Publications (11)73.45 Total impact
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Article: A role for prolyl 3-hydroxylase 2 in post-translational modification of fibril-forming collagens.
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ABSTRACT: The fibrillar collagen types I, II, and V/XI have recently been shown to have partially 3-hydroxylated proline (3Hyp) residues at sites other than the established primary Pro-986 site in the collagen triple helical domain. These sites showed tissue specificity in degree of hydroxylation and a pattern of D-periodic spacing. This suggested a contributory role in fibril supramolecular assembly. The sites in clade A fibrillar α1(II), α2(V), and α1(I) collagen chains share common features with known prolyl 3-hydroxylase 2 (P3H2) substrate sites in α1(IV) chains implying a role for this enzyme. We pursued this possibility using the Swarm rat chondrosarcoma cell line (RCS-LTC) found to express high levels of P3H2 mRNA. Mass spectrometry determined that all the additional candidate 3Hyp substrate sites in the pN type II collagen made by these cells were highly hydroxylated. In RNA interference experiments, P3H2 protein synthesis was suppressed coordinately with prolyl 3-hydroxylation at Pro-944, Pro-707, and the C-terminal GPP repeat of the pNα1(II) chain, but Pro-986 remained fully hydroxylated. Furthermore, when P3H2 expression was turned off, as seen naturally in cultured SAOS-2 osteosarcoma cells, full 3Hyp occupancy at Pro-986 in α1(I) chains was unaffected, whereas 3-hydroxylation of residue Pro-944 in the α2(V) chain was largely lost, and 3-hydroxylation of Pro-707 in α2(V) and α2(I) chains were sharply reduced. The results imply that P3H2 has preferred substrate sequences among the classes of 3Hyp sites in clade A collagen chains.Journal of Biological Chemistry 07/2011; 286(35):30662-9. · 4.77 Impact Factor -
Article: The heterozygous disproportionate micromelia (dmm) mouse: morphological changes in fetal cartilage precede postnatal dwarfism and compared with lethal homozygotes can explain the mild phenotype.
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ABSTRACT: The disproportionate micromelia (Dmm) mouse has a mutation in the C-propeptide coding region of the Col2a1 gene that causes lethal dwarfism when homozygous (Dmm/Dmm) but causes only mild dwarfism observable approximately 1-week postpartum when heterozygous (Dmm/+). The purpose of this study was 2-fold: first, to analyze and quantify morphological changes that precede the expression of mild dwarfism in Dmm/+ animals, and second, to compare morphological alterations between Dmm/+ and Dmm/Dmm fetal cartilage that may correlate with the marked skeletal differences between mild and lethal dwarfism. Light and electron transmission microscopy were used to visualize structure of chondrocytes and extracellular matrix (ECM) of fetal rib cartilage. Both Dmm/+ and Dmm/Dmm fetal rib cartilage had significantly larger chondrocytes, greater cell density, and less ECM per unit area than +/+ littermates. Quantitative RT-PCR showed a decrease in aggrecan mRNA in Dmm/+ vs +/+ cartilage. Furthermore, the cytoplasm of chondrocytes in Dmm/+ and Dmm/Dmm cartilage was occupied by significantly more distended rough endoplasmic reticulum (RER) compared with wild-type chondrocytes. Fibril diameters and packing densities of +/+ and Dmm/+ cartilage were similar, but Dmm/Dmm cartilage showed thinner, sparsely distributed fibrils. These findings support the prevailing hypothesis that a C-propeptide mutation could interrupt the normal assembly and secretion of Type II procollagen trimers, resulting in a buildup of proalpha1(II) chains in the RER and a reduced rate of matrix synthesis. Thus, intracellular entrapment of proalpha1(II) seems to be primarily responsible for the dominant-negative effect of the Dmm mutation in the expression of dwarfism.Journal of Histochemistry and Cytochemistry 09/2008; 56(11):1003-11. · 2.72 Impact Factor -
Article: Type XXVII collagen at the transition of cartilage to bone during skeletogenesis.
