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Human Keratinocytes Respond to Extracellular UTP by Induction of Hyaluronan Synthase 2 Expression and Increased Hyaluronan Synthesis*

  • Tiina Jokela
    Affiliations
    Institute of Biomedicine, University of Eastern Finland, 70211 Kuopio, Finland
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  • Riikka Kärnä
    Affiliations
    Institute of Biomedicine, University of Eastern Finland, 70211 Kuopio, Finland
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  • Leena Rauhala
    Affiliations
    Institute of Biomedicine, University of Eastern Finland, 70211 Kuopio, Finland
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  • Genevieve Bart
    Affiliations
    Institute of Biomedicine, University of Eastern Finland, 70211 Kuopio, Finland
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  • Sanna Pasonen-Seppänen
    Affiliations
    Institute of Biomedicine, University of Eastern Finland, 70211 Kuopio, Finland
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  • Sanna Oikari
    Affiliations
    Institute of Biomedicine, University of Eastern Finland, 70211 Kuopio, Finland
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  • Markku I. Tammi
    Correspondence
    To whom correspondence should be addressed.
    Affiliations
    Institute of Biomedicine, University of Eastern Finland, 70211 Kuopio, Finland
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  • Raija H. Tammi
    Affiliations
    Institute of Biomedicine, University of Eastern Finland, 70211 Kuopio, Finland
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  • Author Footnotes
    * This work was supported by grants from the Sigrid Juselius Foundation (to R. H. T., M. I. T., and S. P. S.), the Special Government Funding of Kuopio University Hospital (to M. I. T.), The Spearhead Funds of the University of Eastern Finland/Cancer Center of Eastern Finland (to M. I. T. and R. H. T.), and the Cancer Foundation of Northern Savo (to L. R.). The authors declare that they have no conflicts of interest with the contents of this article.
    2 The abbreviations used are: P2YG protein-coupled purinergic receptorqRTquantitative RTHAShyaluronan synthaseHYALhyaluronidaseUDP-GlcNAcUDP-N-acetylglucosamineUDP-GlcUAUDP-glucuronic acidUDP-GlcUDP-glucosePTXpertussis toxinCREBcAMP response element-binding proteinCBPCREB-binding proteinCaMKIICa2+/calmodulin-dependent protein kinase IIp38p38 mitogen-activated protein kinaseHaCaTspontaneously transformed aneuploid immortal keratinocyte cell line from human skinREKrat epidermal keratinocytesERendoplasmic reticulumBIMbisindolylmaleimideEGFRepidermal growth factor receptorELSAenzyme-linked sorbent assayDABdiaminobenzidineANOVAanalysis of variance.
Open AccessPublished:February 10, 2017DOI:https://doi.org/10.1074/jbc.M116.760322
      The release of nucleotides into extracellular space is triggered by insults like wounding and ultraviolet radiation, resulting in stimulatory or inhibitory signals via plasma membrane nucleotide receptors. As similar insults are known to activate hyaluronan synthesis we explored the possibility that extracellular UTP or its breakdown products UDP and UMP act as mediators for hyaluronan synthase (HAS) activation in human epidermal keratinocytes. UTP increased hyaluronan both in the pericellular matrix and in the culture medium of HaCaT cells. 10–100 μm UTP strongly up-regulated HAS2 expression, although the other hyaluronan synthases (HAS1, HAS3) and hyaluronidases (HYAL1, HYAL2) were not affected. The HAS2 response was rapid and transient, with the maximum stimulation at 1.5 h. UDP exerted a similar effect, but higher concentrations were required for the response, and UMP showed no stimulation at all. Specific siRNAs against the UTP receptor P2Y2, and inhibitors of UDP receptors P2Y6 and P2Y14, indicated that the response to UTP was mediated mainly through P2Y2 and to a lesser extent via UDP receptors. UTP increased the phosphorylation of p38, ERK, CREB, and Ser-727 of STAT3 and induced nuclear translocation of pCaMKII. Inhibitors of PKC, p38, ERK, CaMKII, STAT3, and CREB partially blocked the activation of HAS2 expression, confirming the involvement of these pathways in the UTP-induced HAS2 response. The present data reveal a selective up-regulation of HAS2 expression by extracellular UTP, which is likely to contribute to the previously reported rapid activation of hyaluronan metabolism in response to tissue trauma or ultraviolet radiation.

