Skip to main content

Advertisement

Log in

Nicotinic acetylcholine receptor alpha7 subunit is time-dependently expressed in distinct cell types during skin wound healing in mice

  • Original Paper
  • Published:
Histochemistry and Cell Biology Aims and scope Submit manuscript

Abstract

Recent studies have shown that nicotinic acetylcholine receptor alpha7 subunit (nAChRα7) plays an important role in regulation of inflammation, angiogenesis and keratinocyte biology, but little is known about its expression after the skin is wounded. A preliminary study on time-dependent expression and distribution of nAChRα7 was performed by immunohistochemistry, Western blotting and RT-PCR during skin wound healing in mice. After a 1-cm-long incision was made in the skin of the central dorsum, mice were killed at intervals ranging from 6 h to 14 days post-injury. In uninjured skin controls, nAChRα7 positive staining was observed in epidermis, hair follicles, sebaceous glands, vessel endothelium and resident dermal fibroblastic cells. In wounded specimens, a small number of polymorphonuclear cells, a large number of mononuclear cells (MNCs) and fibroblastic cells (FBCs) showed positive reaction for nAChRα7 in the wound zones. Simultaneously, nAChRα7 immunoreactivity was evident in endothelial-like cells of regenerated vessels and neoepidermis. By morphometric analysis, an up-regulation of nAChRα7 expression was verified at the inflammatory phase after skin injury and reached a peak at the proliferative phase of wound healing. The expression tendency was further confirmed by Western blotting and RT-PCR assay. By immunofluorescent staining for co-localization, the nAChRα7-positive MNCs and FBCs in skin wounds were identified as macrophages, fibrocytes and myofibroblasts. A number of nAChRα7-positive myofibroblasts were also CD45 positive, indicating that they originated from differentiation of fibrocytes. The results demonstrate that nAChRα7 is time-dependently expressed in distinct cell types, which may be closely involved in inflammatory response and repair process during skin wound healing.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9

Similar content being viewed by others

References

  • Abe R, Donnelly SC, Peng T, Bucala R, Metz CN (2001) Peripheral blood fibrocytes: differentiation pathway and migration to wound sites. J Immunol 166:7556–7562

    PubMed  CAS  Google Scholar 

  • Aiba S, Tagami H (1997) Inverse correlation between CD34 expression and proline-4-hydroxylase immunoreactivity on spindle cells noted in hypertrophic scars and keloids. J Cutan Pathol 24:65–69

    Article  PubMed  CAS  Google Scholar 

  • Alkondon M, Albuquerque EX (2004) The nicotinic acetylcholine receptor subtypes and their function in the hippocampus and cerebral cortex. Prog Brain Res 145:109–120

    Article  PubMed  CAS  Google Scholar 

  • Alkondon M, Pereira EF, Cortes WS, Maelicke A, Albuquerque EX (1997) Choline is a selective agonist of alpha7 nicotinic acetylcholine receptors in the rat brain neurons. Eur J Neurosci 9:2734–2742

    Google Scholar 

  • Anderson AA, Ushakov DS, Ferenczi MA, Mori R, Martin P, Saffell JL (2008) Morphoregulation by acetylcholinesterase in fibroblasts and astrocytes. J Cell Physiol 215:82–100

    Article  PubMed  CAS  Google Scholar 

  • Andersson-Sjöland A, de Alba CG, Nihlberg K, Becerril C, Ramírez R, Pardo A, Westergren-Thorsson G, Selman M (2008) Fibrocytes are a potential source of lung fibroblasts in idiopathic pulmonary fibrosis. Int J Biochem Cell Biol 40:2129–2140

    Article  PubMed  Google Scholar 

  • Arredondo J, Nguyen VT, Chernyavsky AI, Bercovich D, Orr-Urtreger A, Kummer W, Lips K, Vetter DE, Grando SA (2002) Central role of alpha7 nicotinic receptor in differentiation of the stratified squamous epithelium. J Cell Biol 159:325–336

    Article  PubMed  CAS  Google Scholar 

  • Arredondo J, Nguyen VT, Chernyavsky AI, Bercovich D, Orr-Urtreger A, Vetter DE, Grando SA (2003) Functional role of alpha7 nicotinic receptor in physiological control of cutaneous homeostasis. Life Sci 72:2063–2067

