OA-associated catabolic factors up-regulate Regnase-1 in mouse articular chondrocytes
To identify RNases that may be associated with OA pathogenesis, we analyzed the expression levels of RNases (both endonucleases and exonucleases) in primary-culture articular chondrocytes stimulated with OA-associated catabolic factors, including a pro-inflammatory cytokine (IL-1β)  and adenovirus-mediated overexpression of HIF-2α (Ad-HIF-2α)  or ZIP8 (Ad-ZIP8) . Our microarray analysis revealed that Regnase-1 (encoded by Zc3h12a) was exclusively up-regulated in chondrocytes stimulated with the tested catabolic regulators (Fig. 1A, Supplementary Table 2). We further characterized the expression of Regnase-1 in primary-culture chondrocytes. Unstimulated chondrocytes exhibited various mRNA levels of ZC3H12 family members (Fig. 1B), whereas our RT-PCR (Supplementary Fig. 1) and quantitative RT-PCR (qRT-PCR) (Fig. 1B) analyses revealed that Regnase-1 was exclusively up-regulated by the OA-associated catabolic factors (i.e., IL-1β treatment or infection of Ad-HIF-2α or Ad-ZIP8). Regnase-1 protein levels were also increased by Ad-Regnase-1 infection or IL-1β treatment in primary-culture chondrocytes (Fig. 1C).
Overexpression of Regnase-1 in joint tissues ameliorates post-traumatic OA cartilage destruction in mice
Because the above results suggest that up-regulated Regnase-1 in OA-like chondrocytes may play a role in OA pathogenesis, we examined the functions of Regnase-1 in OA development by overexpressing it in mouse knee joint tissues via intra-articular (IA) injection of GFP-tagged Ad-Regnase-1. We previously reported that an adenoviral system can be used to effectively deliver transgenes into joint tissues [7–9]. Consistent with this, detection of GFP fluorescence confirmed the overexpression of Regnase-1 in cartilage tissue (Fig. 2A). However, this overexpression of Regnase-1 did not cause any OA-like change in the cartilage or synovium of the joint tissues (Fig. 2B). Consistently, Regnase-1 overexpression in primary-culture chondrocytes did not affect mRNA levels of matrix-degrading enzymes (MMP3, MMP13, and ADAMTS5), cartilage ECM molecules (type II collagen and aggrecan), or cellular catabolic regulators (HIF-2α and ZIP8) (Fig. 2C).
We further examined the effects of Regnase-1 overexpression in knee joint tissues under DMM-induced post-traumatic OA. Unlikely to the effects of Regnase-1 overexpression alone, adenoviral overexpression of Regnase-1 in joint tissues in DMM-operated mice significantly suppressed post-traumatic OA cartilage destruction (Fig. 2D and E). Other examined manifestations of OA, such as osteophyte formation and thickness of the subchondral bone plate (SBP), also tended to be reduced by Regnase-1 overexpression, but the differences did not reach the level of statistical significance (Fig. 2D and E). These findings suggest that overexpression of Regnase-1 alone does not cause OA pathogenesis; instead, up-regulated Regnase-1 in OA chondrocytes appears to play a protective role in DMM-induced post-traumatic cartilage destruction in mice.
Knockdown of Regnase-1 exacerbates post-traumatic OA cartilage destruction.
To further elucidate the functions of Regnase-1 in OA pathogenesis, we generated Zc3h12a KO mice or by knocking down Regnase-1 in whole-joint tissues via IA injection of an adenovirus expressing shRNA against Regnase-1 (Ad-shRegnase-1). IA injection of Ad-shRegnase-1 alone did not cause cartilage damage (Supplementary Fig. 2A). Additionally, Regnase-1 knockdown in primary-culture chondrocytes did not affect the expression levels of matrix-degrading enzymes or cartilage ECM molecules (Supplementary Fig. 2B). However, knock-down of Regnase-1 via IA injection of Ad-shRegnase-1in DMM-operated mice significantly enhanced DMM-induced OA manifestations such as cartilage destruction, osteophyte formation, and thickening of the SBP plate (Fig. 3A and B). This is consistent with the idea that up-regulated Regnase-1 in OA chondrocytes has chondro-protective effects in DMM-induced OA pathogenesis.
We further validated the functions of Regnase-1 in OA pathogenesis by using a Regnase-1 KO mouse generated by deleting 68 bp from exon 2 of the Zc3h12a gene (Supplementary Fig. 3A and B). Because homozygous KO mice (Zc3h12a−/−) exhibit pre-mature death within 12 weeks of birth , we used heterozygous Zc3h12a+/− mice for our experimental OA studies. Zc3h12a+/− mice exhibited marked decreases in the mRNA levels of Regnase-1 in the chondrocytes of their cartilage tissues, but had normal skeletal development in E18.5 embryos (Supplementary Fig. 3C). Compared with WT littermates, Zc3h12a+/− mice exhibited significant enhancement of DMM-induced cartilage erosion but no significant change in osteophyte formation or SBP thickness (Fig. 3C and D). These results additionally indicate that up-regulated Regnase-1 exerts protective functions in DMM-induced post-traumatic OA cartilage destruction in mice.
