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• W2000832714 abstract "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. 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. adenosine 5′-diphosphate adenosine 5′-triphosphate intracellular free calcium concentration inositol 1,4,5-trisphosphate 2-methylthio-5′-adenosine diphosphate normal human keratinocytes uridine 5′-diphosphate uridine 5′-triphosphate In the stratified squamous epithelium of the epidermis, cells are continually desquamated from the uppermost layer. Cell division occurs predominantly in the basal layer where stem cells are situated at the tips of the rete ridges. Stem cells give rise to transit amplifying cells (Eckert, 1989Eckert R.L. Structure, function and differentiation of the keratinocyte.Physiol Rev. 1989; 69: 1316-1345Crossref PubMed Scopus (199) Google Scholar), which move to the suprabasal layers where they differentiate. During their migration, the cells are subjected to an increasing calcium gradient (Menon et al, 1985). This, in combination with other signals, induces the morphologic and biochemical changes of differentiation, including changes in cytoskeletal filaments, intercellular attachments, lipid content, and formation of the cell envelope. Each layer of the epidermis contains cells at different stages of differentiation, culminating in terminally differentiated keratinocytes in the cornified layer. The maintenance of the balance between desquamation and proliferation in keratinocytes is a complex process, controlled by multiple extracellular factors. These include stimulation by transforming growth factor α (TGFα) (Barrandon and Green, 1984Barrandon Y. Green H. Cell migration is essential for sustained growth of keratinocyte colonies: the roles of transforming growth factor-α and epidermal growth factor.Cell. 1984; 50: 1131-1137Abstract Full Text PDF Scopus (546) Google Scholar), epidermal growth factor (Rheinwald, 1980Rheinwald J.G. Serial cultivation of normal human epidermal keratinocytes.Meth Cell Biol. 1980; 21A: 229-254Crossref PubMed Scopus (236) Google Scholar), and extracellular nucleotides (Pillai and Bikle, 1992Pillai S. Bikle D.D. Adenosine triphosphate stimulates phophoinositide metabolism, mobilises intracellular calcium, and inhibits terminal differentiation of human epidermal keratinocytes.J Clin Invest. 1992; 90: 42-51Crossref PubMed Scopus (105) Google Scholar;Dixon et al., 1999Dixon C.J. Bowler W.B. Littlewood-Evans A. Dillon J.P. Bilbe G. Sharpe G.R. Gallagher J.A. Regulation of epidermal homeostasis through P2Y2 receptors.Br J Pharmacol. 1999; 127: 1680-1686Crossref PubMed Scopus (97) Google Scholar), and inhibition by TGFβ (Shipley et al., 1986Shipley G.D. Pittelkow M.R. Wille J.J. Scott R.E. Moses H.L. Reversible inhibition of normal human prokeratinocyte proliferation by type β transforming growth factor growth inhibitor in serum-free medium.Cancer Res. 1986; 46: 2068-2071PubMed Google Scholar), extracellular calcium (Cao) (Hennings et al., 1980Hennings H. Michael D. Cheng C. Steinert P. Holbrook K. Yuspa S.H. Calcium regulation of growth and differentiation of mouse epidermal cells in culture.Cell. 1980; 19: 245-254Abstract Full Text PDF PubMed Scopus (1450) Google Scholar), and vitamin D3 (Jones and Sharpe, 1994Jones K.T. Sharpe G.R. Intracellular free calcium and growth changes in single human keratinocytes in response to vitamin D and five epi-analogues.Arch Dermatol Res. 1994; 286: 123-129Crossref PubMed Scopus (28) Google Scholar). When the epidermis is damaged, platelets release extracellular nucleotides, as part of the aggregation reaction associated with clotting (Huang et al., 1989Huang N. Wang D. Heppel L.A. Extracellular ATP is a mitogen for 3T3, 3T6, and A431 cells and acts synergistically with other growth factors.Proc Natl Acad Sci USA. 1989; 86: 7904-7908Crossref PubMed Scopus (135) Google Scholar). In fibroblasts, adenosine 5′-triphosphate (ATP) acts synergistically with epidermal growth factor and insulin to increase the rate of DNA synthesis (Wang et al., 1990Wang D. Huang N. Heppel L.A. Extracellular ATP shows synergistic enhancement of DNA synthesis when combined with agents that are active in wound healing or as neurotransmitters.Biochem Biophys Res Commun. 