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Targeted disruption of androgen receptor in mouse osteocytes: The androgen receptor in osteocytes is important for the maintenance of bone structure in males
S60
Abstracts
OPB04 (Recipient of a 2012 ECTS New Investigator Award) Targeted disruption of androgen receptor in mouse osteocytes: The androgen receptor in osteocytes is important for the maintenance of bone structure in males M. Sinnesael⁎, F. Claessens, S. Boonen, D. Vanderschueren Experimental Medicine and endocrinology, K.U.Leuven, Leuven, Belgium Androgens play a key role in the maintenance of male skeletal integrity. However, the role of the androgen receptor as well as the target cell for such androgen signaling in bone remains unclear. The aim of the present study was therefore to investigate the role of classical androgen receptor (AR) signaling specifically in osteocytes using the Cre/loxP system in male mice (driven by dentin matrix protein 1 (ocARKOs)). First, the osteocyte fractions of wild type (WT) and ocARKOs were isolated through sequential collagenase digestion of femora and tibiae. These fractions were characterized by their expression of sclerostin and osteocalcin. Interestingly, relative androgen receptor expression as measured by RT-PCR was the highest in the osteocyte fraction of WT males compared to the more immature fractions. Targeted disruption of AR reduced AR receptor expression in this osteocyte fraction by more than 70%, hereby confirming the knockout. The skeletal phenotype of mutant mice was further assessed by histomorphometry and quantitative μCT at 12 and 32 weeks of age, respectively. The ocARKOs had significantly higher trabecular separation in the distal femur compared to WT, both at 12 and at 32 weeks. At 32 weeks ocARKOs also had significantly lower trabecular bone volume and trabecular number, both in tibia and femur. Also bone turnover as measured by serum osteocalcin was increased in ocARKOs compared to WTs. Three-point bending of the femoral diaphysis revealed that, compared to WT bones, ocARKO bones were stiffer and required a lower ultimate force for failure. However, femoral cortical geometric parameters as well as volumetric density were not significantly different at any time point. In conclusion, in osteocytes of male mice, specific inactivation of the AR increased bone turnover and decreased structural integrity of bone, leading to a reduction of trabecular bone volume at 32 weeks of age. These findings provide further evidence for a direct role of androgens in the maintenance of trabecular bone volume through DNA binding-dependent actions of the AR in osteocytes. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared. doi:10.1016/j.bone.2012.02.164
OPB05 Kuskokwim Disease extends recessive osteogenesis imperfecta type XI phenotypes caused by mutations in FKBP10 A.M. Barnesa,⁎, S.J. Baleb, G. Duncanc, W. Patond, W.A. Cabrala, J.C. Marinia a Bone and Extracellular Matrix Branch, NICHD/NIH, Bethesda, USA b Gene Dx, Gaithersburg, USA c Christchurch Hospital, Christchurch, New Zealand d Alaska Native Medical Center, Anchorage, USA Abstract: Rare recessive forms of osteogenesis imperfecta (type XI OI, MIM #610968) have been shown to be caused by mutations in FKBP10, located on chromosome 17q21.2. FKBP10 encodes a collagen-binding protein with PPIase and chaperone functions. The 12 FKBP10 mutations reported comprised 9 null and one missense alleles and cause severe progressive deforming OI. Subsequently, Bruck syndrome type I (BRKS1), also known as OI with congenital contractures, was shown to be a subtype of FKBP10 mutations, with contractures as a variable manifestation in patients with the same FKBP10 mutation or even in a sibship. Kuskokwim Disease, first reported in 1969, is a rare recessive disorder found only in Yup'ik Eskimos of the Yukon–Kuskokwim river delta, characterized by multiple congenital and progressive contractures of large joints, and skeletal anomalies affecting patellae, spine and feet. Bale et al. (Abst #1580, ASHG 1999) reported a mild bone dysplasia (lumbar vertebral BMD z = − 0.47 to −2.29; femoral sites with BMD in osteopenic range) and occasional fractures in Kuskokwim patients. Using 12 Yup'ik families, the gene defect was localized to ~ 26 cM on 17q12–21 by homozygosity mapping, and excluded mutations in COL1A1 at 17q22. The chromosomal location of this congenital contracture disorder, together with its mild bone dysplasia, led us to test for FKBP10 mutations in these patients. Sequencing of FKBP10 revealed a homozygous in-frame 3 nt deletion in exon 5 (c.875_877delACT), which causes the deletion of p.Tyr293. This mutation occurs in the 3rd PPIase domain of FKBP65 and is highly conserved among species. The same mutation was found in patients from 3 separate Yup'ik pedigrees, and in 3% of Yu'pik control alleles. This result will allow molecular screening for carrier status in this population isolate. Real-time PCR demonstrated that the mutant transcripts are not degraded, with FKBP10 expression ranging from 0.75 to 1.67 of control in the dermal fibroblasts of five patients. FKBP65 protein was present in patient fibroblasts at about ~ 5–10% control levels, suggesting that the protein is substantially destabilized by the single residue deletion. Steady-state type I collagen has near-normal electrophoretic migration. In conclusion, this report extends the phenotypes caused by mutations in
