Discovery of \Alpha-Klotho Unveiled New Insights Into Calcium and Phosphate Homeostasis

$Discovery of \Alpha-Klotho Unveiled New Insights Into Calcium and Phosphate Homeostasis$

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 NABESHIMA1,2;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 1 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 2 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. The major role of NaPi type II has been factor 23 (FGF23), secreted frizzled related protein clearly demonstrated in Npt2-/- mice. Compared to 4, matrix extracellular phosphoglycoprotein, and wild-type mice, the Npt2-/- mice display a higher FGF7 have been demonstrated to inhibit the urinary excretion of Pi and lower NaPi co-transport activity of NaPi-IIa co-transporters in renal epi- activity (mainly mediated by NaPi-IIa and addi- thelial cells.38),42)–46) Furthermore, current studies tionally by NaPi-IIc) in the brush border mem- have strongly suggested the importance of the branes (BBM) of renal proximal tubules.35) This network of proteins interacting with NaPi-IIa co- suggests that the regulation of Pi reabsorption is transporters in apical membrane expression of achieved mainly by controlling the apical expres- NaPi-IIa co-transporters and the fate of endocy- sion of NaPi-IIa in BBM of renal proximal tu- tosed NaPi-IIa co-transporters.47) bules.36),37) Consistently, phosphaturic factors re- Intensive study on calcium homeostasis over duce the expression of NaPi-IIa, whereas factors the past several decades has established a system- that increase renal Pi reabsorption increase NaPi- atized understanding of its roles in living phenom- IIa in BBM. Therefore, control mechanisms that ena, leaving us with the impression that this field is regulate apical expression and membrane retrieval fairly defined and understood. On the contrary, our of NaPi-IIa are essential to our understanding of present knowledge about phosphate homeostasis is how Pi homeostasis is achieved. fragmentary and still in a premature stage. How- NaPi co-transport activity and organ specific ever, the currently demonstrated molecular func- Pi absorptive processes are regulated by PTH and tion of -Kl has given new insights into this field. 1,25(OH)2D, which interact in a coordinate fashion Here, the biological roles and molecular functions to regulate Pi homeostasis. In the kidney, various of -Kl in calcium and phosphate homeostasis are factors, most importantly PTH, influence the effi- summarized. ciency of renal Pi reabsorption.38) Animals fed with The discovery of -Klotho. -kl mutant a low-Pi diet have decreased serum Pi concentra- mice display a variety of accelerated aging-related tions that are associated with a reciprocal increase disorders, including hypoactivity, sterility, skin in circulating Ca2þ concentrations. The increase in thinning, decreased bone mineral density, vascular serum Ca2þ inhibits PTH release, which in turn calcifications, ectopic calcification in various soft reduces the renal excretion of Pi into the urine. In tissues (lung, kidney, stomach, heart, and skin), addition, a low-Pi diet and reductions in serum Pi defective hearing, thymus atrophy, pulmonary areassociatedwithincreased 1,25(OH)2Dsynthe- emphysema, ataxia, and abnormality of pituitary sis,39) which consequently restores serum Pi con- gland, as well as hypoglycemia, hypercalcemia and centrations to normal state by increasing intestinal severe hyperphosphatemia in association with in- 1) (mediated via NaPi-IIb) and renal Pi recoveries. It creased concentrations of 1,25(OH)2D. The re- is, however, difficult to discriminate between direct sponsible gene for these phenotypes was termed - versus indirect effects of 1,25(OH)2DonrenalPi kl, and shown to encode a type I membrane protein 128 Y. NABESHIMA [Vol. 85,

