miR-155 modulates TNF-α-inhibited osteogenic differentiation by targeting SOCS1 expression

Abstract

Bone morphogenetic proteins (BMPs) can induce ectopic bone formation, which is negatively regulated by inflammatory cytokines, such as tumor necrosis factor (TNF)-α. Recently, miR-155 has been reported to regulate the transforming growth factor (TGF)-β signaling pathway and inflammatory responses. However, whether and how miR-155 modulates TNF-α-regulated osteogenic differentiation have not been explored. In this study, we demonstrated that miR-155 was involved in TNF-α-mediated inhibition of osteogenic differentiation. Knockdown of miR-155 partially mitigated the inhibition of TNF-α on BMP-2-induced osteogenic differentiation. Bioinformatic analysis identified the candidate target site in the 3′ untranslated region (3′UTR) of SOCS1. Knockdown of miR-155 increased SOCS1 protein expression during TNF-α stimulation in MC3T3-E1 cells. And transfection with miR-155 inhibited the wild-type, but not the mutant, 3′UTR of SOCS1-regulated luciferase activity, indicating that SOCS1 is a direct target of miR-155 in osteoblast cells. Furthermore, miR-155 expression could be induced by TNF-α through the JNK pathway. As the result of increased SOCS1 expression, knockdown of miR-155 significantly reduced the JNK/c-Jun activation. In addition, transfection of SOCS1 siRNA or overexpression of SOCS1 coding region could narrow the differences of alkaline phosphatase (ALP) and osteocalcin (OSC) expression between the control and miR-155 inhibitor transfected cells. These data indicated that miR-155 modulates TNF-α-regulated osteogenic differentiation by targeting SOCS1, at least partially through the SAPK/JNK pathway. These findings may provide new insights into understanding the regulatory role of miR-155 in the process of osteogenic differentiation in inflammatory condition.

Introduction

Bone morphogenetic proteins (BMPs) are members of the TGF superfamily and can induce ectopic bone formation [1]. BMPs can induce heterogeneous oligomerization of type I and type II receptor serine/threonine kinases and Smad1/5/8 phosphorylation. Subsequently, the phosphorylated Smad1/5/8 are associated with Smad4 and translocate into the nucleus where they cooperate with tissue-specific transcription factors to drive osteogenic target gene expression [2]. However, the efficacy of BMPs in inducing bone formation is limited in clinical use and their osteogenic effects appear to be regulated negatively by other factors [3], [4], [5]. Discovery of the role of those negative regulators in osteogenic differentiation is of great significance.

Inflammatory cytokines, such as TNF-α, have been shown to inhibit BMP-induced bone formation and osteoblast differentiation in multiple models [6], [7]. TNF-α has been considered as one of the major cytokines responsible for bone loss in many bone-related inflammatory diseases [8], [9]. Indeed, TNF-α can inhibit BMP signaling by interfering with the DNA binding of R-Smads through activating the NF-κB pathway or inhibit BMP-induced osteoblast differentiation by activating the stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) signal pathway [10], [11]. In addition, TNF-α can induce Smad7 and Msx2 expression, which mediate the inhibitory action of TNF-α on BMP signaling and related osteoblast differentiation [12], [13].

Recently, miRNAs have emerged as a major class of gene expression regulators linked to many biological functions [14]. miRNAs can bind to the 3´UTR of target mRNAs, inhibiting the translation of target genes or promoting the degradation of mRNAs [15]. Previous studies have shown that miR-26a, -206, -204, -133, -135, -135b, -125b, -141, -200a and ‐208 can inhibit osteogenic differentiation [16], [17], [18], [19], [20], [21], [22]. On the contrary, miR-29b and miR-2861 have been demonstrated to promote osteogenesis [23], [24]. Moreover, miR-23a–27a–24-2 cluster has been reported to regulate the osteoblast differentiation [25]. Apparently, miRNAs are important players during the osteogenic differentiation.

miR-155, induced by TGF-β1, can act through a feedback loop to negatively regulate TGF-β1 signaling pathway [26]. Furthermore, miR-155 is associated with inflammatory responses. Previous studies have shown that inflammatory stimuli, such as lipopolysaccharide (LPS) and TNF-α, can induce miR-155 expression in different types of cells in vitro [27], [28]. However, whether and how miR-155 could modulate TNF-α-regulated osteogenic differentiation have not been explored.

The purpose of the present study is to examine the impact of miR-155 on TNF-α-regulated BMP signaling in MC3T3-E1 cells. In the study, we observed that TNF-α up-regulated miR-155 expression, and knockdown of miR-155 partially mitigated the inhibition of TNF-α on BMP-2-induced osteogenic differentiation. SOCS1 was demonstrated as a target gene of miR-155 using a sensor luciferase reporter assay. Knockdown of miR-155 expression inhibited the JNK activation. Furthermore, transfection of SOCS1 siRNA or overexpression of SOCS1 coding region could narrow the differences of ALP and OSC expression between the control and miR-155 inhibitor transfected cells. These data indicated that miR-155 modulates TNF-α-regulated osteogenic differentiation by targeting SOCS1, and probably augmenting the SAPK/JNK pathway. These findings suggest that miR-155 regulates osteogenic differentiation under the inflammatory condition.