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ABSTRACT: COL27A1 is a member of the collagen fibrillar gene family and is expressed in cartilaginous tissues including the anlage of endochondral bone. To begin to understand its role in skeletogenesis, the temporospatial distributions of its RNA message and protein product, type XXVII collagen, were determined in developing human skeletal tissues. Laser capture microdissection and quantitative reverse-transcription polymerase chain reaction demonstrated that gene expression occurred throughout the growth plate and that it was higher in the resting and proliferative zones than in hypertrophic cartilage. Immunohistochemical analyses showed that type XXVII collagen was most evident in hypertrophic cartilage at the primary ossification center and at the growth plate and that it accumulated in the pericellular matrix. Synthesis of type XXVII collagen overlapped partly with that of type X collagen, a marker of chondrocyte hypertrophy, preceded the transition of cartilage to bone, and was associated with cartilage calcification. Immunogold electron microscopy of extracted ECM components from mouse growth plate showed that type XXVII collagen was a component of long non-banded fibrous structures, filamentous networks, and thin banded fibrils. The timing and location of synthesis suggest that type XXVII collagen plays a role during the calcification of cartilage and the transition of cartilage to bone.Bone 11/2007; 41(4):535-42. · 4.02 Impact Factor -
Article: Collagen XI chain misassembly in cartilage of the chondrodysplasia (cho) mouse.
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ABSTRACT: Molecular mechanisms controlling the assembly of cartilage-specific types II, IX and XI collagens into a heteropolymeric network of uniformly thin, unbanded fibrils are not well understood, but collagen XI has been implicated. The present study on cartilage from the homozygous chondrodysplasia (cho/cho) mouse adds support to this concept. In the absence of alpha1(XI) collagen chains, thick, banded collagen fibrils are formed in the extracellular matrix of cho/cho cartilage. A functional knock-out of the type XI collagen molecule has been assumed. We have re-examined this at the protein level to see if, rather than a complete knock-out, alternative type XI chain assemblies were formed. Mass spectrometry of purified triple-helical collagen from the rib cartilage of cho/cho mice identified alpha1(V) and alpha2(XI) chains. These chains were recovered in roughly equal amounts based on Coomassie Blue staining of SDS-PAGE gels, in addition to alpha1(II)/alpha3(XI) collagen chains. Using telopeptide-specific antibodies and Western blot analysis, it was further shown that type V/XI trimers were present in the matrix cross-linked to each other and to type II collagen molecules to form heteropolymers. Cartilage from heterozygous (cho/+) mice contained a mix of alpha1(V) and alpha1(XI) chains and a mix of thin and thick fibrils on transmission electron microscopy. In summary, the results imply that native type XI collagen molecules containing an alpha1(XI) chain are required to form uniformly thin fibrils and support a role for type XI collagen as the template for the characteristic type II collagen fibril network of developing cartilage.Matrix Biology 11/2007; 26(8):597-603. · 3.30 Impact Factor -
Article: The post-translational phenotype of collagen synthesized by SAOS-2 osteosarcoma cells.
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ABSTRACT: The human osteosarcoma-derived cell line, SAOS-2, exhibits many of the phenotypic characteristics of osteoblasts including the deposition of types I and V collagens in an extracellular matrix. Lesser amounts of collagen XI chains were also detected. The cell layer collagen contains hydroxylysyl pyridinoline cross-links but without the accompanying lysyl pyridinoline typical of human bone collagen. This indicates that the lysine residues at the two helical cross-linking loci are fully hydroxylated. The isoform of lysyl hydroxylase, LH1, known to be required for full hydroxylation at these sites, was shown to be highly expressed by SAOS-2 cells. Our findings provide insight on the mechanism of post-translational overmodification of lysine residues in collagen made by osteosarcoma tumors, and may be relevant for understanding a similar overmodification observed in osteoporotic bone.Bone 06/2007; 40(5):1343-51. · 4.02 Impact Factor -
Article: CRTAP is required for prolyl 3- hydroxylation and mutations cause recessive osteogenesis imperfecta.
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ABSTRACT: Prolyl hydroxylation is a critical posttranslational modification that affects structure, function, and turnover of target proteins. Prolyl 3-hydroxylation occurs at only one position in the triple-helical domain of fibrillar collagen chains, and its biological significance is unknown. CRTAP shares homology with a family of putative prolyl 3-hydroxylases (P3Hs), but it does not contain their common dioxygenase domain. Loss of Crtap in mice causes an osteochondrodysplasia characterized by severe osteoporosis and decreased osteoid production. CRTAP can form a complex with P3H1 and cyclophilin B (CYPB), and Crtap-/- bone and cartilage collagens show decreased prolyl 3-hydroxylation. Moreover, mutant collagen shows evidence of overmodification, and collagen fibrils in mutant skin have increased diameter consistent with altered fibrillogenesis. In humans, CRTAP mutations are associated with the clinical spectrum of recessive osteogenesis imperfecta, including the type II and VII forms. Hence, dysregulation of prolyl 3-hydroxylation is a mechanism for connective tissue disease.Cell 11/2006; 127(2):291-304. · 32.40 Impact Factor -
Article: Sp1 family of transcription factors regulates the human alpha2 (XI) collagen gene (COL11A2) in Saos-2 osteoblastic cells.
