Effect of double knockout of cartilage-specific mitogen-inducible gene 6 and transforming growth factor-alpha on joint homeostasis

Abstract

Purpose: Osteoarthritis (OA) is a prevalent joint disease that involves the degeneration of smooth articular cartilage found at interfacing bone surfaces in synovial joints. The mRNA for transforming growth factor-alpha (Tgfa, a potential ligand of epidermal growth factor receptor (EGFR)), has been seen upregulated in a subset of OA cases, both in rat models and human patients (Appleton et al., 2007). Mitogen-inducible gene 6 (Mig-6) is induced by EGFR signaling and acts as a negative feedback regulator by sterically inhibiting additional downstream signaling and targeting EGFR for internalization. In previous studies, mice with a cartilage-specific knockout (KO) of Mig-6 have exhibited anabolic increases in articular cartilage thickness, suggesting that decreased EGFR signaling can mitigate OA effects (Pest et al, 2014). However, development of chondro-osseous nodules (CONs) within the joint capsule in this phenotype restricts movement of the joint, which can be debilitating. We aim to attenuate EGFR signaling by inhibiting Tgfa production in Mig-6-KO mice to further understand the role of this pathway in joint homeostasis. Methods: Male mice displaying cartilage-specific Mig-6 deletion with both homozygous Tgfa wild type (Mig-6fl/flCol2a1-Cre+ Tgfa+/+) and Tgfa-null (Mig-6fl/flCol2a1-Cre+ Tgfa-/-) genotypes, along with male mice displaying control genotypes (Mig-6fl/flCol2a1-Cre- Tgfa+/+ and Mig-6fl/flCol2a1-Cre- Tgfa-/-), were generated and raised to 12 weeks of age. Mice from different litters were age-matched in order to achieve N ≥ 5. Joint structure was then assessed using toluidine blue histological staining. Average articular cartilage thickness and cell density were obtained from sagittal sections of the elbow and knee joint, as well as from frontal sections of the knee. Immunohistochemical analysis was also performed for SOX9, a transcription factor and key marker for both chondrocyte development and phenotype maintenance. Results: Regardless of the absence or presence of TGFa, a significant increase in articular cartilage thickness was found at the surfaces of the humerus, ulna, femur, and tibia in Mig-6-KO models compared to controls. Similarly, cartilage-specific Mig-6-KO, but not whole-body TGFa KO, significantly increased cell density in mice 12 weeks of age. Formation of CONs were found in the knee joints of Mig-6-KO mice regardless of TGFa presence. Unexpectedly, Tgfa-null Mig-6-KO mice appeared to develop more severe CONs that were larger in area with increased proteoglycan staining compared to Mig-6-KO mice with TGFα. Immunochemical staining for SOX9 also appeared more intense in Mig-6-KO mice without TGFα compared to controls. Conclusions: This study indicates that MIG-6 plays an integral role in regulating articular cartilage growth in synovial joints and deletion of Mig-6 results in an anabolic phenotype. However, at the time point examined, TGFα did not have an effect on articular cartilage thickness or cell density. This suggests that the anabolic phenotype in Mig6-deficient mice arises from the role of MIG-6 in other non-EGFR specific mechanisms, or through alternate ligand activation of EGFR.