      Introduction

      Cells subjected to mechanical pressure, osmotic shock, bacterial infection, chemical irritation, UV radiation, and tissue wounding rapidly release nucleotides like UTP, UDP, ATP, and ADP to the extracellular space. The nucleotide release acts as an alarm signal initiating cellular defense mechanisms (
      • Inoue Inoue
      • Hosoi Hosoi
      • Denda Denda
      Extracellular ATP has stimulatory effects on the expression and release of IL-6 via purinergic receptors in normal human epidermal keratinocytes.
      ,
      • Pastore Pastore
      • Mascia Mascia
      • Gulinelli Gulinelli
      • Forchap Forchap
      • Dattilo Dattilo
      • Adinolfi Adinolfi
      • Girolomoni Girolomoni
      • Di Virgilio Di Virgilio
      • Ferrari Ferrari
      Stimulation of purinergic receptors modulates chemokine expression in human keratinocytes.
      ,
      • Holzer Holzer
      • Granstein Granstein
      Role of extracellular adenosine triphosphate in human skin.
      ,
      • Ho Ho
      • Yang Yang
      • Lin Lin
      • Lin Lin
      Ecto-nucleoside triphosphate diphosphohydrolase 2 modulates local ATP-induced calcium signaling in human HaCaT keratinocytes.
      ,
      • Azorin Azorin
      • Raoux Raoux
      • Rodat-Despoix Rodat-Despoix
      • Merrot Merrot
      • Delmas Delmas
      • Crest Crest
      ATP signalling is crucial for the response of human keratinocytes to mechanical stimulation by hypo-osmotic shock.
      ,
      • Tsutsumi Tsutsumi
      • Inoue Inoue
      • Denda Denda
      • Ikeyama Ikeyama
      • Goto Goto
      • Denda Denda
      Mechanical-stimulation-evoked calcium waves in proliferating and differentiated human keratinocytes.
      ,
      • Yoshida Yoshida
      • Kobayashi Kobayashi
      • Ohkubo Ohkubo
      • Nakahata Nakahata
      ATP stimulates interleukin-6 production via P2Y receptors in human HaCaT keratinocytes.
      ,
      • Boarder Boarder
      • Hourani Hourani
      The regulation of vascular function by P2 receptors: multiple sites and multiple receptors.
      • Takai Takai
      • Tsukimoto Tsukimoto
      • Harada Harada
      • Kojima Kojima
      Involvement of P2Y6 receptor in p38 MAPK-mediated COX-2 expression in response to UVB irradiation of human keratinocytes.
      ). This is particularly important in the epidermis, which forms the protective outer barrier of the body (
      • Inoue Inoue
      • Hosoi Hosoi
      • Denda Denda
      Extracellular ATP has stimulatory effects on the expression and release of IL-6 via purinergic receptors in normal human epidermal keratinocytes.
      ,
      • Ho Ho
      • Yang Yang
      • Lin Lin
      • Lin Lin
      Ecto-nucleoside triphosphate diphosphohydrolase 2 modulates local ATP-induced calcium signaling in human HaCaT keratinocytes.
      ). Extracellular nucleotides have also been recognized as important mediators of other physiological signals such as pain sensation (
      • Pastore Pastore
      • Mascia Mascia
      • Gulinelli Gulinelli
      • Forchap Forchap
      • Dattilo Dattilo
      • Adinolfi Adinolfi
      • Girolomoni Girolomoni
      • Di Virgilio Di Virgilio
      • Ferrari Ferrari
      Stimulation of purinergic receptors modulates chemokine expression in human keratinocytes.
      ,
      • Mandadi Mandadi
      • Sokabe Sokabe
      • Shibasaki Shibasaki
      • Katanosaka Katanosaka
      • Mizuno Mizuno
      • Moqrich Moqrich
      • Patapoutian Patapoutian
      • Fukumi-Tominaga Fukumi-Tominaga
      • Mizumura Mizumura
      • Tominaga Tominaga
      TRPV3 in keratinocytes transmits temperature information to sensory neurons via ATP.
      ), keratinocyte proliferation, migration, and apoptosis, and activation of inflammatory cells (
      • Burrell Burrell
      • Bowler Bowler
      • Gallagher Gallagher
      • Sharpe Sharpe
      Human keratinocytes express multiple P2Y-receptors: evidence for functional P2Y1, P2Y2, and P2Y4 receptors.
      ,
      • Greig Greig
      • Linge Linge
      • Cambrey Cambrey
      • Burnstock Burnstock
      Purinergic receptors are part of a signaling system for keratinocyte proliferation, differentiation, and apoptosis in human fetal epidermis.
      • Burnstock Burnstock
      • Verkhratsky Verkhratsky
      Long-term (trophic) purinergic signalling: purinoceptors control cell proliferation, differentiation and death.
      ). Besides via cellular lysis, nucleotides can also be released by exocytosis, or by diffusion through different plasma membrane channels (
      • Filippini Filippini
      • Taffs Taffs
      • Sitkovsky Sitkovsky
      Extracellular ATP in T-lymphocyte activation: possible role in effector functions.
      ,
      • Jiang Jiang
      • Mak Mak
      • Devidas Devidas
      • Schwiebert Schwiebert
      • Bragin Bragin
      • Zhang Zhang
      • Skach Skach
      • Guggino Guggino
      • Foskett Foskett
      • Engelhardt Engelhardt
      Cystic fibrosis transmembrane conductance regulator-associated ATP release is controlled by a chloride sensor.
      ,
      • Dixon Dixon
      • Bowler Bowler
      • Littlewood-Evans Littlewood-Evans
      • Dillon Dillon
      • Bilbe Bilbe
      • Sharpe Sharpe
      • Gallagher Gallagher
      Regulation of epidermal homeostasis through P2Y2 receptors.
      • Burrell Burrell
      • Wlodarski Wlodarski
      • Foster Foster
      • Buckley Buckley
      • Sharpe Sharpe
      • Quayle Quayle
      • Simpson Simpson
      • Gallagher Gallagher
      Human keratinocytes release ATP and utilize three mechanisms for nucleotide interconversion at the cell surface.
      ). In the extracellular milieu UTP and ATP are rapidly degraded to corresponding diphosphates and monophosphates (
      • Ho Ho
      • Yang Yang
      • Lin Lin
      • Lin Lin
      Ecto-nucleoside triphosphate diphosphohydrolase 2 modulates local ATP-induced calcium signaling in human HaCaT keratinocytes.
      ). As nucleotides are released at relatively high concentrations and metabolized rapidly, they are ideal signaling molecules in tissues (
      • Pellegatti Pellegatti
      • Falzoni Falzoni
      • Pinton Pinton
      • Rizzuto Rizzuto
      • Di Virgilio Di Virgilio
      A novel recombinant plasma membrane-targeted luciferase reveals a new pathway for ATP secretion.
      ).
      Extracellular nucleotides signal via specific ligand-gated or G protein-coupled receptor families (P2X and P2Y, respectively). These receptors are expressed in keratinocytes in a differentiation-dependent manner, members of the P2Y
      The abbreviations used are: P2Y
      G protein-coupled purinergic receptor
      qRT
      quantitative RT
      HAS
      hyaluronan synthase
      HYAL
      hyaluronidase
      UDP-GlcNAc
      UDP-N-acetylglucosamine
      UDP-GlcUA
      UDP-glucuronic acid
      UDP-Glc
      UDP-glucose
      PTX
      pertussis toxin
      CREB
      cAMP response element-binding protein
      CBP
      CREB-binding protein
      CaMKII
      Ca2+/calmodulin-dependent protein kinase II
      p38
      p38 mitogen-activated protein kinase
      HaCaT
      spontaneously transformed aneuploid immortal keratinocyte cell line from human skin
      REK
      rat epidermal keratinocytes
      ER
      endoplasmic reticulum
      BIM
      bisindolylmaleimide
      EGFR
      epidermal growth factor receptor
      ELSA
      enzyme-linked sorbent assay
      DAB
      diaminobenzidine
      ANOVA
      analysis of variance.
      family are mainly found in the basal cell compartment, representing the proliferative cell population (reviewed in Ref.
      • Burnstock Burnstock
      • Verkhratsky Verkhratsky
      Long-term (trophic) purinergic signalling: purinoceptors control cell proliferation, differentiation and death.
      ). UTP signals via P2Y2 and P2Y4, and UDP via P2Y6 and P2Y14 (reviewed in Ref.
      • Glaser Glaser
      • Resende Resende
      • Ulrich Ulrich
      Implications of purinergic receptor-mediated intracellular calcium transients in neural differentiation.
      ). All of the UTP/UDP receptors are expressed in keratinocytes, although the levels of P2Y4 and P2Y14 are probably low (
      • Ho Ho
      • Yang Yang
      • Lin Lin
      • Lin Lin
      Ecto-nucleoside triphosphate diphosphohydrolase 2 modulates local ATP-induced calcium signaling in human HaCaT keratinocytes.
