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Insights into the function of SOX9 and campomelic dysplasia
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Abstracts
whole embryos, we observed similar expression pattern of both transcripts, ubiquitously distributed in basement membrane, prominently in the central nervous system. Immunostaining of whole embryos with antiserum raised against drole-GST fusion protein in mice also showed that Drole protein was accumulated in the central nervous system during embryogenesis. Moreover, loss of function mutants exhibited low surviving ratio during development and mildly abnormal larval locomotion. Thus, drole is widely expressed in basement membrane and may play some important roles in the formation of the central nervous system.
8 weeks of age. Treadmill exercise resulted in decreased whole body and skeletal muscle mass for all muscles and genotypes studied. In addition, after exercise oim/oim mice had altered femoral bone geometry (μCT) and improved biomechanical strength (not yet significant), as seen by increased torsional ultimate strength, tensile strength and energy to failure as compared to non-exercise oim/oim mice. These results suggest that moderate weight-bearing physical activity may improve the amount and strength of bone in patients with OI. doi:10.1016/j.matbio.2008.09.277
doi:10.1016/j.matbio.2008.09.275
61 Abnormal lipid homeostasis in chondrocytes of S1Pcko mice Debabrata Patraa, Xiaoyun Xinga, Jennifer Bryana, Guosheng Liangb, Linda Sandella a Washington University, St. Louis, MO 63110, United States b UT Southwestern Medical Center, Dallas, TX, United States Site-1 protease (S1P) plays an essential role in the regulated processing of endoplasmic reticulum (ER) membrane-bound transcription factors SREBPs and ATF6. SREBPs are involved in cholesterol and fatty acid homeostasis; ATF6 is involved in ER stress signaling (ERSS). Cartilage-specific knockout of S1P in mice (S1Pcko) results in poor cartilage with a drastic decrease of collagen type IIB (Col IIB) in the matrix and consequent lack of endochondral bone formation. Most of the Col IIB appears trapped inside the cell. Ultrastructural analysis of the cartilage shows engorged and fragmented ER characteristic of ER stress. The ER is important for protein synthesis and folding and is the site of fatty acid and cholesterol synthesis. The ER is the essential secretory apparatus of the chondrocyte and therefore maintaining ER lipid composition would be critical for matrix synthesis. In S1Pcko mice, ER stress signaling appears to function normally. However, genome-wide expression profiling followed by real-time PCR analysis demonstrated that fatty acid and cholesterol synthesis is down-regulated in S1Pcko cartilage. This down-regulation may lead to poor ER membrane integrity and therefore ER fragmentation and engorgement presumably due to an inability to secrete Col IIB and therefore ER stress. These data suggest that in embryonic cartilage S1P is involved in maintaining ER membrane integrity by upholding lipid homeostasis and preventing ER stress. This study provides a link to the importance of lipids in cartilage and bone development and may be useful in cartilage diseases and tissue engineering.
63 Insights into the function of SOX9 and campomelic dysplasia Kathryn S. Cheah Department of Biochemistry, University of Hong Kong, Hong Kong Human SOX9 mutations cause the skeletal malformation syndrome campomelic dysplasia (CD). Complete inactivation of the Sox9 gene in mice results in failure of cartilage formation. Studies in zebrafish and Xenopus suggest that Sox9 may be crucial for specification of the otic placode. In mice, loss of Sox9 results in failure of otic placode invagination. Heterozygous mutations in human SOX9 result in conductive and sensorineural deafness in some CD patients, implying a later morphogenetic role but phenotypic details are limited. Sox9−/− null mice die before morphogenesis of the inner ear is complete, precluding investigation of the role of Sox9 later in ear development. Because all the SOX9 mutations are heterozygous and appear to cause loss of function, the CD phenotype has been attributed to haploinsufficency of SOX9. However SOX9 proteins containing an intact HMG box and a truncated activation domain may act dominant negatively by competition with the wild-type for binding to target genes and interfere with interaction with partner factors via the transactivation domain. To assess whether such mutations in SOX9 may act via a dominant interference mechanism we generated transgenic and conditional knock'in mice expressing a mouse equivalent of a CD mutation, a Y440X nonsense mutation causing premature termination within the trans-activation domain of SOX9 (Sox9Y440X). Our data reveal an essential role for Sox9 for sensory and nonsensory morphogenesis of the inner ear. We compared the phenotypic impact of the Sox9 Y440X mutation with a Sox9 null mutation. These studies point to context dependent mechanisms for the Y440X nonsense mutation. doi:10.1016/j.matbio.2008.09.278
