"2006 Merrell, Allyson"
Up-Regulation of DDR2 and MMP13 as a Mechanism of Osteoarthritis Merrell, Allyson and Merrell, Kenneth Faculty Mentor: Robert E. Seegmiller, Physiology and Developmental Biology Background Arthritis is a painful, debilitating disease that affects sixty-six million Americans (The Arthritis Foundation 2005). Osteoarthritis (OA) results in joint degeneration caused by cartilage erosion and synovial inflammation. While the causes of OA remain unclear, current evidence suggests that both mechanical and biochemical factors can initiate OA. Since ethical and practical issues limit the availability of human tissue in OA research it is necessary to use animal models. Mice with the disproportionate micromelia mutation (Dmm) exhibit histological characteristics similar to those of humans with OA. Current research suggests the heterozygous Dmm mouse (D/+) is a valid model for early-onset OA (Bomsta 2005). To use D/+ mice as a model for OA more work is needed to better understand the mechanisms involved in the D/+ pathology. Through this study we have further established the mechanism by which OA onset is initiated in D/+ mice. Articular cartilage is composed of chondrocytes and the extracellular matrix (ECM) that surrounds the cells. Within the ECM collagen fibrils exist as structural support for the cartilage. Collagenases maintain the integrity of the cartilage by remodeling it as collagen fibers age or become damaged. This process requires a delicate balance between collagen production and degradation. The D/+ mutation affects the transport of type II collagen out of chondrocytes. This results in a decrease in this product in the ECM (Seegmiller 1997). Another murine model of OA, chondrodrodysplasia (cho), results from the mutation of the COL11 gene. Mice with this mutation exhibit increased levels of matrix metalloproteinase 13 (MMP13), associated with up- regulation of the Discoidin Domain Receptor 2 (DDR2) at six months (Xu 2004). MMP13 belongs to a family of MMPs that function by degrading proteins in the ECM. DDR2 is a plasma membrane receptor that up-regulates MMP13 through the Ras signal pathway. In an earlier study, tissues stained with antibodies for MMP13 and DDR2 revealed an increase in both MMP13 and DDR2 in cho mice in comparison with wild type mice (Xu 2004). We hypothesized that OA in D/+ mice was initiated by a similar mechanism up-regulating DDR2 and MMP13 to produce OA symptoms. Materials and Methods Knee joints were harvested from previously genotyped D/+ and wild-type +/+ mice, ages three, six and nine months. Knee joints were fixed with a PLP fixative, embedded into paraffin blocks, sectioned and stained. The medial fibular chondyle and its adjacent tibial cartilage, regions where OA lesions have previously been reported, comprised our specific area of observation. Adjacent sections were treated with antigen retrieval reagents to remove paraffin layering and expose tissue samples for staining. One slide per stratum, 30μm of tissue, was stained with Lilies Hematoxylin and Eosin, which allowed visualization of erosion and lesions. Another slide per stratum was incubated with testicular hyaluronidase, hydrogen peroxide, and serum blocking solution. Following the unmasking process the slides were incubated in primary antibody specific to 1 Up-Regulation of DDR2 and MMP13 as a Mechanism of Osteoarthritis DDR2 or MMP13 (goat) and subsequently in secondary antibody specific to the primary (donkey anti-goat). The secondary antibody is tagged with strepaviden, which is observable with light microscopy. Tissue samples were divided into three different wells, representing two negative controls and the target experimental tissue. Proximal to the frosted label of the slide tissue was isolated and treated solely with the chromagen and conjugate reagents in the absence of the primary and secondary antibodies. The central well was incubated in chromagen, conjugate reagents and secondary antibody, which served as a negative control through demonstration of any binding of the secondary antibody to off-target proteins. The distal well was bathed in primary and secondary antibody, conjugate and chromagen, and served as the experimental sections. Results To analyze the differential staining we assessed the stained tissue on a scale of one to three. A section with a score of one demonstrates