The number and activity of chondrocytes are the most important components in cartilage. Various factors can induce the degeneration of chondrocytes and result in damage to cartilage integrity. However, it is difficult for chondrocytes to regenerate themselves and repair the defect site. Many drugs used to treat OA, such as steroids, hyaluronic acid, chondroitin, and nonsteroidal anti-inflammatory drugs (NSAIDs), cannot effectively stop the progression of OA [18]. Accumulating evidence has proven that PDGF-BB has the potential to enhance the proliferation of chondrocytes, which hints at the ability of PDGF-BB to repair damage in OA [8–11]. Therefore, we used PDGF-BB to treat MIA-induced OA, and the results showed that PDGF-BB attenuated the progression of MIA-induced OA by inhibiting inflammation and enhancing the proliferation of cartilage by regulating the JAK2/STAT3, PI3K/AKT, and p38 signaling pathways, and these effects could be inhibited by a PDGFR inhibitor. Additionally, the upregulation of SOX-9 and the downregulation of RunX-2, which were accompanied by PKA activation, were observed after PDGF-BB treatment in vivo and in vitro. The chondroprotective effects of PDGF-BB on MIA-induced chondrocytes could be partly inhibited by SOX-9-siRNA.
The degeneration of chondrocytes and destructive metabolism of cartilage matrix in articular cartilage are thought to play key roles in the initiation and progression of OA. How to enhance the proliferation of chondrocytes and maintain a stable metabolic environment are research directions in the treatment of OA. A previous study proved that PDGF increased human meniscal cell proliferation and the production of matrix proteins accompanied by increased mRNA expression of collagen II and a three-fold increase in SOX-9 gene transcription [19, 20]. Anita Brandl et al. observed significantly increased proliferation in PDGF-BB-treated human chondrocytes [9]. In IL-1β-induced human chondrocytes, PDGF-BB inhibited chondrocyte apoptosis and upregulated the levels of collagen type II, cartilage-specific proteoglycans, β1-integrin and SOX-9 [10]. In the present study, cartilage degeneration was inhibited after MIA induction, as determined by histological changes in cartilage in the PDGF-BB group compared to the NS group. We treated MIA-induced chondrocytes with or without PDGF-BB in vivo and found increased chondrocyte proliferation in these two groups, which was consistent with previous studies [9, 10, 19, 20]. In addition, we also found increases in anabolism markers (aggrecan, type II collagen, and integrin α5β1) and decreases in catabolism markers (MMPS and ADATMS) in PDGF-BB-treated cartilage and chondrocytes. The upregulation of aggrecan, type II collagen, and integrin α5β1 and downregulation of MMPS and ADATMS in our study suggest multiple effects of PDGF-BB on the maintenance of anabolism and catabolism in cartilage in OA.
Osteoarthritis is a chronic degenerative inflammatory process of cartilage in joints, in which cartilage degeneration can stimulate chondrocytes to release inflammatory cytokines, which further damages chondrocytes and accelerates cartilage degeneration [5, 21]. IL-1 is synthesized by abnormal chondrocytes and induces the synthesis of other inflammatory cytokines, such as TNF, IL-6, the chemokine IL-8 and MMPs (MMP 1, MMP 3 and MMP 13), which in turn drive cartilage matrix breakdown and enhance articular chondrocyte destruction [5, 21]. In addition, IL-1 can stimulate the expression of proinflammatory cytokines, such as iNOS and COX-2 [22]. With the abundant accumulation of proinflammatory cytokines, the progression of OA is subsequently exacerbated. Due to its anti-inflammatory potential, PDGF-BB has already exhibited value in the treatment of inflammatory diseases [10, 23]. Therefore, we used PDGF-BB to treat MIA-induced OA and chondrocytes because high expression levels of IL-1, IL-6, IL-8, TNF-α, iNOS, and COX-2 were detected after MIA induction. In the present study, the production of proinflammatory cytokines decreased in the OA + PDGD-BB group and MIC + PDGF-BB group. The results were consistent with studies demonstrating the anti-inflammatory effects of PDGF-BB [10, 23]. Overall, the decreased levels of proinflammatory cytokines proved the anti-inflammatory effect of PDGF-BB on OA- and MIA-induced chondrocytes.
