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Inhibition of UVA‐induced c‐Jun N‐terminal Kinase Activity Results in Caspase‐dependent Apoptosis in Human Keratinocytes
Author(s) -
Silvers Amy L.,
Finch Joanne S.,
Bowden G. Timothy
Publication year - 2006
Publication title -
photochemistry and photobiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.818
H-Index - 131
eISSN - 1751-1097
pISSN - 0031-8655
DOI - 10.1562/2005-08-26-ra-659
Subject(s) - hacat , apoptosis , poly adp ribose polymerase , keratinocyte , programmed cell death , microbiology and biotechnology , caspase , caspase 3 , cell culture , chemistry , kinase , cleavage (geology) , caspase 8 , uvb induced apoptosis , biology , in vitro , biochemistry , dna , polymerase , genetics , paleontology , fracture (geology)
Inhibition of c‐Jun N‐terminal kinase (JNK) with the pharmacologic inhibitor SP600125 in UVA‐irradiated HaCaT cells and human primary keratinocytes resulted in dramatic phenotypic changes indicative of cell death. These phenotypic changes correlated with caspase 8, 9 and 3 activations as well as cleavage of the caspase substrate polyADP‐ribose poly‐merase (PARP). Morphologic analysis and analysis of sub‐G 0 DNA content confirmed apoptotic cell death in these keratinocytes after combination treatment. Addition of the general caspase inhibitor zVAD‐fmk to combination‐treated HaCaT cells was able to completely block caspase activation, PAW cleavage, the increase in sub‐G 0 DNA content and the classic morphologic features of apoptosis, indicating that this combination treatment resulted in caspase‐dependent apoptotic cell death. zVAD‐fmk treatment of primary keratinocytes was able to completely inhibit caspase activation and PARP cleavage, reduce morphologic apoptosis at lower concentrations of SP600125 and decrease the sub‐G 0 DNA content detected after UVA + SP600125 treatment. However, cell death and a significant amount of debris was still detected after caspase inhibitor treatment, particularly with 125 n M SP600125. At subconfluent conditions and low passage, primary keratinocytes were more sensitive to UVA irradiation alone than HaCaT cells. In conclusion, we have observed that inhibition of UVA‐induced JNK activity with the pharmacologic inhibitor SP600125 resulted in caspase‐dependent apoptotic cell death in both the immortalized keratinocyte cell line HaCaT and primary keratinocytes. However, the increased sensitivity of primary keratinocytes to experimental stress may have also resulted in direct cellular injury and caspase‐independent cell death.