FKBP10, ranging from the previously reported severe OI and BRKS1, to Kuskokwim syndrome, a rare Yu'pik disorder with predominantly congenital contractures and mild bone dysplasia. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared. doi:10.1016/j.bone.2012.02.165
OPB06 Cranial and craniovertebral junction anomalies in FGFR3Y367C/+ mice: New insights in normal and pathological skull growth F. Di Rocco⁎, N. Kaci, E. Mugniery, C. Benoist-Lasselin, A. Munnich, L. Legeai-Mallet INSERM U781, Paris, France Abstract: FGFR3 is an important regulator of bone formation. Several pathologies have been described involving this gene in which both skull and craniovertebral junction growth are affected (achondroplasia, Muenke syndrome, Crouzon syndrome with acanthosis nigricans). We have used three fgfr3Y367C/+ mouse models expressing the Y367C mutation ubiquitously (CMV-fgfr3Y367C/+), only in the osteoblasts (Col I-fgfr3Y367C/+) or only in the chondrocytes (Col II-fgfr3Y367C/+) to understand the impact of FGFR3 mutations on the development of the skull and determine the role played by each of these cell types. The three mouse models have been studied at 3 weeks of age using macroscopic histology, radiology and microCT analysis. The CMV-fgfr3Y367C/+ mice display severe morphological changes affecting the long bones but also the cranial vault and skull base. The skull showed a brachycephalic aspect (length severely reduced at 66%, p b 0.001, and high slightly increased, 103%, p b 0.05) with an altered ossification of the vault. The posterior fossa was hypoplastic with a small foramen magnum. In all mice, a prognathism was found with an absence of basal synchondrosis. The size of nasal, frontal, interparietal and occipital bones was significantly altered (55%, 82%, 125%, and 46%, respectively). Col II-fgfr3Y367C/+ mouse model shows also some severe morphological alterations with a brachycephalic shape. (Length was reduced at 75%, p b 0.0001 and high increased 103%, p b 0.05), with a modification of anterior and posterior angles (76%, p b 0.001 and 107% p b 0.05, respectively) and a reduction of the size of the occipital foramen and atlas vertebra. Skull base anomalies were also observed with a complete absence of the synchodrosis and a prognatism. However, no defect in calvaria bones was found. Conversely, Col I-fgfr3Y367C/+ model results in mild morphological changes. No anomalies of the calvarial sutures and no prognathism were observed. An increase in length of the skull (106%, p b 0.01) and alteration in the anterior and occipital foramen angles was found (97%, p b 0.05 and110%, p b 0.01, respectively). The comparative analysis of our three mouse models confirms the importance of endochondral ossification in the growth of the skull and craniovertebral junction. The differences in phenotypes observed between the three mouse models show that FGFR3 plays a role also in membranous ossification. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared. doi:10.1016/j.bone.2012.02.166
OPB07 (Recipient of a 2012 ECTS New Investigator Award) BMP-1 enhances bone fracture repair I. Erjaveca,⁎, L. Grgurevića, I. Dumić-Čulea, J. Brkljačića, D. Đurđevićb, S. Vukičevića a Anatomy, School of Medicine, University of Zagreb, Croatia b Trauma Clinic, Clinical Hospital “Sisters of Mercy”, Zagreb, Croatia Abstract: Bone is the only organ which can be fully regenerated following injury. However, the fracture repair is often limited due to reduced bone regenerative potential. Normal extracellular matrix (ECM) deposition, needed for creating a scaffold for bone mineralization during fracture repair, requires BMP1 a key metalloproteinase involved in ECM formation. In this study we evaluated the role of BMP1 in vitro and on bone repair in a rat fracture model. An osteotomy of the femur was made in male Sprague–Dawley rats. The fragments were fixed by an intramedullary Kirschner pin. The rats were assigned to four groups: control group, group receiving 3 μg of BMP1 3 times a week, group receiving 50 μg BMP-1-3 antibody once a week and the final group receiving 50 μg BMP-1-1 antibody once a week. The effect of treatment was monitored radiographically and femurs were scanned ex vivo by μCT. Bone biomechanical testing was done using a material testing system. In parallel, bone marrow cells harvested from femurs of 8-weekold WT mice were pooled and plated together with mouse osteoblast-like MC3T3-E1 cell line. Cultures were treated with BMP1 (150 ng/ml) or BMP1-3 antibody (1 μg/ml). The von Kossa stain was used to determine the mineralized matrix formation. After treatment total RNA was extracted from MC3T3-E1 cell culture using TRIzol. Gene
