REVIEW
`
`Purinergic Signaling in Healthy and Diseased Skin
`Geoffrey Burnstock1, Gillian E. Knight1 and Aina V.H. Greig2
`
`Adenosine 50-triphosphate and adenosine receptors
`have been identified in adult and fetal keratinocytes,
`fibroblasts, melanocytes, mast cells, Langerhans cells,
`and Meissner’s corpuscles, as well as in hair follicles,
`sweat glands, and smooth muscle and endothelial
`cells of skin vessels. Purinergic signaling is involved in
`skin pathology, including inflammation, wound heal-
`ing, pain, psoriasis, scleroderma, warts, and skin
`cancer.
`
`Journal of Investigative Dermatology (2012) 132, 526–546;
`doi:10.1038/jid.2011.344; published online 8 December 2011
`
`INTRODUCTION
`Extracellular purine nucleotides and nucleosides have
`biological effects in a variety of cell and tissue types. The
`majority of studies have been concerned with short-term
`events that occur in neurotransmission and secretion (Burn-
`stock, 2007a). Now,
`there is increasing evidence that
`purinergic signaling can have long-term, trophic effects on
`embryonic development, cell proliferation, differentiation,
`and apoptosis (Abbracchio and Burnstock, 1998; Burnstock,
`2002a, b; Burnstock and Verkhratsky, 2010).
`
`Purinergic receptors
`Purinergic receptors are classified into two groups: P1
`receptors (selective for adenosine) and P2 receptors (selective
`for adenosine 50-triphosphate (ATP), adenosine 50-diphos-
`phate (ADP), and uridine 50-triphosphate (UTP), which act
`as extracellular
`signaling molecules)
`(Burnstock, 1978).
`Purinergic receptors are expressed in most neural and
`nonneural cells (see Burnstock and Knight, 2004).
`
`1Autonomic Neuroscience Centre, Royal Free and University College Medical
`School, London, UK and 2Department of Plastic Surgery, St Thomas’ Hospital,
`London, UK
`
`Correspondence: Geoffrey Burnstock, Autonomic Neuroscience Centre,
`Royal Free and University College Medical School, Rowland Hill Street,
`London, NW3 2PF, UK. E-mail: g.burnstock@ucl.ac.uk
`Abbreviations: ADP, adenosine 50-diphosphate; AMP, adenosine
`monophosphate; ATP, adenosine 50-triphosphate; BzATP, 20,30-O(benzoyl-4-
`benzoyl-ATP; cAMP, cyclic adenosine monophosphate; CGRP, calcitonin
`gene-related peptide; DMBA, 7,12-dimethyl-benz(a)anthracene; DRG, dorsal
`root ganglion; EC50, half-maximal effective concentration; HPV, human
`papillomavirus; InsP3, inositol 1,4,5-triphosphate; a,b-meATP, a,b-
`methyleneATP; PPADS, pyridoxal-phosphate-6-azophenyl-20,40-disulfonic
`acid; SSc, systemic sclerosis; TPA, 12-O-tetradecanoylphorbol-13-acetate;
`TRPV, transient receptor potential V; UTP, uridine 50-triphosphate
`Received 6 January 2011; revised 3 June 2011; accepted 27 August 2011;
`published online 8 December 2011
`
`P1 receptors. Four members of the adenosine/P1 receptor
`family have now been cloned and characterized from a
`variety of species: A1, A2A, A2B, and A3, and selective
`agonists and antagonists have been identified (Table 1). All
`P1 receptors couple to G-proteins (Figure 1a), and modulate
`adenylate cyclase activity in an inhibitory (A1, A3) or
`stimulatory (A2A, A2B) fashion, resulting in cyclic adenosine
`monophosphate (cAMP) changes.
`
`P2 receptors. P2 receptors are divided into two families: P2X
`and P2Y (Figure 1b and c), based on molecular structure,
`transduction mechanisms, and pharmacological properties
`(Burnstock and Kennedy, 1985; Abbracchio and Burnstock,
`1994).
`P2X receptors are ligand-gated ion channels, and are
`activated by extracellular ATP to elicit a flow of cations
`þ
`þ
`, and Ca2 þ
`, K
`) across the plasma membrane. Seven
`(Na
`subtypes of P2X receptors are recognized (Khakh et al., 2001)
`(Table 2). All P2X receptors mediate fast signaling; however
`their localization, function, and pharmacological character-
`istics are different. Based on agonist efficacy and desensitiza-
`tion characteristics, P2X receptors have been grouped into
`three distinct classes. Group 1 includes P2X1 and P2X3
`receptors with a high affinity for ATP (half-maximal effective
`concentration (EC50) ¼ 1 mM) that are rapidly activated and
`desensitized. Group 2 includes P2X2, P2X4, P2X5, and P2X6
`receptors that have a lower affinity for ATP (EC50 ¼ 10 mM),
`and show slow desensitization and sustained depolarizing
`currents. Group 3 is represented by the P2X7 receptor that has
`very low affinity for ATP (EC50 ¼ 300–400 mM), shows little or
`no desensitization, and, in addition to functioning as an ATP-
`gated ion channel, can also function as a nonselective ion
`pore (Di Virgilio et al., 1999).