1990; 66: 251-258Crossref Scopus (84) Google Scholar). Synergy between ATP and platelet-derived growth factor and TGFα also occurs (Huang et al., 1989Huang N. Wang D. Heppel L.A. Extracellular ATP is a mitogen for 3T3, 3T6, and A431 cells and acts synergistically with other growth factors.Proc Natl Acad Sci USA. 1989; 86: 7904-7908Crossref PubMed Scopus (135) Google Scholar). In normal human keratinocytes (NHK), however, extracellular nucleotides have been directly linked to the proliferation response (Pillai and Bikle, 1992Pillai S. Bikle D.D. Adenosine triphosphate stimulates phophoinositide metabolism, mobilises intracellular calcium, and inhibits terminal differentiation of human epidermal keratinocytes.J Clin Invest. 1992; 90: 42-51Crossref PubMed Scopus (105) Google Scholar;Dixon et al., 1999Dixon C.J. Bowler W.B. Littlewood-Evans A. Dillon J.P. Bilbe G. Sharpe G.R. Gallagher J.A. Regulation of epidermal homeostasis through P2Y2 receptors.Br J Pharmacol. 1999; 127: 1680-1686Crossref PubMed Scopus (97) Google Scholar). Not only are nucleotides released by platelets, but they are also released by dead or damaged cells at sites of trauma, and by NHK during both stress and normal epidermal homeostasis (Dixon et al., 1999Dixon C.J. Bowler W.B. Littlewood-Evans A. Dillon J.P. Bilbe G. Sharpe G.R. Gallagher J.A. Regulation of epidermal homeostasis through P2Y2 receptors.Br J Pharmacol. 1999; 127: 1680-1686Crossref PubMed Scopus (97) Google Scholar). Release from NHK has been proposed to occur via ATP-binding cassette proteins, which have been reported to be in close proximity to subtypes of P2 receptors (Al-Awqati, 1995Al-Awqati Q. Regulation of ion channels by ABC-transporters that secrete ATP.Science. 1995; 269: 805-806Crossref PubMed Scopus (171) Google Scholar). Detection methods, utilizing a real-time firefly luciferin-luciferase assay, have indicated that released concentrations of ATP are physiologically active at the adjacent receptors within the local area of release (Dixon et al., 1999Dixon C.J. Bowler W.B. Littlewood-Evans A. Dillon J.P. Bilbe G. Sharpe G.R. Gallagher J.A. Regulation of epidermal homeostasis through P2Y2 receptors.Br J Pharmacol. 1999; 127: 1680-1686Crossref PubMed Scopus (97) Google Scholar). P2 receptors are a family of transmembrane receptors classified according to the mechanisms of signal transduction (Dubyak and El-Moatassim, 1993Dubyak G.R. El-Moatassim C. Signal transduction via P2-purinergic receptors for extracellular ATP and other nucleotides.Am J Physiol. 1993; 265: 577-C606Crossref PubMed Google Scholar) and the results of molecular cloning (Abbracchio and Burnstock, 1994Abbracchio M.P. Burnstock G. Purinoceptors: are there families of P2X and P2Y purinoceptors?.Pharmacol Ther. 1994; 64: 445-475Crossref PubMed Scopus (963) Google Scholar). There are two main classes: the P2X receptors are ligand-gated ion channels (Bean, 1992Bean B.P. Pharmacology and electrophysiology of ATP-activated ion channels.Trends Pharmacol Sci. 1992; 13: 87-90Abstract Full Text PDF PubMed Scopus (292) Google Scholar) and the P2Y receptors are G-protein-coupled Ca2+ mobilizing ATP receptors (O'Connor et al., 1991O'Connor S.E. Dainty I.A. Leff P. Further subclassification of ATP receptors based