No. 3] Proc. Jpn. Acad., Ser. B 85 (2009) 125 Review Discovery of -Klotho unveiled new insights into calcium and phosphate homeostasis By Yo-ichi NABESHIMAÃ1,Ã2;y (Communicated by Takao SEKIYA, M.J.A.) Abstract: -Klotho was first identified as the responsible gene in a mutant mouse line whose disruption results in a variety of premature aging-related phenotypes. -Klotho has been shown to participate in the regulation of parathyroid hormone secretion and trans-epithelial transport of Ca2þ in the choroid plexus and kidney. -Klotho, acting as a cofactor for FGF23, is also a major regulator of vitamin D biosynthesis and phosphate reabsorption in the kidney. These suggest that -Klotho is a key player that integrates a multi-step regulatory system of calcium and phosphate homeostasis. Collectively, the molecular function of -Klotho reveals a new paradigm that may change current concepts in mineral homeostasis and give rise to new insights in this field. Keywords: mineral homeostasis, -Klotho, FGF23, PTH, Vitamin D, Naþ,Kþ-ATPase multiple phenotypes related to disruption of min- Introduction eral homeostasis including ectopic calcification in In Greek mythology, life span is controlled by a variety of soft tissues, decreased bone mineral the three daughters of Zeus and Themis, namely, density, hypercalcemia and severe hyperphospha- Klotho who combs and spins the thread of life, temia in association with increased concentrations 1),4) 5) Lachesis who determines the length of life by of 1,25(OH)2D and serum FGF23. These led to measuring the length of thread, and Athropos who the prediction that -Klotho ( -Kl) is involved in cuts the string to bring a life to an end. In science, calcium and phosphorus homeostasis. thenameKlothowasconferredtoagenethatwas Calcium ion (Ca2þ) is a key mineral composi- fortuitously discovered in 1997. tion because of its diverse intra-and extracellular From the analysis of insertion mutant mouse roles.6) Among intracellular Ca2þ, the cytosolic free 2þ lines, we generated the klotho mutant mouse, a calcium concentration ([Ca ]i)isanimportant short-lived model mouse that displays a variety of second messenger and is the cofactor for proteins premature aging-related phenotypes.1) Ahomo- and enzymes that regulate a variety of cellular logue was subsequently identified and named - functions, including hormonal secretion, neuro- klotho.2) To avoid confusion, we refer to the transmission, muscle contraction, cell motility, original gene as -klotho ( -kl). The -kl gene is glycogen metabolism and cellular proliferation.6) 2þ predominantly expressed in tissues that are in- The [Ca ]i in resting cells is 100 nM. It is volved in the regulation of calcium and phosphate regulated by a series of channels, pumps, and other homeostasis,1),3) and -kl mutant mice exhibited transport mechanisms that control the movements of Ca2þ into and out of the cell and between various Ã1 Department of Pathology and Tumor Biology, Kyoto intracellular compartments.7) Consonant with its University Graduate School of Medicine, Kyoto, Japan. role as a second messenger, the [Ca2þ] in activated Ã2 Core Research for Evolutional Science and Technology, i Japan Science and Technology Corporation, Saitama, Japan. cells can rise by 10- to 100-fold due to the uptake y Correspondence should be addressed: Y. Nabeshima, of extracellular Ca2þ and/or release of Ca2þ from Department of Pathology and Tumor Biology, Kyoto University cellular stores, such as the endoplasmic reticulum. Graduate School of Medicine, Yoshida Konoe-cho, Sakyo-ku, 2þ Kyoto 606-8501, Japan Despite the importance of intracellular Ca in (e-mail: [email protected]). cellular metabolism, this compartment comprises doi: 10.2183/pjab/85.125 #2009 The Japan Academy 126 Y. NABESHIMA [Vol. 85, less than 1% of the total body calcium content in trigger the response of target cells by binding to mammalian species.8) their receptors and subsequently controlling the 2þ 2þ Extracellular Ca ([Ca ]0)servesasacofac- intake, metabolism, and excretion of calcium. tor for adhesion molecules, clotting factors, and Furthermore, the synthesis and secretion of these other proteins; regulates neuronal excitability; and hormones are mutually activated or suppressed by 2þ is an essential part of the mineral phase of bone. each other, and are also controlled by [Ca ]o which Particularly, the cloning of a G protein-coupled is monitored by calcium sensing receptors.15),16) In calcium sensing receptor (CaR) has proved that response to slight decrements in the level of the 2þ the calcium ion can indeed serve as an extracellular [Ca ]o, PTH release rapidly increases within first messenger.9) In contrast to intracellular Ca2þ, second order.17)–19) Renal actions of PTH include a 2þ the [Ca ]o is tightly controlled at 1{1:3 mM in reduction in tubular reabsorption of phosphate ions 8),10) 2þ mammals. Such accurate control of the [Ca ]o as well as an increase in distal tubular reabsorption ensures a steady supply of Ca2þ for intracellular of Ca2þ, which takes place within minutes.15) PTH functions. The total amount