The JAK-STAT transcriptional regulator, STAT-5, activates the ATM DNA damage pathway to induce HPV 31 genome amplification upon epithelial differentiation

Abstract

High-risk human papillomavirus (HPV) must evade innate immune surveillance to establish persistent infections and to amplify viral genomes upon differentiation. Members of the JAK-STAT family are important regulators of the innate immune response and HPV proteins downregulate expression of STAT-1 to allow for stable maintenance of viral episomes. STAT-5 is another member of this pathway that modulates the inflammatory response and plays an important role in controlling cell cycle progression in response to cytokines and growth factors. Our studies show that HPV E7 activates STAT-5 phosphorylation without altering total protein levels. Inhibition of STAT-5 phosphorylation by the drug pimozide abolishes viral genome amplification and late gene expression in differentiating keratinocytes. In contrast, treatment of undifferentiated cells that stably maintain episomes has no effect on viral replication. Knockdown studies show that the STAT-5β isoform is mainly responsible for this activity and that this is mediated through the ATM DNA damage response. A downstream target of STAT-5, the peroxisome proliferator-activated receptor γ (PPARγ) contributes to the effects on members of the ATM pathway. Overall, these findings identify an important new regulatory mechanism by which the innate immune regulator, STAT-5, promotes HPV viral replication through activation of the ATM DNA damage response.

Figure 1. HPV31 increases the levels of STAT-5 phosphorylation.

A) Western blot analysis of STAT-5, p-STAT-5, and GAPDH levels in HFK, HPV31, and HPV31-positive CIN612 cells grown in monolayer cultures. B) Bar graphs show the relative expression level of target proteins, normalized to GAPDH, from panel A western analysis. The statistical analysis was assayed by 2-tail t-test. Data = mean +/−standard error. * indicates p-value<0.01; ** indicates p-value<0.05. The band intensities were determined by ImageJ64 software. C) Western blot analysis for STAT-5, p-STAT-5, and GAPDH levels in HFK, HPV31 and HPV31-positive CIN612 cells differentiated in high calcium media for indicated times. D) Bar graphs show the relative expression level of target proteins, normalized to GAPDH, from panel C western analysis. The statistical analysis was assayed by 2-tail t-test. Data = mean +/−standard error. * indicates p-value<0.01; ** indicates p-value<0.05. All results are representative of observations from 3 independent experiments.

Figure 2. Suppression of STAT-5 phosphorylation by pimozide blocks HPV genome amplification upon keratinocyte differentiation.

A) Western blot analysis of p-STAT-5, STAT-5α, STAT-5β, STAT-5, p-ATM, ATM, p-CHK2, CHK2 and GAPDH proteins in differentiated CIN612 cells in the presence or absence of pimozide (10 µM) in high calcium media for indicated times. B) Southern blot analysis for HPV31 episomes in CIN612 cells untreated or treated with pimozide (10 µM) following differentiation in high calcium media for indicated times (hrs). C) Northern blot analysis for HPV31 early and late gene expression in CIN612 cells in the presence or absence of pimozide (10 µM) following differentiation in high calcium media for indicated times (hrs). D) Southern blot analysis for HPV31 episomes in monolayer cultures of CIN612 cells untreated or treated with pimozide (10 µM) for indicated times (hrs). E) Western blot analysis of p-STAT-5, STAT-5α, STAT-5β, STAT-5, Bcl-XL, and PARP proteins in monolayer CIN612 cells treated with pimozide (10 µM) for indicated times. F) Cell proliferation assay of monolayer CIN612 cells with or without pimozide (10 µM) for indicated times. P<0.05. All results are representative of observations from 2 or more independent experiments.

Figure 3. STAT-5 knockdown results in a loss of differentiation-dependent HPV genome amplification.

HPV31-positive CIN612 cells were infected with lentiviruses expressing scramble shRNA, STAT-5α shRNA, or STAT-5β shRNA and incubated for 48 or 72 hours postinfection, followed by an additional 72 hours of differentiation in high calcium media. A) Western blot analysis of STAT-5α, STAT-5β, and STAT-5 proteins in monolayer CIN612 cells infected with multiple shRNA lentiviruses targeting STAT-5α or STAT-5β for indicated times. TRC1 and TRC2 refer to the scrambled vector controls for STAT-5α and STAT-5β respectively. B) Western blot analysis of STAT-5α, STAT-5β, and STAT-5 proteins following differentiation of CIN612 cells that had been infected with combined shRNA lentiviruses targeting STAT-5α or STAT-5β for indicated times. C) Bar graphs show the relative expression level of target proteins, normalized to GAPDH, from panel B western analysis. The statistical analysis was assayed by 2-tail t-test. Data = mean+/−standard error. * indicates p-value<0.01; ** indicates p-value<0.05. D). Southern blot analysis of HPV31 genomes in CIN612 cells following infection with shRNA lentiviruses and differentiation in high calcium media for indicated times (hrs). All results are representative of observations from 2 or more independent experiments.

Figure 4. Knockdown of STAT-5 suppresses ATM DNA damage responses.

HPV31-positive CIN612 cells were transduced as described in legend to Figure 3. A) Western blot analysis of p-ATM, ATM, p-CHK2, CHK2, involucrin and GAPDH protein levels in uninfected and shRNA lentivirus infected CIN612 cells upon differentiation in high-calcium media for indicated times. B) Western blot analysis of p-CHK2, CHK2, STAT-5α, STAT-5β, and GAPDH protein levels in shRNA control and shRNA lentivirus infected CIN612 cells upon differentiation in high-calcium media for indicated times. C) Western blot analysis of BRCA-1. BRCA-2, SMC-1, p-SMC-1, RAD51 and GAPDH protein at total or phosphorylation levels in uninfected and shRNA lentivirus infected CIN612 cells upon differentiation for indicated times. The quantification of the band intensities is shown as bar graph figures in Figure S1. All results are representative of observations from 3 independent experiments.

Figure 5. STAT-5-dependent ATM DNA damage signaling may be mediated by PPARγ.

A) Western blot analysis of PPARγ and GAPDH protein levels in HFKs and HPV-31 positive keratinocytes. B) Western blot analysis for PPARγ and GAPDH levels in HFK, HPV31 and HPV31-positive CIN612 cells differentiated in calcium media for indicated times. C) Southern blot analysis for HPV31 episomes in HPV31 cells or the cells exposed to HX531 (2 µM) following differentiation in high calcium media for indicated times (hrs). D) Southern blot analysis for HPV31 episomes in CIN612 cells or the cells exposed to HX531 (2 µM) for indicated times (hrs). E) Western blot analysis of PPARγ protein levels in differentiating CIN612 cells infected with multiple shRNA targeting STAT-5α or STAT-5β for indicated times. F) Western blot analysis of PPARγ, ATM, CHK2, ATR, and CHK1 proteins at total or phosphorylation levels in differentiated CIN612 cells treated with or without HX531 (2 µM) in high calcium media for indicated times. The quantification of the data in bar figures is shown in Figure S2. All results are representative of observations from 3 independent experiments.

Figure 6. HPV E7 protein is responsible for STAT-5 activation.

A) Western blot analysis of p-STAT-5, STAT-5α, STAT-5β, STAT-5, SOCS-1 and CDC25A protein levels in HFK, 31E6, and 31E7cells. B) Bar graphs show the relative expression level of target proteins, normalized to GAPDH, from panel A western analysis. The statistical analysis was assayed by 2-tail t-test. Data = mean +/−standard error. * indicates p-value<0.01; ** indicates p-value<0.05. All results are representative of observations from 3 independent experiments.