Effect of erythropoietin production induced by hypoxia on autophagy in HepG2 cells

https://doi.org/10.1016/j.bbrc.2017.11.167Get rights and content

Highlights

  • Erythropoietin production by hypoxia restrained autophagy induction.

  • Autophagy induction by hypoxia did not affect expression of erythropoietin mRNA.

  • Increase of phosphorylated STAT3 by erythropoietin restrained autophagy induction.

  • Restraint of autophagy induction by erythropoietin did not affect cell viability.

Abstract

The induction of hypoxia inducible factor (HIF) and autophagy are important cellular responses to hypoxia. The production of erythropoietin (EPO) regulated by HIF is increased by hypoxia and participates in cell protection in various organs and tissues. Signal Transducers and Activator of Transcription 3 (STAT3) is a regulatory factor that is common to autophagy induction and EPO-EPO receptor signaling. In this study, we analyzed the promotion of EPO production and autophagy, and the participation of STAT3, in HepG2 cells under hypoxia. Treatment with EPO siRNA (si-EPO) significantly increased autophagy induction by hypoxia, while treatment with recombinant EPO inhibited the effect of si-EPO. NSC74859, an inhibitor of the phosphorylation of STAT3, increased autophagy induction to the same extent as si-EPO treatment. Even when 3-Methyladenine, an inhibitor of autophagy, was added, the increase of EPO mRNA expression due to hypoxia was not affected. Hypoxia-induced EPO restrained autophagy induction through the EPO receptor and phosphorylation of STAT3. Because cell viability with treatment of si-EPO under hypoxia did not increase over the control, our results suggested that EPO produced by hypoxia prevented excess autophagy induction.

Introduction

The decline of oxygen concentrations at high altitudes and the decline of oxygen supply to the body due to anemia lead to systemic hypoxia. Additionally, the lack of blood flow during infarction of blood vessel leads to microenvironmental hypoxia. Cell injury by hypoxia is caused by a decline of ATP production by inhibition of oxidative phosphorylation in intracellular mitochondria [1], [2]. Failure of intracellular Na+ and Ca2+ ion balance occurs by inhibition of ATP-dependent Na and Ca pumps [3]. Oxidative stress increases reactive oxygen species (ROS) production due to an increase of hypoxanthine concentration due to the metabolism of the adenine nucleotides and the inhibition of oxidative phosphorylation in mitochondria [2], [3], [4]. Various protective mechanisms activate to prevent cell injury by hypoxia. These include the restraint of ROS production [5], the induction of hypoxia inducible factor (HIF) that participates in cytoprotection [6], and the induction of autophagy [4], [7], [8].

One of the factors regulated by HIF is erythropoietin (EPO). EPO is indispensable to red blood cell production and participates in cell protection in various organs and cell types [9]. EPO is produced in nerves, myocardium, blood vessel walls, spleen, liver, and kidney, and EPO receptor (EPOR) is expressed in various organs. It has been reported that cytoprotection by EPO-EPOR signaling includes antiapoptotic, antioxidant, and anti-inflammatory activities [9], [10]. Neoplastic cells produce EPO, which is thought to protect these cells from apoptosis [9]. We previously reported that EPO produced in HepG2 cells derived from human liver cancer affects cell protection [11]. Many reports indicate that autophagy is induced by hypoxia, but there are reports that hypoxia downregulates autophagy in neoplastic cells of the pancreas and nervous system [7], [8], [12], [13].

Various regulatory factors participate in autophagy induction in the hypoxic state, including HIF [14], [15], but it is not apparent whether EPO, which is induced by hypoxia, participates in autophagy. STAT3 is a regulatory factor common to both autophagy induction [15] and EPO-EPOR signaling [16], [17], [18], [19]. STAT3 is a transcription factor and oncoprotein known to promote the survival of neoplastic cells [21]. In EPO-EPOR signaling, EPOR promotes the phosphorylation of STAT3 through activation of tyrosine kinases such as JAK2 [9]. Inhibiting phosphorylation of STAT3 upregulates autophagy through Akt/mTOR and Era signaling in head and neck squamous cell carcinoma [22]. However, there is a report that phosphorylation of STAT3 upregulates autophagy in pancreatic cancer [23], and the relationship between STAT3 and autophagy appears to vary by cell type. Therefore, in the present study, we analyzed the relationship between induced EPO and autophagy and the participation of STAT3 in HepG2 cells under hypoxia.

Section snippets

Materials

HepG2 cells were provided by the RIKEN BRC through the National Bio-Resource Project of the MEXT, Japan. Primers for real-time reverse transcription-polymerase chain reaction (RT-PCR) were purchased from Life Technologies Japan Ltd. (Tokyo, Japan). Dulbecco's modified Eagle's medium (DMEM) was purchased from Nissui Phamaceutical Co. (Tokyo, Japan). Recombinant human erythropoietin (rEPO) was purchased from R&D Systems, Inc. (Minneapolis, MN). 3-Methyladenine (3-MA) was purchased from BioGems

Effect of EPO induced by hypoxia on autophagy induction in HepG2 cells

Autophagy due to hypoxia was evaluated by MDC staining (Fig. 1A) and quantification of LC3II (Fig. 1B). When EPO mRNA expression was inhibited by EPO siRNA (si-EPO), the autophagy derived by hypoxia significantly increased compared to the si-control RNA (si-Control). Treatment with si-EPO did not affect autophagy induction under normoxic conditions (data not shown). When recombinant EPO was added to HepG2 cells treated with si-EPO under hypoxia, the promotion of autophagy due to hypoxia

Discussion

In this study, we investigated the relationship between EPO production and autophagy induction during hypoxia. Even when autophagy was restrained by 3MA, EPO mRNA expression under hypoxia did not change, suggesting that autophagy did not influence EPO mRNA expression. On the other hand, autophagy induction by hypoxia was increased by si-EPO addition, suggesting that the increase of EPO production by hypoxia restrained autophagy induction. When rEPO was added when EPO expression was inhibited

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