    Article  PubMed  CAS  Google Scholar 

  • Bandapalli OR, Geheeb M, Kobelt D, Kuehnle K, Elezkurtaj S, Herrmann J, Gressner AM, Weiskirchen R, Beule D, Blüthgen N, Herzel H, Franke C, Brand K (2006) Global analysis of host tissue gene expression in the invasive front of colorectal liver metastases. Int J Cancer 118:74–89

    Article  PubMed  CAS  Google Scholar 

  • Baum CL, Arpey CJ (2005) Normal cutaneous wound healing: clinical correlation with cellular and molecular events. Dermatol Surg 31:674–686

    Article  PubMed  CAS  Google Scholar 

  • Bellemare J, Roberge CJ, Bergeron D, Lopez-Vallé CA, Roy M, Moulin VJ (2005) Epidermis promotes dermal fibrosis: role in the pathogenesis of hypertrophic scars. J Pathol 206:1–8

    Article  PubMed  Google Scholar 

  • Bellini A, Mattoli S (2007) The role of the fibrocyte, a bone marrow-derived mesenchymal progenitor, in reactive and reparative fibroses. Lab Invest 87:858–870

    Article  PubMed  CAS  Google Scholar 

  • Bucala R, Spiegel LA, Chesney J, Hogan M, Cerami A (1994) Circulating fibrocytes define a new leukocyte subpopulation that mediates tissue repair. Mol Med 1:71–81

    PubMed  CAS  Google Scholar 

  • Chernyavsky AI, Arredondo J, Marubio LM, Grando SA (2004) Differential regulation of keratinocyte chemokinesis and chemotaxis through distinct nicotinic receptor subtypes. J Cell Sci 117:5665–5679

    Article  PubMed  CAS  Google Scholar 

  • Chesney J, Metz C, Stavitsky AB, Bacher M, Bucala R (1998) Regulated production of type I collagen and inflammatory cytokines by peripheral blood fibrocytes. J Immunol 160:419–425

    PubMed  CAS  Google Scholar 

  • Choi YH, Burdick MD, Strieter RM (2010) Human circulating fibrocytes have the capacity to differentiate osteoblasts and chondrocytes. Int J Biochem Cell Biol 42:662–671

    Article  PubMed  CAS  Google Scholar 

  • Cooke JP (2007) Angiogenesis and the role of the endothelial nicotinic acetylcholine receptor. Life Sci 80:2347–2351

    Article  PubMed  CAS  Google Scholar 

  • Flaherty M, Chojkier M (1986) Selective inhibition of collagen synthesis by the Ca2+ ionophore A23187 in cultured human fibroblasts. J Biol Chem 261:12060–12065

    PubMed  CAS  Google Scholar 

  • Follonier Castella L, Gabbiani G, McCulloch CA, Hinz B (2010) Regulation of myofibroblast activities: calcium pulls some strings behind the scene. Exp Cell Res 316:2390–2401

    Article  PubMed  CAS  Google Scholar 

  • Funayama E, Chodon T, Oyama A, Sugihara T (2003) Keratinocytes promote proliferation and inhibit apoptosis of the underlying fibroblasts: an important role in the pathogenesis of keloid. J Invest Dermatol 121:1326–1331

    Article  PubMed  CAS  Google Scholar 

  • Gahring LC, Rogers SW (2006) Neuronal nicotinic acetylcholine receptor expression and function on nonneuronal cells. AAPS J 7:E885–E894

    Article  PubMed  Google Scholar 

  • Gomperts BN, Strieter RM (2007) Fibrocytes in lung disease. J Leukoc Biol 82:449–456

    Article  PubMed  CAS  Google Scholar 

  • Grando SA (1997) Biological functions of keratinocyte cholinergic receptors. J Invest Dermatol Symp Proc 2:41–48

    CAS  Google Scholar 

  • Grando SA, Kist DA, Qi M, Dahl MV (1993) Human keratinocytes synthesize, secrete, and degrade acetylcholine. J Invest Dermatol 101:32–36

