Characterizing the TT Cells as a Novel In- Vitro Model for the Calcium-Sensing Receptor and Unveiling the Potential of VIF Peptide as a Novel Endogenous Positive Modulator

Abstract

Chronic Kidney Disease (CKD) represents a significant global health problem. One of CKD’s most serious complications is a mineral and bone disorder (CKD-MBD) which comprises secondary hyperparathyroidism (SHPT) and vascular calcification (VC). The calcium-sensing receptor (CaSR) is the key controller of mineral homeostasis and specifically of parathyroid hormone (PTH) secretion and is thus an ideal therapeutic target for SHPT and CKD-MBD. However, the lack of a reliable, parathyroid gland (PTG)-derived cell line remains a significant experimental challenge, with most CaSR research performed on artificially transfected HEK-293 cells. While CaSR-HEK cells provide valuable insights into the CaSR pharmacology, having a calciotropic cell line naturally expressing the CaSR would significantly advance our comprehension of the CaSR pharmacology. Thus, my first objective was to characterise the CaSR activity in human thyroidal C (TT) cells, that express CaSR endogenously. TT cells were shown to be sensitive to extracellular Ca2+ (Ca2+o) and to other CaSR positive modulators by their eliciting intracellular Ca2+ (Ca2+i) mobilisation and calcitonin secretion. This was abolished by cotreatment with the selective CaSR negative allosteric modulator (NAM) NPS-2143, proving the responses to be CaSR mediated. Moreover, treating TT cells with selective CaSR positive allosteric modulators (PAMs) in Ca2+-free buffer, still elicited an increase in Ca2+i concentration, confirming the Ca2+ mobilisation to be released from intracellular Ca2+ stores. Additionally, raising the extracellular phosphate (Pi) concentration from 0.8 mM (physiological) to 2 mM (CKD-like) elicited reductions in both CaSR-mediated Ca2+i mobilisation, and, calcitonin secretion even when Ca2+o was corrected for likely Ca x Pi precipitation. Furthermore, TT cell CaSR responses were substantially attenuated by the Gq11-selective inhibitor YM254890 confirming signal mediation via the Gq/11 pathway. Collectively, these results validate the use of TT cells as a useful in vitro model for the study of the CaSR pharmacology. Being one of the only human, calciotropic cell-lines to naturally express the CaSR, I next examined whether TT cells could be used to characterise transcriptomic changes downstream of sustained CaSR activation, with or without hyperphosphataemia and calcitriol treatment. Prolonged exposure to high Pi failed to alter the CaSR mRNA and protein expression levels. RNA-seq analysis also showed no significant effect of CaSR inhibition. In contrast, overnight exposure of TT cells to calcitriol significantly upregulated the CaSR mRNA expression and protein abundance. Furthermore, RNA-seq analysis of calcitriol-treated TT cells revealed three novel, and potentially calciotropic, genes namely AMZ1, TSK, and PRSS33. Together, these findings indicate the utility of TT cells for examining transcriptomic changes in mineral homeostasis though perhaps not specifically for the CaSR modulation. Lastly, I investigated the newly identified vasoconstriction inhibiting factor (VIF) peptide, a chromogranin A fragment, on the CaSR signalling in CaSR-HEK cells. When added to subthreshold Ca2+o levels, VIF increased Ca2+i mobilisation but had no direct effect when only basal calcium levels were present. This demonstrates for the first time that VIF may function as a CaSR PAM. This CaSR potentiation of VIF was abolished by NPS-2143, suggesting a CaSR-specific effect. Cells treated with VIF peptide gave Ca2+o concentration curves significantly different to those with Ca2+o treatment alone. Specifically, VIF increased the efficacy of Ca2+o agonism (its Emax) but with little change on the CaSR’s sensitivity to Ca2+o. This is the first evidence of a CaSR modulator endogenous to the parathyroid gland itself and could represent a short-loop feedback modulator of PTH secretion. In summary, these findings contribute to our understanding of the CaSR pharmacology and provide valuable insights that could inform future drug development efforts targeting the most serious complications of CKD-MBD.