- Email: [email protected]
Collagen interacting proteins in fetal bovine cartilage
ASMB Meeting Abstracts / Matrix Biology 25 (2006) S1–S94
Anti-Lamp1 treatment also upregulated M180 mRNA expression. Further, blockade of Lamp1, followed by exogenous r[A4], doubled M180 expression. To confirm that r[A-4] activates intracellular signaling, we analyzed the expression of NOS2 and NOS3. r[A-4] for 4 h upregulated NOS2 3 fold, and doubled NOS3 mRNA. The current study demonstrates that r[A-4] enhances the expression of M180 mRNA, characteristic of the ameloblast maturation phase. This modulation might be partially mediated through the NO signaling pathway. Supported by NIH grant DE-014758.
S73
178 Supramolecular organization of heterotypic fibrils by decorin U. Hansen a, D.G. Seidler b, P. Bruckner
a
a
Dept. Physiol. Chem. and Pathobiochem., Muenster, Germany b Dept. of Pathology, Anatomy and Cell Biology, Philadelphia, USA
doi:10.1016/j.matbio.2006.08.199
177 Scleraxis and NFATc contribute to COL1a1 expression in tendons V. Léjard a , G. Brideau a , F. Blais b, G. Wagner c, M. Roehrl c, D. Duprez b, J. Rossert a a
INSERM U652 and Paris-Descartes University, Paris CNRS UMR 7622, Paris c Harvard Medical School, Boston b
The combinatorial action of separate cis-acting elements controls the cell-specific expression of the two type I collagen genes. In particular, two elements located between −3.2 and −2.3 kb and named TSE1 and TSE2 are needed to induce expression of the mouse COL1a1 gene in tendon fibroblasts. TSE1 contains a consensus binding site for NFATc transcription factors, and in gel shift assays it bound NFATc proteins. In situ hybridization experiments showed that only NFATc4 is expressed in tendons. Similarly, TT-D6 tendon fibroblastic cells selectively expressed the NFATc4 protein. In transfection experiments, overexpression of NFATc transactivated a reporter construct harboring 4 copies of TSE1 cloned upstream of the COL1a1 minimal promoter. Real-time RT-PCR experiments performed using TT-D6 cells showed that inhibition of the nuclear translocation of NFATc using either cyclosporine A or INCA-6 strongly inhibited the expression of the COL1a1 and COL1a2 genes. Scleraxis (SCX) is a basic helix-loop-helix transcription factor that is expressed selectively in tendon fibroblasts. TSE2 contains an E box, and gel shift assays showed that SCX can bind to this E box as an heterodimer with E47. In transfection experiments, overexpression of SCX and E47 strongly enhanced the activity of reporter constructs harboring 4 copies of TSE2 cloned upstream of the COL1a1 minimal promoter or a 3.2-kb segment of the COL1a1 proximal promoter. Taken together, these results suggest that SCX and NFATc4 cooperate to activate the COL1a1 gene in tendon fibroblasts. doi:10.1016/j.matbio.2006.08.200
Articular cartilage fibrils are biochemically heterogenous. All fibrils contain collagen II as their major component but also include further components which determine several crucial fibril properties. Decorin is preferentially associated with wide cartilage fibrils, but is not present in prototypic thin fibrils which, instead, contain collagen IX. To gain further insight into the molecular control of fibrillar architecture we examined the influence of decorin on reconstituted collagen fibrils. Mixtures of soluble collagens II and XI (ƒ=8:1) or collagens II, IX and XI (ƒ=8:1:1) were subjected to in vitro fibrillogenesis. The collagens were allowed to aggregate for 3 h. Then, decorin was added. After additional 12 h and 24 h, the aggregation products were examined by TEM. After 3 h, mixtures of collagen II and XI, with or without collagen IX, generated prototypic fibrils with a diameter of 20nm. After addition of decorin, fibrils of collagens II and XI laterally aggregated into bundles with aligned D-periodic banding. These aggregates were absent in controls, and also in reconstitution products of collagens II, IX, and XI. In further experiments, decorin was present in the beginning of fibrillogenesis and end products were examined after 3h. However, decorin interacts only with already formed fibrils but not with monomeric collagens. Thus, decorin could organize a lateral alignment of fibrils containing collagens II and XI which was prevented in fibrils containing collagen II, IX and XI. The results show that decorin and collagen IX can modulate fibril morphology and maintain interfibrillar spacing. doi:10.1016/j.matbio.2006.08.201
179 Collagen interacting proteins in fetal bovine cartilage N. Hazeki-Taylor, K.M. Irwin, R.J. Brown, J.T. Oxford Department of Biology and Biomolecular Research Center, Boise State University, Boise, ID 83725, United States Cartilage is a dense connective tissue composed of chondrocytes and a firm gel-like substance known as the extracellular matrix (ECM). The components of the ECM interact strongly with each other and provide a scaffold for
S74
ASMB Meeting Abstracts / Matrix Biology 25 (2006) S1–S94