      ,
      • Yoshida Yoshida
      • Kobayashi Kobayashi
      • Ohkubo Ohkubo
      • Nakahata Nakahata
      ATP stimulates interleukin-6 production via P2Y receptors in human HaCaT keratinocytes.
      ,
      • Burrell Burrell
      • Bowler Bowler
      • Gallagher Gallagher
      • Sharpe Sharpe
      Human keratinocytes express multiple P2Y-receptors: evidence for functional P2Y1, P2Y2, and P2Y4 receptors.
      ,
      • Jokela Jokela
      • Kärnä Kärnä
      • Makkonen Makkonen
      • Laitinen Laitinen
      • Tammi Tammi
      • Tammi Tammi
      Extracellular UDP-glucose activates P2Y14 receptor and induces signal transducer and activator of transcription 3 (STAT3) Tyr705 phosphorylation and binding to hyaluronan synthase 2 (HAS2) promoter, stimulating hyaluronan synthesis of keratinocytes.
      ). The P2Y2, P2Y4, and P2Y6 subtypes are coupled to Gq/G11 proteins and activate phospholipase C, thus inducing inositol 1,4,5-trisphosphate-mediated Ca2+ release from the ER, and activation of PKC, whereas the P2Y14 receptors inhibit adenylyl cyclase activity via Gi/o proteins (reviewed in Ref.
      • Glaser Glaser
      • Resende Resende
      • Ulrich Ulrich
      Implications of purinergic receptor-mediated intracellular calcium transients in neural differentiation.
      ).
      Hyaluronan is a linear, high molecular mass polysaccharide composed of glucuronic acid and N-acetylglucosamine. The hyaluronan chain is built at the inner surface of the plasma membrane by hyaluronan synthase (HAS) enzymes, which also form a pore for hyaluronan transport into the extracellular space (
      • Weigel Weigel
      • Hascall Hascall
      • Tammi Tammi
      Hyaluronan synthases.
      ). The newly synthesized hyaluronan chain remains associated with the pericellular matrix, either bound to the synthase, or plasma membrane receptors like CD44, but is later released into the extracellular matrix, where it is often associated with aggregating proteoglycans or other extracellular membrane molecules like IαI or TSG6 (reviewed in Ref.
      • Wang Wang
      • de la Motte de la Motte
      • Lauer Lauer
      • Hascall Hascall
      Hyaluronan matrices in pathobiological processes.
      ). There are three hyaluronan synthase enzymes in mammals (HAS1–3). The distribution of the isoenzymes in tissues and the regulation of their expression as well as their synthetic activity differ (
      • Tammi Tammi
      • Passi Passi
      • Rilla Rilla
      • Karousou Karousou
      • Vigetti Vigetti
      • Makkonen Makkonen
      • Tammi Tammi
      Transcriptional and post-translational regulation of hyaluronan synthesis.
      ) allowing context-dependent regulation of hyaluronan synthesis.
      Enhanced hyaluronan metabolism occurs with tissue injury and inflammation (reviewed in Refs.
      • Oikari Oikari
      • Jokela Jokela
      • Tammi Tammi
      • Tammi Tammi
      ,
      • Lee-Sayer Lee-Sayer
      • Dong Dong
      • Arif Arif
      • Olsson Olsson
      • Brown Brown
      • Johnson Johnson
      The where, when, how, and why of hyaluronan binding by immune cells.
      • Petrey Petrey
      • de la Motte de la Motte
      Hyaluronan, a crucial regulator of inflammation.
      ), situations that are also associated with extracellular release of nucleotides. However, practically nothing is known about the possible influence of these signaling molecules on hyaluronan synthesis. In gingival fibroblasts (
      • Murakami Murakami
      • Hashikawa Hashikawa
      • Saho Saho
      • Takedachi Takedachi
      • Nozaki Nozaki
      • Shimabukuro Shimabukuro
      • Okada Okada
      Adenosine regulates the IL-1 α-induced cellular functions of human gingival fibroblasts.
      ) and smooth muscle cells (
      • Grandoch Grandoch
      • Hoffmann Hoffmann
      • Röck Röck
      • Wenzel Wenzel
      • Oberhuber Oberhuber
      • Schelzig Schelzig
      • Fischer Fischer
      Novel effects of adenosine receptors on pericellular hyaluronan matrix: implications for human smooth muscle cell phenotype and interactions with monocytes during atherosclerosis.
      ), HAS1 expression is up-regulated by adenosine, a breakdown product of ATP, leading to the formation of hyaluronan-rich pericellular matrices. In human keratinocytes the sugar nucleotide UDP-glucose stimulates HAS2 expression and hyaluronan synthesis (
      • Jokela Jokela
      • Kärnä Kärnä
      • Makkonen Makkonen
      • Laitinen Laitinen
      • Tammi Tammi
      • Tammi Tammi
      Extracellular UDP-glucose activates P2Y14 receptor and induces signal transducer and activator of transcription 3 (STAT3) Tyr705 phosphorylation and binding to hyaluronan synthase 2 (HAS2) promoter, stimulating hyaluronan synthesis of keratinocytes.
      ).
      In skin epidermis hyaluronan content is rapidly increased after tissue wounding (
      • Tammi Tammi
      • Pasonen-Seppänen Pasonen-Seppänen
      • Kolehmainen Kolehmainen
      • Tammi Tammi
      Hyaluronan synthase induction and hyaluronan accumulation in mouse epidermis following skin injury.
      ), exposure to chemical irritants (
      • Maytin Maytin
      • Chung Chung
      • Seetharaman Seetharaman
      Hyaluronan participates in the epidermal response to disruption of the permeability barrier in vivo.
      ), and exposure to ultraviolet B radiation (UVB) (
      • Rauhala Rauhala
      • Hämäläinen Hämäläinen
      • Salonen Salonen
      • Bart Bart
      • Tammi Tammi
      • Pasonen-Seppänen Pasonen-Seppänen
      • Tammi Tammi
      Low dose ultraviolet B irradiation increases hyaluronan synthesis in epidermal keratinocytes via sequential induction of hyaluronan synthases Has1–3 mediated by p38 and Ca2+/calmodulin-dependent protein kinase II (CaMKII) signaling.
      ). Elevations of HAS2 and HAS3 mRNA are seen after skin wounding (
      • Tammi Tammi
      • Pasonen-Seppänen Pasonen-Seppänen
      • Kolehmainen Kolehmainen
      • Tammi Tammi
      Hyaluronan synthase induction and hyaluronan accumulation in mouse epidermis following skin injury.
      ,
      • Monslow Monslow
      • Sato Sato
      • Mack Mack
      • Maytin Maytin
      Wounding-induced synthesis of hyaluronic acid in organotypic epidermal cultures requires the release of heparin-binding egf and activation of the EGFR.
      ), and UVB radiation also induces HAS1 (
      • Rauhala Rauhala
      • Hämäläinen Hämäläinen
      • Salonen Salonen
      • Bart Bart
      • Tammi Tammi
      • Pasonen-Seppänen Pasonen-Seppänen
      • Tammi Tammi
      Low dose ultraviolet B irradiation increases hyaluronan synthesis in epidermal keratinocytes via sequential induction of hyaluronan synthases Has1–3 mediated by p38 and Ca2+/calmodulin-dependent protein kinase II (CaMKII) signaling.
      ). The mechanisms of HAS up-regulation after trauma remain unresolved at the moment, although activation of the EGF family growth factors by an insult may at least partly explain the up-regulation of HAS2 and HAS3 (
      • Monslow Monslow
      • Sato Sato
      • Mack Mack
      • Maytin Maytin
      Wounding-induced synthesis of hyaluronic acid in organotypic epidermal cultures requires the release of heparin-binding egf and activation of the EGFR.
      ,
      • Pasonen-Seppänen Pasonen-Seppänen
      • Karvinen Karvinen
      • Törrönen Törrönen
      • Hyttinen Hyttinen
      • Jokela Jokela
      • Lammi Lammi
      • Tammi Tammi
      • Tammi Tammi
      EGF up-regulates, whereas TGF-α down-regulates, the hyaluronan synthases Has2 and Has3 in organotypic keratinocyte cultures: correlations with epidermal proliferation and differentiation.
      ).
      The present work explores the hypothesis that the extracellular nucleotide UTP and its breakdown products UDP and UMP contribute to the rapid HAS expression and hyaluronan accumulation after various skin traumas. We establish for the first time that extracellular UTP causes a pulse of hyaluronan synthesis via a strong, specific and rapid up-regulation of HAS2 expression, mediated by the activation of p38, ERK, STAT3, CaMKII and CREB, whereas the expressions of HAS1, HAS3, HYAL1, and HYAL2 are unaffected. High concentrations of UDP reproduce the effect, whereas UMP had no significant influence on HAS2 expression.

