Klotho: The Ageing-Klotho: The Ageing-Suppressor GeneSuppressor Gene

ByBy Naglaa Fathy AlhusseiniNaglaa Fathy AlhusseiniAssistant prof. of Medical Assistant prof. of Medical

BiochemistryBiochemistry Faculty of MedicineFaculty of MedicineBenha UniversityBenha University

20132013

OverviewOverview

• IntroductionIntroduction

• Klotho-deficient miceKlotho-deficient mice

• Klotho-overexpressing transgenic Klotho-overexpressing transgenic micemice

• Klotho protein functionsKlotho protein functions

• ConclusionsConclusions

IntroductionIntroductionKlotho Klotho • in Greek mythology, in Greek mythology,

three goddesses three goddesses determine life span of determine life span of every one by controlling every one by controlling the thread of life. They the thread of life. They are Klotho, Lachesis, are Klotho, Lachesis, and Atropos who spins, and Atropos who spins, measures, and cuts the measures, and cuts the thread of life, thread of life, respectively. respectively.

– Control the life (spins the thread of life) Control the life (spins the thread of life) and destiny of everyoneand destiny of everyone

– She is the goddess who helps life to She is the goddess who helps life to unfold, in contrast to The (apoptotic) unfold, in contrast to The (apoptotic) Atropos, who cuts the thread of lifeAtropos, who cuts the thread of life

IntroductionIntroductionGene Gene • Identified app. 16 years ago, by the Japanese group of Kuro-o Identified app. 16 years ago, by the Japanese group of Kuro-o

et alet al.*.*• Named after Greek goddess,As prolonging lifespan is Named after Greek goddess,As prolonging lifespan is

probably the most important role of this ageing-suppressor probably the most important role of this ageing-suppressor genegene

• The The klothoklotho gene is composed of 5 exons gene is composed of 5 exons

• The The klothoklotho gene is expressed in limited tissues and cell types. gene is expressed in limited tissues and cell types. The highest expression is observed in distal convoluted The highest expression is observed in distal convoluted tubules in the kidney and choroid plexus in the brain .tubules in the kidney and choroid plexus in the brain .

• Lower expression is detected in pituitary gland, brain, Lower expression is detected in pituitary gland, brain, parathyroid gland pancreas, ovary, testis, placenta, skeletal parathyroid gland pancreas, ovary, testis, placenta, skeletal muscle, urinary bladder, colon , inner ear , and breast muscle, urinary bladder, colon , inner ear , and breast epithelial cells epithelial cells

*Nature 1997; 390: 45–51

IntroductionIntroduction

• Japanese researchers reported Japanese researchers reported – Defect in Defect in Klotho Klotho gene expression in the gene expression in the

mouse resulted in a syndrome that mouse resulted in a syndrome that resembled resembled •Human ageing, including Human ageing, including

– Short lifespan, infertility, arteriosclerosis, skin Short lifespan, infertility, arteriosclerosis, skin atrophy, osteoporosis and emphysemaatrophy, osteoporosis and emphysema

•The gene encoded a membrane protein that The gene encoded a membrane protein that – Shared sequence similarity with the beta-Shared sequence similarity with the beta-

glucosidase enzymesglucosidase enzymes

Nature 1997; 390: 45–51

Klotho- deficient miceKlotho- deficient mice

7-week-old normal mouse (left) and a klotho mouse, an animal model that shows multiple phenotypes resembling human aging

Ageing Research Reviews 2009;8:43–51

Ageing Research Reviews 2009;8:43–51

IntroductionIntroduction

• The Klotho gene encodes The Klotho gene encodes – Single-pass Single-pass

transmembrane protein transmembrane protein •Belongs to a family 1 Belongs to a family 1

glycosidase glycosidase

•Expressed primarily in renal Expressed primarily in renal tubules (distal tubules)tubules (distal tubules)

– Present in the circulation Present in the circulation and urine and urine

Blood Cells Mol Dis 1998; 24: 83–100

• Klotho-deficient mice and FGF23-deficient Klotho-deficient mice and FGF23-deficient mice have an identical phenotype including mice have an identical phenotype including – Hyperphosphataemia, hypercalcaemia, Hyperphosphataemia, hypercalcaemia,

elevated plasma calcitriol and vascular elevated plasma calcitriol and vascular calcification, in addition to premature calcification, in addition to premature ageingageing