the cell, while the organization of this scaffold plays a crucial role in regulating the function of chondrocytes. Several cartilage ECM components have been identified, however the interaction between these components in vivo remains unknown. In order to further identify these proteins and describe their interaction with collagen, cartilage was solublized into several buffers and the molecular weights of the components were examined both before and after collagenase treatment. The cartilage was suspended in a neutral buffer containing 0.15 M NaCl, 1 M NaCl, or 4 M GuHCl sequentially. This study focuses on the 4 M GuHCl insoluble material, where type II, IX, and XI collagens were predominantly recovered after pepsin treatment. Upon collagenase treatment of this insoluble material several proteins were released, including thrombospondin-1 (Tsp-1), aggrecan, and TGF-β induced protein (βig-h3). In addition, the Nterminal domain of the α1 chain of type XI collagen and the NC4 domain of the α1 chain of type IX collagen were identified. These results suggest that Tsp-1, βig-h3, and aggrecan strongly interact with collagen fibrils in vivo. In addition, this data suggests that the N-terminal domain of the α1 chain of type XI collagen may be retained on the mature collagen fibrils found in cartilage.
revealed dilated secretory vesicles in cells expressing R519C Pro-GFP and R789C Pro-GFP variants, suggesting intracellular accumulation. In our recent studies we focused on how these alterations of the ECM and/or the intracellular accumulation of mutant procollagen II changed the behavior of the embedded cells. We found evidence that cells expressing some of the mutant collagens were prone to apoptosis probably caused by endoplasmatic reticulum (ER) stress. doi:10.1016/j.matbio.2006.08.203
181 Collagen crosslink profile relates to tendon material properties H.L. Birch a, T.J. Smith a, E.R. Draper a, A.J. Bailey b, N.C. Avery b, A.E. Goodship c a
IOMS, UCL, London, UK Collagen Research Group, University of Bristol, Bristol, UK c Royal Veterinary College, London, UK b
doi:10.1016/j.matbio.2006.08.202
180 Intracellular accumulation of mutant collagen leads to apoptosis V. Hintze, A. Steplewski, A. Fertala Thomas Jefferson University, Department of Dermatology and Cutaneous Biology, Philadelphia, PA 19107, USA A number of single-nucleotide mutations in the COL2A1 gene encoding procollagen II, a main structural component of cartilage, have been reported in patients with cartilage disorders referred to as chondrodysplasias. To study the effects of collagen II mutations in an experimental system we created a model consisting of SW-1353 cells expressing various cartilaginous macromolecules including wild-type procollagen II and recombinant R75C, R519C, R789C and G853E procollagen tagged with a green fluorescent protein (GFP). The cells expressing Pro-GFP variants were grown in suspension culture forming spherical nodules. Over time they increased their mass and deposited ECM components. Some but not all of the investigated mutations caused significant alterations in the characteristics of single molecules, the morphology of fibrils and spherical nodules as well as in the deposition of collagen II in the extracellular matrix (ECM). The expression of the R789C Pro-GFP variant even proved to change the fibril formation of fibronectin, another important ECM component. Electron microscopy of cell layers also
Tendon mechanical properties appropriate for the specific function of that tendon during locomotion are an essential requirement. We have shown previously that this is achieved by variation in material properties of the matrix in addition to differences in gross properties of the tendon and associated muscle. Collagen is the major fibrous component of tendon matrix and responsible for providing tensile strength. In our previous work we found that although the energy storing equine superficial digital flexor tendon (SDFT) had a lower elastic modulus (material stiffness) than the positional common digital extensor tendon (CDET); total collagen content was not significantly different between the tendon types. In this study we hypothesise that organisation of the collagen component and the nature of collagen crosslinks differs between the functionally distinct SDFT and CDET. We found that the mass average collagen fibril diameter was significantly (p<0.001, n=6) lower in the SDFT (169±19 nm) than the CDET (229±36 nm). The collagen crosslink profile showed considerable variation between tendon types. Hydroxylysylpyridinoline (HP) was present at significantly (p<0.001, n=11) higher levels in the SDFT while the CDET contained both histidinohydroxylysinonorleucine and histidinohydroxymerodesmosine which were not detected in the SDFT tissue. Levels of HP in the SDFT showed a significant (p=0.032, n=32) positive correlation with tendon ultimate stress values. In conclusion, collagen crosslinking differs between tendons allowing variation in tendon matrix material properties. doi:10.1016/j.matbio.2006.08.204
Anti-Lamp1 treatment also upregulated M180 mRNA expression. Further, blockade of Lamp1, followed by exogenous r[A4], doubled M180 expression. To confirm that r[A-4] activates intracellular signaling, we analyzed the expression of NOS2 and NOS3. r[A-4] for 4 h upregulated NOS2 3 fold, and doubled NOS3 mRNA. The current study demonstrates that r[A-4] enhances the expression of M180 mRNA, characteristic of the ameloblast maturation phase. This modulation might be partially mediated through the NO signaling pathway. Supported by NIH grant DE-014758.