Abstracts
OPB04 (Recipient of a 2012 ECTS New Investigator Award) Targeted disruption of androgen receptor in mouse osteocytes: The androgen receptor in osteocytes is important for the maintenance of bone structure in males M. Sinnesael⁎, F. Claessens, S. Boonen, D. Vanderschueren Experimental Medicine and endocrinology, K.U.Leuven, Leuven, Belgium Androgens play a key role in the maintenance of male skeletal integrity. However, the role of the androgen receptor as well as the target cell for such androgen signaling in bone remains unclear. The aim of the present study was therefore to investigate the role of classical androgen receptor (AR) signaling specifically in osteocytes using the Cre/loxP system in male mice (driven by dentin matrix protein 1 (ocARKOs)). First, the osteocyte fractions of wild type (WT) and ocARKOs were isolated through sequential collagenase digestion of femora and tibiae. These fractions were characterized by their expression of sclerostin and osteocalcin. Interestingly, relative androgen receptor expression as measured by RT-PCR was the highest in the osteocyte fraction of WT males compared to the more immature fractions. Targeted disruption of AR reduced AR receptor expression in this osteocyte fraction by more than 70%, hereby confirming the knockout. The skeletal phenotype of mutant mice was further assessed by histomorphometry and quantitative μCT at 12 and 32 weeks of age, respectively. The ocARKOs had significantly higher trabecular separation in the distal femur compared to WT, both at 12 and at 32 weeks. At 32 weeks ocARKOs also had significantly lower trabecular bone volume and trabecular number, both in tibia and femur. Also bone turnover as measured by serum osteocalcin was increased in ocARKOs compared to WTs. Three-point bending of the femoral diaphysis revealed that, compared to WT bones, ocARKO bones were stiffer and required a lower ultimate force for failure. However, femoral cortical geometric parameters as well as volumetric density were not significantly different at any time point. In conclusion, in osteocytes of male mice, specific inactivation of the AR increased bone turnover and decreased structural integrity of bone, leading to a reduction of trabecular bone volume at 32 weeks of age. These findings provide further evidence for a direct role of androgens in the maintenance of trabecular bone volume through DNA binding-dependent actions of the AR in osteocytes. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared. doi:10.1016/j.bone.2012.02.164
OPB05 Kuskokwim Disease extends recessive osteogenesis imperfecta type XI phenotypes caused by mutations in FKBP10 A.M. Barnesa,⁎, S.J. Baleb, G. Duncanc, W. Patond, W.A. Cabrala, J.C. Marinia a Bone and Extracellular Matrix Branch, NICHD/NIH, Bethesda, USA b Gene Dx, Gaithersburg, USA c Christchurch Hospital, Christchurch, New Zealand d Alaska Native Medical Center, Anchorage, USA Abstract: Rare recessive forms of osteogenesis imperfecta (type XI OI, MIM #610968) have been shown to be caused by mutations in FKBP10, located on chromosome 17q21.2. FKBP10 encodes a collagen-binding protein with PPIase and chaperone functions. The 12 FKBP10 mutations reported comprised 9 null and one missense alleles and cause severe progressive deforming OI. Subsequently, Bruck syndrome type I (BRKS1), also known as OI with congenital contractures, was shown to be a subtype of FKBP10 mutations, with contractures as a variable manifestation in patients with the same FKBP10 mutation or even in a sibship. Kuskokwim Disease, first reported in 1969, is a rare recessive disorder found only in Yup'ik Eskimos of the Yukon–Kuskokwim river delta, characterized by multiple congenital and progressive contractures of large joints, and skeletal anomalies affecting patellae, spine and feet. Bale et al. (Abst #1580, ASHG 1999) reported a mild bone dysplasia (lumbar vertebral BMD z = − 0.47 to −2.29; femoral sites with BMD in osteopenic range) and occasional fractures in Kuskokwim patients. Using 12 Yup'ik families, the gene defect was localized to ~ 26 cM on 17q12–21 by homozygosity mapping, and excluded mutations in COL1A1 at 17q22. The chromosomal location of this congenital contracture disorder, together with its mild bone dysplasia, led us to test for FKBP10 mutations in these patients. Sequencing of FKBP10 revealed a homozygous in-frame 3 nt deletion in exon 5 (c.875_877delACT), which causes the deletion of p.Tyr293. This mutation occurs in the 3rd PPIase domain of FKBP65 and is highly conserved among species. The same mutation was found in patients from 3 separate Yup'ik pedigrees, and in 3% of Yu'pik control alleles. This result will allow molecular screening for carrier status in this population isolate. Real-time PCR demonstrated that the mutant transcripts are not degraded, with FKBP10 expression ranging from 0.75 to 1.67 of control in the dermal fibroblasts of five patients. FKBP65 protein was present in patient fibroblasts at about ~ 5–10% control levels, suggesting that the protein is substantially destabilized by the single residue deletion. Steady-state type I collagen has near-normal electrophoretic migration. In conclusion, this report extends the phenotypes caused by mutations in
