Unique Matrix Structure in the Rough Endoplasmic Reticulum Cisternae of Pseudoachondroplasia Chondrocytes
2007; Elsevier BV; Volume: 170; Issue: 1 Linguagem: Inglês
10.2353/ajpath.2007.060530
ISSN1525-2191
AutoresThomas M. Merritt, Roger J. Bick, Brian J. Poindexter, Joseph L. Alcorn, Jacqueline T. Hecht,
Tópico(s)Cell Adhesion Molecules Research
ResumoMutations in cartilage oligomeric matrix protein (COMP) cause two skeletal dysplasias, pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED/EDM1). Because COMP exists as a homopentamer, only one mutant COMP subunit may result in an abnormal complex that is accumulated in expanded rough endoplasmic reticulum (rER) cisternae, a hallmark of PSACH. Type IX collagen and matrilin-3 (MATN3), also accumulate in the rER cisternae of PSACH chondrocytes, but it is unknown how mutant COMP interacts with these proteins. The studies herein focus on defining the organization of these intracellularly retained proteins using fluorescence deconvolution microscopy. A unique matrix organization was identified in which type II procollagen formed a central core surrounded by a protein network of mutant COMP, type IX collagen, and MATN3. This pattern of matrix organization was found in multiple cisternae from single chondrocytes and in chondrocytes with different COMP mutations, indicating a common pattern of interaction. This suggests that stalling of mutant COMP and an interaction between mutant COMP and type II procollagen are initiating events in the assembly of matrix in the rER, possibly explaining why the material is not readily cleared from the rER. Altogether, these data suggest that mutant COMP initiates and perhaps catalyzes premature intracellular matrix assembly. Mutations in cartilage oligomeric matrix protein (COMP) cause two skeletal dysplasias, pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED/EDM1). Because COMP exists as a homopentamer, only one mutant COMP subunit may result in an abnormal complex that is accumulated in expanded rough endoplasmic reticulum (rER) cisternae, a hallmark of PSACH. Type IX collagen and matrilin-3 (MATN3), also accumulate in the rER cisternae of PSACH chondrocytes, but it is unknown how mutant COMP interacts with these proteins. The studies herein focus on defining the organization of these intracellularly retained proteins using fluorescence deconvolution microscopy. A unique matrix organization was identified in which type II procollagen formed a central core surrounded by a protein network of mutant COMP, type IX collagen, and MATN3. This pattern of matrix organization was found in multiple cisternae from single chondrocytes and in chondrocytes with different COMP mutations, indicating a common pattern of interaction. This suggests that stalling of mutant COMP and an interaction between mutant COMP and type II procollagen are initiating events in the assembly of matrix in the rER, possibly explaining why the material is not readily cleared from the rER. Altogether, these data suggest that mutant COMP initiates and perhaps catalyzes premature intracellular matrix assembly. Cartilage oligomeric matrix protein (COMP/TSP5), a large extracellular glycoprotein, is a pentameric member of the thrombospondin gene family.1Adams J Tucker RP Lawler J The Thrombospondin Gene Family. Springer-Verlag, New York1995Google Scholar COMP is primarily found in musculoskeletal tissues and has recently been identified as a marker for osteoarthritis.2DiCesare PE Morgelin M Mann K Paulsson M Cartilage oligomeric matrix protein and thrombospondin 1. 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Even though the role of these proteins in the extracellular matrix (ECM) seems to be structural, the individual loss of either COMP or type IX collagen or MATN3 does not produce a dwarf phenotype in mice.38Svensson L Aszodi A Heinegard D Hunziker EB Reinholt FP Fassler R Oldberg A Cartilage oligomeric matrix protein-deficient mice have normal skeletal development.Mol Cell Biol. 2002; 22: 4366-4371Crossref PubMed Scopus (141) Google Scholar, 39Fässler R Schnegelsberg PN Dausman J Shinya T Muragaki Y McCarthy MT Olsen BR Jaenisch R Mice lacking alpha 1 (IX) collagen develop noninflammatory degenerative joint disease.Proc Natl Acad Sci USA. 1994; 91: 5070-5074Crossref PubMed Scopus (265) Google Scholar, 40Nakata K Ono K Miyazaki J Olsen BR Muragaki Y Adachi E Yamamura K Kimura T Osteoarthritis associated with mild chondrodysplasia in transgenic mice expressing alpha 1(IX) collagen chains with a central deletion.Proc Natl Acad Sci USA. 1993; 90: 2870-2874Crossref