Experimental Lung Research, Early Online, 1–10, 2011 Copyright © Informa Healthcare USA, Inc. ISSN: 0190-2148 print / 1521-0499 online DOI: 10.3109/01902148.2010.545471 An exon 5-less splice variant of the extracellular calcium-sensing receptor rescues absence of the full-length receptor in the developing mouse lung Brenda Finney, 1,2 William J. Wilkinson, 2 Lydia Searchfield, 2 Martin Cole, 2 Stacey Bailey, 2 Paul J. Kemp, 2 and Daniela Riccardi 2 1 Center for Cardiovascular Sciences, Institute for Biomedical Research, University of Birmingham, Edgbaston, United Kingdom 2 Division of Pathophysiology and Repair, Cardiff University, Cardiff, United Kingdom ABSTRACT The authors have recently demonstrated that, in the developing mouse lung, fetal plasma Ca 2+ suppresses branching morphogenesis and cell proliferation while promoting fluid secretion via activation of the extracellular Ca 2+ -sensing receptor (CaSR). The aim of the current study was to further elucidate the role of Ca 2+ in lung development by studying the effects of extracellular Ca 2+ on fetal lung development in mice lacking the CaSR. These mice were produced by exon 5 deletion in the CaSR gene. Since such a maneuver has been known to induce the expression of an exon 5-less splice variant of the CaSR in some tissues, the molecular and functional expression of this splice variant in the developing mouse lung was also investigated. Whereas there was a mild in vivo phenotype observed in these mice, in vitro sensitivity of Casr −/− lung explants to specific activators of the CaSR was unaffected. These results imply that compensatory expression of an exon 5-less splice variant rescues CaSR function in this mouse model and therefore a lung-specific, complete CaSR knockout model must be developed to fully appreciate the role for this receptor in lung development and the contribution of its ablation to postnatal respiratory disease. KEYWORDS branching morphogenesis, exon 5-less splice variant, extracellular calcium-sensing receptor, lung development Using the mouse lung explant culture model and chemically defined, serum-free conditions, we have recently demonstrated that extracellular calcium ions (Ca 2+ o ) are an important extrinsic factor that profoundly affects the intrinsic lung developmental program [1]. Indeed, we have shown that concen- trations of Ca 2+ o reminiscent of those seen during gestation (i.e., 1.7 mM) exert a physiological brake on lung branching morphogenesis and cellular pro- liferation while promoting lung fluid secretion. In contrast, exposing the developing lungs to concen- trations of Ca 2+ o similar to those seen in the adult (i.e., 1.05 to 1.2 mM) produced diametrically opposite effects, with an increase in branching Received 5 October 2010; accepted 28 November 2010. Address correspondence to Brenda Finney, PhD, Center for Cardiovascular Sciences, Institute for Biomedical Research, University of Birmingham, Edgbaston, B15 2TT United Kingdom. E-mail: b.a.finney@bham.ac.uk morphogenesis and a suppression of fluid secretion. Furthermore, we have shown that these effects are mediated by the G protein–coupled, extracel- lular Ca 2+ -sensing receptor (CaSR), through phospholipase C and phosphoinositide 3 kinase signaling. Strikingly, CaSR expression in the mouse lung is developmentally regulated and restricted to between embryonic day 10.5 (E10.5) and E16.5, coinciding with times when lung branching morphogenesis occurs and, consistent with other species [2, 3], it is absent from the postnatal lung [1]. Yet, in human subjects carrying inactivating mutations in the CaSR gene, the incidence of interstitial lung disease is above that of the normal population [4, 5], suggesting that partial loss of CaSR could somehow have a negative impact on respiratory function. Given the role of the CaSR in the developing lung and its absence in the adult tissue, we hypothesized that altered CaSR function 1 Exp Lung Res Downloaded from informahealthcare.com by University of Birmingham on 03/04/11 For personal use only.