This proposal is a mentored training fellowship that combines clinical exposure and research efforts with an integrated research and clinical training plan to ensure the PI?s transition to a research-focused dermatology residency. The PI?s clinical and research interests are in the inflammatory and immune-related molecular mechanisms of skin disease. The research focus of this proposal investigates the role of UVB exposure in driving an epithelial to mesenchymal (EMT)-like phenotype in normal human keratinocytes. This proposal will use 2D and 3D human keratinocyte cell cultures, in vivo mouse models, and patient samples to test the overall hypothesis that UVB exposure results in Toll-Like Receptor 3 (TLR3)-dependent activation of NF-?B, leading to an EMT-like phenotype in keratinocytes through the induction of EMT-associated genes and MMP-9.
In Aim 1, we hypothesize that TLR3-mediated NF-?B activation is required for the UVB-induced EMT-like morphologic change in keratinocytes. To test this, keratinocytes exposed to UVB or PIC in combination with inhibition of NF-?B will be assayed for NF-?B-dependent signaling changes, EMT associated gene and protein changes, and changes in keratinocyte morphology, migration, and invasion properties. We will use both WT and TLR3KO mice as well as human patient samples of cutaneous squamous cell carcinoma and normal skin to investigate the co-expression of NF-?B and TLR3 proteins in skin.
In Aim 2, we will test the hypothesis that TLR3 activation and subsequent NF-?B stimulation are required for MMP-9 induction and activation following UVB exposure. Keratinocytes exposed to UVB or PIC in combination with MMP-9 inhibition will be monitored for changes in morphology and alterations in migration and invasion potential. The effect of UVB exposure on MMP-9 will also be studied in vivo using WT and TLR3KO mice. This proposal challenges current understanding which focuses on UVB as a DNA-damaging agent and instead investigates TLR3 as a sensor of UVB-induced cell damage and activator of inflammatory pathways that can have profound effects on keratinocyte morphology and function. Our proposal is significant in that it will advance the field of skin biology by increasing our understanding of UVB-mediated activation of the innate immune system and skin tumorigenesis.
Aside from the DNA-damaging effects of UVB, UVB induces a significant inflammatory response in skin. We observe that normal human keratinocytes exposed to UVB leads to increased Toll-Like Receptor 3 (TLR3) expression and a morphology change reminiscent of epithelial to mesenchymal transition. Deciphering the downstream mediators of this EMT-like morphology change is key to understanding whether the TLR3 signaling pathway is involved in promoting skin tumorigenesis.
