Pflügers Archiv - European Journal of Physiology

, Volume 459, Issue 2, pp 333–343

Klotho

Authors

    • Department of PathologyThe University of Texas Southwestern Medical Center at Dallas
Integrative Physiology

DOI: 10.1007/s00424-009-0722-7

Cite this article as:
Kuro-o, M. Pflugers Arch - Eur J Physiol (2010) 459: 333. doi:10.1007/s00424-009-0722-7

Abstract

The klotho gene was identified as an “aging-suppressor” gene in mice that accelerates aging when disrupted and extends life span when overexpressed. It encodes a single-pass transmembrane protein and is expressed primarily in renal tubules. The extracellular domain of Klotho protein is secreted into blood and urine by ectodomain shedding. The two forms of Klotho protein, membrane Klotho and secreted Klotho, exert distinct functions. Membrane Klotho forms a complex with fibroblast growth factor (FGF) receptors and functions as an obligate co-receptor for FGF23, a bone-derived hormone that induces phosphate excretion into urine. Mice lacking Klotho or FGF23 not only exhibit phosphate retention but also display a premature-aging syndrome, revealing an unexpected link between phosphate metabolism and aging. Secreted Klotho functions as a humoral factor that regulates activity of multiple glycoproteins on the cell surface, including ion channels and growth factor receptors such as insulin/insulin-like growth factor-1 receptors. Potential contribution of these multiple activities of Klotho protein to aging processes is discussed.

Keywords

KlothoFGF23PhosphateVitamin DβKlotho

Introduction

The klotho gene was originally identified as a gene disrupted in a mouse strain that inherited a syndrome resembling human aging in an autosomal recessive manner [48]. This strain, named after a Greek goddess Klotho who spins the thread of life, was serendipitously generated during an attempt to make transgenic mice by conventional pronuclear microinjection of a transgene [47]. Unfortunately, the transgene was not expressed in the klotho mouse. However, integration of the transgene into the mouse genome disrupted a promoter region of a certain gene and shuts down its expression, which was later identified as the klotho gene.

A defect in klotho gene expression in mice causes no visible phenotypes until 3–4 weeks of age, but thereafter leads to multiple aging-like phenotypes [48], including growth retardation, hypogonadotropic hypogonadism, rapid thymic involution [62], skin atrophy, sarcopenia, vascular calcification, osteopenia [38], pulmonary emphysema [33, 81, 92], cognition impairment [67], hearing disturbance [37], and motor neuron degeneration [1], and die prematurely around 2 months of age. In contrast, transgenic mice that overexpress the klotho gene live longer than wild-type mice [54]. Thus, the klotho gene may function as an aging-suppressor gene that extends life span when overexpressed and accelerates aging when disrupted [46].

The klotho gene is composed of five exons [59, 90] and encodes a type I single-pass transmembrane protein of 1,014 amino acid long. The intracellular domain is very short (10 amino acid long) and has no known functional domains. The extracellular domain is composed of two internal repeats with weak homology to family 1 glycosidases that hydrolyze β-glucosidic linkage in saccharides, glycoproteins, and glycolipids [48, 61].

The klotho gene is expressed predominantly in distal convoluted tubules in the kidney and choroid plexus in the brain [48]. It is also expressed in several endocrine organs, including pituitary, parathyroid [5], pancreas, ovary, testis, and placenta [48]. Despite the tissue-specific expression of the klotho gene, a defect in klotho expression causes systemic phenotypes, suggesting that Klotho may be involved in the regulation of an endocrine system(s).

Recent studies have revealed multiple functions of Klotho protein in endocrine regulation of mineral metabolism and growth factor signaling. The purpose of this review was to summarize Klotho protein function and to discuss its potential effects on aging processes in mammals.

Function of membrane Klotho protein

Klotho as a co-receptor for FGF23

Klotho protein function was not clear until it was realized that phenotypes of Klotho-deficient mice were almost identical with those of mice lacking fibroblast growth factor-23 (FGF23). FGF23 had been identified as a gene mutated in patients with autosomal dominant hypophosphatemic rickets (ADHR) [107]. ADHR is one of the hereditary disorders that exhibit a phosphate wasting into urine, indicating the involvement of FGF23 in phosphate handling in the kidney.

