Extracellular calcium-sensing receptor mediates human bronchial epithelial wound repair Javier Milara a, *, Manuel Mata a , Adela Serrano a , Teresa Peiro ´ a , Esteban J. Morcillo b,c,d , Julio Cortijo a,b,c a Research Unit, University General Hospital Consortium, Av. Tres Cruces s/n E-46014 Valencia, Spain b Department of Pharmacology, Faculty of Medicine, University of Valencia, Av. Blasco Ibanez 17, E-46010 Valencia, Spain c CIBERES, Health Institute Carlos III, Valencia, Spain d Clinical Pharmacology Unit, University Clinic Hospital, Av. Blasco Ibanez 17, E-46010 Valencia, Spain 1. Introduction The airway epithelium acts as a protective barrier preventing the exposure of the underlying tissue to noxious particles. The epithelium is routinely challenged by allergens and inhaled air pollutants, resulting in a damage that requires repair to restore barrier integrity. Damage is also commonly seen in diseases such as asthma or chronic obstructive pulmonary disease (COPD) among others [1,2]. Furthermore, the epithelium is not only a passive barrier, but also a source of inflammatory cytokines [2]. The mechanisms and regulators of epithelial repair are poorly understood. In this process a common sequence of injury and wound repair have been described in vivo [2]. The first step in epithelial repair is the migration of the basal cells neighboring the wound, followed by proliferation and active mitosis and squamous metaplasia. Finally, progressive redifferentiation with the emergence of preciliated cells (mixed phenotype of ciliated cells with mucous secretory granules) followed by ciliogenesis and complete regeneration of a pseudostratified mucociliary epithelium complete epithelial repair [3]. Numer- ous cellular and molecular factors are involved in the repair and regeneration of the airway epithelium. These factors are modulated by the matrix metalloproteinases (MMPs), cytokines, and growth factors released by the epithelial and mesenchymal cells [2]. In the initial damage subsequent to an inflammatory context, epithelial cells may communicate with each other through the fast propagation of intracellular Ca 2+ waves helping neighbouring cells to induce migration and proliferation in order to wounding repair [4,5]. Among the factors and mechanisms that produce Ca 2+ waves after airway epithelial injury, extracellular Ca 2+ ([Ca 2+ ] out ), and Biochemical Pharmacology 80 (2010) 236–246 ARTICLE INFO Article history: Received 27 January 2010 Accepted 30 March 2010 Keywords: Calcium-sensing receptor Airway epithelial wound repair Migration Proliferation ABSTRACT The airway epithelium routinely undergoes damage that requires repair to restore epithelial barrier integrity. Cell migration followed by proliferation are necessary steps to achieve epithelial repair. Calcium-sensing receptor (CaSR) is implicated in cell migration and proliferation processes. Thus we hypothesized that CaSR mediates lung epithelial wound repair. We detected CaSR expression in human lung and in well-differentiated human bronchial epithelial cells (HBEC). To test the CaSR functionality, HBEC loaded with fura-2 were stimulated with extracellular Ca 2+ ([Ca 2+ ] out ) which resulted in a concentration-dependent intracellular Ca 2+ ([Ca 2+ ] i ) increase (potency  5.6 mM [Ca 2+ ] out ). Further- more, increasing [Ca 2+ ] out induced phosphorylation of the extracellular signal-regulated kinase (ERK1/2) which was blocked by siRNA-CaSR and the specific inhibitor of CaSR, NPS2390. Epithelial repair after mechanical injury of differentiated HBEC was a process dependent of [Ca 2+ ] out since it accelerated wound repair and HBEC proliferation being highest at 5 mM [Ca 2+ ] out . Furthermore, U73122 (an inhibitor of phospholipase C (PLC)) and PD 98059 (an inhibitor of ERK1/2) as well as siRNA- CaSR and NPS2390 partially inhibited wound repair and HBEC proliferation. On the other hand, mechanical injury produced an [Ca 2+ ] i wave propagation that was partially inhibited by siRNA-CaSR, NPS2390 and the extracellular Ca 2+ chelator EGTA, which suggest a link of CaSR between cell–cell communication and wound repair in differentiated HBEC. Our data, for the first time, shows that CaSR plays an important role in airway epithelial repair, which may help to develop novel regenerative therapeutics allowing the rapid repair of lung damaged epithelium. ß 2010 Elsevier Inc. All rights reserved. * Corresponding author at: Unidad de Investigacio ´ n, Consorcio Hospital General Universitario, Avenida Tres Cruces s/n, E-46014 Valencia, Spain. Tel.: +34 620231549, fax: +34 961972145. E-mail addresses: xmilara@hotmail.com (J. Milara), manuel.mata@uv.es (M. Mata), adela_serrano@hotmail.com (A. Serrano), tepeisal@alumni.uv.es (T. Peiro ´ ), Esteban.Morcillo@uv.es (E.J. Morcillo), julio.cortijo@uv.es (J. Cortijo). Contents lists available at ScienceDirect Biochemical Pharmacology journal homepage: www.elsevier.com/locate/biochempharm 0006-2952/$ – see front matter ß 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bcp.2010.03.035