Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Clinical Trial
. 2012 Aug;86(15):7943-52.
doi: 10.1128/JVI.00132-12. Epub 2012 May 23.

Human papillomavirus type 8 E6 oncoprotein inhibits transcription of the PDZ protein syntenin-2

Daliborka Lazić  1 , Martin Hufbauer, Paola Zigrino, Stephanie Buchholz, Siamaque Kazem, Mariet C W Feltkamp, Cornelia Mauch, Gertrud Steger, Herbert Pfister, Baki Akgül
Affiliations
Clinical Trial

Human papillomavirus type 8 E6 oncoprotein inhibits transcription of the PDZ protein syntenin-2

Daliborka Lazić et al. J Virol. 2012 Aug.

Abstract

The E6 proteins from high-risk alpha human papillomavirus (HPV) types (e.g., HPV16) are characterized by the presence of a PDZ-binding motif through which they interact with a number of cellular PDZ domain-containing substrates and cooperate in their degradation. The ability of these E6 proteins to bind to PDZ domain proteins correlates with the oncogenic potential of the virus. The E6 proteins of oncogenic HPV from the genus Betapapillomavirus (betaPV, e.g., HPV8) do not encode a PDZ-binding motif. We found that the PDZ domain protein syntenin-2 is transcriptionally downregulated in primary human epidermal keratinocytes (PHEK) by HPV8 E6. The mRNA levels of the known HPV16 E6 PDZ protein targets Dlg, Scribble, Magi-1, Magi-3, PSD95, and Mupp1 were not changed by HPV8 E6. Decreased protein levels of syntenin-2 were observed in cell extracts from PHEK expressing HPV5, -8, -16, -20, and -38 E6 but not in HPV1 and -4 E6-positive keratinocytes. Surprisingly, HPV16 E6 also repressed transcription of syntenin-2 but with a much lower efficiency than HPV8 E6. In healthy human skin, syntenin-2 expression is localized in suprabasal epidermal layers. In organotypic skin cultures, the differentiation-dependent expression of syntenin-2 was absent in HPV8 E6- and E6E7-expressing cells. In basal cell carcinomas of the skin, syntenin-2 was not detectable, whereas in squamous cell carcinomas, expression was located in differentiated areas. Short hairpin RNA-mediated knockdown of syntenin-2 led to an inhibition of differentiation and an increase in the proliferation capacity in PHEK. These results identified syntenin-2 as the first PDZ domain protein controlled by HPV8 and HPV16 at the mRNA level.

