Potentiation of ATP- and bradykinin-induced [Ca2+]c responses by PTHrP peptides in the HaCaT cell line.

In the epidermis, local and systemic factors including extracellular nucleotides and parathyroid hormone-related protein (PTHrP) regulate keratinocyte proliferation and differentiation. Extracellular nucleotides increase proliferation via activation of P2 receptors and induction of calcium transients, while endoproteases cleave PTHrP, resulting in fragments with different cellular functions. We investigated the effects of adenosine 5'-triphosphate (ATP) alone and in combination with synthetic PTHrP peptides on calcium transients in HaCaT cells. ATP induced calcium transients, while PTHrP peptides did not. C-terminal and mid-molecule PTHrP peptides (1-100 pM) potentiated ATP-induced calcium transients independently of calcium influx. 3-Isobutyl-1-methylxanthine potentiated ATP-induced calcium transients, suggesting that a cyclic monophosphate is responsible. Cyclic AMP is not involved, but cyclic GMP is a likely candidate since the protein kinase G inhibitor, KT5823, inhibited potentiation. Co-stimulation with ATP and either PTHrP (43-52) or PTHrP (70-77) increased proliferation, suggesting that this is important in the regulation of cell turnover and wound healing and may be a mechanism for hyperproliferation in skin disorders such as psoriasis. Finally, PTHrP fragments potentiated bradykinin-induced calcium transients, suggesting a role in inflammation in the skin. Since PTHrP is found in many normal and malignant cells, potentiation is likely to have a wider role in modulating signal transduction events.

Human keratinocytes release ATP and utilize three mechanisms for nucleotide interconversion at the cell surface.

Nucleotide activation of P2 receptors is important in autocrine and paracrine regulation in many tissues. In the epidermis, nucleotides are involved in proliferation, differentiation, and apoptosis. In this study, we have used a combination of luciferin-luciferase luminometry, pharmacological inhibitors, and confocal microscopy to demonstrate that HaCaT keratinocytes release ATP into the culture medium, and that there are three mechanisms for nucleotide interconversion, resulting in ATP generation at the cell surface. Addition of ADP, GTP, or UTP to culture medium elevated the ATP concentration. ADP to ATP conversion was inhibited by diadenosine pentaphosphate, oligomycin, and UDP, suggesting the involvement of cell surface adenylate kinase, F(1)F(0) ATP synthase, and nucleoside diphosphokinase (NDPK), respectively, which was supported by immunohistochemistry. Simultaneous addition of ADP and GTP elevated ATP above that for each nucleotide alone indicating that GTP acts as a phosphate donor. However, the activity of NDPK, F(1)F(0) ATP synthase or the forward reaction of adenylate kinase could not fully account for the culture medium ATP content. We postulate that this discrepancy is due to the reverse reaction of adenylate kinase utilizing AMP. In normal human skin, F(1)F(0) ATP synthase and NDPK were differentially localized, with mitochondrial expression in the basal layer, and cell surface expression in the differentiated layers. We and others have previously demonstrated that keratinocytes express multiple P2 receptors. In this study we now identify the potential sources of extracellular ATP required to activate these receptors and provide better understanding of the role of nucleotides in normal epidermal homeostasis and wound healing.

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 P2Y2 subtype is known to elevate the intracellular free calcium concentration (Cai) 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 P2Y1, P2Y2, P2Y4, and P2Y6 receptors was demonstrated in HaCaT cells and differentiated and undifferentiated normal human keratinocytes. Cai was monitored in response to a panel of P2Y receptor agonists. To couple mobilized Cai 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 P2Y2 receptors. In HaCaT cells, additional characteristic responses were observed in a subpopulation of cells; adenosine 5'-triphosphate failed to elevate Cai in some cells responding to uridine 5'-triphosphate, indicating the presence of P2Y4 receptors, whereas the P2Y1-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 P2Y6 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 (P2Y1, P2Y2, and P2Y4 subtypes) are differentially involved in the regulation of proliferation in human keratinocytes and therefore may be important in wound healing.