/-) osteoblasts, but only FAK(+/+) osteoblasts demonstrated the resulting degradation of NF-kappaB inhibitors IkappaBalpha and IkappaBbeta. OFF did not induce the degradation of IkappaBepsilon or the processing of p105 in either FAK(+/+) and FAK(-/-) osteoblasts. To compare the role of FAK in mediating OFF-induced mechanotransduction to the well characterized activation of NF-kappaB by inflammatory cytokines, we exposed FAK(+/+) and FAK(-/-) osteoblasts to TNF-alpha. Interestingly, FAK was not required for TNF-alpha induced NF-kappaB activation in osteoblasts. In addition we determined that TNF-alpha treatment did not induce the degradation of IkappaBbeta as did OFF. These data indicate a novel relationship between FAK and NF-kappaB activation in osteoblast mechanotransduction and demonstrates that the mechanism of FSS-induced NF-kappaB activation in osteoblasts differs from the well characterized TNF-alpha-induced activation.

Nmp4/CIZ contributes to fluid shear stress induced MMP-13 gene induction in osteoblasts

The expression of matrix metalloproteinase‐13 (MMP‐13), involved in bone turnover, is elevated in stretched MC3T3‐E1 osteoblast‐like cells. Strain‐mediated forces impact bone remodeling due in large part to the movement of fluid through the canalicular‐lacunar network. The resulting fluid shear stress (FSS) over the surface membranes of bone cells initiates bone remodeling. Although the nuclear events mediating putative FSS‐induced changes in osteoblast MMP‐13 transcription are unknown, previous studies with bone cells suggest an overlap between osteoblast FSS‐ and PTH‐induced signal response pathways. MMP‐13 PTH response is regulated by a 110 bp 5′ regulatory region, conserved across the mouse, rat, and human genes, that supports the binding of numerous transcription factors including Runx2, c‐fos/c‐jun, Ets‐1, and nuclear matrix protein 4/cas interacting zinc finger protein (Nmp4/CIZ) a nucleocytoplasmic shuttling trans‐acting protein that attenuates PTH‐driven transcription. Nmp4/CIZ also binds p130cas, an adaptor protein implicated in mechanotransduction. Here we sought to determine whether Nmp4/CIZ contributes to FSS‐induced changes in MMP‐13 transcription. FSS (12 dynes/cm2, 3–5 h) increased MMP‐13 promoter‐reporter activity approximately two‐fold in MC3T3‐E1 osteoblast‐like cells attended by a comparable increase in mRNA expression. This was accompanied by a decrease in Nmp4/CIZ binding to its cis‐element within the PTH response region, the mutation of which abrogated the MMP‐13 response to FSS. Interestingly, FSS enhanced Nmp4/CIZ promoter activity and induced p130cas nuclear translocation. We conclude that the PTH regulatory region of MMP‐13 also contributes to FSS response and that Nmp4/CIZ plays similar but distinct roles in mediating hormone‐ and FSS‐driven induction of MMP‐13 in bone cells. J. Cell. Biochem. 102: 1202–1213, 2007.

Nmp4/CIZ inhibits mechanically induced β-catenin signaling activity in osteoblasts

Cellular mechanotransduction, the process of converting mechanical signals into biochemical responses within cells, is a critical aspect of bone health. While the effects of mechanical loading on bone are well recognized, elucidating the specific molecular pathways involved in the processing of mechanical signals by bone cells represents a challenge and an opportunity to identify therapeutic strategies to combat bone loss. In this study we have for the first time examined the relationship between the nucleocytoplasmic shuttling transcription factor nuclear matrix protein‐4/cas interacting zinc finger protein (Nmp4/CIZ) and β‐catenin signaling in response to a physiologic mechanical stimulation (oscillatory fluid shear stress, OFSS) in osteoblasts. Using calvaria‐derived osteoblasts from Nmp4‐deficient and wild‐type mice, we found that the normal translocation of β‐catenin to the nucleus in osteoblasts that is induced by OFSS is enhanced when Nmp4/CIZ is absent. Furthermore, we found that other aspects of OFSS‐induced mechanotransduction generally associated with the β‐catenin signaling pathway, including ERK, Akt, and GSK3β activity, as well as expression of the β‐catenin‐responsive protein cyclin D1 are also enhanced in cells lacking Nmp4/CIZ. Finally, we found that in the absence of Nmp4/CIZ, OFSS‐induced cytoskeletal reorganization and the formation of focal adhesions between osteoblasts and the extracellular substrate is qualitatively enhanced, suggesting that Nmp4/CIZ may reduce the sensitivity of bone cells to mechanical stimuli. Together these results provide experimental support for the concept that Nmp4/CIZ plays an inhibitory role in the response of bone cells to mechanical stimulation induced by OFSS. J. Cell. Physiol. 223: 435–441, 2010.