PubMed Disclaimer

Figures

Fig 1
Fig 1
Downregulation of syntenin-2 protein expression in keratinocytes expressing HPV E6. (A) Western blot analysis of syntenin-1 and -2 in keratinocytes infected with retrovirus coding for HPV8 E6, E6E7, and E7. (B) Western blot analysis of syntenin-2 in keratinocytes infected with retrovirus coding for E6 of HPV type 1, 4, 5, 8, 16, 20, or 38 or with empty retrovirus pLXSN. GAPDH was used as loading control.
Fig 2
Fig 2
Syntenin-2 is downregulated at the transcriptional level in HPV8 E6-expressing cells. (A) Syntenin-1 mRNA expression in keratinocytes retrovirally infected with the empty construct pLXSN or constructs coding for HPV8 E6, E7, or E6E7. (B) Syntenin-2 mRNA expression was quantified in keratinocytes positive for pLXSN, HPV8 E6, E7, or E6E7. (C and D) PHEK expressing HPV8 E6 wt or HPV8 E6Δ132-136 were studied for E6 (C) and for syntenin-2 (D) mRNA and protein expression. (E) MRNA levels of syntenin-2, Dlg1, Scribble, Magi-1 and -3, Mupp1, and PSD95 in pLXSN- or HPV8 E6-positive primary keratinocytes. MRNA levels of gene targets were normalized to HPRT1 mRNA levels. Error bars show standard deviations.
Fig 3
Fig 3
Syntenin-2 is targeted by HPV16 E6 at the transcriptional level. (A) PHEK expressing HPV16 E6 wt were studied for E6 mRNA expression by qRT-PCR. (B) HPV16 E6-positive PHEK were incubated in the presence of either MG132 or solvent before harvesting and then analyzed by Western blotting. (C) Coimmunoprecipitation (Co-IP) showed that the HPV16 E6P59V mutant does not bind to E6AP (left). Extracts from C33a cells, transiently transfected into 10-cm culture dishes with empty vectors or vectors for HPV16 E6-Flag or HPV16 E6P59V-Flag (15 μg each) were incubated with anti-Flag–M2 affinity beads. Bound E6AP was detected in the subsequent Western blot analysis by using a monoclonal E6AP antibody. To verify the presence of Flag-tagged protein, an aliquot of the extract was used as input control in a Western blot developed with the M2 anti-Flag antibody. In vivo degradation assay showed that the HPV16 E6P59V mutant does not degrade p53 (right). C33a cells were transiently cotransfected into 6-cm culture dishes with expression vectors for Flag-p53 and vectors coding for HPV16 E6-Flag or HPV16 E6P59V-Flag (2.5 μg each), and then p53 levels were detected by Western blotting using the Do-1 anti-p53 antibody. (D) Transient transfection of C33a with HPV16 E6 expression vectors was performed as indicated. Additionally, transfected HA-NAP was included as an internal transfection control. (E) Syntenin-2 mRNA expression was quantified in pLXSN- and HPV16 E6-positive PHEK by qRT-PCR, and protein levels detected by Western blotting. MRNA levels of gene targets were normalized to HPRT1 mRNA levels. Error bars show standard deviations.
Fig 4
Fig 4
Syntenin-1 and -2 mRNA levels in control or HPV8 E6-expressing primary keratinocytes after UVB irradiation. Syntenin-1 and -2 mRNA levels were normalized to HPRT1 mRNA levels. Error bars show standard deviations.
Fig 5
Fig 5
Syntenin-2 expression patterns in healthy human skin and skin malignancies. Immunohistochemical analysis of syntenin-2 expression in healthy human skin and BCC and SCC specimens. Sections of paraffin-embedded tissue were stained with anti-syntenin-2 antibody (brown staining) and counterstained with hematoxylin. The SCC1 specimen is representative of poorly differentiated SCC, and the SCC2 and SCC3 specimens are representative for highly differentiated SCCs.
Fig 6
Fig 6
Expression of syntenin-2 in organotypic skin cultures of N/TERT keratinocytes expressing HPV8 E6, E7, or E6E7. Sections of paraffin-embedded cultures were stained with anti-syntenin-2 antibody (brown staining) and counterstained with hematoxylin. Magnification, ×200.
Fig 7
Fig 7
Specific knockdown of syntenin-2 led to changes in keratinocyte differentiation and proliferation. (A) Western blot analysis of syntenin-2, syntenin-1, and involucrin in keratinocytes infected with the empty retroviral construct pSuperior or retroviral constructs coding for shSyntenin-2. Involucrin levels were also analyzed in keratinocytes expressing pLXSN or HPV8 E6. GAPDH was used as the loading control. (B) Organotypic skin cultures of PHEK expressing the empty retroviral construct pSuperior or the construct coding for shSyntenin-2. These were repeated three times, and sections of representative cultures are shown which were stained with hematoxylin and eosin and analyzed for histological changes. Magnification, ×200. (C) Flow cytometric analysis of pSuperior- and shSyntenin-2-expressing cells. Dot plots (top; gated on cells in the S-phase of the cell cycle), and histogram illustrations (bottom; gated on polyploid cells) of BrdU- and propidium iodide-treated cells.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Accardi R, et al. 2011. E6 and E7 from human papillomavirus type 16 cooperate to target the PDZ protein Na/H exchange regulatory factor 1. J. Virol. 85:8208–8216 - PMC - PubMed
    1. Akgül B, et al. 2011. Upregulation of lipocalin-2 in human papillomavirus-positive keratinocytes and cutaneous squamous cell carcinomas. J. Gen. Virol. 92:395–401 - PubMed
    1. Akgül B, et al. 2010. Human papillomavirus 5 and 8 E6 downregulate interleukin-8 secretion in primary human keratinocytes. J. Gen. Virol. 91:888–892 - PubMed
    1. Akgül B, Cooke JC, Storey A. 2006. HPV-associated skin disease. J. Pathol. 208:165–175 - PubMed
    1. Akgül B, et al. 2005. The E7 protein of cutaneous human papillomavirus type 8 causes invasion of human keratinocytes into the dermis in organotypic cultures of skin. Cancer Res. 65:2216–2223 - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources