Limits...
HPV16 E5 deregulates the autophagic process in human keratinocytes.

Belleudi F, Nanni M, Raffa S, Torrisi MR - Oncotarget (2015)

Bottom Line: Here we investigated the possible effects of 16E5 on autophagy in human keratinocytes expressing the viral protein.The 16E5 presence strongly inhibited the autophagic process, while forced expression and activation of KGFR counteracted this effect, demonstrating that the viral protein and the receptor exert opposite and interplaying roles not only on epithelial differentiation, but also in the control of autophagy.In W12 cells, silencing of the 16E5 gene in the context of the viral full length genome confirmed its role on autophagy inhibition.

View Article: PubMed Central - PubMed

Affiliation: Istituto Pasteur-Fondazione Cenci Bolognetti, Dipartimento di Medicina Clinica e Molecolare, Sapienza Università di Roma, Rome, Italy.

ABSTRACT
Autophagy plays key roles during host defense against pathogens, but viruses have evolved strategies to block the process or to exploit it for replication and successful infection. The E5 oncoprotein of human papillomavirus type 16 (HPV16 E5) perturbs epithelial homeostasis down-regulating the expression of the keratinocyte growth factor receptor (KGFR/FGFR2b), whose signaling induces autophagy. Here we investigated the possible effects of 16E5 on autophagy in human keratinocytes expressing the viral protein. The 16E5 presence strongly inhibited the autophagic process, while forced expression and activation of KGFR counteracted this effect, demonstrating that the viral protein and the receptor exert opposite and interplaying roles not only on epithelial differentiation, but also in the control of autophagy. In W12 cells, silencing of the 16E5 gene in the context of the viral full length genome confirmed its role on autophagy inhibition. Finally, molecular approaches showed that the viral protein interferes with the transcriptional regulation of autophagy also through the impairment of p53 function, indicating that 16E5 uses parallel mechanisms for autophagy impairment. Overall our results further support the hypothesis that a transcriptional crosstalk among 16E5 and KGFR might be the crucial molecular driver of epithelial deregulation during early steps of HPV infection and transformation.

No MeSH data available.


Related in: MedlinePlus

16E5 inhibits KGF-induced autophagy(a) HaCaT cells were transiently transfected with pCI-neo E5-HA expression vector (HaCaT E5) or with the empty vector alone (HaCaT pCI-neo). The 16E5 mRNA transcripts, quantitated by real-time relative RT-PCR and normalized with respect to those detected in the HPV16-positive cervical epithelial cell line W12 at the passage 6 (W12p6), are highly expressed only in HaCaT E5 cells. (b, c) HaCaT E5 and HaCaT pCI-neo cells were serum-starved in the presence or absence of KGF 100 ng/ml for 24 h. Western blot analysis shows that, upon KGF stimulation, the LC3-II band is reduced (b), while the SQSTM1 band is enhanced (c), in HaCaT E5 cells compared to HaCaT pCI-neo cells. The equal loading was assessed using anti-β actin antibody. For the densitometric analysis, the values from 3 independent experiments were normalized, expressed as fold increase and reported in graph as mean values ± standard deviation (SD). Student t test was performed and significance levels have been defined as p < 0.05: (b, c) *p < 0.05 vs the corrisponding unstimulated cells, **p < 0.05 vs the corresponding HaCaT pCI-neo cells. (d) HaCaT cells were transiently cotransfected with pEGFP-C2-LC3 construct and pCI-neo E5-HA (HaCaT EGFP-LC3/E5) or pCI-neo empty vector (HaCaT EGFP-LC3) before stimulation with KGF as above. Immunofluorescence was performed using anti-HA monoclonal antibody (red), to visualize 16E5, and cell nuclei were stained with DAPI. Upon KGF treatment, the number of LC3-positive dots per cell is increased in HaCaT EGFP-LC3 cells and in HaCaT EGFP-LC3/E5 cells not showing 16E5 staining (arrows), but is reduced in HaCaT EGFP-LC3/E5 cells strongly labeled for 16E5 (arrowhead) if compared to serum-starved HaCaT EGFP-LC3 cells. The quantitative analysis was performed as described in the materials and methods and results are expressed as mean values ± standard errors (SE). Student t test was performed and significance levels have been defined as p < 0.05: *p < 0.001 vs the corresponding serum starved-cells, **p < 0.001 vs the corresponding HaCaT EGFP-LC3 cells. Bar: 10 μm
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4496223&req=5

