Heat shock and arsenite induce expression of the nonclassical class I histocompatibility HLA-G gene in tumor cell lines

Abstract

The nonclassical histocompatibility class I gene HLA-G has a tissue-restricted expression. To explore mechanisms involved in HLA-G transcriptional regulation, we have investigated the effect of stress, including heat shock and arsenite treatment, on HLA-G expression in tumor cell lines. We show that stress induces an increase of the level of the different HLA-G alternative transcripts without affecting other MHC class I HLA-A, -B, -E, and -F transcripts. A heat shock element (HSE) that binds to heat shock factor 1 (HSF1) on stress conditions was further identified within the HLA-G promoter. Considering the ability of HLA-G to modulate the function of immunocompetent cells, we hypothesize a new feature of HLA-G as a signal regulating the immune response to stress.

Fig 1.

Induced expression of all the known HLA-G transcripts in stress-treated cells. (A and B) Pan-HLA-G primers G.257 (exon 2) and GA.3U (3′-untranslated region) were used for PCR amplification of HLA-G transcripts corresponding to all the known HLA-G isoforms (HLA-G1: 874 bp; HLA-G2: 598 bp; HLA-G3: 325 bp; HLA-G4: 601 bp; HLA-G5: 996 bp; HLA-G6: 720 bp) and obtained from heat-shocked cells, 42°C, 2 h (left panels), or from arsenite-treated cells, 100 μM As₂O₃, 2 h (right panels). HLA-G–specific transcripts were revealed by hybridization with either the exon 2–specific G.R probe (A) or an intron 4–specific G.I4F probe (B). (C) Specific RT-PCR amplification of HLA-G5 transcripts (489 bp), which corresponds to a soluble isoform, was performed with primers G.526 (exon 3) and G.i4b (intron 4). cDNAs were revealed by hybridization with a G.I4F probe. (D) Specific RT-PCR amplification of HLA-G2 and -G6 transcripts was performed with primers G.-3 (exon 2/exon 4) and G.1216 (3′-untranslated region). HLA-G6 cDNAs (745 bp) were revealed by hybridization with a G.I4F probe. (E) RT-PCR analysis of the effect of actinomycin D on heat shock–mediated HLA-G mRNA expression. M8 cells were untreated or heat shocked (42°C, 2 h) in the absence (−) or the presence (+) of actinomycin D and allowed to recover for various times. Pan-HLA-G primers G.257 (exon 2) and G.1004R (3′-untranslated region) were used for PCR amplification of HLA-G transcripts corresponding to all the known HLA-G isoforms (HLA-G1: 764 bp; HLA-G2: 488 bp; HLA-G3: 215 bp; HLA-G4: 491 bp; HLA-G5: 886 bp; HLA-G6: 610 bp). (F) Induced expression of HLA-G transcripts in a stress-treated glioblastoma cell line (T98G). RT-PCR analysis of HLA-G mRNAs obtained from untreated, heat-shocked cells (42°C, 2 h) or arsenite-treated (100 μM As₂O₃, 2 h) T98G cells and allowed to recover for 4 h. Pan-HLA-G primers G.257 (exon 2) and GA.3U (3′-untranslated region) were used for PCR amplification of HLA-G transcripts corresponding to all the known HLA-G isoforms. The JEG-3 choriocarcinoma cell line was used as a control for high HLA-G transcription. Absence of contaminant DNA was controlled by concomitant amplification of the PCR mixture without a template (H₂O). Bands corresponding to HLA-G1, -G2, -G3, -G4, -G5, and -G6 are indicated by arrows. PCR products coamplified in the same reaction by β-actin primers were detected on the same membrane by a β-actin probe

Fig 2.

Stress does not modify levels of HLA-A, -B, -E, and -F class I transcripts. (A) Northern blot analysis of mRNAs isolated from heat-shocked (42°C, 2 h) and arsenite-treated (100 μM As₂O₃, 2 h) cells at various times after recovery at 37°C. The filters were hybridized with HLA-A, HLA-B locus-specific probes, or GAPDH probe used as a control to quantify RNA in each sample. RNAs extracted from PBMCs were used as a positive control for HLA class Ia expression. (B) primers E.251 (exon 2) and E.1272 (3′-untranslated region) were used for PCR amplification of HLA-E transcripts from heat-shocked cells, 42°C, 2 h (left panel), or from arsenite-treated cells, 100 μM As₂O₃, 2 h (right panel). HLA-E–specific transcripts were revealed by hybridization with the E.R-specific probe located in exon 3. (C) RT-PCR amplification of HLA-F transcripts was performed with primers Pan clI (F) located in exon2 and Pan clI (R) located in exon 4. HLA-F-specific transcripts were revealed by hybridization with the F.R-specific probe located in exon 2. The JEG-3 choriocarcinoma cell line was used as a control for high HLA-G transcription. Absence of contaminant DNA was controlled by concomitant amplification of the PCR mixture without a template (H₂O). PCR products coamplified in the same reaction by β-actin primers were detected on the same membrane by a β-actin probe

Fig 3.

Sequences of the oligonucleotides used as probe and/or cold competitor in EMSA experiments. Sequences used are HSEHSP derived from the human hsp70 proximal promoter (position -119 to -87 upstream of the transcription start site), the wild-type HSEG (position -487 to -466 upstream of the HLA-G translation initiation codon) and mutant MUTHSEG, and an oligonucleotide corresponding to positions -1285 to -1254 of HLA-G promoter IR used as an irrelevant competitor. The residues altered in MUTHSEG are indicated in bold type. NGAAN sequences, comprising an array of inverted units characteristic of HSEs, are underlined. Palindromes formed by alignment of HSEs lying face to face on the DNA helix are framed

Fig 4.

Heat shock induces binding of HSF1 to HLA-G–and hsp70-derived HSEs. (A) EMSA analysis of the kinetic of stress-induced HSE-binding activities using ³²P-labeled HSEHSP (upper panel) or HSEG probes (lower panel) in nuclear extracts prepared from either untreated or heat-shocked M8 cells (42°C, 2h) at various recovery time at 37°C or arsenite-treated cells (100 μM As₂O₃, 2 h) in absence or presence of a 100-fold excess of cold oligonucleotide competitors (COC). (B) nuclear extracts, prepared from stressed cells immediately after the end of stress treatment, were incubated either alone or in presence of various dilutions of anti-HSF1 (α-HSF1) serum or in presence a nonspecific rabbit polyclonal control antiserum (CAS) for 15 min at room temperature before addition of the binding solution. SC and NSC denote specific and nonspecific DNA-protein complexes, respectively