    Article  PubMed  CAS  Google Scholar 

  • Grando SA, Horton RM, Pereira EF, Diethelm-Okita BM, George PM, Albuquerque EX, Conti-Fine BM (1995) A nicotinic acetylcholine receptor regulating cell adhesion and motility is expressed in human keratinocytes. J Invest Dermatol 105:774–781

    Article  PubMed  CAS  Google Scholar 

  • Guan DW, Ohshima T, Kondo T (2000) Immunohistochemical study on Fas and Fas ligand in skin wound healing. Histochem J 32:85–91

    Article  PubMed  CAS  Google Scholar 

  • Hashimoto N, Jin H, Liu T, Chensue SW, Phan SH (2004) Bone marrow derived progenitor cells in pulmonary fibrosis. J Clin Invest 113:243–252

    PubMed  CAS  Google Scholar 

  • Herber DL, Severance EG, Cuevas J, Morgan D, Gordon MN (2004) Biochemical and histochemical evidence of nonspecific binding of alpha7nAChR antibodies to mouse brain tissue. J Histochem Cytochem 52:1367–1376

    Article  PubMed  CAS  Google Scholar 

  • Hogg RC, Raggenbass M, Bertrand D (2003) Nicotinic acetylcholine receptors: from structure to brain function. Rev Physiol Biochem Pharmacol 147:1–46

    Article  PubMed  CAS  Google Scholar 

  • Hong KM, Burdick MD, Phillips RJ, Heber D, Strieter RM (2005) Characterization of human fibrocytes as circulating adipocyte progenitors and the formation of human adipose tissue in SCID mice. FASEB J 19:2029–2031

    PubMed  CAS  Google Scholar 

  • Karlin A (2002) Emerging structure of the nicotinic acetylcholine receptors. Nat Rev Neurosci 3:102–114

    Article  PubMed  CAS  Google Scholar 

  • Karrer S, Bosserhoff AK, Weiderer P, Landthaler M, Szeimies RM (2004) Keratinocyte-derived cytokines after photodynamic therapy and their paracrine induction of matrix metalloproteinases in fibroblasts. Br J Dermatol 151:776–783

    Article  PubMed  CAS  Google Scholar 

  • Kawashima K, Fujii T (2008) Basic and clinical aspects of non-neuronal acetylcholine: overview of non-neuronal cholinergic systems and their biological significance. J Pharmacol Sci 106:167–173

    Article  PubMed  CAS  Google Scholar 

  • Klapproth H, Reinheimer T, Metzen J, Munch M, Bittinger F, Kirkpatrick CJ, Hohle KD, Schemann M, Racke K, Wessler I (1997) Non-neuronal acetylcholine, a signalling molecule synthesized by surface cells of rat and man. Naunyn-Schmiedebergs Arch Pharmacol 355:515–523

    Article  PubMed  CAS  Google Scholar 

  • Kummer W, Lips KS, Pfeil U (2008) The epithelial cholinergic system of the airways. Histochem Cell Biol 130:219–234

    Article  PubMed  CAS  Google Scholar 

  • Kurzen H, Wessler I, Kirkpatrick CJ, Kawashima K, Grando SA (2007) The non-neuronal cholinergic system of human skin. Horm Metab Res 39:125–135

    Article  PubMed  CAS  Google Scholar 

  • Liu RH, Mizuta M, Matsukura S (2004) The expression and functional role of nicotinic acetylcholine receptors in rat adipocytes. J Pharmacol Exp Ther 310:52–58

    Article  PubMed  CAS  Google Scholar 

  • Mann A, Breuhahn K, Schirmacher P, Blessing M (2001) Keratinocyte-derived granulocyte-macrophage colony stimulating factor accelerates wound healing: stimulation of keratinocyte proliferation, granulation tissue formation, and vascularization. J Invest Dermatol 117:1382–1390

    Article  PubMed  CAS  Google Scholar 

  • Metz CN (2003) Fibrocytes: a unique cell population implicated in wound healing. Cell Mol Life Sci 60:1342–1350

    Article  PubMed  CAS  Google Scholar 

  • Metz CN, Tracey KJ (2005) It takes nerve to dampen inflammation. Nat Immunol 6:756–757