of soluble extracellular also acts on bone cells to enhance the release of Ca2þ, however, constitutes only a minute fraction of Ca2þ from the skeleton within 1–2 h.8),10),11),20),21) the total calcium content (e.g. mammals 0:1%). When hypocalcemia is more prolonged, elevation The highest fraction of total body calcium resides as of PTH levels persist for several hours or more calcium phosphate salts within the skeleton where and then the circulating 25-hydroxyvitamin D it provides a structural framework that protects [25(OH)D] is activated by the renal 25-hydroxyvi- critical bodily structures and facilitates locomotion tamin D-1 -hydroxylase (Cyp27b1) to form 22)–24) as well as constituting a large reservoir of calcium 1,25(OH)2D that in turn acts on specific and phosphate ions for times when intestinal receptors in the intestine25),26) to promote gastro- absorption and renal conservation of these ions intestinal absorption of Ca2þ and phosphate ions. are not sufficient for maintenance of constancy of Hypercalcemia produces the opposite series of the extracellular Ca2þ and phosphate concentra- changes in the Ca2þ homeostatic system. Reduc- tions.8),10),11) tions in the circulating levels of PTH promote renal A complex homeostatic system has evolved in Ca2þ wasting and phosphate retention as well as mammalian species, which is designed to maintain diminished skeletal release of mineral ions and 2þ 8),10)–12) near constancy of the [Ca ]o. This system eventually leads to reduction of gastrointestinal includes two essential components. The first com- absorption of these ions through decreased syn- ponent is comprised of cell types that sense changes thesis of 1,25(OH)2D. Therefore, as with hypocal- 2þ in the [Ca ]o and respond with appropriate alter- cemia, the response of the homeostatic mechanisms 2þ 2þ ations in their secretion of Ca -regulating hor- to hypercalcemia tends to restore [Ca ]o concen- mones. The parathyroid glands are key sensors of trations toward the normal state. 2þ variations in the [Ca ]o in mammalian species, The maintenance of normal phosphate (Pi) responding with changes in parathyroid hormone homeostasis is critical for diverse physiologic proc- (PTH) secretion that are inversely related to the esses including cell signaling, nucleic acid synthesis, ambient ionized calcium concentration.13) In con- energy homeostasis, formation of lipid bilayers, 2þ 27)–31) trast to the parathyroid cell, high [Ca ]o stimulate and bone formation. Owingtotheimportance hormonal release from calcitonin (CT)-secreting of Pi in diverse biological processes, decrements in parafollicular or C-cells of the thyroid gland.14) The serum Pi concentration and negative Pi balance second essential component of the homeostatic lead to serious diseases. Acute decreases in serum Pi system is comprised of the effecter systems, speci- concentration result in myopathy, cardiac dysfunc- alized cells in the kidneys, bone and intestine that tion, abnormal neutrophil function, platelet dys- respond to these calcitropic hormones with changes function and red-cell membrane fragility.32) Its in the transport of mineral ions so as to restore the importance is also illustrated by chronic disorders 2þ [Ca ]o toward the normal state. characterized by hypophosphatemia due to exces- Calcium homeostasis is largely regulated by sive renal phosphate loss.31) In fact, affected pa- the actions of two major hormones, namely PTH tients develop rickets with diminished bone and 1,25-dihydroxyvitamin D (1,25(OH)2D) that strength, deformity, short structure, and bone pain. No. 3] Discovery of -Klotho unveiled new insights into calcium and phosphate homeostasis 127 Conversely, high phosphate levels may have ad- reabsorption, as in vivo, the vitamin D status is verse physiologic effects. Patients with chronic closely associated with alterations in plasma cal- kidney disease often develop hyperphosphatemia cium and PTH concentrations. Conversely, when due to impaired renal clearance, and subsequently animals are fed a high-Pi diet, serum Ca2þ concen- progress to hyperparathyroidism and renal osteo- trations decrease, and PTH release is increased. dystrophy.33) Recently, Martin et al. suggested that changes in In mammals, absorption and reabsorption of Pi PTH secretion in response to dietary Pi occur take place primarily in the intestine and kidney, rapidly (within 10 min) and independently of respectively. Because the movement of Pi into changes in serum Pi or Ca2þ concentrations,40) the cell does not occur by simple diffusion, various suggesting that a signal emanating from the intes- Hþ-orNaþ-coupled Pi cotransporters mediate the tine may affect PTH secretion.41) An elevation in transport of Pi across the cell membrane.34) The serum Pi after a high-Pi meal also reduces þ Na -coupled Pi (NaPi) co-transporters that are 1,25(OH)2D synthesis and intestinal Pi absorption. important in Pi uptake in vertebrates belong to two Other than PTH and 1,25(OH)2D, several largefamilies,theNaPitypeIIandtheNaPitype phospaturic peptides such as fibroblast growth III families.