55) with an extra-cellular domain that exhibits signifi- and metabolism of 1,25(OH)2D. GALNT 3 is a cant similarity to -glycosidases, enzymes involved Golgi-associated enzyme that selectively O-glyco- in digestion of the sugar moiety of substrates.48) - sylates a furin-like convertase recognition sequence Kl was therefore predicted to be present on the cell in FGF23, thereby allowing secretion of intact surface, however large amounts of -Kl are detect- FGF23 by preventing proteolytic processing.56) able as dual forms in the cytoplasm. The 120 kDa Therefore, dysfunction of either FGF23 or GALNT premature form of -Kl resides mainly in the ER, 3 decreases circulating bioactive FGF23. Surpris- while the 135 kDa is a mature protein that is ingly, the -kl deficient phenotype largely overlaps cleaved and secreted into the blood, cerebrospinal with the phenotype of Fgf23-null mice.1),55) These fluid (CSF), and urine.49),50) This suggests -Kl may indicate functional crosstalk between -Kl and have dual actions depending on its intracellular and FGF23 and lead us to speculate that -kl muta- secreted forms. tion(s) might also be found in the genome of The -kl gene is predominantly expressed in patients with tumoral calcinosis. the parathyroid glands, kidney and the choroid Ichikawa et al. reported a homozygous mis- plexus.1),3) The parathyroid glands play a key role in sense mutation (H193R) in the -kl gene of a 13- systemic calcium homeostasis by monitoring the year-old girl who presented with severe tumoral 2þ concentration of [Ca ]o through the CaR and calcinosis with dural and carotid artery calcifica- secreting appropriate levels of PTH to maintain tions.57) Mapping of H193R mutation onto the 2þ 9),15),16) normal [Ca ]o concentrations. In the kidney, crystal structure of myrosinase, a plant homolog -Kl is exclusively co-expressed with calcium per- of -Kl, revealed that this histidine residue was at meable Transient Receptor Potential V5 (TRPV5) thebaseofthedeepcatalyticcleftandmutationof channels, Naþ/Ca2þ exchanger 1 (NCX1) and this histidine to arginine should destabilize -Kl. calbindin-D28K (a vitamin D sensitive intracellular Intra-cellular abundance and secretion of H193R - Ca2þ transporting protein) in a specialized region of Kl were thus markedly reduced, resulting in the the nephron segments where transepithelial Ca2þ diminished ability of FGF23. Similar to mice reabsorption is actively regulated. This co-local- deficient in -kl or Fgf23, this patient exhibited ization is believed to be important for the homeo- defects in mineral ion homeostasis with hyper- 2þ 2þ static control of [Ca ]e by regulating Ca re- calcemia, marked hyperphosphatemia and subse- absorption in the kidney. Indeed, mice lacking quent ectopic calcifications in soft tissues, as well as TRPV5 display diminished renal Ca2þ reabsorp- the elevation of circulating FGF23, PTH and tion, which causes severe hypercalciuria and com- 1,25(OH)2Dinserum. pensatory intestinal uptake of dietary Ca2þ.51) The Conversely, a mutation that causes an increase choroid plexus in many respects functions as ‘‘the in soluble -Kl shows adverse defects in mineral ion kidney of the brain’’.52) Its main function is the homeostasis. Recently, our collaborator Brownstein secretion of CSF, which involves the movement of et al. at Yale University discovered a new human water by osmosis and the unidirectional transport disease featuring hypophosphatemic rickets with of ions including calcium.53) The expression of -Kl marked parathyroid hyperplasia and decrease of in tissues involved in calcium metabolism, led to the circulating 1,25(OH)2D, as well as the elevation of prediction that -Kl might be involved in calcium FGF23 and PTH.58) In this patient, higher circu- homeostasis.54) latory FGF23, in combination with -Kl, might Discovery of -Klotho mutations in hu- have led to remarkable suppressions both of man. Familial tumoral calcinosis is characterized 1,25(OH)2D synthesis and renal phosphate re- by ectopic calcifications and hyperphosphatemia absorption, resulting in the vast wasting of phos- due to inactivating mutations in Fgf23 or UDP-N- phate into urine and the decrease of serum acetyl--D-galactosamine: polypeptide N-acetyl- 1,25(OH)2D levels. Furthermore, the increase in galactosaminyltransferase 3 (GALNT3). FGF23 is serum PTH appears to have led to severe hypo- a hormone that promotes renal phosphate excretion phosphatemia by promoting the rapid removal of by decreasing phosphate reabsorption in the NaPi-IIa from the apical membrane followed by its proximal tubule and also reduces circulating degradation. As for the possible explanation for the 1,25(OH)2D by decreasing both the biosynthesis cause of marked increase in serum levels of -Kl, a No. 3] Discovery of -Klotho unveiled new insights into calcium and phosphate homeostasis 129 de novo translocation with a breakpoint adjacent to FGF23. Notably elevated serum phosphate has -kl gene was suggested. The inference that the been reported to promote proliferation of para- translocation is the cause of the patient’s syndrome thyroid cells and enhances both PTH secretion and is supported by (i) the de novo appearance of this mRNA stability; these effects have been speculated translocation in conjunction with the appearance of to be mediated by reduced serum Ca2þ levels, but a rare syndrome, (ii) the marked alteration in -Kl the mechanism is still uncertain.59)–62) Conversely, levels in association with the mutation, and (iii) high Ca2þ observed in the patient with missense corroborating prior evidence that genetic deficiency mutation and 1,25(OH)2D are well known to inhibit for -kl in mice produces the reverse phenotypes. PTH production, and FGF23 has recently been And additionally, (iv) a likely loss of function shown to inhibit expression of PTH mRNA and mutation in humans (H193R missense mutation)57) secretion of PTH from parathyroid cells.63),64) (iii) It was associated with adverse symptoms. should be also noted that subtotal parathyroidec- Thediscoveryoftheabovetwopatientsraised tomy normalized serum calcium and PTH levels several issues (i) The first issue is the elevation even in a homozygous missense mutation in a-kl of circulating FGF23 levels in both patients. In gene.57) How can we reconcile this evidence with response to severe hyperphosphatemia and increas- current observations? Serum PTH levels are main- ed 1,25(OH)2D concentrations, the patient with the tained at basal levels under normocalcemic condi- 2þ missense mutation had appropriately elevated tions, and are quickly increased when [Ca ]o is FGF23 levels. It is also possible that FGF23 lowered by the active and regulated secretion of signaling in this patient was severely compromised PTH. In this context, FGF23 is predicted to be as a result of the diminished ability of mutant -Kl involved in the regulation of PTH storage and basal to form a ternary FGF23--Kl-FGFR1c complex, levels of PTH in serum63),64) and the active and thereby suggesting that the increase in FGF23 regulated PTH secretion is characterized by its production was compensation for impaired FGF23 reliance on the -Kl/Naþ,Kþ-ATPase complex, low 2þ signaling. Notably, circulatory levels of FGF23 [Ca ]o mediated stimuli and a rapid time-course have been shown to increase in -kl mutant mice.5) response.54) However,theroleofFGF23inthe Unexpectedly, the circulating FGF23 levels are also active and regulated secretion of PTH has yet to be markedly elevated in the patient with high serum - defined.63),64) As for the PTH secretion in this Kl. Since elevation of FGF23 has been demonstrat- patient, while the extent of functional disorder of ed to be closely associated with hyperphosphate- -Kl in the parathyroid glands is not clear, it is mia, Brownstein et al. proposed that the elevated possible that these two PTH regulatory pathways -Kl level mimics aspects of the normal response are malfunctioning since they are dependent on - to hyperphosphatemia and implicates -Kl in the Kl. Indeed, serum PTH concentrations are main- selective regulation of phosphate levels. However, tained at very low levels after subtotal parathyr- increased PTH has also been demonstrated to oidectomy. However, serum calcium levels remain increase FGF23 levels. Therefore it is difficult to at nearly the upper limit of normal range.57) Since discern whether the elevated FGF23 is the result of calcium metabolism is known to be regulated by increased -Kl or is part of a negative-feedback loop many factors such as dietary calcium intake, responding to the hyperparathyroidism observed in influence by other regulatory factors and phosphate this patient. It should be also noted that FGF23 concentrations, an unknown mechanism that com- levels are markedly elevated with -kl deficiency.5) pensates for impaired PTH secretion is assumed to These suggest complex regulation of FGF23, which exist, but evidence of such a system remains to be remain to be clarified. (ii) The second issue is why found. parathyroid hyperplasia is observed in both pa- -Klotho/Naþ,Kþ-ATPase complex for- tients. It is difficult to make a consistent explan- mation. The Naþ,Kþ-adenosine triphosphatase ation from the serum data of both patients, since (Naþ,Kþ-ATPase) is a well-studied heteromer con- serum concentrations of phosphate and 1,25(OH)2D sisting of a larger subunit responsible for ion are high in the patient with missense mutation, but transport and catalytic activity, and of a smaller low in the patient with high serum -Kl, while both glycosylated subunit required for the regulation of patients commonly showed high serum levels of the catalytic action of the subunit and for the 130 Y. NABESHIMA [Vol. 85,