on agonist studies.Trends Pharmacol Sci. 1991; 12: 137-141Abstract Full Text PDF PubMed Scopus (329) Google Scholar). Whereas some P2X receptor subtypes are rapidly desensitized by agonists, the P2Y receptors, in particular the P2Y1 and P2Y2 subtypes, are not (reviewed byRalevic and Burnstock, 1998Ralevic V. Burnstock G. Receptors for purines and pyrimidine.Pharmacol Rev. 1998; 50: 413-492PubMed Google Scholar). The classes are subdivided and characterized according to the potencies of various agonists (Burnstock and King, 1996Burnstock G. King B.F. Numbering of cloned P2 purinoceptors.Drug Dev Res. 1996; 38: 67-71Crossref Scopus (150) Google Scholar;Kunapuli and Daniel, 1998Kunapuli S.P. Daniel J.L. P2 receptor subtypes in the cardiovascular system.Biochem J. 1998; 336: 513-523Crossref PubMed Scopus (214) Google Scholar). To date, members of the P2Y subgroup that have been cloned from human tissues include the P2Y1, P2Y2, P2Y4, and P2Y6 subtypes. Selective agonists include 2-methylthio-adenosine-5′-diphosphate (2MeSADP) at the P2Y1 receptor (Kunapuli and Daniel, 1998Kunapuli S.P. Daniel J.L. P2 receptor subtypes in the cardiovascular system.Biochem J. 1998; 336: 513-523Crossref PubMed Scopus (214) Google Scholar;Kyle Palmer et al., 1998Kyle Palmer R. Boyer J.C. Schachter J.B. Nicholas R.A. Harden T.K. Agonist action of adenosine triphosphate at the human P2Y1 receptor.Mol Pharmacol. 1998; 54: 1118-1123PubMed Google Scholar) and uridine 5′-diphosphate (UDP) at the P2Y6 receptor (Nicholas et al., 1996Nicholas R.A. Watt W.C. Lazarowski E.R. Li Q. Harden T.K. Uridine nucleotide selectivity of three phospholipase C-activating P2 receptors: identification of a UDP-selective, a UTP-selective, and an ATP- and UTP-specific receptor.Mol Pharmacol. 1996; 50: 224-229PubMed Google Scholar). The P2Y2 receptor is characterized by ATP and uridine 5′-triphosphate (UTP) acting as equipotent agonists, whereas the P2Y4 receptor can be identified when UTP has greater potency than ATP (Boarder and Hourani, 1998Boarder M.R. Hourani S.M. The regulation of vascular function by P2 receptors: multiple sites and multiple receptors.Trends Pharmacol Sci. 1998; 19: 99-107Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). Whereas reverse transcription polymerase chain reaction (RT-PCR) techniques enable mRNA expression within tissues to be determined, functional responses are indicated by intracellular changes in signaling molecules, such as inositol phosphates and intracellular calcium (Cai), and cell outcome, such as proliferation or differentiation. Caution must be used during identification of functional receptors, however, as agonist potency orders may be altered in cultured tissues (Ralevic and Burnstock, 1998Ralevic V. Burnstock G. Receptors for purines and pyrimidine.Pharmacol Rev. 1998; 50: 413-492PubMed Google Scholar) and may be influenced by agonist stability and the presence of interconverting ecto-nucleotidases (Zimmermann, 1996Zimmermann H. Extracellular purine metabolism.Drug Dev Res. 1996; 39: 337-352Crossref Scopus (146) Google Scholar). P2Y receptor activation is coupled, via a G protein, to production of inositol 1,4,5-trisphosphate (InsP3), which initiates transient increases in Cai, and production of diacylglycerol (DAG). The Cai transients last for a period of minutes and are linked to an increase in DNA synthesis and proliferation (Pillai and Bikle, 1992Pillai S. Bikle D.D. Adenosine triphosphate stimulates phophoinositide metabolism, mobilises intracellular calcium, and inhibits terminal differentiation of human epidermal keratinocytes.J Clin Invest. 1992; 90: 42-51Crossref PubMed Scopus (105) Google Scholar;Dixon et al., 1999Dixon C.J. Bowler W.B. Littlewood-Evans A. Dillon J.P. Bilbe G. Sharpe G.R. Gallagher J.A. Regulation of epidermal homeostasis through P2Y2 receptors.Br J Pharmacol. 