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ABSTRACT: Genes encoding type XI collagen, normally associated with chondrogenesis, are also expressed by osteoblasts. By studying Saos-2 cells, we showed that the transcription factors, Sp1, Sp3, and Sp7 (Osterix), regulate COL11A2 expression through its proximal promoter. The findings indicate both ubiquitous and osteoblast-specific mechanisms of collagen gene regulation. Type XI collagen is essential for skeletal morphogenesis. Collagen XI gene regulation has been studied in chondrocytes but not in osteoblasts. We cultured Saos-2 cells, a human osteosarcoma-derived line of osteoblasts, and analyzed them for alpha2(XI) protein and COL11A2 regulatory mechanisms. Although types I and V were the dominant collagens deposited by Saos-2 cells, they expressed COL11A2 mRNA, and alpha2(XI) chains were present in the extracellular matrix. The COL11A2 promoter region (from -149 to -40) containing three Sp1 binding sites was required for promoter activity in transient transfection assays. All three Sp1 sites were critical for binding by nuclear proteins in electrophoretic mobility shift assays. Further analysis using consensus oligonucleotides and specific antibodies as well as chromatin immunoprecipitation assay implicated Sp1 and Sp3 in binding to this promoter region. Overexpressing Sp1 or Sp3 significantly increased COL11A2 promoter activity and endogenous COL11A2 gene expression, an effect that was suppressed by the Sp1-binding inhibitor mithramycin A. Further experiments showed that Sp1, Sp3, CREB-binding protein (CBP), p300, and histone deacetylase (HDAC) were physically associated and HDAC inhibitors (trichostatin A or NaB) upregulated COL11A2 promoter activity and endogenous gene expression. Another Sp1 family member, Sp7 (Osterix), was expressed in Saos-2 cells, but not in chondrocytes, and was shown by chromatin immunoprecipitation to occupy the COL11A2 promoter. Overexpressing Sp7 increased COL11A2 promoter activity and endogenous gene expression, an effect also blocked by mithramycin A. Using siRNA to knockdown Sp1, Sp3, or Sp7, it was shown that depression of any of them decreased COL11A2 promoter activity and endogenous gene expression. Finally, primary cultures of osteoblasts expressed COL11A2 and Sp7, upregulated COL11A2 promoter activity and endogenous gene expression when Sp1, Sp3, or Sp7 were overexpressed, and downregulated them when Sp1, Sp3, or Sp7 were selectively depressed. The results establish that Sp1 proteins regulate COL11A2 transcription by binding to its proximal promoter and directly interacting with CBP, p300, and HDAC.Journal of Bone and Mineral Research 06/2006; 21(5):661-73. · 6.37 Impact Factor -
Article: Sp1 Family of Transcription Factors Regulates the Human α2 (XI) Collagen Gene (COL11A2) in Saos‐2 Osteoblastic Cells
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ABSTRACT: Genes encoding type XI collagen, normally associated with chondrogenesis, are also expressed by osteoblasts. By studying Saos-2 cells, we showed that the transcription factors, Sp1, Sp3, and Sp7 (Osterix), regulate COL11A2 expression through its proximal promoter. The findings indicate both ubiquitous and osteoblast-specific mechanisms of collagen gene regulation.Introduction: Type XI collagen is essential for skeletal morphogenesis. Collagen XI gene regulation has been studied in chondrocytes but not in osteoblasts.Materials and Methods: We cultured Saos-2 cells, a human osteosarcoma-derived line of osteoblasts, and analyzed them for 2(XI) protein and COL11A2 regulatory mechanisms.Results and Conclusions: Although types I and V were the dominant collagens deposited by Saos-2 cells, they expressed COL11A2 mRNA, and 2(XI) chains were present in the extracellular matrix. The COL11A2 promoter region (from −149 to −40) containing three Sp1 binding sites was required for promoter activity in transient transfection assays. All three Sp1 sites were critical for binding by nuclear proteins in electrophoretic mobility shift assays. Further analysis using consensus oligonucleotides and specific antibodies as well as chromatin immunoprecipitation assay implicated Sp1 and Sp3 in binding to this promoter region. Overexpressing Sp1 or Sp3 significantly increased COL11A2 promoter activity and endogenous COL11A2 gene expression, an effect that was suppressed by the Sp1-binding inhibitor mithramycin A. Further experiments showed that Sp1, Sp3, CREB-binding protein (CBP), p300, and histone deacetylase (HDAC) were physically associated and HDAC inhibitors (trichostatin A or NaB) upregulated COL11A2 promoter activity and endogenous gene expression. Another Sp1 family member, Sp7 (Osterix), was expressed in Saos-2 cells, but not in chondrocytes, and was shown by chromatin immunoprecipitation to occupy the COL11A2 promoter. Overexpressing Sp7 increased COL11A2 promoter activity and endogenous gene expression, an effect also blocked by mithramycin A. Using siRNA to knockdown Sp1, Sp3, or Sp7, it was shown that depression of any of them decreased COL11A2 promoter activity and endogenous gene expression. Finally, primary cultures of osteoblasts expressed COL11A2 and Sp7, upregulated COL11A2 promoter activity and endogenous gene expression when Sp1, Sp3, or Sp7 were overexpressed, and downregulated them when Sp1, Sp3, or Sp7 were selectively depressed. The results establish that Sp1 proteins regulate COL11A2 transcription by binding to its proximal promoter and directly interacting with CBP, p300, and HDAC.Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research 04/2006; 21(5):661 - 673. · 6.04 Impact Factor -
Article: Protein consequences of the Col2a1 C-propeptide mutation in the chondrodysplastic Dmm mouse.