`Functional P2X channels are homomultimers or hetero-
`multimers formed by the association of at least three subunits.
`For a summary of possible P2X subunit combinations, see
`Burnstock and Kennedy (2011). It is not yet known how the
`properties of different P2X subunits influence the phenotype
`of
`the heteromultimeric receptors. Alternative splicing
`and species differences may increase heterogeneity of P2X
`receptors.
`The P2Y family of receptors is a subclass of the super-
`family of G-protein-coupled receptors, each having seven
`transmembrane domains. The third intracellular loop and the
`C-terminus are thought to be involved in G-protein coupling,
`whereas
`the third,
`sixth, and seventh transmembrane
`domains have been implicated in nucleotide binding (Burn-
`stock and Kennedy, 2011). The principal signal transduction
`pathway of P2Y receptors involves phospholipase C, which
`leads to the formation of inositol 1,4,5-triphosphate (InsP3)
`
`526
`
`Journal of Investigative Dermatology (2012), Volume 132
`
`& 2012 The Society for Investigative Dermatology
`
`L'OREAL USA, INC. EX. 1015
`
`

`

`G Burnstock et al.
`Purinergic Signaling in Healthy and Diseased Skin
`
`Table 1. Summary of properties of P1 receptor subtypes in the skin
`
`Receptor Site
`
`Agonist
`
`Antagonist
`
`Receptor properties
`
`A1
`
`A2A
`
`A2B
`
`Endothelial cells
`
`CCPA adenosine
`
`DPCPX, theophylline
`
`Endothelial cells; neutrophils;
`fibroblasts
`
`CGS21680 adenosine
`
`Theophylline
`
`Keratinocytes; vascular smooth
`muscle cells; human retinal
`endothelial cells
`
`Adenosine, NECA
`
`MRS1751, theophylline
`
`Reduces bradykinin and histamine-induced
`vascular leakage
`
`Endothelial cell proliferation; reduces
`bradykinin and histamine-induced vascular
`leakage; decreases oxygen free radical
`production of neutrophils; increases
`angiogenesis in wound healing; promotes
`dermal collagen production by fibroblasts
`
`Antiproliferative in keratinocytes and vascular
`smooth muscle cells; mediates VEGF expression
`in retinal endothelial cells; promotes wound
`healing
`
`A3
`Cl-IB-MECA
`MRE 3008F20
`Not established
`Abbreviations: CCPA, 2-chloro-N6-cyclopentyladenosine; DPCPX, 8-cyclopentyl-1,3-dipropylxanthine; NECA, 50-N-ethylcarboxamidoadenosine; VEGF,
`vascular endothelial growth factor.
`
`Agonist and antagonist
`recognition site
`
`S
`
`S
`
`S
`
`S
`
`V
`
`VI
`H
`
`VII
`H
`
`IV
`
`III
`
`II
`
`I
`
`NH2
`
`Extracellular
`
`Intracellular
`
`COOH
`
`NH2
`
`Extracellular
`surface
`
`S
`
`S
`
`Plasma
`membrane
`
`S–S
`
`S–S
`
`M1
`
`M2
`
`COOH
`
`Intracellular
`surface
`
`NH2
`
`COOH
`Figure 1. Membrane receptors for extracellular adenosine and adenosine 50-
`triphosphate (ATP). (a) The P1 family of receptors for extracellular adenosine
`are G-protein-coupled receptors (S–S; disulfide bond). (b) The P2X family of
`receptors are ligand-gated ion channels (S–S; disulfide bond; M1 and M2,
`transmembrane domains), and (c) the P2Y family are G-protein-coupled
`receptors (S–S; disulfide bond; green circles represent amino-acid residues
`that are conserved between P2Y1, P2Y2, and P2Y3 receptors; fawn circles
`represent residues that are not conserved; and red circles represent residues
`that are known to be functionally important in other G-protein-coupled
`receptors). Panel a is from Ralevic and Burnstock (1998); reproduced with
`permission from the American Society for Pharmacology and Experimental
`Therapeutics. Panel b is from Brake et al. (1994); reproduced with permission
`from Nature. Panel c is modified from Barnard et al. (1994); reproduced with
`permission from Elsevier.