(A) Conventional α-Kl dependent (B) Pathway Pathway α-Kl

WT α-Kl dependent pathway α -/- -ATPase -Kl + ,K +

Conventional pathway

RE NaK Surface Na EE α-Kl 2+ low [Ca ]o high Degradation Golgi EE: Early Endosome ER RE: Recycling Endosome

Fig. 1. Surface recruitment of Naþ,Kþ-ATPase in correlation with the cleavage and secretion of -Kl. In -Kl expressing cells, Naþ,Kþ-ATPase is recruited to the cell surface by a combination of ‘‘the conventional pathway’’ and ‘‘the -Kl dependent pathway’’. Low Ca2þ induces the massive recruitment of Naþ,Kþ-ATPase to the plasma membrane. In contrast, high Ca2þ leads 2þ þ þ to the decline of such additional recruitment. Accordingly, [Ca ]e regulates this additional recruitment of Na ,K -ATPase to the plasma membrane in correlation with the cleavage and secretion of -Kl. control of cell surface recruitment of Naþ,Kþ- accumulates in the endosome fraction including the ATPase complexes.65) Although Naþ,Kþ-ATPase recycling endosome, in preparation for the recruit- is ubiquitously expressed, its expression level varies ment to the cell surface (Fig. 1A).54) among tissues; the expression level of Naþ,Kþ- Surface recruitment of Naþ,Kþ-ATPase in 2þ ATPase is significantly higher in the kidney DCT response to [Ca ]o. The interaction of -Kl and cells, choroid plexus and the parathyroid glands. Naþ,Kþ-ATPaseraisedanhypothesisthat-Kl This suggests that highly expressed Naþ,Kþ-AT- directly affects Naþ,Kþ-ATPase activity. Based on Pase in these -Kl expressing cells plays a specific the facts that (i) the -Kl and Naþ,Kþ-ATPase role in cooperation with -Kl. complexes are preferentially localized intracellular- Naþ,Kþ-ATPase was isolated as a binding ly and (ii) the catalytic action of Naþ,Kþ-ATPase is protein of two intracellular forms of -Kl, namely, an event that is regulated on the cell surface, the 120 kDa premature form and 135 kDa mature form. regulation of catalytic efficiency by -Kl was ruled Sub-cellular fractionation revealed that the com- out as unlikely, and instead focus was put on its plexes of premature -Kl and Naþ,Kþ-ATPase are regulatory role in the recruitment of Naþ,Kþ- found in the ER fraction, while mature -Kl and ATPase to the cell surface. Naþ,Kþ-ATPase complexes accumulate in the en- The activity of Naþ,Kþ-ATPase in the choroid dosome and Golgi apparatus/cell membrane frac- plexus epithelial cells is inversely correlated with 2þ 2þ tions. From cell surface biotinylation analyses, it [Ca ]o. When incubated in a low Ca solution, was also confirmed that although 1-and 1-sub- the activity of Naþ,Kþ-ATPase increased rapidly, units of Naþ,Kþ-ATPase are readily detectable while it decreased in a high Ca2þ solution. Consis- both in the cell surface and cytoplasmic fractions, tent with this observation, low Ca2þ levels in media -Kl is primarily detected in the cytoplasm but not significantly increased the surface amount of Naþ, in the cell surface fraction. Taken together, this Kþ-ATPase and conversely high Ca2þ levels de- suggests that a subset of NaþKþ-ATPase traffics creased the amount of Naþ,Kþ-ATPase (Fig. 1B). from the ER to the cell surface in conjunction with The fluctuation of the amount of Naþ,Kþ-ATPase -Kl and -Kl/Naþ,Kþ-ATPase complexes located at the epithelial cell surface rapidly changes and in the ER and Golgi apparatus, and abundantly becomes detectable within 30 sec after the shift of No. 3] Discovery of -Klotho unveiled new insights into calcium and phosphate homeostasis 131

