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Estrogen receptor alpha expression in cartilage is important for the ameliorating effects of estrogen on synovitis, but not joint destruction
S64
Abstracts
expressing (hSOST+/tg) and RPTPμ-/-*hSOST+/tg mice was cultured under osteogenic conditions. Differentiation, mineralization and osteocyte formation were determined by ALP activity in culture medium, alizarin Red staining and expression of osteocalcin, DMP1 and sclerostin. In addition, in mice subjected to 3 weeks of 3 G gravitation by centrifugation, sclerostin expression was determined by immunohistochemical staining of sections of the ulna and radius and compared to stationary controls. Our results show that RPTPμ−/−-derived cultures have increased differentiation, mineralization and osteocyte formation, while hSOST+/tg cultures show opposite effects. Interestingly, over-expressing human sclerostin in RPTPμ−/− mice completely overruled the effects of RPTPμ deficiency, since RPTPμ−/−*hSOST+/tg cultures showed a similar degree of differentiation as hSOST+/tg cultures. Centrifugation increased the expression of sclerostin in the ulna/radius. This may be secondary to increased osteogenic differentiation and osteocyte formation that might have occurred in this period, as opposed to earlier studies reporting opposite effects after short-term mechanical stimulation (1). Our data indicate that RPTPmu and sclerostin are both involved in the regulation of bone formation and suggest that they act in the same pathway. Additional research is currently being conducted to support these findings in addition to determining the effect of mechanical loading on the expression of RPTPμ, to confirm its proposed role in bone mechanosensing. (1) Robling, A.G., et al., J Biol Chem, 2008. 283(9): p. 5866-75. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared. doi:10.1016/j.bone.2012.02.176
OPB17 Lack of the chondrocyte IGF-I receptor leads to perinatal death and alterations in extracellular matrix protein expression in vitro J. Heilig⁎, M. Paulsson, F. Zaucke Institute for Biochemistry II, University of Cologne, Medical Faculty, Cologne, Germany Abstract: Introduction: Chondrocytes express both IGF-I as well as its receptor IGFIR. As the deficiency of both IGF-I and the IGF-IR resulted in a high degree of perinatal lethality in the respective mouse models, the postnatal role of IGF-I in skeletal development could not be investigated so far. Therefore, we aimed at generating a cartilage-specific deletion of the IGF-IR in mice. In addition an in vitro model should be established where the effects of IGF-I can be investigated by deleting the IGF-IR in primary mouse chondrocytes by treatment with recombinant Cre recombinase. Methods: To assess the function of the IGF-IR in skeletal development we crossed mice carrying the floxed IGF-IR gene with mice expressing the Cre recombinase under the control of the murine collagen II promoter. The deletion of the IGF-IR in primary mouse chondrocytes is achieved by treating the cells with a recombinantly expressed Cre recombinase analog (HTNC). Results: Despite of a deletion of the IGF-IR exclusively in cartilage all homozygous IGF-IR deficient mice died perinatally. Skeletal analysis with alcian blue/alizarin red staining revealed a reduction in limb and whole body length as well as a reduction in tracheal diameter. In the cell culture model an efficient deletion of the IGF-IR could be achieved in primary chondrocytes by the use of the recombinant Cre recombinase HTNC. After this treatment with HTNC approximately 50% of the cells died. The surviving cells showed not only a deletion of the IGF-IR, and as a consequence a reduced activity of signaling downstream elements, but also reduced levels of extracellular matrix (ECM) proteins in the pericellular matrix. Discussion: Mice lacking the IGF-IR in cartilage die perinatally as the respective models for the global deficiency of IGF-I or the IGF-IR. The specific reason for this early death remains to be determined. In addition, further studies on the structure of the growth plate of the IGF-IR deficient animals will be performed to investigate the short-stature phenotype. In general, treatment of primary chondrocytes with HTNC is a promising tool for the deletion of floxed genes and represents a good alternative to knock-out approaches. In the future, RNA isolated from cells lacking the IGF-IR will be investigated in a microarray approach to identify novel target genes of the IGF-I signaling in chondrocytes. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared. doi:10.1016/j.bone.2012.02.177
OPB18 Estrogen receptor alpha expression in cartilage is important for the ameliorating effects of estrogen on synovitis, but not joint destruction M.K. Lagerquista,⁎, C. Engdahla, A.E. Börjessona, A. Anderssona, A. Stubeliusa, A. Krustb, P. Chambonb, U. Islanderc, C. Ohlssona, H. Carlstena
