HPV8-E6 Interferes with Syntenin-2 Expression through Deregulation of Differentiation, Methylation and Phosphatidylinositide-Kinase Dependent Mechanisms

Abstract

The E6 oncoproteins of high-risk human papillomaviruses (HPV) of genus alpha contain a short peptide sequence at the carboxy-terminus, the PDZ binding domain, with which they interact with the corresponding PDZ domain of cellular proteins. Interestingly, E6 proteins from papillomaviruses of genus beta (betaPV) do not encode a comparable PDZ binding domain. Irrespective of this fact, we previously showed that the E6 protein of HPV8 (betaPV type) could circumvent this deficit by targeting the PDZ protein Syntenin-2 through transcriptional repression (Lazic et al., 2012). Despite its high binding affinity to phosphatidylinositol-4,5-bisphosphate (PI(4,5)P₂), very little is known about Syntenin-2. This study aimed to extend the knowledge on Syntenin-2 and how its expression is controlled. We now identified that Syntenin-2 is expressed at high levels in differentiating and in lower amounts in keratinocytes cultured in serum-free media containing low calcium concentration. HPV8-E6 led to a further reduction of Syntenin-2 expression only in cells cultured in low calcium. In the skin of patients suffering from Epidermodysplasia verruciformis, who are predisposed to betaPV infection, Syntenin-2 was expressed in differentiating keratinocytes of non-lesional skin, but was absent in virus positive squamous tumors. Using 5-Aza-2'-deoxycytidine, which causes DNA demethylation, Syntenin-2 transcription was profoundly activated and fully restored in the absence and presence of HPV8-E6, implicating that E6 mediated repression of Syntenin-2 transcription is due to promoter hypermethylation. Since Syntenin-2 binds to PI(4,5)P₂, we further tested whether the PI(4,5)P₂ metabolic pathway might govern Syntenin-2 expression. PI(4,5)P₂ is generated by the activity of phosphatidylinositol-4-phosphate-5-kinase type I (PIP5KI) or phosphatidylinositol-5-phosphate-4-kinase type II (PIP4KII) isoforms α, β and γ. Phosphatidylinositide kinases have recently been identified as regulators of gene transcription. Surprisingly, transfection of siRNAs directed against PIP5KI and PIP4KII resulted in higher Syntenin-2 expression with the highest effect mediated by siPIP5KIα. HPV8-E6 was able to counteract siPIP4KIIα, siPIP4KIIβ and siPIP5KIγ mediated Syntenin-2 re-expression but not siPIP5KIα. Finally, we identified Syntenin-2 as a key factor regulating PIP5KIα expression. Collectively, our data demonstrates that Syntenin-2 is regulated through multiple mechanisms and that downregulation of Syntenin-2 expression may contribute to E6 mediated dedifferentiation of infected skin cells.

Keywords: 5-bisphosphate; E6 oncoprotein; Syntenin-2; differentiation; human papillomavirus (HPV); methylation; phosphatidylinositol-4.

FIGURE 1

Evolution of the Syntenin gene family. (A) Phylogram of major opisthokont lineages [tree simplified after (Torruella et al., 2015)]. Host range of papillomaviruses corresponds to Syntenin-1/Syntenin-2 evolutionary split. (B) Maximum likelihood tree for the Syntenin gene family. Representative Syntenin sequences were obtained from UniProt (The UniProt Consortium, 2017), aligned by the L-Ins-I algorithm of the MAFFT package (Katoh et al., 2002), and subjected to tree calculation by the T-Rex server (Boc et al., 2012) using the RAxML method (Stamatakis, 2006) (Syn-1: Syntenin-1; Syn-2: Syntenin-2).

