miR-146a, an IL-1β responsive miRNA, induces vascular endothelial growth factor and chondrocyte apoptosis by targeting Smad4

Abstract

IntroductionmiR-146a is one of the first identified miRNAs expressed differentially in osteoarthritis (OA) cartilage. However, the role it plays in OA pathogenesis is not clear. The aim of this study is to identify a molecular target of miR-146a, thereby elucidating its function in chondrocytes during OA pathogenesis.MethodsPrimary chondrocytes from Sprague-Dawley rats were treated with IL-1β before the expression levels of miR-146a, Smad4 and vascular endothelial growth factor (VEGF) were quantified by real-time PCR and/or western blotting. The effect of miR-146a on cellular response to transforming growth factor (TGF)-β1 was quantified by a luciferase reporter harboring TGF-β1 responsive elements and by extracellular signal-regulated kinase assay. The effect of miR-146a on apoptosis was quantified by the TUNEL assay. OA pathogenesis was surgically induced with joint instability in rats, evaluated by histopathological analysis with safranin O staining, and the expression levels of miR-146a, Smad4, and VEGF were quantified using real-time PCR and/or immunohistochemistry.ResultsIL-1β treatment of chondrocytes increased the expression levels of miR-146a and VEGF and decreased the levels of Smad4 in a time-dependent manner. miR-146a upregulated VEGF expression and downregulated Smad4 expression in chondrocytes, while a miR-146a inhibitor acted in a converse manner. Smad4, a common mediator of the TGF-β pathway, is identified as a direct target of miR-146a by harboring a miR-146a binding sequence in the 3'-UTR region of its mRNA. Mutation of the binding sequence significantly relieved the inhibition of the Smad4 reporter activity by miR-146a. Furthermore, miR-146a upregulation of VEGF is mediated by Smad4. Expression of miR-146a led to a reduction of cellular responsiveness to TGF-β and an increase of apoptosis rate in chondrocytes. In vivo, cartilage from surgically induced OA rats displayed higher levels of miR-146a and VEGF compared with the sham group. In contrast, Smad4 expression level was lower in the OA group than the sham group.ConclusionIL-1β responsive miR-146a is overexpressed in an experimentally induced OA model, accompanied by upregulation of VEGF and downregulation of Smad4 in vivo. miR-146a may contribute to OA pathogenesis by increasing VEGF levels and by impairing the TGF-β signaling pathway through targeted inhibition of Smad4 in cartilage.

Evidence that miR‐146a attenuates aging‐ and trauma‐induced osteoarthritis by inhibiting Notch1, IL‐6, and IL‐1 mediated catabolism

“…We were the first to identify miR‐146a as a mechanoresponsive microRNA along with miR‐365 through cytomechanical screening of microRNA microarray (Guan, Yang, Wei & Chen, 2011). Since then, multiple studies have supported the role of miR‐146a as a mechano‐miR (Huang, Crawford, Higuita‐Castro, Nana‐Sinkam & Ghadiali, 2012; Li et al., 2012). In this study, we further show that, while cyclic load of human articular chondrocytes in 3D culture in physiological range stimulated miR‐146a expression, mechanical overload inhibited miR‐146a expression.…”

“…However, its role during OA pathogenesis is controversial with some studies suggesting a protective function while other studies indicating a destructive role in cartilage homeostasis. For example, expression of miR‐146a in chondrocytes has been proposed to contribute to OA pathogenesis by diminishing the response to TGF‐β (Li et al., 2012). A most recent study seemed to correlate with this hypothesis in vivo (Zhang et al., 2017).…”

Identification of microRNA-181a-5p and microRNA-4454 as mediators of facet cartilage degeneration

“…This is the first report to our knowledge that identifies miR-181a-5p and miR-4454 as mediators of cartilage degeneration in FJs and potential therapeutic targets for stopping cartilage degeneration. miR-146 is another miRNA that is highly expressed in low-grade OA cartilage and is induced by IL-1β stimulation; additionally, it mediates chondrocyte apoptosis (13,14). In serum, Beyer et al recently reported miRNA let-7e as a potential predictor for severe knee or hip OA (15).…”

MicroRNAs: Important Epigenetic Regulators in Osteoarthritis

“…Taganov et al [26] proposed that miR-146, which is induced by IL-1 at the early-stage of OA, may control cytokine signaling pathways by down-regulating IL-1 receptorassociated kinase 1 (IRAK1) and the TNF receptorassociated factor 6 (TRAF6) levels ( Table 1). Recent analysis of IL-1 induced miR-146a in rat OA chondrocytes revealed that this miR induces VEGF expression and inhibits expression of SMAD4 (Table 1), which plays a significant role in anabolic TGF-pathway [27]. Results indicate that chondrocyte apoptosis was triggered in response to changes in expression profiles of genes regulated by miR-146a [27].…”

“…Recent analysis of IL-1 induced miR-146a in rat OA chondrocytes revealed that this miR induces VEGF expression and inhibits expression of SMAD4 (Table 1), which plays a significant role in anabolic TGF-pathway [27]. Results indicate that chondrocyte apoptosis was triggered in response to changes in expression profiles of genes regulated by miR-146a [27].…”