2þ þ þ [Ca ]o. This result demonstrates that Na ,K - channel, TRPV4, since (i) TRPV4 is highly ex- ATPase activity is correlated with its amount on pressed in the apical membrane of choroid plexus 2þ 2þ the cell surface, both of which respond to [Ca ]o, epithelial cells and (ii) low Ca concentrations in 2þ leading us to conclude that the shift of [Ca ]o the extracellular fluid induce larger cation perme- triggers a rapid response that recruits Naþ,Kþ- ability when activated with agonist. To analyze the ATPase to the plasma membrane (Fig. 1A,B). As role of TRPV4, 4-PDD (4-phorbol 12,13 dideca- expected, -Kl is required for the rapid and noate), a specific agonist for TRPV4, was used to þ þ 2þ regulated recruitment of Na ,K -ATPase to the activate TRPV4 at normal [Ca ]o (1.25 mM). In cell surface. Furthermore, secretion of -Kl is choroid plexuses prepared from WT mice, 4-PDD 2þ induced in response to low [Ca ]o in the kidney, administration induced a rapid increase of the cell parathyroid glands and the choroid plexus, suggest- surface expression of Naþ,Kþ-ATPase. However, in 2þ -/- ing that [Ca ]o mediates cleavage of -Kl in -kl mice, the administration of 4-PDD did conjunction with the rapid response that recruits not induce such a phenomenon. Therefore, -Kl Naþ,Kþ-ATPase to the cell surface (Fig. 1A, appears to be required for this TRPV4 signaling Fig. 3A).54) pathway. Consistent with this result, the amount of Novel Naþ,Kþ-ATPase recruiting sys- secreted -Kl was induced up to 2–3 fold over the tem. The surface expression of Naþ,Kþ-ATPase control upon 4-PDD administration. Moreover, is controlled by the balance of recruitment to the addition of 2 mM EGTA diminished the effect of 4- plasma membrane and internalization of Naþ,Kþ- PDD, indicating that extracellular Ca2þ is essential ATPase (the conventional pathway).66) In -Kl forthissystem.54) Based on this evidence, we re- expressing cells, Naþ,Kþ-ATPase is recruited to the constructed the Naþ,Kþ-ATPase recruitment sys- cell surface by a combination of ‘‘the conventional tem in HeLa cells by transfection of plasmids pathway’’ and ‘‘the -Kl dependent pathway’’ expressing TRPV4 together with various forms of (Fig. 1A). The latter is characterized by its Ca2þ- -Kl. Naþ,Kþ-ATPase recruitment and its activity 2þ dependency and rapid response. A low [Ca ]o was found to be induced by the expression of intact induces further recruitment of Naþ,Kþ-ATPase to -Kl and TRPV4, and by 4-PDD administration. the plasma membrane. In contrast, high Ca2þ leads Consistently, secretion of the extracellular domain to the decline of such additional recruitment. Under of intact -Kl was rapidly induced by the admin- normocalcemic conditions, a certain amount of istration of 4-PDD.54) Although these results do Naþ,Kþ-ATPase is additionally recruited by the not prove that TRPV4 per se plays a role in sensing -Kl dependent pathway. However, the recruitment Ca2þ levels, TRPV4 and the success in reconstruc- to the plasma membrane of Naþ,Kþ-ATPase in - tion provide a good tool for studying the role of - kl -/- mice is solely dominated by the conventional Kl in Naþ,Kþ-ATPase recruitment. pathway and probably represents ‘basal’ recruit- -Klotho/Naþ,Kþ-ATPase complex in the ment. Therefore, the amount/activity of surface transepithelial Ca2þ transport. Naþ,Kþ-AT- Naþ,Kþ-ATPase in wild type mice is significantly Pase is a membrane-bound pump that transports higher than that of -kl -/- mice. Accordingly, two Kþ in and three Naþ out of the cell.65) Naþ,Kþ- 2þ [Ca ]o regulates this additional recruitment of ATPase activity influences many kinds of cellular Naþ,Kþ-ATPase to the plasma membrane in corre- and physiological events which take place near lation with the cleavage and secretion of -Kl the cell membrane by the regulation of electro- (Fig. 1A,B).54) chemical gradients in the plasma membrane, since Reconstruction of Naþ,Kþ-ATPase recruit- the fluctuations of Naþ,Kþ-ATPase activity recon- ment system. To define the components of the cile the activities of other ATPases, ion channels, cell surface machinery that links the fluctuation of and ion exchangers including Ca2þ/cation anti- 2þ [Ca ]o and the efficiency of additional recruitment porter superfamily members such as NCX and of Naþ,Kþ-ATPase to the plasma membrane, we NCKX.67),68) Particularly, NCXs comprise three analyzed candidate molecules that fulfilled the members (NCX1; most extensively studied, and following criteria: (i) expressed in the choroid broadly expressed with particular abundance in plexus and (ii) active at low Ca2þ concentrations. heart, brain and kidney, NCX2 expressed in brain, Among several candidates, we focused on a cation and NCX3 expressed in brain and muscle) and the 132 Y. NABESHIMA [Vol. 85,