FIGURE 2

Differentiation dependent expression of Syntenin-2 in skin keratinocytes. (A) Representative Western blot showing Syntenin-2 levels in total cell extracts of the keratinocyte cell lines PM1, HaCaT, RTS3b and isogenic N/TERT cells either cultured in RM+ (N/TERT^RM+) or in KGM-Gold (N/TERT^KGM). Equal loading was confirmed by immunoblotting for Tubulin. (B) Representative Western blot of Syntenin-2 levels in total cell extracts of N/TERT^KGM treated with 2 mM CaCl₂ for up to 8 days. Calcium induced keratinocyte differentiation was confirmed by Western blotting for Loricrin. Equal loading was confirmed by immunoblotting for Tubulin. (C) Representative immunofluorescence staining images detecting Syntenin-2 in EV-derived skin lesions. The skin area with productive betaPV infection was identified by anti-E4 staining. Nuclear counterstain was performed using DAPI. Syntenin-2 expression is present in suprabasal keratinocytes of non-lesional/E4-negative EV skin and absent in an E4-positive skin tumor. Top lane: H&E staining of the EV tissue analyzed.

FIGURE 3

Syntenin-2 expression is repressed by HPV8-E6 in keratinocytes with basal cell characteristics through promoter hypermethylation. (A) Quantification of Syntenin-2 mRNA expression by qRT-PCR in empty vector and HPV8-E6 expressing RTS3b, HaCaT, PM1 and isogenic N/TERT keratinocytes cultured either in RM+ or KGM. (B) Representative immunocytochemical staining of Syntenin-2 in N/TERT^KGM-control (Left) and N/TERT^KGM-8E6 (Right) demonstrating reduction of mainly cytoplasmic Syntenin-2 in HPV8-E6 positive cells (blue: DAPI; green: Syntenin-2). (C) Representative Western blot showing Syntenin-2 in total cell extracts of N/TERT^KGM expressing either HPV8-E6 wild type or the E6 mutants L61A, W63A, L61A/W63A, V68A, D96A, D126A or K136N. Loading was confirmed by immunoblotting for Tubulin. (D) Quantification of Syntenin-2 protein levels from (C) normalized to Tubulin protein levels. (E) Quantification of Syntenin-2 mRNA expression in N/TERT^KGM-control and N/TERT^KGM-8E6 cells after treatment with 10 μM 5-Aza or DMSO (n = 3 independent experiments, measured in duplicate, Upper). Data are presented as mean ± SEM (^∗∗∗p < 0.001). Western blot analysis of Syntenin-2 in extracts from cells treated with 5-Aza. Equal loading was confirmed by immunoblotting for Tubulin (Lower).

FIGURE 4

PI(4,5)P₂ generating kinases are involved in control of Syntenin-2 expression. (A) Control (siScr), siPIP4KIIα, siPIP4KIIβ, siPIP5KIα or siPIP5KIγ transfected N/TERT^KGM-control and N/TERT^KGM-8E6 cells were harvested 48 h post transfection and mRNA expression of kinase isoforms was determined by qRT-PCR (n = 3 independent experiments, measured in duplicate). Data are presented as mean ± SEM (^∗∗∗p < 0.001). (B) Quantification of Syntenin-2 mRNA expression by qRT-PCR in N/TERT^KGM-control and N/TERT^KGM-8E6, in which the kinase isoforms PIP4KIIα, PIP4KIIβ, PIP5KIα and PIP5KIγ were silenced by siRNA transfection. (C) Representative Western blot showing Syntenin-2 expression in total cell extracts from N/TERT^KGM-control and N/TERT^KGM-8E6, in which kinase isoforms were silenced by siRNA transfection. Equal loading was confirmed by immunoblotting for Tubulin. (D) Quantification of Syntenin-2 protein levels in cells treated with siRNAs for specific kinase isoforms (n = 3 independent experiments). Data are presented as mean ± SEM. (E) Representative Western blot demonstrating downregulation of PIP5KIα protein levels in cells transfected with specific siRNA against Syntenin-2 (Left). Bars presenting the quantification of PIP5KIα and Syntenin-2 protein levels are shown in the Middle and Right (n = 3 independent experiments). Data are presented as mean ± SEM.

FIGURE 5

Graphical summary of the regulatory pathways identified in this study controlling Syntenin-2 expression in normal keratinocytes (Left). Known HPV8-E6 mediated interference with these mechanisms is shown on the Right.