Recent studies have identified FGF23 as a bone-derived hormone that acts on kidney to promote phosphate excretion into urine (phosphaturia) [75]. The amount of phosphate excreted into urine is primarily determined by the amount of phosphate reabsorbed at renal proximal tubules. Phosphate in the luminal fluid in the proximal tubules is taken up mainly through sodium–phosphate co-transporter type-2a (NaPi-2a) expressed on the apical brush border membrane of proximal tubules [83, 84, 89]. FGF23 exerts its phosphaturic activity by suppressing NaPi-2a [57, 83]. Administration of recombinant FGF23 protein to rodents reduces the number of NaPi-2a inserted in the apical brush border membrane of proximal tubules and induces phosphaturia within hours, although the precise signaling pathway through which FGF23 regulates NaPi-2a trafficking and/or expression remains to be determined.

ADHR patients carry missense mutations in the FGF23 gene that confer resistance to proteolytic inactivation on the mutant FGF23 protein, resulting in high serum FGF23 levels and phosphate-wasting phenotypes including hypophosphatemia and defects in bone mineralization (rickets) [88]. Furthermore, several mutations that affect expression and/or proteolytic degeneration of FGF23 have been identified in mice and humans, in which phosphate-wasting phenotypes are associated with increased serum FGF23 levels [51, 75]. These observations have established that FGF23 functions as a phosphaturic hormone.

In contrast, mice lacking FGF23 (Fgf23−/− mice) develop phosphate-retention phenotypes characterized by extensive soft tissue calcification and hyperphosphatemia [87]. In addition to these predictable phenotypes, Fgf23−/− mice exhibit unexpected phenotypes, including growth retardation, hypogonadism, premature thymic involution, sarcopenia, osteopenia, skin atrophy, and pulmonary emphysema, which are reminiscent of the premature-aging syndrome in Klotho-deficient mice. Likewise, Klotho-deficient mice not only exhibit premature aging but also suffer extensive soft tissue calcification and hyperphosphatemia, which are reminiscent of the phosphate-retention phenotype in Fgf23−/− mice. These observations revealed an unexpected link between FGF23 and Klotho, suggesting that FGF23 and Klotho might function in a common endocrine system that regulates phosphate metabolism. In support of this notion, Klotho-deficient mice have extremely high serum FGF23 levels, indicating that loss of Klotho induces resistance to FGF23 [103].

Consistent with these genetic studies, biochemical and cell biological studies have demonstrated that Klotho protein functions as an obligate co-receptor for FGF23 [44, 53, 103]. FGF23 has very low affinity to FGF receptors (FGFRs) and cannot activate its cognate receptors without Klotho under physiological concentration [112]. Klotho protein forms a binary complex with several FGF receptor isoforms (FGFR1c, 3c, and 4) and significantly increases their affinity to FGF23 [53]. The fact that FGF23 requires Klotho for activating FGF receptors explains why FGF23-deficient mice and Klotho-deficient mice develop identical phenotypes and why Klotho-deficient mice are resistant to FGF23. Also, kidney-specific expression of Klotho explains why FGF23 can identify kidney as its target organ among many other tissues that express multiple FGF receptor isoforms. Thus, Klotho and FGF23 have emerged as essential components of this newly identified bone–kidney endocrine axis that maintains phosphate homeostasis [45].

Another important function of this endocrine axis is to regulate serum levels of vitamin D. Vitamin D has an activity that promotes absorption of dietary phosphate from intestine [16]. The active form of vitamin D (1,25-dihydroxyvitamin D3) is synthesized in the kidney from its inactive precursor (25-hydroxyvitamin D3) with 1α-hydroxylase encoded by the Cyp27b1 gene and is inactivated with 24-hydroxylase encoded by the Cyp24 gene. FGF23 suppresses Cyp27b1 gene expression and increases Cyp24 gene expression, leading to reduction of serum 1,25-dihydroxyvitamin D3 levels [86]. Thus, in addition to functioning as a phosphaturic hormone, FGF23 functions as a counter-regulatory hormone for vitamin D [58]. These two distinct activities of FGF23 collectively induce a negative phosphate balance in a Klotho-dependent manner.

