Advertisement

Astrocytic STAT3 activation and chronic itch require IP3R1/TRPC-dependent Ca2+ signals in mice

Published:August 08, 2020DOI:https://doi.org/10.1016/j.jaci.2020.06.039

      Background

      Chronic itch is a debilitating symptom of inflammatory skin diseases, but the underlying mechanism is poorly understood. We have recently demonstrated that astrocytes in the spinal dorsal horn become reactive in models of atopic and contact dermatitis via activation of the transcription factor signal transducer and activator of transcription 3 (STAT3) and critically contribute to chronic itch. In general, STAT3 is transiently activated; however, STAT3 activation in reactive astrocytes of chronic itch model mice persistently occurs via an unknown mechanism.

      Objective

      We aimed to determine the mechanisms of persistent activation of astrocytic STAT3 in chronic itch conditions.

      Methods

      To determine the factors that are required for persistent activation of astrocytic STAT3, Western blotting and calcium imaging with cultured astrocytes or spinal cord slices were performed. Thereafter, chronic itch model mice were used for genetic and behavioral experiments to confirm the role of the factors determined to mediate persistent STAT3 activation from in vitro and ex vivo experiments in chronic itch.

      Results

      IP3 receptor type 1 (IP3R1) knockdown in astrocytes suppressed IL-6–induced persistent STAT3 activation and expression of lipocalin-2 (LCN2), an astrocytic STAT3-dependent inflammatory factor that is required for chronic itch. IP3R1-dependent astrocytic Ca2+ responses involved Ca2+ influx through the cation channel transient receptor potential canonical (TRPC), which was required for persistent STAT3 activation evoked by IL-6. IL-6 expression was upregulated in dorsal root ganglion neurons in a mouse model of chronic itch. Dorsal root ganglion neuron–specific IL-6 knockdown, spinal astrocyte–specific IP3R1 knockdown, and pharmacologic spinal TRPC inhibition attenuated LCN2 expression and chronic itch.

      Conclusion

      Our findings suggest that IP3R1/TRPC channel–mediated Ca2+ signals elicited by IL-6 in astrocytes are necessary for persistent STAT3 activation, LCN2 expression, and chronic itch, and they may also provide new targets for therapeutic intervention.

      Graphical abstract

      Key words

      Abbreviations used:

      AAV (Adeno-associated virus), 2APB (2-aminoethoxydiphenylborane), CNS (Central nervous system), DCP (Diphenylcyclopropenone), DRG (Dorsal root ganglion), ESYN (Enhanced synapsin), GRP (Gastrin-releasing peptide), IP3R (Inositol 1,4,5-triphosphate receptor), IP3R1 (IP3 receptor type 1), IP3R2 (IP3 receptor type 2), JAK (Janus kinase), KO (Knockout), LCN2 (Lipocalin-2), PYK2 (Proline-rich tyrosine kinase 2), Pyr3 (Pyrazole-3), SDH (Spinal dorsal horn), shRNA (Short hairpin RNA), siRNA (Small interfering RNA), STAT3 (Signal transducer and activator of transcription 3), TRPC (Transient receptor potential canonical), WT (Wild-type)
      To read this article in full you will need to make a payment

