Effects of UV, 4NQO and TPA on gene expression in cultured human epidermal keratinocytes

Abstract

In an approach to study effects of UV light on gene expression in human epidermal keratinocytes, a cDNA library was constructed from poly(A)RNA isolated after UV irradiation from cultured keratinocytes. The cDNA library was differentially screened with labelled cDNA probes synthesized on poly(A)RNA isolated from UV irradiated or nonirradiated keratinocytes. Forty clones were selected and subjected to further analysis, 31 of them are described in this report. Whereas total mRNA synthesis is reduced after UV irradiation or treatment with 4-NQO Northern blot analysis revealed that there is an at least relative increase in the level of mRNAs corresponding to the majority of the isolated cDNA clones. Among these 15 were identified as corresponding to mRNAs for 50K and 56K keratins and for 50K- and 46K-related keratin. In addition, clones were found corresponding to the proteinase inhibitor cystatin A and to the glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Treatment of keratinocytes with the tumor promoter TPA had no effect on the mRNA level for most of the clones except those corresponding to keratins. Our results indicate that in keratinocytes UV irradiation leads to a relative increase in the level of some mRNAs.

Functional expression of recombinant human stefin A in mammalian and bacterial cells

“…A 5′ oligonucleotide [5′-A GCC ACC ATG GAC CTG GAG GC TTA containing an NcoI recognition sequence (underlined)] and a 3′ oligonucleotide [5′-C ACT TTG GAT CCA TGC TGC TA containing a BamHI recognition sequence (underlined)] were designed based upon the published human cDNA sequence (21). The upstream primer changes the isoleucine codon immediately after the ATG initiation codon to a valine codon.…”

S100A11, S100A10, Annexin I, Desmosomal Proteins, Small Proline-rich Proteins, Plasminogen Activator Inhibitor-2, and Involucrin Are Components of the Cornified Envelope of Cultured Human Epidermal Keratinocytes

“…The fact that the amino terminus was not released by CNBr, and that subsequent proteinase K digestion released peptide P9a, suggests that the NH 2 terminus of annexin I segment contains a cross-link(s). Possible sites for cross-link formation include Lys 9 , Lys 26 , and Lys 29 , and Gln 10 , Gln 19 , and Gln 23 . Three of these sites, Gln 19 , Gln 23 , and Lys 26 , have been previously identified as cross-linking sites in vitro (30,31).…”

“…Possible sites for cross-link formation include Lys 9 , Lys 26 , and Lys 29 , and Gln 10 , Gln 19 , and Gln 23 . Three of these sites, Gln 19 , Gln 23 , and Lys 26 , have been previously identified as cross-linking sites in vitro (30,31). No peptides matching the annexin I COOH terminus, Tyr 319 -Asn 346 , were recovered in this study.…”

“…Transglutaminase enzymes catalyze the assembly of this structure via formation of ⑀-(␥-glutamyl)lysine bonds between envelope precursors (4 -7). The proteins that have been identified as constituents of the CE include loricrin (8 -10), involucrin (11)(12)(13)(14), the small proline-rich (SPR) family of proteins (15)(16)(17)(18), cystatin A (19,20), elafin (21)(22)(23), filaggrin (24 -26), keratin (27,28), desmosomal components (27), annexin I (29 -31), plasminogen activator inhibitor-2 (PAI-2) (32), and the 195-kDa and 210-kDa proteins (33,34). Cross-links have been identified within loricrin, elafin, filaggrin, keratin, SPR1, SPR2, and desmoplakin (35).…”

Opposing activities of c-Fos and Fra-2 on AP-1 regulated transcriptional activity in mouse keratinocytes induced to differentiate by calcium and phorbol esters

“…Transcripts encoding K1 and K10 are subsequently down-regulated in the granular layer, where the late marker genes loricrin and ®laggrin are induced (Roop et al, 1988;Fuchs, 1990). In addition, keratinocyte transglutaminases are activated in the granular layer to catalyze the formation of corni®ed envelopes (reviewed in Polakowska and Goldsmith, 1991), insoluble structures composed of precursor proteins involucrin (Rice and Green, 1979), small proline rich (spr) proteins (Kartasova et al, 1987;Marvin et al, 1992), loricrin (Mehrel et al, 1990), and other components (Steinert and Merekov, 1995).…”