Osteoblastic ephrinB2 is required for normal osteoblast and osteoclast differentiation

Bone 48 (2011) S65–S66

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Bone j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b o n e

Orals 1: Bone cells OC05 The CREB transcription factor regulates bone mass in humans and mice, activates BMP2 and contributes to PTH action in osteoblasts J.R. Edwards a,⁎, H. Liu b, R. Zhang b, S. Dong b, S.-Y. Ko b, H.-W. Deng b, M. Zhao b a NDORMS, University of Oxford, Oxford, UK b University of Missouri at Kansas City, Kansas City, USA Abstract: A strong transcriptional element mediates skeletal modeling and remodeling. The cAMP-response element binding protein (CREB) acts downstream of many pathways, such as calcium signaling in osteoblasts. However, the role of CREB in human skeletal health or CREB targets in bone remains unclear, despite strong data indicating a role mediating the anabolic effect of PTH. To investigate the physiological role of CREB in bone biology, we performed human genome-wide association studies complemented with functional studies in CREB-deficient animals and molecular and pharmacological manipulation of the CREB signaling axis. Genetic analysis of US Caucasian subjects identified polymorphisms at multiple positions within the CREB gene which were significantly associated with variations in vertebral (p < 0.01) and radial BMD (p < 0.05). Osteoblast-specific CREB knockout mice were created using the Col1a1(3.6)-Cre promoter. μCT and histological characterization demonstrated a significant reduction in trabecular bone volume of CREB-deficient mice (8 weeks), along with decreased trabecular number and thickness compared to controls. These results indicate that CREB plays a role in normal skeletal development and homeostasis as we age, however the molecular mechanism by which CREB functions in bone remained unknown. Following isolation and culture of primary osteoblasts from WT and CREB-deficient mice, we identified that BMP2 expression was substantially decreased. Furthermore, overexpression of CREB up-regulated BMP2 in MC3T3 cells. DNA sequence analysis revealed multiple cAMP response elements (CREs) in the BMP2 promoter and promoter deletion, EMSA and mutation studies confirmed CREB binding and BMP2 activation. Moreover, CREB transfection into various osteoblast cell lines increased alkaline phosphatase activity and could be blocked by the BMP antagonist noggin. Also, treatment of osteoblasts with cAMP/PKA activator IBMX or inhibitor KT5720 significantly altered CREB transactivation and BMP2 expression. In addition, BMP2 promoter activity was significantly enhanced following PTH treatment. Together, these complementary studies suggest that CREB plays an important role in controlling the development and maintenance of the skeleton and may mediate the anabolic effect of PTH by regulating BMP2 production and consequent osteoblast differentiation. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: None declared. doi:10.1016/j.bone.2011.03.064

OC06 (recipient of a 2011 ECTS/IBMS New Investigator Award) Osteoblastic ephrinB2 is required for normal osteoblast and osteoclast differentiation F.M. Takyar a,b,⁎, B. Crimeen-Irwin a, T.J. Martin a,b, N.A. Sims a,b a Bone Cell Biology and Disease Unit, St. Vincent's Institute of Medical Research, Australia b Department of Medicine at St. Vincent's Hospital, The University of Melbourne, Melbourne, Australia Abstract: Bidirectional signaling between EphB4 and ephrinB2 in osteoblasts leads to increased osteoblast differentiation in vitro, and interference with this interaction inhibits osteoblast activity. Furthermore, injection of PTH rapidly enhances ephrinB2 expression in mouse and rat bone and isolated osteoblasts (EH Allan et al., JBMR 2008). To determine the role of osteoblastic ephrinB2 in bone metabolism, we crossed ephrinB2-flox and osteoblast-specific tetracycline-regulatable Osx1Cre mice to delete ephrinB2 from an early stage of osteoblast differentiation in vivo. Two groups of mice were studied: one where ephrinB2 was deleted constitutively, and one where mothers and pups were treated with tetracycline until 4 weeks of age, to allow ephrinB2 expression during

