Down‐Regulation of Rac GTPase‐Activating Protein OCRL1 Causes Aberrant Activation of Rac1 in Osteoarthritis Development

Abstract

Objective. Chondrocyte hypertrophy and mineralization are considered to be important pathologic factors in osteoarthritis (OA). We previously reported that Rac1 was aberrantly activated to promote chondrocyte hypertrophy, mineralization, and expression of matrix metalloproteinase 13 and ADAMTS in OA. However, the underlying mechanism of aberrant Rac1 activation in OA is unclear. The present study was undertaken to identify the specific molecular regulator controlling Rac1 activity in OA, as well as to investigate its function in chondrocyte hypertrophy, mineralization, and OA development.Methods. Expression levels of 28 upstream regulators of Rac1 activity, including 8 GTPase-activating proteins (GAPs) and 20 guanine nucleotide exchange factors, in OA and normal cartilage were assessed by quantitative polymerase chain reaction. Chondrocytes were transduced with lentiviral vectors encoding OCRL1, GAP, non-GAP, CA-Rac1, and DN-Rac1, either alone or in combination. Alkaline phosphatase staining was used as a marker of chondrocyte hypertrophy. Rac1 activity was analyzed by pulldown assay. Finally, OA was established in mice by surgical transection of the anterior cruciate ligament and cutting of the medial meniscus. The mice were injected intraarticularly with OCRL1-encoding lentivirus, and whole joints were assessed histologically 6 weeks after surgery.Results. OCRL1 was abundantly expressed in normal cartilage and was the only significantly downregulated RacGAP in OA cartilage. Overexpression of OCRL1 inhibited interleukin-1b-induced Rac1 activity, chondrocyte hypertrophy, and expression of hypertrophyrelated genes. Conversely, knockdown of OCRL1 elevated Rac1 activity and promoted chondrocyte hypertrophy and mineralization. Further, OCRL1 modulated Rac1 activity via its GAP domain. Finally, intraarticular injection of OCRL1-encoding lentivirus protected against destruction and degeneration of cartilage in the mouse OA model.Conclusion. OCRL1 acts as a RacGAP in cartilage to impede chondrocyte hypertrophy and OA development through modulating Rac1 activity. This regulatory pathway might provide potential targets for the development of new therapies for OA.Osteoarthritis (OA), characterized by cartilage degradation, synovial inflammation, and subchondral bone remodeling, is the most common form of degenerative joint disease (1). There is now a general consensus that chondrocytes in OA undergo hypertrophy and mineralization (characterized by expression of matrix metalloproteinase 13 [2], type X collagen [3],

Kdm6b regulates cartilage development and homeostasis through anabolic metabolism

“…First, we evaluated the knockdown efficiency of Kdm6b shRNA in the murine MSC line C3H10T1/2, and it was shown that the two shRNAs considerably reduced Kdm6b mRNA expression, with Kdm6b-sh2 displaying better efficiency (figure 4A). Then, PLKO and Kdm6b-sh2 were packaged into lentivirus for intra-articular injection of knee joint after DMM surgery, as described previously 11. At the same time, 8-week-old Col2a1-CreER T2 ;Kdm6b f/f mice and Kdm6b f/f littermates were intraperitoneally injected with TM daily for 5 days.…”

“…Destabilisation of medial meniscus (DMM) surgery was performed on the knee joints of 10-week-old male mice as previously reported 24. We performed intra-articular injection of lentivirus to C57BL/6J wild-type mice after surgical induction according to previously described procedures 11. All animal experiments were approved by the Zhejiang University Ethics Committee (NO.ZJU20160427).…”

Intra-articular Delivery of Antago-miR-483-5p Inhibits Osteoarthritis by Modulating Matrilin 3 and Tissue Inhibitor of Metalloproteinase 2

“…34 Similarly, intra-articular injection of antago-miR would infect not only articular cartilage but also the synovium, which plays an important role in OA development. However, it is technically difficult to identify the role of miR-483-5p in synovium tissue, articular chondrocytes, or other cells in this model.…”

“…With regard to lentivirus (GenePharma) injection, 10 mL lentivirus-mediated miR-483-5p (5 0 -AAGACGGGAGAAGAGAAGG GAG-3 0 ) (4 Â 10 8 TU/ml), siTimp2 (5 0 -GGAATGACATCTATGG CAA-3 0 ) (1 Â 10 9 TU/mL), or NC (5 0 -TTCTCCGAACGTGT CACGTTTC-3 0 ), Matn3 (NM_010770.4) (Cyagen Biosciences), TIMP2(NM_011594.3) (Cyagen Biosciences), and corresponding negative controls were injected into the knee joint 34 of male mice (n = 5/group) using a 33G needle and a micro-syringe (Hamilton). For the antago-miR-483-5p injection, 250 mM antago-miR-483-5p (5 0 -CUCCCUUCUCUUCUCCCGUCUU-3 0 ) or antago-mir NC (5 0 -UUGUACUACACAAAAGUACUG-3 0 ) (GenePharma) were injected.…”

The oculocerebrorenal syndrome of Lowe: an update

“…Synovial biopsy shows rubbery tissue without an inflammatory infiltrate and fibrous tissue containing fibrillary material [70]. Zhu et al have recently demonstrated abundant expression of OCRL in normal cartilage, which was downregulated in a mouse model of osteoarthritis and could be restored by intraarticular injection of ORCL -encoding lentivirus [71]. Musculoskeletal complications have not yet been reported in Dent-2 patients.…”