• In contrast, over-expression of the In contrast, over-expression of the KlothoKlotho gene gene – Extends the lifespan and increases Extends the lifespan and increases

resistance to oxidative stressresistance to oxidative stress

Nephrol Dial Transplant 2007; 22: 1524–1526

Regulation of FGF23 signaling Regulation of FGF23 signaling by Klothoby Klotho

• The common phenotypes of Klotho and The common phenotypes of Klotho and FGF23 overexpression and deletion, FGF23 overexpression and deletion, respectively, led to the postulate of a respectively, led to the postulate of a – Common signal transduction pathwayCommon signal transduction pathway– Kuro Kuro et alet al.* showed that .* showed that – Klotho protein directly bound to multiple Klotho protein directly bound to multiple

FGFRsFGFRs– The Klotho–FGFR complex bound to FGF23 The Klotho–FGFR complex bound to FGF23

with higher affinity than FGFR or Klotho with higher affinity than FGFR or Klotho alonealone

*J Biol Chem 2006; 281: 6120–6123

Regulation of FGF23 signaling Regulation of FGF23 signaling by Klothoby Klotho

• Conversion by Klotho of canonical Conversion by Klotho of canonical FGF receptor into FGF23-specific FGF receptor into FGF23-specific receptorreceptor

Nephrol Dial Transplant 2007; 22: 1524–1526

Regulation of FGF23 signaling Regulation of FGF23 signaling by Klothoby Klotho

• The fact that FGF23 requires Klotho The fact that FGF23 requires Klotho as a co-receptor explains as a co-receptor explains – Why Klotho-deficient mice develop Why Klotho-deficient mice develop

phenotypes identical with those phenotypes identical with those observed in FGF23-deficient mice and observed in FGF23-deficient mice and

– Why Klotho-deficient mice had Why Klotho-deficient mice had extremely high serum FGF23 levelsextremely high serum FGF23 levels

Nephrol Dial Transplant 2009;24: 1705–1708

Function of the Function of the transmembrane form of transmembrane form of KlothoKlotho

1- Regulation of phosphate metabolism 1- Regulation of phosphate metabolism • Recent studies have identified FGF23 as a novel Recent studies have identified FGF23 as a novel

endocrine factor that lowers blood phosphate endocrine factor that lowers blood phosphate and vitamin D levels .and vitamin D levels .

• FGF23 is secreted from osteocytes in response FGF23 is secreted from osteocytes in response to high blood phosphate and vitamin D levels . to high blood phosphate and vitamin D levels . FGF23 acts on kidney to induce phosphaturia .FGF23 acts on kidney to induce phosphaturia .

• FGF23 induces a negative phosphate balance FGF23 induces a negative phosphate balance by functioning as a phosphaturic hormone as by functioning as a phosphaturic hormone as well as a counter-regulatory hormone for well as a counter-regulatory hormone for vitamin Dvitamin D

Kuro-o 2009, Klotho and aging NIHKuro-o 2009, Klotho and aging NIH

Function of the secreted Function of the secreted form of Klothoform of Klotho

1- Suppression of insulin/IGF-1 signaling 1- Suppression of insulin/IGF-1 signaling

• Klotho-deficient mice are hypoglycemic and extremely Klotho-deficient mice are hypoglycemic and extremely sensitive to insulin. In contrast, Klotho-overexpressing sensitive to insulin. In contrast, Klotho-overexpressing transgenic mice are moderately resistant to insulin and IGF-transgenic mice are moderately resistant to insulin and IGF-1, although they maintain normal fasting blood glucose 1, although they maintain normal fasting blood glucose levels and are not diabetic. levels and are not diabetic.

• These observations suggest that Klotho may inhibit the These observations suggest that Klotho may inhibit the insulin/IGF-1 signaling pathway. insulin/IGF-1 signaling pathway.

