title: Mouse keratinocytes express c98, a novel gene homologous to bcl-2, that is stimulated by insulin-like growth factor 1 and prevents dexamethasone-induced apoptosis.
abstract: Many studies have been undertaken to investigate the mechanisms of skin differentiation. In particular, growth factors and hormones are believed to play important roles in skin proliferation, differentiation and survival. Insulin-like growth factor-1 (IGF-1) has been identified as a survival factor in many tissues including the skin, but the molecular mechanism of IGF-1 in epidermal differentiation is not completely understood. Neonatal mouse skin is useful for studying changes in gene expression, as the mitotic activity of skin cells changes shortly after birth. Using RNA differential display (DD), a 357-nt message that is specifically expressed in the epidermal keratinocytes of IGF-1-injected newborn mice but not in controls, has been identified. Confirmation of expression of this gene by ribonuclease protection assay (RPA) showed that its mRNA expression in the epidermal keratinocytes is induced by IGF-1. Using RNA ligase-mediated rapid amplification of 5' cDNA ends (RLM-5'-RACE), we have successfully isolated a 3473-bp full-length gene, c98, that has 97% sequence homology to a bcl-2-like gene, bcl-w. The latter has been identified as a proto-oncogene in several murine myeloid cell lines. Amino acid sequence analysis of the c98 showed that it has 97% sequence identity to the bcl-w protein and possesses bcl-2 homology domains (BH) 1, 2 and 3. Immunoblotting data revealed similar increases of c98 protein expression to its mRNA expression in the keratinocytes of IGF-1-injected animals. Weak expression of other bcl-2 family member proteins, bax, bcl-2 and bcl-xL, were also found in the immunoblots. Additionally, IGF-1 was found to be able to protect epidermal keratinocytes from dexamethasone (DEX)-induced apoptosis, based on the findings that after the cells were treated with DEX, DNA laddering was present in the control mice but not in those injected with IGF-1. Further, using a photometric enzyme-linked immunoassay to quantitate keratinocyte death, we found that after addition of DEX, the amounts of cytoplasmic histone-associated DNA fragments were not significantly (P>0.05) different in IGF-1-treated cells compared with untreated control cells during the high mitotic stage of skin epidermis. To assess the role of c98 in these anti-apoptotic processes, we have generated a recombinant plasmid that contains an expression vector and c98 and transfected this plasmid into the keratinocytes from mice without IGF-1-treatment. Expression of the c98 protein was found to completely (P>0.05) block DEX-induced apoptosis after cell transfection. Taken together, our current data demonstrated that IGF-1 plays an anti-apoptotic role in the DEX-induced apoptosis in epidermal keratinocytes and this, at least in part, may be mediated through expression of c98.
authors.affiliation: Graduate Institute of Biotechnology, National Pingtung University of Science and Technology, 1, Hseuh-Fu, Road, Pingtung 91201, Taiwan, ROC. suhy@mail.npust.edu.tw
authors.forename: Hung-Yi
authors.initials: HY
authors.lastname: Su
authors.forename: Winston T K
authors.initials: WT
authors.lastname: Cheng
authors.forename: Shih-Chu
authors.initials: SC
authors.lastname: Chen
authors.forename: Chen-Tse
authors.initials: CT
authors.lastname: Lin
authors.forename: Yi-Yang
authors.initials: YY
authors.lastname: Lien
authors.forename: Hung-Jen
authors.initials: HJ
authors.lastname: Liu
authors.forename: R Stewart
authors.initials: RS
authors.lastname: Gilmour
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000096:biosynthesis* / Q000737:chemistry; D003907:Dexamethasone / Q000037:antagonists & inhibitors / Q000494:pharmacology; D019254:Genes, bcl-2; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology*; D015603:Keratinocytes / Q000187:drug effects* / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D012313:RNA / Q000032:analysis / Q000302:isolation & purification; D012689:Sequence Homology, Nucleic Acid; D012867:Skin / Q000254:growth & development / Q000378:metabolism
chemical_list: D018076:DNA, Complementary; D012313:RNA; D007334:Insulin-Like Growth Factor I; D003907:Dexamethasone