A defect in Klotho gene expression in mice accelerates the degeneration of multiple age-sensitive traits. Here, we show that overexpression of Klotho in mice extends life span. Klotho protein functions as a circulating hormone that binds to a cell-surface receptor and represses intracellular signals of insulin and insulin-like growth factor 1 (IGF1), an evolutionarily conserved mechanism for extending life span. Alleviation of aging-like phenotypes in Klotho-deficient mice was observed by perturbing insulin and IGF1 signaling, suggesting that Klotho-mediated inhibition of insulin and IGF1 signaling contributes to its anti-aging properties. Klotho protein may function as an anti-aging hormone in mammals.Klotho was originally identified as a mutated gene in a mouse strain that accelerates agedependent loss of function in multiple age-sensitive traits (1). An insertional mutation that disrupts the 5′ promoter region of the Klotho gene resulted in a strong hypomorphic allele. Mice homozygous for the mutated allele (KL −/− mice) appeared normal until 3 to 4 weeks old but then began to manifest multiple age-related disorders observed in humans, including ectopic calcification, skin atrophy, muscle atrophy, osteoporosis, arteriosclerosis, and pulmonary emphysema. KL −/− mice suffered premature death around two months of age.

The Klotho gene encodes a 130-kDa single-pass transmembrane protein with a short cytoplasmic domain (10 amino acids) and is expressed predominantly in the kidney. Mice carrying a loss-of-function mutation in the Klotho gene develop a syndrome resembling human aging, including shortened life span, skin atrophy, muscle atrophy, osteoporosis, arteriosclerosis, and pulmonary emphysema (1). Conversely, overexpression of the Klotho gene extends the life span and increases resistance to oxidative stress in mice (2-4). These observations suggest that the Klotho gene functions as an aging suppressor gene. The extracellular domain of Klotho protein is shed and secreted in the blood (2, 5), potentially functioning as a humoral factor that signals suppression of intracellular insulin/IGF1 signaling, which partly contributes to its anti-aging properties (2). However, a signaling pathway(s) directly activated by Klotho protein, including the identity of the Klotho receptor, has not been determined. The function of the transmembrane form of Klotho protein also remains to be determined.Fibroblast growth factor-23 (FGF23) 2 was originally identified as a gene mutated in patients with autosomal dominant hypophosphatemic rickets (6), where mutations in the FGF23 gene conferred resistance to inactivation by protease cleavage, resulting in elevated serum levels of FGF23 (7-12). FGF23 inhibits phosphate transport in renal proximal tubular cells and in proximal tubules perfused in vitro (13). Consistent with these findings, mice defective in FGF23 expression show increased renal phosphate reabsorption and hyperphosphatemia (14). Although FGF23 binds to multiple FGF receptors (FGFRs) (15), it has modest receptor affinity (K D ϭ 200 -700 nM) and often requires cofactors such as heparin or glycosaminoglycan (15, 16) to activate FGF signaling in cultured cells and to inhibit phosphate transport in proximal tubules perfused in vitro (13).Klotho-deficient mice (Klotho Ϫ/Ϫ mice) and FGF23 deficient mice (Fgf23 Ϫ/Ϫ mice) develop many common phenotypes, including shortened life span, growth retardation, infertility, muscle atrophy, hypoglycemia, and vascular calcification in the kidneys. Notably, they both have increased serum levels of phosphate (14, 17). These observations have led us to the hypothesis that Klotho and FGF23 may function via a common signal transduction pathway. In this report we show that Klotho binds to multiple FGFRs and functions as a cofactor necessary for FGF signaling activation by FGF23. MATERIALS AND METHODSExpression Vectors-Complementary DNA containing the mouse FGFRs coding region (IMAGE Clone, Invitrogen, supplemental Fig. 1) were cloned into pcDNA3.1(ϩ) expression vector (Invitrogen). Before subcloning, a V5-epitope tag was added to the C terminus and appropriate restriction enzyme sites to the both ends using synthetic oligonucleotides and polymerase chain reaction. Expression vectors for the mouse FGF23 resistant to proteolytic inactivation (R179Q) (18), the transmembrane form of mouse Klotho, and the extracel...

