Biochemical and Biophysical Research Communications
IL-17A regulates the autophagic activity of osteoclast precursors through RANKL-JNK1 signaling during osteoclastogenesis in vitro
Introduction
Interleukin-17 (IL-17) is distinguishingly produced by T helper 17 cells (Th17). Like other pro-inflammatory cytokines derived from activated T cells, IL-17 can influence osteoclastogenesis and subsequent bone resorption in many inflammation-mediated bone diseases, such as postmenopausal osteoporosis (PMO) [1]. The IL-17 cytokine family includes at least six members: IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, and IL-17F, while IL-17 is usually referred to IL-17A. It is important to note that the role of IL-17 in PMO remains controversial so far. Some studies suggested that IL-17 contributed to the development of PMO [1,2], while others believed that IL-17 had protective effects against osteoporosis induced by estrogen deficiency [3].
Consistently, the studies of osteoclastogenesis under direct interference of IL-17A showed different results. Based on the previous studies [[4], [5], [6]], the following rules can be revealed: a low concentration of IL-17A stimulates the differentiation and activity of osteoclasts, which are inhibited by a high concentration of IL-17A. Nevertheless, the underlying mechanisms remain unclarified.
Previous studies have revealed that a series of autophagy proteins, including ATG5, ATG7, LC3 and ATG4B, are involved in polarization of osteoclasts, and associated with the formation of osteoclast ruffled border, H+ secretion by lysosomes, and protein hydrolysation [7]. In addition, both ATG7 and Beclin1 play a crucial role in osteoclastogenesis as well as the following activity of bone absorption [8]. Besides, during the osteoclastogenesis induced by RANKL, knockout of P62 can also lead to inhibition of osteoclastogenesis [9]. The studies above disclose that protective autophagy is involved in multiple stages of osteoclast proliferation and differentiation, and bone absorption activity of osteoclasts. In addition, the prior research has shown that IL-17A could promote the process of pulmonary fibrosis via the change of autophagic activity [10]. Similarly, the changes of autophagic activity induced by IL-17A are also reported in relevant studies about other system lesions [[11], [12], [13]]. However, it remains unclear whether or how IL-17A alters the autophagic activity of osteoclast precursors(OCPs) in osteoclastogenesis.
It has been expounded that there is a synergistic effect between IL-17A and RANKL in promoting osteoclast formation, based on the reality that compared with treatment of RANKL only, combination of the two cytokines could result in an obviously increased maturation of osteoclast [5]. Moreover, It has been reported that RANKL plays a significant role in autophagy activation during osteoclastogenesis [9,[14], [15], [16]]. In addition, JNK, a downstream signaling molecule of RANKL-mediated osteoclastogenesis [[17], [18], [19], [20], [21]], can also activate autophagy in the active form [[22], [23], [24]]. Our team has demonstrated that RANKL could activate autophagy through JNK signaling, thereby promoting osteoclast formation (unpublished results). Collectively, it was presumed that IL-17A might enhance autophagic activity of OCPs via RANKL-JNK pathway during osteoclastogenesis, which deserves confirmation as well.
The present study disclosed the effect of autophagy on IL-17A-regulated osteoclast formation under different cytokine levels. In addition, it was clarified that JNK signal was likely to mediate OCPs autophagy induced by a low level of IL-17A.
Section snippets
Cell culture
RAW264.7 macrophage cell line's incubations were performed in Dulbecco's modified Eagle's medium(DMEM; Hyclone; Logan, Utah,USA) with 10% Fetal bovine serum (FBS; Gibco; Grand Island,NY,USA), 100IU/ml penicillin, and 100 μg/ml streptomycin. The cells were kept under 37 °C and 5% CO2. Before all trials, RAW264.7 cells were added into α-MEM along with 10% FBS, antibiotics and M-CSF(20 ng/ml in all experiments; Peprotech; Rocky Hill,NJ,USA) for 2 days to induce the OCPs.
Small interfering RNA (siRNA) transfection
The control siRNA or siRNA
A low concentration of IL-17A could enhance OCPs autophagy, whereas a high level of IL-17A is contrary
The autophagic activity is generally observed by the LC3 conversion rate (LC3II/I) [25]. The administration of IL-17A at a concentration of 0.5 ng/ml enhanced the LC3 conversion. However, with the increase of IL-17A levels, the LC3 conversion decreased gradually, and was inhibited to the greatest extent at 50 ng/ml (Fig. 1A and 1B). The detection of autolysosome under TEM is another approach to assess autophagic activity. Consequently, autolysosomes were significantly increased in 0.5 ng/ml
Discussion
Our study indicated that IL-17A could promote OCPs autophagic activity and osteoclastogenesis at a low concentration, and the promoting effect was gradually changed to an inhibiting effect in response to increased IL-17A levels, which reached the minimum level at the peak concentration. The result above is consistent with the study by Satoshi et al. [4]. They found that IL-17A enhanced osteoclastogenesis only at the lowest concentration, while IL-17A gradually exerted an increasingly powerful
Conflicts of interest
All authors have no conflicts of interest.
Acknowledgement
This work was supported by The Fourth Round of Fujian Provincial Joint Tackling Project between Health and Education_Joint Research Fund of National Health and Family Planning Commission of China (wkj2016-2-02) and Fujian Provincial Natural Science Foundation (2016J01500).We thank Professor Lin Chen for providing support of experimental technique and Professor Weibo Xia for reviewing article.
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