The bone–kidney endocrine axis mediated by Klotho and FGF23

Vitamin D positively regulates expression of the FGF23 gene in the bone. Injection of 1,25-dihydroxyvitamin D3 increases serum FGF23 levels within hours in rodents [86]. Binding of 1,25-dihydroxyvitamin D3 to nuclear vitamin D receptor (VDR) induces heterodimerization with another nuclear receptor RXR. The VDR-RXR heterodimer in turn binds to the promoter region of the FGF23 gene and transactivates its expression [45]. FGF23 secreted from osteocytes reaches the Klotho–FGFR complex in the kidney and transmits signal to suppress synthesis and promote inactivation of 1,25-dihydroxyvitamin D3, thereby closing a negative feedback loop (Fig. 1). This negative feedback loop is essential for vitamin D homeostasis because defects in either FGF23 or Klotho result in elevated serum 1,25-dihydroxyvitamin D3 levels (hypervitaminosis D) in mice and humans.
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Fig. 1

The bone–kidney endocrine axis that regulates phosphate and vitamin D homeostasis. High serum phosphate increases FGF23 expression in the bone. FGF23 secreted from the bone binds to the Klotho–FGFR complex expressed on the kidney and suppresses phosphate reabsorption by inhibiting NaPi-2a (a negative feedback loop for phosphate homeostasis). High serum vitamin D increases FGF23 expression. Active vitamin D (1,25-dihydroxyvitamin D3) binds to vitamin D receptor (VDR) in osteocytes, which forms a heterodimer with a nuclear receptor RXR and directly binds to a promoter region of the FGF23 gene to transactivate its expression. FGF23 acts on the Klotho–FGFR complex in the kidney and suppresses expression of Cyp27b1 gene that encodes 1α-hydroxylase (a negative feedback loop for vitamin D homeostasis)

Phosphate also positively regulates FGF23 expression, although the mechanism behind this regulation remains to be determined. Phosphate overload by high phosphate diet increases serum FGF23 levels in mice and humans [73, 79, 95]. Increased FGF23 in turn induces phosphaturia to excrete excess phosphate into urine, thereby closing another negative feedback loop for phosphate homeostasis (Fig. 1). Suppression of serum vitamin D levels by increased FGF23 also contributes to prevention of phosphate retention through reducing phosphate absorption from intestine. However, these two negative feedback loops for phosphate and vitamin D homeostasis can function independently. Although VDR-deficient mice (VDR−/− mice) have low serum levels of phosphate and FGF23, the “rescue” diet (rich in calcium and phosphate) restores serum FGF23 levels in VDR−/− mice, indicating that phosphate can increase FGF23 independently of vitamin D [113]. Also, administration of low-dose vitamin D increases serum FGF23 levels without significant increase in serum phosphate levels in mice, indicating that vitamin D can increase FGF23 independently of phosphate [86].

Disruption of these negative feedback loops by ablating either FGF23 or Klotho leads to hyperphosphatemia and hypervitaminosis D. Hypercalcemia is also observed because vitamin D promotes both phosphate and calcium absorption from intestine. The question is whether this metabolic state (hyperphosphatemia, hypercalcemia, and/or hypervitaminosis D) is primarily responsible for the premature-aging syndrome. Several studies have addressed this question. First, Klotho- and FGF23-deficient mice, when placed on vitamin D-deficient diet, exhibited normal serum phosphate and calcium levels and no longer developed aging-like phenotypes [91, 100]. Second, ablation of vitamin D action in Klotho-deficient mice and FGF23-deficient mice by disrupting the Cyp27b1 gene [69, 77] or vitamin D receptor gene [25] rescued hyperphosphatemia and hypercalcemia, as well as the premature aging syndrome. Lastly, low phosphate diet rescued shortened life span and vascular calcification in FGF23- and Klotho-deficient mice [64, 91]. These studies have clearly demonstrated that the premature aging syndrome caused by defects in the Klotho-FGF23 endocrine axis is attributed to retention of phosphate, calcium, and/or vitamin D. It should be noted that low phosphate diet significantly increases serum levels of calcium and vitamin D [91]. This is considered as an adaptation for limited phosphate availability to maximize phosphate absorption from intestine by increasing serum vitamin D levels. However, vitamin D promotes calcium absorption at the same time and results in hypercalcemia. In fact, low phosphate diet further increased already high serum calcium and vitamin D levels in FGF23-deficeint mice but still rescued multiple aging-like phenotypoes [91]. These findings suggest that phosphate, but not calcium or vitamin D, is primarily responsible for the aging-like phenotypes. It is likely that low vitamin D diet and ablation of vitamin D activity rescued accelerated aging through reducing serum phosphate levels, although it is possible that high serum vitamin D and/or calcium levels may be a prerequisite for phosphate to induce the premature aging syndrome.

