DEHP suppresses erythropoiesis
To rapidly test whether DEHP influenced erythropoiesis, we used human chronic myelogenous leukaemia K562 and erythroleukemia HEL 92.1.7 (HEL) cell lines. The erythroid and megakaryocytic differentiation potentials of the two cell lines were comparable to those of human CD34+ HSPCs [20, 21]. Sodium butyrate (NaB) was used to induce erythroid differentiation in both cell lines [21]. The addition of DEHP in the culture medium did not affect K562 cell proliferation (Fig. 1A) and survival (Fig. 1B). Based on previous reports on DEHP plasma concentrations in chronic kidney disease (CKD) patients undergoing haemodialysis [22, 23], 1 µg/ml of DEHP was selected for application in the experiments in the present study.
DEHP treatment substantially decreased NaB-induced erythroid differentiation, which was evaluated based on Hgb production, in both K562 and HEL cells. Without NaB treatment, DEHP also decreased spontaneous erythroid differentiation, which was resulted from increasing cell numbers, in both cell lines (Fig. 1C). Erythropoietin (EPO) is the primary cytokine that regulates erythropoiesis [24, 25]. An increase in DEHP concentration gradually and significantly suppressed the formation of EPO-induced colony-forming unit-erythroids (CFU-Es) in BM (Fig. 1D) and SP cells (Fig. 1E). CFU-Es are differentiated form the earlier erythroid progenitor cells, burst-forming unit-erythroids (BFU-Es) [26]. DEHP also suppressed BFU-E formation (Fig. 1F). The results not only demonstrated that DEHP suppressed erythropoiesis but also indicated that the two human cell lines were comparable cell models that could be adopted in subsequent investigations.
DEHP suppresses the differentiation of HSPCs, whereas removal of DEHP restores HSPC self-renewal activity
To examine the effect of DEHP in the proliferation and differentiation of HSPCs, BM and SP cells were cultured in a methylcellulose-based medium. Consequently, HSPCs proliferate and differentiate to form discrete colonies, including the oligopotential progenitor CFU-granulocyte, erythrocyte, monocyte/macrophage, and megakaryocyte (CFU-GEMM), lineage-restricted progenitor CFU-granulocyte and monocyte/macrophage (CFU-GM) and the precursors of granulocytes (CFU-G) and monocytes/macrophages (CFU-M). DEHP decreased the formation of CFU-GEMM, CFU-GM, CFU-G and CFU-M in both BM and SP cells in a dose-dependent manner (Fig. 2). The results clearly indicated that DEHP influenced HSPC differentiation.
HSPC self-renewal can be examined by re-plating cells in methylcellulose-based medium. HSPCs retained a similar capacity to generate haematopoietic cells in the second plating, while the potential decreased gradually in the third and fourth iterations (Fig. 3A), which is consistent with our previous observations [27]. The experimental design used to examine whether DEHP removal restored HSPC potential is presented in Fig. 3B. In the first plating, DEHP inhibited total colony number growth significantly. Subsequently, a cell number similar to the number of the first-generation colony cells (2 × 104 cells) was re-plated with or without DEHPs (1 µg/ml). Like normal HSPCs in BM cells retained similar ability to generate haematopoietic cells in the first and second plating, removal of DEHP in the second plating keep similar self-renewal capability. However, continuous exposure to DEHP significantly decreased HSPC function (Fig. 3C). Overall, the results suggested that DEHP interfered transiently with HSPC functions.
