Browsing by Subject "Bone formation"
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Item Effects of PYK2-Deficiency on Midpalatal Suture Expansion in Mice(2015-08) Sun, Jun; Bruzzaniti, Angela; Liu, Sean Shih-Yao; Brown, David T.; Chu, Tien Min; Levon, John A.Background: Suture expansion is a very important clinical approach to correct maxillary width deficiency, but it has a high potential for treatment relapse. Accelerating bone formation and mineralization in the midpalatal suture during suture expansion is beneficial in preventing relapse of the arch width and reducing the retention period. Pyk2 is tyrosine kinase which has been shown to mediate signaling pathways that are involved in the process of bone remodeling. Pyk2 knock-out (KO) mice have augmented bone formation and increased bone mass, suggesting that therapeutic strategies that inhibit Pyk2 may be useful to enhance bone remodeling and prevent suture relapse during suture expansion. Objectives: To determine if Pyk2-deficiency affects midpalatal suture bone mass and bone remodeling with or without suture expansion in mice. Methods: Thirty-six Pyk2-KO and thirty-six wild type (WT) 6 week-old male mice were randomly assigned into three groups: receiving no expansion force (0 g), 10 g or 20 g force of rapid maxillary expansion for 14 days. Half of the mice in each group were used for histology analysis; the other half was assigned for fluorescence analysis. Suture width, maxilla width and bone volume/tissue volume around suture bone edges were measured using micro-CT. Histological analyses of osteoclasts (tartrate resistant acid phosphatase, TRAP), osteoblasts (alkaline phosphatase, ALP) and chondrocytes (alcian blue) were performed. Results: The BV/TV ratio was significantly higher in Pyk2-KO control mice compared to WT control mice. Suture expansion in WT and Pyk2-KO mice led to an increase in bone marrow spaces around the suture edge and significantly reduced BV/TV. Expansion also led to a significant increase in suture width, suture fibrous area, osteoclast number, cartilage area and hypertrophic chondrocyte number. However, BV/TV in Pyk2-KO mice was significantly higher than in WT mice at both the 10 g and 20 g force levels. In addition, Pyk2-KO exhibited reduced suture width, maxilla width, fibrous area and osteoclast number per bone surface (OC.S/BS) compared to WT mice under expansion forces. Cartilage area and hypertrophic chondrocyte number were increased by force but were independent of mouse genotypes. Conclusion: Pyk2-KO mice have higher BV/TV and narrower suture width compared to WT mice, which may be due to decreased osteoclast activity. The higher BV/TV of the midpalatal sutures of Pyk2-KO mice following suture expansion may suggest the presence of a more stable suture that has a reduced potential for relapse. Therapeutic strategies to inhibit Pyk2 during RME may be beneficial in increasing bone mass and preventing relapse of the suture.Item The essential role of Stat3 in bone homeostasis and mechanotransduction(2014) Zhou, Hongkang; Li, Jiliang; Marrs, James; Stocum, David L.; Atkinson, Simon; Aguilar, R. Claudio; Yokota, Hiroki, 1955-Signal Transducer and Activator of Transcription 3 (Stat3) is a transcription factor expressed in bone and joint cells that include osteoblasts, osteocytes, osteoclasts, and chondrocytes. Stat3 is activated by a variety of cytokines and growth factors, including IL-6/gp130 family cytokines. These cytokines not only regulate the differentiation of osteoblasts and osteoclasts, but also regulate proliferation of chondrocytes through Stat3 activation. In 2007, mutations of Stat3 have been confirmed to cause a rare human immunodeficiency disease – Job syndrome which presents skeletal abnormalities like: reduced bone density (osteopenia), scoliosis, hyperextensibility of joints, and recurrent pathological bone fractures. Changes in the Stat3 gene alter the structure and function of the Stat3 proteins, impairing its ability to control the activity of other genes. However, little is known about the effects of Stat3 mutations on bone cells and tissues. To investigate the in vivo physiological role of Stat3 in bone homeostasis, osteoblast/osteocyte-specific Stat3 knockout (KO) mice were generated via the Cre-LoxP recombination system. The osteoblast/osteocyte-specific Stat3 KO mice showed bone abnormalities and an osteoporotic phenotype because of a reduced bone formation rate. Furthermore, inactivation of Stat3 decreased load-driven bone formation, and the disruption of Stat3 in osteoblasts suppressed load-driven mitochondrial activity, which led to an elevated level of reactive oxygen species (ROS) in cultured primary osteoblasts. Stat3 has been found to be responsive to mechanical stimulation, and might play an important role in mechanical signal transduction in osteocytes. To investigate the role Stat3 plays in mechanical signaling transduction, osteocyte-specific Stat3 knockout (KO) mice were created. Inactivation of Stat3 in osteocytes presented a significantly reduced load-driven bone formation. Decreased osteoblast activity indicated by reduced osteoid surface was also found in osteocyte-specific Stat3 KO mice. Moreover, sclerostin (SOST) protein which is a critical osteocyte-specific inhibitor of bone formation, its encoded gene SOST expression has been found to be enhanced in osteocyte-specific Stat3 KO mice. Thus, these results clearly demonstrated that Stat3 plays an important role in bone homeostasis and mechanotransduction, and Stat3 is not only involved in bone-formation-important genes regulation in the nucleus but also in mediation of ROS and oxidative stress in mitochondria.Item Increased S1P expression in osteoclasts enhances bone formation in an animal model of Paget's disease(Wiley, 2021-04) Nagata, Yuki; Miyagawa, Kazuaki; Ohata, Yasuhisa; Petrusca, Daniela N.; Pagnotti, Gabriel M.; Mohammad, Khalid S.; Guise, Theresa A.; Windle, Jolene J.; Roodman, G. David; Kurihara, Noriyoshi; Medicine, School of MedicinePaget's disease (PD) is characterized by increased numbers of abnormal osteoclasts (OCLs) that drive exuberant bone formation, but the mechanisms responsible for the increased bone formation remain unclear. We previously reported that OCLs from 70% of PD patients express measles virus nucleocapsid protein (MVNP), and that transgenic mice with targeted expression of MVNP in OCLs (MVNP mice) develop bone lesions and abnormal OCLs characteristic of PD. In this report, we examined if OCL-derived sphingosine-1-phosphate (S1P) contributed to the abnormal bone formation in PD, since OCL-derived S1P can act as a coupling factor to increase normal bone formation via binding S1P-receptor-3 (S1PR3) on osteoblasts (OBs). We report that OCLs from MVNP mice and PD patients expressed high levels of sphingosine kinase-1 (SphK-1) compared with wild-type (WT) mouse and normal donor OCLs. SphK-1 production by MVNP-OCLs was interleukin-6 (IL-6)-dependent since OCLs from MVNP/IL-6-/- mice expressed lower levels of SphK-1. Immunohistochemistry of bone biopsies from a normal donor, a PD patient, WT and MVNP mice confirmed increased expression levels of SphK-1 in OCLs and S1PR3 in OBs of the PD patient and MVNP mice compared with normal donor and WT mice. Further, MVNP-OCLs cocultured with OBs from MVNP or WT mice increased OB-S1PR3 expression and enhanced expression of OB differentiation markers in MVNP-OBs precursors compared with WT-OBs, which was mediated by IL-6 and insulin-like growth factor 1 secreted by MVNP-OCLs. Finally, the addition of an S1PR3 antagonist (VPC23019) to WT or MVNP-OBs treated with WT and MVNP-OCL-conditioned media (CM) blocked enhanced OB differentiation of MVNP-OBs treated with MVNP-OCL-CM. In contrast, the addition of the SIPR3 agonist, VPC24191, to the cultures enhanced osterix and Col-1A expression in MVNP-OBs treated with MVNP-OCL-CM compared with WT-OBs treated with WT-OCL-CM. These results suggest that IL-6 produced by PD-OCLs increases S1P in OCLs and S1PR3 on OBs, to increase bone formation in PD.Item Pyk2 and Megakaryocytes Regulate Osteoblast Differentiation and Migration Via Distinct and Overlapping Mechanisms(Wiley, 2016-06) Eleniste, Pierre P.; Patel, Vruti; Posritong, Sumana; Zero, Odette; Largura, Heather; Cheng, Ying-Hua; Himes, Evan R.; Hamilton, Matthew; Baughman, Jenna; Kacena, Melissa A.; Bruzzaniti, Angela; Department of Biomedical and Applied Sciences, School of DentistryOsteoblast differentiation and migration are necessary for bone formation during bone remodeling. Mice lacking the proline-rich tyrosine kinase Pyk2 (Pyk2-KO) have increased bone mass, in part due to increased osteoblast proliferation. Megakaryocytes (MKs), the platelet-producing cells, also promote osteoblast proliferation in vitro and bone-formation in vivo via a pathway that involves Pyk2. In the current study, we examined the mechanism of action of Pyk2, and the role of MKs, on osteoblast differentiation and migration. We found that Pyk2-KO osteoblasts express elevated alkaline phosphatase (ALP), type I collagen and osteocalcin mRNA levels as well as increased ALP activity, and mineralization, confirming that Pyk2 negatively regulates osteoblast function. Since Pyk2 Y402 phosphorylation is important for its catalytic activity and for its protein-scaffolding functions, we