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Molecular based structure and ligation of the collagen fibril
Abstracts
mass is a common finding in both Marfan and Beals syndrome (caused by Fbn1 and Fbn2 mutations respectively), and that osteoblasts are unique examples of cells secreting both small and large latent TGFβ complexes, we investigated the role of microfibrils in primary calvarial osteoblast (COb) cultures from Fbn1 or Fbn2 null mice. Loss of Fbn1 or Fbn2 was correlated with elevated TGFβ signaling and normative TGFβ gene expression. TGFβ activity was 2 fold higher in Fbn2 than Fbn1 null COb cultures, implying that the two proteins differentially regulate local TGFβ bioavailabilty in these cells. Consistent with this conclusion, Fbn2 null COb assembled seemingly normal Fbn1 and LTBP1, whereas LTBP1 was somewhat reduced in Fbn1 null cultures. Higher TGFβ activity translated into reduced matrix deposition and mineralization in Fbn2 null compared to Fbn1 null cultures. Impaired differentiation of Fbn2 null COb was rescued by TGFβ antagonism or BMP2 addition ruling out an earlier change in cell fate. In line with this observation, RNA interference in primary COb recapitulated the phenotypes of Fbn1 and Fbn2 null cultures. Finally, co-culture experiments revealed that both Fbn1 and Fbn2 null Cob stimulate osteoclastogenesis as the result of ALK5 independent increase in RANKL expression. Collectively, our findings demonstrate the differential roles of fibrillins in local control of matrix assembly, TGFβ bioavailability, and resident cell differentiation during bone remodeling. doi:10.1016/j.matbio.2008.09.426
212 Collagen X is required for proper hematopoietic development Elizabeth Sweeney, Olena Jacenko University of Pennsylvania, Philadelphia, PA, United States During endochondral ossification (EO), the hypertrophic cartilage (HC)containing growth plate is replaced by trabecular bone and a hematopoietic marrow. We propose that EO establishes the hematopoietic stem cell (HSC) niche. This is based on the skeleto-hematopoietic disease phenotypes in mice where the function of collagen X, a unique HC matrix protein, was disrupted either by transgenesis (Tg mice), or by gene inactivation (KO mice). Murine defects manifested as aberrant growth plates, reduced trabecular bone, marrow hypoplasia, altered lymphocyte profiles throughout life, and compromised immunity. A fraction of all mice succumbed to perinatal lethality at week-3 due to inability to suppress opportunistic infections, and displayed marrow aplasia and lymphatic organ atrophy. Serum cytokine arrays revealed mis-expression of inflammatory and hematopoietic cytokines in all collagen X mice. Of note, many of these cytokines bind heparan sulfate proteoglycans (HSPGs), which are reduced or lacking in the chondro-osseous junctions (coj) of the collagen X mice. Neonatal bone marrow transplantations confirmed that the hematopoietic defects result from an altered coj and marrow environment in the collagen
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X mice, rather than from defects in their HSCs. Finally, in vitro co-cultures with wild type HSCs and different cell types from the coj demonstrated an altered ability of osteoblasts and HC from collagen X mice to support B lymphopoiesis. We propose that the collagen X/HSPG network sequesters hematopoietic cytokines and growth factors at the coj, and that disruption of collagen X function causes an imbalance in cytokine metabolism, leading to impaired hematopoiesis and immunity. doi:10.1016/j.matbio.2008.09.427
213 Molecular based structure and ligation of the collagen fibril Joseph P. Orgela,b, Olga Antipovaa,b, Aya Eida, Rong Wanga, Dengli Qiua, Shawn Sweeneyc, John Scottd, James San Antonioc a Illinois Institute of Technology, Chicago IL, United States b BioCAT, APS, Argonne National lab, IL, United States c Thomas Jefferson University, Philadelphia, PA, United States d Manchester University, Manchester UK The fibrous collagens are the fundamental constituents of the Extracellular Matrix (ECM) of animals, forming the structural basis of all known mammalian connective tissues and organ systems. Yet, despite the fundamental biological importance of collagen, many of us are perplexed by the complexity of the assemblies that the collagens form. This is particularly true at what may be the most significant aspect of collagen structure from a cellular point of view, at the intermediate sub-fibrillar (and or surface) level where many important biological processes occur in growth, development and disease. These include but are not limited to: fibrillogenesis, tissue remolding and in forming the scaffolding upon which organ systems, bones, cartilage, etc., i.e. the animal body, are built upon. Clearly, obtaining an unambiguous and contextualized visualization of collagen molecules would be of significant value to the scientific community. We have recently determined the structure of the type I collagen microfibril and fibril at the molecular level from whole intact rat-tail tendons. Using this data it is possible to map the amino acid chemistry, ligand binding data and other observations onto the defining shape modality of the fibrillar collagen ECM. In so doing, we have been able to propose the first fibrillar based mechanism of collagenolysis and provide a number of illuminating observations regarding other collagen fibril–ligand interactions. doi:10.1016/j.matbio.2008.09.428
