Elsevier

Human Immunology

Volume 68, Issue 4, April 2007, Pages 277-285
Human Immunology

Hypoxia Modulates HLA-G Gene Expression in Tumor Cells

https://doi.org/10.1016/j.humimm.2006.10.016Get rights and content

Abstract

Human leukocyte antigen G (HLA-G) molecules are expressed in cytotrophoblasts and play a key role in maintaining immune tolerance at the maternal-fetal interface. HLA-G expression was also reported in inflammatory diseases, organ transplantation, and malignant tumors. The regulatory mechanisms of HLA-G gene expression differ from those of classical HLA class I genes and are still only partially elucidated. Focusing on tumor cells, we previously demonstrated a tight control of HLA-G gene expression by cis-acting epigenetic mechanisms. In the present study, we hypothesized that these processes are dependent of microenvironment conditions, and more particularly, stress conditions like hypoxia. Cellular response to hypoxia is mainly driven by a key transcription factor, hypoxia-inducible factor 1 (HIF-1), and other factors, such as NF-κB, involved in angiogenesis and cell survival. Here we confirmed the influence of hypoxia on HLA-G gene induction in the HLA-G–negative M8 melanoma cell line. Moreover, upon treatment with the hypoxia-mimicking desferrioxamine, we demonstrated a decrease in HLA-G gene expression in melanoma FON and choriocarcinoma JEG-3 cell lines, both expressing constitutively HLA-G. Finally, we demonstrated for the first time that the modulation of HLA-G gene expression is dependent of HIF-1 stabilization and thus might be relevant for the control of HLA-G gene expression in hypoxic tumors.

Introduction

The nonclassical major histocompatibility complex class I molecule human leukocyte antigen G (HLA-G) is a key regulator of immune responses by modulating natural killer, cytotoxic T lymphocytes, and antigen-presenting cell functions. HLA-G plays a crucial role in fetal-maternal tolerance, maintenance of transplanted organs, and escape from tumor immunosurveillance [1]. The mechanisms leading to restricted HLA-G expression in normal tissues and to ectopic HLA-G expression in cancer have to be investigated more precisely [2]. Among them, one important level of regulation is HLA-G gene transcription, which is specifically controlled by unique HLA-G gene promoter because almost all known regulatory sequences for classical HLA class I genes are disrupted [3]. Nevertheless, only a few transcription factors and regulatory sequences have been identified until now. Regulation may also act on HLA-G protein expression through the efficiency of translation and the modulation of antigen processing machinery component expression [4, 5]. Moreover, in vivo stimulators are likely to be key factors controlling one or several levels of regulation. Some of them have been identified, such as interleukin-10 [6, 7], interferons [8, 9, 10], leukemia inhibitory factor [11], hormones [12, 13], and stress [14, 15], but how they work is generally still under investigation.

By treating tumoral cells with 5-aza-2′-deoxycytidine demethylating agent or inhibitors of histone deacetylase in vitro, we and other groups demonstrated that HLA-G gene activity is controlled by cis-acting epigenetic mechanisms, such as DNA methylation/demethylation [16, 17, 18] and histone deacetylation/acetylation [19]. An emerging hypothesis is that these processes are dependent of microenvironment conditions—and more particularly—stress conditions [20]. Indeed, although HLA-G silencing by DNA methylation appears to be a common feature of tumor cell lines cultured in the absence of stressful conditions, HLA-G is expressed in numerous tumor lesions in vivo [2]. To further explore the regulatory mechanisms that participate in tumor-specific activation of HLA-G transcription under physiologic conditions, we thus focused on the effect of hypoxia—a physiologically relevant tumor-related stress—that is a common feature of rapidly growing malignant tumors and their metastases [21]. In such a situation, the balance between cell proliferation and oxygen supply is affected, leading to a decrease in oxygen partial pressure. However, cancer cells can undergo genetic and adaptive changes that allow them to survive and proliferate for better perfusion and oxygen partial pressure. A large part of the coordinated cellular response to hypoxia is driven by a transcriptional program initiated via stabilization of the hypoxia-inducible factor 1 (HIF-1), promoting angiogenesis and cell survival [22]. HIF-1 consists of α and β subunits. Under normoxic conditions, HIF-1α hydroxylation enables the von Hippel-Lindau protein to bind to HIF-1α. This binding causes ubiquitylation of HIF-1α, resulting in its degradation by proteasome. Under hypoxic conditions, stabilized HIF-1α translocates into the nucleus, where it binds to HIF-1β [23]. The HIF-1 complex binds to hypoxia-responsive element sequences (5′-RCGTG-3′) [24] in the promoters of target genes, such as vascular endothelial growth factor [25] and erythropoietin [26], causing activation of transcription. While HIF-1 plays a major role in controlling the ubiquitous transcriptional response to hypoxia, it is clear that other transcription factors are also activated [22]. In particular, upon hypoxic stimulation, NF-κB translocates into the nucleus following ubiquitylation and degradation of the repressor molecule IκB [27].