Pyk2 regulates megakaryocyte-induced increases in osteoblast number and bone formation

Preclinical and clinical evidence from megakaryocyte (MK)‐related diseases suggests that MKs play a significant role in maintaining bone homeostasis. Findings from our laboratories reveal that MKs significantly increase osteoblast (OB) number through direct MK‐OB contact and the activation of integrins. We, therefore, examined the role of Pyk2, a tyrosine kinase known to be regulated downstream of integrins, in the MK‐mediated enhancement of OBs. When OBs were co‐cultured with MKs, total Pyk2 levels in OBs were significantly enhanced primarily because of increased Pyk2 gene transcription. Additionally, p53 and Mdm2 were both decreased in OBs upon MK stimulation, which would be permissive of cell cycle entry. We then demonstrated that OB number was markedly reduced when Pyk2−/− OBs, as opposed to wild‐type (WT) OBs, were co‐cultured with MKs. We also determined that MKs inhibit OB differentiation in the presence and absence of Pyk2 expression. Finally, given that MK‐replete spleen cells from GATA‐1–deficient mice can robustly stimulate OB proliferation and bone formation in WT mice, we adoptively transferred spleen cells from these mice into Pyk2−/− recipient mice. Importantly, GATA‐1–deficient spleen cells failed to stimulate an increase in bone formation in Pyk2−/− mice, suggesting in vivo the important role of Pyk2 in the MK‐induced increase in bone volume. Further understanding of the signaling pathways involved in the MK‐mediated enhancement of OB number and bone formation will facilitate the development of novel anabolic therapies to treat bone loss diseases.

Interferon Gamma, but not Calcitriol Improves the Osteopetrotic Phenotypes in ADO2 Mice.

ADO2 is a heritable osteosclerotic disorder that usually results from heterozygous missense dominant negative mutations in the chloride channel 7 gene (CLCN7). ADO2 is characterized by a wide range of features and severity, including multiple fractures, impaired vision due to secondary bony overgrowth and/or the lack of the optical canal enlargement with growth, and osteonecrosis/osteomyelitis. The disease is presently incurable, although anecdotal evidence suggests that calcitriol and interferon gamma‐1b (IFN‐G) may have some beneficial effects. To identify the role of these drugs for the treatment of ADO2, we utilized a knock‐in (G213R mutation in Clcn7) ADO2 mouse model that resembles the human disease. Six‐week‐old ADO2 heterozygous mice were administered vehicle (PBS) or calcitriol or IFN‐G 5 times per week for 8 weeks. We determined bone phenotypes using DXA and μCT, and analyzed serum biochemistry and bone resorption markers. ADO2 mice treated with all doses of IFN‐G significantly (p<0.05) attenuated the increase of whole body aBMD and distal femur BV/TV gain in both male and female compared to the vehicle group. In contrast, mice treated with low and medium doses of calcitriol showed a trend of higher aBMD and BV/TV whereas high dose calcitriol significantly (p<0.05) increased bone mass compared to the vehicle group. The calcium and phosphorus levels did not differ between vehicle and IFN‐G or calcitriol treated mice; however, we detected significantly (p<0.05) elevated levels of CTX/TRAP5b ratio in IFN‐G treated mice. Our findings indicate that while IFN‐G at all doses substantially improved the osteopetrotic phenotypes in ADO2 heterozygous mice, calcitriol treatment at any dose did not improve the phenotype and at high dose further increased bone mass. Thus, use of high dose calcitriol therapy in ADO2 patients merits serious reconsideration. Importantly, our data support the prospect of a clinical trial of IFN‐G in ADO2 patients.

Blockade of TNFR1 signaling: A role of oscillatory fluid shear stress in osteoblasts

