Investigating the Impact of UVB and Cyclosporin A on Epithelial and Mesenchymal Cell Behavior in Monolayer Culture

Abstract

Cyclosporin A (CsA) is a keystone immunosuppressant used in organ transplantation and autoimmune disease management. However, long-term administration is linked to an elevated risk of cutaneous squamous cell carcinoma (cSCC), particularly in UV-exposed skin. Despite extensive knowledge about its immunoregulatory mechanisms, the direct cellular responses of skin epithelial and stromal cells remain less understood. This study investigated the early molecular and functional effects of CsA, UVB irradiation, and the Akt inhibitor Triciribine (TCN) in twodimensional (2D) monolayer cultures of HaCaT keratinocytes and dermal fibroblasts. Gene expression profiling focused on key pathways central to epithelial-mesenchymal transition (EMT), extracellular matrix (ECM) remodeling, and stress signaling. In keratinocytes, CsA enhanced ATF3 expression under UVB and kinase inhibition (TCN), while E-cadherin (CDH1) remained consistently expressed, preserving epithelial identity. Migration assays revealed impaired collective motility, particularly after UVB exposure. Interestingly, CsA appeared to buffer UVB-induced cytotoxicity and detachment, suggesting a partially protective role and potential involvement in counteracting EMT progression during acute stress. In fibroblasts, transcriptional activation of the TGF-β pathway and MMP9 under TCN treatment highlighted early fibroblast activation and dynamic ECM remodeling. Fibroblasts exhibited robust motility and resilience to CsA and UVB, and showed only mild delays after TCN exposure without significant cytotoxicity. These findings underscore distinct adaptive strategies: keratinocytes prioritize stress adaptation and epithelial preservation, while fibroblasts engage remodeling programs to sustain tissue integrity. Importantly, TCN alone or in combination with UVB and/or CsA, emerged as a key modulator of fibroblast behavior, influencing mesenchymal traits and ECM dynamics. Although 2D assays are limited in fully capturing invasive transformation compared to 3D systems, they effectively reflect early-stage adaptation and cell-matrix interactions under pharmacological and environmental stress. Overall, this work provides new insights into CsA- and UVB-mediated regulation of skin cell behavior, with implication for understanding cSCC development in immunosuppressed patients, and lays the foundation for future studies on chronic adaptation, invasive transformation, and dermal-epidermal dynamics in cancer and wound healing contexts.