`
`and mobilization of intracellular calcium. InsP3 regulates cell
`growth and DNA replication (Berridge, 1987). Eight subtypes
`of P2Y receptors have been described so far (Burnstock,
`2007b) (Table 3).
`
`Nucleotide ligands
`ATP acts as a cotransmitter in many nerves of both peripheral
`and central nervous systems. ATP is released together
`with noradrenaline and neuropeptide Y from sympathetic
`nerves. It is released as a cotransmitter with acetylcholine
`from parasympathetic nerves
`supplying
`the bladder,
`developing skeletal neuromuscular
`junctions and some
`neurons in the brain. It is released with nitric oxide and
`vasoactive intestinal polypeptide from nonadrenergic inhibi-
`tory enteric nerves; with glutamate from primary afferent
`sensory nerves and in different subpopulations of neurons in
`the brain; with dopamine, noradrenaline, acetylcholine,
`glutamate, g-aminobutyric acid, and 5-hydroxytryptamine.
`Cotransmission offers subtle, local variations in neurotrans-
`mission and neuromodulation mechanisms
`(Burnstock,
`2009a).
`Adenosine, AMP, ADP, and ATP can be released into the
`extracellular environment during inflammation, wounding,
`hypoxia, and other pathological states. There is a complex
`relationship between these nucleotides. Extracellular ATP has
`a very short half-life before it is degraded to adenosine (from
`milliseconds to seconds depending on the site of release and
`the level of activity of ectonucleotidases). AMP, ADP, and
`ATP can be converted to adenosine through spontaneous
`hydrolysis and/or through the activity of 50-ectonucleotidases,
`ecto-ADPases, or ecto-ATPases. Adenosine can be converted
`to inosine via the action of adenosine deaminase, or by
`uptake into cells through nucleoside transporters. When
`externalized, adenosine and its nucleotides can participate
`in physiological processes. The rapid breakdown of ATP to
`adenosine results in a multiplicity of different receptor sub-
`types being activated, which can mediate different physiolog-
`ical processes (e.g., proliferation, differentiation, migration,
`
`www.jidonline.org
`
`527
`
`

`

`G Burnstock et al.
`Purinergic Signaling in Healthy and Diseased Skin
`
`Table 2. Summary of properties of P2X receptor subtypes in the skin
`
`Receptor Site
`
`Smooth muscle; platelets
`
`Smooth muscle; autonomic and sensory
`ganglia
`
`Nociceptive sensory neurons (trigeminal,
`nodose, and dorsal root ganglia);
`endothelial and epithelial cells
`
`Agonist
`
`a,b-meATP
`
`Weak agonists: 2-
`MeSATP, b,g-meATP,
`ATPgS
`
`a,b-MeATP
`
`Antagonist
`
`Receptor properties
`
`TNP-ATP, isoPPADS
`
`Rapidly desensitizing
`
`isoPPADS, Reactive Blue 2 Significant permeability to Ca2+; sensitive to
`extracellular acidification
`
`TNP-ATP, isoPPADS
`
`Rapidly desensitizing
`
`P2X1
`
`P2X2
`
`P2X3
`
`P2X4
`
`P2X5
`
`P2X6
`
`P2X7
`
`CNS; testis; colon
`
`Keratinocytes; growing hair follicles
`
`ATP
`
`ATPgS
`
`TNP-ATP
`
`Slowly desensitizing
`
`PPADS, suramin
`(nonselective)
`
`Proliferating and differentiating cells in
`keratinized and nonkeratinized epithelia
`
`CNS; motor neurons in spinal cord
`
`2-MeSATP
`
`None
`
`Minimal desensitizing
`
`Apoptotic cells—e.g., keratinized
`Bifunctional: either acts as cation channel or
`epithelium, macrophages, monocytes,
`forms a large pore and allows calcium entry
`lymphocytes, granulocytes
`and cell death
`Abbreviations: ADP, adenosine 50-diphosphate; ATP, adenosine 50-triphosphate; BzATP, 20,30-O-(4-benzoyl-benzoyl)-ATP; CNS, central nervous system;
`a,b-meATP, a,b-methyleneATP; isoPPADS, pyridoxal-phosphate-6-azophenyl-20,50-disulfonic acid; 2-MeSADP, 2-methylthio ADP; 2-MeSATP, 2-methylthio
`ATP; PPADS, pyridoxal-phosphate-6-azophenyl-20,40-disulfonic acid; TNP-ATP, 20,30-O-(2,4,6-trinitrophenyl)-ATP.