Phosphate and aging

Phosphate and insulin sensitivity

Inorganic phosphate is one of the essential nutrients and a major structural component of DNA, cell membrane (phospholipids), and bone. In addition, phosphate participates in a myriad of biological processes including energy metabolism (ATP production) and signal transduction mediated by kinases. It is also involved in pathophysiology of various common disorders such as bone diseases (osteoporosis and osteomalasia), vascular calcification, and chronic kidney disease among others. Although direct effects of phosphate on aging have not been explored extensively, several studies have demonstrated that inorganic phosphate affects glucose metabolism and oxidative stress, which potentially modifies aging processes as well.

Animals placed on low phosphate diet were reported to exhibit increased gluconeogenesis and decreased glycolysis [109, 110]. This metabolic state is reminiscent of that induced by diet restriction, which delays aging and extends life span in diverse organisms. To adapt reduced food availability, animals under diet restriction reduces blood insulin levels and attenuates overall activity of intracellular insulin signaling in tissues, which triggers changes in the expression of insulin-responsive genes and leads to global changes in glucose metabolism, including increased gluconeogenesis and decreased glycolysis [9, 39, 55, 106]. Although phosphate restriction does not induce hypoinsulinemia like diet restriction, low phosphate diet induces moderate insulin resistance by unknown mechanisms [23, 72]. The moderate insulin resistance induced by phosphate restriction also attenuates overall activity of intracellular insulin signaling in tissues, leading to changes in expression of insulin-responsive genes and metabolic state similar to those induced by hypoinsulinemia upon diet restriction. Conversely, phosphate retention is associated with increased insulin sensitivity and accelerated aging as observed in Klotho-deficient mice and FGF23-deficient mice [77, 87, 104]. They exhibit hypoglycemia, hypoinsulinemia, and extremely increased insulin sensitivity, which can be rescued by resolving hyperphosphatemia [25]. Thus, serum phosphate levels positively correlate with insulin sensitivity.

Increased insulin resistance does not necessarily mean diabetes and short life span. Rather, it has become increasingly clear that partial or tissue-specific inhibition of insulin-like signaling pathway is a mechanism for anti-aging and life span extension evolutionarily conserved from worms to mammals. Hypomorphic alleles of the genes encoding orthologs of insulin receptor, insulin receptor substrates (IRS), and PI3 kinase have been associated with extended life span in Caenorhabditis elegans and Drosophila [14, 40, 41, 65, 96]. In rodents, extended longevity was reported in mice lacking insulin receptor in adipose tissues [6], mice heterozygous for a null allele of the insulin-like growth factor-1 (IGF-1) receptor gene [28], mice lacking IRS-1 [85], mice lacking IRS-2 in the brain [94], and dwarf mice with impaired somatotrophic endocrine axis (the growth hormone–IGF-1 endocrine axis) [4, 8, 19]. In humans, some centenarians carry loss-of-function mutations in the IGF-1 receptor gene and show resistance to IGF-1, short statue, and high serum IGF-1 levels [93]. In addition, some long-lived mice exhibit insulin resistance [54, 85], indicating that increased insulin sensitivity is not a prerequisite for long life span and anti-aging. Although life span extension by diet restriction is associated with increased insulin sensitivity, it is associated with hypoinsulinemia and the metabolic state compatible with attenuated insulin signaling activity in tissues.

Phosphate and oxidative stress

In addition to its involvement in glucose metabolism and insulin sensitivity, inorganic phosphate increases oxidative stress both in vivo and in vitro. Phosphate retention caused by Klotho deficiency in mice is associated with cognition impairment due to increased oxidative damage to hippocampus neurons, which can be rescued by administration of an antioxidant [67]. Furthermore, human vascular endothelial cells exposed to high phosphate medium (2.5 mM) have higher levels of cellular reactive oxygen species (ROS) than those cultured in normal phosphate medium (1.0 mM) [15]. These observations have raised the possibility that hyperphosphatemia by itself may be a cause of endothelial dysfunction and cardiovascular disease in chronic kidney disease (CKD) patients because blood phosphate levels higher than 2.5 mM are often observed in these patients. Indeed, hyperphosphatemia has been identified as a potent risk of death in CKD patients [21, 99]. Of note, the National Kidney Foundation task force has indicated that the cardiovascular mortality of a 35-year-old patient on dialysis is equivalent to that of an 80-year-old “healthy” individual, rendering CKD to be a premature vascular aging [60]. Also, the American Heart Association identified CKD patients as the highest-risk group for cardiovascular disease [80]. Importantly, Klotho expression is significantly reduced in CKD patients [43]. Thus, CKD may be viewed as a segmental progeroid syndrome associated with Klotho deficiency and phosphate retention.