little or no staining at the articular cartilage. A score of two represents patched or scattered staining throughout the articular surface. The final score of three reveals tissue samples that are universally stained in the articular cartilage. Representative samples are shown in figure one. Score-1 Score-2 Score-3 Figure 1. Scoring of tissue sections based upon degree of intensity of stain and thickness of staining in articular cartilage. At three months we observed relatively no difference in staining between the wild type and mutant animals with both DDR2 and MMP13 (figures two and three, respectively). The DDR2 samples were both scored with a two, while the MMP13 samples were scored with a one. Sixth month animals varied more in tissue staining. With both antibodies, wild type animals displayed staining with a score of two. Mutant mice had more intense staining and received a score of three. This increased staining appeared to be more intense in the matrix immediately surrounding the cells of the articular cartilage. The space decreases as the chondrocytes of D/+ mice lose their columnar organization in the cartilage and become randomly distributed. Nine month mice also had variance in tissue staining. Staining in +/+ mice appeared to be more intense and scored a three. In the D/+ animals, which scored a two, the articular cartilage had already undergone erosion. This erosion resulted in a decreased number of chondrocytes and therefore less intense DDR2 and MMP13 staining. 2 Up-Regulation of DDR2 and MMP13 as a Mechanism of Osteoarthritis DDR2 3 Month +/+ 3 Month D/+ 6 Month +/+ 6 Month D/+ 9 Month +/+ 9 Month D/+ Figure 2. Tibial sections of immunihistochemistry with anti-DDR2 antibodies. Arrows indicate areas of intense staining. 3 Up-Regulation of DDR2 and MMP13 as a Mechanism of Osteoarthritis MMP13 3 Month +/+ 3 Month D/+ 6 Month +/+ 6 Month D/+ 9 Month +/+ 9 Month D/+ Figure 3. Tibial sections of immunohistochemical staining with anti-MMP13 antibodies. Arrows indicate areas of intense staining. 4 Up-Regulation of DDR2 and MMP13 as a Mechanism of Osteoarthritis Discussion Based on our results, we believe that DDR2 and MMP13 play a role in normal and mutant progression of osteoarthritis. It appears in wild type mice that the onset of OA involving the DDR2/MMP13 pathway is initiated around 9 months of age. Based on earlier findings, wild type mice develop OA symptoms around this age, so it is possible that this pathway is a possible cause, as the staining in wild type mice increased between each successive age group. Supporting this is our data from the mutant mice. Intensity of staining in the mutants peaked at six months which coincides with findings of early onset of OA in D/+ mice. It appears that OA in D/+ mice could be initiated by the same pathway implicated in the onset of OA in cho mice. Perhaps a decrease in functional type II collagen results in a decrease in chondrocyte pericellular space. This would allow the type II collagen in the matrix to contact DDR2 at a more frequent rate. Subsequent up-regulation of DDR2 and MMP13 would result in further weakening of the matrix and increased collagen signaling. This would exacerbate the predisposition to OA. From our studies, it appears that this mechanism is occurring in D/+ mice around six months of age where we saw a peak in both DDR2 and MMP13 staining. By nine months, a decline in staining could be due to degeneration of the cartilage, which parallels previous data on the destruction of the cartilage. Our study confirms a plausible mechanism for early onset OA in Dmm mice similar to that found in cho mice (Xu 2004). References Arthritis Foundation. 2005. The Arthritis Foundation. October 7 2005 http://www.arthritis.org Bomsta, Brandon D., Laura C. Bridgewater, and Robert E. Seegmiller. “Premature osteoarthritis in the Disproportionate micromelia (Dmm) mouse.” Submitted for publication. 2005. Seegmiller, Robert. et al. “Disproportionate Micromelia (Dmm) in Mice Caused by a Mutation in the C-Propeptide Coding Region of COL2A1.” Developmental Dynamics. Vol 208 (1997). Xu, Lin et al. “Activation of the Discoidin Domain Receptor 2 Induces Expression of Matrix Metalloproteinase 13 Associated with Osteoarthritis in Mice.” Journal of Biological Chemistry. Vol 280, No. 1 (2004). 5 Up-Regulation of DDR2 and MMP13 as a Mechanism of Osteoarthritis