      Discussion

      The present study shows for the first time that the extracellular uridine nucleotides UTP and UDP strongly up-regulate HAS2 expression, leading to the accumulation of hyaluronan in the pericellular matrix and later in the culture medium. As these nucleotides are often released after tissue trauma, the present findings provide a novel signaling mechanism to explain the rapid increase in hyaluronan synthesis detected after various insults (
      • Tammi Tammi
      • Pasonen-Seppänen Pasonen-Seppänen
      • Kolehmainen Kolehmainen
      • Tammi Tammi
      Hyaluronan synthase induction and hyaluronan accumulation in mouse epidermis following skin injury.
      ,
      • Maytin Maytin
      • Chung Chung
      • Seetharaman Seetharaman
      Hyaluronan participates in the epidermal response to disruption of the permeability barrier in vivo.
      ,
      • Rauhala Rauhala
      • Hämäläinen Hämäläinen
      • Salonen Salonen
      • Bart Bart
      • Tammi Tammi
      • Pasonen-Seppänen Pasonen-Seppänen
      • Tammi Tammi
      Low dose ultraviolet B irradiation increases hyaluronan synthesis in epidermal keratinocytes via sequential induction of hyaluronan synthases Has1–3 mediated by p38 and Ca2+/calmodulin-dependent protein kinase II (CaMKII) signaling.
      • Monslow Monslow
      • Sato Sato
      • Mack Mack
      • Maytin Maytin
      Wounding-induced synthesis of hyaluronic acid in organotypic epidermal cultures requires the release of heparin-binding egf and activation of the EGFR.
      ). It also links hyaluronan to the defense mechanisms activated by danger signals such as extracellular nucleotides. The UTP-induced increase in HAS2 activation is transient, however, indicating that a more sustained response observed after wounding and UVR (
      • Tammi Tammi
      • Pasonen-Seppänen Pasonen-Seppänen
      • Kolehmainen Kolehmainen
      • Tammi Tammi
      Hyaluronan synthase induction and hyaluronan accumulation in mouse epidermis following skin injury.
      ,
      • Rauhala Rauhala
      • Hämäläinen Hämäläinen
      • Salonen Salonen
      • Bart Bart
      • Tammi Tammi
      • Pasonen-Seppänen Pasonen-Seppänen
      • Tammi Tammi
      Low dose ultraviolet B irradiation increases hyaluronan synthesis in epidermal keratinocytes via sequential induction of hyaluronan synthases Has1–3 mediated by p38 and Ca2+/calmodulin-dependent protein kinase II (CaMKII) signaling.
      ,
      • Monslow Monslow
      • Sato Sato
      • Mack Mack
      • Maytin Maytin
      Wounding-induced synthesis of hyaluronic acid in organotypic epidermal cultures requires the release of heparin-binding egf and activation of the EGFR.
      ) requires subsequent activation of other signaling routes, such as growth factors or cytokines (
      • Pasonen-Seppänen Pasonen-Seppänen
      • Karvinen Karvinen
      • Törrönen Törrönen
      • Hyttinen Hyttinen
      • Jokela Jokela
      • Lammi Lammi
      • Tammi Tammi
      • Tammi Tammi
      EGF up-regulates, whereas TGF-α down-regulates, the hyaluronan synthases Has2 and Has3 in organotypic keratinocyte cultures: correlations with epidermal proliferation and differentiation.
      ,
      • Pienimaki Pienimaki
      • Rilla Rilla
      • Fulop Fulop
      • Sironen Sironen
      • Karvinen Karvinen
      • Pasonen Pasonen
      • Lammi Lammi
      • Tammi Tammi
      • Hascall Hascall
      • Tammi Tammi
      Epidermal growth factor activates hyaluronan synthase 2 in epidermal keratinocytes and increases pericellular and intracellular hyaluronan.
      ,
      • Karvinen Karvinen
      • Pasonen-Seppänen Pasonen-Seppänen
      • Hyttinen Hyttinen
      • Pienimäki Pienimäki
      • Törrönen Törrönen
      • Jokela Jokela
      • Tammi Tammi
      • Tammi Tammi
      Keratinocyte growth factor stimulates migration and hyaluronan synthesis in the epidermis by activation of keratinocyte hyaluronan synthases 2 and 3.
      ).