a
Centre for Bone and Arthritis Research, MEDICINE, Gothenburg, Sweden IGBMC, Illkirch, France c Centre for Bone and Arthritis Research, MEDICINE, Illkirch, Sweden b
Abstract: Objective: Estrogen ameliorates incidence and progression of rheumatoid arthritis. The aim of this study was to investigate the importance of cartilagespecific estrogen receptor alpha (ERα) expression for the ameliorating effects of estrogen on arthritis. Methods: Mice with total (total ERα−/−) or cartilage-specific (Col2α1-ERα−/−) inactivation of ERα and wild-type mice were ovariectomized, treated with estradiol (0.83 μg/day) or placebo and induced with antigen-inducedarthritis (AIA). At termination, knees were collected for histology, synovial cells and splenic T cells were investigated using flow cytometry and T cell proliferation and cytokine production was examined. Results: Estrogen protected against AIA-induced synovitis and joint destruction in wild-type mice and this was associated with decreased frequencies of inflammatory cells (neutrophils, monocytes/macrophages and T cells) in synovial tissue and decreased systemic T cell proliferation. No estrogenic effect was seen in total ERa deficient mice. In cartilage-specific ERa inactivated mice, estrogen protected against joint destruction and decreased systemic T cell proliferation to a similar extent as in wild-type mice. In contrast, estrogen did not ameliorate synovitis in mice lacking ERα in cartilage. Conclusions: Estrogen ameliorates both synovitis and joint destruction in AIA via ERα and this decreased severity in arthritis is associated with a decrease in synovial inflammatory cells and decreased T cell proliferation. Interestingly, ERa expression in cartilage is required for the ameliorating estrogenic effects on synovitis but not joint destruction, suggesting different target cells and mechanisms for the estrogenic protection of synovial inflammation and joint destruction. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared.
doi:10.1016/j.bone.2012.02.178
OPB19 Mutations in Sequestosome-1 result in inclusion body formation E. Azzam, M.H. Helfrich, L.J. Hocking⁎ University of Aberdeen, Aberdeen, UK Abstract: Paget's disease of bone (PDB) is characterised by focal lesions of increased bone turnover, driven by overactive osteoclasts. A pathognomonic feature of PDB is the presence of inclusion bodies in the nucleus and cytoplasm of osteoclasts within the affected areas of the bone. Similar inclusions are seen in related high bone turnover disorders such as Familial Expansile Osteolysis and the syndrome that combines Inclusion Body Myositis, PDB and Frontotemporal Dementia. Mutations in Sequestosome-1 (SQSTM1) have been identified as a cause of PDB, with one transgenic mouse model displaying intranuclear inclusions within osteoclasts; and we have shown that autophagic protein degradation is impaired by SQSTM1 mutations. The aim of this study was to determine whether inclusion bodies arise due to mutations in SQSTM1 in a model cell system and whether these are exacerbated by pharmacological inhibition of autophagy. HEK293 cell lines stably expressing exogenous wild-type (WT) and mutated SQSTM1 (P392L, E396X and G425R) were generated using the Flp-In System (Invitrogen). These cells also express endogenous SQSTM1, approximating the heterozygous mutations found in most patients. Cells were examined by transmission electron microscopy, both under normal growth conditions and following bafilomycin treatment to inhibit autophagic protein degradation. One hundred cells were examined and classified according to whether or not they contained paracrystalline inclusions. Under normal conditions, paracrystalline inclusions (cytoplasmic and nuclear) were identified in 2% WT cells, 4% P392L cells and 10% G425R cells. There was no dramatic increase in the number of cells containing inclusions upon pharmacological inhibition of autophagy (2% WT, 8% P392L; 8% G425R). No inclusions were identified in the parent cell line or in E396X cells, with or without bafilomycin treatment. Some smaller inclusions appeared inside autophagosomes, indicating that these can be degraded by autophagy. We have generated a simple cell-based system that recapitulates a characteristic feature of Pagetic osteoclasts, demonstrating that SQSTM1 mutations alone promote inclusion body formation. This model will be useful to further examine the mechanisms that cause PDB, and is easy to manipulate for the development and testing of novel treatments. Furthermore, the lack of inclusions in the truncated mutant E396X reveals the importance of the C-terminal UBA domain of SQSTM1 in the formation of inclusion bodies. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared.