Subsequently, we investigated the potential anti-inflammatory and metabolic mechanisms of PDGF-BB-mediated chondroprotection in OA. PDGF-BB activates PDGFR-β, a receptor tyrosine kinase (RTK), and induces cell proliferation, migration, and angiogenesis by initiating various signaling pathways [24, 25]. PDGFR-β binds to PDGF-BB and remains active following internalization and may thus continue signaling from within endosomes [25]. Subsequent signal transduction and gene regulation were involved in multiple effects. Various studies have proven that the activation of JAK/STAT and PI3K/AKT and the p38 signaling pathway are associated with OA and play important roles in inflammation and the pathogenesis of chondrocyte and extracellular matrix degradation [14, 15]. In view of this important link, we next investigated whether PDGF-BB binding to PDGFR-β participated in the regulation of the JAK2/STAT3, PI3K/AKT, and p38 signaling pathways to prevent OA progression. The activation of PDGFR-β by PDGF-BB was proven in the present study to be accompanied by suppressed activation of JAK2/STAT3, PI3K/AKT, and p38 in MIA-induced OA. Furthermore, chondrocyte proliferation and cartilage anabolism markers, such as aggrecan, collagen II, integrin α5β1, and SOX-9, were significantly increased, while catabolism markers, such as collagen Ⅹ, MMPs, and ADATMs, were significantly decreased after PDGF-BB treatment. The anti-inflammatory effects of suppressing the activation of JAK2/STAT3, PI3K/AKT, and p38 have already been proven in previous studies [14, 15]. In combination with the results showing that the downregulation of JAK2/STAT3, PI3K/AKT, and p38 was followed by the downregulation of inflammatory cytokines, we concluded that the anti-inflammatory effect of PDGF-BB on OA via the suppression of the JAK2/STAT3, PI3K/AKT, and p38 signaling pathways also played a pivotal role in this process. However, intracellular signal transduction is a complex process and with overlapping interactions. It has been proven that activated JAK2 can regulate PI3K, which indicates that there is crosstalk between JAK2 and the PI3K signaling pathway [26]. In addition, JAK-mediated signaling critically relies on not only STAT transcription factors but also the activation of the MAPK and PI3K/Akt signaling pathways [27]. Furthermore, there is evidence that cAMP-PKA induction blocks inflammatory signaling pathways [28, 29] and that cAMP/PKA activation interacts with JAK2, PI3K and ERK signaling [30–32]. The PKA inhibitor abolished the anti-inflammatory effect of PDGF-BB in the PKAi group compared to the PDGF-BB group, while it did not have a significant influence on the phosphorylation of JAK2/STAT3, PI3K/AKT, or p38. Additionally, a PDGFR-β inhibitor attenuated the activation of PKA in this study. Although we could not identify the order in which JAK, PI3K, p38, and PKA act, PDGF-BB binding to PDGFR-β enhanced chondrocyte proliferation and inhibited inflammation, at least in part due to the downregulation of the JAK2/STAT3, PI3K/AKT, and p38 signaling pathways and the upregulation of PKA. The regulation of JAK, which is an upstream regulatory factor of PI3K/Akt and MAPK, may play a key role in PDGF-BB-mediated chondroprotective effects.
A stable chondrocyte phenotype is essential for maintaining the balance of anabolism and catabolism in cartilage. Hypertrophic chondrocytes are the main cell type in the lesion area in cartilage, in which the SOX-9/RunX-2 balance is a major contributor to the chondrocyte phenotype. RunX-2 is known to be a master transcription factor involved in chondrocyte hypertrophy, is markedly elevated following stimulation and regulates aggrecan loss by upregulating MMP13 and ADAMTS-4/5 expression in OA [33, 34]. Inhibiting RunX-2 leads to decreases in OA severity and the expression of type X collagen and MMP-13 [35, 36]. Our results were consistent with the previous studies showing that a high increase in RunX-2 in MIA-induced OA and chondrocytes was accompanied by the upregulation of MMP-3, MMP-13, and ADAMTS. In our study, the negative regulation of RunX-2 was reversed by PDGF-BB treatment. It was previously proven that the inhibition of MAPK and the downregulation of RunX-2 to treat OA could be a measurement of degenerative diseases [37]. Combined with the results that PDGF-BB inhibited the activation of the PI3K and p38 signaling pathways in OA, we hypothesized that PDGF-BB induced the downregulation of RunX-2 at least partly via the regulation of the PI3K/AKT and p38 pathways.
SOX-9 acts as a master regulator of aggrecan and collagen and is highly expressed in normal chondrocytes, thus maintaining cartilage and chondrocyte homeostasis [38, 39]. High expression of SOX-9 could inhibit ADAMTS-induced cartilage degeneration, while SOX-9 mRNA expression was reduced in osteoarthritic cartilage [40]. In this study, SOX-9 phosphorylation was suppressed in the OA model, but PDGF-BB induced a positive response by enhancing SOX-9 phosphorylation. Moreover, our results showed that inhibiting PKA activation by a PKA inhibitor and SOX-9 mRNA expression by transfection with siRNA upregulated RunX-2 phosphorylation in chondrocytes. As a result, the downregulation of RunX-2 could be partly mediated by the PKA/SOX-9 signaling pathway.
We next investigated the molecular mechanisms by which PDGF-BB mediates the proliferation of chondrocytes and enhances the phosphorylation of SOX-9. Activated PKA plays an essential role in chondrocyte differentiation, and inhibiting PKA with a PKA antagonist efficiently blocks chondrogenic differentiation [41, 42]. Additionally, PKA-induced SOX9 phosphorylation promoted SOX9 binding to gene enhancer elements and stimulated SOX9 transcriptional activity [43]. With strong evidence of the positive effects of PKA on SOX-9, we sought to explore the underlying mechanism of chondroprotection mediated by PDGF-BB. In this study, we observed increased levels of PKA and SOX-9 in MIA-induced cartilage and chondrocytes in the presence of PDGF-BB. Treatment of MIA-induced chondrocytes with PDGF-BB and a PKA inhibitor suppressed SOX-9 activity, which was also found after treatment with a PDGFR-β antagonist. Consistently, the expression of aggrecan and collagen was upregulated after PDGF-BB treatment. In addition, activation of STAT3 is also sufficient to promote SOX-9 activity for the acquisition of the chondroblast phenotype, whereas inhibiting STAT3 prevents cartilage formation and attenuates SOX9 reporter activity [43]. However, the phosphorylation of STAT3 in this study was significantly increased in MIA-induced OA, which was consistent with previous studies showing that STAT3 activation promoted inflammation and cartilage degradation in OA [16]. A likely explanation is that the main cells in the lesion area are hypertrophic chondrocytes, which tend to undergo apoptosis and lose metabolic capacity compared to normal chondrocytes. Another possibility is that the upstream regulatory pathway may be involved in crosstalk with other proteins induced by inflammation, which in turn suppresses the STAT3-induced activation of SOX-9. Nevertheless, our findings suggested that PDGF-BB enhanced SOX-9 activity, in part, via the PKA signaling pathway and played a critical role in enhancing anabolism in MIA-induced chondrocytes.