FKBP10, ranging from the previously reported severe OI and BRKS1, to Kuskokwim syndrome, a rare Yu'pik disorder with predominantly congenital contractures and mild bone dysplasia. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared. doi:10.1016/j.bone.2012.02.165
OPB06 Cranial and craniovertebral junction anomalies in FGFR3Y367C/+ mice: New insights in normal and pathological skull growth F. Di Rocco⁎, N. Kaci, E. Mugniery, C. Benoist-Lasselin, A. Munnich, L. Legeai-Mallet INSERM U781, Paris, France Abstract: FGFR3 is an important regulator of bone formation. Several pathologies have been described involving this gene in which both skull and craniovertebral junction growth are affected (achondroplasia, Muenke syndrome, Crouzon syndrome with acanthosis nigricans). We have used three fgfr3Y367C/+ mouse models expressing the Y367C mutation ubiquitously (CMV-fgfr3Y367C/+), only in the osteoblasts (Col I-fgfr3Y367C/+) or only in the chondrocytes (Col II-fgfr3Y367C/+) to understand the impact of FGFR3 mutations on the development of the skull and determine the role played by each of these cell types. The three mouse models have been studied at 3 weeks of age using macroscopic histology, radiology and microCT analysis. The CMV-fgfr3Y367C/+ mice display severe morphological changes affecting the long bones but also the cranial vault and skull base. The skull showed a brachycephalic aspect (length severely reduced at 66%, p b 0.001, and high slightly increased, 103%, p b 0.05) with an altered ossification of the vault. The posterior fossa was hypoplastic with a small foramen magnum. In all mice, a prognathism was found with an absence of basal synchondrosis. The size of nasal, frontal, interparietal and occipital bones was significantly altered (55%, 82%, 125%, and 46%, respectively). Col II-fgfr3Y367C/+ mouse model shows also some severe morphological alterations with a brachycephalic shape. (Length was reduced at 75%, p b 0.0001 and high increased 103%, p b 0.05), with a modification of anterior and posterior angles (76%, p b 0.001 and 107% p b 0.05, respectively) and a reduction of the size of the occipital foramen and atlas vertebra. Skull base anomalies were also observed with a complete absence of the synchodrosis and a prognatism. However, no defect in calvaria bones was found. Conversely, Col I-fgfr3Y367C/+ model results in mild morphological changes. No anomalies of the calvarial sutures and no prognathism were observed. An increase in length of the skull (106%, p b 0.01) and alteration in the anterior and occipital foramen angles was found (97%, p b 0.05 and110%, p b 0.01, respectively). The comparative analysis of our three mouse models confirms the importance of endochondral ossification in the growth of the skull and craniovertebral junction. The differences in phenotypes observed between the three mouse models show that FGFR3 plays a role also in membranous ossification. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared. doi:10.1016/j.bone.2012.02.166
OPB07 (Recipient of a 2012 ECTS New Investigator Award) BMP-1 enhances bone fracture repair I. Erjaveca,⁎, L. Grgurevića, I. Dumić-Čulea, J. Brkljačića, D. Đurđevićb, S. Vukičevića a Anatomy, School of Medicine, University of Zagreb, Croatia b Trauma Clinic, Clinical Hospital “Sisters of Mercy”, Zagreb, Croatia Abstract: Bone is the only organ which can be fully regenerated following injury. However, the fracture repair is often limited due to reduced bone regenerative potential. Normal extracellular matrix (ECM) deposition, needed for creating a scaffold for bone mineralization during fracture repair, requires BMP1 a key metalloproteinase involved in ECM formation. In this study we evaluated the role of BMP1 in vitro and on bone repair in a rat fracture model. An osteotomy of the femur was made in male Sprague–Dawley rats. The fragments were fixed by an intramedullary Kirschner pin. The rats were assigned to four groups: control group, group receiving 3 μg of BMP1 3 times a week, group receiving 50 μg BMP-1-3 antibody once a week and the final group receiving 50 μg BMP-1-1 antibody once a week. The effect of treatment was monitored radiographically and femurs were scanned ex vivo by μCT. Bone biomechanical testing was done using a material testing system. In parallel, bone marrow cells harvested from femurs of 8-weekold WT mice were pooled and plated together with mouse osteoblast-like MC3T3-E1 cell line. Cultures were treated with BMP1 (150 ng/ml) or BMP1-3 antibody (1 μg/ml). The von Kossa stain was used to determine the mineralized matrix formation. After treatment total RNA was extracted from MC3T3-E1 cell culture using TRIzol. Gene