Figure 1: 16E5 inhibits KGF-induced autophagy(a) HaCaT cells were transiently transfected with pCI-neo E5-HA expression vector (HaCaT E5) or with the empty vector alone (HaCaT pCI-neo). The 16E5 mRNA transcripts, quantitated by real-time relative RT-PCR and normalized with respect to those detected in the HPV16-positive cervical epithelial cell line W12 at the passage 6 (W12p6), are highly expressed only in HaCaT E5 cells. (b, c) HaCaT E5 and HaCaT pCI-neo cells were serum-starved in the presence or absence of KGF 100 ng/ml for 24 h. Western blot analysis shows that, upon KGF stimulation, the LC3-II band is reduced (b), while the SQSTM1 band is enhanced (c), in HaCaT E5 cells compared to HaCaT pCI-neo cells. The equal loading was assessed using anti-β actin antibody. For the densitometric analysis, the values from 3 independent experiments were normalized, expressed as fold increase and reported in graph as mean values ± standard deviation (SD). Student t test was performed and significance levels have been defined as p < 0.05: (b, c) *p < 0.05 vs the corrisponding unstimulated cells, **p < 0.05 vs the corresponding HaCaT pCI-neo cells. (d) HaCaT cells were transiently cotransfected with pEGFP-C2-LC3 construct and pCI-neo E5-HA (HaCaT EGFP-LC3/E5) or pCI-neo empty vector (HaCaT EGFP-LC3) before stimulation with KGF as above. Immunofluorescence was performed using anti-HA monoclonal antibody (red), to visualize 16E5, and cell nuclei were stained with DAPI. Upon KGF treatment, the number of LC3-positive dots per cell is increased in HaCaT EGFP-LC3 cells and in HaCaT EGFP-LC3/E5 cells not showing 16E5 staining (arrows), but is reduced in HaCaT EGFP-LC3/E5 cells strongly labeled for 16E5 (arrowhead) if compared to serum-starved HaCaT EGFP-LC3 cells. The quantitative analysis was performed as described in the materials and methods and results are expressed as mean values ± standard errors (SE). Student t test was performed and significance levels have been defined as p < 0.05: *p < 0.001 vs the corresponding serum starved-cells, **p < 0.001 vs the corresponding HaCaT EGFP-LC3 cells. Bar: 10 μm

Mentions: Since we have recently demonstrated that 16E5 down-regulates KGFR [12, 13], whose ligand-specific activation triggers autophagy in keratinocytes [16], here we analyzed the effects of 16E5 ectopic expression on KGF-triggered autophagy in the human keratinocyte HaCaT cell line, spontaneously immortalized from a primary culture of keratinocytes [17]. To this aim, cells were transiently transfected with pCI-neo E5-HA expression vector [18] (HaCaT E5) or with the empty vector alone (HaCaT pCI-neo). The expected high expression of 16E5 mRNA transcript levels in HaCaT E5 [13] was first confirmed by real-time relative RT-PCR and normalized with respect to the levels of the viral protein transcript in the HPV16-positive cervical epithelial cell line W12 [19] at the passage 6 (W12p6) (Figure 1a). Then, to investigate the possible effects of 16E5 expression on KGF-induced autophagy, HaCaT pCI-neo and HaCaT E5 cells were serum-starved in the presence or absence of KGF for 24 h. Both the growth factor concentration and the single time point of treatment have been previously selected as optimal experimental conditions for an efficient autophagic induction in HaCaT cells [16]. The amount of the well-established autophagosome marker membrane-associated microtubule associated protein 1 light chain 3-II (LC3-II) was monitored by western blot analysis. The results showed that, after KGF stimulation, the increase of the 16 kDa band corresponding to LC3-II marker, evident in HaCaT pCI-neo cells (Figure 1b), appeared significantly reduced in HaCaT E5 cells (Figure 1b), indicating that the KGF-induced autophagosome formation was counteracted by the presence of 16E5.


