Effect of Cartilage Oligomeric Matrix Protein (comp) on Chondrogenesis in Vitro

Introduction: Cartilage oligomeric matrix protein (COMP) is an N-terminal disulfide-linked, 524 kd homopentameric glycoprotein found primarily surrounding chondrocytes in the cartilage extracellular matrix. COMP gene mutations have been identified in two types of autosomal dominant chondrodysplasias that exhibit osteoarthritic phenotypes; pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED). Currently, the normal function of COMP is unknown. Understanding the normal function of COMP is crucial to deciphering its role in cartilage function and in forms of osteoarthritis such as MED and PSACH. We have recently cloned the full-length cDNA of mouse COMP. To learn more about the role of normal COMP and the effect of ectopic COMP expression on chondrogenesis, we examined the effect of COMP expression in an in vitro model cell system consisting of C3H10T1/2 mouse embryonic mesenchymal cells. C3H10T1/2 cells are pluripotential cells that are able to differentiate in culture along multiple pathways depending on the stimulus. When maintained as high density micromass cultures in the presence of bone morphogenetic protein-2 (BMP2), 10T1/2 cells preferentially undergo chondrogenesis, evident by Alcian Blue staining, metabolic sulfate incorporation, and expression of cartilage matrix genes. Methods: Wildtype human COMP cDNA was cloned into the pBABE retroviral vector. This pBABE-COMP construct was then transfected into the Phoenix-eco packaging cell line to make infectious virus. The control construct consists of the retroviral plasmid alone. Following transfection, cells were placed at 32C for 48 hours and the medium containing the infectious virus was then harvested, filtered and used to infect subconfluent (20%), proliferating 10T1/2 cells, which were then grown to confluence for an additional 72 hours. RNA was isolated for RT-PCR confirmation of COMP expression. Cells infected with either the control virus or the COMP construct were examined as follows. Proliferation rate was analyzed in low-density cultures based on cell counts and flow cytometry. For chondrogenesis, cells were placed into high-density (1x10 cells/ml) micromass cultures and treated with or without BMP2 (100ng/mL). The cultures were harvested at days 4, 7, and 10 and examined for 1) collagen type II expression by RT-PCR; 2) cartilage matrix deposition by alcian blue staining; 3) metabolic sulfate incorporation relative to leucine incorporation. Results and Discussion: Cultures of infected cells were monitored for expression of COMP by RT-PCR as they reached confluence. COMP is expressed at a high level in the cells infected with pBABE-COMP, in contrast to the control-infected cultures, which showed no COMP expression. Thus retroviral transduction of COMP leads to a detectable level of ectopic expression. Cell counting and flow cytometry revealed that COMP expressing cells had a demonstrable slow growth phenotype of nearly a two-fold decrease in cell number over a 4-day period. This reduction in the level of cell proliferation consisted of a two-fold decrease in the percent cells in both S phase and G2/M with a corresponding increase in the percent cells in G1 phase. Thus COMP appears to reduce the extent of cell proliferation in monolayer cultures by changing the cell cycle distribution pattern, leading to a partial cell cycle arrest. COMP expression in micromass cultures enhanced BMP2 induced chondrogenesis. Alcian blue staining was more intense in cultures of BMP2 treated cells expressing COMP compared to the control cells treated with BMP2 (Figure 1A), which was confirmed by scanning densitometry (Figure 1B). The relative level of SO4 incorporation into the extracellular matrix in COMP expressing cells was also nearly two times higher than that in the control cells by day 10, following BMP2 treatment (Figure 1C). This is a time dependent effect since the levels of SO4 incorporation were nearly equal between the control and COMP expressing cells prior to day 10. Finally, COMP expressing cells also showed an increase in the levels of collagen type II mRNA by day 7 as shown by RT-PCR analysis of RNA from the control and COMP expressing cells that were treated with BMP2 (Figure 1D). Taken together these results indicate that expression of COMP in 10T1/2 cells significantly enhanced BMP2 induced chondrogenesis. This effect is accompanied by a partial cell cycle arrest by day 4, significant stimulation of collagen type II gene expression, and sulfate proteoglycan expression by and after day 7 respectively. Since the first overt signs of BMP-2 induction of chondrogenesis in 10T1/2 cells become apparent by day 4-5, the COMP effect is likely to act in concert with and is in fact dependent on the BMP-2 effects (Note: COMP expression done without BMP-2 treatment shows minimal chondrogenesis). This is the first report on the effect of COMP, a compound of matrix cartilage on mesenchymal chondrogenesis, and studies suggest that COMP may play an important role in early cartilage development.