IRF-1 and p65 mediate upregulation of constitutive HLA-A antigen expression by hepatocellular carcinoma cells

Abstract

Malignant transformation of hepatocytes is frequently associated with upregulation of HLA-A expression. Currently there is no information available regarding the mechanisms underlying this phenotypic change. We investigated HLA-A expression in 165 paraffin embedded tissues and 21 fresh tissues from liver cancer patients. Utilizing truncated HLA-A promoter-reporter constructs and gel-shift assay we had identified the regulatory elements and transcription factors required for HLA-A upregulation. 54% of the paraffin embedded tissues showed increased HLA-A expression in their cancerous part. 43% of the fresh liver cancer tissues had increased HLA-A complex expression with the HLA-A heavy chain gene demonstrating the highest level of upregulation (62%). Enhanced HLA-A expression in the liver cell lines QGY7701 and BEL7402 was found to be mediated by binding of interferon regulatory factor 1 (IRF-1) to interferon stimulated response element, and of nuclear transcription factor p65 binding to enhancer A element in the HLA-A promoter of these cell lines. The in vivo relevance of these findings was indicated by the association of the enhanced expression of IRF-1 and accumulation of nuclear p65 with HLA-A upregulation in 8 of the 21 liver cancer lesions investigated. Our results indicated that HLA-A upregulation in liver cancer was mediated by both increased nuclear aggregation of transcription factor p65 and upregulation of transcription factor IRF-1.

Fig. 1

HLA-A expression was elevated in liver cancer. (A) Typical cell surface expression of HLA-A antigen in paraffin embedded liver cancer tissue (left panel) with negative expression in its normal compartment (right panel, positive staining of infiltrate lymphocyte and endothelial cells, Immunohistochemistry, 200x). (B) Increased expression of HLA-A complex in fresh liver cancer tissue (left panel) with negative expression in its normal compartment (right panel, positive staining of infiltrate lymphocyte and endothelial cells, Immunohistochemistry, 200x). (C) The expression of several molecules in the antigen processing pathway was detected by RT-PCR in liver cancer tissue (T) compared to its noncancerous part (N), with increased expression of HLA-A heavy chain in all the 3 samples.

Fig. 2

The ISRE and enhancer A were important elements for the constitutive HLA-A promoter activity in liver cancer cell lines. (A) Schematic illustration of constructs used for HLA-A heavy chain promoter-luciferase reporter assay. AP: HLA-A heavy chain promoter (from −217 bp to the transcriptional initiation site); AP-ISRE del: HLA-A heavy chain promoter without ISRE; AP1: HLA-A heavy chain promoter without enhancer A; AP2: HLA-A heavy chain promoter without enhancer A and ISRE; AP3: HLA-A heavy chain promoter without enhancer A, ISRE, siteα and enhancer B. All of them were constructed into pGL3-Basic luciferase reporter plasmid. (B) Increased HLA-A expression in HCC cell lines QGY7701 and BEL7402 compared to BEL7405. Total cellular RNA from the three cells were extracted and subjected to Northern blot analyses. The expression of the β-actin gene was served as the internal control. (C) Luciferase activity of different promoter–reporter plasmids in the three HCC cell lines. For each cell line, mean luciferase activity was calculated from three independent experiments and shown with the standard error. (D) Comparison of the activity of enhancer A and ISRE in different HCC cell lines. The activity of enhancer A, ISRE, or enhancer A + ISRE was calculated by dividing the mean luciferase activity of each reporter construct with the element by that of the comparable construct without the element. Both of the enhancer A and ISRE activity were obviously increased in QGY7701 and BEL7402 cells compared to BEL7405. Besides that, the combined transcriptional activity of enhancer A and ISRE was more than that of the individual one.