mass is a common finding in both Marfan and Beals syndrome (caused by Fbn1 and Fbn2 mutations respectively), and that osteoblasts are unique examples of cells secreting both small and large latent TGFβ complexes, we investigated the role of microfibrils in primary calvarial osteoblast (COb) cultures from Fbn1 or Fbn2 null mice. Loss of Fbn1 or Fbn2 was correlated with elevated TGFβ signaling and normative TGFβ gene expression. TGFβ activity was 2 fold higher in Fbn2 than Fbn1 null COb cultures, implying that the two proteins differentially regulate local TGFβ bioavailabilty in these cells. Consistent with this conclusion, Fbn2 null COb assembled seemingly normal Fbn1 and LTBP1, whereas LTBP1 was somewhat reduced in Fbn1 null cultures. Higher TGFβ activity translated into reduced matrix deposition and mineralization in Fbn2 null compared to Fbn1 null cultures. Impaired differentiation of Fbn2 null COb was rescued by TGFβ antagonism or BMP2 addition ruling out an earlier change in cell fate. In line with this observation, RNA interference in primary COb recapitulated the phenotypes of Fbn1 and Fbn2 null cultures. Finally, co-culture experiments revealed that both Fbn1 and Fbn2 null Cob stimulate osteoclastogenesis as the result of ALK5 independent increase in RANKL expression. Collectively, our findings demonstrate the differential roles of fibrillins in local control of matrix assembly, TGFβ bioavailability, and resident cell differentiation during bone remodeling. doi:10.1016/j.matbio.2008.09.426
212 Collagen X is required for proper hematopoietic development Elizabeth Sweeney, Olena Jacenko University of Pennsylvania, Philadelphia, PA, United States During endochondral ossification (EO), the hypertrophic cartilage (HC)containing growth plate is replaced by trabecular bone and a hematopoietic marrow. We propose that EO establishes the hematopoietic stem cell (HSC) niche. This is based on the skeleto-hematopoietic disease phenotypes in mice where the function of collagen X, a unique HC matrix protein, was disrupted either by transgenesis (Tg mice), or by gene inactivation (KO mice). Murine defects manifested as aberrant growth plates, reduced trabecular bone, marrow hypoplasia, altered lymphocyte profiles throughout life, and compromised immunity. A fraction of all mice succumbed to perinatal lethality at week-3 due to inability to suppress opportunistic infections, and displayed marrow aplasia and lymphatic organ atrophy. Serum cytokine arrays revealed mis-expression of inflammatory and hematopoietic cytokines in all collagen X mice. Of note, many of these cytokines bind heparan sulfate proteoglycans (HSPGs), which are reduced or lacking in the chondro-osseous junctions (coj) of the collagen X mice. Neonatal bone marrow transplantations confirmed that the hematopoietic defects result from an altered coj and marrow environment in the collagen
S61
X mice, rather than from defects in their HSCs. Finally, in vitro co-cultures with wild type HSCs and different cell types from the coj demonstrated an altered ability of osteoblasts and HC from collagen X mice to support B lymphopoiesis. We propose that the collagen X/HSPG network sequesters hematopoietic cytokines and growth factors at the coj, and that disruption of collagen X function causes an imbalance in cytokine metabolism, leading to impaired hematopoiesis and immunity. doi:10.1016/j.matbio.2008.09.427
213 Molecular based structure and ligation of the collagen fibril Joseph P. Orgela,b, Olga Antipovaa,b, Aya Eida, Rong Wanga, Dengli Qiua, Shawn Sweeneyc, John Scottd, James San Antonioc a Illinois Institute of Technology, Chicago IL, United States b BioCAT, APS, Argonne National lab, IL, United States c Thomas Jefferson University, Philadelphia, PA, United States d Manchester University, Manchester UK The fibrous collagens are the fundamental constituents of the Extracellular Matrix (ECM) of animals, forming the structural basis of all known mammalian connective tissues and organ systems. Yet, despite the fundamental biological importance of collagen, many of us are perplexed by the complexity of the assemblies that the collagens form. This is particularly true at what may be the most significant aspect of collagen structure from a cellular point of view, at the intermediate sub-fibrillar (and or surface) level where many important biological processes occur in growth, development and disease. These include but are not limited to: fibrillogenesis, tissue remolding and in forming the scaffolding upon which organ systems, bones, cartilage, etc., i.e. the animal body, are built upon. Clearly, obtaining an unambiguous and contextualized visualization of collagen molecules would be of significant value to the scientific community. We have recently determined the structure of the type I collagen microfibril and fibril at the molecular level from whole intact rat-tail tendons. Using this data it is possible to map the amino acid chemistry, ligand binding data and other observations onto the defining shape modality of the fibrillar collagen ECM. In so doing, we have been able to propose the first fibrillar based mechanism of collagenolysis and provide a number of illuminating observations regarding other collagen fibril–ligand interactions. doi:10.1016/j.matbio.2008.09.428