In this study, we observed increased IL-6 secretion and mIL-6R expression during osteogenic differentiation in BM-MSCs. In response to stimulation with exogenous IL-6, IL-6R or both, ARS and ALP staining, osteoblastic gene expression and STAT3 phosphorylation were increased. In addition, treatment with IL-6- or IL-6R-neutralizing antibodies exerted opposite effects. Moreover, we observed a positive feedback loop between IL-6 and IL-6R, through which these two factors stimulated each other’s expression in BM-MSCs. Therefore, IL-6 and mIL-6R form complexes to activate the downstream STAT3 signaling pathway, which subsequently promotes osteogenic differentiation in BM-MSCs in an autocrine/paracrine manner.

IL-6 is an important cytokine that is involved in various physiological and pathological processes [5]. Specifically, IL-6 plays an important role in maintaining the dynamic equilibrium between bone formation and resorption [12]. A previous study has reported that IL-6 secreted by osteoblasts promotes the activation and differentiation of osteoclasts [13]. MSCs are the predominant cell of origin of osteoblasts in vivo, and these cells both secrete and respond to IL-6 [6, 14]. Kondo et al. have demonstrated that although IL-6 expression decreases during differentiation, IL-6 can accelerate chondrogenic differentiation in MSCs [7]. Huh et al. also demonstrated that in response to Toll-like receptor activation, IL-6 secretion from adipose MSCs can increase, and this promotes the osteogenic differentiation of adipose-derived MSCs [15]. In this study, we found that the level of IL-6 secreted by BM-MSCs increased during osteogenic differentiation and activated the STAT3 signaling pathway, which further facilitated osteogenic differentiation in BM-MSCs. These results were mostly consistent with previous reports. However, several differences between the results of other studies and ours should be mentioned. On one hand, we found that BM-MSCs could continually secrete IL-6 during osteogenic differentiation without requiring other stimuli. On the other hand, IL-6 secreted by BM-MSCs gradually increases, rather than decreases, with the progression of osteogenic differentiation. These results suggest that during osteogenic differentiation of BM-MSCs, IL-6 acts as an autocrine/paracrine feedback loop to promote differentiation. We speculate that these differences may arise due to differences in the tissue source of MSCs.

Both mIL-6R and sIL-6R bind to IL-6 and phosphorylate STAT3 to regulate various cellular functions [8]. As previously reported, IL-6R, together with IL-6, participates in bone metabolism in vivo [12]. In our study, BM-MSCs expressed mIL-6R rather than sIL-6R during osteogenic differentiation, which further promoted osteogenic differentiation in BM-MSCs through the STAT3 signaling pathway. However, whether MSCs can express both forms of IL-6R is still controversial. Some studies have claimed that IL-6R is not expressed on the membrane of MSCs and that IL-6 alone cannot promote osteogenic differentiation in MSCs without exogenous sIL-6R [15, 16]. In contrast, recent studies have demonstrated that IL-6R expression is not only detected in MSCs but also gradually increased during differentiation [7]. Moreover, IL-6R levels in MSCs have been associated with disease prognosis [17]. It has been reported that IL-6R expression is regulated by various endogenous and exogenous factors, including dexamethasone, which was used in our study [18, 19]. Therefore, we conclude that controversies regarding IL-6R expression may have resulted from differences in experimental conditions in vitro and differentiation environments in vivo.

Because both IL-6 and IL-6R form complexes to regulate the function of various cells that participate in many physiological and pathological processes, these two molecules are indispensable for bodily functions [5]. IL-6 and IL-6R are known to tightly regulate each other’s expression to establish a dynamic equilibrium [19]. For example, IL-6 can stimulate IL-6R expression in hepatocytes and bronchial epithelial cells, which is indicative of a positive feedback loop in these cells [20, 21]. Nevertheless, the regulatory relationship between IL-6 and IL-6R in MSCs remains unclear. To elucidate the relevant mechanism, in this study, we showed a positive correlation between IL-6 and IL-6R levels because the expression of both proteins was upregulated/downregulated via relative exogenous proteins/neutralizing antibodies. In addition, IL-6 and IL-6R were found to stimulate each other’s expression in BM-MSCs during osteogenic differentiation. Therefore, IL-6 and IL-6R form complex to promote osteogenic differentiation of BM-MSCs as an autocrine/paracrine positive feedback loop. However, definitive regulatory mechanisms need to be addressed in the future.

The IL-6/IL-6R/STAT3 signaling pathway has been shown to play a critical role in bone metabolism [22]. On one hand, IL-6 affects bone mass by modulating the balance between osteocytes and osteoclasts [23]. On the other hand, IL-6R is maintained at a relatively high level in the serum to modulate bone formation, and polymorphisms in the IL-6R gene are related to bone mineral density [24]. Dysregulation of these two molecules can lead to abnormal bone metabolism and have been linked with the pathogenesis of diseases [25]. Previous studies have demonstrated that abnormal differentiation of MSCs is one of the important mechanism defects in bone metabolism in diseases, but the definitive mechanism still needs to be elucidated [10, 26, 27]. In this study, we demonstrated the critical role of the IL-6/IL-6R complex in the osteogenic differentiation of BM-MSCs. We further concluded that a dysregulated IL-6/IL-6R/STAT3 signaling pathway may lead to abnormal bone metabolism under pathological conditions by affecting the differentiation of MSCs. Besides, tocilizumab, an anti-IL-6R antibody, has been widely used for various diseases and was shown to mitigate dysregulated bone metabolism [5, 28]. Our results suggest that anti-IL-6 antibodies could also have potential to be used in the clinic as a novel treatment.

However, several limitations still exist in this study, including the lack of understanding of the regulatory mechanism of IL-6 and IL-6R expression in BM-MSCs. Besides, the expressions and functions of IL-6 and IL-6R during osteogenic differentiation of MSCs in vivo are also still unclear. These limitations should be addressed in future studies.