doi:10.1016/j.bone.2012.02.179
Abstracts
expressing (hSOST+/tg) and RPTPμ-/-*hSOST+/tg mice was cultured under osteogenic conditions. Differentiation, mineralization and osteocyte formation were determined by ALP activity in culture medium, alizarin Red staining and expression of osteocalcin, DMP1 and sclerostin. In addition, in mice subjected to 3 weeks of 3 G gravitation by centrifugation, sclerostin expression was determined by immunohistochemical staining of sections of the ulna and radius and compared to stationary controls. Our results show that RPTPμ−/−-derived cultures have increased differentiation, mineralization and osteocyte formation, while hSOST+/tg cultures show opposite effects. Interestingly, over-expressing human sclerostin in RPTPμ−/− mice completely overruled the effects of RPTPμ deficiency, since RPTPμ−/−*hSOST+/tg cultures showed a similar degree of differentiation as hSOST+/tg cultures. Centrifugation increased the expression of sclerostin in the ulna/radius. This may be secondary to increased osteogenic differentiation and osteocyte formation that might have occurred in this period, as opposed to earlier studies reporting opposite effects after short-term mechanical stimulation (1). Our data indicate that RPTPmu and sclerostin are both involved in the regulation of bone formation and suggest that they act in the same pathway. Additional research is currently being conducted to support these findings in addition to determining the effect of mechanical loading on the expression of RPTPμ, to confirm its proposed role in bone mechanosensing. (1) Robling, A.G., et al., J Biol Chem, 2008. 283(9): p. 5866-75. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared. doi:10.1016/j.bone.2012.02.176
OPB17 Lack of the chondrocyte IGF-I receptor leads to perinatal death and alterations in extracellular matrix protein expression in vitro J. Heilig⁎, M. Paulsson, F. Zaucke Institute for Biochemistry II, University of Cologne, Medical Faculty, Cologne, Germany Abstract: Introduction: Chondrocytes express both IGF-I as well as its receptor IGFIR. As the deficiency of both IGF-I and the IGF-IR resulted in a high degree of perinatal lethality in the respective mouse models, the postnatal role of IGF-I in skeletal development could not be investigated so far. Therefore, we aimed at generating a cartilage-specific deletion of the IGF-IR in mice. In addition an in vitro model should be established where the effects of IGF-I can be investigated by deleting the IGF-IR in primary mouse chondrocytes by treatment with recombinant Cre recombinase. Methods: To assess the function of the IGF-IR in skeletal development we crossed mice carrying the floxed IGF-IR gene with mice expressing the Cre recombinase under the control of the murine collagen II promoter. The deletion of the IGF-IR in primary mouse chondrocytes is achieved by treating the cells with a recombinantly expressed Cre recombinase analog (HTNC). Results: Despite of a deletion of the IGF-IR exclusively in cartilage all homozygous IGF-IR deficient mice died perinatally. Skeletal analysis with alcian blue/alizarin red staining revealed a reduction in limb and whole body length as well as a reduction in tracheal diameter. In the cell culture model an efficient deletion of the IGF-IR could be achieved in primary chondrocytes by the use of the recombinant Cre recombinase HTNC. After this treatment with HTNC approximately 50% of the cells died. The surviving cells showed not only a deletion of the IGF-IR, and as a consequence a reduced activity of signaling downstream elements, but also reduced levels of extracellular matrix (ECM) proteins in the pericellular matrix. Discussion: Mice lacking the IGF-IR in cartilage die perinatally as the respective models for the global deficiency of IGF-I or the IGF-IR. The specific reason for this early death remains to be determined. In addition, further studies on the structure of the growth plate of the IGF-IR deficient animals will be performed to investigate the short-stature phenotype. In general, treatment of primary chondrocytes with HTNC is a promising tool for the deletion of floxed genes and represents a good alternative to knock-out approaches. In the future, RNA isolated from cells lacking the IGF-IR will be investigated in a microarray approach to identify novel target genes of the IGF-I signaling in chondrocytes. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared. doi:10.1016/j.bone.2012.02.177
OPB18 Estrogen receptor alpha expression in cartilage is important for the ameliorating effects of estrogen on synovitis, but not joint destruction M.K. Lagerquista,⁎, C. Engdahla, A.E. Börjessona, A. Anderssona, A. Stubeliusa, A. Krustb, P. Chambonb, U. Islanderc, C. Ohlssona, H. Carlstena
a
