Parathyroid Hormone-Related Protein (1-40) Enhances Calcium Uptake in Rat Enterocytes Through PTHR1 Receptor and Protein Kinase Cα/β Signaling

Abstract

Background/Aims: Parathyroid hormone-related protein (PTHrP) is implicated in regulating calcium homeostasis in vertebrates, including sea bream, chick, and mammals. However, the molecular mechanism underlying the function of PTHrP in regulating calcium transport is still not fully investigated. This study aimed to investigate the effect of PTHrP on the calcium uptake and its underlying molecular mechanism in rat enterocytes. Methods: The rat intestinal epithelial cell line (IEC-6) was used. Calcium uptake was determined by using the fluo-4 acetoxymethyl ester fluorescence method. The expression levels of RNAs and proteins was assessed by RT-PCR and Western-blot, respectively. Results: PTHrP (1-40) induced rapid calcium uptake in enterocytes in a dose-dependent manner. PTHrP (1-40) up-regulated parathyroid hormone 1 receptor (PTHR1) protein but not 1,25D3-MARRS receptor. Pre-treatment of anti- PTHR1 antibody abolished the PTHrP (1-40)-induced calcium uptake. PTHrP (1-40) significantly up-regulated four transcellular calcium transporter proteins, potential vanilloid member 6 (TRPV6), calbindin-D9k (CaBP-D9k), sodium-calcium exchanger 1 (NCX1) and plasma membrane calcium ATPase 1 (PMCA1), in a dose- and time-dependent manner. Pre-treatment with TRPV6 or CaBP-D9k antibodies or knockout of rat TRPV6 or CaBP-D9k markedly inhibited PTHrP (1-40)-induced calcium uptake, whereas inhibition of NCX or PMCA1 by antibodies or inhibitors had no effect on PTHrP(1-40)-induced calcium uptake. Furthermore, PTHrP (1-40) treatment up-regulated protein levels of protein kinase C (PKC α/β) and protein kinase A (PKA). Pretreatment of PKC α/β inhibitor (but not PKA inhibitor) inhibited PTHrP (1-40)-induced calcium uptake. Conclusion: PTHrP (1-40) stimulates calcium uptake via PTHR1 receptor and PKC α/β signaling pathway in rat enterocytes, and calcium transporters TRPV6 and CaBP-D9k are necessary for this stimulatory effect.