Extracellular phosphate enters into the cell primarily through sodium-phosphate co-transporter type-3 (NaPi-3, also known as Pit-1/2) [105]. Cytoplasmic phosphate is transported into mitochondria to be utilized for oxidative phosphorylation. It has been known that extra-mitochondrial phosphate concentration (or cytoplasmic phosphate concentration) positively correlates with mitochondrial membrane potential (∆Ψ) [7], and ∆Ψ positively correlates with ROS production in mitochondria [70]. In addition, phosphate enhances delivery of reducing equivalent to cytochrome c in complex III in the electron transport chain [7]. These effects of phosphate on mitochondrial function potentially increase ROS production in the cell and may affect aging processes.

Phosphate and longevity

The fact that the premature aging syndrome in Klotho-deficient mice and FGF23-deficient mice is rescued by resolving hyperphosphatemia has raised the possibility that aging process may be accelerated by inorganic phosphate that plays multiple roles in glucose metabolism, insulin sensitivity, and oxidative stress in health and disease. Several lines of circumstantial evidence support this notion. First, serum phosphate levels positively correlate with all-cause mortality in humans. An epidemiological study using patients whose serum phosphate levels are within normal range demonstrated that those with high serum phosphate levels (≥4.0 mg/dL) exhibited ~70% higher mortality rate than those with low serum phosphate levels (<2.5 mg/dL) [99]. Furthermore, the longevity of various species in mammals inversely correlates with the serum phosphate level, but not with the serum calcium level (Table 1, Fig. 2), indicating an association between phosphate metabolism and aging processes.
Table 1

Relation between longevity and serum phosphate level in mammals

Species

Longevity (year)

Serum phosphate (mg/dL)a

Serum calcium (mg/dL)a

Reference

Mouse (Klotho−/−)

0.15

14.0

10.3

[48, 84]

Mouse

3

7.8

9.2

[27, 84]

Rat

3

9.0

11.8

[27, 97]

Hamster

4

6.5

9.7

[18, 22, 97]

Gerbil

6

5.4

4.8

[18, 22]

Nutria

9

7.5

9.6

[17, 22]

Rabbit

10

7.4

14

[27, 97]

Guinea pig

12

5.3

10.2

[22, 97]

Sheep

21

5.5

8.4

[27, 74]

Squirrel

24

5.4

7.5

[2, 22]

Porcupine

27

4.4

9.6

[22, 63]

Naked mole rat

28

4.6

9.6

[22, 111]

Flying fox

30

4.4

9.4

[24, 27]

Bear

50

4.9

9.6

[27, 76]

Rhinoceros

50

3.7

12.4

[27, 66]

Camel

50

4.6

9.3

[27, 78]

Elephant

70

4.3

9.5

[27, 102]

Human

75

3.6

9.5

[27, 71]

Human (centenarian)

100

3.1

8.6

[71]

aAverage or median values, whichever available in the literatures

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Fig. 2

Relation between longevity and serum levels of phosphate and calcium in mammals (based on Table 1). The longevity is inversely correlated with serum phosphate levels (closed circles, \( y = - 1.5183{\text{Ln}}(x) + 10.015 \), R2 = 0.8945). On the other hand, there is no significant correlation between longevity and serum calcium levels (open circles)

Function of secreted Klotho protein

Regulation of ion channel activity

In addition to functioning as a co-receptor for FGF23, Klotho protein functions as a humoral factor. The extracellular domain of Klotho protein is clipped on the cell surface (ectodomain shedding) by membrane-anchored proteases ADAM10 and ADAM17 to generate a secreted form of Klotho protein [13]. In fact, the entire extracellular domain of ~120–130 kDa (Klotho ectodomain) is detected in the blood, urine, and cerebrospinal fluid (CSF) [29, 54]. Thus, Klotho protein exists at least in two forms. One is membrane Klotho expressed primarily in renal tubular cells, and the other is secreted Klotho that exists in the blood, urine, and CSF.