2þ 2þ NCX proteins sub-serve a variety of roles depending of [Ca ]o. Indeed, transepithelial Ca transport in upon the site of expression. These include cardiac the DCT nephron and choroid plexus is efficiently 2þ excitation-contraction coupling, neuronal signaling induced in response to low [Ca ]o concentrations and Ca2þ reabsorption in the kidney,69) raising the and suppressed when Ca2þ concentrations are possibility that the higher Naþ gradient created by increased. This indicates that the -Kl/Naþ,Kþ- the Naþ,Kþ-ATPase activity may drive the trans- ATPase system regulates Ca2þ transport, at least in epithelial calcium transport in the choroid plexus part, in a CaR independent manner. Taken togeth- and kidney (Fig. 3A). er, all of the above suggest the importance of In the choroid plexus, the importance of the calcium sensor machinery that is distinct from CaR. -Kl/Naþ,Kþ-ATPase complex in Ca2þ transport These studies suggested that Ca2þ reabsorp- þ þ 2þ was suggested by (i) surface amounts of Na ,K - tion is enhanced directly in response to low [Ca ]o ATPase in -kl -/- mice were lower than that of WT, stimuli in the distal nephron as well in a cell and consistently, (ii) the concentration of total autonomous manner independent of calcium-regu- calcium in CSF in -kl -/- mice was considerably lating hormones. Kronenberg et al. described their lower than that of WT mice. These results imply prediction in a textbook as follows: ‘‘Ca2þ reab- that calcium homeostasis is impaired in the CSF sorption is enhanced directly by tendency to of -kl -/- mice and that -Klisinvolvedinthe hypocalcemia, which is detected by calcium-sensing regulation of calcium concentrations in the CSF receptors in Henle’s loop and possibly also in the (Fig. 3A). Similarly, the responsive increase of distal nephron that control transepithelial calcium þ þ 81) surface Na ,K -ATPase expression may play a movements independent of PTH or 1,25(OH)2D’’. pivotal role in Ca2þ reabsorption in the kidney DCT Although its molecular basis has long remained cells (Fig. 3A). The calcium transport across var- unknown, the following mechanism may be the first ious nephron segments was actively examined in satisfactory explanation for the prediction proposed the second half of the last century.70)–75) In parallel, by Kronenberg et al. That is, the trans-epithelial the involvement of Ca2þ-ATPase76) and Ca2þ/Naþ transport of Ca2þ is directly triggered and proc- antiport77) in Ca2þ transport was suggested, and essed by the increased Naþ gradient in cooperation 2þ Shareghi and Stoner found that Ca reabsorption with TRPV5, Calbindin D28k, and NCX-1, all of in the DCT and the granular portion of the cortical which are exclusively co-expressed with -Kl in the collecting duct was significantly enhanced by nephron segment responsible for the active and PTH.78) In 1993, Brown et al. proposed a molecular regulated Ca2þ reabsorption (Fig. 3A). Indeed, basis for the above observations by the discovery of regulated reabsorption of calcium in the kidney is the calcium sensing receptor (CaR).9) CaR senses impaired in -kl -/- mice, resulting in the excess 2þ 82) high [Ca ]o concentrations and inhibits tubular excretion of calcium into urine. 2þ þ þ reabsorption of calcium when [Ca ]o is increased. -Klotho/Na ,K -ATPase complex in the Thus, serum Ca2þ levels are excessively elevated in regulated PTH secretion. In the parathyroid CaR knockout mice due to enhanced renal tubular glands, PTH secretion is stimulated by the decline 2þ calcium reabsorption in addition to marked increase of [Ca ]o. In fact, a substantial decrease in serum of serum PTH and resultant stimulated bone Ca2þ concentration corresponded to a marked resorption.79) However, as for the transcellular increase in serum PTH in WT mice. However, the Ca2þ transport in the DCT nephron and choroid serum PTH response in -kl -/- mice was signifi- plexus, the involvement of CaR seems to be limited cantly lower than that of WT mice, suggesting that or unlikely, if any, because the major site of intra- -Kl is essential for the regulated secretion of PTH. renal CaR expression is the thick ascending limb of Furthermore, the extent of PTH release in WT Henle’s loop but not in DCT cells,80) and because samples treated with ouabain, a specific inhibitor of CaR is not detectable in the choroid plexus.54) Naþ,Kþ-ATPase, is intriguingly similar to that seen Furthermore, the response of CaR to increasing in the specimens from -kl -/- mice without ouabain, 2þ [Ca ]o concentration is well recognized, but the and ouabain treatment induced no additional response of CaR to lowered [Ca2+]o is not as well inhibitory effects on PTH secretion in the para- defined.16) On the other hand, -Kl and Naþ,Kþ- thyroid glands from -kl -/- mice.54) Collectively, ATPase are involved in the response to a wide range current and previous observations suggest the No. 3] Discovery of -Klotho unveiled new insights into calcium and phosphate homeostasis 133 following regulatory scheme of PTH secretion. diffuses to the basolateral cell surface where Ca2þ is 2þ þ þ When [Ca ]o is lowered, Na ,K -ATPase is quick- extruded into the blood compartment via NCX1 ly recruited to the plasma membrane through the and PMCA1b (Plasma Membrane Calcium ATPase ‘‘-Kl dependent pathway’’ in correlation with the 1b) (Fig. 3A).51) High Ca2þ influx is possibly cleavage and secretion of -Kl (Fig. 1A). An obtained by prolonged channel durability at the 2þ electrochemical gradient across the cell membrane cell surface, because, regardless of [Ca ]o,both created by increased Naþ,Kþ-ATPase may cause TRPV5 and TRPV6 are constitutively active as the activation of unidentified signal(s), leading to cation channels. The question is how the TRPV5 the release of PTH. In the PTH-secreting system, - and TRPV6 channel abundance is controlled. Kl and Naþ,Kþ-ATPase work as players of a Chang et al. reported a novel mechanism that common signaling pathway and thus, if this path- regulates the abundance of TRPV5 at the luminal way is disrupted either by -kl deficiency or by cell surface:50) -Klinurine,asa-glucuronidase, administration of ouabain, the regulated secretion increases TRPV5 channel abundance at the luminal of PTH is equivalently suppressed (Fig. 3A).54) In cell surface by hydrolyzing the N-linked extracel- turn, when parathyroid CaR is activated in re- lular sugar residues of TRPV5 without affecting 2þ 2þ sponse to the increase of [Ca ]o, inositol-1,4,5- the Ca uptake activity of the TRPV5 channel triphosphate (IP3) accumulates and intracellular (Fig. 3A). More recently, it was also reported that calcium rises because of the release of calcium from -Kl in urine increases TRPV5 channel abundance intracellular stores and of the opening of plasma at the luminal cell surface by participating in membrane calcium channels. This increase in the specific removal of 2,6-linked sialic acids from intracellular calcium subsequently leads to a de- glycan chains of TRPV5.85) crease in PTH secretion.16) Since Brown et al. first This maintains a durable calcium channel suggested the importance of Naþ,Kþ-ATPase in activity and membrane calcium permeability in regulating the secretion of PTH, the crucial ques- the kidney, resulting in an increased Ca2þ influx tion of how Naþ,Kþ-ATPase activity is