Serpin squamous cell carcinoma antigen inhibits UV-induced apoptosis via suppression of c-JUN NH2-terminal kinase

Abstract

Protection from ultraviolet (UV) irradiation is a fundamental issue for living organisms. Although melanin's critical role in the protection of basal keratinocytes is well understood, other factors remain essentially unknown. We demonstrate that up-regulation of squamous cell carcinoma antigen-1 (SCCA1) suppresses c-Jun NH2-terminal kinase-1 (JNK1) and thus blocks UV-induced keratinocyte apoptosis. We found that serpin SCCA1 is markedly elevated in the top layers of sun-exposed or UV-irradiated epidermis. UV-induced apoptosis was significantly decreased when SCCA was overexpressed in 3T3/J2 cells. It was significantly increased when SCCA was down-regulated with small interfering RNA in HaCaT keratinocytes. A search for SCCA-interacting molecules showed specific binding with phosphorylated JNK. Interestingly, SCCA1 specifically suppressed the kinase activity of JNK1. Upon exposure of keratinocytes to UV, SCCA1 was bound to JNK1 and transferred to the nucleus. Involucrin promoter-driven SCCA1 transgenic mice showed remarkable resistance against UV irradiation. These findings reveal an unexpected serpin function and define a novel UV protection mechanism in human skin.

Figure 1.

SCCA is up-regulated by UV irradiation in vivo and in vitro. (A) Immunostaining for SCCA1 and SCCA2 in sun-protected buttock skin, sun-exposed (cheek), and UV-irradiated buttock skin (two minimal erythema doses of radiation; biopsy taken after 48 h). The inset shows nuclear localization of SCCA1 at high magnification. Arrows indicate heavy SCCA1 staining in the nuclei. (B) In situ mRNA hybridization of SCCA1. The sense probe did not show any positive reaction. The dark brown color seen in basal cells is caused by melanin. (C) Quantitative real-time PCR analysis of SCCA1 and SCCA2 mRNA levels in cultured human keratinocytes. Values given are SCCA1 or SCCA2 mRNA levels normalized to the amount of G3PDH. Error bars represent the mean of five wells ± SD. **, P < 0.01. Bars, 100 μm.

Figure 2.

Overexpression or down-regulation of SCCAs significantly affected UV-induced apoptosis. (A) FACS analyses of apoptotic cells using SCCA1- or SCCA2-transfected 3T3/J2 cells, 48 h after UV irradiation (30 mJ/cm²). Cells were stained with FITC-conjugated Annexin V and propidium iodide. (B) Analyses of five experiments are summarized. (C) 12 clones whose expression levels of SCCA1 mRNA distributed from 1 to 2,772-fold were established. Using these clones, the effects of UV irradiation were examined. Cells were harvested 48 h after UV irradiation (50 mJ/cm²) and FACS analyses were performed. The antiapoptotic activity correlated with SCCA1 expression. r = 0.734. (D) Using pSilencer vector, an siRNA construct targeted to a homologous sequence of SCCAs was stably transfected into HaCaT keratinocytes. Typical FACS analyses of nonirradiated (UV−) and UV-irradiated (UV+) siSCCA/HaCaT cells were shown. Apoptotic cells were analyzed 48 h after UV irradiation (75 mJ/cm²). (E) Statistical analyses of five experiments. Error bars represent the mean of five wells ± SD.

Figure 3.

SCCA1 binds with JNK1 and translocates into nucleus after UV irradiation. (A) Localization of SCCA1 before and 24 h after UV irradiation (50 mJ/cm²). NHK cells were stained for SCCA1, nucleus, and F-actin with (UV+) or without UV treatment (UV−). (B) Interaction of SCCAs with p-JNK. HSC-4 cell extract was applied to a signal transduction antibody array, followed by incubation with HRP-conjugated antibody against either SCCA1 or SCCA2. Black spots indicate positive binding to p-JNK. Arrows indicate the spots representing antibodies to JNK and p-JNK. (C) HaCaT cell extracts transfected with control pSilencer vector or siSCCA were immunoprecipitated with anti-JNK1 antibody, followed by blotting with indicated antibodies. Normal rabbit IgG was used as a nonspecific control (middle). Effects of UV irradiation were also examined (N, not irradiated; +UV, 5 min after 100 mJ/cm² UV irradiation). (D) Effects of JIP1 peptide on SCCA1 localization. NHK cells were cultured in the presence of 5 μM of JIP1 peptide and stained for SCCA1, nucleus, and F-actin with (UV+, 50 mJ/cm²) or without (UV−) UV treatment. (E) Statistical analysis of SCCA1 translocation. In each case (i.e., control [nontreated], UV-irradiated [UV], control + JIP1 peptide [5 μM; Cont. + JIP], and UV-irradiated [50 mJ/cm²] + JIP1 peptide [5 μM; UV + JIP]), a minimum of 300 cells were counted three times. Error bars represent the mean of three experiments ± SD. ***, P < 0.001. Bars, 20 μm.

Figure 4.

SCCA1 specifically inhibits kinase activity of JNK1. (A) Inhibition of JNK1 kinase activity by SCCA1. An in vitro kinase pull-down assay of the phosphorylation of c-Jun by JNK1 was performed using an SAPK/JNK assay kit. A constant amount of recombinant SCCA1 was incubated with various concentrations of active JNK1, and changes in c-Jun phosphorylation were analyzed with Western blots. (B) Quantitative analysis of the results shown in A. Representative data from five independent experiments are shown. Each band was scanned and the relative intensity was measured with or without SCCA1 (control) and plotted against the amount of p-JNK1. (C) Effects of SCCA1 on p38α kinase activity. Various concentrations of p38α MAPK were incubated with or without 10 times the excess amount of SCCA1, and changes in ATF2 phosphorylation were analyzed with Western blot using anti–phospho-ATF2 antibody. (D) Effects of SCCA1 on ERK1 and ERK2 kinase activity. Kinase activities of ERK1 or ERK2 with or without 20 ng SCCA1 were measured usinga MAPK (ERK1/2) activity assay kit. (E) Changes in c-Jun phosphorylation in HaCaT cells. Extracts from the control or siSCCA/HaCaT cells were obtained 0, 5, and 15 min after UV irradiation (100 mJ/cm²), and levels of c-Jun or phospho-c-Jun were compared with immunoblots. Densitmetric analysis of the ratio between c-Jun and phospho-c-Jun from three independent assays was also shown. Error bars represent the mean of five experiments ± SD. (F) Effects of SCCA knockdown on ERK1/2, p38α, and MAPKAPK2 after UV irradiation. The same extracts used in E were immunoblotted for each form of kinases.

Figure 5.

Effect of UV irradiation on involucrin promoter-driven SCCA1 transgenic mice. (A) Immunostaining of SCCAs in wild-type and transgenic epidermis. Paraffin-embedded skin sections were stained with antibodies to SCCA1 or SCCA2. (B) Histological appearance of wild-type and transgenic mice 24 h after UV irradiation (200 mJ/cm²/d for 2 d). Wild-type (HR-1) mice demonstrated degeneration of the epidermis, including spongiosis (arrows) in the suprabasal layer. Involucrin-SCCA1 transgenic mice did not show any notable changes in UV-irradiated skin. Bars, 50 μm.