Expression of NF-kappaB in epidermis and the relationship between NF-kappaB activation and inhibition of keratinocyte growth.
BACKGROUND
Nuclear factor-kappaB (NF-kappaB) is a transcription factor involved in a number of signalling pathways in many cell types. NF-kappaB in mice has been implicated as an important regulator of keratinocyte proliferation and differentiation.


OBJECTIVE
To evaluate the role of NF-kappaB in keratinocyte growth in human beings, we examined its expression in keratinocytes both in culture and in situ, and studied the relationship between NF-kappaB activation and the inhibition of keratinocyte proliferation induced by known modulators of keratinocyte growth.


METHODS
The expression of subunits of the NF-kappaB family was examined in human skin, primary cultured keratinocytes and an immortalized keratinocyte line by immunohistochemistry and reverse transcriptase-polymerase chain reaction analysis. NF-kB activation was examined in keratinocytes treated with various modulating agents by electrophoretic mobility shift assay (for DNA-binding activity) and by immunocytochemistry (nuclear translocation). The proliferative capacity of treated keratinocytes was also examined by 3H-thymidine incorporation, cell cycle analysis, and expression of Ki-67, a nuclear marker for cell proliferation. The involvement of NF-kappaB was assessed using sodium salicylate, which inhibits NF-kappaB activation.


RESULTS
The NF-kappaB subunits, p50, p65, RelB, and c-Rel (but not p52), were detected in keratinocytes and in normal epidermis at mRNA and protein levels. The four subunits were expressed in a cytoplasmic (rather than a nuclear) pattern in both basal and suprabasal keratinocytes. Phorbol myristate acetate (PMA), tumour necrosis factor alpha, and interferon gamma each activated NF-kappaB and inhibited keratinocyte proliferation. Lipopolysaccharide and dexamethasone did not activate NF-kappaB and had the least effect on proliferation. Finally, a high concentration of calcium (Ca2+) and retinoic acid each failed to activate NF-kappaB, but were potent inhibitors of keratinocyte proliferation, respectively. PMA-induced cell cycle arrest of keratinocytes was blocked by pretreatment with sodium salicylate.


CONCLUSIONS
NF-kappaB is constitutively expressed in a resting state in both human cultured keratinocytes and the epidermis. Activation of NF-kappaB is required for PMA-induced keratinocyte growth arrest.
Takao
J
J
Department of Dermatology, The University of Texas Southwestern Medical Center, and the Dallas Veterans Affairs Medical Center, Dallas, TX 75390, USA.
Yudate
T
T
Das
A
A
Shikano
S
S
Bonkobara
M
M
Ariizumi
K
K
Cruz
P D
PD
D002453:Cell Cycle / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D004817:Epidermis / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D007124:Immunoenzyme Techniques; D007231:Infant, Newborn; D015603:Keratinocytes / Q000166:cytology / Q000378:metabolism*; D008297:Male; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012980:Sodium Salicylate / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000037:antagonists & inhibitors / Q000494:pharmacology
D016328:NF-kappa B; D012333:RNA, Messenger; D013755:Tetradecanoylphorbol Acetate; D012980:Sodium Salicylate