regulated from the lumen to preserve normal blood Ca2þ in response to low Ca2þ stimuli in the parathyroid levels by reduction of Ca2þ loss via urine (Fig. 3A). glands has remained unanswered.83) The identifica- Ca2þ enters at the luminal membrane and is tion of -Kl as a key regulator in the surface sequestered by calcium binding proteins. The recruitment of Naþ,Kþ-ATPase in response to low bound Ca2þ is then extruded into the blood Ca2þ stimuli offers an answer (Fig. 3A). However, compartment (Fig. 3A). In this scheme, Ca2þ efflux the intra-cellular mechanism that actively induces at basolateral membrane is quickly controlled in 2þ 2þ 2þ PTH secretion when [Ca ]o is lowered remains to response to the fluctuations of [Ca ]o,andCa be clarified. efflux at the basolateral membrane might be -Kl as an enzyme -glucuronidase. The mutually balanced by Ca2þ influx at the apical high similarity of -Kl with the -glucosidase membrane since the excessive influx of Ca2þ over family, suggested that it may possess a -glucosi- the efflux ability results in a cytotoxic overflow of dase activity. However, this has been questioned Ca2þ in the cytoplasm. Importantly, -Kl plays because -Kl lacks the glutamic acid residues that critical roles in both the influx and efflux of Ca2þ by are responsible for the catalytic activity of this regulating the abundance of TRPV5 channels on enzyme family. Nonetheless, -glucuronidase activ- the luminal cell surface50),85) and the recruitment ity for -Kl was demonstrated.84) In addition to - of Naþ,Kþ-ATPase to the basolateral cell sur- glucuronides, -Kl was found to hydrolyze the face,54) respectively (Fig. 3A). This leads to a extracellular sugar residues of TRPV5.50) As de- prediction that -Kl may play the critical role in scribed above, TRPV5 is exclusively co-expressed the coordinate and balanced regulation of influx 2þ with calbindin D28k,NCX-1and-Kl in the DCT and efflux of Ca that takes place at both sides of epithelial cells which are responsible for the active DCT cells. and regulated Ca2þ reabsorption in the kidney. -Kl in combination with FGF23 regulates Ca2þ enters into the cell at the luminal membrane vitamin D metabolism. FGF23, in combination (apical) via TRPV5 and/or TRPV6 and is seques- with -Kl, negatively regulates the metabolism of 2þ tered by calbindin-D28k or -D9k. Bound Ca then vitamin D in the kidney. In this pathway, -Kl is 134 Y. NABESHIMA [Vol. 85, assumed to be necessary for the recognition of to be the major regulator of NaPi-IIa and NaPi-IIc FGF23 by target cells. As Urakawa et al. reported, abundance in the apical membranes of PCT cells, -Kl binds to FGF23 and converts the canonical promoting the rapid removal of NaPi-II from the FGF receptor 1c (FGFR1c) to a receptor specific membrane and its subsequent degradation.88)–90) As for FGF23.5) This enables the high affinity binding a feedback loop, an increment in Pi is known to ofFGF23tothecellsurfaceofDCTcellswhere- promote (i) proliferation of parathyroid cells, (ii) Kl is expressed. Subsequently, -Kl enhances the enhancement of PTH secretion, and (iii) stabiliza- ability of FGF23 to induce phosphorylation of FGF tion of PTH mRNA, and thereby Pi concentration receptor substrate and extracellular signal-regulat- can be enhanced. However, this evidence does not ed kinase (ERK) in DCT cells. As for the regulation suggest the presence of a Pi receptor, since the of Cyp27b1 (1-hydroxylase) gene expression, it is effects of high Pi concentrations are possibly reasonable to speculate that either (i) one or more indirect and have been speculated to be mediated signal mediators from the distal to the proximal by means of reduced serum Ca2þ levels.59)–62) 2þ tubule would be required or (ii) some paracrine [Ca ]o and Pi levels are inversely regulated and action of secreted -Kl would be necessary for this thus the increase in serum Pi results in the decrease 2þ signal transduction to take place because the in [Ca ]o that trigger the increase of PTH activity. Cyp27b1 gene is preferentially expressed in prox- Other than PTH, FGF23 also down-regulates imal convoluted tubule (PCT) cells, but not in DCT serum Pi levels, due to decreased NaPi-IIa abun- cells where FGF23/-Kl signal is transduced. dance in the apical membrane (Fig. 2). In fact, 1,25(OH)2D, the end-product of this pathway, homozygous loss of function of FGF23 results in has prominent effects on the kidney, intestine and severe hyperphosphatemia and conversely, gain of bone (Fig. 3B). 1,25(OH)2D enhances calcium up- mutation of FGF23 results in hypophosphate- 91)–93) take in the intestine. In the kidney, 1,25(OH)2D mia. Of particular interest, -Kl is essential activates the vitamin D receptor (VDR) by binding for the above two pathways since FGF23 signaling to its ligand binding domain and negatively regu- and regulated secretion of PTH depend on the lating the expression of Cyp27b1, while positively actions of -Kl (Fig. 2). Thus, renal and intestinal regulating Cyp24 and -Kl expression.4) In the NaþPi transporter activities and expression levels bone, 1,25(OH)2D binds to VDR and induces of the type IIa, IIb, and IIc NaPi transporter FGF23 synthesis in osteocytes86) and osteoblasts.87) proteins were significantly increased in the -kl/-kl In turn, secreted FGF23 suppresses 1,25(OH)2D mice (originally established mutant line) and the synthesis and the expression of -Kl in the kidney NaPi IIa transporter was localized exclusively to to adjust extra-cellular concentrations of Ca2þ and the apical membrane of PCT cells in -kl/-kl Pi. Based on these findings, we proposed a compre- mice.94),95) hensive regulatory scheme of mineral homeostasis The roles of -Klotho and FGF23 in multi- that is illustrated by the mutually regulated posi- step regulation of calcium homeostasis. The tive/negative feedback actions of -Kl, FGF23 and comprehensive scheme of calcium homeostasis and 1,25(OH)2D. aglobalimageof-Kl function in the regulatory FGF23, in combination with -Kl, regulates network of calcium homeostasis are illustrated in renal phosphate reabsorption. The kidney plays Fig. 3B. As shown in Fig. 3B, calcium homeostasis a predominant role in the regulation of serum Pi is maintained by a ‘‘multi-step response system’’ levels. The majority of Pi reabsorption takes place categorized according to the time course, into (I) in the proximal tubule, and is mainly mediated by seconds to minutes, (II) minutes to hours and (III) NaPi-IIa and additionally by the related protein hours to day(s) order regulations. In response to NaPi-IIc.88) Serum Pi levels are also regulated by hypocalcemic stimuli, transepithelial Ca2þ trans- intestinal absorption of Pi that is mediated via port in the choroid plexus and the -Kl-expressing NaþPi-IIb transporter, the actions of which are nephron segments, and PTH secretion in the para- affected by dietary Pi content and 1,25(OH)2D3. thyroid glands are all triggered by electrochemical The regulated Pi reabsorption in the kidney is gradients created by Naþ,Kþ-ATPase (Fig. 3B- affected by various factors such as PTH, dietary Pi, I).54) Because these responses begin immediately 2þ 1,25(OH)2D and FGF23. PTH has been considered after a decline in Ca concentration and persist for No. 3] Discovery of -Klotho unveiled new insights into calcium and phosphate homeostasis 135

