Abstract
Major biological effects of UVB are attributed to cyclobutane pyrimidine dimers (CPDs), the most common photolesions formed on DNA. To investigate the contribution of CPDs to UVB-induced changes of gene expression, a model system was established by transfecting keratinocytes with pseudouridine-modified mRNA (Ψ-mRNA) encoding CPD-photolyase. Microarray analyses of this model system demonstrated that more than 50% of the gene expression altered by UVB was mediated by CPD photolesions. Functional classification of the gene targets revealed strong effects of CPDs on the regulation of the cell cycle and transcriptional machineries. To confirm the microarray data, cell cycle-regulatory genes, CCNE1 and CDKN2B that were induced exclusively by CPDs were selected for further investigation. Following UVB irradiation, expression of these genes increased significantly at both mRNA and protein levels, but not in cells transfected with CPD-photolyase Ψ-mRNA and exposed to photoreactivating light. Treatment of cells with inhibitors of c-Jun N-terminal kinase (JNK) blocked the UVB-dependent upregulation of both genes suggesting a role for JNK in relaying the signal of UVB-induced CPDs into transcriptional responses. Thus, photolyase mRNA-based experimental platform demonstrates CPD-dependent and -independent events of UVB-induced cellular responses, and, as such, has the potential to identify novel molecular targets for treatment of UVB-mediated skin diseases.
Highlights
The incidence of keratinocyte-derived skin cancer, which is the most common human malignancy, continues to increase worldwide, presenting a serious challenge to healthcare systems [1]
Top functional categories were determined among these genes, and we found that Cyclobutane pyrimidine dimers (CPDs)-related gene expression changes were mainly associated with regulation of the cell cycle and transcriptional machineries in response to Ultraviolet B (UVB) irradiation of human keratinocytes
We demonstrated that in vitro-transcribed mRNA encoding non-human CPD-photolyase can efficiently be translated into a functional protein in cultured human keratinocytes
Summary
The incidence of keratinocyte-derived skin cancer, which is the most common human malignancy, continues to increase worldwide, presenting a serious challenge to healthcare systems [1]. Ultraviolet B (UVB) (290–320 nm) radiation is the main environmental risk factor for sunburn, skin carcinogenesis and premature skin ageing [2,3]. Cyclobutane pyrimidine dimers (CPDs) are the predominant photolesions caused by UVB radiation, and primarily they are responsible for these adverse effects [4]. The pathogenetic role of CPDs is further substantiated by presence of CPD-related signature mutations in genes involved in the formation of skin cancers [7], as well as, by the correlation between the action spectrum value for the induction of CPD photolesions and development of UV-induced skin cancer in animal models [8,9]. DNA lesions, including CPDs are excised by the nucleotide excision repair (NER) system of human keratinocytes [13]. The rate and accuracy of DNA repair by NER are suboptimal [14]
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