QS289. RGS5 Modulates Human Calcium Sensing Receptor and Is Variably Expressed in Primary Hyperparathyroidism

Abstract

Introduction: Primary hyperparathyroidism (pHPT) is associated with dysregulated calcium metabolism leading to hypercalcemia, hypercalciuria, kidney stones and osteoporosis. Parathyroid chief cells seem to have a diminished capacity to sense calcium resulting in inappropriate secretion of PTH. While several mechanisms of abnormal parathyroid cell growth have been described, the molecular etiology of calcium dysregulation in pHPT is not understood. Mutations in the g-protein coupled calcium sensing receptor (CASR) have not been described in sporadic pHPT. Dysregulated downstream signaling from CASR are a plausible explanation for inappropriate PTH secretion in pHPT. To investigate abnormal mechanisms of CASR signaling in pHPT we sought to identify parathyroid expression of CASR pathway genes using microarray followed by molecular characterization of candidates. Methods: Gene expression data (Operon Biotechnologies Inc., Huntsville AL) was generated on 41 parathyroid adenomas and 12 normal paired parathyroid biopsies. K-means clustering was carried after lowess data normalization. Quantitative-PCR and western blotting was used to validate array data observations. For in vitro gene experiments, transfections were carried out in a human embryonic kidney cell line (HEK 293). CASR-stimulated inositol phosphate mobilization was measured using an anion exchange chromatography assay. Results: K-means clustering of the adenoma array data identified regulator of g-protein signaling 5 (RGS5) to co-express with CASR. In paired adenoma-normal specimens, lowess normalized RGS5 expression was higher in the adenomas (Figure 1). Quantitative PCR studies confirmed the presence and over expression of RGS5 in parathyroid tumors. Western blotting confirmed RGS5 protein expression in parathyroid tissue lysates. To examine how RGS5 expression affected calcium sensing, RGS5 was co-transfected with CASR. CASR-stimulated inositol phosphate mobilization was significantly less in the RGS5/CASR co-transfected cells as compared to empty vector/CASR transfected cells (Figure 2). Conclusions: These results indicate that RGS5 is expressed in parathyroid adenomas and has the ability to attenuate calcium sensing through CASR. Inappropriate RGS5 expression may represent a novel mechanism of disease in pHPT patients. View Large Image Figure Viewer