307 INVOLVEMENT OF THE CALCIUM SENSING RECEPTOR IN GROWTH AND PROLIFERATION OF STEM CELLS FROM EQUINE UMBILICAL CORD MATRIX

Abstract

The calcium sensing receptor (CaSR) plays a key role in cells involved in calcium (Ca²⁺) homeostasis by directly sensing changes in extracellular Ca²⁺ concentration ([Ca²⁺]o), and external Ca²⁺ is a potent mediator of cell proliferation. The present study investigated the effects of high [Ca²⁺]o and of the CaSR agonist NPS R-467 on growth and proliferation of equine size-sieved umbilical cord matrix mesenchymal stem cells (UCM-MSC). The involvement of CaSR on observed cell response was analysed at the mRNA and protein level. Two subpopulations of UCM-MSC, isolated using multi-dishes with transwell inserts of 8-μm pores and expressing MSC markers (CD105, CD44, CD29; Corradetti et al. 2010 Reprod. Fertil. Dev. 22, 347–348), were analysed. Cells were cultured in medium containing: (A) low [Ca²⁺]o (0.37 mM), (B) high [Ca²⁺]o (2.87 mM), (C) NPS R-467 (3 μm) in the presence of high [Ca²⁺]o, and (D) the CaSR antagonist NPS 2390 (10 μm for 30′) followed by NPS R-467 in the presence of high [Ca²⁺]o. Growth and proliferation rates were compared among treatments (Student’s t-test). The CaSR expression and subcellular localization were investigated by real-time quantitative RT-PCR, immunofluorescence, and confocal microscopy. In the >8-μm cell line, the addition of NPS R-467, in the presence of [Ca²⁺]o, significantly increased cell growth after day 7 of culture (C v. A and B; P < 0.001). Increasing [Ca²⁺]o was not effective in this cell line (B v. A; not significant). In the <8-μm cell line, NPS R-467 increased cell growth, even at a lower extent (C v. A; P < 0.05), as observed on day 9 of culture. In this cell line, an increased proliferation rate was observed upon [Ca²⁺]o increase (B v. A; P < 0.05). In both cell lines, preincubation with NPS 2390 significantly inhibited the agonistic effect of NPS R-467. In both cell lines, a stimulatory effect of additional calcium and NPS R-467 on cell proliferation, in terms of reduced DT values, was observed. In the 2 cell lines, CaSR expression was down-regulated in the presence of high calcium and in NPS R-467-treated cells compared with controls (B and C v. A cells; P < 0.001). Treatment with high calcium or NPS R-467 reduced CaSR labelling in the cytosol and increased it at the cortical level. We found that CaSR is expressed at mRNA and protein levels in equine UCM-MSC, and it is functionally active because the selective CaSR agonist NPS R-467 induced a stimulatory effect on cell growth and proliferation, which was reversed by the CaSR antagonist NPS 2390. The different responses to treatments between the 2 UCM-MSC subpopulations suggest that CaSR could be differentially activated in these cell lines. The calcimimetic NPS R-467 might be useful as an adjunctive component of media for UCM-MSC culture to obtain enough cells for down-stream purposes.