      UTP Selectively Activates HAS2 in Keratinocytes

      Although HAS2 underwent a marked increase, the effect of UTP on HAS3 was slight and inconsistent, and completely non-existent on HAS1. The absence of influence on HYAL1 and HYAL2 and the unchanged levels of the UDP-sugar substrates also support the idea that the accumulation of hyaluronan was specifically due to HAS2 up-regulation. A similar HAS2-specific response of keratinocytes was previously found for the growth factors EGF and KGF (
      • Pasonen-Seppänen Pasonen-Seppänen
      • Karvinen Karvinen
      • Törrönen Törrönen
      • Hyttinen Hyttinen
      • Jokela Jokela
      • Lammi Lammi
      • Tammi Tammi
      • Tammi Tammi
      EGF up-regulates, whereas TGF-α down-regulates, the hyaluronan synthases Has2 and Has3 in organotypic keratinocyte cultures: correlations with epidermal proliferation and differentiation.
      ,
      • Pienimaki Pienimaki
      • Rilla Rilla
      • Fulop Fulop
      • Sironen Sironen
      • Karvinen Karvinen
      • Pasonen Pasonen
      • Lammi Lammi
      • Tammi Tammi
      • Hascall Hascall
      • Tammi Tammi
      Epidermal growth factor activates hyaluronan synthase 2 in epidermal keratinocytes and increases pericellular and intracellular hyaluronan.
      ,
      • Karvinen Karvinen
      • Pasonen-Seppänen Pasonen-Seppänen
      • Hyttinen Hyttinen
      • Pienimäki Pienimäki
      • Törrönen Törrönen
      • Jokela Jokela
      • Tammi Tammi
      • Tammi Tammi
      Keratinocyte growth factor stimulates migration and hyaluronan synthesis in the epidermis by activation of keratinocyte hyaluronan synthases 2 and 3.
      ), whereas HAS2 and HAS3 contribute to keratinocyte hyaluronan production at approximately equal proportions under basal culture conditions (
      • Rauhala Rauhala
      • Hämäläinen Hämäläinen
      • Salonen Salonen
      • Bart Bart
      • Tammi Tammi
      • Pasonen-Seppänen Pasonen-Seppänen
      • Tammi Tammi
      Low dose ultraviolet B irradiation increases hyaluronan synthesis in epidermal keratinocytes via sequential induction of hyaluronan synthases Has1–3 mediated by p38 and Ca2+/calmodulin-dependent protein kinase II (CaMKII) signaling.
      ). In addition to direct transcriptional regulation of HAS2, the effect of UTP could arise from the induction of HAS2-AS1, which in turn can regulate HAS2 expression via chromatin remodeling (
      • Vigetti Vigetti
      • Deleonibus Deleonibus
      • Moretto Moretto
      • Bowen Bowen
      • Fischer Fischer
      • Grandoch Grandoch
      • Oberhuber Oberhuber
      • Love Love
      • Hanover Hanover
      • Cinquetti Cinquetti
      • Karousou Karousou
      • Viola Viola
      • D'Angelo D'Angelo
      • Hascall Hascall
      • De Luca De Luca
      • Passi Passi
      Natural antisense transcript for hyaluronan synthase 2 (HAS2-AS1) induces transcription of HAS2 via protein O-GlcNAcylation.
      ).
      Although the UTP-induced HAS2 induction was rapid and transient, it was associated with a significant increase in the amount of hyaluronan in the pericellular matrix and in the culture medium, indicating that the elevated mRNA levels were reflected in HAS2 protein activity. The possible importance of producing hyaluronan specifically by HAS2 is not known, but it has been found that the appearance of the cell surface coat produced by overexpressed HAS2 is slightly different from that made by HAS3 (
      • Rilla Rilla
      • Oikari Oikari
      • Jokela Jokela
      • Hyttinen Hyttinen
      • Kärnä Kärnä
      • Tammi Tammi
      • Tammi Tammi
      Hyaluronan synthase 1 (HAS1) requires higher cellular UDP-GlcNAc concentration than HAS2 and HAS3.
      ). Although the increase in hyaluronan followed the increase in HAS2 mRNA, it is possible that post-translational modifications such as phosphorylation, ubiquitination, or O-GlcNAcylation of HAS2 were involved in the UTP-induced hyaluronan synthesis (
      • Vigetti Vigetti
      • Genasetti Genasetti
      • Karousou Karousou
      • Viola Viola
      • Clerici Clerici
      • Bartolini Bartolini
      • Moretto Moretto
      • De Luca De Luca
      • Hascall Hascall
      • Passi Passi
      Modulation of hyaluronan synthase activity in cellular membrane fractions.
      ,
      • Karousou Karousou
      • Kamiryo Kamiryo
      • Skandalis Skandalis
      • Ruusala Ruusala
      • Asteriou Asteriou
      • Passi Passi
      • Yamashita Yamashita
      • Hellman Hellman
      • Heldin Heldin
      • Heldin Heldin
      The activity of hyaluronan synthase 2 is regulated by dimerization and ubiquitination.
      ,
      • Vigetti Vigetti
      • Deleonibus Deleonibus
      • Moretto Moretto
      • Karousou Karousou
      • Viola Viola
      • Bartolini Bartolini
      • Hascall Hascall
      • Tammi Tammi
      • De Luca De Luca
      • Passi Passi
      Role of UDP-N-acetylglucosamine (GlcNAc) and O-GlcNAcylation of hyaluronan synthase 2 in the control of chondroitin sulfate and hyaluronan synthesis.
      ). However, the involvement of O-GlcNAcylation is less likely, because UTP did not influence the level of UDP-GlcNAc, an important substrate for O-GlcNAcylation.
      The increase of hyaluronan surrounding the cells was detectable within 2 to 4 h, whereas reaching a significant change in the released hyaluronan required 6 h, probably due to the relatively long half-life (8 h) of pericellular hyaluronan in keratinocytes (
      • Tammi Tammi
      • MacCallum MacCallum
      • Hascall Hascall
      • Pienimäki Pienimäki
      • Hyttinen Hyttinen
      • Tammi Tammi
      Hyaluronan bound to CD44 on keratinocytes is displaced by hyaluronan decasaccharides and not hexasaccharides.
      ). Treatment of mesenchymal cells with a viral mimetic, or subjecting them to ER stress or hyperglycemia leads to the formation of cable-like structures of hyaluronan that attract inflammatory cells (
      • Petrey Petrey
      • de la Motte de la Motte
      Hyaluronan, a crucial regulator of inflammation.
      ). Although not as prominent as in mesenchymal cells, keratinocytes can also form monocyte adhesive hyaluronan cables in response to inflammatory cytokines (
      • Jokela Jokela
      • Lindgren Lindgren
      • Rilla Rilla
      • Maytin Maytin
      • Hascall Hascall
      • Tammi Tammi
      • Tammi Tammi
      Induction of hyaluronan cables and monocyte adherence in epidermal keratinocytes.
      ). UTP appears to differ from the above mentioned conditions in this respect, as no signs of cable formation were observed.

      Receptors Involved in the UTP-induced HAS2 Response

      UTP signals via the P2Y2 and P2Y4 receptors, however, the expression level of P2Y4 appears to be very low in keratinocytes (
      • Ho Ho
      • Yang Yang
      • Lin Lin
      • Lin Lin
      Ecto-nucleoside triphosphate diphosphohydrolase 2 modulates local ATP-induced calcium signaling in human HaCaT keratinocytes.
      ,
      • Yoshida Yoshida
      • Kobayashi Kobayashi
      • Ohkubo Ohkubo
      • Nakahata Nakahata
      ATP stimulates interleukin-6 production via P2Y receptors in human HaCaT keratinocytes.
      ,
      • Burrell Burrell
      • Bowler Bowler
      • Gallagher Gallagher
      • Sharpe Sharpe
      Human keratinocytes express multiple P2Y-receptors: evidence for functional P2Y1, P2Y2, and P2Y4 receptors.
      ), suggesting that it has a minor role in the UTP-induced responses in these cells. On the other hand, a P2Y2-specific siRNA reduced the UTP-induced HAS2 response by 62%, which considering only a partial (59%) silencing of the mRNA expression, indicates that the P2Y2 receptor mediates a major proportion of the effect.
      As UTP released into the extracellular space is actively metabolized to UDP also by the HaCaT cells (
      • Ho Ho
      • Yang Yang
      • Lin Lin
      • Lin Lin
      Ecto-nucleoside triphosphate diphosphohydrolase 2 modulates local ATP-induced calcium signaling in human HaCaT keratinocytes.
      ) and UDP enhanced HAS2 mRNA expression, it was possible that a part of the HAS2 induction after UTP release might occur via the P2Y6 and P2Y14 receptors for UDP (Fig. 6). Indeed, under our experimental conditions, inhibitors of P2Y6 and P2Y14, which reduced the UDP-induced HAS2 response, caused a slight, although nonsignificant down-regulation of the UTP response. The higher concentration of UDP required to achieve a stimulation comparable with that by UTP, together with the modest effect of the UDP-receptor inhibitors on UTP-induced HAS2 response, suggest that UTP degradation to UDP is not necessary for HAS2 up-regulation, although it may contribute to the response. In contrast to UTP and UDP, the corresponding monophosphate UMP failed to induce HAS2 expression. This might be explained by the fact that no known receptor for this nucleotide exists. The finding also suggests that no significant reversal of extracellular UMP to UDP or UTP occurs in the HaCaT cell cultures.
      Figure thumbnail gr6
      FIGURE 6Schematic representation of the signaling pathways involved in the UTP-induced HAS2 up-regulation. Extracellular UTP and its breakdown product UDP activate the P2Y receptors that increase HAS2 expression via the indicated signaling steps. Those steps positively verified in the current study are marked green, whereas the pathways excluded are indicated by an “x.”

