Calcium Sensing Receptor Activation by Calcimimetic R-568 in Human Amniotic Fluid Mesenchymal Stem Cells: Correlation with Osteogenic Differentiation

Abstract

Human amniotic fluid mesenchymal stem cells (hAFMSCs) are promising for therapeutic applications in bone damage. Calcium sensing receptor (CaSR), a G protein-coupled receptor, plays a physiological role in the regulation of bone metabolism. Thus, the bone CaSR could be targeted by calcimimetic agonists, which may be potentially helpful in treating bone diseases. The aim of our study was to characterize CaSR expression in hAFMSCs and to assess the activity of calcimimetic R-568 during in vitro osteogenesis. Using western blotting, immunofluorescence, and flow cytometry, we consistently observed constitutive CaSR in osteo-differentiating hAFMSCs. Notably, both R-568 and calcium significantly enhanced hAFMSC osteogenic differentiation after exposure to osteogenic medium. To provide further evidence of the involvement of CaSR in osteogenesis, we correlated its expression with that of established osteogenic markers, that is, alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), and osteopontin (OPN), and novel, not yet completely defined regulators of osteogenesis. Among these are β-catenin and Slug, which are mediators of Wnt signaling, and nuclear factor of activated T cells c1 (NFATc1), which plays a critical role in calcium/calcineurin signaling. Taken together, our results demonstrate that CaSR is expressed in hAFMSCs, positively correlates with osteogenic markers, and is activated by R-568. Notably, downregulation of CaSR by RNA interference supports the conclusion that CaSR activation plays a central role in hAFMSC osteogenesis. Thus, this study provides significant information on the mechanisms of hAFMSC osteogenesis, which could provide additional molecular basis for the use of calcimimetics in bone regenerative medicine.

Osteogenic differentiation of amniotic fluid mesenchymal stromal cells and their bone regeneration potential

“…This effect was abolished by CaSR allosteric inhibitor Calhex231 and by selective inhibitor NPS2143. Importantly, downregulation of CaSR by a genesilencing approach confirmed the crucial role of CaSR in supporting osteogenic differentiation [44] . These findings support the role of calcimimetics in the osteogenic differentiation of AFMSCs, and suggest a strategy to develop therapy against bone injury.…”

“…They do not display any hematopoietic molecular markers (CD14, CD31 and CD45), while they express the intracellular stemness markers TERT, SOX2 and Nanog and the surface adhesion molecules CD29, CD58, CD166 and CD90 [44] . The absence of HLADR and presence of HLAABC suggest that these cells may be applicable in immune mediated disorders as well as in the treatment of graft versushost disease [43] .…”

“…Attempts have been made to produce osteoblastic cells from amniotic fluid samples in a very short time, using a single step culture procedure, which allows a 20d reduction in culture time [60] . One notes that several studies have demonstrated how such osteoinductive molecules are able to improve the osteogenic differentiation process [44,6368] . In detail, then, AFMSCs transfected with a defective adenoviral vector expressing human lim mineralization protein 3, an intracellular positive regulator of osteoblast differentiation, showed downregulation of stemness kruppellike factor4 and then increased osteogenic differentiation [63] .…”

“…Some studies have reported the great potential of ckit selected amniotic fluid cells prior to osteogenic differentiation [40] , while others have demonstrated that unselected cells are also able to properly differentiate into osteospecific cells [13,41,44,60] . In this review we mainly discuss the in vitro osteogenic differentiation of AFMSCs with occasional mention of papers in which authors use ckit selected cells, named AFSCs (Table 1).…”

“…Wnt signaling is also involved in AFMSC commitment toward osteogenesis [13] . Recently, we identified for the first time, the presence of Calcium Sensing Receptor (CaSR) in ovine and human AFMSCs [44,68] . CaSR, originally cloned from parathyroid glands, acts by controlling the secretion of parathyroid hormone in response to changes in extracellular calcium levels [75] .…”