Browsing by Subject "dermatology"

Ubiquitination is a post-translational modification that mediates protein stability and function, and it is implicated in almost every aspect of cellular activities. UBE2N is an E2 conjugating enzyme that mediates Lys-63 (K63) polyubiquitination. It plays essential roles in signal transduction and DNA repair mechanisms. In this thesis, we utilized two conditional mouse models to investigate the role of UBE2N in the skin: Rosa26CreER.Ube2nfl/fl and Krt5CreER.Ube2nfl/fl. We found that the conditional deletion of Ube2n in adult skin resulted in inflammatory skin defects. Using histology, single-cell RNA-sequencing, RT-qPCR, flow cytometry, immunofluorescent staining, and western blotting, we learned that the inflammatory skin accompanied epidermal and dermal thickening, parakeratosis, and increased immune cell infiltration. The skin expressed a pro-inflammatory gene signature including elevated levels of cytokines such as Il1 and Il24 and chemokines such as Cxcl1 and Cxcl2. The immune infiltration was primarily of myeloid origin including neutrophils and M1-like proinflammatory macrophages. Using the basal keratinocyte-specific Krt5CreER.Ube2nfl/fl transgenic mouse model, we also learned that loss of UBE2N in keratinocytes is sufficient to induce inflammatory skin defects that was observed in the Rosa26CreER.Ube2nfl/fl mice. The Ube2n knockout (KO) keratinocytes also showed increased levels of pro-inflammatory cytokine and myeloid cell chemokine genes. Consistently, the Ube2n KO skin showed infiltration of myeloid cells. Furthermore, we discovered that genes in the IL-1 signaling pathway are highly upregulated in both the Rosa26CreER.Ube2nfl/fl and the Krt5CreER.Ube2nfl/fl. Of them, interleukin-1 receptor-associated kinase 1 (IRAK1) was activated in the Ube2n KO skin. To test the hypothesis that the loss of Ube2n induced inflammatory phenotypes are mediated by IL-1 signaling pathway, we treated Krt5CreER.Ube2nfl/fl mice with an IRAK1 and 4 (IRAK1/4)-specific pharmacological inhibitor. The IRAK1/4 inhibitor reduced the level of inflammatory phenotypes of the Ube2n KO skin. This suggested that IRAK1/4 signaling pathway plays an important role in mediating the inflammation caused by loss of Ube2n. Many chronic inflammatory skin disorders are accompanied by cutaneous dysbiosis. Since loss of UBE2N led to inflammatory skin lesions with disrupted epidermis and increased pro-inflammatory immune signature, we also investigated whether UBE2N plays a role in cutaneous microbial regulation. In order to assess the cutaneous microbiome of the Ube2n KO skin, we performed an amplicon sequencing analysis of the V4 region of 16S rRNA gene from skin swabs of the and the control mice. The sequencing data showed that there was a dramatic shift in microbial population: an increased abundance of Corynebacterium in the inflamed Ube2n KO skin. In summary, our investigation of the role of UBE2N and its function in the skin revealed that it is critical for maintaining epidermal homeostasis and suppression of skin inflammation. We also learned that UBE2N loss-of-function-mediated skin inflammation leads to dysbiosis. This suggests that the restoration of UBE2N signaling and/or IRAK1/4 inhibition could be potential therapeutic strategies for inflammatory skin disorders and controlling healthy skin microbiome.