Fluid shear stress protects cells from TNF‐α‐induced apoptosis. Oscillatory fluid shear stress (OFSS) is generally perceived as physiologically relevant biophysical signal for bone cells. Here we identify several cellular mechanisms responsible for mediating the protective effects of OFSS against TNF‐α‐induced apoptosis in vitro. We found that exposure of MC3T3‐E1 osteoblast‐like cells to as little as 5 min of OFSS suppressed TNF‐α‐induced activation of caspase‐3, cleavage of PARP and phosphorylation of histone. In contrast, H2O2‐induced apoptosis was not inhibited by OFSS suggesting that OFSS might not be protecting cells from TNF‐α‐induced apoptosis via stimulation of global pro‐survival signaling pathways. In support of this speculation, OFSS inhibition of TNF‐α‐induced apoptosis was unaffected by inhibitors of several pro‐survival signaling pathways including pI3‐kinase (LY294002), MAPK/ERK kinase (PD98059 or U0126), intracellular Ca2+ release (U73122), NO production (L‐NAME), or protein synthesis (cycloheximide) that were applied to cells during exposure to OFSS and during TNF‐α treatment. However, TNF‐α‐induced phosphorylation and degradation of IκBα was blocked by pre‐exposure of cells to OFSS suggesting a more specific effect of OFSS on TNF‐α signaling. We therefore focused on the mechanism of OFSS regulation of TNF‐receptor 1 (TNFR1) signaling and found that OFSS (1) reduced the amount of receptor on the cell surface, (2) prevented the association of ubiquitinated RIP in TNFR1 complexes with TRADD and TRAF2, and (3) reduced TNF‐α‐induced IL‐8 promoter activity in the nucleus. We conclude that the anti‐apoptotic effect of OFSS is not mediated by activation of universal pro‐survival signaling pathways. Rather, OFSS inhibits TNF‐α‐induced pro‐apoptotic signaling which can be explained by the down‐regulation of TNFR1 on the cell surface and blockade of TNFR1 downstream signaling by OFSS. J. Cell. Physiol. 226: 1044–1051, 2011.

Loss of VHL in mesenchymal progenitors of the limb bud alters multiple steps of endochondral bone development

Adaptation to low oxygen tension (hypoxia) is a critical event during development. The transcription factors Hypoxia Inducible Factor-1α (HIF-1α) and HIF-2α are essential mediators of the homeostatic responses that allow hypoxic cells to survive and differentiate. Von Hippel-Lindau protein (VHL) is the E3 ubiquitin ligase that targets HIFs to the proteasome for degradation in normoxia. We have previously demonstrated that the transcription factor HIF-1α is essential for survival and differentiation of growth plate chondrocytes, whereas HIF-2α is not necessary for fetal growth plate development. We have also shown that VHL is important for endochondral bone development, since loss of VHL in chondrocytes causes severe dwarfism. In this study, in order to expand our understanding of the role of VHL in chondrogenesis, we conditionally deleted VHL in mesenchymal progenitors of the limb bud, i.e. in cells not yet committed to the chondrocyte lineage. Deficiency of VHL in limb bud mesenchyme does not alter the timely differentiation of mesenchymal cells into chondrocytes. However, it causes structural collapse of the cartilaginous growth plate as a result of impaired proliferation, delayed terminal differentiation, and ectopic death of chondrocytes. This phenotype is associated to delayed replacement of cartilage by bone. Notably, loss of HIF-2α fully rescues the late formation of the bone marrow cavity in VHL mutant mice, though it does not affect any other detectable abnormality of the VHL mutant growth plates. Our findings demonstrate that VHL regulates bone morphogenesis as its loss considerably alters size, shape and overall development of the skeletal elements.

Immortalization and characterization of osteoblast cell lines generated from wild-type and Nmp4-null mouse bone marrow stromal cells using murine telomerase reverse transcriptase (mTERT).

Intermittent parathyroid hormone (PTH) adds new bone to the osteoporotic skeleton; the transcription factor Nmp4/CIZ represses PTH‐induced bone formation in mice and as a consequence is a potential drug target for improving hormone clinical efficacy. To explore the impact of Nmp4/CIZ on osteoblast phenotype, we immortalized bone marrow stromal cells from wildtype (WT) and Nmp4‐knockout (KO) mice using murine telomerase reverse transcriptase. Clonal lines were initially chosen based on their positive staining for alkaline phosphatase and capacity for mineralization. Disabling Nmp4/CIZ had no gross impact on osteoblast phenotype development. WT and KO clones exhibited identical sustained growth, reduced population doubling times, extended maintenance of the mature osteoblast phenotype, and competency for differentiating toward the osteoblast and adipocyte lineages. Additional screening of the immortalized cells for PTH‐responsiveness permitted further studies with single WT and KO clones. We recently demonstrated that PTH‐induced c‐fos femoral mRNA expression is enhanced in Nmp4‐KO mice and in the present study we observed that hormone stimulated either an equivalent or modestly enhanced increase in c‐fos mRNA expression in both primary null and KO clone cells depending on PTH concentration. The null primary osteoblasts and KO clone cells exhibited a transiently enhanced response to bone morphogenetic protein 2 (BMP2). The clones exhibited lower and higher expressions of the PTH receptor (Pthr1) and the BMP2 receptor (Bmpr1a, Alk3), respectively, as compared to primary cells. These immortalized cell lines will provide a valuable tool for disentangling the complex functional roles underlying Nmp4/CIZ regulation of bone anabolism. J. Cell. Physiol. 227: 1873–1882, 2012.