      The UTP-induced Activation of HAS2 Expression Is Mediated via Pathways Dependent on CaMKII and PKC

      As P2Y2, P2Y4, and P2Y6 are associated with the same G-protein, Gq/11, it is likely that their downstream signaling is similar, involving intracellular Ca2+ release and phospholipase C. MAP kinases, calmodulin, and related kinases as well as the transcription factors STAT3 and CREB are known to be activated by these receptors, reviewed in Ref.
      • Burnstock Burnstock
      • Verkhratsky Verkhratsky
      Long-term (trophic) purinergic signalling: purinoceptors control cell proliferation, differentiation and death.
      and outlined in Fig. 6. UDP, on the other hand, via its association with P2Y14 and its inhibitory partner Gi, can suppress cAMP formation thereby reducing CREB activity. However, P2Y14 can also activate STAT3 via ERK and JAK2 (
      • Jokela Jokela
      • Kärnä Kärnä
      • Makkonen Makkonen
      • Laitinen Laitinen
      • Tammi Tammi
      • Tammi Tammi
      Extracellular UDP-glucose activates P2Y14 receptor and induces signal transducer and activator of transcription 3 (STAT3) Tyr705 phosphorylation and binding to hyaluronan synthase 2 (HAS2) promoter, stimulating hyaluronan synthesis of keratinocytes.
      ,
      • Fricks Fricks
      • Carter Carter
      • Lazarowski Lazarowski
      • Harden Harden
      Gi-dependent cell signaling responses of the human P2Y14 receptor in model cell systems.
      ). In general, our findings on the activation of MAPK, CaMKII, STAT3, and CREB in HaCaT cells by UTP stimulation are in line with previous reports. The experiments applying specific inhibitors of these signaling molecules showed markedly reduced HAS2 responses to UTP, indicating involvement of PKC, p38, ERK, CaMKII, STAT3, and CREB. However, the true role of pERK in the HAS2 response remains uncertain due to the strong effect of the inhibitor on the basal HAS2 expression level. In any case, co-activation of both ERK and p38 signaling has been demonstrated in HaCaT cells after nucleotide exposure (
      • Giltaire Giltaire
      • Lambert Lambert
      • Poumay Poumay
      HB-EGF synthesis and release induced by cholesterol depletion of human epidermal keratinocytes is controlled by extracellular ATP and involves both p38 and ERK1/2 signaling pathways.
      ). The inability of the JAK2/EGFR inhibitor AG490 to block the UTP-dependent HAS2 response rules out EGFR activation after extracellular nucleotide exposure (
      • Li Li
      • Qiu Qiu
      • Zhang Zhang
      • Tian Tian
      • Fang Fang
      P2Y2 receptor and EGFR cooperate to promote prostate cancer cell invasion via ERK1/2 pathway.
      ).
      Although CREB is a target of several of the indicated upstream signaling pathways, STAT3 is regulated by MAP kinases and JAK2 (
      • Bromberg Bromberg
      • Chen Chen
      STAT proteins: signal tranducers and activators of transcription.
      ). p38 and JNK have been associated with the Ser-727 phosphorylation of STAT3, whereas JAK2 is associated with Tyr-705 phosphorylation (reviewed in Ref.
      • Qi Qi
      • Yang Yang
      Regulation and function of signal transducer and activator of transcription 3.
      ). UTP induced STAT3 phosphorylation in Ser-727, but not in Tyr-705, the former correlating temporally with p38 activation. These findings are in line with the inhibitor experiments indicating the involvement of p38 but not JAK2 in HAS2 mRNA induction. Although the specific significance of Ser-727 phosphorylation is unsettled, it is needed for full transcriptional activation of STAT-regulated gene expression, for example, by recruiting co-activators to the promoter (
      • Qi Qi
      • Yang Yang
      Regulation and function of signal transducer and activator of transcription 3.
      ,
      • Schuringa Schuringa
      • Schepers Schepers
      • Vellenga Vellenga
      • Kruijer Kruijer
      Ser727-dependent transcriptional activation by association of p300 with STAT3 upon IL-6 stimulation.
      ). Although Tyr-705 phosphorylation has been classically regarded as necessary for STAT3 activity, recent findings suggest that STAT3 may also influence gene expression independently of its phosphorylation status (reviewed Ref.
      • Qi Qi
      • Yang Yang
      Regulation and function of signal transducer and activator of transcription 3.
      ).
      Blocking CREB binding to CBP, a necessary cofactor for CREB activity (
      • Zhang Zhang
      • Odom Odom
      • Koo Koo
      • Conkright Conkright
      • Canettieri Canettieri
      • Best Best
      • Chen Chen
      • Jenner Jenner
      • Herbolsheimer Herbolsheimer
      • Jacobsen Jacobsen
      • Kadam Kadam
      • Ecker Ecker
      • Emerson Emerson
      • Hogenesch Hogenesch
      • Unterman Unterman
      • Young Young
      • Montminy Montminy
      Genome-wide analysis of cAMP-response element binding protein occupancy, phosphorylation, and target gene activation in human tissues.
      ), and inhibition of STAT3 activity both reduced the UTP-induced HAS2 up-regulation. Both of these transcription factors have active binding sites on the HAS2 promoter and regulate HAS2 expression in keratinocytes (
      • Makkonen Makkonen
      • Pasonen-Seppänen Pasonen-Seppänen
      • Törrönen Törrönen
      • Tammi Tammi
      • Carlberg Carlberg
      Regulation of the hyaluronan synthase 2 gene by convergence in cyclic AMP response element-binding protein and retinoid acid receptor signaling.
      ,
      • Saavalainen Saavalainen
      • Pasonen-Seppänen Pasonen-Seppänen
      • Dunlop Dunlop
      • Tammi Tammi
      • Tammi Tammi
      • Carlberg Carlberg
      The human hyaluronan synthase 2 gene is a primary retinoic acid and epidermal growth factor responding gene.
      ). The lower efficiency of CREB blocking may be due to a lower potency of the inhibitors used, or a weaker activity of CREB on the HAS2 promoter. CREB alone, even with CBP, has been suggested to be insufficient to activate transcription (
      • Zhang Zhang
      • Odom Odom
      • Koo Koo
      • Conkright Conkright
      • Canettieri Canettieri
      • Best Best
      • Chen Chen
      • Jenner Jenner
      • Herbolsheimer Herbolsheimer
      • Jacobsen Jacobsen
      • Kadam Kadam
      • Ecker Ecker
      • Emerson Emerson
      • Hogenesch Hogenesch
      • Unterman Unterman
      • Young Young
      • Montminy Montminy
      Genome-wide analysis of cAMP-response element binding protein occupancy, phosphorylation, and target gene activation in human tissues.
      ). However, as both STAT3 and CREB inhibitors influenced UTP-induced HAS2 expression, it is also possible that their effect is cooperative as shown with some other promoters (
      • Le Goff Le Goff
      • Zheng Zheng
      • Brubaker Brubaker
      • Smith Smith
      Identification of the cAMP-responsive enhancer of the murine ABCA1 gene: requirement for CREB1 and STAT3/4 elements.
      ,
      • Ichiba Ichiba
      • Nakajima Nakajima
      • Yamanaka Yamanaka
      • Kiuchi Kiuchi
      • Hirano Hirano
      Autoregulation of the Stat3 gene through cooperation with a cAMP-responsive element-binding protein.
      ).

