Research paperFFAs-ROS-ERK/P38 pathway plays a key role in adipocyte lipotoxicity on osteoblasts in co-culture
Introduction
Obesity and osteoporosis are two common complex diseases, the relationship between which has been widely studied. Although contrasting evidence indicates that fat mass may have beneficial or harmful effects on bone [1], the latest study shows that body fat mass is negatively correlated with bone mass when the mechanical loading effect of body weight is statistically removed [2]. In the process of aging, body fat is redistributed [3] and appears to accumulate in depots where it should not, including bone marrow [4]. In addition, the accumulation of fat in bone marrow is also observed in anorexia, a state of pathophysiological reduction of body fat tissue [5].
The accumulation of reactive lipids in bone marrow leads to lipotoxicity. Previous studies have found that co-culture of osteoblasts with adipocytes leads to a decrease of osteoblasts proliferation [6], [7]. Further studies have demonstrated that the adverse effect of adipocytes on osteoblast differentiation, proliferation, and function was associated with lipolysis [8] and the increase of free fatty acids (FFAs) [7], [9]. In vitro studies have found that co-culturing of osteoblasts with adipocytes decreases osteoblast associated with the presence of FFA in the media. Moreover, age-related changes in bone marrow fat and osteoblast differentiation in vivo are related with increased levels of FFAs oxidation [10]. Literature has shown that under certain experimental conditions, FFAs exhibit toxic effect to various cell lines [11], [12], [13].
In the last decades, steroid-induced diseases, such as osteoporosis and osteonecrosis, is arising due to the wide use of glucocorticoids. Steroid-induced osteoporosis represents one of the most important secondary causes of osteoporosis [14]. The observation that steroid-induced osteoporosis and osteonecrosis is accompanied by bone marrow lipid deposition indicates that disorder of fat metabolism is associated with the effect of steroid on bone [15], [16], [17]. During early steroid-associated osteonecrosis development, adipogenesis of marrow mesenchymal stem cells is elevated [18]. Moreover, glucocorticoids directly induce and enhance lipolysis in adipocytes [19], and glucocorticoid treatment is associated with increased levels of circulating FFAs in humans [20], [21]. Recently, a study found that dexamethasone (Dex) increased the adverse effect of adipocytes on osteoblasts through lipolysis. However, the molecular mechanism underlying lipotoxic effect of adipocytes on osteoblasts is still unclear.
The present study was designed to elucidate the signaling pathway responsible for lipotoxic effect of adipocytes on osteoblasts. Using a co-culture system, we have identified that FFAs released by the adipocytes inhibited osteoblasts proliferation and function and induced osteoblasts apoptosis. Dex promoted the inhibitory effect of adipocytes on osteoblasts through stimulating FFAs release. In addition, reactive oxygen species (ROS)-activated extracellular signal-regulated kinase (ERK)/P38 signaling pathway was responsible for adipocytes-induced and Dex-increased lipotoxic effect on osteoblasts. The evidence provides a novel clue for the lipotoxic effect of adipocytes on bone within the marrow microenvironment and prevention of lipotoxicity on bone metabolism.
Section snippets
Cell culture
Mouse MC3T3-E1 cells (an osteoblast-like cell line from C57BL/6 mouse calvaria) were obtained from ATCC. MC3T3-E1 cells were cultured at 37 °C in 5% CO2 atmosphere in α-modified minimal essential medium (α-MEM; GIBCO). Unless otherwise specified, the medium contained 10% heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin, and 100 U/ml streptomycin. The culture medium was changed every 3 days. When MC3T3-E1 cells was more than 80% confluent (designated as day 0), differentiation
Dex promotes the inhibitory effect of adipocytes on osteoblasts in co-culture
In the present study, to investigate the effect of adipocytes on osteoblasts function, osteoblasts were co-cultured with adipocytes (AD/OB) or osteoblasts (OB). In the co-culture systems, MC3T3-E1 were differentiated into osteoblasts on the bottom of the wells, whereas fully 3T3-L1 pre-adipocytes were differentiated into adipocytes and cultured on the inserts of the transwell, without any direct contact between the two cell types. In the present study, we conducted MTT assay. Since MTT assay
Discussion
Obesity, anorexia and age-related conditions are accompanied by a notable increase of adipocytes in bone marrow tissue [1], [5], [25]. In addition, world-wide use of exogenous glucocorticoid and prolonged endogenous overproduction of cortisol are associated with remarkable accumulation of adipocytes in bone marrow [26], [27]. These conditions make adipocytes accumulation and lipotoxicity in bone marrow a severe problem in the whole world. In the current study, using a co-culture system of
Conflict of interest
The authors declare that there are no conflicts of interest.
Acknowledgment
This work was supported by 863 High-Tech Project (No. 2012AA020502-6)
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