1999; 127: 1680-1686Crossref PubMed Scopus (97) Google Scholar). Differentiation and Ca2+ mobilization are also linked, although release must be sustained over a longer period of time and must include influx of extracellular calcium (Sharpe et al., 1989Sharpe G.R. Gillespie J.I. Greenwell J.R. An increase in intracellular free calcium is an early event during differentiation of cultured human keratinocytes.FEBS Lett. 1989; 254: 25-28Abstract Full Text PDF PubMed Scopus (98) Google Scholar). The main target for DAG is stimulation of protein kinase C, which in turn stimulates the mitogen-activated protein kinase cascade. This has been observed to increase DNA synthesis and cell division, and has been linked to P2 receptors, particularly the P2Y1 and P2Y2 subtypes (Boarder et al., 1995Boarder M.R. Weisman G.A. Turner J.T. Wilkinson G.F. G protein-coupled P2 purinoceptors: from molecular biology to functional responses.Trends Pharmacol Sci. 1995; 16: 133-139Abstract Full Text PDF PubMed Scopus (181) Google Scholar). P2Y2 receptors, present in a wide range of cell types, have been linked to proliferation in porcine smooth muscle cells (Wang et al., 1992Wang D. Huang N. Heppel L.A. Extracellular ATP and ADP stimulate proliferation of porcine aortic smooth muscle cells.J Cell Physiol. 1992; 153: 221-223Crossref PubMed Scopus (119) Google Scholar), rat astroglial cells (Abbracchio et al., 1994Abbracchio M.P. Saffrey M.J. Hopker V. Burnstock G. Modulation of astroglial cell-proliferation by analogs of adenosine and ATP in primary cultures of rat striatum.Neuroscience. 1994; 59: 67-76Crossref PubMed Scopus (132) Google Scholar), and MCF-7 breast cancer cells (Dixon et al., 1997Dixon C.J. Bowler W.B. Fleetwood P. Ginty A.F. Gallagher J.A. Carron J.A. Extracellular nucleotides stimulate proliferation in MCF-7 breast cancer cells via P2 purinoceptors.B J Cancer. 1997; 75: 34-39Crossref PubMed Scopus (72) Google Scholar). Differential expression of multiple subtypes is species, cell type and differentiation dependent (Pillai and Bikle, 1992Pillai S. Bikle D.D. Adenosine triphosphate stimulates phophoinositide metabolism, mobilises intracellular calcium, and inhibits terminal differentiation of human epidermal keratinocytes.J Clin Invest. 1992; 90: 42-51Crossref PubMed Scopus (105) Google Scholar;Reimer and Dixon, 1992Reimer W.J. Dixon S.J. Extracellular nucleotides elevate [Ca2+]i in rat osteoblastic cells by interaction with 2 receptor subtypes.Am J Physiol. 1992; 263: 1040-C1048PubMed Google Scholar;Ralevic and Burnstock, 1998Ralevic V. Burnstock G. Receptors for purines and pyrimidine.Pharmacol Rev. 1998; 50: 413-492PubMed Google Scholar). For example, in NHK, Cai elevation has been observed with adenosine 5′-diphosphate (ADP), an agonist at both the P2Y1 and P2Y6 receptors, and ATP and UTP, which are agonists at P2Y2 and P2Y4 receptors (Pillai and Bikle, 1992Pillai S. Bikle D.D. Adenosine triphosphate stimulates phophoinositide metabolism, mobilises intracellular calcium, and inhibits terminal differentiation of human epidermal keratinocytes.J Clin Invest. 1992; 90: 42-51Crossref PubMed Scopus (105) Google Scholar). A full characterization of P2Y receptors and their functions in NHK has not previously been published. In this study, we have investigated the presence and function of P2Y receptors in both NHK and the HaCaT cell line. The use of the human keratinocyte cell line HaCaT (Boukamp et al., 1988Boukamp P. Petrussevska R.T. Breitkreutz D. Hornung J. Markham A. Fusenig N.E. Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line.J Cell Biol. 1988; 106: 761-771Crossref PubMed Scopus (3276) Google Scholar) was also investigated as a model of keratinocyte proliferation and was compared with NHK in order to validate its use. HaCaT cells are immortal, but retain some ability to differentiate (Ryle et al., 1989Ryle C.M. Breitkreutz D. Stark H.J. Leigh I.M. Steinert P.M. Roop D. Fusenig N.E. Density-dependent modulation of synthesis of keratins 1 and 10 in the human keratinocyte line HaCaT and in ras-transfected tumorigenic clones.Differentiation. 1989; 40: 42-54Crossref PubMed Scopus (133) Google Scholar;Breitkreutz et al., 1993Breitkreutz D. Stark H.J. Plein P. Baur M. Fusenig N.E. Differential modulation of epidermal keratinization in immortalized (HaCaT) and tumorigenic human skin keratinocytes (HaCaT-ras) by retinoic acid and extracellular Ca2+.Differentiation. 1993; 54: 201-217Crossref PubMed Scopus (88) Google Scholar) and have the capacity to produce normal epidermis in athymic mice (Boukamp et al., 1988Boukamp P. Petrussevska R.T. Breitkreutz D. Hornung J. Markham A. Fusenig N.E. Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line.J Cell Biol. 1988; 106: 761-771Crossref PubMed Scopus (3276) Google Scholar). Using P2Y receptor agonists, we have investigated the functional receptors involved in the regulation of proliferation in NHK and the HaCaT cell line by observation of changes in Cai and the measurement of proliferation by cell counting. Presence of the receptors identified has been confirmed using RT-PCR. Although the mRNA expression profile appeared to be the same for both cell types, there were clear differences in the functional receptors expressed, which probably reflect the transformation of the cell line and/or the changes in differentiation status. Results indicate that, whereas only P2Y2 receptors appear to be functional in NHK, the HaCaT cell line expresses multiple functional receptors, including P2Y1, P2Y2, and P2Y4 receptors, which are involved in the proliferation response. Furthermore, changes in receptor mRNA expression due to differentiation were analyzed by culturing NHK in elevated concentrations of Ca2+. Although there was no apparent change in expression of P2Y1 or P2Y6 receptors, P2Y2 and P2Y4 mRNA expression was downregulated in the cells treated in 200 μM or 1 mM Ca2+ for 24 h. NHK were isolated from neonatal foreskins or from retroauricular tissue. Cells were cultured in MCDB153 (Sigma, Poole, U.K.), using the method previously described byJones and Sharpe, 1994Jones K.T. Sharpe G.R. Intracellular free calcium and growth changes in single human keratinocytes in response to vitamin D and five epi-analogues.Arch Dermatol Res. 1994; 286: 123-129Crossref PubMed Scopus (28) Google Scholar, and were passaged at approximately 70% confluence with addition of 100 μg per ml bovine hypothalamic extract (Pel-Freez, U.S.A.) to aid cell adhesion. Cells were used between passages 1 and 4. The HaCaT cell line was a gift from Professor Fusenig (Division of Differentiation and Carcinogenesis in Vitro, Institute of Biochemistry, German Carcinogenesis Research Center). HaCaT cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% fetal bovine serum (FBS) and 2 mM L-glutamine. DMEM and L-glutamine were purchased from Sigma, Poole, U.K., whereas FBS was obtained from Seralabs, U.K. Cells passaged at approximately 70% confluence were preincubated with 0.02% ethylenediamine tetraacetic acid (EDTA; Sigma, Poole, U.K.) for 5–10 min before incubation with 0.02% EDTA in 0.05% trypsin (ICN-Flow, U.K.) for 5 min. Cells were resuspended in fresh DMEM and split at a ratio of 1:10. Cells were used between passages 45 and 65. Cells were seeded onto 6 cm Petri dishes (Costar, High Wycombe, U.K.), at a concentration of 1×105 cells per dish, and allowed to adhere for 72 and 24 h for NHK and HaCaT cells, respectively, prior to