Elsevier

Biochemical Pharmacology

Volume 80, Issue 2, 15 July 2010, Pages 236-246
Biochemical Pharmacology

Extracellular calcium-sensing receptor mediates human bronchial epithelial wound repair

https://doi.org/10.1016/j.bcp.2010.03.035Get rights and content

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 Ca2+ ([Ca2+]out) which resulted in a concentration-dependent intracellular Ca2+ ([Ca2+]i) increase (potency  5.6 mM [Ca2+]out). Furthermore, increasing [Ca2+]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 [Ca2+]out since it accelerated wound repair and HBEC proliferation being highest at 5 mM [Ca2+]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 [Ca2+]i wave propagation that was partially inhibited by siRNA-CaSR, NPS2390 and the extracellular Ca2+ 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.

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]. Numerous 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 Ca2+ waves helping neighbouring cells to induce migration and proliferation in order to wounding repair [4], [5]. Among the factors and mechanisms that produce Ca2+ waves after airway epithelial injury, extracellular Ca2+ ([Ca2+]out), and intracellular Ca2+ ([Ca2+]i) play a key role in these orchestrating communications [4], [5], [6].

It is well established in several cell types that the majority of Ca2+ released during intracellular signalling events is exported to the extracellular space, usually through the activity of the plasma membrane Ca2+ ATPase [7], [8]. As internal Ca2+ stores maintain a total [Ca2+] of several mM [9], and the ratio of extracellular space volume to cell volume is very small in a structurally intact tissue, substantial fluctuations in [Ca2+]out are expected to occur as a consequence.

Cell-surface proteins that can act as sensors of [Ca2+]out, have been observed in a number of biological systems. In particular, calcium-sensing receptor (CaSR) has been implicated as an important factor mediating the propagation of intercellular signals between cells [10]. CaSR was originally cloned from the parathyroid gland, but now it is known that it is expressed in a wide variety of mammalian tissues and cell types although its function remains to be elucidated [11]. The CaSR is cooperatively activated by Ca2+ ions over a concentration range of 0.5–10 mM resulting in a cell-type-specific coupling to intracellular signalling cascades such as phospholipase C (PLC)/InsP3/Ca2+, adenosine 3′,5′-cyclic monophosphate (cAMP) and MAP-kinase pathways [12]. CaSR has been identified in rat lung [13], and recently we have observed that nickel triggers intracellular Ca2+ mobilization and inflammatory mediators in human airway epithelial cells through the activation of CaSR [14], however its physiological role in human airway epithelium remains unclear.

Since CaSR mediates cell–cell communications through [Ca2+]out local levels, we hypothesized that the expression of CaSR on human bronchial epithelial cells (HBEC) may be related with epithelial repair. We found that the lack of expression or the inhibition of the CaSR may delay the process of airway epithelial wound repair in well-differentiated HBEC cultures which may contribute to the understanding of airway epithelial repair following lung injury.

Section snippets

Materials and methods

Unless otherwise stated, all reagents used were obtained from Sigma (Chemical Co., Madrid, Spain). U73122, NPS 2390, PD 98059, BAPTA-acetoxymethyl ester (AM) and Fura-2AM were disolved in dimethyl sulfoxide (DMSO) at 10 mM stock concentration. Several dilutions of the stocks were performed with cell culture medium. The final concentration of DMSO in the culture cell did not exceed 0.1% and had no significant pharmacological activity.

CaSR is expressed in human lung tissue

CaSR protein expression was detected in the human lung, mainly in epithelial cells from human bronchus (Fig. 1, panels a–d, representative of four patients).

The CaSR pattern of expression found in healthy lung tissue (Fig. 1b) corresponded with those patients with lung carcinoma suggesting that CaSR expression was not exclusively induced by tumour microenvironment. The ontogeny was similar when any of the two antibodies defined in Section 2 were employed. For brevity, only the data using

Discussion

The present study demonstrates that CaSR is expressed in human lung tissue and that it partially mediates wound repair process in well-differentiated HBEC. The evidence of CaSR in wound repair was assessed by the use of specific inhibitor and transient siRNA-CaSR. Furthermore, we observed that CaSR mediates [Ca2+]i waves after epithelial injury through local [Ca2+]out increases inducing PLC activation, [Ca2+]i elevation as well as ERK1/2 phosphorylation which in turn activates cell migration

Conclusions

The present study provides cellular evidence that CaSR actively participates in the repair of the bronchus epithelium after acute injury through rapid cell migration and posterior proliferation of HBEC. In this process CaSR activation promotes cell–cell communication through [Ca2+]i wave propagation which may be translated in a novel type of intercellular communication in the human airway epithelium.

Conflict of interest

The authors declare that they do not have any actual or potential conflict of interest including any financial, personal or other relationships with other people or organizations within three years of beginning this submitted work that could inappropriately influence, or be perceived to influence, our work.

Acknowledgements

This work was supported by grants SAF2005-00669/SAF2008-03113 (JC), SAF2006-01002/SAF2009-08913 (EJM), CIBERES (CB06/06/0027) and CAIBER (CAI08/01/0039) from Ministry of Science and Innovation and Health Institute ‘Carlos III’ of Spanish Government, and research grants from Regional Government (Prometeo/2008/045; ‘Generalitat Valenciana’). JM has a research contract from ‘Fondo de Investigaciones Sanitarias’ (FIS) of Health Institute ‘Carlos III’ of Ministry of Health (Spain).

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