expressed the phosphorylation-mutant (Pyk2(Y402F) ) and kinase-mutant (Pyk2(K457A) ) in Pyk2-KO osteoblasts. Both Pyk2(Y402F) and Pyk2(K457A) reduced ALP activity, whereas only kinase-inactive Pyk2(K457A) inhibited Pyk2-KO osteoblast migration. Consistent with a role for Pyk2 on ALP activity, co-culture of MKs with osteoblasts led to a decrease in the level of phosphorylated Pyk2 (pY402) as well as a decrease in ALP activity. Although, Pyk2-KO osteoblasts exhibited increased migration compared to wild-type osteoblasts, Pyk2 expression was not required necessary for the ability of MKs to stimulate osteoblast migration. Together, these data suggest that osteoblast differentiation and migration are inversely regulated by MKs via distinct Pyk2-dependent and independent signaling pathways. Novel drugs that distinguish between the kinase-dependent or protein-scaffolding functions of Pyk2 may provide therapeutic specificity for the control of bone-related diseases.Item Pyk2 deficiency potentiates osteoblast differentiation and mineralizing activity in response to estrogen or raloxifene(Elsevier, 2018-10-15) Posritong, Sumana; Hong, Jung Min; Eleniste, Pierre P.; McIntyre, Patrick W.; Wu, Jennifer L.; Himes, Evan R.; Patel, Vruti; Kacena, Melissa A.; Bruzzaniti, Angela; Biomedical Sciences and Comprehensive Care, School of DentistryBone remodeling is controlled by the actions of bone-degrading osteoclasts and bone-forming osteoblasts (OBs). Aging and loss of estrogen after menopause affects bone mass and quality. Estrogen therapy, including selective estrogen receptor modulators (SERMs), can prevent bone loss and increase bone mineral density in post-menopausal women. Although investigations of the effects of estrogen on osteoclast activity are well advanced, the mechanism of action of estrogen on OBs is still unclear. The proline-rich tyrosine kinase 2 (Pyk2) is important for bone formation and female mice lacking Pyk2 (Pyk2-KO) exhibit elevated bone mass, increased bone formation rate and reduced osteoclast activity. Therefore, in the current study, we examined the role of estrogen signaling on the mechanism of action of Pyk2 in OBs. As expected, Pyk2-KO OBs showed significantly higher proliferation, matrix formation, and mineralization than WT OBs. In addition we found that Pyk2-KO OBs cultured in the presence of either 17β-estradiol (E2) or raloxifene, a SERM used for the treatment of post-menopausal osteoporosis, showed a further robust increase in alkaline phosphatase (ALP) activity and mineralization. We examined the possible mechanism of action and found that Pyk2 deletion promotes the proteasome-mediated degradation of estrogen receptor α (ERα), but not estrogen receptor β (ERβ). As a consequence, E2 signaling via ERβ was enhanced in Pyk2-KO OBs. In addition, we found that Pyk2 deletion and E2 stimulation had an additive effect on ERK phosphorylation, which is known to stimulate cell differentiation and survival. Our findings suggest that in the absence of Pyk2, estrogen exerts an osteogenic effect on OBs through altered ERα and ERβ signaling. Thus, targeting Pyk2, in combination with estrogen or raloxifene, may be a novel strategy for the prevention and/or treatment of bone loss diseases.Item Regulation of osteoblast activity by Pyk2-targeted approaches(2016-11-15) Posritong, Sumana; Bruzzaniti, Angela; Chu, Tien-Min G.; Bottino, Marco C.; Li, Jiliang; Main, Russell P.The hormonal and cellular mechanisms controlling bone formation are not completely understood. The proline-rich tyrosine kinase 2 (Pyk2) is important for osteoblast (OB) activity and bone formation. However, female mice lacking Pyk2 (Pyk2-KO) exhibit elevated bone volume/total volume. Previously, our laboratory found ovariectomized Pyk2-KO mice supplemented with 17β-estradiol (E2) exhibited a greater increase in bone volume than WT mice treated with E2. The overall hypotheses of our studies are that Pyk2 regulates OB activity by modulating the E2-signaling cascade and that a Pyk2-inhibitor will promote OB activity and be suitable for bone regeneration applications. In Aim1, we determined the mechanism of action of Pyk2 and E2 in OBs. Pyk2-KO OBs showed significantly higher proliferation, matrix formation, and mineralization than WT OBs. In the presence of E2 or raloxifene, a selective estrogen receptor (ER) modulator, both matrix formation and mineralization were further increased in Pyk2-KO OBs, but not WT OBs. Consistent with a role of Pyk2 in E2 signaling, Pyk2-depletion led to the proteasome-mediated degradation of ERα, but not ERβ. Finally, we found Pyk2-depletion and E2 have an additive effect on ERK phosphorylation, known