Centre for Bone and Arthritis Research, MEDICINE, Gothenburg, Sweden IGBMC, Illkirch, France c Centre for Bone and Arthritis Research, MEDICINE, Illkirch, Sweden b
Abstract: Objective: Estrogen ameliorates incidence and progression of rheumatoid arthritis. The aim of this study was to investigate the importance of cartilagespecific estrogen receptor alpha (ERα) expression for the ameliorating effects of estrogen on arthritis. Methods: Mice with total (total ERα−/−) or cartilage-specific (Col2α1-ERα−/−) inactivation of ERα and wild-type mice were ovariectomized, treated with estradiol (0.83 μg/day) or placebo and induced with antigen-inducedarthritis (AIA). At termination, knees were collected for histology, synovial cells and splenic T cells were investigated using flow cytometry and T cell proliferation and cytokine production was examined. Results: Estrogen protected against AIA-induced synovitis and joint destruction in wild-type mice and this was associated with decreased frequencies of inflammatory cells (neutrophils, monocytes/macrophages and T cells) in synovial tissue and decreased systemic T cell proliferation. No estrogenic effect was seen in total ERa deficient mice. In cartilage-specific ERa inactivated mice, estrogen protected against joint destruction and decreased systemic T cell proliferation to a similar extent as in wild-type mice. In contrast, estrogen did not ameliorate synovitis in mice lacking ERα in cartilage. Conclusions: Estrogen ameliorates both synovitis and joint destruction in AIA via ERα and this decreased severity in arthritis is associated with a decrease in synovial inflammatory cells and decreased T cell proliferation. Interestingly, ERa expression in cartilage is required for the ameliorating estrogenic effects on synovitis but not joint destruction, suggesting different target cells and mechanisms for the estrogenic protection of synovial inflammation and joint destruction. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared.
doi:10.1016/j.bone.2012.02.178
OPB19 Mutations in Sequestosome-1 result in inclusion body formation E. Azzam, M.H. Helfrich, L.J. Hocking⁎ University of Aberdeen, Aberdeen, UK Abstract: Paget's disease of bone (PDB) is characterised by focal lesions of increased bone turnover, driven by overactive osteoclasts. A pathognomonic feature of PDB is the presence of inclusion bodies in the nucleus and cytoplasm of osteoclasts within the affected areas of the bone. Similar inclusions are seen in related high bone turnover disorders such as Familial Expansile Osteolysis and the syndrome that combines Inclusion Body Myositis, PDB and Frontotemporal Dementia. Mutations in Sequestosome-1 (SQSTM1) have been identified as a cause of PDB, with one transgenic mouse model displaying intranuclear inclusions within osteoclasts; and we have shown that autophagic protein degradation is impaired by SQSTM1 mutations. The aim of this study was to determine whether inclusion bodies arise due to mutations in SQSTM1 in a model cell system and whether these are exacerbated by pharmacological inhibition of autophagy. HEK293 cell lines stably expressing exogenous wild-type (WT) and mutated SQSTM1 (P392L, E396X and G425R) were generated using the Flp-In System (Invitrogen). These cells also express endogenous SQSTM1, approximating the heterozygous mutations found in most patients. Cells were examined by transmission electron microscopy, both under normal growth conditions and following bafilomycin treatment to inhibit autophagic protein degradation. One hundred cells were examined and classified according to whether or not they contained paracrystalline inclusions. Under normal conditions, paracrystalline inclusions (cytoplasmic and nuclear) were identified in 2% WT cells, 4% P392L cells and 10% G425R cells. There was no dramatic increase in the number of cells containing inclusions upon pharmacological inhibition of autophagy (2% WT, 8% P392L; 8% G425R). No inclusions were identified in the parent cell line or in E396X cells, with or without bafilomycin treatment. Some smaller inclusions appeared inside autophagosomes, indicating that these can be degraded by autophagy. We have generated a simple cell-based system that recapitulates a characteristic feature of Pagetic osteoclasts, demonstrating that SQSTM1 mutations alone promote inclusion body formation. This model will be useful to further examine the mechanisms that cause PDB, and is easy to manipulate for the development and testing of novel treatments. Furthermore, the lack of inclusions in the truncated mutant E396X reveals the importance of the C-terminal UBA domain of SQSTM1 in the formation of inclusion bodies. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared.
doi:10.1016/j.bone.2012.02.179