The role of osteoclastic miR-214-3p in early osteoarthritis development

Osteoarthritis (OA) is a highly prevalent degenerative joint disease that affects 7% of the men and 13% of the women aged 50 and above in Hong Kong and places large economic burden on Hong Kong health care system. The pathological features of OA include aberrant subchondral bone (SCB) remodeling, articular cartilage degradation, synovium inflammation and osteophyte formation. Currently, there is no proven disease-modifying drugs for OA due to the limited knowledge on the underlying mechanism responsible for the initiation and progression of OA. Although a primary role of articular cartilage in OA development has been extensively investigated, emerging evidences suggest that the active osteoclast-mediated SCB remodeling precedes the articular cartilage degradation and plays a critical role in the initiation and progression of OA. Interestingly, although several studies in the last decades showed that the anti-resorptive agents (bisphosphonates) could reduce cartilage degradation in the animal models of OA, a series of recent studies in turn have proved that the bisphosphonates failed to prevent cartilage degradation, or even exert a negative effect on articular cartilage, in OA animal models. More importantly, results from a phase III clinical trial has demonstrated that the anti-resorptive agents neither lessen the symptoms nor improve the radiographic progression in OA patients. These controversial results underscore the importance in identifying the early molecular events responsible for the activated SCB remodeling in early OA development.

In our previous studies, we have documented the pro-osteoclastogenic role of microRNA-214-3p (miR- 214-3p). Moreover, we have demonstrated that osteoclast could release exosomal miR-214-3p that could be detected in circulation. Interestingly, our recent preliminary real-time quantitative-PCR (qPCR) data showed that the SCB miR-214-3p expression levels were significantly higher in OA patients as compared to the non-OA controls. Furthermore, the SCB miR-214-3p levels were significantly higher in mild and moderate OA patients than those in severe OA patients. In a mouse model with surgery-induced OA, we observed the upregulation of miR-214-3p level within the SCB osteoclasts at the knee joint together with the increasing miR-214-3p level in serum from day 0 day to day 14 after anterior cruciate ligament transection (ACLT), which preceded the increase in the serum bone resorption marker CTX-I (C-terminal crosslinked telopeptide type I collagen) and cartilage degradation marker CTX-II (C-terminal crosslinked telopeptide type II collagen). Furthermore, we found that the OA osteoclasts differentiated from bone marrow monocytes/macrophages (BMMs) of the ACLT mice showed enhanced resorption activity in cartilage explant in vitro as compared to the control osteoclasts differentiated from BMMs of sham-operated mice. Consistently, the OA osteoclasts also expressed remarkably higher level of miR-214-3p than the control osteoclasts. In addition, we found that miR-214-3p could directly target TIMP2 (Tissue inhibitor of metalloproteinases 2, an endogenous MMP inhibitor) to promote OA osteoclast differentiation and enhance their resorption activity on cartilage explant in vitro. In our preliminary study, we also observed highly activated osteoclast-mediated SCB remodeling and accelerated articular cartilage degradation as well as lowly expressed TIMP2 in our established osteoclast-specific miR-214-3p conditional knockin (CKI) mice in the early period after ACLT. Thus, we hypothesized that the elevated osteoclastic miR-214-3p could target TIMP2 to promote the SCB remodeling to contribute to the early OA development. To facilitate testing the hypothesis, we have generated the osteoclast-specific miR-214-3p conditional knockout (CKO) mice and developed an osteoclast-targeting delivery system (OC-TDS) for evaluating the role of osteoclastic miR-214-3p in early OA development by genetic and pharmacological approaches, respectively.

In this proposal, we have the following three specific aims:
(1) To study the serum miR-214-3p level, SCB remodeling, TIMP2 expression and cartilage degradation by qPCR, micro-CT, bone histomorphometry, histology, immunohistochemistry (IHC) and enzyme-linked immunosorbent assay (ELISA) in CKO mice after ACLT.
(2) To investigate the SCB remodeling, TIMP2 expression and cartilage degradation by micro-CT, bone histomorphometry, histology, IHC and ELISA in CKI mice treated with TIMP2-3’UTR encapsulated in the OC-TDS after ACLT.
(3) To evaluate the serum miR-214-3p level, SCB remodeling, TIMP2 expression and cartilage degradation by qPCR, micro-CT, bone histomorphometry, histology, IHC and ELISA in ACLT mice after treatment with antagomir-214-3p encapsulated in the OC-TDS.

If our hypothesis could be supported, it would not only identify miR-214-3p as a novel therapeutic target for early treatment of OA, but also indicate a new serum biomarker for early diagnosis of OA.