doi:10.1016/j.matbio.2008.09.276 64 Expression of ADAMTS-9 in mouse growth plate cartilage 62 Impact of exercise on skeletal muscle and bone in oim mice S. Carletona, B. Weberb, A. McCambridgea, J. Ferrierab, M.B. Brownb, C. Phillipsa a Department of Biochemistry/Child Health, United States b Veterinary Pathobiology, University of Missouri, United States Osteogenesis imperfecta (OI), a heritable connective tissue disorder commonly due to type I procollagen gene defects, is characterized by bone deformity and fragility. The homozygote oim mouse models moderately severe OI type III (oim/oim; homozygous for a functional null COL1A2 gene defect) with reduced bone mineral density and strength. Heterozygous mice (oim/+) model mild OI type I and have an intermediate phenotype between oim/oim and wildtype (Wt) mice. Normally, physical activity increases exercise tolerance, muscle strength and bone biomechanical integrity. We investigated whether treadmill (resistance) exercise will impact skeletal muscle and improve bone biomechanical integrity in Wt, oim/+ and oim/oim mice. We first characterized the hind limb skeletal muscles of 4 month old Wt, oim/+, and oim/oim mice with normal cage activity and determined that there was no evidence of muscle weakness or pathology. Mice exercised on a treadmill 30 min/day, 5 days/week at 10 m/min for 8 weeks starting at
Kanae Kumagishia, Keiichiro Nishidaa, Ryusuke Momotaa, Yuichiro Yamaaib, Satoshi Hirohatac, Kadir Demircanc, Ichiro Naitoa, Yoshifumi Ninomiyac, Aiji Ohtsukaa a Department of Human Morphology, Japan b Department of Oral Function and Anatomy, Japan c Department of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Japan Expression of ADAMTS-9 during the course of chondrocyte differentiation was examined by RT-PCR using ATDC-5 cells. Knee joints from 15 male ICR mice were obtained at 0, 7 and 14 weeks after birth (n = 5 each). They were fixed, decalcified and embedded in paraffin. The expression of ADAMTS-9 in growth plate cartilage of proximal tibia were examined by in situ hybridization (ISH) and immunohistochemistry (IHC). Proliferative and hypertrophic chondrocytes were labeled in adjacent sections by IHC for PCNA and type X collagen, respectively.ADAMTS-9 expression was noted at day 8 and 14 of ATDC5 cell culture, temporally decreased at day 21, and then increased up-to day 35. In the mouse growth plate cartilage, proliferative and hypertrophic chondrocytes showed strong expression for ADAMTS-9, but it was weaker in mature chondrocytes. There was no expression of ADAMTS-9 in undifferentiated mesenchymal cells. The strength of the signal in ISH and
Abstracts
whole embryos, we observed similar expression pattern of both transcripts, ubiquitously distributed in basement membrane, prominently in the central nervous system. Immunostaining of whole embryos with antiserum raised against drole-GST fusion protein in mice also showed that Drole protein was accumulated in the central nervous system during embryogenesis. Moreover, loss of function mutants exhibited low surviving ratio during development and mildly abnormal larval locomotion. Thus, drole is widely expressed in basement membrane and may play some important roles in the formation of the central nervous system.
8 weeks of age. Treadmill exercise resulted in decreased whole body and skeletal muscle mass for all muscles and genotypes studied. In addition, after exercise oim/oim mice had altered femoral bone geometry (μCT) and improved biomechanical strength (not yet significant), as seen by increased torsional ultimate strength, tensile strength and energy to failure as compared to non-exercise oim/oim mice. These results suggest that moderate weight-bearing physical activity may improve the amount and strength of bone in patients with OI. doi:10.1016/j.matbio.2008.09.277
doi:10.1016/j.matbio.2008.09.275
61 Abnormal lipid homeostasis in chondrocytes of S1Pcko mice Debabrata Patraa, Xiaoyun Xinga, Jennifer Bryana, Guosheng Liangb, Linda Sandella a Washington University, St. Louis, MO 63110, United States b UT Southwestern Medical Center, Dallas, TX, United States Site-1 protease (S1P) plays an essential role in the regulated processing of endoplasmic reticulum (ER) membrane-bound transcription factors SREBPs and ATF6. SREBPs are involved in cholesterol and fatty acid homeostasis; ATF6 is involved in ER stress signaling (ERSS). Cartilage-specific knockout of S1P in mice (S1Pcko) results in poor cartilage with a drastic decrease of collagen type IIB (Col IIB) in the matrix and consequent lack of endochondral bone formation. Most of the Col IIB appears trapped inside the cell. Ultrastructural analysis of the cartilage shows engorged and fragmented ER characteristic of ER stress. The ER is important for protein synthesis and folding and is the site of fatty acid and cholesterol synthesis. The ER is the essential secretory apparatus of the chondrocyte and therefore maintaining ER lipid composition would be critical for matrix synthesis. In S1Pcko mice, ER stress signaling appears to function normally. However, genome-wide expression profiling followed by real-time PCR analysis demonstrated that fatty acid and cholesterol synthesis is down-regulated in S1Pcko cartilage. This down-regulation may lead to poor ER membrane integrity and therefore ER fragmentation and engorgement presumably due to an inability to secrete Col IIB and therefore ER stress. These data suggest that in embryonic cartilage S1P is involved in maintaining ER membrane integrity by upholding lipid homeostasis and preventing ER stress. This study provides a link to the importance of lipids in cartilage and bone development and may be useful in cartilage diseases and tissue engineering.