Fig. 3

Transcription factor IRF-1 regulated HLA-A promoter activity through binding to ISRE in liver cancer cell lines. (A) The intensity of band A was increased in QGY7701 and BEL7402 cells than in BEL7405 cells. Band A was formed by incubating labeled ISRE probes with nuclear extracts of QGY7701, BEL7402 or BEL7405. Incubating labeled ISRE probes with nuclear extracts of IFN-γ treated BEL7405 was used as the positive control (lane 2). (B) Band A could be competed by cold ISRE probe. Nuclear extracts of QGY7701 were incubated with labeled ISRE probes either in the presence of 200-fold excess of unlabeled OCT-1 fragments (lane 2) or of unlabeled IRF-1 fragments (lane 3). (C) Nuclear extracts of QGY7701 were incubated with labeled ISRE probes (lane 2) and band A was decreased in the presence of anti-IRF-1 antibody (lane 1), but not of anti-IRF-2 antibody (lane 3). (D) The expression of HLA-A heavy chain and IRF-1 protein was detected in HCC cell lines by Western analysis. IRF-1 and HLA-A were both increased in QGY7701 and BEL7402 cells compare to BEL7405 cells. The amount of HLA-A was calculated as the ratio of the density of the HLA-A band to the density of the β-actin band by using Software Image 7. Other bands in the following figures were calculated by the same way except the result of Western blot analysis of nuclear protein. (E) Elevated expression of HLA-A heavy chain was detected by Western analysis after transfection of full length of IRF-1 gene into BEL7405 cells. The expression of HLA-A heavy chain in QGY7701 cells was added as the positive control. (F) Increased expression of surface HLA-A complex in BEL7405 cells transfected with IRF-1 was demonstrated by indirect flow cytometry analysis with antibody LGIII-147.4.1. The gray area represented background staining of the cells with the isotype-matched control antibody. The dotted line and the thin solid line represented HLA-A expression of BEL7405 cells transfected with an empty plasmid and with IRF-1, respectively. (G) Detected by Western analysis, the expression of HLA-A heavy chain was decreased after transfection of dominant negative form of IRF-1 or IRF-2 in BEL7405 cell.

Fig. 4

Transcription factors p65 binding to enhancer A regulated HLA-A promoter activity in liver cancer cell lines. (A) P65 binding to enhancer A was increased in QGY7701 and BEL7402 cells compared to BEL7405. Nuclear extracts of QGY7701 was incubated with labeled enhancer A probes (lane 5, band B). In the presence of 50-, 100-, or 200-fold excess of unlabeled enhancer A probes (lanes 1, 2, and 3), band B was weakened in response to competitive inhibition in a dose depended manner. Band B was supershift by anti-p65 mAbs (lane 4) and the intensity of band B was increased in the nuclear extracts of QGY7701 and BEL7402 compared to BEL7405 (lanes 5, 6, and 7). (B) The expression of total p65, IκB, and nuclear p65 was examined in HCC cell lines by Western blot. The expression of p65 was increased only in the nuclear extracts of QGY7701 and BEL7402 cells compared to that of BEL7405 cells (The number below each band was calculated as the ratio of the density of the p65 band in the BEL7402 cells and BEL7405 cells to that in the QGY7701 cells). In contrast, the expression of IκB was decreased in QGY7701 and BEL7402 cells. (C) Detected by Western analysis, HLA-A heavy chain proteins were strongly induced by transfection of p65 expression plasmid. (D) Increased cell surface expression of HLA-A complex in BEL7405 upon p65 transfection was detected by flow cytometry analysis with antibody LGIII-147.4.1. The gray area represented background staining of the cells with the control antibody. The dotted line and the thin solid line represented HLA-A expression of BEL7405 cells transfected with an empty plasmid and with p65, respectively. (E) The expression of HLA-A protein was specifically decreased in BEL7405 by transfection of IkB or of an IkB mutant plasmid, which degrades slowly in cell cytoplasm than wild type IkB.

Fig. 5

Synergistic induction of HLA-A expression by IRF-1 and p65. (A) Detected by luciferase assay, p65 transactivated HLA-A promoter–reporter (from −217 bp to the transcription initiation site) more strongly than IRF-1 in BEL7405 cell, while the highest induction was seen when the cell was co-transfected with IRF-1 and p65. (B) Simultaneously transfection of the two factors led to an obviously increased HLA-A complex expression over the level of expression achieved by transfection with IRF-1 or p65 separately. The gray area showed background staining of the cells with the control antibody. HLA-A expression in BEL7405 cells transfected with empty plasmid was represented by dotted line; transfected with IRF-1, thin solid line; transfected with p65, black line; transfected with both IRF-1 and p65, shallow gray area.

Fig. 6

Enhanced HLA-A expression in liver cancer tissues was associated with IRF-1 upregulation and activation of NF-ΚB. (A) Upregulation of IRF-1 and increased HLA-A expression in the same HCC tissue (number 10). Beta-actin was included as an internal control. The expression of IRF-1 and HLA-A in QGY7701 cells was used as a positive control (pc). (B) Increased expression of nuclear p65 with enhanced expression of HLA-A heavy chain in the same HCC tissue (number 21).