    Article  PubMed  CAS  Google Scholar 

  • Mielke JG, Mealing GA (2009) Cellular distribution of the nicotinic acetylcholine receptor alpha7 subunit in rat hippocampus. Neurosci Res 65:296–306

    Article  PubMed  CAS  Google Scholar 

  • Misery L (2004) Nicotine effects on skin: are they positive or negative? Exp Dermatol 13:665–670

    Article  PubMed  CAS  Google Scholar 

  • Moore BB, Kolodsick JE, Thannickal VJ, Cooke K, Moore TA, Hogaboam C, Wilke CA, Toews GB (2005) CCR2-mediated recruitment of fibrocytes to the alveolar space after fibrotic injury. Am J Pathol 166:675–684

    Article  PubMed  CAS  Google Scholar 

  • Mori L, Bellini A, Stacey MA, Schmidt M, Mattoli S (2005) Fibrocytes contribute to the myofibroblast population in wounded skin and originate from the bone marrow. Exp Cell Res 304:81–90

    Article  PubMed  CAS  Google Scholar 

  • Moser N, Mechawar N, Jones I, Gochberg-Sarver A, Orr-Urtreger A, Plomann M, Salas R, Molles B, Marubio L, Roth U, Maskos U, Winzer-Serhan U, Bourgeois JP, Le Sourd AM, De Biasi M, Schröder H, Lindstrom J, Maelicke A, Changeux JP, Wevers A (2007) Evaluating the suitability of nicotinic acetylcholine receptor antibodies for standard immunodetection procedures. J Neurochem 102:479–492

    Article  PubMed  CAS  Google Scholar 

  • Neumann S, Razen M, Habermehl P, Meyer CU, Zepp F, Kirkpatrick CJ, Wessler I (2007) The non-neuronal cholinergic system in peripheral blood cells: effects of nicotinic and muscarinic receptor antagonists on phagocytosis, respiratory burst and migration. Life Sci 80:2361–2364

    Article  PubMed  CAS  Google Scholar 

  • Ong CT, Khoo YT, Tan EK, Mukhopadhyay A, Do DV, Han HC, Lim IJ, Phan TT (2007) Epithelial–mesenchymal interactions in keloid pathogenesis modulate vascular endothelial growth factor expression and secretion. J Pathol 211:95–108

    Article  PubMed  CAS  Google Scholar 

  • Orr-Urtreger A, Göldner FM, Saeki M, Lorenzo I, Goldberg L, De Biasi M, Dani JA, Patrick JW, Beaudet AL (1997) Mice deficient in the alpha7 neuronal nicotinic acetylcholine receptor lack alpha-bungarotoxin binding sites and hippocampal fast nicotinic currents. J Neurosci 17:9165–9171

    PubMed  CAS  Google Scholar 

  • Osborne-Hereford AV, Rogers SW, Gahring LC (2008) Neuronal nicotinic alpha7 receptors modulate inflammatory cytokine production in the skin following ultraviolet radiation. J Neuroimmunol 193:130–139

    Article  PubMed  CAS  Google Scholar 

  • Papke RL, Bencherif M, Lippiello P (1996) An evaluation of neuronal nicotinic acetylcholine receptor activation by quaternary nitrogen compounds indicates that choline is selective for the alpha 7 subtype. Neurosci Lett 213:201–204

    PubMed  CAS  Google Scholar 

  • Phillips RJ, Burdick MD, Hong K, Lutz MA, Murray LA, Xue YY, Belperio JA, Keane MP, Strieter RM (2004) Circulating fibrocytes traffic to the lungs in response to CXCL12 and mediate fibrosis. J Clin Invest 114:438–446

    PubMed  CAS  Google Scholar 

  • Quan TE, Cowper S, Wu SP, Bockenstedt LK, Bucala R (2004) Circulating fibrocytes: collagen-secreting cells of the peripheral blood. Int J Biochem Cell Biol 36:598–606

    Article  PubMed  CAS  Google Scholar 

  • Rehan VK, Wang Y, Sugano S, Romero S, Chen X, Santos J, Khazanchi A, Torday JS (2005) Mechanism of nicotine-induced pulmonary fibroblast transdifferentiation. Am J Physiol Lung Cell Mol Physiol 289:L667–L676