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ABSTRACT: The Disproportionate micromelia (Dmm) mouse has a three nucleotide deletion in Col2a1 in the region encoding the C-propeptide which results in the substitution of one amino acid, Asn, for two amino acids, Lys-Thr. Western blot and immunohistochemical analyses failed to detect type II collagen in the cartilage matrix of the homozygous mice and showed reduced levels in the matrix of heterozygous mice. Type II collagen chains localized intracellularly within the chondrocytes of homozygote and heterozygote tissues. These findings provide evidence that the expression of type II procollagen chains containing the defective C-propeptide results in an intracellular retention and faulty secretion of type II procollagen molecules. A complete absence of mature type II collagen from the homozygote cartilage and an insufficiency of type II collagen in the heterozygote cartilage explains the Dmm mouse phenotype. The integrity of the C-propeptide is thus crucial for the biosynthesis of normal type II collagen by chondrocytes.Matrix Biology 10/2003; 22(5):449-53. · 3.30 Impact Factor -
Article: Assembly of collagen types II, IX and XI into nascent hetero-fibrils by a rat chondrocyte cell line.
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ABSTRACT: The cell line, RCS-LTC (derived from the Swarm rat chondrosarcoma), deposits a copious extracellular matrix in which the collagen component is primarily a polymer of partially processed type II N-procollagen molecules. Transmission electron microscopy of the matrix shows no obvious fibrils, only a mass of thin unbanded filaments. We have used this cell system to show that the type II N-procollagen polymer nevertheless is stabilized by pyridinoline cross-links at molecular sites (mediated by N- and C-telopeptide domains) found in collagen II fibrils processed normally. Retention of the N-propeptide therefore does not appear to interfere with the interactions needed to form cross-links and mature them into trivalent pyridinoline residues. In addition, using antibodies that recognize specific cross-linking domains, it was shown that types IX and XI collagens, also abundantly deposited into the matrix by this cell line, become covalently cross-linked to the type II N-procollagen. The results indicate that the assembly and intertype cross-linking of the cartilage type II collagen heteropolymer is an integral, early process in fibril assembly and can occur efficiently prior to the removal of the collagen II N-propeptides.European Journal of Biochemistry 09/2003; 270(15):3243-50. · 3.58 Impact Factor -
Article: Tissue response and Msx1 expression after human fetal digit tip amputation in vitro.
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ABSTRACT: Regeneration of mammalian digit tips is well described; however, associated cellular or molecular events have not been studied in humans. We describe an in vitro human fetal model of response to digit tip amputation, and report expression of the transcription repressor Msx1 in the developing and regrowing human digit tip. Human fetal digits from specimens ranging from 53 to 117 days' estimated gestational age (EGA) were cultured in a defined serum-free medium with supplemented oxygen for time periods from 4 days to 4 weeks. Histology and immunohistochemistry were performed on paired control and tip-amputated digits. Regrowing tissue covered the cut end of the distal phalanx in digits up to 80 days' EGA. Msx1 expression was detected beneath the nail field in control digits to at least 70 days' EGA and at the regrowing tip of 57-day digits at 4 and 7 days post-amputation. Our results show that human fetal digits regrow tissue in vitro in response to tip amputation. This process appears spatially associated with Msx1 expression. Msx1 expression appears increased at the regrowing tip of 57-day digits by 4 days after amputation.Wound Repair and Regeneration 14(4):398-404. · 2.91 Impact Factor
Top co-authors
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Institutions
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2003–2011
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University of Washington Seattle
- Department of Orthopaedics and Sports Medicine
Seattle, WA, USA
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2008
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Brigham Young University - Provo Main Campus
- Department of Physiology and Developmental Biology
Provo, UT, USA
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