Chang and Ferrone gave the first evidence for the induction of HLA-G transcription in tumoral cells upon treatment with hypoxia mimetic desferrioxamine (DFX) using HLA-G–negative melanoma cells [15]. In the present study, we reproduced their data using classical and real-time reverse-transcriptase–polymerase chain reaction (RT-PCR), and we confirmed the influence of hypoxia on HLA-G gene induction in the M8 melanoma cell line. We also assessed the effect of hypoxic treatment on HLA-G–positive tumor cell lines, revealing a downregulation of HLA-G expression. In both cases, we demonstrated that the modulation of HLA-G gene expression is dependent on HIF-1 stabilization.

Section snippets

Human Cell Line Cultures

The choriocarcinoma cell line JEG-3 (American Type Culture Collection, Rockville, MD) was maintained in Dulbecco’s modified Eagle’s medium with Glutamax-I supplemented with 4500 mg/l glucose (Invitrogen, Cergy Pontoise, France). The melanoma cell lines M8 [28], 1074mel, and FON [29] were maintained in RPMI 1640 (Sigma-Aldrich, St. Quentin Fallavier, France) supplemented with 2 mM L-glutamine (Sigma). All cultures were also supplemented with 10% heat-inactivated fetal calf serum, gentamicin (10

HLA-G Gene Transcription is Activated by Hypoxia in HLA-G–Negative Cell Lines

According to the experiments of Chang et al. [15, 20], HLA-G gene transcription in melanoma cell line 1074mel is activated by the hypoxia-mimicking reagent, DFX, used at 250 μM for 24 hours (Figure 1A).

To evaluate the extent of HLA-G gene modulation by hypoxia in other HLA-G–negative cell lines, we first analyzed the effect of DFX, for 24 hours, on the activation of HLA-G gene transcription in different human HLA-G–negative malignant cell lines from a melanoma (M8), a choriocarcinoma (JAR), a

Discussion

In an attempt to identify physiologically relevant factors that may modulate HLA-G expression in tumor cells, we investigated hypoxia conditions on cells expressing or not expressing HLA-G transcripts. We noted that the response in HLA-G expression after cell exposure to DFX varied according to the HLA-G positive or HLA-G negative status of the cells.

First, we observed that after incubation with the hypoxia mimetic DFX, HLA-G mRNA was significantly induced in two melanoma cell lines that do not

Acknowledgments

This work was supported by the Commissariat à l’Energie Atomique and by the Association pour la Recherche sur le Cancer (grants 7611 and 7806). We thank Dr. S. Ferrone, Roswell Park Cancer Institute, Buffalo, NY for providing the 1074mel and TP25-99 mAb, and Dr. F. Faure, Institut Curie, Paris, France for providing the FON cells.

References (41)

  • T. Nagamatsu et al.

    Hypoxia does not reduce HLA-G expression on extravillous cytotrophoblasts

    J Reprod Immunol

    (2004)
  • N. Denko et al.

    Hypoxia actively represses transcription by inducing negative cofactor 2 (Dr1/DrAP1) and blocking preinitiation complex assembly

    J Biol Chem

    (2003)
  • L. Liu et al.

    Hypoxia-induced energy stress regulates mRNA translation and cell growth

    Mol Cell

    (2006)
  • H.J. Jeong et al.

    Hypoxia-induced IL-6 production is associated with activation of MAP kinase, HIF-1, and NF-kappaB on HEI-OC1 cells

    Hear Res

    (2005)
  • N. Rouas-Freiss et al.

    HLA-G proteins in cancer: do they provide tumor cells with an escape mechanism?

    Cancer Res

    (2005)
  • C. Solier et al.

    HLA-G unique promoter region: functional implications

    Immunogenetics

    (2001)
  • P. Moreau et al.

    HLA-G protein processing and transport to the cell surface

    Cell Mol Life Sci

    (2002)
  • W.H. Yan et al.

    Induction of HLA-G expression in a melanoma cell line OCM-1A following the treatment with 5-aza-2′-deoxycytidine

    Cell Res

    (2005)
  • P. Moreau et al.

    IL-10 selectively induces HLA-G expression in human trophoblasts and monocytes

    Int Immunol

    (1999)
  • S. Ugurel et al.

    Soluble human leukocyte antigen–G serum level is elevated in melanoma patients and is further increased by interferon-alpha immunotherapy

    Cancer

    (2001)
  • Cited by (97)

    • Controlled hypobaric hypoxia increases immunological tolerance by modifying HLA-G expression, a potential therapy to inflammatory diseases

      2020, Medical Hypotheses
      Citation Excerpt :

      In this sense, hypoxia should be considered a double-edged sword concerning medical conditions. HLA-G is an immunosuppressive molecule which can be regulated by Hypoxia-Inducible Factors (HIFs) [22]. High levels of this molecule have already been measured, and proposed to act as regulators, in climbers exposed to high altitudes [27].

    View all citing articles on Scopus
    View full text