      References

        • Miller G.
        Biomedicine. Grasping for clues to the biology of itch.
        Science. 2007; 318: 188-189
        • Sun Y.G.
        • Chen Z.F.
        A gastrin-releasing peptide receptor mediates the itch sensation in the spinal cord.
        Nature. 2007; 448: 700-703
        • Sun Y.G.
        • Zhao Z.Q.
        • Meng X.L.
        • Yin J.
        • Liu X.Y.
        • Chen Z.F.
        Cellular basis of itch sensation.
        Science. 2009; 325: 1531-1534
        • Mishra S.K.
        • Hoon M.A.
        The cells and circuitry for itch responses in mice.
        Science. 2013; 340: 968-971
        • Ikoma A.
        • Rukwied R.
        • Stander S.
        • Steinhoff M.
        • Miyachi Y.
        • Schmelz M.
        Neuronal sensitization for histamine-induced itch in lesional skin of patients with atopic dermatitis.
        Arch Dermatol. 2003; 139: 1455-1458
        • Tsuda M.
        Astrocytes in the spinal dorsal horn and chronic itch.
        Neurosci Res. 2018; 126: 9-14
        • Haydon P.G.
        • Carmignoto G.
        Astrocyte control of synaptic transmission and neurovascular coupling.
        Physiol Rev. 2006; 86: 1009-1031
        • Halassa M.M.
        • Haydon P.G.
        Integrated brain circuits: astrocytic networks modulate neuronal activity and behavior.
        Annu Rev Physiol. 2010; 72: 335-355
        • Ceyzeriat K.
        • Abjean L.
        • Carrillo-de Sauvage M.A.
        • Ben Haim L.
        • Escartin C.
        The complex STATes of astrocyte reactivity: how are they controlled by the JAK-STAT3 pathway?.
        Neuroscience. 2016; 330: 205-218
        • Sofroniew M.V.
        Molecular dissection of reactive astrogliosis and glial scar formation.
        Trends Neurosci. 2009; 32: 638-647
        • Shiratori-Hayashi M.
        • Koga K.
        • Tozaki-Saitoh H.
        • Kohro Y.
        • Toyonaga H.
        • Yamaguchi C.
        • et al.
        STAT3-dependent reactive astrogliosis in the spinal dorsal horn underlies chronic itch.
        Nat Med. 2015; 21: 927-931
        • Koga K.
        • Yamagata R.
        • Kohno K.
        • Yamane T.
        • Shiratori-Hayashi M.
        • Kohro Y.
        • et al.
        Sensitization of spinal itch transmission neurons in a mouse model of chronic itch requires an astrocytic factor.
        J Allergy Clin Immunol. 2020; 145: 183-191.e10
        • Ivashkiv L.B.
        • Hu X.
        Signaling by STATs.
        Arthritis Res Ther. 2004; 6: 159-168
        • Herrmann J.E.
        • Imura T.
        • Song B.
        • Qi J.
        • Ao Y.
        • Nguyen T.K.
        • et al.
        STAT3 is a critical regulator of astrogliosis and scar formation after spinal cord injury.
        J Neurosci. 2008; 28: 7231-7243
        • Tsuda M.
        • Kohro Y.
        • Yano T.
        • Tsujikawa T.
        • Kitano J.
        • Tozaki-Saitoh H.
        • et al.
        JAK-STAT3 pathway regulates spinal astrocyte proliferation and neuropathic pain maintenance in rats.
        Brain. 2011; 134: 1127-1139
        • Kohro Y.
        • Sakaguchi E.
        • Tashima R.
        • Tozaki-Saitoh H.
        • Okano H.
        • Inoue K.
        • et al.
        A new minimally-invasive method for microinjection into the mouse spinal dorsal horn.
        Sci Rep. 2015; 5: 14306
        • O'Callaghan J.P.
        • Kelly K.A.
        • VanGilder R.L.
        • Sofroniew M.V.
        • Miller D.B.
        Early activation of STAT3 regulates reactive astrogliosis induced by diverse forms of neurotoxicity.
        PLoS One. 2014; 9e102003
        • Ben Haim L.
        • Carrillo-de Sauvage M.A.
        • Ceyzeriat K.
        • Escartin C.
        Elusive roles for reactive astrocytes in neurodegenerative diseases.
        Front Cell Neurosci. 2015; 9: 278
        • Kuchibhotla K.V.
        • Lattarulo C.R.
        • Hyman B.T.
        • Bacskai B.J.