Although membrane Klotho functions as a co-receptor for FGF23, secreted Klotho cannot function as a soluble receptor for FGF23 because it is the Klotho–FGFR complex that has high affinity to FGF23, but not Klotho protein or FGFR alone [53]. These findings suggest that secreted Klotho may have function independent of FGF23. Recent studies have revealed novel function of secreted Klotho distinct from that of membrane Klotho, which includes regulation of multiple ion channels and growth factor receptors on the cell surface.

Transient receptor potential cation channel, subfamily V, member 5 (TRPV5) is one of the ion channels regulated by secreted Klotho protein [11, 12]. TRPV5 is a calcium channel expressed on the apical side of the renal tubular cells and is primarily responsible for the Ca2+ entry in transepithelial Ca2+ reabsorption in the kidney [26]. Secreted Klotho protein inhibits internalization of TRPV5 and increases the number of TRPV5 on the cell surface, thereby increasing TRPV5-mediated Ca2+ influx and renal Ca2+ reabsorption. In fact, Klotho-deficient mice exhibit higher calcium excretion into urine than wild-type mice [101]. The Klotho-induced retention of TRPV5 on the plasma membrane was associated with modification of the glycans of TRPV5 [11, 12]. Consistent with the fact that Klotho belongs to the family 1 glycosidases that cleave glycosidic bonds, secreted Klotho was reported to have weak β-glucuronidase activity in vitro [98]. However, the β-glucuronidase activity of Klotho may not be involved in the modification of TRPV5 glycans because typical N-linked glycans of mammalian glycoproteins do not contain glucuronic acids. Instead, sialic acids cap the terminals of branched N-linked glycans. Secreted Klotho removes these terminal sialic acids though its putative sialidase activity [11]. Removal of sialic acids exposes underlying galactose or disaccharide N-acetyllactosamine in the glycans. Once exposed, these sugars can bind to galectin-1, a lectin (sugar-binding protein) abundant in the extracellular matrix. Thus, removal of terminal sialic acids from the glycans of TRPV5 by secreted Klotho promotes interaction between TRPV5 glycans and galectin-1, which traps TRPV5 on the cell surface and prevents its endocytosis, resulting in accumulation of TRPV5 on the plasma membrane (Fig. 3) [11].
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Fig. 3

Klotho inhibits internalization of cell-surface calcium channel TRPV5. The number of TRPV5 on the plasma membrane is determined by counterbalance between insertion by forward trafficking from Golgi and removal by endocytosis to endosomes. Terminals of sugar chains of many cell-surface glycoproteins are capped with sialic acids (red). Secreted Klotho protein removes these sialic acids through its putative α2→6 sialidase activity and exposes underlying galactose residues (green) in the glycans. The exposed galactose then binds to galectin-1 (blue) in the extracellular matrix. Galectin-1 traps TRPV5 on the cell surface and prevents its endocytosis, leading to accumulation of TRPV5 on the plasma membrane and increase in calcium influx

Furthermore, secreted Klotho regulates renal outer medullary potassium channel (ROMK1) through the mechanism identical with that for TRPV5 regulation [10]. ROMK1 is a potassium channel expressed on the apical side of renal tubular cells and is primarily responsible for potassium secretion into urine. Secreted Klotho removes terminal sialic acids from the glycans of ROMK1, which triggers interaction between the ROMK1 glycans and galectin-1, tethers ROMK1 to extracellular matrices, and prevents its internalization. Thus, secreted Klotho increases the number of ROMK1 on the plasma membrane and increases potassium secretion into urine. Indeed, injection of secreted Klotho into mice promotes potassium excretion into urine [10]. This action of secreted Klotho represents a novel mechanism for regulation of internalization of cell-surface glycoproteins.