Regnase-1 modulates the expression levels of matrix-degrading enzymes in chondrocytes
To elucidate possible mechanisms underlying the protective effects of Regnase-1, we examined whether Regnase-1 modulates the expression levels of matrix-degrading enzymes and cartilage ECM molecules in chondrocytes. For this purpose, Regnase-1 was overexpressed in primary-culture chondrocytes via Ad-Regnase-1 infection, and the chondrocytes were stimulated with IL-1β. Among the examined molecules, the mRNA level of MMP3 (but not those of MMP13, ADAMTS5, or cartilage ECM molecules) was slightly but significantly decreased by the overexpression of Regnase-1 in IL-1β-treated chondrocytes (Fig. 4A and B). Similarly, the HIF-2α-induced increase in the mRNA level of MMP3 was also slightly but significantly decreased by Regnase-1 overexpression, whereas the mRNA levels of the other examined catabolic and anabolic factors were not modulated (Fig. 4C and D).
While Regnase-1 overexpression caused a relatively small modulation of MMP3 expression, Zc3h12a−/− chondrocytes exhibited a more dramatic modulation of the expression levels of matrix-degrading enzymes: Zc3h12a−/− chondrocytes exhibited significantly increased mRNA levels of MMP3, MMP13, and ADAMTS5 under IL-1β treatment, compared to IL-1β-treated WT chondrocytes (Fig. 5A). The up-regulations of MMP3, MMP13, and ADAMTS5 in HIF-2α-overexpressing chondrocytes were also significantly enhanced in Zc3h12a−/− chondrocytes (Fig. 5B). The enhanced expression levels of matrix-degrading enzymes in Zc3h12a−/− chondrocytes were well consistent with the enhanced OA cartilage destruction seen in DMM-operated Zc3h12a−/− mice.
Although overexpression or knockdown of Regnase-1 alone did not modulate expression of matrix-degrading enzymes in chondrocytes (Fig. 2C, Supplementary Fig. 2B), up-regulation of matrix-degrading enzymes by OA-associated catabolic factor such as IL-1β and HIF-2α was significantly inhibited by Regnase-1 overexpression (Fig. 4) and potentiated by Regnase-1 knock-out in chondrocytes (Fig. 5). Therefore, it is likely that up-regulated Regnase-1 in OA chondrocytes may suppresse cartilage destruction by forming negative feedback loop of catabolic signaling such as expression of matrix-degrading enzymes in chondrocytes.
Identification and characterization of Regnase-1 targets in chondrocytes
Regnase-1 directly binds to stem-loop structures in the 3'-UTRs of target mRNAs to cause their decay (11, 12). To identify target mRNAs of Regnase-1 in chondrocytes, we infected primary-culture chondrocytes with Ad-Regnase-1 or Ad-shRegnase-1 to up- and down-regulate Regnasse-1, respectively, and conducted microarray analyses. From among the 161 genes found to be down-regulated (< 0.6-fold) by Regnase-1 overexpression, only nine were up-regulated (> 1.5-fold) by Regnase-1 knockdown (Fig. 6A, Supplementary Table 3). In contrast, of the 67 genes that were up-regulated (> 1.5-fold) by Regnase-1 knockdown, 10 genes were down-regulated (< 0.7-fold) by Regnase-1 overexpression (Fig. 6A, Supplementary Table 4). The genes that were up-regulated by Regnase-1 overexpression or down-regulated by Regnase-1 knockdown are listed in Supplementary Tables 5 and 6, respectively.
Of the putative targets of Regnase-1, serum amyloid A3 (SAA3) was the most markedly up-regulated by Regnase-1 knockdown and down-regulated by Regnase-1 overexpression (Fig. 6B). SAA3 is a member of the SAA protein family of apolipoproteins. Different isoforms of SAA are expressed constitutively (constitutive SAAs) at different levels or in response to inflammatory stimuli (acute-phase SAAs) . We first used the SAA3-3´-UTR reporter plasmid to test whether SAA3 was a bona fide direct target of Regnase-1. We found that the expression of WT Regnase-1 reduced luciferase activity, whereas that of D141N Regnase-1, which lacks RNase activity , did not (Fig. 6C). In addition to SAA3, we also found that SAA1, SAA2, and SAA4 also tended to be up-regulated by Ad-shRegnase-1 and down-regulated by Ad-Regnase-1 (Fig. 6B, Supplementary Fig. 4). Among the SAA family members, SAA3 was the most abundant in chondrocytes (Fig. 6D) and the most significantly modulated by Regnase-1 expression (Supplementary Fig. 4). We therefore selected SAA3 as a Regnase-1 target for functional analysis in OA pathogenesis.
SAA3, a Regnase-1 target, does not modulate OA pathogenesis
The function of SAA3 in OA pathogenesis was examined by its overexpression in joint tissues via IA injection of Ad-SAA3. Compared with Ad-C injection, IA injection of Ad-SAA3 in mice did not affect cartilage homeostasis or cause synovial inflammation (Fig. 6E). Consistently, overexpression of SAA3 in primary-culture chondrocytes did not modulate the mRNA levels of matrix-degrading enzymes (MMP3, MMP13, and ADAMTS5) or cartilage ECM molecules (type II collagen and aggrecan) (Fig. 6F). Furthermore, IA injection of Ad-SAA3 in sham- or DMM-operated mice did not affect post-traumatic OA manifestations, such as cartilage destruction, osteophyte formation, and synovitis (Fig. 6G and H). Our results collectively indicate that SAA3 overexpression in joint tissues did not cause or modulate OA pathogenesis in mice, suggesting that the chondro-protective functions of Regnase-1 in post-traumatic OA cartilage destruction is mediated by unidentified other target(s) of Regnase-1, rather than SAA3, in chondrocytes.