`
`BzATP
`
`KN-62, Coomassie Brilliant
`Blue G
`
`Table 3. Summary of properties of P2Y receptor subtypes in the skin
`
`Receptor Site
`
`Agonist
`
`Antagonist
`
`Receptor properties
`
`P2Y1
`
`P2Y2
`
`P2Y4
`
`P2Y6
`
`P2Y11
`
`P2Y12
`
`P2Y13
`
`Epithelial and endothelial cells;
`platelets; immune cells
`
`Epithelial cells; endothelial vascular
`smooth muscle cells; immune cells
`
`ADP, ATP, 2-MeSADP
`
`MRS2179
`
`UTPgS, UTP, ATP
`
`PPADS, suramin
`
`Platelet aggregation; vascular relaxation;
`endothelial cell proliferation
`
`
`secretion, vascular relaxation;
`Epithelial Cl
`keratinocyte proliferation
`
`Endothelial cells
`
`UTP, ATP
`
`PPADS
`
`Not established
`
`Some epithelial cells; T cells
`
`UDP, UTP
`
`PPADS, suramin
`
`Not established
`
`Granulocytes
`
`Platelets
`
`Lymph nodes
`
`ATP
`
`ADP
`
`Suramin
`
`Suramin
`
`Not established
`
`Platelet aggregation
`
`ADP, 2-MeSADP
`
`MRS2211, 2-MeSAMP
`
`Stimulation of MAPK; inhibition of adenylate
`cyclase
`
`P2Y14
`
`Adipose tissue
`
`Chemoattractant and neuroimmune
`functions
`Abbreviations: ADP, adenosine 50-diphosphate; ATP, adenosine 50-triphosphate; MAPK, mitogen-activated protein kinase; 2-MeSADP, 2-methylthio ADP;
`2-MeSAMP, 2-methylthio AMP; PPADS, pyridoxal-phosphate-6-azophenyl-20,40-disulfonic acid; UTP, uridine 50-triphosphate.
`
`UDP glucose, UDP galactose Not known
`
`and cell death), and thus purines may act as important local
`messengers in the skin.
`This review article aims to give an overview of purinergic
`signaling in cells in the skin, skin appendages, and then in
`pathological processes affecting the skin.
`
`KERATINOCYTES
`P1 receptors
`P1 receptors were first hypothesized to be present in the
`epidermis in the 1970s. Adenosine, AMP, ADP, and ATP
`activated adenylate cyclase in pig epidermis, resulting in
`accumulation of cAMP. Theophylline, an adenosine receptor
`antagonist, blocked the response to adenosine as well as to
`the nucleotides (Iizuka et al., 1976), and hence it was thought
`
`likely that responses to nucleotides were mediated by P1
`receptors after enzymatic breakdown to adenosine.
`Human keratinocytes express A2B receptor mRNA, but not
`significant levels of A1, A2A, or A3 receptor mRNA. A2A
`receptor stimulation promotes both proliferation and apo-
`ptosis, whereas A3 stimulation arrests proliferation and
`apoptosis (Merighi et al., 2002). Both primary cultures of
`mouse keratinocytes and the murine keratinocyte cell line,
`MSC-P5, express A2A, A2B, and A3 receptors, with A2B
`receptors having the strongest expression. Stimulation of A2B
`receptors resulted in enhanced growth (Braun et al., 2006).
`Other studies have reported the antiproliferative effects of
`adenosine nucleotides for both human and porcine kerati-
`nocyte explants (see, e.g., Flaxman and Harper, 1975).
`
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`Journal of Investigative Dermatology (2012), Volume 132
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`

`G Burnstock et al.
`Purinergic Signaling in Healthy and Diseased Skin
`
`Adenosine, ATP, and ADP were also shown to be anti-
`proliferative for normal human epidermal keratinocytes
`cultured in the absence or presence of exogenous epidermal
`growth factor (Cook et al., 1995), probably via A2B receptors
`(Brown et al., 2000). In this study, ATP had a more potent
`effect than adenosine. The antiproliferative effect of adeno-
`sine was not blocked by the A1/A2 receptor antagonist,
`8-(p-sulfophenyl)theophylline, confirming the findings in a
`previous study that used theophylline (Brown et al., 2000).
`The adenosine transport blocking agent, dipyridamole, had
`an inhibitory effect of its own and did not potentiate the effect
`of adenosine. This suggested that extracellular adenosine
`receptors were not being activated in this system. The effects
`of adenosine might be mediated via an intracellular site of
`action (Brown et al., 2000). 8-Chloro-adenosine, presumably
`acting via P1 receptors,
`induced ‘‘growth arrest without
`differentiation of primary mouse epidermal keratinocytes’’
`(Dransfield et al., 2001).
`In a study of the effect of various purinoceptor agonists on
`primary human keratinocyte cultures, the response to ATP
`was not blocked by 8-(p-sulfophenyl)theophylline, which
`meant that the effect of ATP was not due to breakdown of
`ATP to adenosine and activation of extracellular adenosine
`receptors
`(Greig et al., 2003a). Dipyridamole did not
`potentiate the ATP response, and hence blocking adenosine
`transport into the cell, and thereby increasing local extra-
`cellular adenosine concentrations, did not have any effect.