        Synchronous hyperactivity and intercellular calcium waves in astrocytes in Alzheimer mice.
        Science. 2009; 323: 1211-1215
        • Kanemaru K.
        • Kubota J.
        • Sekiya H.
        • Hirose K.
        • Okubo Y.
        • Iino M.
        Calcium-dependent N-cadherin up-regulation mediates reactive astrogliosis and neuroprotection after brain injury.
        Proc Natl Acad Sci U S A. 2013; 110: 11612-11617
        • Tian G.F.
        • Azmi H.
        • Takano T.
        • Xu Q.
        • Peng W.
        • Lin J.
        • et al.
        An astrocytic basis of epilepsy.
        Nat Med. 2005; 11: 973-981
        • Bezzi P.
        • Carmignoto G.
        • Pasti L.
        • Vesce S.
        • Rossi D.
        • Rizzini B.L.
        • et al.
        Prostaglandins stimulate calcium-dependent glutamate release in astrocytes.
        Nature. 1998; 391: 281-285
        • Navarrete M.
        • Araque A.
        Endocannabinoids potentiate synaptic transmission through stimulation of astrocytes.
        Neuron. 2010; 68: 113-126
        • Panatier A.
        • Vallee J.
        • Haber M.
        • Murai K.K.
        • Lacaille J.C.
        • Robitaille R.
        Astrocytes are endogenous regulators of basal transmission at central synapses.
        Cell. 2011; 146: 785-798
        • Petravicz J.
        • Fiacco T.A.
        • McCarthy K.D.
        Loss of IP3 receptor-dependent Ca2+ increases in hippocampal astrocytes does not affect baseline CA1 pyramidal neuron synaptic activity.
        J Neurosci. 2008; 28: 4967-4973
        • Cao X.
        • Li L.P.
        • Wang Q.
        • Wu Q.
        • Hu H.H.
        • Zhang M.
        • et al.
        Astrocyte-derived ATP modulates depressive-like behaviors.
        Nat Med. 2013; 19: 773-777
        • Wang F.
        • Xu Q.
        • Wang W.
        • Takano T.
        • Nedergaard M.
        Bergmann glia modulate cerebellar Purkinje cell bistability via Ca2+-dependent K+ uptake.
        Proc Natl Acad Sci U S A. 2012; 109: 7911-7916
        • Li H.
        • Xie Y.
        • Zhang N.
        • Yu Y.
        • Zhang Q.
        • Ding S.
        Disruption of IP(3)R2-mediated Ca(2)(+) signaling pathway in astrocytes ameliorates neuronal death and brain damage while reducing behavioral deficits after focal ischemic stroke.
        Cell Calcium. 2015; 58: 565-576
        • Petravicz J.
        • Boyt K.M.
        • McCarthy K.D.
        Astrocyte IP3R2-dependent Ca(2+) signaling is not a major modulator of neuronal pathways governing behavior.
        Front Behav Neurosci. 2014; 8: 384
        • Takata N.
        • Nagai T.
        • Ozawa K.
        • Oe Y.
        • Mikoshiba K.
        • Hirase H.
        Cerebral blood flow modulation by basal forebrain or whisker stimulation can occur independently of large cytosolic Ca2+ signaling in astrocytes.
        PLoS One. 2013; 8e66525
        • Agulhon C.
        • Fiacco T.A.
        • McCarthy K.D.
        Hippocampal short- and long-term plasticity are not modulated by astrocyte Ca2+ signaling.
        Science. 2010; 327: 1250-1254
        • Sherwood M.W.
        • Arizono M.
        • Hisatsune C.
        • Bannai H.
        • Ebisui E.
        • Sherwood J.L.
        • et al.
        Astrocytic IP3 Rs: contribution to Ca(2+) signalling and hippocampal LTP.
        Glia. 2017; 65: 502-513
        • Okubo Y.
        • Kanemaru K.
        • Suzuki J.
        • Kobayashi K.
        • Hirose K.
        • Iino M.
        Inositol 1,4,5-trisphosphate receptor type 2-independent Ca(2+) release from the endoplasmic reticulum in astrocytes.
        Glia. 2019; 67: 113-124
        • Machida A.
        • Kuwahara H.
        • Mayra A.
        • Kubodera T.
        • Hirai T.
        • Sunaga F.
        • et al.
        Intraperitoneal administration of AAV9-shRNA inhibits target gene expression in the dorsal root ganglia of neonatal mice.
        Mol Pain. 2013; 9: 36
        • Liddelow S.A.
        • Guttenplan K.A.
        • Clarke L.E.
