A) Proliferation rate as determined by Ki67 index was unaltered inPTH-KL/mice (40 magnification). localization of NFATC2, was constitutively activated inPTH-KL/mice. Treatment with the calcineurin-inhibitor cyclosporine A abolished FGF23-mediated PTH suppression inPTH-KL/mice whereas wild-type mice remained responsive. Comparable results were observed in thyro-parathyroid explantsex vivo. Collectively, we present genetic and functional evidence for a novel, Klotho-independent, calcineurin-mediated FGF23 signaling pathway in parathyroid glands that mediates suppression of PTH. The presence of Klotho-independent FGF23 effects in a Klotho-expressing target organ represents a paradigm shift in the conceptualization of FGF23 endocrine action. == Author Summary == Inorganic calcium is a critical element for a diverse range of cellular processes ranging from cell signaling to energy metabolism, and its extracellular concentration is usually controlled by parathyroid hormone (PTH). Klotho is usually expressed in parathyroid chief cells and reported to facilitate PTH secretion during hypocalcemia and mediate FGF23 suppression of PTH synthesis and secretion. To dissect the role of parathyroid Klotho in health and disease, we generated parathyroid-specificKlothoknockout mice. The mutant mice had normal serum levels of PTH and calcium. Further, their parathyroid sensitivity to acute fluctuations in serum calcium and response to FGF23 treatment were preserved, and mutant mice developed secondary hyperparathyroidism of comparable magnitude as wild-type mice when challenged with renal failure. A Kaempferol-3-O-glucorhamnoside previously unknown parathyroid FGF23 signaling pathway involving calcineurin was constitutively activated in the mutant mice, and blocking this pathway abolished FGF23-induced suppression of PTH secretion. Our data challenges the concepts of Klotho as a mandatory factor for the acute hypocalcemic PTH response and decreased Klotho abundance as a pathogenic factor in secondary hyperparathyroidism. Finally, the presence of Klotho-independent FGF23 effects in a Klotho-expressing target organ represents a paradigm shift in the conceptualization of FGF23 endocrine action. == Introduction == Calcium plays a pivotal role in many biological processes, such as intra-cellular signaling, cell membrane depolarization and excitation, energy metabolism and skeletal mineralization. Accordingly, a fine-tuned regulation of serum calcium level is usually a prerequisite for normal cellular and organ function in most organisms. Parathyroid hormone (PTH) is the principal hormonal regulator of circulating calcium as it rapidly increases its renal tubular reabsorption and mobilization from bone deposits in response to a decrease in serum calcium[1]. In turn, free calcium ions can efficiently inhibit PTH secretion as part of Kaempferol-3-O-glucorhamnoside an endocrine feedback loop mediated by the calcium-sensing receptor (CaSR) located on parathyroid chief cells[2]. Type I membrane-bound alpha-Klotho (Klotho) defines tissue specificity for the phosphaturic hormone fibroblast growth factor-23 (FGF23) by acting as a permissive co-receptor[3]. Klotho is usually predominantly expressed in organs requiring abundant calcium transport such as kidneys, parathyroid glands and choroid plexus[4]. In the parathyroids, FGF23 binds to binary complexes of an FGF receptor (FGFR) and Klotho to suppress PTH secretion[5],[6]. Klotho activity on the other hand has been implicated as fundamental for the stimulation of PTH secretion during hypocalcemic conditions[7], although the underlying mechanism has been challenged[8]. Secondary hyperparathyroidism (sHPT) is usually a common manifestation in chronic kidney disease (CKD) despite markedly increased serum FGF23 concentrations. This presumably reflects parathyroid resistance to FGF23 action, which was also supported by lack of response to FGF23 injections in a rat model of CKD[9],[10]. The proposed mechanism underlying such FGF23 resistance is usually decreased abundance of parathyroid Klotho and FGFRs[11],[12]. To dissect the role of parathyroid gland resident Klotho in physiology and in pathophysiological says such as CKD, we generated a novel mouse strain harboring a Kaempferol-3-O-glucorhamnoside parathyroid-specific deletion of theKlothogene. The present study sheds new light around the function of parathyroid Klotho and identifies a novel, Klotho-independent signaling pathway of FGF23 that is involved in the regulation of PTH secretion. == Results == == Generation ofPTH-KL/mice == Mice with a parathyroid specific deletion of Klotho (PTH-KL/) were generated using Cre-LoxP recombination (Physique S1). Floxed Klotho mice were crossed with mice expressing Rabbit Polyclonal to CNTN2 Cre recombinase driven by the human PTH promoter, which was previously shown to have Cre activity exclusively in the parathyroid glands[13]. Successful deletion of parathyroid Klotho protein was confirmed with immunohistochemical staining of thyro-parathyroid tissue (Physique 1). Overall efficiency of deletion varied, and was up to >90% in investigated samples. Subanalyses of mice with the most efficient deletion showed similar results to the full analyses. == Physique 1. Tissue-specific deletion of theKlothogene. == Upper left panel. Immunohistochemical staining confirmed successful deletion of Klotho in parathyroid glands ofPTH-KL/mice. 20 magnification.Upper right panel. The gross appearance.