treatment with extracellular nucleotides (Sigma). UTP and ATP were added to the dishes (n=4 for treatments of extracellular nucleotides, and n=8 for controls) for a further 24 h, before cell numbers were determined using a Coulter counter (model ZM). Proliferation was determined as the initial seeded number subtracted from the final cell number. Changes in proliferation when treated with extracellular nucleotides were then compared to controls. Cell contents were extracted from confluent cultures using 0.5 ml Tri-Reagent™ (Sigma) per 6 cm Petri dish. Total RNA was separated using chloroform (BDH-Merck, Poole, U.K.) and isopropanol (Sigma) according to the manufacturer's guidelines (Sigma). The RNA was treated with RNAse-free DNase I (35 U per μl; Qiagen, Crawley, U.K.) for 30 min before being stored at –20°C as an ethanolic precipitate. An aliquot of 5 μg total RNA was used as a template for first strand cDNA synthesis in a 50 μl reaction incubated at 37°C for 1 h. The reaction mixture contained 20 units RNAse inhibitor (Roche Molecular Biochemicals, U.K.), 50 mM Tris–HCl pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 μM dithiothreitol, 1.5 μl oligo dT, 0.5 mM of each dNTP (dATP, dCTP, dGTP, dTTP), and 800 units of Superscript™ II RT (Gibco BRL, Paisley, U.K.). Oligo dT and dNTPs were obtained from Roche Molecular Biochemicals. PCR reactions were carried out using a 50 μl reaction containing 0.5 mM of each dNTP (dATP, dCTP, dGTP, dTTP), 20 mM Tris–HCl pH 8.4, 50 mM KCl, 1.5 mM MgCl2, 1 μM each of sense and antisense primers, 1 unit Thermoprime+(Gibco BRL, Paisley, U.K.), and 2 μl cDNA. For the glyceraldehyde phosphate dehydrogenase (GAPDH) primers, the initial denaturation stage was at a temperature of 94°C for 2 min. This was followed by 35 cycles (Dixon et al., 1999Dixon C.J. Bowler W.B. Littlewood-Evans A. Dillon J.P. Bilbe G. Sharpe G.R. Gallagher J.A. Regulation of epidermal homeostasis through P2Y2 receptors.Br J Pharmacol. 1999; 127: 1680-1686Crossref PubMed Scopus (97) Google Scholar) of denaturation at 94°C for 30 s, annealing at 60°C for 30 s, and an initial extension period of 30 s at 72°C. Finally, an extension period of 5 min at 72°C was performed. For the P2Y receptor primers, the annealing temperature was altered to 58°C. Primer sequences (Maier et al., 1997Maier R. Glatz A. Mosbacher J. Bilbe G. Cloning of P2Y6 cDNAs and identification of a pseudogene: comparison of P2Y receptor subtype expression in bone and brain tissues.Biochem Biophys Res Commun. 1997; 237: 298-302Crossref Scopus (69) Google Scholar) used were as follows: GAPDH sense 5′-GGTGAAGGTCGGAGTCAACGG, anti-sense 5′-GGTCATGAGTCC-TTCCACGAT; P2Y1 sense 5′-TGTGGTGTACCCCCTCAAGTCCC, anti-sense 5′-ATCCGTAACAGCCCAGAATCAGCA; P2Y2 sense 5′-CCAGGCCCCCGTGCTCTACTTTG, anti-sense 5′-CATGTTGATGG-CGTTGAGGGTGTG; P2Y4 sense 5′-CGTCTTCTCGCCTCCGCT-CTCT, anti-sense 5′-GCCCTGCACTCATCCCCTTTTCT; P2Y6 sense 5′-CCGCTGAACATCTGTGTC, anti-sense 5′-AGAGCCATGCCATAG-GGC. Cells were grown on 22 mm round glass coverslips (BDH-Merck) for 24 h at seeding densities of approximately 1×104 cells per coverslip. Cai measurements were taken from single cells within colonies of approximately four to eight cells and were performed at 30°C. Cells were loaded with the fluorescent calcium dye fura-2 by incubation with the acetoxymethyl ester (Molecular Probes, Leiden, The Netherlands) at a concentration of 1–5 μM for 60 min at 37°C in calcium-free phosphate-buffered saline. Cells were bathed in calcium-free phosphate-buffered saline and were stimulated by addition of agonists, and the ratio of emitted light (measured at 520 nm), after excitation at 350 nm and 380 nm, was plotted to provide a measure of the concentration changes in Cai (Sharpe et al., 1993Sharpe