    Article  PubMed  CAS  Google Scholar 

  • Sakai N, Wada T, Yokoyama H, Lipp M, Ueha S, Matsushima K, Kaneko S (2006) Secondary lymphoid tissue chemokine (SLC/CCL21)/CCR7 signaling regulates fibrocytes in renal fibrosis. Proc Natl Acad Sci USA 103:14098–14103

    Article  PubMed  CAS  Google Scholar 

  • Schmidt M, Sun G, Stacey MA, Mori L, Mattoli S (2003) Identification of circulating fibrocytes as precursors of bronchial myofibroblasts in asthma. J Immunol 171:380–389

    PubMed  CAS  Google Scholar 

  • Sekhon HS, Keller JA, Proskocil BJ, Martin EL, Spindel ER (2002) Maternal nicotine exposure upregulates collagen gene expression in fetal monkey lung. Association with alpha7 nicotinic acetylcholine receptors. Am J Respir Cell Mol Biol 26:31–41

    PubMed  CAS  Google Scholar 

  • Shephard P, Martin G, Smola-Hess S, Brunner G, Krieg T, Smola H (2004) Myofibroblast differentiation is induced in keratinocyte-fibroblast co-cultures and is antagonistically regulated by endogenous transforming growth factor-beta and interleukin-1. Am J Pathol 164:2055–2066

    Article  PubMed  CAS  Google Scholar 

  • Su X, Lee JW, Matthay ZA, Mednick G, Uchida T, Fang X, Gupta N, Matthay MA (2007) Activation of the alpha7 nAChR reduces acid-induced acute lung injury in mice and rats. Am J Respir Cell Mol Biol 37:186–192

    Article  PubMed  CAS  Google Scholar 

  • Tracey KJ (2002) The inflammatory reflex. Nature 420:853–859

    Article  PubMed  CAS  Google Scholar 

  • Wang H, Yu M, Ochani M, Amella CA, Tanovic M, Susarla S, Li JH, Wang H, Yang H, Ulloa L, Al-Abed Y, Czura CJ, Tracey KJ (2003) Nicotinic acetylcholine receptor alpha7 subunit is an essential regulator of inflammation. Nature 421:384–388

    Article  PubMed  CAS  Google Scholar 

  • Werner S, Grose R (2003) Regulation of wound healing by growth factors and cytokines. Physiol Rev 83:835–870

    PubMed  CAS  Google Scholar 

  • Xiu J, Nordberg A, Zhang JT, Guan ZZ (2005) Expression of nicotinic receptors on primary cultures of rat astrocytes and up-regulation of the alpha7, alpha4 and beta2 subunits in response to nanomolar concentrations of the beta-amyloid peptide(1–42). Neurochem Int 47:281–290

    Article  PubMed  CAS  Google Scholar 

  • Yang L, Scott PG, Dodd C, Medina A, Jiao H, Shankowsky HA, Ghahary A, Tredget EE (2005) Identification of fibrocytes in postburn hypertrophic scar. Wound Repair Regen 13:398–404

    Article  PubMed  Google Scholar 

  • Yu TS, Cheng ZH, Li LQ, Zhao R, Fan YY, Du Y, Ma WX, Guan DW (2010) The cannabinoid receptor type 2 is time-dependently expressed during skeletal muscle wound healing in rats. Int J Legal Med 124:397–404

    Article  PubMed  Google Scholar 

  • Zia S, Ndoye A, Lee TX, Webber RJ, Grando SA (2000) Receptor mediated inhibition of keratinocyte migration by nicotine involves modulations of calcium influx and intracellular concentration. J Pharmacol Exp Ther 293:973–981

    PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This study was financially supported in part by grants from the Research Fund for the Doctoral Program funded by the Ministry of Education of China (200801590020) and National Natural Science Foundation of China (30271347).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Da-Wei Guan.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Fan, YY., Yu, TS., Wang, T. et al. Nicotinic acetylcholine receptor alpha7 subunit is time-dependently expressed in distinct cell types during skin wound healing in mice. Histochem Cell Biol 135, 375–387 (2011). https://doi.org/10.1007/s00418-011-0798-y

Download citation

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00418-011-0798-y

Keywords

Navigation