`This
`suggests
`that
`the main trophic agent
`in human
`keratinocyte cultures is ATP, working via P2 receptors,
`whereas adenosine has a minor effect, which is largely
`obscured by the ATP response.
`
`P2 receptors
`The presence of P2 purinergic receptors was suggested by
`a study on canine keratinocytes
`(Suter et al., 1991).
`Extracellular ATP stimulates P2 receptors to transiently
`increase intracellular calcium levels. An increase in intracel-
`lular calcium in cultured human keratinocytes is an early
`event in terminal differentiation (Sharpe et al., 1989), which
`is of primary importance during this process.
`
`Proliferation. The first clue to a role for ATP in proliferation
`came from the finding that ATP stimulates phosphoinositide,
`which mobilizes [Ca2 þ
`]i, stimulates thymidine incorpora-
`tion,
`thus inhibiting differentiation in cultures of human
`epidermal keratinocytes (Pillai and Bikle, 1992). ‘‘In whole-
`cell recordings from HaCaT cells,’’ an immortalized human
`keratinocyte cell
`line, ATP ‘‘caused a bipolar change in
`membrane potential,
`transient depolarisation followed by
`long-lasting hyperpolarisation’’
`(Koegel and Alzheimer,
`2001). ‘‘Extracellular ATP stimulates HaCaT cell proliferation
`via purinoceptor-mediated [Ca2 þ
`]i mobilization’’ (Lee et al.,
`2001).
`Reverse transcription-PCR and in situ hybridization studies
`identified P2Y2 receptors on the basal layer of the epidermis,
`which is the site of cell proliferation, and also identified P2Y2
`receptors in primary cultured human keratinocytes. ATP and
`UTP were equally effective in stimulating proliferation,
`
`consistent with the pharmacological profile of P2Y2 receptors
`(Dixon et al., 1999). This study also showed that cultured
`human keratinocytes released ATP under static conditions.
`Double labeling of human skin with proliferation markers
`Ki-67, proliferating cell nuclear antigen, and P2Y1 and P2Y2
`receptors, identified a proliferating subpopulation of basal
`and parabasal keratinocytes. Cells positive for these two
`markers were also positive for P2Y1 and P2Y2 receptors
`(Greig et al., 2003a) (Figure 2). Low concentrations of ATP,
`UTP, and 2-methylthio ADP caused an increase in keratino-
`cyte cell number in primary human keratinocyte cultures,
`also suggesting a role for P2Y1 and P2Y2 receptors in
`keratinocyte proliferation (Greig et al., 2003a). Further
`evidence has demonstrated functional P2Y1, P2Y2, and
`P2Y4 receptors in human keratinocytes, which are involved
`in the regulation of cell proliferation (Burrell et al., 2003).
`Costimulation of HaCaT cells by ATP acting on P2Y2
`receptors and parathyroid hormone–related protein increased
`proliferation (Burrell et al., 2008). Confocal optical sectioning
`through mulilayered HaCaT cultures
`showed that
`the
`responsiveness to ATP differs dramatically between prolifer-
`ating cells and cells undergoing partial differentiation
`(Burgstahler et al., 2003). It was claimed that UTP caused
`IL-6 production in HaCaT keratinocytes via P2Y2 and/or P2Y4
`receptors (Kobayashi et al., 2006; Yoshida et al., 2006). This
`finding might be important as studies have shown that IL-6
`has a physiological role in the repair processes of wounds
`(Gallucci et al., 2000). Calcium-permeable transient receptor
`potential canonical 7 is a purinoceptor-operated 1,2-diacyl-
`gycerol-activated channel in HaCaT cells (Beck et al., 2006),
`thought to be an additional mediator of calcium influx into
`keratinocytes, which in turn can lead to differentiation.
`‘‘Calcium-independent phospholipase A is required for Ca2 þ
`entry into HaCaT keratinocytes following ATP or UTP
`stimulation’’ (Ross et al., 2007, 2008), and Ca2 þ
`movement
`is involved in the control of numerous cellular process.
`‘‘Retinoids, vitamin A derivatives, are important regulators
`of growth and differentiation of skin cells’’ and have been
`used therapeutically for aging skin. An agonist for the retinoic
`receptor was shown to enhance expression of mRNA for P2Y2
`receptors in basal keratinocytes involved in proliferation of
`the epidermis (Fujishita et al., 2006).