PubMed Scopus (168) Google Scholar, 41Ko Y Kobbe B Nicolae C Miosge N Paulsson M Wagener R Aszodi A Matrilin-3 is dispensable for mouse skeletal growth and development.Mol Cell Biol. 2004; 24: 1691-1699Crossref PubMed Scopus (57) Google Scholar However, recent work suggests that correct integration and amount of COMP and MATN3 into the matrix depends on the presence of type IX collagen.12Budde B Blumbach K Ylostalo J Zaucke F Ehlen HW Wagener R Ala-Kokko L Paulsson M Bruckner P Grassel S Altered integration of matrilin-3 into cartilage extracellular matrix in the absence of collagen IX.Mol Cell Biol. 2005; 25: 10465-10478Crossref PubMed Scopus (110) Google Scholar This finding also suggests that the interaction of these proteins is important for correct matrix assembly, but individual loss of one protein is not sufficient to disrupt normal cartilage development and growth. In vivo and in vitro immunostaining studies have shown that the PSACH matrix is deficient in COMP, type IX collagen, and MATN3 and that these proteins are abundant in the rER cisternae of PSACH chondrocytes.26Hecht JT Hayes E Haynes R Cole WG COMP mutations, chondrocyte function and cartilage matrix.Matrix Biol. 2005; 23: 525-533Crossref PubMed Scopus (89) Google Scholar, 28Hecht JT Makitie O Hayes E Haynes R Susic M Montufar-Solis D Duke PJ Cole WG Chondrocyte cell death and intracellular distribution of COMP and type IX collagen in the pseudoachondroplasia growth plate.J Orthop Res. 2004; 22: 759-767Crossref PubMed Scopus (70) Google Scholar In contrast, type II collagen and aggrecan, the large aggregating proteoglycan, are efficiently secreted with little being retained in the rER.26Hecht JT Hayes E Haynes R Cole WG COMP mutations, chondrocyte function and cartilage matrix.Matrix Biol. 2005; 23: 525-533Crossref PubMed Scopus (89) Google Scholar, 28Hecht JT Makitie O Hayes E Haynes R Susic M Montufar-Solis D Duke PJ Cole WG Chondrocyte cell death and intracellular distribution of COMP and type IX collagen in the pseudoachondroplasia growth plate.J Orthop Res. 2004; 22: 759-767Crossref PubMed Scopus (70) Google Scholar It is unknown why type II collagen is not affected by COMP mutations. In this study, we use fluorescence deconvolution microscopy with image reconstruction and protein modeling to further investigate the effect of different COMP mutations on type II and IX collagens and MATN3, both in the large rER cisternae and the PSACH matrix. We find that type II collagen is the primary structural protein in PSACH pericellular matrix, just as in the wild-type ECM but, importantly, that type II procollagen plays a role in the accumulation of COMP, type IX collagen, and MATN3 into a matrix in the rER cisternae. The samples used in this study were previously described.26Hecht JT Hayes E Haynes R Cole WG COMP mutations, chondrocyte function and cartilage matrix.Matrix Biol. 2005; 23: 525-533Crossref PubMed Scopus (89) Google Scholar, 28Hecht JT Makitie O Hayes E Haynes R Susic M Montufar-Solis D Duke PJ Cole WG Chondrocyte cell death and intracellular distribution of COMP and type IX collagen in the pseudoachondroplasia growth plate.J Orthop Res. 2004; 22: 759-767Crossref PubMed Scopus (70) Google Scholar Normal growth plate (NGP) was used as the control standard to compare the immunostaining of our cultured chondrocyte nodules. Costochondral control and three different PSACH chondrocyte cell lines, with D469del, G427E, and D511Y mutations, were used in these experiments to generate cartilage nodules using our standard laboratory protocol.26Hecht JT Hayes E Haynes R Cole WG COMP mutations, chondrocyte function and cartilage matrix.Matrix Biol. 2005; 23: 525-533Crossref PubMed Scopus (89) Google Scholar, 28Hecht JT Makitie O Hayes E Haynes R Susic M Montufar-Solis D Duke PJ Cole WG Chondrocyte cell death and intracellular distribution of COMP and type IX collagen in the pseudoachondroplasia growth plate.J Orthop Res. 2004; 22: 759-767Crossref PubMed Scopus (70) Google Scholar All cartilage nodules were grown in nonadherent culture conditions for 56 days. Nodules were fixed in 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO), embedded in paraffin, and sectioned using standard techniques.26Hecht JT Hayes E Haynes R Cole WG COMP mutations, chondrocyte function and cartilage matrix.Matrix Biol. 2005; 23: 525-533Crossref PubMed Scopus (89) Google Scholar Tissue sections were deparaffinized and then rinsed in dH2O for 5 minutes. The sections were serially digested with 1) collagenase (type IA) (Sigma-Aldrich) at 1 mg/ml in phosphate-buffered saline (PBS) for 