Intracellular calcium pool emptying induces DNA synthesis in HaCaT keratinocytes

Calcium signaling provides a central control mechanism for 2 growth,. differentiation and apoptosis of epidermal keratinocytes. Moreover, calcium signaling is important for carcinogenesis in view of the observations suggesting that emptying of intracellular stores in keratinocytes [e.g. by a selective blocker of calcium pump in the endoplasmic reticulum (ER), thapsigargin] facilitates skin cancer development. In this work, we analyzed whether calcium content in the intracellular stores is linked to HaCaT keratinocyte growth and apoptosis control. Treatment with thapsigargin caused calcium release from the intracellular pool and permanent pool depletion (up to 24 h) could be achieved using a high dose (1 muM) of this inhibitor. HaCaT cells cultured in these conditions exhibited an increased rate of DNA synthesis, assessed by the BrdU incorporation assay. Moreover, a weak stimulation of involucrin (terminal differentiation marker) was observed. Studies where intracellular free calcium (Ca-i(2+)) was chelated with BAPTA [1,2-bis(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid] revealed that abrogation of thapsigargin-induced Ca-i(2+) elevation did not counteract its effects on DNA synthesis, but blocked thapsigargin-induced involucrin expression. Apoptosis was readily achieved by extracellular calcium chelation using EGTA [ethylene glycol-bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid], but was not observed after thapsigargin or BAPTA alone or in combination. In conclusion, depletion of intracellular calcium stores causes stimulation of keratinocyte proliferation independently of the elevation of Ca-i(2+).