S73
178 Supramolecular organization of heterotypic fibrils by decorin U. Hansen a, D.G. Seidler b, P. Bruckner
a
a
Dept. Physiol. Chem. and Pathobiochem., Muenster, Germany b Dept. of Pathology, Anatomy and Cell Biology, Philadelphia, USA
doi:10.1016/j.matbio.2006.08.199
177 Scleraxis and NFATc contribute to COL1a1 expression in tendons V. Léjard a , G. Brideau a , F. Blais b, G. Wagner c, M. Roehrl c, D. Duprez b, J. Rossert a a
INSERM U652 and Paris-Descartes University, Paris CNRS UMR 7622, Paris c Harvard Medical School, Boston b
The combinatorial action of separate cis-acting elements controls the cell-specific expression of the two type I collagen genes. In particular, two elements located between −3.2 and −2.3 kb and named TSE1 and TSE2 are needed to induce expression of the mouse COL1a1 gene in tendon fibroblasts. TSE1 contains a consensus binding site for NFATc transcription factors, and in gel shift assays it bound NFATc proteins. In situ hybridization experiments showed that only NFATc4 is expressed in tendons. Similarly, TT-D6 tendon fibroblastic cells selectively expressed the NFATc4 protein. In transfection experiments, overexpression of NFATc transactivated a reporter construct harboring 4 copies of TSE1 cloned upstream of the COL1a1 minimal promoter. Real-time RT-PCR experiments performed using TT-D6 cells showed that inhibition of the nuclear translocation of NFATc using either cyclosporine A or INCA-6 strongly inhibited the expression of the COL1a1 and COL1a2 genes. Scleraxis (SCX) is a basic helix-loop-helix transcription factor that is expressed selectively in tendon fibroblasts. TSE2 contains an E box, and gel shift assays showed that SCX can bind to this E box as an heterodimer with E47. In transfection experiments, overexpression of SCX and E47 strongly enhanced the activity of reporter constructs harboring 4 copies of TSE2 cloned upstream of the COL1a1 minimal promoter or a 3.2-kb segment of the COL1a1 proximal promoter. Taken together, these results suggest that SCX and NFATc4 cooperate to activate the COL1a1 gene in tendon fibroblasts. doi:10.1016/j.matbio.2006.08.200
Articular cartilage fibrils are biochemically heterogenous. All fibrils contain collagen II as their major component but also include further components which determine several crucial fibril properties. Decorin is preferentially associated with wide cartilage fibrils, but is not present in prototypic thin fibrils which, instead, contain collagen IX. To gain further insight into the molecular control of fibrillar architecture we examined the influence of decorin on reconstituted collagen fibrils. Mixtures of soluble collagens II and XI (ƒ=8:1) or collagens II, IX and XI (ƒ=8:1:1) were subjected to in vitro fibrillogenesis. The collagens were allowed to aggregate for 3 h. Then, decorin was added. After additional 12 h and 24 h, the aggregation products were examined by TEM. After 3 h, mixtures of collagen II and XI, with or without collagen IX, generated prototypic fibrils with a diameter of 20nm. After addition of decorin, fibrils of collagens II and XI laterally aggregated into bundles with aligned D-periodic banding. These aggregates were absent in controls, and also in reconstitution products of collagens II, IX, and XI. In further experiments, decorin was present in the beginning of fibrillogenesis and end products were examined after 3h. However, decorin interacts only with already formed fibrils but not with monomeric collagens. Thus, decorin could organize a lateral alignment of fibrils containing collagens II and XI which was prevented in fibrils containing collagen II, IX and XI. The results show that decorin and collagen IX can modulate fibril morphology and maintain interfibrillar spacing. doi:10.1016/j.matbio.2006.08.201
179 Collagen interacting proteins in fetal bovine cartilage N. Hazeki-Taylor, K.M. Irwin, R.J. Brown, J.T. Oxford Department of Biology and Biomolecular Research Center, Boise State University, Boise, ID 83725, United States Cartilage is a dense connective tissue composed of chondrocytes and a firm gel-like substance known as the extracellular matrix (ECM). The components of the ECM interact strongly with each other and provide a scaffold for
S74
ASMB Meeting Abstracts / Matrix Biology 25 (2006) S1–S94
the cell, while the organization of this scaffold plays a crucial role in regulating the function of chondrocytes. Several cartilage ECM components have been identified, however the interaction between these components in vivo remains unknown. In order to further identify these proteins and describe their interaction with collagen, cartilage was solublized into several buffers and the molecular weights of the components were examined both before and after collagenase treatment. The cartilage was suspended in a neutral buffer containing 0.15 M NaCl, 1 M NaCl, or 4 M GuHCl sequentially. This study focuses on the 4 M GuHCl insoluble material, where type II, IX, and XI collagens were predominantly recovered after pepsin treatment. Upon collagenase treatment of this insoluble material several proteins were released, including thrombospondin-1 (Tsp-1), aggrecan, and TGF-β induced protein (βig-h3). In addition, the Nterminal domain of the α1 chain of type XI collagen and the NC4 domain of the α1 chain of type IX collagen were identified. These results suggest that Tsp-1, βig-h3, and aggrecan strongly interact with collagen fibrils in vivo. In addition, this data suggests that the N-terminal domain of the α1 chain of type XI collagen may be retained on the mature collagen fibrils found in cartilage.