        • Bennett F.C.
        • Bohlen C.J.
        • Schirmer L.
        • et al.
        Neurotoxic reactive astrocytes are induced by activated microglia.
        Nature. 2017; 541: 481-487
        • Adebiyi A.
        • Narayanan D.
        • Jaggar J.H.
        Caveolin-1 assembles type 1 inositol 1,4,5-trisphosphate receptors and canonical transient receptor potential 3 channels into a functional signaling complex in arterial smooth muscle cells.
        J Biol Chem. 2011; 286: 4341-4348
        • Salido G.M.
        • Sage S.O.
        • Rosado J.A.
        TRPC channels and store-operated Ca(2+) entry.
        Biochim Biophys Acta. 2009; 1793: 223-230
        • Prakriya M.
        • Feske S.
        • Gwack Y.
        • Srikanth S.
        • Rao A.
        • Hogan P.G.
        Orai1 is an essential pore subunit of the CRAC channel.
        Nature. 2006; 443: 230-233
        • Chen X.
        • Lu M.
        • He X.
        • Ma L.
        • Birnbaumer L.
        • Liao Y.
        TRPC3/6/7 knockdown protects the brain from cerebral ischemia injury via astrocyte apoptosis inhibition and effects on NF-κB translocation.
        Mol Neurobiol. 2017; 54: 7555-7566
        • Kwon J.
        • An H.
        • Sa M.
        • Won J.
        • Shin J.I.
        • Lee C.J.
        Orai1 and Orai3 in combination with Stim1 mediate the majority of store-operated calcium entry in astrocytes.
        Exp Neurobiol. 2017; 26: 42-54
        • Shigetomi E.
        • Kracun S.
        • Khakh B.S.
        Monitoring astrocyte calcium microdomains with improved membrane targeted GCaMP reporters.
        Neuron Glia Biol. 2010; 6: 183-191
        • Yoshihara K.
        • Matsuda T.
        • Kohro Y.
        • Tozaki-Saitoh H.
        • Inoue K.
        • Tsuda M.
        Astrocytic Ca(2+) responses in the spinal dorsal horn by noxious stimuli to the skin.
        J Pharmacol Sci. 2018; 137: 101-104
        • Gordon G.R.
        • Iremonger K.J.
        • Kantevari S.
        • Ellis-Davies G.C.
        • MacVicar B.A.
        • Bains J.S.
        Astrocyte-mediated distributed plasticity at hypothalamic glutamate synapses.
        Neuron. 2009; 64: 391-403
        • Di Castro M.A.
        • Chuquet J.
        • Liaudet N.
        • Bhaukaurally K.
        • Santello M.
        • Bouvier D.
        • et al.
        Local Ca2+ detection and modulation of synaptic release by astrocytes.
        Nat Neurosci. 2011; 14: 1276-1284
        • Srinivasan R.
        • Huang B.S.
        • Venugopal S.
        • Johnston A.D.
        • Chai H.
        • Zeng H.
        • et al.
        Ca(2+) signaling in astrocytes from Ip3r2(-/-) mice in brain slices and during startle responses in vivo.
        Nat Neurosci. 2015; 18: 708-717
        • Kesherwani V.
        • Agrawal S.K.
        Regulation of inositol 1,4,5-triphosphate receptor, type 1 (IP3R1) in hypoxic/reperfusion injury of white matter.
        Neurol Res. 2012; 34: 504-511
        • Feske S.
        • Giltnane J.
        • Dolmetsch R.
        • Staudt L.M.
        • Rao A.
        Gene regulation mediated by calcium signals in T lymphocytes.
        Nat Immunol. 2001; 2: 316-324
        • Foley J.
        • Blutstein T.
        • Lee S.
        • Erneux C.
        • Halassa M.M.
        • Haydon P.
        Astrocytic IP3/Ca(2+) signaling modulates theta rhythm and REM sleep.
        Front Neural Circuits. 2017; 11: 3
        • Adebiyi A.
        • Zhao G.
        • Narayanan D.
        • Thomas-Gatewood C.M.
        • Bannister J.P.
        • Jaggar J.H.
        Isoform-selective physical coupling of TRPC3 channels to IP3 receptors in smooth muscle cells regulates arterial contractility.
        Circ Res. 2010; 106: 1603-1612
        • Hofmann T.
        • Schaefer M.
        • Schultz G.
        • Gudermann T.
        Subunit composition of mammalian transient receptor potential channels in living cells.
        Proc Natl Acad Sci U S A. 2002; 99: 7461-7466
