Mechanistic target of rapamycin (mTOR) dependent regulation of thioredoxin interacting protein (TXNIP) transcription in hypoxia

Abstract

Thioredoxin interacting protein (TXNIP), first identified as an inhibitor of thioredoxin, is also a tumor suppressor as well as an inhibitor of lipogenesis. TXNIP is known to be transcriptionally regulated in response to nutrients such as glucose and stress signals, including endoplasmic reticulum stress and lactic acidosis. In this study, we characterized the transcriptional regulation of TXNIP in response to hypoxia. Using a hepatocellular carcinoma cell line, we have found that TXNIP mRNA expression is regulated in a biphasic manner in hypoxia whereby TXNIP expression showed an initial rapid decrease, followed by an increase under prolonged hypoxia. Interestingly, we have shown that TXNIP induction in prolonged hypoxia is independent of the Hypoxia-Inducible Factor (HIF) transcription factor. The effect of hypoxia on TXNIP expression is mediated via the inhibition of the 4E-BP1/eIF4E axis of mechanistic target of rapamycin (mTORC1). Thus, we found that inhibiting mTORC1-dependent 4E-BP1 phosphorylation mimics the effect of hypoxia on TXNIP expression. Furthermore, overexpressing eIF4E prevents the induction of TXNIP in hypoxia. Our results suggest that mTORC1 may be an important regulator of hypoxia-dependent gene expression.

Introduction

Hypoxia is a type of microenvironmental stress prevalent in solid tumors, alongside with deprivation of glucose and other nutrients, as well as extracellular acidosis. To mediate adaptive responses to reduced oxygen levels, protective mechanisms at the transcriptional level are activated. These responses facilitate adjustments in cellular physiology and metabolism for survival and growth under hypoxic conditions. The major regulator of the cellular response to hypoxic stress is the transcription factor Hypoxia-Inducible Factor 1 (HIF-1). HIF-1 exists as a heterodimer of a constitutively expressed nuclear subunit, HIF-1β, and a α-subunit, HIF-1α or HIF-2α, that is controlled by cellular oxygen levels. As a result, HIF-1 transcriptional activity is regulated in an oxygen-dependent manner. In the presence of oxygen, HIF-1α is unstable and is rapidly degraded. This is due to the hydroxylation of proline residues in HIF-1α by HIF prolyl hydroxylase domain (PHD) enzymes. The hydroxylation of HIF-1α leads to the binding of the von Hippel-Lindau tumor suppressor protein (pVHL), a substrate adaptor component of an E3 ubiquitin ligase complex. Subsequent ubiquitination of the HIF-1α protein by the pVHL E3 ligase complex triggers a rapid proteasome-dependent degradation of HIF-1α [1]. Under hypoxic conditions, the activity of PHD enzymes is inhibited because in addition to 2-oxoglutarate and iron, oxygen is required as a substrate to catalyze the hydroxylation reaction. Therefore, hypoxia prevents the binding of pVHL and significantly prolongs the half-life of HIF-1α. This allows HIF-1α accumulation and translocation into the nucleus where it dimerizes with HIF-1β, followed by binding to the Hypoxia-Response Element (HRE) of hypoxia-inducible genes. In the face of oxygen deficits, HIF orchestrates cellular adaptations by inducing the expression of a wide array of genes, including pro-angiogenic factors and genes involved in glycolysis and glucose transport [2].

Thioredoxin-interacting protein (TXNIP) is an important gene that is known to be transcriptionally regulated in response to hypoxia. TXNIP, also known as Vitamin D3 up-regulated protein (VDUP1), was first identified as an endogenous inhibitor of thioredoxin [3]. TXNIP has also been demonstrated to function as an important regulator of cellular glucose [4], [5], [6] and lipid metabolism [7], [8]. Furthermore, TXNIP is believed to function as a tumor suppressor gene, and is often suppressed in various human tumors. Importantly, TXNIP-deficient mice have markedly increased incidence of hepatocellular carcinoma as well a number of other cancers [6], [9].

TXNIP has also been shown to be transcriptionally upregulated by glucose [10]. In addition, various types of cellular stress have been shown to cause an upregulation of TXNIP. These include oxidative damage, heat shock, UV irradiation, endoplasmic-reticulum stress, lactic acidosis and hypoxia [11], [12], [13]. TXNIP was shown to be a hypoxia-induced gene in human microendothelial cells [14] and in murine heart [15]. TXNIP has also been reported to be induced during hypoxia in a HIF-dependent manner in pancreatic cancer [16]. In contrast, we have recently reported that hypoxia results in a rapid decrease in TXNIP mRNA and protein expression via a HIF-independent mechanism in Hela cells [17]. Therefore, the effect of hypoxia on TXNIP expression and the role of HIF remain controversial.

Given the importance of TXNIP as a major regulator of cellular redox state and metabolism and the well-established link between TXNIP and human diseases such as diabetes and cancers, a thorough understanding of the transcriptional regulation of TXNIP in hypoxia is important. In this study, we focus on the mechanistic aspect of hypoxia-dependent regulation of TXNIP. We found that hypoxia caused a biphasic response to TXNIP expression, whereby TXNIP is initially downregulated followed by a transcriptional induction under prolonged hypoxia. Interestingly, hypoxia-dependent induction of TXNIP expression is independent of the HIF pathway. We also provide evidence that the upregulation of TXNIP under prolonged hypoxia is a consequence of the inhibition of the 4EBP1-eIF4E axis of the mTOR signaling pathway.