THE ROLE OF OSTEOACTIVIN IN MUSCULOSKELETAL TISSUES AS A REPAIR AND ANABOLIC FACTOR

Abstract

Osteoactivin (OA) is a novel osteogenic and repair factor. It has the ability to regulate cell proliferation, adhesion, differentiation, and synthesis and regulation of extracellular matrix proteins in various cell types under both normal and pathological conditions. Initial identification of osteoactivin (OA)/glycoprotein non-melanoma clone B (gpnmb) was demonstrated in an osteopetrotic rat model, where OA expression was increased 3-fold in mutant bones, compared to normal. OA mRNA and protein expression increase during active bone regeneration post-fracture, and primary rat osteoblasts show increased OA expression during differentiation ex vivo. To further examine OA/gpnmb as an osteoinductive agent, we characterized the skeletal phenotype of transgenic mouse overexpressing OA/gpnmb under the CMV-promoter (OA-Tg). Western blot analysis showed increased OA/gpnmb in OA-Tg osteoblasts, compared to wild-type (WT). In OA-Tg mouse femurs versus WT littermates, micro-CT analysis showed increased trabecular bone volume and thickness, and cortical bone thickness; histomorphometry showed increased osteoblast numbers, bone formation and mineral apposition rates in OA-Tg mice; and biomechanical testing showed higher peak moment and stiffness. Given that OA/gpnmb is also over-expressed in osteoclasts in OA-Tg mice, we evaluated bone resorption by ELISA and histomorphometry, and observed decreased serum CTX-1 and RANK-L, and decreased osteoclast numbers in OA-Tg, compared to WT mice, indicating decreased bone remodeling in OA-Tg mice. The proliferation rate of OA-Tg osteoblasts ex vivo was higher, compared to WT, as was alkaline phosphatase staining and activity, the latter indicating enhanced differentiation of OA-Tg osteoprogenitors. Quantitative RT-PCR analysis showed increased TGF-β1 and TGF-β receptors I and II expression in OA-Tg osteoblasts, compared to WT. Together, these data suggest that OA overexpression has an osteoinductive effect on bone mass in vivo and stimulates osteoprogenitor differentiation ex vivo. OA expression increases during tissue degeneration and regeneration, fracture repair, and after denervation-induced disuse atrophy, concomitant with increased matrix metalloproteinases (MMPs). However, OA’s expression with repetitive overuse injuries is unknown. We sought to evaluate in an animal model of upper extremity repetitive overuse, at low force loads: 1) OA expression in an operant rat model of repetitive overuse; 2) expression of MMPs; 3) inflammatory cytokines indicative of injury or inflammation; and 4) the inducible form of heat shock protein 70 (HSPA1A/HSP72), a protein known to increase during metabolic stress and be involved in cellular repair. We hypothesized that OA is functioning as a growth factor during periods of tissue repair. Young adult, female Sprague-Dawley rats performed a high repetition negligible force (HRNF) food retrieval task for up to 6 weeks, and were compared to control rats. Quantitative PCR, Western blot analyses and immunohistochemistry showed increased OA mRNA and protein expression in flexor digitorum muscles of 6-week HRNF rats, compared to controls. OA protein levels increased similarly in 6-week HRNF flexor digitorum tendons. Increased OA immunostaining was localized to the myofiber sarcolemma, macrophage-like cells and tenocytes. In muscles, Western blot analyses showed progressive increases in MMP-1, -2 and -3, whereas tendons had increased MMP-1 and -3, with HRNF task performance. ELISA and immunohistochemistry showed increased HSP72 in 6-week HRNF muscles, and co-localization with OA in the myofiber sarcolemma. HSP72 increased in 6-week HRNF tenocytes, compared to controls. Inflammatory cytokines IL-1alpha or beta showed transient increases at 3 weeks in muscles and tendons, while IL-1alpha was significantly decreased in 6-week HRNF muscles. The simultaneous increases of MMPs and HSP72 with OA, factors involved in tissue repair, supports a role of OA in tissue regeneration after repetitive overuse. We extended the study above to examine the expression of OA during high repetition high force loading in our animal model of upper extremity overuse, in combination with anti-inflammatory drug, to evaluate OA’s link to inflammatory processes. Young adult female rats underwent an initial training period to learn the task (10 min/day, 5 days/wk, for 6 wks), before then performing a high repetition high force (HRHF) task for 11 weeks (2 hours/day, 3 days/week). Results were compared to age-matched control (C) rats. At the end of HRHF task week 3, two cohorts of HRHF rats received 5 intraperitoneal injections of saline (HRHF+Veh) or anti-rat TNF-a (HRHF+anti-TNF) across 4-7 weeks, as did controls (C+Veh and C+anti-TNF). Two other cohorts rested during weeks 4-7 with or without treatment (HRHF+anti-TNF/Rest and HRHF+Veh/Rest), to parallel its partner group. Motor behavior was assessed and revealed decreased grip strength in HRHF+Veh rats beginning immediately post training (HRHF task week 0), and that anti-TNF-α treatment prevented this grip strength decline. The 4-week anti-TNF-α therapy extended maintenance of grip strength near control levels through week 9, despite no further treatment after week 7. By experimental week 11, ELISA showed no significant differences in OA levels in forearm flexor digitorum muscles, and histomorphometry showed no difference in the circumference of this muscle in any HRHF group, compared to controls, matching findings of no gain in grip strength above control levels in any HRHF group. However, ELISA of distal radius and ulna homogenates showed increased OA levels in HRHF+anti-TNF rats, as well as increased IL-18 in bones of both anti-TNF treated HRHF groups (HRHF+anti-TNF and HRHF+anti-TNF/Rest rats), compared to controls. Micro-CT analysis showed that rats receiving anti-TNF-α treatment, with or without rest, had increased bone mass (detected as increased trabecular bone volume, thickness, and number and reduced trabecular separation), compared to the other groups. Histomorphometry showed increased osteoblast numbers in HRHF+anti-TNF rats, compared controls, yet decreased osteoclast numbers, compared to HRHF+Veh rats, indicative of increased bone anabolism in anti-TNF-a treated rats. Thus, these findings suggest that TNF-α blocks OA expression in bones, and that its increase when combined with prolonged repetitive loading, enhances osteoblast activity and bone formation.