`Differentiation. Changes in intracellular Ca2 þ
`were found to
`regulate differentiation of keratinocytes in the granular layer
`of the epidermis (Menon et al., 1985), which is an interesting
`observation in view of the later discovery of high expression
`the epidermis,
`the
`of P2X5 receptors in this layer of
`purinoceptor subtype that is known to mediate cell differ-
`entiation (Gro¨ schel-Stewart et al., 1999; Greig et al., 2003a).
`Double labeling of P2X5 receptors with cytokeratin K10 or
`involucrin showed that P2X5 receptors were expressed
`in differentiating keratinocytes within the epidermis (Greig
`et al., 2003a). The increase in [Ca2 þ
`in response to
`]i
`ATP varied in each layer of the epidermis and was greater
`in the basal than in the outer layers (Tsutsumi et al., 2009a).
`Both extracellular ATP and UTP ‘‘induce transient rises in
`cytosolic free Ca2 þ
`’’ in both human and canine keratinocytes
`
`www.jidonline.org
`
`529
`
`

`

`G Burnstock et al.
`Purinergic Signaling in Healthy and Diseased Skin
`
`(Sharpe et al., 1989; Suter et al., 1991), suggesting in
`retrospect that P2Y2 and/or P2Y4 receptors were involved.
`Multiple P2X receptor subtypes were identified later in
`cultured human epidermal keratinocytes using whole-cell
`patch clamp techniques,
`reverse transcription-PCR, and
`intracellular Ca2 þ
`measurements (Inoue et al., 2005). P2X2,
`P2X3, P2X5, and P2X7 receptor mRNA was increased in
`differentiated cells, whereas P2Y2 receptor mRNA was
`downregulated in differentiated cells.
`Protein kinase C-a has been shown to decrease cell
`proliferation and augment cell differentiation of human
`keratinocytes (Hegemann et al., 1994). The expression of
`P2X7 receptors was significantly increased in keratinocytes
`overexpressing protein kinase C-a (Go¨ nczi et al., 2008).
`
`Terminal differentiation/apoptosis. An immunohistochem-
`ical study of rat stratified squamous epithelium showed that
`P2X7 receptors were clearly associated with the keratiniza-
`tion process in the outer layer (Gro¨ schel-Stewart et al., 1999).
`This was confirmed in human epidermis with colocalization
`with markers for keratinocyte apoptosis: P2X7 receptors
`control death of keratinocytes in the outer stratum corneum
`(Greig et al., 2003a). The P2X7 receptor is unlike other P2X
`receptors because it is a bifunctional molecule that can be
`
`triggered to act as a channel permeable to small cations, or
`on prolonged stimulation can form a cytolytic pore perme-
`able to large hydrophilic molecules up to 900 Da (Surprenant
`et al., 1996). The opening of this pore results in the increase
`in intracellular cytosolic free calcium ions and the induction
`of cell death (Zheng et al., 1991; Ferrari et al., 1996). There is
`evidence that
`this process is dependent on the caspase
`signaling cascade (Coutinho-Silva et al., 1999; Ferrari et al.,
`1999). Double labeling of P2X7 receptors with anti-caspase-3
`showed colocalization within the stratum corneum (Greig
`et al., 2003a) (Figure 2f). High concentrations of ATP and
`20,30-O(benzoyl-4-benzoyl-ATP)
`(BzATP, a P2X7 receptor
`agonist) caused a significant decrease in keratinocyte cell
`number in primary human keratinocyte cultures, providing
`evidence for a functional role for P2X7 receptors (Greig et al.,
`2003a).
`
`Fetal keratinocytes
`The expression of P2X5, P2X7, P2Y1, and P2Y2 receptors in
`8–11-week-old human fetal epidermis was investigated using
`immunohistochemistry (Greig et al., 2003b). P2 purinergic
`receptors are likely to be involved in fetal keratinocyte
`proliferation via P2Y1 receptors found on basal cells and fetal
`keratinocyte differentiation via activation of P2X5 receptors.