DEHP did not reprogram cellular bioenergetics during erythroid differentiation
Previous studies have indicated that DEHP treatment influences cellular glucose metabolism [13], and erythropoiesis is highly associated with reprogramming of glucose metabolism and energy consumption [19]. Mammalian cells generate ATP by mitochondrial (oxidative phosphorylation) and non-mitochondrial (glycolysis) metabolism. Cellular oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) are key indicators of mitochondrial respiration and glycolysis, respectively. The indices could provide a systemic perspective of cellular metabolic function in live cells and can be evaluated using a Seahorse Extracellular Flux Analyzer [28, 29]. Here, we firstly examined whether DEHP influenced energy metabolism in undifferentiated K562 cells. K562 cells were treated with 1 µg/ml DEHP for 24 h, and then collected for OCR and ECAR assays. OCR is measured before and after the addition of various inhibitors to derive several mitochondrial respiration parameters. The cellular OCR baseline is measured before the addition of oligomycin, a complex V inhibitor. The ATP-linked respiration can be evaluated following treatment with oligomycin. Subsequently, FCCP, a protonophore, is added to collapse the inner membrane gradient, allowing the electron transport chain (ETC) to function at its maximum potential, and then maximal respiratory capacity is measured. Finally, antimycin A and rotenone, inhibitors of complex III and I, are added to shut down ETC function and reveal the non-mitochondrial respiration. Mitochondrial spare respiratory capacity is calculated by subtracting basal respiration from maximal respiratory capacity (Fig. 4A). DEHP treatment did not influence mitochondrial respiration parameters in undifferentiated K562 cells (Fig. 4B and 4C).
For the ECAR assay, cells were cultured in the assay buffer without glucose for 1 h. Glucose is used to elicit glycolytic activity. Afterward, oligomycin injection blocks mitochondrial respiration to allow glycolysis to function at its maximum rate. Finally, the addition of 2-DG, a glucose analogue, inhibits glycolysis, and, therefore, provides a baseline ECAR measurement. Glycolytic reserve capacity is calculated by subtracting basal ECAR from maximal ECAR (Fig. 4D). Similar to the OCR analysis case, DEHP treatment did not influence any glycolysis parameter in undifferentiated K562 cells (Fig. 4E and 4F). Subsequently, we analysed whether DEHP reprogramed cellular bioenergetics in the course of erythroid differentiation.
K562 cells were treated with NaB with or without DEHP (1 µg/ml) for 24 h, and then subjected to OCR and ECAR assays. Induction of erythroid differentiation increased basal and maximal mitochondrial respiration dramatically, in addition to ATP production (Fig. 5A and 5B). In addition, erythroid differentiation induction decreased glycolytic activity significantly (Fig. 5C and 5D). The results were consistent with previous findings on the rearrangement of glucose metabolism and energy consumption during erythropoiesis in chicken T2EC cells [19]. However, the addition of DEHP into differentiated cells did not influence cellular bioenergetics further (Fig. 5). The results indicated that the effects of DEHP on erythroid differentiation are not via modulating cellular bioenergetics.
DEHP induces Klotho expression, which affects erythroid differentiation
Previous studies have demonstrated that loss of Klotho, which was initially considered an anti-ageing gene, disrupted HSPC homing, and increased erythropoiesis [30]. Therefore, Klotho expression was analysed in K562 cells under different treatments. Compared to the wild-type (WT) K562 cells (Control, C), the expression of Klotho was decreased in NaB-treated K562 cells. DEHP addition in NaB-treated K562 cells induced Klotho expression markedly (Fig. 6A). The association between DEHP and Klotho expression was further examined in CFU-E cells. DEHP exposure induced Klotho expression in CFU-E cells significantly (Fig. 6B). To confirm the influence of Klotho in DEHP-mediated suppression of erythropoiesis, the knockdown approach was used. Three Klotho short-hair (shRNA) were expressed transiently in K562 cells for 3 days, and then endogenous Klotho protein expression was analysed using Western blot analysis. The C2 and E2 shRNAs exhibited better knockdown efficiency (Fig. 6C). Afterward, C2 and E2 shRNA-expressed K562 cells were treated with NaB with or without DEHP. Knockdown of Klotho increased erythroid differentiation significantly, which was consistent with the findings of a previous report [30]. Notably, Klotho knockdown abolished DEHP-mediated suppression of erythroid differentiation (Fig. 6D). Overall, the results indicated that DEHP downregulated erythroid differentiation via Klotho expression induction.