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skeletal development. Tibiae from adult (12 week old) Osx1Cre.efnB2LoxP mice and controls (Osx1Cre.efnB2wt) were analyzed by histomorphometry. Results were the same regardless of whether EphrinB2 was deleted throughout life or only after skeletal development. No change in bone structure was observed in Osx1Cre.efnB2LoxP mice compared with Osx1Cre.efnB2wt controls. Nonetheless, osteoblast number and osteoid surface/bone surface were both significantly increased (by 50% and 63%, respectively). Despite this, osteoid thickness was significantly decreased (by 36%) in Osx1Cre.efnB2LoxP mice compared to controls, indicating that even in the presence of many osteoblasts, ephrinB2-deficient osteoblasts have reduced ability to form new bone. Despite increased osteoblast formation, osteoclast number was significantly decreased (by 65%) in Osx1Cre.efnB2LoxP mice compared to controls. This finding of impaired osteoclast formation in mice null for ephrinB2 in osteoblasts is being investigated in ex vivo experiments. As an alternative to genetic deletion of ephrinB2 in osteoblasts, we treated 8-week-old male C57Bl/6 mice for 4 weeks with soluble extracellular domain of EphB4 (sEphB4), a specific antagonist of ephrinB2–EphB4 binding. Consistent with the phenotype of Osx1Cre. efnB2LoxP mice, sEphB4 treatment caused a significant decrease in trabecular number compared to vehicle treated animals (P = 0.03). These results strongly suggest that ephrinB2 signaling through the EphB4 receptor within the osteoblast lineage is required for normal differentiation of both osteoblasts and osteoclasts in bone remodeling. These in vivo experiments do not yet resolve the question whether this is achieved through forward or reverse signaling. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: None declared. doi:10.1016/j.bone.2011.03.065

OC07 The p38α MAPK is required for regulating osteoblast function and bone formation C. Thouverey ⁎, J. Caverzasio Service of Bone Diseases, Department of Rehabilitation and Geriatrics, University Hospital of Geneva, Geneva, Switzerland Abstract: Various osteogenic ligands that stimulate osteoblast differentiation and function act, in part, through the p38 mitogen-activated kinase (MAPK) pathway. A recent in vivo investigation has highlighted the physiological role of the TAK1-MKK3/6-p38 pathway in osteoblastogenesis and bone formation. Interestingly, the authors have shown that p38ß is critical for late osteoblast differentiation and that loss of p38ß is not compensated by p38α, thus suggesting that p38α and p38ß may have different functions in bone formation. To elucidate the in vivo role of p38α in regulating osteoblast function, we generated mice lacking p38α in mature osteoblasts. Mice expressing Cre recombinase under the control of the osteocalcin promoter (Ocn–Cre) were crossed with mice harboring floxed p38α-encoding gene (p38αf/f). The bone phenotype of control (p38αf/f) and mutant (Ocn–Cre;p38αf/f) mice was assessed by dual energy X-ray absorptiometry, microcomputed tomography and gene expression analyses at 3 months of age (n= 6 per group). Data were analyzed by unpaired t-test. Mutant mice exhibited lower bone mineral density compared to control mice (−7.1%, p= 0.009). Ocn–Cre;p38αf/f mice displayed an important reduction in trabecular bone volume at the distal femoral metaphysis (−25.0%, p =0.03) associated with low trabecular thickness (−13.0%, p= 0.02). A similar pattern of low trabecular bone mass was observed at the fifth lumbar vertebral body. In addition, Ocn–Cre;p38αf/f mice also showed decreased cortical thickness at the femoral midshaft (−16.1%, p= 0.003). Consistent with this low bone mass phenotype, Osx, Col1a1, Alp and Ocn expressions were reduced by 35% to 55% in long bones of mutant mice. Finally, primary p38α knockout osteoblasts demonstrated lower Osx, Col1a1, Alp and Ocn expressions (p≤0.01) and reduced capacity to mineralize in vitro, indicating a defective function of osteoblasts lacking p38α. These findings indicate that p38α is an essential regulator of osteoblast function and bone formation in vivo. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: None declared. doi:10.1016/j.bone.2011.03.066