p68 DEAD box RNA helicase expression in keratinocytes. Regulation, nucleolar localization, and functional connection to proliferation and vascular endothelial growth factor gene expression.
Nitric oxide (NO) represents a short lived mediator that pivotally drives keratinocyte movements during cutaneous wound healing. In this study, we have identified p68 DEAD box RNA helicase (p68) from an NO-induced differential keratinocyte cDNA library. Subsequently, we have analyzed regulation of p68 by wound-associated mediators in human and murine keratinocytes. NO, serum, growth factors, and pro-inflammatory cytokines were potent inducers of p68 expression in the cells. p68 was constitutively expressed in the epithelial compartment of murine skin. Upon injury, we found a transient down-regulation of overall p68 protein in wound tissue. However, p68 did not completely disappear during early wound repair, as we found an expression of p68 protein in isolated wound margin tissue 24 h after wounding. Moreover, immunohistochemistry and cell fractionation analysis revealed a restricted localization of p68 in keratinocyte nuclei of the developing epithelium. Accordingly, cultured keratinocytes also showed a nuclear localization of the helicase. Moreover, confocal microscopy revealed a strong localization of p68 protein within the nucleoli of the cells. Functional analyses demonstrated that p68 strongly participated in keratinocyte proliferation and gene expression. Keratinocytes that constitutively overexpressed p68 protein were characterized by a marked increase in serum-induced proliferation and vascular endothelial growth factor expression, whereas down-regulation of endogenous p68 using small interfering RNA markedly attenuated serum-induced proliferation and vascular endothelial growth factor expression. Altogether, our results suggest a tightly controlled expression and nucleolar localization of p68 in keratinocytes in vitro and during skin repair in vivo that functionally contributes to keratinocyte proliferation and gene expression.
Kahlina
Kornelija
K
Pharmazentrum Frankfurt, Klinikum der Johann Wolfgang Goethe-Universität, Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Germany.
Goren
Itamar
I
Pfeilschifter
Josef
J
Frank
Stefan
S
D000818:Animals; D045744:Cell Line, Tumor; D002466:Cell Nucleolus / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D049109:Cell Proliferation; D002478:Cells, Cultured; D018388:Codon, Terminator; D053487:DEAD-box RNA Helicases; D018076:DNA, Complementary / Q000378:metabolism; D015536:Down-Regulation; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005786:Gene Expression Regulation; D015723:Gene Library; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006133:Growth Substances / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D015603:Keratinocytes / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D011494:Protein Kinases / Q000096:biosynthesis*; D012313:RNA / Q000378:metabolism; D020365:RNA Helicases / Q000096:biosynthesis*; D034741:RNA, Small Interfering / Q000378:metabolism; D012343:RNA, Transfer / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism; D012867:Skin / Q000473:pathology; D013347:Subcellular Fractions / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis* / Q000378:metabolism; D014945:Wound Healing
D018388:Codon, Terminator; D018076:DNA, Complementary; D006133:Growth Substances; D034741:RNA, Small Interfering; D042461:Vascular Endothelial Growth Factor A; D049452:Green Fluorescent Proteins; D012313:RNA; D012343:RNA, Transfer; D011494:Protein Kinases; D012260:Ribonucleases; C506749:Ddx5 protein, human; C506751:Ddx5 protein, mouse; D053487:DEAD-box RNA Helicases; D020365:RNA Helicases