      The Interplay between UTP and Hyaluronan

      The rapid nature of the HAS2 response suggests that it may be a part of the cellular defense reactions induced by UTP signaling. Indeed, the UTP-induced HAS2 expression occurred with a similar temporal pattern as the influence of UTP on the expression of the proinflammatory cytokines IL-6 and IL-8 in HaCaT cells (
      • Yoshida Yoshida
      • Kobayashi Kobayashi
      • Ohkubo Ohkubo
      • Nakahata Nakahata
      ATP stimulates interleukin-6 production via P2Y receptors in human HaCaT keratinocytes.
      ,
      • Kobayashi Kobayashi
      • Ohkubo Ohkubo
      • Nakahata Nakahata
      Contribution of extracellular signal-regulated kinase to UTP-induced interleukin-6 biosynthesis in HaCaT keratinocytes.
      ). The simultaneous response rules out the possibility of the cytokines controlling HAS2 expression or vice versa; rather, it is likely that the early HAS2 and cytokine responses are coordinated. Later on, breakdown of hyaluronan to low molecular mass fragments can potentiate IL-6 signaling, resulting in further IL-6 production (
      • Vistejnova Vistejnova
      • Safrankova Safrankova
      • Nesporova Nesporova
      • Slavkovsky Slavkovsky
      • Hermannova Hermannova
      • Hosek Hosek
      • Velebny Velebny
      • Kubala Kubala
      Low molecular weight hyaluronan mediated CD44 dependent induction of IL-6 and chemokines in human dermal fibroblasts potentiates innate immune response.
      ), whereas intact hyaluronan dampens the inflammatory reaction by counteracting the IL-6 signaling (
      • Austin Austin
      • Gilchrist Gilchrist
      • Fehlings Fehlings
      High molecular weight hyaluronan reduces lipopolysaccharide mediated microglial activation.
      ,
      • Wood Wood
      • Breitschwerdt Breitschwerdt
      • Gookin Gookin
      Autocrine effects of interleukin-6 mediate acute-phase proinflammatory and tissue-reparative transcriptional responses of canine bladder mucosa.
      • Rooney Rooney
      • Srivastava Srivastava
      • Watson Watson
      • Quinlan Quinlan
      • Pandit Pandit
      Hyaluronic acid decreases IL-6 and IL-8 secretion and permeability in an inflammatory model of interstitial cystitis.
      ). Apart from regulating cytokine release, hyaluronan itself can protect cells from apoptosis by scavenging reactive oxygen species. This has been shown in cultured corneal epithelial cells and keratinocytes treated with either UV radiation or toxic substances (
      • Ye Ye
      • Wu Wu
      • Wu Wu
      • Wang Wang
      • Zhang Zhang
      • Shi Shi
      • Yang Yang
      High molecular weight hyaluronan decreases oxidative DNA damage induced by EDTA in human corneal epithelial cells.
      ,
      • Pauloin Pauloin
      • Dutot Dutot
      • Joly Joly
      • Warnet Warnet
      • Rat Rat
      High molecular weight hyaluronan decreases UVB-induced apoptosis and inflammation in human epithelial corneal cells.
      ). Furthermore, Wang and co-workers (
      • Wang Wang
      • Lauer Lauer
      • Anand Anand
      • Mack Mack
      • Maytin Maytin
      Hyaluronan synthase 2 protects skin fibroblasts against apoptosis induced by environmental stress.
      ) showed that high levels of hyaluronan and Has2 expression were associated with resistance to UVB radiation and serum starvation in mouse fibroblasts. Thus, tissue stress that liberates UTP is followed by a rapid build-up of a hyaluronan matrix, which might protect the cells from further damage in case the harmful insult continues.
      HAS2 activation and hyaluronan may also have signaling functions in their own right. Via its cell surface receptor CD44, hyaluronan has been shown to modulate the signaling of several growth factor receptors like EGFR and PDGFR, as reviewed in Ref.
      • Misra Misra
      • Hascall Hascall
      • Markwald Markwald
      • Ghatak Ghatak
      Interactions between hyaluronan and its receptors (CD44, RHAMM) regulate the activities of inflammation and cancer.
      , often potentiating the effects of the native ligands. Interestingly, in keratinocytes and fibroblasts, synergism was found in the motogenic response between extracellular nucleotides and EGF, TGFα, PDGF, and TGFα (
      • Wang Wang
      • Huang Huang
      • Heppel Heppel
      Extracellular ATP shows synergistic enhancement of DNA synthesis when combined with agents that are active in wound healing or as neurotransmitters.
      ). Further studies are needed to check whether hyaluronan is involved in this cooperation.
      UTP stimulates the migration of endothelial cells, arterial smooth muscle cells, corneal epithelial cells, prostate cancer cells, and schwannoma cells (
      • Kaczmarek Kaczmarek
      • Erb Erb
      • Koziak Koziak
      • Jarzyna Jarzyna
      • Wink Wink
      • Guckelberger Guckelberger
      • Blusztajn Blusztajn
      • Trinkaus-Randall Trinkaus-Randall
      • Weisman Weisman
      • Robson Robson
      Modulation of endothelial cell migration by extracellular nucleotides: involvement of focal adhesion kinase and phosphatidylinositol 3-kinase-mediated pathways.
      ,
      • Pillois Pillois
      • Chaulet Chaulet
      • Belloc Belloc
      • Dupuch Dupuch
      • Desgranges Desgranges
      • Gadeau Gadeau
      Nucleotide receptors involved in UTP-induced rat arterial smooth muscle cell migration.
      ,
      • Li Li
      • Qiu Qiu
      • Zhang Zhang
      • Liu Liu
      • You You
      • Tian Tian
      • Fang Fang
      P2Y2 receptor promotes cell invasion and metastasis in prostate cancer cells.
      • Lamarca Lamarca
      • Gella Gella
      • Martiañez Martiañez
      • Segura Segura
      • Figueiro-Silva Figueiro-Silva
      • Grijota-Martinez Grijota-Martinez
      • Trullas Trullas
      • Casals Casals
      Uridine 5′-triphosphate promotes in vitro Schwannoma cell migration through matrix metalloproteinase-2 activation.
      ), whereas in keratinocytes it inhibits cell spreading and motility (
      • Taboubi Taboubi
      • Milanini Milanini
      • Delamarre Delamarre
      • Parat Parat
      • Garrouste Garrouste
      • Pommier Pommier
      • Takasaki Takasaki
      • Hubaud Hubaud
      • Kovacic Kovacic
      • Lehmann Lehmann
      Gα(q/11)-coupled P2Y2 nucleotide receptor inhibits human keratinocyte spreading and migration.
      ,
      • Faure Faure
      • Garrouste Garrouste
      • Parat Parat
      • Monferran Monferran
      • Leloup Leloup
      • Pommier Pommier
      • Kovacic Kovacic
      • Lehmann Lehmann
      P2Y2 receptor inhibits EGF-induced MAPK pathway to stabilise keratinocyte hemidesmosomes.
      ). The influence of hyaluronan on cell motility is obviously complex. It has been shown to stimulate cell migration and in turn its removal inhibits migration (reviewed in Ref.
      • Evanko Evanko
      • Tammi Tammi
      • Tammi Tammi
      • Wight Wight
      Hyaluronan-dependent pericellular matrix.
      ). However, hyaluronan can also inhibit cell motility, especially when present in excessive amounts such as during artificial overexpression induced by HAS transfection (
      • Brinck Brinck
      • Heldin Heldin
      Expression of recombinant hyaluronan synthase (HAS) isoforms in CHO cells reduces cell migration and cell surface CD44.
      ,
      • Takabe Takabe
      • Bart Bart
      • Ropponen Ropponen
      • Rilla Rilla
      • Tammi Tammi
      • Tammi Tammi
      • Pasonen-Seppänen Pasonen-Seppänen
      Hyaluronan synthase 3 (HAS3) overexpression down-regulates MV3 melanoma cell proliferation, migration and adhesion.
      ). The physiological response of the cells to hyaluronan depends on its molecular mass (
      • Oikari Oikari
      • Jokela Jokela
      • Tammi Tammi
      • Tammi Tammi
      ,
      • Ruppert Ruppert
      • Hawn Hawn
      • Arrigoni Arrigoni
      • Wight Wight
      • Bollyky Bollyky
      Tissue integrity signals communicated by high-molecular weight hyaluronan and the resolution of inflammation.
      ). UTP induced mainly HAS2, which is known to synthesize high molecular mass hyaluronan (
      • Brinck Brinck
      • Heldin Heldin
      Expression of recombinant hyaluronan synthase (HAS) isoforms in CHO cells reduces cell migration and cell surface CD44.
      ,
      • Itano Itano
      • Sawai Sawai
      • Yoshida Yoshida
      • Lenas Lenas
      • Yamada Yamada
      • Imagawa Imagawa
      • Shinomura Shinomura
      • Hamaguchi Hamaguchi
      • Yoshida Yoshida
      • Ohnuki Ohnuki
      • Miyauchi Miyauchi
      • Spicer Spicer
      • McDonald McDonald
      • Kimata Kimata
      Three isoforms of mammalian hyaluronan synthases have distinct enzymatic properties.
      ). Whether hyaluronan breakdown was activated by UTP was not investigated here. However, we did not observe any increase in the intracellular hyaluronan, which is often associated with hyaluronan breakdown (
      • Rauhala Rauhala
      • Hämäläinen Hämäläinen
      • Salonen Salonen
      • Bart Bart
      • Tammi Tammi
      • Pasonen-Seppänen Pasonen-Seppänen
      • Tammi Tammi
      Low dose ultraviolet B irradiation increases hyaluronan synthesis in epidermal keratinocytes via sequential induction of hyaluronan synthases Has1–3 mediated by p38 and Ca2+/calmodulin-dependent protein kinase II (CaMKII) signaling.
      ,
      • Tammi Tammi
      • Rilla Rilla
      • Pienimaki Pienimaki
      • MacCallum MacCallum
      • Hogg Hogg
      • Luukkonen Luukkonen
      • Hascall Hascall
      • Tammi Tammi
      Hyaluronan enters keratinocytes by a novel endocytic route for catabolism.
      ). Moreover, the expression of hyaluronidases was not changed. However, because the inhibition of cell motility occurs already within 15 to 30 min after the UTP exposure (
      • Taboubi Taboubi
      • Milanini Milanini
      • Delamarre Delamarre
      • Parat Parat
      • Garrouste Garrouste
      • Pommier Pommier
      • Takasaki Takasaki
      • Hubaud Hubaud
      • Kovacic Kovacic
      • Lehmann Lehmann
      Gα(q/11)-coupled P2Y2 nucleotide receptor inhibits human keratinocyte spreading and migration.
      ,
      • Faure Faure
      • Garrouste Garrouste
      • Parat Parat
      • Monferran Monferran
      • Leloup Leloup
      • Pommier Pommier
      • Kovacic Kovacic
      • Lehmann Lehmann
      P2Y2 receptor inhibits EGF-induced MAPK pathway to stabilise keratinocyte hemidesmosomes.
      ), it unlikely relates to hyaluronan synthesis, which takes place later.