G.R. Fisher C. Gillespie J.I. Greenwell J.R. Growth and differentiation stimuli induce different and distinct increases in intracellular free calcium in human keratinocytes.Arch Dermatol Res. 1993; 284: 445-450Crossref PubMed Scopus (34) Google Scholar). Ratios were converted into calcium concentrations by comparison with a calibration curve obtained using standard calcium concentrations in the form of a kit [Molecular Probes; range 0–1350 nM Ca2+ in 10 mM K2 ethyleneglycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (K2EGTA), 0–10 mM CaEGTA, 50 μM fura-2, 100 mM KCl, 10 mM 3-(N-morpholino) propanesulfonic acid, pH 7.2]. The increase in Cai upon activation was then determined by subtracting the baseline concentrations from the peak values. Previous studies using NHK have concentrated on the P2Y2 receptor alone (Dixon et al., 1999Dixon C.J. Bowler W.B. Littlewood-Evans A. Dillon J.P. Bilbe G. Sharpe G.R. Gallagher J.A. Regulation of epidermal homeostasis through P2Y2 receptors.Br J Pharmacol. 1999; 127: 1680-1686Crossref PubMed Scopus (97) Google Scholar), and a more complete P2Y receptor profile for this cell type has not previously been reported. Furthermore, expression of mRNA for P2Y receptors has not previously been investigated in the HaCaT cell line, which is a transformed cell line, routinely used to investigate keratinocyte responses. Therefore, RT-PCR techniques were utilized in order to determine whether these two cell types expressed mRNA for the P2Y1, P2Y2, P2Y4, and P2Y6 subtypes of P2Y receptors. PCR products of the correct molecular weights were amplified for P2Y1 (237 bp), P2Y2 (344 bp), P2Y4 (411 bp), and P2Y6 (463 bp) receptors in both NHK (Fig 1) and HaCaT cells (Fig 2), indicating that keratinocytes express mRNA for multiple P2Y receptor subtypes. Amplification of GAPDH (520 bp) was used, in both cell types, to confirm the integrity of the sample cDNA.Figure 2HaCaT cells express mRNA for P2Y1, P2Y2, P2Y4, and P2Y6 receptors. PCR products for P2Y1 (237 bp), P2Y2 (344 bp), P2Y4 (411 bp), and P2Y6 (463 bp) after 35 cycles at 58°C were obtained using cDNA from HaCaT cells, and cDNA integrity was confirmed by amplification of GAPDH (520 bp) after 35 cycles at 60°C.View Large Image Figure ViewerDownload (PPT) To identify whether P2Y receptor mRNA expression was altered during differentiation, NHK were cultured in the presence of elevated Ca2+ (200 μM and 1 mM). NHK were seeded onto 6 cm Petri dishes and allowed to adhere and settle in standard medium containing 70 μM Ca2+ for 36 h. cDNA, synthesized from these cells, acted as a 0 h control and PCR products for P2Y1, P2Y2, P2Y4, and P2Y6 receptors were successfully obtained after 35 cycles and for GAPDH after 30 cycles (Fig 3). NHK, cultured in medium containing 70 μM, 200 μM, or 1 mM Ca2+ for a further 24 h, again expressed mRNA for P2Y1, P2Y2, P2Y4, and P2Y6 receptors and GAPDH; however, there was a dose-dependent decrease in the expression of P2Y2 and P2Y4 receptors after 24 h incubation in 200 μM or 1 mM Ca2+ in comparison with the cells that had been cultured in 70 μM Ca2+ (Fig 3). In contrast, there was no apparent change in the expression of the P2Y1 or P2Y6 receptors in cells that had been cultured in elevated Ca2+versus 7" @default.
• W2000832714 created "2016-06-24" @default.
• W2000832714 creator A5036613562 @default.
• W2000832714 creator A5055628541 @default.
• W2000832714 creator A5072463585 @default.
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• W2000832714 date "2003-03-01" @default.
• W2000832714 modified "2023-10-16" @default.
• W2000832714 title "Human Keratinocytes Express Multiple P2Y-Receptors: Evidence for Functional P2Y1, P2Y2, and P2Y4 Receptors" @default.
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