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Evaluation of Keratinocyte Proliferation on Two- and Three-dimensional Type I Collagen Substrates
Published on: April 22, 2019
Conditionally activated E7 proteins of high-risk and low-risk human papillomaviruses induce S phase in postmitotic,
N Sanjib Banerjee1, Nicholas J Genovese, Francisco Noya
1Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35294-0005, USA.
Abstract:
The productive program of human papillomaviruses (HPVs) in epithelia is tightly linked to squamous differentiation. The E7 proteins of high-risk HPV genotypes efficiently inactivate the pRB family of proteins that control the cell cycle, triggering S phase in suprabasal keratinocytes. This ability has until now not been demonstrated for the low-risk HPV-6 or HPV-11 E7 proteins. An inducible system in which HPV-16 E7 is fused to the ligand binding domain of the human estrogen receptor (ER) was described by Smith-McCune et al. (K. Smith-McCune, D. Kalman, C. Robbins, S. Shivakumar, L. Yuschenkoff, and J. M. Bishop, Proc. Natl. Acad. Sci. USA 96:6999-7004, 1999). In the absence of hormone, E7ER is cytoplasmic, and upon addition of 17beta-estradiol, it translocates to the nucleus. Using organotypic epithelial raft cultures developed from primary human keratinocytes, we show that 16E7ER promotes either S-phase reentry or p21cip1 accumulation in differentiated keratinocytes in a stochastic manner as early as 6 h postinduction with 17beta-estradiol. A vector expressing the ER moiety alone had no effect. These observations prove unequivocally that the E7 protein drives S-phase reentry in postmitotic, differentiated keratinocytes rather than preventing S-phase exit while the cells ascend through the epithelium. HPV-11 E7ER and, much less efficiently, HPV-6 E7ER also promoted S-phase reentry by differentiated cells upon exposure to 17beta-estradiol. S-phase induction required the consensus pRB binding motif. We propose that the elevated nuclear levels of the low-risk HPV E7 protein afforded by the inducible system account for the positive results. These observations are entirely consistent with the fact that low-risk HPV genotypes replicate in the differentiated strata in patient specimens, as do the high-risk HPVs.
Insights
High-risk human papillomaviruses (HPVs) E7 proteins drive cell cycle reentry in differentiated keratinocytes. Low-risk HPV-6 and HPV-11 E7 proteins also induce S-phase reentry, demonstrating a conserved mechanism for HPV replication.
Area of Science:
- Virology
- Cell Biology
- Molecular Biology
Background:
- Human papillomaviruses (HPVs) productive replication is linked to epithelial squamous differentiation.
- High-risk HPV E7 proteins inactivate cell cycle regulators (pRB family), forcing suprabasal keratinocytes into S phase.
- This S-phase-inducing ability of E7 proteins from low-risk HPV types (HPV-6, HPV-11) was not previously demonstrated.
Purpose of the Study:
- To investigate whether low-risk HPV E7 proteins can induce S-phase reentry in differentiated keratinocytes.
- To compare the efficacy of high-risk HPV-16 E7 with low-risk HPV-6 and HPV-11 E7 proteins in inducing S-phase reentry.
- To elucidate the mechanism by which HPV E7 proteins affect the cell cycle in differentiated epithelial cells.
Main Methods:
- Utilized an inducible system with HPV-16 E7 fused to the estrogen receptor (ER) ligand-binding domain (E7ER).
- Employed organotypic epithelial raft cultures derived from primary human keratinocytes.
- Administered 17beta-estradiol to induce nuclear translocation of E7ER and analyzed for S-phase reentry and p21cip1 accumulation.
Main Results:
- HPV-16 E7ER induced S-phase reentry or p21cip1 accumulation in differentiated keratinocytes stochastically within 6 hours of induction.
- HPV-11 E7ER and, less efficiently, HPV-6 E7ER also promoted S-phase reentry in differentiated cells upon 17beta-estradiol exposure.
- S-phase induction by E7 proteins required the consensus pRB binding motif, and elevated nuclear levels of low-risk E7 likely explained the observed effects.
Conclusions:
- The E7 protein directly drives S-phase reentry in postmitotic, differentiated keratinocytes, not merely preventing cell cycle exit.
- Low-risk HPV types (HPV-6, HPV-11) possess the functional capacity to induce S-phase reentry in differentiated cells, similar to high-risk types.
- The findings support the replication of both high-risk and low-risk HPVs within the differentiated strata of patient epithelia.
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