        • Elg S.
        • Marmigere F.
        • Mattsson J.P.
        • Ernfors P.
        Cellular subtype distribution and developmental regulation of TRPC channel members in the mouse dorsal root ganglion.
        J Comp Neurol. 2007; 503: 35-46
        • Dong P.
        • Guo C.
        • Huang S.
        • Ma M.
        • Liu Q.
        • Luo W.
        TRPC3 is dispensable for beta-alanine triggered acute itch.
        Sci Rep. 2017; 7: 13869
        • Liu T.
        • Han Q.
        • Chen G.
        • Huang Y.
        • Zhao L.X.
        • Berta T.
        • et al.
        Toll-like receptor 4 contributes to chronic itch, alloknesis, and spinal astrocyte activation in male mice.
        Pain. 2016; 157: 806-817
        • Vanderheyden V.
        • Devogelaere B.
        • Missiaen L.
        • De Smedt H.
        • Bultynck G.
        • Parys J.B.
        Regulation of inositol 1,4,5-trisphosphate-induced Ca2+ release by reversible phosphorylation and dephosphorylation.
        Biochim Biophys Acta. 2009; 1793: 959-970
        • Rothaug M.
        • Becker-Pauly C.
        • Rose-John S.
        The role of interleukin-6 signaling in nervous tissue.
        Biochim Biophys Acta. 2016; 1863: 1218-1227
        • Lev S.
        • Moreno H.
        • Martinez R.
        • Canoll P.
        • Peles E.
        • Musacchio J.M.
        • et al.
        Protein tyrosine kinase PYK2 involved in Ca(2+)-induced regulation of ion channel and MAP kinase functions.
        Nature. 1995; 376: 737-745
        • Verma N.
        • Keinan O.
        • Selitrennik M.
        • Karn T.
        • Filipits M.
        • Lev S.
        PYK2 sustains endosomal-derived receptor signalling and enhances epithelial-to-mesenchymal transition.
        Nat Commun. 2015; 6: 6064
        • Meads M.B.
        • Fang B.
        • Mathews L.
        • Gemmer J.
        • Nong L.
        • Rosado-Lopez I.
        • et al.
        Targeting PYK2 mediates microenvironment-specific cell death in multiple myeloma.
        Oncogene. 2016; 35: 2723-2734
        • Du L.
        • Hu X.
        • Yang W.
        • Yasheng H.
        • Liu S.
        • Zhang W.
        • et al.
        Spinal IL-33/ST2 signaling mediates chronic itch in mice through the astrocytic JAK2-STAT3 cascade.
        Glia. 2019; 67: 1680-1693
        • Moulin D.
        • Donze O.
        • Talabot-Ayer D.
        • Mezin F.
        • Palmer G.
        • Gabay C.
        Interleukin (IL)-33 induces the release of pro-inflammatory mediators by mast cells.
        Cytokine. 2007; 40: 216-225
        • Rank M.A.
        • Kobayashi T.
        • Kozaki H.
        • Bartemes K.R.
        • Squillace D.L.
        • Kita H.
        IL-33-activated dendritic cells induce an atypical TH2-type response.
        J Allergy Clin Immunol. 2009; 123: 1047-1054
        • Ikoma A.
        • Steinhoff M.
        • Stander S.
        • Yosipovitch G.
        • Schmelz M.
        The neurobiology of itch.
        Nat Rev Neurosci. 2006; 7: 535-547
        • Sawada M.
        • Imamura K.
        • Nagatsu T.
        Role of cytokines in inflammatory process in Parkinson's disease.
        J Neural Transm Suppl. 2006; : 373-381
        • Benveniste E.N.
        Cytokine actions in the central nervous system.
        Cytokine Growth Factor Rev. 1998; 9: 259-275
        • Ben Haim L.
        • Ceyzeriat K.
        • Carrillo-de Sauvage M.A.
        • Aubry F.
        • Auregan G.
        • Guillermier M.
        • et al.
        The JAK/STAT3 pathway is a common inducer of astrocyte reactivity in Alzheimer's and Huntington's diseases.
        J Neurosci. 2015; 35: 2817-2829
        • Tang T.S.
        • Tu H.
        • Chan E.Y.
        • Maximov A.
        • Wang Z.
        • Wellington C.L.
        • et al.
        Huntingtin and huntingtin-associated protein 1 influence neuronal calcium signaling mediated by inositol-(1,4,5) triphosphate receptor type 1.
        Neuron. 2003; 39: 227-239