Papillomavirus type 16 oncogenes downregulate expression of interferon-responsive genes and upregulate proliferation-associated and NF-kappaB-responsive genes in cervical keratinocytes - PubMed (original) (raw)
Papillomavirus type 16 oncogenes downregulate expression of interferon-responsive genes and upregulate proliferation-associated and NF-kappaB-responsive genes in cervical keratinocytes
M Nees et al. J Virol. 2001 May.
Abstract
Infection with high-risk human papillomaviruses (HPV) is a major risk factor for development of cervical cancer. Expression of the HPV E6 and E7 oncoproteins increases in differentiating keratinocytes, resulting in inactivation of the p53 and retinoblastoma proteins, two important transcriptional regulators. We used cDNA microarrays to examine global alterations in gene expression in differentiating cervical keratinocytes after infection with retroviruses encoding HPV type 16 (HPV-16) E6 and E7. Expression of 80 cellular genes (approximately 4% of the genes on the array) was altered reproducibly by E6 and/or E7. Cluster analysis classified these genes into three functional groups: (i) interferon (IFN)-responsive genes, (ii) genes stimulated by NF-kappaB, and (iii) genes regulated in cell cycle progression and DNA synthesis. HPV-16 E6 or a dominant negative p53 protein downregulated multiple IFN-responsive genes. E6 decreased expression of IFN-alpha and -beta, downregulated nuclear STAT-1 protein, and decreased binding of STAT-1 to the IFN-stimulated response element. E7 alone was less effective; however, coexpression of E6 and E7 downregulated IFN-responsive genes more efficiently than E6. The HPV-16 E6 protein also stimulated expression of multiple genes known to be inducible by NF-kappaB and AP-1. E6 enhanced expression of functional components of the NF-kappaB signal pathway, including p50, NIK, and TRAF-interacting protein, and increased binding to NF-kappaB and AP-1 DNA consensus binding sites. Secretion of interleukin-8, RANTES, macrophage inflammatory protein 1alpha, and 10-kappaDa IFN-gamma-inducible protein were increased in differentiating keratinocytes by E6. Thus, high-level expression of the HPV-16 E6 protein in differentiating keratinocytes directly alters expression of genes that influence host resistance to infection and immune function.
Figures
FIG. 1
Cluster analysis of cellular gene expression. Data from 48 independent hybridization experiments examined multiple experimental conditions and different combinations of HPV-16 E6 and/or E7 genes or p53DN. Data were organized and displayed using cluster analyses (K-Means clustering algorithm). The four treatment groups are indicated at the top. Group 1 included cells expressing p53DN, E6, E7, or E6/E7 and maintained in the absence of growth factors with 1.4 mM Ca2+ to induce differentiation. Three experiments were performed using cells that were made quiescent but that were not induced to differentiate with calcium (qui). Group 2 included HPV-immortalized keratinocytes in Ca2+ to induce differentiation. Group 3 denotes a subset of experiments performed in parallel with group 1 but with inversion of Cy3 and Cy5 dyes as a control for labeling. Group 4 includes HPV-infected, differentiating keratinocytes treated with 300 to 1,000 of U IFN-α, -β, or -γ per ml. In groups 1 to 3, vector-only-infected cells are compared to HPV-infected cells; in group 4, E6/E7-expressing cells with and without IFN are compared. Clusters of differentially expressed mRNAs are shown on the left (I to IV); gene names and IMAGE clone IDs are given at the right. Color coding: green, downregulation of gene expression; red, induction; black, no significant change; grey, no data available.
FIG. 2
RT-PCR and ELISA of differential expression of IFN-inducible genes in cluster II. (A) RT-PCR analysis of IFN-regulated genes after infection of keratinocytes with HPV E6 and/or E7. Duplicate data are from two independent experiments. 2′-5′-OAS, 2′-5′ oligoadenylate synthase. (B) RT-PCR analysis of IFN-α and -β RNAs using consensus primer pairs for IFN. NC indicates the negative control in which no enzyme was added to the RT reaction. (C) ELISA analysis of intracellular IFN-α from one representative experiment. Keratinocytes were infected with HPV-16 E6, E6/E7, or p53DN. (D) ELISA analysis of intracellular IFN-α from three independent experiments. Cells were infected with HPV-16 oncogenes or p53DN mutant as above, with low-risk HPV-6 E6 or E7, or with sense p53 (p53+). Bars depict the means of three experiments, and brackets indicate standard errors. Changes other than HPV-6 E7 were statistically significant (Fisher's exact test).
FIG. 3
Western analysis showing altered expression and activity of STAT-1 in keratinocytes expressing E6 and E7. (A) Western analysis of total, cytoplasmic, nuclear, and phosphorylated STAT-1 protein in keratinocytes after infection with retroviruses containing HPV-16 E6 and/or E7. (B) EMSA for binding of STAT transcription factors to oligonucleotides containing the ISRE consensus sequence. Nuclear extracts were derived from cells infected with E6 and/or E7 or p53DN mutant and grown in the presence of dsRNA (which induces STAT activation), basal medium to induce quiescence (basal), or the same medium with Ca2+ to induce differentiation. HeLa, control nuclear extract from HeLa cell line; 100x comp., competition for specific DNA binding with a 100-fold molar excess of unlabeled oligonucleotide; 100x unsp. comp, unspecific competition with unrelated oligonucleotide.
FIG. 4
RT-PCR and EMSA demonstrate that E6 increased expression of NF-κB-regulated genes and enhanced activity of NF-κB and AP-1. (A) RT-PCR analysis of several NF-κB-regulated genes after infection with HPV-16 E6 and/or E7. Duplicate data are from two independent experiments. (B) Western analysis of p65 RelA, p50 (NF-κB1), IκBα, and p38 MAPK proteins in keratinocytes after infection with retroviruses containing HPV-16 E6 and/or E7. (C) EMSA analysis for binding of CREB, AP-1, and NF-κB transcription factors in nuclear extracts to oligonucleotides with the respective DNA consensus sequences. Nuclear extracts were derived from cells growing in the presence of dsRNA (which induces STAT activation), and quiescent (basal) or differentiating keratinocytes (Ca2+). HeLa, control nuclear extract from HeLa cell line; 100x comp., competition for specific DNA binding with a 100-fold molar excess of unlabeled oligonucleotide; 100x unsp. comp, unspecific competition with unrelated oligonucleotide. D. EMSA analysis of NF-κB DNA binding activity in keratinocytes expressing wild-type HPV-16 E6 and mutants with variable p53-degrading activities. E6mut F2V does not bind and degrade p53 in vitro. Mutants L110Q and F125V retain p53 binding activity.
FIG. 5
RNase protection analysis of chemokine gene expression. Cervical keratinocytes were infected with empty vector (pLXSN) or E6, E7, and E6/E7 retroviruses and induced to undergo squamous differentiation. Raw data were normalized using glyceraldehyde-3-phosphate dehydrogenase and L32 expression. Data were analyzed by densitometry using a PhosphorImager.
FIG. 6
ELISA analysis of chemokine secretion. Keratinocytes were infected with HPV-16 E6 and/or E7 retroviruses or p53DN mutant. Cells were induced to undergo differentiation, and some cultures were treated with TNF-α. ELISA was performed on supernatants of E6/E7-infected cultures; in other experiments, cells were examined after several (three to eight) subpassages or after immortalization. Data are averages of four individual ELISA readings from experiments and were normalized for absolute cell number or total cellular protein.
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