HPV16 E5 deregulates the autophagic process in human keratinocytes.

Belleudi F, Nanni M, Raffa S, Torrisi MR - Oncotarget (2015)

16E5 inhibits KGF-induced autophagy(a) HaCaT cells were transiently transfected with pCI-neo E5-HA expression vector (HaCaT E5) or with the empty vector alone (HaCaT pCI-neo). The 16E5 mRNA transcripts, quantitated by real-time relative RT-PCR and normalized with respect to those detected in the HPV16-positive cervical epithelial cell line W12 at the passage 6 (W12p6), are highly expressed only in HaCaT E5 cells. (b, c) HaCaT E5 and HaCaT pCI-neo cells were serum-starved in the presence or absence of KGF 100 ng/ml for 24 h. Western blot analysis shows that, upon KGF stimulation, the LC3-II band is reduced (b), while the SQSTM1 band is enhanced (c), in HaCaT E5 cells compared to HaCaT pCI-neo cells. The equal loading was assessed using anti-β actin antibody. For the densitometric analysis, the values from 3 independent experiments were normalized, expressed as fold increase and reported in graph as mean values ± standard deviation (SD). Student t test was performed and significance levels have been defined as p < 0.05: (b, c) *p < 0.05 vs the corrisponding unstimulated cells, **p < 0.05 vs the corresponding HaCaT pCI-neo cells. (d) HaCaT cells were transiently cotransfected with pEGFP-C2-LC3 construct and pCI-neo E5-HA (HaCaT EGFP-LC3/E5) or pCI-neo empty vector (HaCaT EGFP-LC3) before stimulation with KGF as above. Immunofluorescence was performed using anti-HA monoclonal antibody (red), to visualize 16E5, and cell nuclei were stained with DAPI. Upon KGF treatment, the number of LC3-positive dots per cell is increased in HaCaT EGFP-LC3 cells and in HaCaT EGFP-LC3/E5 cells not showing 16E5 staining (arrows), but is reduced in HaCaT EGFP-LC3/E5 cells strongly labeled for 16E5 (arrowhead) if compared to serum-starved HaCaT EGFP-LC3 cells. The quantitative analysis was performed as described in the materials and methods and results are expressed as mean values ± standard errors (SE). Student t test was performed and significance levels have been defined as p < 0.05: *p < 0.001 vs the corresponding serum starved-cells, **p < 0.001 vs the corresponding HaCaT EGFP-LC3 cells. Bar: 10 μm
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4496223&req=5

