Human papillomaviruses modulate expression of microRNA 203 upon epithelial differentiation to control levels of p63 proteins

Abstract

Human papillomaviruses (HPV) link their life cycles to epithelial differentiation and induce productive replication of viral DNA in suprabasal cells. Viral-DNA amplification requires cells to remain active in the cell cycle upon differentiation. This is in contrast to normal cells, which lose proliferative capability upon differentiation. One factor that negatively regulates proliferative capability upon differentiation is microRNA 203 (miR-203), which is expressed primarily in suprabasal epithelial cells. Although HPVs do not encode their own microRNAs (miRNAs), they modulate expression of cellular miRNAs to regulate the activities of cellular proteins. We show that the HPV E7 protein downregulates miR-203 expression upon differentiation, which may occur through the mitogen-activated protein (MAP) kinase/protein kinase C (PKC) pathway. One target of miR-203 is the p63 family of transcription factors, and we demonstrate that HPV-positive cells maintain significantly higher levels of these factors upon differentiation than do normal keratinocytes. Several downstream targets of p63, CARM-1, p21, and Bax, were also increased in E7-expressing cells, and their levels were inversely correlated with amounts of miR-203. Introduction of expression vectors for miR-203 into keratinocytes that stably maintain HPV episomes resulted in short-term elevation of HPV genome copy numbers, but these were rapidly lost upon subsequent passage. When HPV-positive cells expressing high levels of miR-203 were induced to differentiate in methylcellulose, impaired genome amplification was observed. We conclude that high levels of miR-203 are inhibitory to HPV amplification and that HPV proteins act to suppress expression of this microRNA to allow productive replication in differentiating cells.

FIG. 1.

miR-203 levels increase upon differentiation of normal keratinocytes but are downregulated in HPV-positive cells. Northern blot analysis of miR-203 levels in HPV-positive and normal keratinocytes differentiated in 1.5% methylcellulose for 24 and 48 h. (A) NHKs and keratinocytes stably expressing HPV-31 episomes (NHK-31). (B) NHKs and keratinocytes stably expressing HPV-31 E6/E7 and E7. (C) NHKs and keratinocytes expressing HPV-31 E6. 28S rRNA was a loading control. nt, nucleotides.

FIG. 2.

p63 protein levels are inversely correlated with miR-203 levels upon keratinocyte differentiation. (A) Total p63 protein levels in NHKs and NHK-31 cells upon differentiation. (B) Western blot analysis for total p63, ΔNp63α, p53, and involucrin in NHKs and keratinocytes stably expressing HPV-31 E6/E7 and E7. Levels in the undifferentiated state and following differentiation in methylcellulose for 48 h are shown. (C) Total p63 and p53 protein levels in undifferentiated and 48-h-differentiated NHKs and keratinocytes stably expressing HPV-16 E6/E7, E7, and E6. (D) Relative changes in miR-203 RNA and p63 protein levels in HPV-positive and -negative keratinocytes upon differentiation in methylcellulose for 48 h. miR-203 levels were normalized to NHK-E7 cell levels (set to 1) and p63 levels to those seen in NHKs. The results are from 4 independent experiments ± standard errors of the mean.

FIG. 3.

miR-203 targets p63 mRNA in human keratinocytes. (A) NHKs were cotransfected with a luciferase reporter containing a wild-type or mutated p63 3′-UTR fragment, along with an expression vector for the pre-miR-203 precursor, and analyzed after 48 h. The luciferase construct lacking the p63 3′-UTR fragment, as well as mutated 3′ UTR, served as negative controls. Relative luciferase levels were calculated by comparison to transfections without the pre-miR-203 precursor. Transfection efficiency was normalized using Renilla luciferase. The error bars indicate standard errors. (B) qRT-PCR analysis of miR-203 and ΔNp63α mRNA levels following transfection of NHKs with anti-miR-203 inhibitors after 24, 48, and 72 h. Levels were normalized to U6 mRNA. (C) NHKs were transfected with antisense miR-203 RNA for 24 and 48 h and analyzed for ΔNp63α and CARM-1 protein levels. NHKs transfected with nonspecific RNA served as negative controls. (D) NHKs were transfected with antisense miR-203 RNA, and the levels of ΔNp63α, p53, p21, and Bax were determined by Western blot analysis. Negative-control cells were transfected with nonspecific RNA. (E) Western blot analysis of NHKs and NHK-E6 and NHK-E7 cells for levels of ΔNp63α, p21, and Bax following differentiation in methylcellulose for 24 and 48 h.

FIG. 4.

The MAP kinase pathway regulates miR-203 and p63 expression. (A) Northern blot analysis for pre-miR-203 in NHKs and NHK-E7 cells treated with 100 ng/ml PMA for 0, 3, 6, and 9 h. Shown is quantitation of the average fold increase of pre-miR-203 relative to 0 h in NHKs (1.0, 1.8, 3.1, and 3.7) and in NHK-E7 cells (1.0, 1.0,1.3, and 1.6). (B) Western blot analysis of p63, phospho-ERK-1/2, and total ERK-1/2 in NHKs and NHK-E7 cells following treatment with 100 ng/ml PMA for 0, 3, 6, and 9 h.

FIG. 5.

High levels of miR-203 induce increased HPV genomes and transcripts but block amplification. (A) Northern blot analysis of miR-203 levels in CIN612 cells expressing constitutive high levels of miR-203, Southern blot analysis of linearized HPV-31 genomes in CIN612 control cells and CIN612-miR-203 cells, and Northern blot analysis of HPV-31 transcripts in CIN612 cells and CIN612 cells expressing high levels of miR-203 (CIN612-miR-203). Loading controls indicated equal loading in each lane (not shown). E1^E4 is a viral fusion protein expressed from a spliced transcript. (B) Western blot analysis of ΔNp63α and CARM-1 lysates from the CIN612 cell line and CIN612 cells stably transfected with miR-203 overexpression vector. (C) Representative Southern blots showing levels of linearized HPV-31 DNA genomes in methylcellulose-differentiated CIN612 cells and CIN612 miR-203 cells three passages (p3) and nine passages (p9) after transfection.

FIG. 6.

Model for the regulation of miR-203 expression in differentiating normal and HPV-positive epithelia. miR-203 expression is increased upon differentiation of normal keratinocytes, leading to suppression of p63 translation in suprabasal cells. In HPV-infected epithelia, E7 blocks miR-203 upregulation through the MAP kinase pathway, leading to increased levels of p63, cells remaining active in the cell cycle, and HPV genome amplification. EGF, epidermal growth factor.