`
`Figure 2. Double labeling of P2Y1 and P2Y2 receptors with markers of
`proliferation show colocalization within a subpopulation of basal and parabasal
`keratinocytes. Double labeling of P2X5 receptors with markers of differentiated
`keratinocytes show colocalization within the stratum spinosum, and double
`labeling of P2X7 receptors with markers of apoptosis in human leg skin show
`colocalization within the stratum corneum. (a) Ki-67 immunolabeling (a marker
`for proliferation) stained the nuclei (green) of a subpopulation of keratinocytes in
`the basal and parabasal layers of the epidermis. P2Y1 receptor immunostaining
`(red) was found in the basal layer on cells also staining for Ki67. Scale
`bar¼ 30 mm. (b) Proliferating cell nuclear antigen (PCNA) immunolabeling
`(a marker for proliferation) stained the nuclei (green) of a subpopulation of
`keratinocytes. These nuclei were often distributed in clusters and found in the basal
`and parabasal layers of the epidermis. P2Y2 receptor immunostaining (red) was
`also expressed in basal and parabasal epidermal cells. Scale bar¼ 30 mm. (c) P2X5
`receptor immunostaining (red) showed overlap (yellow) with cytokeratin K10
`(green), an early marker of keratinocyte differentiation. P2X5 receptors were
`present in the basal layer of the epidermis up to the mid-granular layer. Cytokeratin
`K10 was distributed in most suprabasal keratinocytes. The stratum basale stained
`only for P2X5 receptors, indicating that no differentiation was taking place in these
`cells. The colocalization of P2X5 receptors and cytokeratin K10 appeared mainly
`in the cytoplasm of differentiating cells within the stratum spinosum and partly in
`the stratum granulosum. Note that the stratum corneum also stained for cytokeratin
`K10, which labeled differentiated keratinocytes, even in dying cells. Scale
`bar¼ 30 mm. (d) P2X5 receptor immunostaining (red) showed overlap (yellow)
`with involucrin (green). P2X5 receptors were present in the basal layer of the
`epidermis up to the midgranular layer. Note that the pattern of staining with
`involucrin was similar to that seen with cytokeratin K10, except that cells from the
`stratum basale up to the midstratum spinosum were not labeled with involucrin,
`which is a late marker of keratinocyte differentiation. Scale bar¼ 30 mm.
`(e) TUNEL (green) labeled the nuclei of cells at the uppermost level of the stratum
`granulosum and P2X7 antibody (red) mainly stained cell fragments within the
`stratum corneum. Scale bar¼ 15 mm. (f) Anti-caspase-3 (green) colocalized with
`areas of P2X7 receptor immunostaining (red) both at the junction of the stratum
`granulosum and within the stratum corneum. Areas of colocalization are yellow.
`Note that the differentiating keratinocytes in the upper stratum granulosum were
`also positive for anti-caspase-3. Bar¼ 15 mm (reproduced from Greig et al., 2003a,
`with permission of the Nature Publishing Group).
`
`530
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`Journal of Investigative Dermatology (2012), Volume 132
`
`

`

`G Burnstock et al.
`Purinergic Signaling in Healthy and Diseased Skin
`
`Double labeling of P2X7 receptors with anti-caspase-3
`showed colocalization within the periderm. The periderm is
`known to exhibit characteristics consistent with apoptosis
`(Kerr et al., 1972) before its loss from the epidermis
`(Polakowska et al., 1994), which occurs at midgestation
`(20–24 weeks). P2X7 receptors are likely to be part of this
`apoptotic process and the expression of P2X7 receptors is
`important in the regulation of apoptosis intrinsic to correct
`development. P2Y2 receptors were also found only in
`periderm cells and there was no colocalization with
`proliferation markers proliferating cell nuclear antigen and
`Ki67 in the periderm cells. The role of P2Y2 receptors is
`currently unknown (Greig et al., 2003b).
`
`Trophic effects in response to mechanical stimulation and heat
`Mechanical stimulation of human epidermal keratinocytes
`causes release of ATP, which then activates P2Y2 receptors to
`induce Ca2 þ
`waves (Koizumi et al., 2004; Tsutsumi et al.,
`2009b; Azorin et al., 2011) (Figure 3). When keratinocytes
`are cocultured with dorsal root ganglion (DRG) neurons, an
`increase in [Ca2 þ
`]i occurs in adjacent neurons, suggesting
`ATP-mediated crosstalk between keratinocytes and sensory
`nerves. The epidermis acts as a physical barrier between the
`organism and its environment. It is innervated by unmyeli-
`nated sensory nerve fibers conveying nociceptive and
`thermoceptive information. ‘‘Trophic effects of keratinocytes
`(perhaps releasing ATP) on axonal development of sensory
`
`A
`
`–2 Seconds
`
`2 Seconds
`
`10 Seconds
`
`20 Seconds
`
`ATP 100 µM
`
`B
`
`–2 Seconds
`
`2 Seconds
`
`UTP 100 µM
`
`10 Seconds
`
`20 Seconds
`
`(nM)
`600
`
`0
`
`100 µm
`
`UTP
`
`100
`
`50
`
`0
`
`response
`
`B
`
`Normalized Ca2+
`
`ATP
`
`100
`
`50
`
`0
`
`response
`
`A
`
`Normalized Ca2+
`
`–7
`
`–6
`
`–4
`
`–3
`
`–7
`
`–6
`
`–4
`–5
`–5
`Log [UTP], M
`Log [ATP], M
`Figure 3. Increases in [Ca2 þ
`]i evoked by both applied adenosine 50-triphosphate (ATP) and uridine 50-triphosphate (UTP) in normal human epidermal
`keratinocytes (NHEKs). (a) Sequential pseudo-color images of Ca2 þ
`responses to 100 mM ATP (A) and UTP (B) in NHEKs. Images were obtained from a
`confocal laser microscope, showing self-ratios of fluo-4 fluorescence. Images were recorded 2 seconds before (2 seconds) and 2, 10, and 20 seconds after
`ATP or UTP application. (b) Concentration–response curves for (A) ATP- and (B) UTP-evoked increases in [Ca2 þ
`]i in NHEKs. Increases in [Ca2 þ
`]i in NHEKs
`were monitored by ratiometric fura 2 fluorescence (DF340/F360) and were then converted into absolute value of [Ca2 þ
`]i using a standard calibration curve.