to increase cell differentiation and survival. In Aim2, we developed a Pyk2-inhibitor loaded hydrogel and evaluated its viscosity, gelation time, swelling, degradation, and release behavior. We found that a hydrogel composed of PEGDA1000 plus 10% gelatin exhibited viscosity and shear-thinning behavior suitable for use as an injectable-carrier. Importantly, the Pyk2-inhibitor-hydrogel was cytocompatible, retained its inhibitory activity against Pyk2 leading to an increase in OB activity. In conclusion, therapeutic strategies targeting Pyk2 may improve systemic bone formation, while Pyk2-inhibitor loaded hydrogels may be suitable for targeted bone regeneration in craniofacial and/or the other skeletal defects.Item Stat3 in osteocytes mediates osteogenic response to loading(Elsevier, 2019-07-29) Corry, Kylie A.; Zhou, Hongkang; Brustovetsky, Tatiana; Himes, Evan R.; Bivi, Nicoletta; Horn, M. Ryne; Kitase, Yukiko; Wallace, Joseph M.; Bellido, Teresita; Brustovetsky, Nickolay; Li, Jiliang; Biology, School of ScienceSignal transducer and activator of transcription 3 (Stat3) is a member of the Stat family of proteins involved in signaling in many different cell types, including osteocytes. Osteocytes are considered major mechanosensing cells in bone due to their intricate dendritic networks able to sense changes in physical force and to orchestrate the response of osteoclasts and osteoblasts. We examined the role of Stat3 in osteocytes by generating mice lacking Stat3 in these cells using the Dmp-1(8kb)-Cre promoter (Stat3cKO mice). Compared to age-matched littermate controls, Stat3cKO mice of either sex (18 weeks old) exhibit reduced bone formation indices, decreased osteoblasts and increased osteoclasts, and altered material properties, without detectable changes in bone mineral density (BMD) or content of either trabecular or cortical bone. In addition, Stat3cKO mice of either sex show significantly decreased load-induced bone formation. Furthermore, pharmacologic inhibition of Stat3 in osteocytes in vitro with WP1066 blocked the increase in cytosolic calcium induced by ATP, a mediator of the cellular responses to sheer stress. WP1066 also increased reactive oxygen species (ROS) production in cultured MLO-Y4 osteocytes. These data demonstrate that Stat3 is a critical mediator of mechanical signals received by osteocytes and suggest that osteocytic Stat3 is a potential therapeutic target to stimulate bone anabolism.Item STING Contributes to Abnormal Bone Formation Induced by Deficiency of DNase II in Mice(Wiley, 2017-02) Baum, Rebecca; Sharma, Shruti; Organ, Jason M.; Jakobs, Christopher; Hornung, Veit; Burr, David B.; Marshak-Rothstein, Ann; Fitzgerald, Katherine A.; Gravallese, Ellen M.; Anatomy and Cell Biology, School of MedicineOBJECTIVE: Cytosolic DNA sensors detect microbial DNA and promote type I interferon (IFN) and proinflammatory cytokine production through the adaptor stimulator of IFN genes (STING) to resolve infection. Endogenous DNA also engages the STING pathway, contributing to autoimmune disease. This study sought to identify the role of STING in regulating bone formation and to define the bone phenotype and its pathophysiologic mechanisms in arthritic mice double deficient in DNase II and IFN-α/β/ω receptor (IFNAR) (DNase II-/- /IFNAR-/- double-knockout [DKO] mice) compared with controls. METHODS: Bone parameters were evaluated by micro-computed tomography and histomorphometry in DKO mice in comparison with mice triple deficient in STING, DNase II, and IFNAR and control mice. Cell culture techniques were employed to determine the parameters of osteoclast and osteoblast differentiation and function. NanoString and Affymetrix array analyses were performed to identify factors promoting ectopic bone formation. RESULTS: Despite the expression of proinflammatory cytokines that would be expected to induce bone loss in the skeleton of DKO mice, the results, paradoxically, demonstrated an accumulation of bone in the long bones and spleens, sites of erythropoiesis and robust DNA accrual. In addition, factors promoting osteoblast recruitment and function were induced. Deficiency of STING significantly inhibited bone accrual. CONCLUSION: These data reveal a novel role for cytosolic DNA sensor pathways in bone in the setting of autoimmune disease. The results demonstrate the requirement of an intact STING pathway for bone formation in this model, a finding that may have relevance to autoimmune diseases in which DNA plays a pathogenic role. Identification of pathways linking innate immunity and bone could reveal novel targets for the treatment of bone abnormalities in human autoimmune diseases.