63 Insights into the function of SOX9 and campomelic dysplasia Kathryn S. Cheah Department of Biochemistry, University of Hong Kong, Hong Kong Human SOX9 mutations cause the skeletal malformation syndrome campomelic dysplasia (CD). Complete inactivation of the Sox9 gene in mice results in failure of cartilage formation. Studies in zebrafish and Xenopus suggest that Sox9 may be crucial for specification of the otic placode. In mice, loss of Sox9 results in failure of otic placode invagination. Heterozygous mutations in human SOX9 result in conductive and sensorineural deafness in some CD patients, implying a later morphogenetic role but phenotypic details are limited. Sox9−/− null mice die before morphogenesis of the inner ear is complete, precluding investigation of the role of Sox9 later in ear development. Because all the SOX9 mutations are heterozygous and appear to cause loss of function, the CD phenotype has been attributed to haploinsufficency of SOX9. However SOX9 proteins containing an intact HMG box and a truncated activation domain may act dominant negatively by competition with the wild-type for binding to target genes and interfere with interaction with partner factors via the transactivation domain. To assess whether such mutations in SOX9 may act via a dominant interference mechanism we generated transgenic and conditional knock'in mice expressing a mouse equivalent of a CD mutation, a Y440X nonsense mutation causing premature termination within the trans-activation domain of SOX9 (Sox9Y440X). Our data reveal an essential role for Sox9 for sensory and nonsensory morphogenesis of the inner ear. We compared the phenotypic impact of the Sox9 Y440X mutation with a Sox9 null mutation. These studies point to context dependent mechanisms for the Y440X nonsense mutation. doi:10.1016/j.matbio.2008.09.278
doi:10.1016/j.matbio.2008.09.276 64 Expression of ADAMTS-9 in mouse growth plate cartilage 62 Impact of exercise on skeletal muscle and bone in oim mice S. Carletona, B. Weberb, A. McCambridgea, J. Ferrierab, M.B. Brownb, C. Phillipsa a Department of Biochemistry/Child Health, United States b Veterinary Pathobiology, University of Missouri, United States Osteogenesis imperfecta (OI), a heritable connective tissue disorder commonly due to type I procollagen gene defects, is characterized by bone deformity and fragility. The homozygote oim mouse models moderately severe OI type III (oim/oim; homozygous for a functional null COL1A2 gene defect) with reduced bone mineral density and strength. Heterozygous mice (oim/+) model mild OI type I and have an intermediate phenotype between oim/oim and wildtype (Wt) mice. Normally, physical activity increases exercise tolerance, muscle strength and bone biomechanical integrity. We investigated whether treadmill (resistance) exercise will impact skeletal muscle and improve bone biomechanical integrity in Wt, oim/+ and oim/oim mice. We first characterized the hind limb skeletal muscles of 4 month old Wt, oim/+, and oim/oim mice with normal cage activity and determined that there was no evidence of muscle weakness or pathology. Mice exercised on a treadmill 30 min/day, 5 days/week at 10 m/min for 8 weeks starting at
Kanae Kumagishia, Keiichiro Nishidaa, Ryusuke Momotaa, Yuichiro Yamaaib, Satoshi Hirohatac, Kadir Demircanc, Ichiro Naitoa, Yoshifumi Ninomiyac, Aiji Ohtsukaa a Department of Human Morphology, Japan b Department of Oral Function and Anatomy, Japan c Department of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Japan Expression of ADAMTS-9 during the course of chondrocyte differentiation was examined by RT-PCR using ATDC-5 cells. Knee joints from 15 male ICR mice were obtained at 0, 7 and 14 weeks after birth (n = 5 each). They were fixed, decalcified and embedded in paraffin. The expression of ADAMTS-9 in growth plate cartilage of proximal tibia were examined by in situ hybridization (ISH) and immunohistochemistry (IHC). Proliferative and hypertrophic chondrocytes were labeled in adjacent sections by IHC for PCNA and type X collagen, respectively.ADAMTS-9 expression was noted at day 8 and 14 of ATDC5 cell culture, temporally decreased at day 21, and then increased up-to day 35. In the mouse growth plate cartilage, proliferative and hypertrophic chondrocytes showed strong expression for ADAMTS-9, but it was weaker in mature chondrocytes. There was no expression of ADAMTS-9 in undifferentiated mesenchymal cells. The strength of the signal in ISH and