Figure 1: 16E5 inhibits KGF-induced autophagy(a) HaCaT cells were transiently transfected with pCI-neo E5-HA expression vector (HaCaT E5) or with the empty vector alone (HaCaT pCI-neo). The 16E5 mRNA transcripts, quantitated by real-time relative RT-PCR and normalized with respect to those detected in the HPV16-positive cervical epithelial cell line W12 at the passage 6 (W12p6), are highly expressed only in HaCaT E5 cells. (b, c) HaCaT E5 and HaCaT pCI-neo cells were serum-starved in the presence or absence of KGF 100 ng/ml for 24 h. Western blot analysis shows that, upon KGF stimulation, the LC3-II band is reduced (b), while the SQSTM1 band is enhanced (c), in HaCaT E5 cells compared to HaCaT pCI-neo cells. The equal loading was assessed using anti-β actin antibody. For the densitometric analysis, the values from 3 independent experiments were normalized, expressed as fold increase and reported in graph as mean values ± standard deviation (SD). Student t test was performed and significance levels have been defined as p < 0.05: (b, c) *p < 0.05 vs the corrisponding unstimulated cells, **p < 0.05 vs the corresponding HaCaT pCI-neo cells. (d) HaCaT cells were transiently cotransfected with pEGFP-C2-LC3 construct and pCI-neo E5-HA (HaCaT EGFP-LC3/E5) or pCI-neo empty vector (HaCaT EGFP-LC3) before stimulation with KGF as above. Immunofluorescence was performed using anti-HA monoclonal antibody (red), to visualize 16E5, and cell nuclei were stained with DAPI. Upon KGF treatment, the number of LC3-positive dots per cell is increased in HaCaT EGFP-LC3 cells and in HaCaT EGFP-LC3/E5 cells not showing 16E5 staining (arrows), but is reduced in HaCaT EGFP-LC3/E5 cells strongly labeled for 16E5 (arrowhead) if compared to serum-starved HaCaT EGFP-LC3 cells. The quantitative analysis was performed as described in the materials and methods and results are expressed as mean values ± standard errors (SE). Student t test was performed and significance levels have been defined as p < 0.05: *p < 0.001 vs the corresponding serum starved-cells, **p < 0.001 vs the corresponding HaCaT EGFP-LC3 cells. Bar: 10 μm
Mentions: Since we have recently demonstrated that 16E5 down-regulates KGFR [12, 13], whose ligand-specific activation triggers autophagy in keratinocytes [16], here we analyzed the effects of 16E5 ectopic expression on KGF-triggered autophagy in the human keratinocyte HaCaT cell line, spontaneously immortalized from a primary culture of keratinocytes [17]. To this aim, cells were transiently transfected with pCI-neo E5-HA expression vector [18] (HaCaT E5) or with the empty vector alone (HaCaT pCI-neo). The expected high expression of 16E5 mRNA transcript levels in HaCaT E5 [13] was first confirmed by real-time relative RT-PCR and normalized with respect to the levels of the viral protein transcript in the HPV16-positive cervical epithelial cell line W12 [19] at the passage 6 (W12p6) (Figure 1a). Then, to investigate the possible effects of 16E5 expression on KGF-induced autophagy, HaCaT pCI-neo and HaCaT E5 cells were serum-starved in the presence or absence of KGF for 24 h. Both the growth factor concentration and the single time point of treatment have been previously selected as optimal experimental conditions for an efficient autophagic induction in HaCaT cells [16]. The amount of the well-established autophagosome marker membrane-associated microtubule associated protein 1 light chain 3-II (LC3-II) was monitored by western blot analysis. The results showed that, after KGF stimulation, the increase of the 16 kDa band corresponding to LC3-II marker, evident in HaCaT pCI-neo cells (Figure 1b), appeared significantly reduced in HaCaT E5 cells (Figure 1b), indicating that the KGF-induced autophagosome formation was counteracted by the presence of 16E5.

Bottom Line: Here we investigated the possible effects of 16E5 on autophagy in human keratinocytes expressing the viral protein.The 16E5 presence strongly inhibited the autophagic process, while forced expression and activation of KGFR counteracted this effect, demonstrating that the viral protein and the receptor exert opposite and interplaying roles not only on epithelial differentiation, but also in the control of autophagy.In W12 cells, silencing of the 16E5 gene in the context of the viral full length genome confirmed its role on autophagy inhibition.

View Article: PubMed Central - PubMed

Affiliation: Istituto Pasteur-Fondazione Cenci Bolognetti, Dipartimento di Medicina Clinica e Molecolare, Sapienza Università di Roma, Rome, Italy.

ABSTRACT
Autophagy plays key roles during host defense against pathogens, but viruses have evolved strategies to block the process or to exploit it for replication and successful infection. The E5 oncoprotein of human papillomavirus type 16 (HPV16 E5) perturbs epithelial homeostasis down-regulating the expression of the keratinocyte growth factor receptor (KGFR/FGFR2b), whose signaling induces autophagy. Here we investigated the possible effects of 16E5 on autophagy in human keratinocytes expressing the viral protein. The 16E5 presence strongly inhibited the autophagic process, while forced expression and activation of KGFR counteracted this effect, demonstrating that the viral protein and the receptor exert opposite and interplaying roles not only on epithelial differentiation, but also in the control of autophagy. In W12 cells, silencing of the 16E5 gene in the context of the viral full length genome confirmed its role on autophagy inhibition. Finally, molecular approaches showed that the viral protein interferes with the transcriptional regulation of autophagy also through the impairment of p53 function, indicating that 16E5 uses parallel mechanisms for autophagy impairment. Overall our results further support the hypothesis that a transcriptional crosstalk among 16E5 and KGFR might be the crucial molecular driver of epithelial deregulation during early steps of HPV infection and transformation.

No MeSH data available.


Related in: MedlinePlus