• The ability of Klotho to inhibit insulin/IGF-1 signaling may be The ability of Klotho to inhibit insulin/IGF-1 signaling may be associated with anti-aging properties of Klotho, since associated with anti-aging properties of Klotho, since numerous lines of genetic evidence indicate that moderate numerous lines of genetic evidence indicate that moderate inhibition of insulin-like signaling pathway is one of the inhibition of insulin-like signaling pathway is one of the evolutionarily conserved mechanisms for suppressing aging evolutionarily conserved mechanisms for suppressing aging

Kuro-o 2009, Klotho and aging NIHKuro-o 2009, Klotho and aging NIH

2- Suppression of oxidative stress 2- Suppression of oxidative stress

• Activation of insulin/IGF-1 signaling increases Activation of insulin/IGF-1 signaling increases activity of a serine-threonine kinase, which activity of a serine-threonine kinase, which inactivate the transcription factors FOXOs (up-inactivate the transcription factors FOXOs (up-regulate expression of multiple target genes, regulate expression of multiple target genes, including antioxidant enzymes such as catalase and including antioxidant enzymes such as catalase and mitochondrial manganese-superoxide dismutase mitochondrial manganese-superoxide dismutase (SOD2) .(SOD2) .

• Catalase and SOD2 detoxify harmful reactive oxygen Catalase and SOD2 detoxify harmful reactive oxygen species (ROS) hydrogen peroxide and superoxide, species (ROS) hydrogen peroxide and superoxide, respectively, and reduce oxidative stress. respectively, and reduce oxidative stress.

• Thus, activation of FOXOs by inhibiting insulin/IGF-1 Thus, activation of FOXOs by inhibiting insulin/IGF-1 signaling can increase cellular protection against signaling can increase cellular protection against oxidative stress and may contribute to the oxidative stress and may contribute to the suppression of aging.suppression of aging.

3- Nitric oxide production3- Nitric oxide production

• Klotho deficiency causes a reduction Klotho deficiency causes a reduction of NO synthesis in vascular of NO synthesis in vascular endothelial cells.endothelial cells.

• Consistent with this finding, Klotho-Consistent with this finding, Klotho-deficient mice exhibit impaired deficient mice exhibit impaired angiogenesis, which is dependent on angiogenesis, which is dependent on endothelium-derived NO endothelium-derived NO

4- Klotho as a suppressor of breast cancer4- Klotho as a suppressor of breast cancer • clinical and laboratory data indicate a critical role clinical and laboratory data indicate a critical role

for insulin/IGF-1 signaling in breast cancer. Notably: for insulin/IGF-1 signaling in breast cancer. Notably: • 1) increased serum insulin levels are associated 1) increased serum insulin levels are associated

with adverse prognosis in breast cancer with adverse prognosis in breast cancer • 2) High circulating IGF-1 levels are associated with 2) High circulating IGF-1 levels are associated with

increased risk of pre-menopausal breast cancer and increased risk of pre-menopausal breast cancer and • 3) Inhibition of insulin/IGF-1 signaling inhibits growth 3) Inhibition of insulin/IGF-1 signaling inhibits growth

of breast cancer cells.of breast cancer cells.• Since secreted Klotho protein inhibits activation of Since secreted Klotho protein inhibits activation of

insulin/IGF-1 receptors, Klotho may function as a insulin/IGF-1 receptors, Klotho may function as a suppressor of breast cancer. suppressor of breast cancer.

Kuro-o 2009, Klotho and aging NIHKuro-o 2009, Klotho and aging NIH

• In addition, several lines of experimental In addition, several lines of experimental evidence support the notion that Klotho evidence support the notion that Klotho functions as a tumor suppressor. functions as a tumor suppressor.

• 1) Forced expression of Klotho in breast 1) Forced expression of Klotho in breast cancer cell lines reduces their proliferation. cancer cell lines reduces their proliferation.

• Inhibition of IGF-1 signaling by Klotho Inhibition of IGF-1 signaling by Klotho increased expression of CCAAT/enhancer-increased expression of CCAAT/enhancer-binding proteins (C/EBP) binding proteins (C/EBP)

• Klotho may be a suppressor of breast Klotho may be a suppressor of breast cancer. It remains to be determined cancer. It remains to be determined whether the anti-breast cancer activity of whether the anti-breast cancer activity of Klotho depends primarily on its ability to Klotho depends primarily on its ability to suppress IGF-1 signaling or on other suppress IGF-1 signaling or on other unknown mechanisms.unknown mechanisms.