Regulation of growth factor signaling

Klotho-deficient mice are hypoglycemic, hypoinsulinemic, and extremely sensitive to insulin [25, 104]. In contrast, Klotho-overexpressing transgenic mice are resistant to insulin and IGF-1, although they maintain normal fasting blood glucose levels and are not diabetic [54]. These observations suggest that Klotho may have an inhibitory effect on insulin/IGF-1 activity. In fact, secreted Klotho has an activity that inhibits insulin- and IGF-1-induced autophosphorylation of insulin receptor and IGF-1 receptor when applied to cultured cells [54, 108]. Although the mechanism by which secreted Klotho inhibits activity of insulin/IGF-1 receptors remains to be determined, it is possible that secreted Klotho may alter internalization and cell surface abundance of insulin/IGF-1 receptors by modifying their glycans. The ability of Klotho to inhibit insulin/IGF-1 receptors may contribute to the anti-aging properties of Klotho since numerous lines of genetic evidence indicate that partial inhibition of insulin-like signaling pathway is one of the evolutionarily conserved mechanisms for suppressing aging as discussed above. The fact that insulin promotes Klotho shedding [13] is of potential importance in the regulation of insulin activity in vivo. Insulin may increase serum levels of secreted Klotho protein, which in turn inhibits insulin signaling in peripheral tissues and prevents prolonged insulin action. This may represent a novel negative feedback mechanism of insulin signaling by secreted Klotho through its endocrine mode of action and may partly explain why Klotho-deficient mice are extremely insulin sensitive.

It was reported that secreted Klotho protein bound to several Wnt ligands and inhibited Wnt signaling through preventing Wnt binding to its cognate cell-surface receptor [56]. Although activation of Wnt signaling is essential for stem cell proliferation and survival, continuous and prolonged Wnt signaling activation can cause exhaustion and depletion of stem cells [42, 82]. Since stem cell dysfunction limits tissue regeneration and potentially affects aging processes, the ability of secreted Klotho protein to inhibit Wnt signaling may also contribute to aging-like phenotypes in Klotho-deficient mice. The number of epidermal stem cells in hair follicles in Klotho-deficient mice was much lower than that in wild-type mice. Enhanced Wnt signaling activity and expression of SAβ-galactosidase, a marker for cell senescence, were observed in epidermal stem cells in Klotho-deficient mice. These findings can be explained by assuming that Klotho deficiency caused continuous activation of Wnt signaling and senescence of stem cells. The reduced number of epidermal stem cells may also explain poor wound healing observed in Klotho-deficient mice.

Concluding remarks

The discovery of the klotho gene has led to identification of a novel bone–kidney endocrine axis that maintains phosphate and vitamin D homeostasis: FGF23 is secreted from bone and acts on kidney where Klotho is expressed to induce phosphaturia and to counteract vitamin D. Although FGF23 belongs to the FGF ligand superfamily, amino acid sequence analysis has segregated FGF23 and two additional FGFs (FGF19 and FGF21) from the other classic FGF ligands [36]. These atypical FGFs (FGF19, FGF21, and FGF23) are collectively called endocrine FGFs [20] because they function as hormones unlike the other classic FGFs that primarily act as paracrine and/or autocrine factors. FGF19 is secreted from intestine upon feeding and acts on liver to suppress bile acid synthesis [30]. FGF21 is secreted from liver upon fasting and acts on white adipose tissue to promote lipolysis and to mediate fasting metabolic responses [3, 31]. Like FGF23 requires Klotho, FGF19 and FGF21 require βKlotho as an obligate co-receptor for high affinity binding to their cognate FGF receptors [49, 68]. βKlotho was identified based on its sequence similarity to Klotho [35]. Unlike Klotho, βKlotho is expressed predominantly in the liver and white adipose tissue [35, 49], the target organs of FGF19 and FGF21, respectively. Consistent with the fact that βKlotho is an obligate co-receptor for FGF19, mice lacking either βKlotho or FGF15 (the mouse ortholog of human FGF19) exhibited increased bile acid synthesis [30, 34]. Thus, Klotho gene family may have evolved in the regulation of endocrine FGFs to confine their target organs in the redundant receptor-ligand system [45, 5052].

Multiple novel endocrine axes mediated by endocrine FGFs and Klothos have emerged that regulate various metabolic processes. FGF23 and Klotho affect aging processes through regulating phosphate metabolism. Interestingly, transgenic mice that overexpress FGF21 exhibit short statue and resistance to growth hormone, which is reminiscent of dwarf mice [32]. It remains to be determined whether FGF19, FGF21, and βKlotho may also affect aging processes through regulating metabolic responses to feeding and fasting.

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© Springer-Verlag 2009