45 minutes at 37°C; 2) hyaluronidase (type I-S: bovine) (Sigma-Aldrich) at 1 mg/ml in PBS, pH 5.0, for 30 minutes at 37°C; and 3) pepsin A (Sigma-Aldrich) at 1 mg/ml in 0.1 N HCL for 30 minutes at 37°C with 5-minute dH2O rinses between digestions. Sections were washed in PBS containing 0.05% Tween 20 (PBS-T) for 5 minutes, followed by a 20-minute incubation with 10% goat serum in PBS-T to reduce nonspecific antibody binding. Subsequently, the antibodies were tested individually (data not shown) and then simultaneously incubated on sections at room temperature for 120 minutes: 1) COMP rabbit polyclonal antibody (Kamiya Biomedical, Seattle, WA) at 1:1000, 2) COL2 goat polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at 1:200, and 3) COL9 mouse monoclonal antibody (Iowa Hybridoma Bank, Iowa City, IA) at 1:200. Three fluorescently tagged secondary polyclonal antibodies were chosen from the following: Alexa Fluor 488, 594, 647, and 750 and used at a 1:250 dilution for 30 minutes at room temperature. The sections were washed in PBS-T and incubated with MATN3 antibody (generously provided by Drs. D.A. Hanson and D.R. Eyre, University of Washington, Seattle, WA) at 1:1500 dilution for 120 minutes at room temperature using the Zenon Labeling Alexa Fluor 488, 594, or 750 kits (Molecular Probes, Eugene, OR) following the manufacturer's instructions. Wavelength combinations were selected to visualize all of the proteins in each image acquisition sequence. 4′-6′-Diamidino-2-phenylindole, 100 mg/ml; Molecular Probes) was used to visualize nuclei but was removed in the analysis. Coverslips were mounted using ProLong Gold anti-fade reagent (Molecular Probes) and secured to glass slides. The sections were visualized by scanning from bottom to top of each tissue section on a DeltaVision scanning fluorescence microscope system (Applied Precision, Issaquah, WA) fitted with an Olympus IX70 microscope inverted microscope using a 100-W mercury arc lamp for illumination (Olympus America, Melville, NY) using appropriate excitation/emission filter sets (Chroma Technology Corp., Brattleboro, VT) specific for each of the fluorescent antibodies). Acquisitions were made at a constant gain, set for maximum fluorescent capture. The acquired images (PXL CCD camera; Photometrics, Tucson, AZ) were all taken at ×40 magnification with an aperture of 1.35 (UAPO 340). All images were then deconvoluted for 10 to 15 iterations to remove extraneous fluorescence (SoftWoRx software; Applied Precision), stacked, volume-rendered, and computer modeled with SoftWoRx imaging software as previously described.42Poindexter BJ Immunofluorescence, deconvolution microscopy and image reconstruction of human defensins in normal and burned skin.J Burns Wounds. 2005; 4: 128-140Google Scholar, 43Bick RJ Poindexter BJ Schiess MC Localization of calcitonin gene-related peptide in cardiomyocytes.Peptides. 2005; 26: 331-336Crossref PubMed Scopus (8) Google Scholar, 44Poindexter BJ Pereira-Smith OM Smith JR Buja LM Bick RJ 3-Dimensional reconstruction and localization of mortalin by deconvolution microscopy.Microsc Analysis. 2001; 89: 21-23Google Scholar Previously, we found that COMP mutations affect the secretion of type IX collagen and MATN3 in PSACH chondrocytes.26Hecht JT Hayes E Haynes R Cole WG COMP mutations, chondrocyte function and cartilage matrix.Matrix Biol. 2005; 23: 525-533Crossref PubMed Scopus (89) Google Scholar, 28Hecht JT Makitie O Hayes E Haynes R Susic M Montufar-Solis D Duke PJ Cole WG Chondrocyte cell death and intracellular distribution of COMP and type IX collagen in the pseudoachondroplasia growth plate.J Orthop Res. 2004; 22: 759-767Crossref PubMed Scopus (70) Google Scholar, 45Chen TL Stevens JW Cole WG Hecht JT Vertel BM Cell-type specific trafficking of expressed mutant COMP in a cell culture model for PSACH.Matrix Biol. 2004; 23: 433-444Crossref PubMed Scopus (25) Google Scholar The focus of the current studies was to use a novel imaging approach, fluorescence deconvolution microscopy, to determine 1) whether there is a difference in ECM produced by chondrocytes with PSACH mutations compared with normal chondrocytes, 2) whether there is a specific pattern to the ECM protein retention, and 3) whether it varied by type of COMP. For these studies, we used NGP and control chondrocyte nodules as standards to compare to PSACH chondrocyte nodules with D469del, G427E, and D511Y mutations.26Hecht JT Hayes E Haynes R Cole WG COMP mutations, chondrocyte function and cartilage matrix.Matrix Biol. 2005; 23: 525-533Crossref PubMed Scopus (89) Google Scholar, 