Calcium and Vitamin D increase mRNA levels for the growth control hIK1 channel in human epidermal keratinocytes but functional channels are not observed.
BACKGROUND
Intermediate-conductance, calcium-activated potassium channels (IKs) modulate proliferation and differentiation in mesodermal cells by enhancing calcium influx, and they contribute to the physiology of fluid movement in certain epithelia. Previous reports suggest that IK channels stimulate proliferative growth in a keratinocyte cell line; however, because these channels indirectly promote calcium influx, a critically unique component of the keratinocyte differentiation program, an alternative hypothesis is that they would be anti-proliferative and pro-differentiating. This study addresses these hypotheses.


METHODS
Real-time PCR, patch clamp electrophysiology, and proliferation assays were used to determine if human IK1 (hIK1) expression and function are correlated with either proliferation or differentiation in cultured human skin epidermal keratinocytes, and skin biopsies grown in explant culture.


RESULTS
hIK1 mRNA expression in human keratinocytes and skin was increased in response to anti-proliferative/pro-differentiating stimuli (elevated calcium and Vitamin D). Correspondingly, the hIK1 agonist 1-EBIO inhibited keratinocyte proliferation suggesting that the channel could be anti-proliferative and pro-differentiating. However, this proliferative inhibition by 1-EBIO was not reversed by a panel of hIK1 blockers, calling into question the mechanism of 1-EBIO action. Subsequent patch clamp electrophysiological analysis failed to detect hIK1 channel currents in keratinocytes, even those expressing substantial hIK1 mRNA in response to calcium and Vitamin D induced differentiation. Identical electrophysiological recording conditions were then used to observe robust IK1 currents in fibroblasts which express IK1 mRNA levels comparable to those of keratinocytes. Thus, the absence of observable hIK1 currents in keratinocytes was not a function of the electrophysiological techniques.


CONCLUSIONS
Human keratinocyte differentiation is stimulated by calcium mobilization and influx, and differentiation stimuli coordinately upregulate mRNA levels of the calcium-activated hIK1 channel. This upregulation is paradoxical in that functional hIK1 channels are not observed in cultured keratinocytes. It appears, therefore, that hIK1 does not contribute to the functional electrophysiology of primary human keratinocytes, nor intact human skin. Further, the results indicate caution is required when interpreting experiments utilizing pharmacological hIK1 modulators in human keratinocytes.
Manaves
Vlasios
V
Molecular Human Physiology Group, Fujisawa Research Institute of America, Evanston, IL 60201 USA. val.manaves@fujisawa.com
Qin
Wuxuan
W
Bauer
Amy L
AL
Rossie
Sandra
S
Kobayashi
Masakazu
M
Rane
Stanley G
SG
D000328:Adult; D001483:Base Sequence; D001562:Benzimidazoles / Q000494:pharmacology; D001706:Biopsy; D000001:Calcimycin / Q000494:pharmacology; D002117:Calcitriol / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002454:Cell Differentiation / Q000502:physiology; D002478:Cells, Cultured; D018999:Charybdotoxin / Q000494:pharmacology; D003022:Clotrimazole / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D000078404:Epidermal Cells; D006801:Humans; D051740:Ikaros Transcription Factor; D015603:Keratinocytes / Q000166:cytology / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D011720:Pyrazoles / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014807:Vitamin D / Q000378:metabolism*
D001562:Benzimidazoles; D004268:DNA-Binding Proteins; D011720:Pyrazoles; D012333:RNA, Messenger; C411671:TRAM 34; D014157:Transcription Factors; D018999:Charybdotoxin; D014807:Vitamin D; D051740:Ikaros Transcription Factor; D000001:Calcimycin; D002117:Calcitriol; D003022:Clotrimazole; C102635:1-ethyl-2-benzimidazolinone; D002118:Calcium