DCT Bone Intestine Kidney Proximal Cells

Ca2+ Ca2+ Pi Degradation NaPi II NaPi II

V5 V6 NaPi Pi Pi 1,25(OH)2D

Ca2+ Ca2+ Ca2+ Pi Pi 25(OH)D

Cyp27b1

Ca2+ Pi

1,25(OH) D α 2 FGF23 -Kl Parathyroid + − FGFR1 Glands α-Kl Na/K DCT Cell Na/K PTH PTH

Fig. 2. PTH and FGF23/-Kl suppress renal phosphate reabsorption. The majority of phosphate reabsorption occurs in the PCT cells in the kidney. PTH and FGF23/-Kl promote the rapid removal of NaPi-IIa from the membrane. An increase in serum phosphate promotes proliferation of parathyroid cells, PTH secretion and stabilization of PTH mRNA. The increased phosphate signal might be mediated by means of reduced serum Ca2þ levels. þ, : indicate positive and negative regulations, respectively.

(A) (B)

2+ Kidney, CP (I) α-Kl/NaK Low[Ca2+] α-Kl/NaK [Ca ]o o Seconds to CP Kidney Ca2+ transport Ca 2+ transport Minutes α to CSF -Kl/NaK Na+ Parathyroid Ca2+ α-kl-/- 2+ 2+ (II) Ca PTH Ca α resorption reabsorption -Kl α-Kl Minutes to Hours Bone TRPV5 K+ Na/K High[Ca2+] o Kidney Na/K (III) α-Kl + Na Hours to Ca2+ absorption FGF23 Na/K Days CT Bone Intestine Ouabain 25(OH)D α-Kl

Cyp27b1 1,25(OH) D PTH 2 Kidney Parathyroid (IV) FGF23/ Pathology : NCX : PMCA : Calb α Cyp27b1 1,25(OH)2D of -Kl 2+ α -/- KO : TRPV5 (Kidney) : Ca -kl mice VD deficient KO Multiple : Na+/K+-ATPase diet Improved VDR Phenotypes

Fig. 3. Roles of -Kl and FGF23 in calcium homeostasis. -Kl binds to Naþ,Kþ-ATPase and regulates the recruitment of Naþ,Kþ- 2þ ATPase to the cell surface membrane in response to lowered [Ca ]o. -Kl controls calcium re-absorption in the kidney DCT cells, calcium transportation across the choroid plexus into the CSF, and PTH secretion in the parathyroid glands. -Kl is also involved in the signal transduction of Fgf23 that suppresses the gene expression of Cyp27b1 in the DCT nephrons that leads to negative regulation of 1,25(OH)2D3 synthesis. Calcium metabolism is governed by complicated reciprocal actions and feedback mechanisms and thus calcium concentrations in serum, body ﬂuid and CSF are maintained within strictly narrow ranges. 136 Y. NABESHIMA [Vol. 85,