28Hecht JT Makitie O Hayes E Haynes R Susic M Montufar-Solis D Duke PJ Cole WG Chondrocyte cell death and intracellular distribution of COMP and type IX collagen in the pseudoachondroplasia growth plate.J Orthop Res. 2004; 22: 759-767Crossref PubMed Scopus (70) Google Scholar First, we stained sections from NGP, control, and PSACH cartilage nodules to visualize the distribution of four ECM proteins, COMP, types II and IX collagens, and MATN3, using fluorescence deconvolution microscopy. As expected, in the NGP and control cartilage matrices, all of the proteins co-localized to the pericellular matrix and to a lesser extent to the interterritorial and territorial matrices (Figure 1, A–J). For these normal (nonmutant) chondrocytes, no intracellular retention could be appreciated. Only COMP, type IX collagen, and MATN3 are shown in Figure 1, E, J, O, T, and Y, because more than three channel acquisitions renders the images difficult to interpret. In comparison, the PSACH nodule showed minimal immunostaining of COMP, type IX collagen, and MATN3 in the matrices surrounding the cells (Figure 1, L–O, Q–T, and V–Y, respectively). In contrast, type II collagen was seen in the matrices of all of the nodules (Figure 1, A, F, K, P, and U), although less was appreciated in all of the PSACH matrices (Figure 1, K, P, and U). Intracellular retention of all four proteins, COMP, types II and IX collagens, and MATN3, was observed in the PSACH chondrocytes with different mutations (Figure 1, K–Y), and the proteins co-localized in cisternae (Figure 1, O, T, and Y) (type II collagen localization not shown). We have previously reported that these proteins also co-localize with calreticulin, a rER resident chaperone protein, demonstrating that these proteins reside in the rER.27Hecht JT Hayes E Snuggs M Decker G Montufar-Solis D Doege K Mwalle F Poole R Stevens J Duke PJ Calreticulin, PDI, Grp94 and BiP chaperone proteins are associated with retained COMP in pseudoachondroplasia chondrocytes.Matrix Biol. 2001; 20: 251-262Crossref PubMed Scopus (81) Google Scholar These results are similar to those observed in PSACH patient chondrocyte samples in vivo and de-monstrate that our nonadherent nodule culture system recapitulates the PSACH chondrocyte cellular phenotype.26Hecht JT Hayes E Haynes R Cole WG COMP mutations, chondrocyte function and cartilage matrix.Matrix Biol. 2005; 23: 525-533Crossref PubMed Scopus (89) Google Scholar, 28Hecht JT Makitie O Hayes E Haynes R Susic M Montufar-Solis D Duke PJ Cole WG Chondrocyte cell death and intracellular distribution of COMP and type IX collagen in the pseudoachondroplasia growth plate.J Orthop Res. 2004; 22: 759-767Crossref PubMed Scopus (70) Google Scholar To more fully realize the spatial arrangements and interactions of these proteins in the rER cisternae, we captured chondrocyte images from each sample and deconvolved the images. These models are shown as wire-frame renditions to allow visualization of the distribution of the individual proteins in the models (Figure 2). Again, both the NGP and control nodule chondrocytes showed the expected pericellular and territorial matrix distribution of the ECM proteins (Figure 2, A and B), as discerned previously26Hecht JT Hayes E Haynes R Cole WG COMP mutations, chondrocyte function and cartilage matrix.Matrix Biol. 2005; 23: 525-533Crossref PubMed Scopus (89) Google Scholar, 28Hecht JT Makitie O Hayes E Haynes R Susic M Montufar-Solis D Duke PJ Cole WG Chondrocyte cell death and intracellular distribution of COMP and type IX collagen in the pseudoachondroplasia growth plate.J Orthop Res. 2004; 22: 759-767Crossref PubMed Scopus (70) Google Scholar and in Figure 1. In contrast, the matrix surrounding the PSACH chondrocytes was primarily composed of type II collagen (Figure 2, C–E); COMP, type IX collagen, and MATN3 were markedly diminished in the PSACH matrices compared with the controls. Previously, it has been shown that type II collagen in PSACH chondrocytes is secreted into the ECM and not significantly retained in the rER.26Hecht JT Hayes E Haynes R Cole WG COMP mutations, chondrocyte function and cartilage matrix.Matrix Biol. 2005; 23: 525-533Crossref PubMed Scopus (89) Google Scholar, 28Hecht JT Makitie O Hayes E Haynes R Susic M Montufar-Solis D Duke PJ Cole WG Chondrocyte cell death and intracellular distribution of COMP and type IX collagen in the pseudoachondroplasia growth plate.J Orthop Res. 2004; 22: 759-767Crossref PubMed Scopus (70) Google Scholar Here, we found that type II procollagen wa
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