`The maximum [Ca2 þ
`]i increase was observed when cells were stimulated with 300 mM ATP (A) or UTP (B). The increase in [Ca2 þ
`]i at each ATP or UTP
`concentration was normalized by the maximum increase in [Ca2 þ
`]i. Results are the means±SEM for 28–73 cells tested. Both the ATP- and UTP-evoked
`concentration–response curves were almost identical with the half-maximal effective dose (ED50) values of 21 and 20 mM, respectively (reproduced from Koizumi
`et al., 2004, with permission of Portland Press Limited).
`
`–3
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`www.jidonline.org
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`

`G Burnstock et al.
`Purinergic Signaling in Healthy and Diseased Skin
`
`neurons have been described in a coculture model’’ (Ulmann
`et al., 2007).
`is
`information detected by the skin layers
`Thermal
`converted into electrical signals and transmitted to the central
`nervous system. Transient receptor potential V (TRPV) 3 and
`TRPV4 are heat-activated cation channels expressed by
`keratinocytes. Evidence has been presented that ATP is
`‘‘released from keratinocytes upon heating’’ and that ‘‘ATP is
`a messenger molecule for TRPVR3-mediated thermotrans-
`duction in skin to sensory nerve activation’’ (Mandadi et al.,
`2009).
`
`into Langerhans cells in wild-type mice and this was used as a
`measure of P2X7
`function. BzATP did not
`stimulate
`þ
`uptake into Langerhans cells in P2X7-deficient
`ethidium
`mice (Tran et al., 2010). ATP has a role in the involvement
`of Langerhans cells in adaptive immunity (Holzer and
`Granstein, 2004). ATPgS has been found to enhance the
`antigen-presenting ability of mouse Langerhans cells, and a
`Langerhans cell line (XS106) was shown to express mRNA
`for P2X1, P2X7, P2Y1, P2Y2, P2Y4, and P2Y11 receptors
`(Granstein et al., 2005), although it was not clear which
`receptor subtype(s) was responsible for this effect.
`
`Chronic venous insufficiency
`In chronic venous insufficiency, there is a thinning of the
`epidermis and changes in purinoceptor subtype expression
`have been found. P2Y1 and P2Y2 receptor expression was
`found to increase in the basal and spinosal layers, whereas
`P2X5 receptor expression increased mainly in the spinosal
`layer and extending further into the stratum granulosum. In
`contrast, P2X7 receptor expression was shown to be reduced
`in the stratum corneum (Metcalfe et al., 2006). These
`receptors are possible targets for manipulating skin to avoid
`ulcers, one of the consequences of venous insufficiency.
`
`DERMAL FIBROBLASTS
`It was shown that upon exposure to ATP, dermal fibroblasts
`contract (Ehrlich et al., 1986). Subsequently, dermal fibro-
`blasts have been shown to express P2X7 and a P2Y-like
`receptor (Solini et al., 1999). Adenosine, probably acting via
`A2 receptors, increased proliferation of human skin fibro-
`blasts in primary cultures (Thellung et al., 1999). Adenosine
`has been used as a topical application to reduce wrinkles
`(Legendre et al., 2007), perhaps by promoting collagen
`production by dermal
`fibroblasts via the adenosine A2A
`receptor (Chan et al., 2006).
`
`ENDOTHELIAL CELLS
`There is evidence for P2Y1, P2Y2, and P2Y4 receptor subtypes
`on vascular endothelial cells, which are involved in mediat-
`ing the release of nitric oxide, and causing subsequent
`vasodilatation (Burnstock, 2002a). Endothelial cells release
`ATP (Pearson and Gordon, 1979; Bodin et al., 1991), and it
`has been suggested that ATP released from vascular
`endothelial cells causes an autocrine mitogenic stimulation
`of endothelial cells (Burnstock, 2002a). Adenosine can also
`stimulate vascular endothelial cell proliferation (Van Daele
`et al., 1992) and DNA synthesis (Ethier and Dobson, 1997).
`Adenosine A2A rece