      Coordination and Convergence of Signaling in Keratinocyte Hyaluronan Responses Triggered by Injury and Environmental Stress

      Interestingly, the UTP-induced signaling pathways regulating HAS2 expression partially overlap with those occurring after UVB exposure, an insult known to cause nucleotide release in keratinocytes (
      • Takai Takai
      • Tsukimoto Tsukimoto
      • Harada Harada
      • Kojima Kojima
      Involvement of P2Y6 receptor in p38 MAPK-mediated COX-2 expression in response to UVB irradiation of human keratinocytes.
      ). Although UVB induces a multitude of signaling events, in rat epidermal keratinocytes (REK) the induction of Has2 and Has3 appears to depend mainly on p38 and CaMKII (
      • Rauhala Rauhala
      • Hämäläinen Hämäläinen
      • Salonen Salonen
      • Bart Bart
      • Tammi Tammi
      • Pasonen-Seppänen Pasonen-Seppänen
      • Tammi Tammi
      Low dose ultraviolet B irradiation increases hyaluronan synthesis in epidermal keratinocytes via sequential induction of hyaluronan synthases Has1–3 mediated by p38 and Ca2+/calmodulin-dependent protein kinase II (CaMKII) signaling.
      ). However, in REK cells UVB also activates the expression of other hyaluronan-related genes, such as Has1, Hyal1, and Hyal2 (
      • Rauhala Rauhala
      • Hämäläinen Hämäläinen
      • Salonen Salonen
      • Bart Bart
      • Tammi Tammi
      • Pasonen-Seppänen Pasonen-Seppänen
      • Tammi Tammi
      Low dose ultraviolet B irradiation increases hyaluronan synthesis in epidermal keratinocytes via sequential induction of hyaluronan synthases Has1–3 mediated by p38 and Ca2+/calmodulin-dependent protein kinase II (CaMKII) signaling.
      ), which did not respond to UTP. Furthermore, the UVB-induced activation of p38 and Has2 expression in rat keratinocytes is long-lasting (36 h), suggesting that for a more sustained response other signaling pathways need to be activated. One of the UVB-activated cascades originates from HB-EGF/EGFR (
      • Seo Seo
      • Juhnn Juhnn
      Gq protein mediates UVB-induced cyclooxygenase-2 expression by stimulating HB-EGF secretion from HaCaT human keratinocytes.
      ), shown to regulate Has2 and Has3 expression in REK cultures (
      • Monslow Monslow
      • Sato Sato
      • Mack Mack
      • Maytin Maytin
      Wounding-induced synthesis of hyaluronic acid in organotypic epidermal cultures requires the release of heparin-binding egf and activation of the EGFR.
      ,
      • Pasonen-Seppänen Pasonen-Seppänen
      • Maytin Maytin
      • Törrönen Törrönen
      • Hyttinen Hyttinen
      • Hascall Hascall
      • MacCallum MacCallum
      • Kultti Kultti
      • Jokela Jokela
      • Tammi Tammi
      • Tammi Tammi
      All-trans retinoic acid-induced hyaluronan production and hyperplasia are partly mediated by EGFR signaling in epidermal keratinocytes.
      ). Although the intracellular signaling mediators triggered by nucleotides and EGFR activation are partially different, they show convergence, suggesting that they can act synergistically (
      • Grimm Grimm
      • Messemer Messemer
      • Stanke Stanke
      • Gachet Gachet
      • Zimmermann Zimmermann
      Coordinate pathways for nucleotide and EGF signaling in cultured adult neural progenitor cells.
      ).
      In conclusion, nucleotides UTP and UDP, but not UMP, released into the extracellular space activate HAS2 mRNA expression in human keratinocytes. The expression of the other hyaluronan synthases or hyaluronidases are not significantly influenced by UTP. The signaling pathway for UTP involves the purinergic P2Y2 receptor, and to a smaller extent the UDP receptors P2Y6 and P2Y14, and their downstream cascades recruiting PKC, and the MAP kinases p38 and ERK, CaMKII, STAT3, and CREB (Fig. 6). The effect of UTP on HAS2 expression is strong and, although transient, causes a significant increase in hyaluronan accumulation in the pericellular matrix and culture medium. This rapid induction of a hyaluronan coat may be a good way to provide an instant response to a potentially damaging signal, whereas making sure a stronger, more sustained response is only activated if required by the circumstances.

      Author Contributions

      T. J. conceived and designed the study, analyzed the results, and contributed to paper writing and prepared all figures. R. H. T. designed and coordinated the study, analyzed results, and wrote the paper. R. K. provided technical expertise for all experiments. G. B. took part in study conception. L. R., S. O., and S. P. S. performed and analyzed part of the experiments, and critically revised the manuscript. M. I. T. took part in the study conception and critically revised the manuscript. All authors approved the manuscript.

      Acknowledgment

      We thank Dr. Jarmo T. Laitinen for critical review of the manuscript.

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