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INVESTIGATIVE REPORT ActaDV ActaDV Advances in dermatology and venereology Acta Dermato-Venereologica
Pre-elafin is Involved in Ultraviolet-induced Keratinocyte Apoptosis via Pro-caspase-3 Activation Associated with Cystatin-A Downregulation
Hyangmi KIM 1 , Minsoo NOH 2 , Ok-Nam BAE 3 , Chang-Hoon LEE 4 and Ai-Young LEE 1
1
Department of Dermatology , Dongguk University Ilsan Hospital , Gyeonggi-do , 2 College of Pharmacy , Natural products Research Institute , Seoul National University , Seoul , 3 College of Pharmacy , Institute of Pharmaceutical Science and Technology , Hanyang University , Ansan , and 4 College of Pharmacy , Dongguk University , Seoul , South Korea
Pre-elafin controls keratinocyte integrity via cornified envelope formation and inhibition of desquamation , but its role in ultraviolet ( UV ) -induced keratinocyte apoptosis is unknown . This study examined the role of pre-elafin in volunteer skin samples and primary cultured normal human keratinocytes irradiated with phototoxic doses of UVA / narrow-band UVB , and in keratinocytes with pre-elafin overexpression / knockdown , under conditions of low and high calcium . Phototoxic doses of UV increased pre-elafin mRNA and protein expression in inverse proportion to keratinocyte survival . Pre-elafin overexpression under conditions of low calcium , which , in contrast to conditions of high calcium , was localized to the cytoplasm , increased keratinocyte apoptosis , whereas knockdown inhibited UV-induced apoptosis . Pre-elafin was co-localized with , but not bound to , cleaved caspase-3 . Pre-elafin reduced cystatin-A expression , which was bound to pro-caspase-3 . In conclusion , UV phototoxicity-induced pre-elafin inside keratinocytes prior to cornified envelope formation could be involved in UV-induced keratinocyte apoptosis via cystatin-A downregulation resulting in pro-caspase-3 activation .
Key words : UV ; keratinocyte apoptosis ; pre-elafin ; cellular location ; cystatin-A ; pro-caspase-3 .
Accepted Jan 24 , 2017 ; Epub ahead of print Jan 25 , 2017 Acta Derm Venereol 2017 ; 97 : 578 – 585 .
Corr : Ai-Young Lee , Department of Dermatology , Dongguk University Ilsan Hospital , 814 Siksa-dong , Ilsandong-gu , Goyang-si , Gyenggi-do 410- 773 , South Korea . E-mail : lay5604 @ naver . com
Overexposure to ultraviolet radiation ( UVR ) induces acute and long-term phototoxicities on human skin . UVR-induced DNA damage is important in the development of these harmful effects . If DNA damage is not completely removed , growth arrest or apoptosis occur to protect the cells from carcinogenesis ( 1 ). The sunburn cell , which is characterized by pyknotic nuclei and eosinophilic cytoplasm , is an apoptotic cell with positive staining on terminal deoxynucelotidyl transferasemediated dUTP nick end labelling ( TUNEL ) assay ( 2 , 3 ). Considering that these cells are keratinocytes , sunburn cells could be a representative example of UV-induced keratinocyte apoptosis . Although molecular pathways have been mentioned as the mechanism of UVR-induced keratinocyte apoptosis ( 4 , 5 ), the detailed picture of UVinduced apoptosis remains to be clarified .
Apoptosis pathways converge to activate pro-caspases . However , there is increasing evidence that proteases other than caspases , such as cathepsins and endogenous cathepsin inhibitor cystatin-A , are also involved in apoptosis of keratinocytes ( 6 – 9 ). The role of serine protease and inhibitors in apoptosis has been suggested , despite differing results ( 10 – 12 ). In a microarray analysis of human skin samples , which were irradiated by a single phototoxic dose of UVA and narrow-band UVB ( NB-UVB ), the serine protease inhibitor pre-elafin was found to be an upregulated differentially expressed gene ( DEG ) common to both UVA and NB-UVB-induced phototoxicities . Preelafin , also known as skin-derived anti-leukoproteinase or skin-derived peptidase inhibitor 3 ( PI3 ), is a secretory protein derived from the precursor pro-pre-elafin . Preelafin is formed by cleavage of a signal sequence from the precursor and contains a putative substrate domain for epidermal transglutaminase ( TG ) at the N-terminus and a proteinase-inhibiting domain at the C-terminus ( 13 ). Elafin is released from pre-elafin by proteolytic cleavage ( 14 ), although the term elafin has often been used regardless of precursor form ( pre-elafin ) and cleaved form ( real elafin ).
Pre-elafin in human skin has previously been shown to play at least 2 different roles in the control of epithelial integrity , depending on keratinocyte differentiation : cornified envelope ( CE ) formation in terminally differentiated keratinocytes and desquamation inhibition in keratinocytes before terminal differentiation ( 15 , 16 ). In this study , another role of pre-elafin in the apoptosis of keratinocytes damaged by UV phototoxicity was examined in primary cultured human keratinocytes and in skin samples from volunteers irradiated with phototoxic doses of UVA and NB-UVB , as well as in cultured keratinocytes subject to pre-elafin overexpression or knockdown . Calcium is an important regulator of keratinocyte differentiation ( 17 ). For CE formation in terminal differentiation , constituting proteins of keratinocytes should be cross-linked by TG . TG activity is also regulated by calcium . Therefore , the role of pre-elafin in cultured keratinocytes under low or high calcium concentrations was examined in order to identify whether cellular location of pre-elafin involved in the apoptosis was different from that involved in CE formation or in inhibition of desquamation . doi : 10.2340 / 00015555-2621 Acta Derm Venereol 2017 ; 97 : 578 – 585
This is an open access article under the CC BY-NC license . www . medicaljournals . se / acta Journal Compilation © 2017 Acta Dermato-Venereologica .