An intact actin-containing cytoskeleton is required for capacitative calcium entry, but not for ATP-induced calcium-mediated cell signaling in cultured human keratinocytes.
BACKGROUND
The present study was focused on structural relationship between intracellular calcium stores and plasma membrane store-operated calcium channels in cultured normal and NF1 keratinocytes.


METHODS
Calcium mobilization induced by thapsigargin or extracellular ATP was studied in control and cytochalasin D-treated human keratinocytes.


RESULTS
Treatment of keratinocytes with cytochalasin D disrupted the actin cytoskeleton and changed the cells from a planar, extended morphology, to a rounded shape. In normal control keratinocytes, thapsigargin induced a marked increase in intracellular calcium concentration ([Ca2+]i). The capacitative calcium influx of cytochalasin D-treated normal keratinocytes was significantly weaker compared to normal control cells. In normal keratinocytes, ATP induced a rapid and transient increase in [Ca2+]i. Thus disruption of the cytoskeleton blocked thapsigargin-induced calcium mobilization, but had no effect on ATP-induced [Ca2+]i mobilization in keratinocytes. The results suggest that microfilaments play crucial role for functional capacitative Ca2+ entry in cultured keratinocytes. The cytoskeleton and calcium mediated cell signaling have been demonstrated to be abnormal in keratinocytes cultured from patients with neurofibromatosis type 1 (NF1). In NF1 keratinocytes, thapsigargin induced a slow and moderate increase in [Ca2+]i. The effect of cytochalasin D on NF1 keratinocytes was less pronounced compared to normal keratinocytes. In NF1 keratinocytes, ATP induced a rapid and transient increase in [Ca2+]i.


CONCLUSIONS
The actin microfilaments play a crucial role for functional capacitative Ca2+ entry in cultured keratinocytes, and that aberrant organization of cytoskeleton may partly explain altered calcium-mediated cell signaling in NF1.
Korkiamäki
Timo
T
Department of Anatomy and Cell Biology, University of Oulu, Oulu, Finland. tkorkiam@paju.oulu.fi
Ylä-Outinen
Heli
H
Koivunen
Jussi
J
Peltonen
Juha
J
D000199:Actins / Q000502:physiology*; D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000328:Adult; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology / Q000502:physiology; D002478:Cells, Cultured; D015638:Cytochalasin D / Q000494:pharmacology; D003599:Cytoskeleton / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D008875:Middle Aged; D009456:Neurofibromatosis 1 / Q000473:pathology; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D012867:Skin / Q000166:cytology / Q000187:drug effects; D019284:Thapsigargin / Q000494:pharmacology
D000199:Actins; D015638:Cytochalasin D; D019284:Thapsigargin; D000255:Adenosine Triphosphate; D002118:Calcium