revealed dilated secretory vesicles in cells expressing R519C Pro-GFP and R789C Pro-GFP variants, suggesting intracellular accumulation. In our recent studies we focused on how these alterations of the ECM and/or the intracellular accumulation of mutant procollagen II changed the behavior of the embedded cells. We found evidence that cells expressing some of the mutant collagens were prone to apoptosis probably caused by endoplasmatic reticulum (ER) stress. doi:10.1016/j.matbio.2006.08.203
181 Collagen crosslink profile relates to tendon material properties H.L. Birch a, T.J. Smith a, E.R. Draper a, A.J. Bailey b, N.C. Avery b, A.E. Goodship c a
IOMS, UCL, London, UK Collagen Research Group, University of Bristol, Bristol, UK c Royal Veterinary College, London, UK b
doi:10.1016/j.matbio.2006.08.202
180 Intracellular accumulation of mutant collagen leads to apoptosis V. Hintze, A. Steplewski, A. Fertala Thomas Jefferson University, Department of Dermatology and Cutaneous Biology, Philadelphia, PA 19107, USA A number of single-nucleotide mutations in the COL2A1 gene encoding procollagen II, a main structural component of cartilage, have been reported in patients with cartilage disorders referred to as chondrodysplasias. To study the effects of collagen II mutations in an experimental system we created a model consisting of SW-1353 cells expressing various cartilaginous macromolecules including wild-type procollagen II and recombinant R75C, R519C, R789C and G853E procollagen tagged with a green fluorescent protein (GFP). The cells expressing Pro-GFP variants were grown in suspension culture forming spherical nodules. Over time they increased their mass and deposited ECM components. Some but not all of the investigated mutations caused significant alterations in the characteristics of single molecules, the morphology of fibrils and spherical nodules as well as in the deposition of collagen II in the extracellular matrix (ECM). The expression of the R789C Pro-GFP variant even proved to change the fibril formation of fibronectin, another important ECM component. Electron microscopy of cell layers also
Tendon mechanical properties appropriate for the specific function of that tendon during locomotion are an essential requirement. We have shown previously that this is achieved by variation in material properties of the matrix in addition to differences in gross properties of the tendon and associated muscle. Collagen is the major fibrous component of tendon matrix and responsible for providing tensile strength. In our previous work we found that although the energy storing equine superficial digital flexor tendon (SDFT) had a lower elastic modulus (material stiffness) than the positional common digital extensor tendon (CDET); total collagen content was not significantly different between the tendon types. In this study we hypothesise that organisation of the collagen component and the nature of collagen crosslinks differs between the functionally distinct SDFT and CDET. We found that the mass average collagen fibril diameter was significantly (p<0.001, n=6) lower in the SDFT (169±19 nm) than the CDET (229±36 nm). The collagen crosslink profile showed considerable variation between tendon types. Hydroxylysylpyridinoline (HP) was present at significantly (p<0.001, n=11) higher levels in the SDFT while the CDET contained both histidinohydroxylysinonorleucine and histidinohydroxymerodesmosine which were not detected in the SDFT tissue. Levels of HP in the SDFT showed a significant (p=0.032, n=32) positive correlation with tendon ultimate stress values. In conclusion, collagen crosslinking differs between tendons allowing variation in tendon matrix material properties. doi:10.1016/j.matbio.2006.08.204