klotho is an aging suppressor gene and extends life span when overexpressed in mice. Klotho protein was recently demonstrated to function as a hormone that inhibits insulin/insulin-like growth factor-1 (IGF-1) signaling. Here we show that Klotho protein increases resistance to oxidative stress at the cellular and organismal level in mammals. Klotho protein activates the FoxO forkhead transcription factors that are negatively regulated by insulin/IGF-1 signaling, thereby inducing expression of manganese superoxide dismutase. This in turn facilitates removal of reactive oxygen species and confers oxidative stress resistance. Thus, Klotho-induced inhibition of insulin/IGF-1 signaling is associated with increased resistance to oxidative stress, which potentially contributes to the anti-aging properties of klotho.A defect in klotho gene expression in mice leads to a syndrome closely resembling human aging, including shortened life span, infertility, growth arrest, hypoactivity, skin atrophy, premature thymic involution, arteriosclerosis, osteoporosis, and pulmonary emphysema (1). Conversely, overexpression of the klotho gene extends the life span in the mouse (2), indicating that the klotho gene functions as an aging suppressor gene in mammals that delays aging when overexpressed and accelerates the development of aging-like disorders when disrupted. The klotho gene encodes a single-pass transmembrane protein and is expressed only in limited tissues, notably the distal convoluted tubules in the kidney and the choroid plexus in the brain (1). The extracellular domain of Klotho protein is shed and secreted into the blood. It then binds to a high affinity but as yet unidentified cell-surface Klotho receptor and signals suppression of tyrosine phosphorylation of insulin/ IGF-1 2 receptors and insulin receptor substrates (IRS), association of IRS with phosphatidylinositol 3-kinase (PI3K), and serine phosphorylation of Akt/PKB (2). Thus, Klotho protein is a hormone that inhibits the intracellular insulin/IGF-1 signaling cascade. This activity likely contributes to the suppression of aging by Klotho, because inhibition of insulin-like signaling is an evolutionarily conserved mechanism for extending life span (see Ref. 3 for review). Indeed, we observed alleviation of aging-like phenotypes in Klotho deficient mice (KL Ϫ/Ϫ mice) by genetic perturbation of insulin/IGF-1 signaling, indicating that the activity of Klotho that inhibits insulin/IGF-1 signaling accounts for its anti-aging properties, at least in part (2). It remains to be determined whether or not the inhibitory effect of Klotho protein on IRS, PI3K, and Akt is entirely dependent on its ability to inhibit insulin/IGF-1 receptors. Notably, single nucleotide polymorphisms in the human KLOTHO gene are associated with longevity (4) and common age-related diseases including coronary artery disease (5), osteoporosis (6 -8), and stroke (9), strongly arguing that Klotho regulates aging in humans. Increased resistance to oxidative stress is associated with increased longevi...

The mammalian fibroblast growth factor (FGF) family comprises 18 polypeptides (FGF1 to FGF10 and FGF16 to FGF23) which participate in a myriad of biological processes during embryogenesis, including but not limited to gastrulation, body plan formation, somitogenesis, and morphogenesis of essentially every tissue/organ such as limb, lung, brain and kidney (3,30). FGFs execute their biological actions by binding to, dimerizing, and activating FGF receptor (FGFR) tyrosine kinases, which are encoded by four distinct genes (Fgfr1 to Fgfr4). Prototypical FGFRs consist of an extracellular domain composed of three immunoglobulin-like domains, a single-pass transmembrane domain, and an intracellular domain responsible for the tyrosine kinase activity (16). The number of principal FGFRs is increased from four to seven due to a major tissue-specific alternative splicing event in the second half of the immunoglobulin-like domain 3 of FGFR1 to FGFR3, which creates epithelial lineage-specific b and mesenchymal lineage-specific c isoforms (16, 21). Generally, the receptor-binding specificity of FGFs is divided along this major alternative splicing of receptors whereby FGFRb-interacting FGFs are produced by mesenchymal cells and FGFRc-interacting FGFs are produced by epithelial cells (21). These reciprocal expression patterns of FGFs and FGFRs result in the establishment of a paracrine epithelial-mesenchymal signaling which is essential for proper organogenesis and patterning during development as well as tissue homeostasis in the adult organism.Based on phylogeny and sequence identity, FGFs are grouped into seven subfamilies (21). The FGF core homology domain (approximately 120 amino acids long) is flanked by Nand C-terminal sequences that are highly variable in both length and primary sequence, particularly among different FGF subfamilies. The core region of FGF19 shares the highest sequence identity with FGF21 (38%) and FGF23 (36%), and therefore, these ligands are considered to form a subfamily. However, the degree of identity within the FGF19 subfamily is only 2 to 3% greater than that between FGF19 subfamily members and members of other FGF subfamilies, making this subfamily the most divergent one. FGF19 subfamily members regulate diverse physiological processes uncommon to classical FGFs, namely, energy (32) and bile acid homeostasis (FGF19) (5,8,13), glucose and lipid metabolism (FGF21) (10), and phosphate and vitamin D homeostasis (FGF23) (27). Moreover, unlike classical FGFs, FGF19 subfamily members achieve their unconventional activities in an endocrine fashion.To date, only a single structure from the endocrine-acting FGF19 subfamily has been reported (4), whereas there are crystal structures available for eight classical, paracrine-acting FGFs (2,20,22,37,38,40). The structures from the paracrine class of FGFs (FGF1, show that the core homology region folds into a globular domain composed of 12 antiparallel ␤-strands (␤1 to ␤12) known as the * Corresponding author. Mailing address:

Fibroblast growth factor 21 (FGF21) is a liver-derived endocrine factor that stimulates glucose uptake in adipocytes. Here, we show that FGF21 activity depends on ␤Klotho, a single-pass transmembrane protein whose expression is induced during differentiation from preadipocytes to adipocytes. ␤Klotho physically interacts with FGF receptors 1c and 4, thereby increasing the ability of these FGF receptors to bind FGF21 and activate the MAP kinase cascade. Knockdown of ␤Klotho expression by siRNA in adipocytes diminishes glucose uptake induced by FGF21. Importantly, administration of FGF21 into mice induces MAP kinase phosphorylation in white adipose tissue and not in tissues without ␤Klotho expression. Thus, ␤Klotho functions as a cofactor essential for FGF21 activity.Klotho ͉ glucose ͉ adipocyte ͉ siRNA ͉ GLUT1

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Sphingolipid-cholesterol rafts are preferred platforms for membrane-linked actin polymerization. This is mediated by in situ PIP(2) synthesis and tyrosine kinase signaling through the WASP-Arp2/3 pathway. Actin comets may provide a novel mechanism for raft-dependent vesicle transport and apical membrane trafficking.

Overexpression of phosphatidylinositol phosphate 5-kinase (PIP5KI) isoforms α, β, or γ in CV-1 cells increased phosphatidylinositol 4,5-bisphosphate (PIP2) levels by 35, 180, and 0%, respectively. Endocytosis of transferrin receptors, association of AP-2 proteins with membranes, and the number of clathrin-coated pits at the plasma membrane increased when PIP2 increased. When expression of PIP5KIβ was inhibited with small interference RNA in HeLa cells, expression of PIP5KIα was also reduced slightly, but PIP5KIγ expression was increased. PIP2 levels and internalization of transferrin receptors dropped 50% in these cells; thus, PIP5KIγ could not compensate for loss of PIP5KIβ. When expression of PIP5KIα was reduced, expression of both PIP5KIβ and PIP5KIγ increased and PIP2 levels did not change. A similar increase of PIP5KIα and PIP5KIβ occurred when PIP5KIγ was inhibited. These results indicate that constitutive endocytosis in CV-1 and HeLa cells requires (and may be regulated by) PIP2 produced primarily by PIP5KIβ.