Human keratinocytes express multiple P2Y-receptors:: Evidence for functional P2Y1, P2Y2, and P2Y4 receptors

Extracellular nucleotides are agonists at the family of receptors known as the P2 receptors, and in keratinocytes the P2Y(2) subtype is known to elevate the intracellular free calcium concentration (Ca-i) and stimulate proliferation. In this study, we have investigated the presence of other functional members of the P2Y subgroup in both normal human keratinocytes and the HaCaT cell line. Using reverse transcription polymerase chain reaction, the expression of mRNA for P2Y(1), P2Y(2), P2Y(4), and P2Y(6) receptors was demonstrated in HaCaT cells and differentiated and undifferentiated normal human keratinocytes. Ca-i was monitored in response to a panel of P2Y receptor agonists. To couple mobilized Ca-i to a downstream cellular response, cell proliferation was also addressed. In both cell types, adenosine 5'-triphosphate and uridine 5'-triphosphate induced Cai transients of approximately equal duration, magnitude, and shape, confirming the presence of functional P2Y(2) receptors. In HaCaT cells, additional characteristic responses were observed in a subpopulation of cells; adenosine 5'-triphosphate failed to elevate Ca-i in some cells responding to uridine 5'-triphosphate, indicating the presence of P2Y4 receptors, whereas the P2Y(1)-specific agonist 2-methylthio-5'-adenosine diphosphate was, again, only effective in a small subpopulation. Uridine 5'-diphosphate was ineffective, indicating the absence of functional P2Y(6) receptors. Adenosine 5'-triphosphate and uridine 5'-triphosphate equally promoted cell growth in normal human keratinocytes in comparison with the control. In HaCaT cells, adenosine 5'-triphosphate, uridine 5'-triphosphate, and adenosine 5'-diphosphate significantly increased proliferation in comparison to the controls, with a 30% higher response to uridine 5'-triphosphate than with adenosine 5'-triphosphate. These data demonstrate that multiple P2Y receptors (P2Y(1), P2Y(2), and P2Y(4) subtypes) are differentially involved in the regulation of proliferation in human keratinocytes and therefore may be important in wound healing.