In this study, we demonstrated that SGK1 was upregulated and activated in MC3T3-E1 cells incubated with low dose of Dex(10-8M), an inducer which commonly used in osteoblasts differentiation. SGK1 knockdown decreased the cell viability, autophagic level and p-FOXO3/FOXO3a level induced by Dex exposure, confirming the protective role of SGK1 on osteoblasts differentiation. In addition, we showed that inhibition of autophagy aggravated the damaging effects of SGK1 knockdown as mentioned upon low dose of Dex exposure, suggesting that SGK1 might protect osteoblasts survival during the differentiation process by promoting autophagy and the FOXO3a signaling pathway.
Dexamethasone is a commonly used anti-inflammatory and an immunosuppressive drug used in clinical practice to treat a variety of diseases. Glucocorticoid-induced osteoporosis (GIO) has considered to be the most common form of secondary osteoporosis for the widely application of GCs. Interestingly, endogenous GCs are essential for the maintenance of bone homeostasis, whereas excess GCs may impede bone formation or facilitate bone resorption [12]. So, it is necessary to uncover the mechanism by which GCs maintains the balance of bone homeostasis under physiological condition and further develop effective strategies for treating or preventing GIO.
Bone homeostasis is a dynamic balance which mediated by osteoblasts, osteoclasts, bone marrow mesenchymal stem cells, and osteocytes. Recent studies demonstrated that different dose of GCs led to different bone cells fate [7, 13]. Osteoblasts, which are derived from mesenchymal stem cells, are mainly responsible for bone formation and development. Zhang S et al found a high dose of Dex (≥ 10− 6 M) accelerated osteoblasts apoptosis, while a low dose of Dex (10− 8 M) increased osteoblasts viability in the early stage [14]. This is consistent with our experimental results. Unfortunately the underlying mechanism is still complex and require further more investigation.
Autophagy is a highly conserved catabolic process that is essential for cell growth, survival, differentiation, development and homeostasis [15]. It has been identified that autophagy play a critical role in both physiological processes and numerous pathological conditions including inflammation, neurodegeneration, cancer and bone metabolic diseases [16]. Under physiological conditions, autophagy is responsible for the removal of damaged or excessive organelles, whereas under pathological conditions, autophagy helps in the redistribution of intracellular nutrients to meet the substance and energy requirement for survival. Despite an appropriate autophagy level is prerequisite for the maintenance of homeostasis and survival of cell, but the excessive activation of autophagy generally leads to a programmed cell death. Therefore, autophagy has been reported as a ‘double-edged sword’ [17]. Over the past ten years, many studies have suggested that autophagy in osteoblasts, plays a critical role in the bone homeostasis and GIO [18]. In the present study, we found that 10-8M Dex induced osteoblast autophagy and maintained osteoblast viability. The positive effect of 10-8M Dex on the occurrence of autophagy was confirmed, as reflected by the increased expression of beclin-1 and LC3II and the decreased expression of P62. Furthermore, our studies found that 10-8M Dex increased LC3II and Beclin-1 levels and decreased SQSTM/p62 level in a time-dependent manner, which could be attenuated by pretreatment with autophagy inhibitor 3-MA, and 3-MA significantly inhibited the cell viability-promoting effect of Dex. Using knockdown of autophagy-
essential genes and osteoblasts-specific autophagy- deficient mice, researchers demonstrated that autophagy deficiency increased oxidative stress and reduced mineralization capacity, which showed that autophagy in osteoblasts is involved in bone homeostasis [19]. However the precise signal pathway of autophagy affecting the viability of osteoblasts exposed to low dose of GCs has rarely been reported.
SGK1 related to Akt (also called PKB), is a serine/threonine kinase that can be activated by PI3k, which is known for decades for its role in ion channel modulation and cell survival in response to stress stimuli. SGK1 participates in the regulation of transport, hormone release, tumor growth, neurodegeneration, cell proliferation, and apoptosis in multiple cell lines [20, 21]. In vitro studies indicated that protein levels and SGK1 activation were significantly stimulated by H2O2 exposure. Human proximal tubular cells with SGK1 inhibition were much more sensitive to H2O2-induced oxidative stress injury than control group cells, as they exhibited increased apoptotic cell death and mitochondrial dysfunction [22]. Li J et al reported overexpression of SGK1 significantly attenuated A549 cell apoptosis and reduced the reactive oxygen species (ROS) generation induced by PM2.5 [23]. Our studies showed that treating MC3T3-E1cells with 10− 8M Dex induced a significant time-dependent increase in the expression of SGK1 both at mRNA levels and protein levels, which began to increase 1h after Dex stimulation and peaked at 3 h. We supposed that SGK1 participates in the cellular survival of low dose Dex. So, we inhibited SGK1 expression using shRNA. The results showed that SGK1 knockdown weakened the promotional role of Dex on cell viability.
In addition to the above functions, SGK1 is now known to act as a switch for autophagy homeostasis. However, there is controversy regarding the effect of SGK1 on autophagy in different cell lines [24, 25]. Notably, we found that the effect of SGK1 on autophagy paralleled its significant increase in cell viability of MC3T3-E1 cells exposed to low dose of Dex. Morover, inhibiting SGK1 signifcantly decreased the expression of autophagy markers LC3II and Beclin-1, promoted the expression of p62 and increase in apoptosis in MC3T3-E1cells under Dex exposure compared to the control. 3-MA further aggravated the effects of SGK1 inhibition on cell viability and apoptosis.
FOXO3a, as a member of the forkhead transcriptionfactor family, is an downstream target of SGK1. SGK1 phosphorylates FOXO3a leading to FOXO3a translocation from the nucleus to the cytoplasm and to the inhibition of FOXO3a-dependent transcription, thereby preventing FOXO3a from inducing apoptosis and/or cell cycle arrest [10]. FOXO3a has been reported to actively promote apoptosis by inducing Bcl-2 family members [26]. Similarly, Chen et al revealed that SGK1 activation significantly increased FOXO3a phosphorylation, and then inhibited the subsequent expression of Bcl-2 interacting mediator of cell death (Bim). SGK1 mediates the hypotonic protective effect against H2O2-induced apoptosis of rat basilar artery smooth muscle cells by inhibiting the FOXO3a/Bim signaling pathway [27]. Recent studies indicated that FOXO3a overexpression not only decreased HIF-1α protein level but also inhibited HIF-1α transcriptional activity, as evidenced by decreased expressions of HIF-1 target genes EPO, HO-1, and Bnip3 known to regulate autophagy [11, 28]. We show here that knocking down SGK1 before Dex exposure significantly downregulated phosphorylated-FOXO3a (p-FOXO3a)/FOXO3, suppressed LC3II and Beclin-1 levels and increased SQSTM/p62 levels in MC3T3-E1 cells, which could be amplifies by 3-MA. The results revealed that a low dose of GCs increased osteoblasts viability by activating autophagy via SGK1/FOXO3a pathway.