a short time, they can be placed into the ‘‘seconds to 1,25(OH)2D and altered mineral-ion homeostasis minutes order regulation’’ (Fig. 3B-I). The PTH- are believed to be the major causes of the premature mediated increase of Ca2þ,suchasCa2þ reabsorp- aging-like phenotypes observed in -kl -/- and 2þ -/- tion in the kidney and Ca reabsorption in the Fgf23 mice, because the lowering of 1,25(OH)2D bone continues for hours and thus belong to the activity by (i) dietary restriction (a regimen in ‘‘minutes to hours order regulation’’ (Fig. 3B-II). which -kl -/- mice are fed a vitamin D-deficient 4) The production of 1,25(OH)2D and subsequent diet), (ii) genetic ablation of 1-hydroxylase in - -/- -/- 97) 1,25(OH)2D-mediated intestinal calcium uptake kl mice (unpublished data) or in Fgf23 mice, are in the order of ‘‘hours to day(s) regulation’’ or (iii) genetic ablation of the VDR gene in -kl -/- (Fig. 3B-III). In addition, 1,25(OH)2D enhances the mice (unpublished data) are all able to rescue the expression and function of TRPV5 present on the premature aging-like phenotypes and enable these apical membrane of DCT cells, and thereby up- mice to survive normally without obvious abnor- regulates Ca2þ reabsorption in the kidney.96) In malities (Fig. 3B-IV). These independently estab- turn, increased calcium suppresses the transepithe- lished conclusions consistently indicate that -Kl lial Ca2þ transport and PTH secretion. The increas- is the key regulator of calcium and phosphate ed VDR ligand, 1,25(OH)2D, suppresses Cyp27b1 homeostasis and clearly explain the major cause of gene expression and enhances the expression of phenotypes observed in -kl -/- mice and the reason FGF23. Subsequently, secreted FGF23, in combi- why -Kl is expressed in the tissues related to nation with -Kl, suppresses Cyp27b1 gene expres- calcium and phosphate regulation. Furthermore, sion in the kidney (Fig. 3B-III). the reduced blood glucose and insulin concentra- Taken together, calcium metabolism is gov- tions observed in -kl -/- mice can be strikingly erned by complicated reciprocal actions along with improved in both male and female -kl -/- mice when feedback mechanisms in the time course from they are fed a vitamin D-deficient diet, suggesting seconds to minutes, hours and day(s) order regu- that the impaired glucose metabolism in -kl -/- mice lations. Thus, extra-cellular calcium concentration is a secondary effect caused by increased vitamin D is rapidly adjusted and continuously maintained activity.4),98) Importantly, the patient carrying a within strictly narrow ranges. In this system, -Kl is missense mutation of -kl gene and the patient with involved in ‘‘seconds to minutes order regulation’’ of high serum -Kl exhibited severe defects in mineral transepithelial Ca2þ transport and secretion of ion homeostasis, but normal fasting glucose and PTH. Subsequently, -Kl participates in ‘‘minutes insulin concentrations.57),58) These studies provide to hours order regulation’’ and ‘‘hours to day(s) compelling evidence that -Kl has a minimal, if order regulation’’ through the action of secreted any, effect on glucose metabolism and does not PTH and PTH-mediated production of 1,25(OH)2D, support a previous study reporting that -Kl respectively. -Kl also participates in the signal interferes with insulin or insulin-like growth factor transduction of FGF23 to adjust calcium concen- signal transduction.99) The next question that needs tration by down-regulating the production of to be resolved is how hypervitaminosis D and the 1,25(OH)2D (Fig. 3B). Furthermore, -Kl in urine subsequently altered mineral-ion balance lead to increases TRPV5 channel abundance at the luminal the multiple premature ageing-like phenotypes, as cell surface by hydrolyzing the sugar residues of documented in both -kl and Fgf23 null mice. TRPV5, which also increase Ca2þ reabsorption in the kidney (Fig. 3B).50),85),96) Therefore, -Kl is the Conclusions key player that integrates a ‘‘multi-step calcium Recent advances that have given rise to control system’’ and should be categorized into a marked progress in mineral homeostasis can be class of protein distinct from the known regulatory summarized as follows; (i) -Kl binds to Naþ,Kþ- þ þ molecules (PTH, 1,25(OH)2D and CT) and from the ATPase, and Na ,K -ATPase is recruited to the direct handling molecules for transepithelial calcium plasma membrane by a novel -Kl dependent transport such as TRPV5, Calbindin D28k,and pathway in correlation with cleavage and secretion 2þ NCX-1. of -Kl in response to [Ca ]o fluctuation. (ii) The Major cause of phenotypes observed in a- increased Naþ gradient created by Naþ,Kþ-ATPase kl -/- and Fgf23 -/- mice. 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Proﬁle

Yo-ichi Nabeshima was born in 1946 and started his research career in 1972 with studies on the biosynthesis of proteins in the liver in the Department of Biochemistry, after graduating from the School of Medicine at the Niigata University. He obtained his Doctor in Medical Science from The Niigata University in 1976, working on the metabolism of ribosomal proteins. He was promoted to assistant professor in 1976 and lecturer in 1979 at The Niigata University, and researcher of the Department of Biochemistry, Japanese Foundation of Cancer Research, Cancer Research Institute. In 1988, he moved to the National Institute of Neuroscience, National Center for Neurology and Psychiatry to start his own research group as Head of Division of Molecular Genetics. Just after moving to the Molecular and Cellular Biology Institute Osaka University in 1997, he was promoted to professor of Department of Pathology and Tumor Biology Graduate School of Medicine Kyoto University in 1998. He was appointed Vice Dean of Graduate School of Medicine Kyoto University (2005–2008), the Head of Genome Research Center Kyoto University (2005–2008), and the Head of Career Path Promotion Unit for Young Life Scientists Kyoto University (2007–2008). Over the past 30 years, he has focused on molecular genetics and his works are summarized into two main thesis; firstly, genetic program which regulate animal development and maturation modeling with skeletal muscles and neural cells, and secondary, the discovery of Klotho mutant mice and identification of its causative gene which contributed to a clarification of genetic program of homeostasis maintenance. The elucidations of alternative splicing mechanism (1984), the roles of myogenin in muscle development (1993), the molecular mechanism of asymmetric cell division of neuronal stem cells (1995, 1997), the roles of STEF, a member of Rho family small G protein family in neuronal cell migration (1997, 2003, 2006), the molecular functions of Ptf1a in neuronal cell fate determination (2005), induction of skeletal muscle cells from bone marrow stromal cells (2005), the establishment of live-imaging systems of undifferentiated spermatogonia cells (2007), and discoveries and characterizations of -Klotho and -Klotho genes (1997, 2005, 2007) are the highlights of his study. He was awarded the Baelz Prize in 1998, the Uehara Prize in 2005, and the TAKEDA Medical Award in 2007. At present he is a Member of Science Council of Japan.