A total of nine relevant ECM proteins genes were found to be differentially expressed in the OVX group compared to the sham group (Figure 2A). Only one ECM gene were significantly up-regulated; Dentin matrix protein 1 (DMP1). DMP1 is expressed in mineralized tissues such as hypertrophic cartilage and bone, where it can bind Ca²⁺ and regulate matrix mineralization [¹⁰]. There were eight ECM genes down-regulated. Fibromodulin (FMOD) is known to interact with both type I and II collagen in bone and cartilage, respectively [¹¹]. Through its binding of collagens it aids stabilization of the tissue. Several collagens (Col) were down-regulated; type IVa2, VIa2, IXa and XV. Type VI and IX are some of the minor collagens of the mature cartilage that are known to interact with other matrix components, such as hyaluronan, heperan sulfate and Chondroitin sulfate proteoglycans. Type XV collagen is member of the FACIT collagen family (fibril-associated collagens with interrupted helices) and mainly expressed by fibroblastic cells [¹²]. Type IV collagen, is a collagen of the basal lamina and widely distributed throughout the body, like many other collagens it is involved in tissue stabilization and flexibility [¹²]. Two chondroitin sulfate proglycans were also down-regulated in the joint tissue of OVX rats: CSPG2 (Versican) and 4 (NG2) (Figure 2A). Both have been shown to be involved in the regulations of bone development, chondrogenesis and angiogenesis, but only present in low amounts in mature cartilage and bone [¹³]. Laminin alpha 5 (LAMa5), a subunit of laminin 5, which is observed in the basement membrane. It has been shown to promote bone growth and regeneration through cell adhesion and motility [¹⁴].

Eight ECM degrading proteases were up-regulated in the joints of the OVX rats (Figure 2B). Metalloproteinase (MMP) 8 (collagenase-2), 9 (gelatinase B), 11 (stromelysin-3) and 16 (MT3-MMP) are collagen and proteoglycan degrading protease, which have been implicated as drivers of joint degradation [¹⁵]. Cathepsins (Cat) C, Z, K and H, which are all cysteine proteases, have been shown to play vital role the regulation of tissue turnover. CatC is a dipeptidyl aminopeptidase, which is involved in the activation of serine proteases such as CatG and elastase [¹⁶]. CatZ is also a dipeptidyl peptidase, but its function in cartilage and bone turnover is unclear. CatK is mainly expressed by osteoclast and are responsible for the release of the bone turnover marker CTX-I. It has been found to be elevated in OVX animals and post-menopausal women. CatH cleaves osteocalcin in bone and might therefore be an important protease in bone turnover [¹⁷]. The A Disintegrin And Metallopeptidase 10 (ADAM10) is expressed by chondrocyte in bone development and in OA chondrocytes. It is therefore believed to be involved in the remodeling of cartilage [¹⁸].

Six proteases were down-regulated; MMP3 and the aggrecanases ADAMTS-like 4, ADAM15 and ADAMTS5, as well as bone morphogenic protein (BMP) 1 and tissue inhibitor metalloproteinase 3 (TIMP3) (Figure 2B). MMP3 can degrade fibrillar collagens such as type II and I collagen [¹⁹]. MMP3 has been shown to be elevated in inflammatory diseases, such as rheumatoid arthritis; however it is mainly expressed by the connective tissue like the synovial membrane and to a lesser degree by bone and cartilage. ADAM15 has been shown to cleave ECM components such as gelatin and type IV collagen. It is has been found to be upregrulated in OA chondrocytes [²⁰]. ADAMTS-like 4 and ADAMTS5 (ADAM with thrombin mortif) are known for their capability to degrade proteoglycans such as aggrecan and versican [²¹]. It intriguing that ADAMTS5 is down-regulated, since previous studies have indicated it to be a primary protease in OA cartilage degradation [²²]. In contrast, others have shown ADAMTS' are involved in both degradation and remodeling of the tissue [²³,²⁴]. The endogenous MMP and aggrecanase inhibitor TIMP3 was also down-regulated. TIMP3 is believed to be an important regulator of cartilage and bone turnover, through its inhibition of MMPs and aggrecanases [²¹]. In contrast to other BMPs, BMP1 does not belong to the TGF-β family of proteins. It is a metalloproteinase that acts on procollagen I, II, and III and has been implicated in cartilage development [²⁵].

Next we searched for factors directly involved in bone and/or cartilage regulation. We found that five genes were significantly up-regulated and 11 were significantly down-regulated in the OVX animals (Figure 2C). Sclerostin (SOST) was markedly over-expressed in the OVX rats. It is expressed by osteoclast and is a negative regulator of bone formation through activation of the Wnt pathway [²⁶]. BMP2 through BMP7 belong to the transforming growth factor beta superfamily of proteins. Whereas BMP6, -5 and -2 were down-regulated, BMP3 was up-regulated. BMP3 is known to induce bone formation and cartilage development and to have antagonistic effect on other BMPs [²⁷]. Transforming growth factors beta-2 (TGFb2) expression was also elevated. TGFb2 is a multi-factorial regulator of cellular growth in developing systems and in repair of adult bone and cartilage [²⁸]. TNFsf11, also known as RANKL, is a natural and necessary surface-bound molecule that activates osteoclasts, cells involved in bone resorption. Overproduction of RANKL is implicated in a variety of degenerative bone diseases, such as arthritis [²⁹]. Osteoclast stimulating factor 1 (OSTF1) expression was also increased in the OVX rats' joints (Figure 2C). It is known to increase osteoclast formation and bone resorption through signal transduction, however only little is known about the family member of osteoblast stimulation factors [³⁰]. Insulin-like growth factor 2 (IGF2), IGF binding protein 2 (IGFBP) and IGFBP6 were all down-regulated in the OVX rats. IGF2 is known to bind both to IGFBP2 and IGFBP6, and its effect on bone cells is thereby prevented (e.g. osteoblast proliferation) [³¹]. Connective tissue growth factor (CTGF) is a potent stimulator for proliferation and differentiation of osteoblast and chondrocytes through regulation of cellular associated genes [³²]. The tightly related proteins Fibroblast growth factor receptor 3 (FGFR3) and the parathyroid hormone-like hormone (PTHLH), which were both down-regulated, play central roles in the physiological regulation of bone formation, by promoting recruitment and survival of osteoblasts, and probably plays a role in the physiological regulation of bone resorption, by enhancing osteoclast formation. Signaling by FGFR3 and PTHLH coordinates cartilage and bone development. PTHLH is also an essential physiological regulator of adult bone mass [³³]. The calcitropic hormone Stanniocalcin 1 (STC1) was down-regulated in the OVX rats. It is involved in the regulation of longitudinal bone growth [³⁴]. Nitric oxide synthase 3 (NOS3, eNOS) was also down-regulated. It has been shown that estrogen induces NOS3 expression in endothelial cells and osteoblasts, raising the possibility that NO derived from the NOS3 pathway plays a role in mediating the effects of sex hormones on bone [³⁵]. BMP2, -5 and -6, which were down-regulated in the OVX rats (Figure 2C), all induces cartilage and bone formation and play role in joint integrity [²⁷].