• Defects in Klotho expression can lead to Defects in Klotho expression can lead to

underexpression of FGF23 and accumulation underexpression of FGF23 and accumulation of phosphatesof phosphates.  .  Accumulated phosphates Accumulated phosphates can accelerate agingcan accelerate aging.  .  Phosphate retention Phosphate retention can lead to an aging phenotypecan lead to an aging phenotype. .

• FGF23 and its relationship to Klotho are FGF23 and its relationship to Klotho are linked to a number of bone and joint linked to a number of bone and joint diseasesdiseases. .

• Klotho is a regulator of oxidative stress and Klotho is a regulator of oxidative stress and cell senescencecell senescence. .

• Klotho inhibits growth and promotes Klotho inhibits growth and promotes apoptosis in some cancer linesapoptosis in some cancer lines. .

• Klotho protein protects against endothelial Klotho protein protects against endothelial dysfunction and hypertensiondysfunction and hypertension. .

• Control of Klotho expression comes about Control of Klotho expression comes about through epigenetic mechanisms; some through epigenetic mechanisms; some cancers can silence Klotho expressioncancers can silence Klotho expression. .

• Consuming cola drinks rich in phosphoric Consuming cola drinks rich in phosphoric acid when coupled with Klotho acid when coupled with Klotho insufficiency may exert a proinsufficiency may exert a pro--aging effectaging effect. .

• In humans, studies suggest that Klotho KLIn humans, studies suggest that Klotho KL--VS gene polymorphisms may be VS gene polymorphisms may be associated with stroke, coronary artery associated with stroke, coronary artery disease and longevitydisease and longevity. .

• Klotho pathways might be targets for antiKlotho pathways might be targets for anti--aging interventionsaging interventions. .

Human KLOTHO gene polymorphism Human KLOTHO gene polymorphism

and mutationsand mutations • . . A functional variant of human Klotho A functional variant of human Klotho

protein contains six sequence variations .protein contains six sequence variations .• Interestingly, heterozygotes for this variant Interestingly, heterozygotes for this variant

have advantage for longevity, while have advantage for longevity, while homozygotes are disadvantageous for homozygotes are disadvantageous for survival, because homozygotes are survival, because homozygotes are significantly under-represented in the aged. significantly under-represented in the aged. In contrast, heterozygotes have longer life In contrast, heterozygotes have longer life span than wild-types in multiple populations, span than wild-types in multiple populations, suggesting that the suggesting that the KLOTHOKLOTHO gene variation gene variation affects human life span.affects human life span.

Kuro-o 2009, Klotho and aging NIHKuro-o 2009, Klotho and aging NIH

• In addition, homozygosity of this variant is In addition, homozygosity of this variant is associated with traditional associated with traditional cardiovascular risk factors including low high-density lipoprotein factors including low high-density lipoprotein cholesterol and high systolic blood pressure cholesterol and high systolic blood pressure and newly identified as an independent risk and newly identified as an independent risk factor for stroke and early-onset coronary factor for stroke and early-onset coronary artery disease.artery disease.

• In contrast, heterozygosity is associated with In contrast, heterozygosity is associated with lower risk for stroke and coronary artery lower risk for stroke and coronary artery disease, which is consistent with its disease, which is consistent with its association with long life span. In addition to association with long life span. In addition to this this KLOTHOKLOTHO variant, several single-nucleotide variant, several single-nucleotide polymorphisms (SNPs) are associated with polymorphisms (SNPs) are associated with bone mineral density glucose metabolism and bone mineral density glucose metabolism and cognitive functioncognitive function

Additional observations gleaned Additional observations gleaned

from morefrom more--recent publicationsrecent publications • Low levels of Klotho may serve as an Low levels of Klotho may serve as an

early warning biomarker for kidney early warning biomarker for kidney disease and cardiovascular complicationsdisease and cardiovascular complications

• Klotho suppresses renal fibrosisKlotho suppresses renal fibrosis..

• inflammatory cytokines, such as TWEAK and inflammatory cytokines, such as TWEAK and TNFTNFαα, downregulate Klotho expression through , downregulate Klotho expression through an NFan NFκκBB--dependent mechanismdependent mechanism. . These results These results may partially explain the relationship between may partially explain the relationship between inflammation and diseases characterized by inflammation and diseases characterized by accelerated aging of organs, including CKDaccelerated aging of organs, including CKD..

Klotho expression can be Klotho expression can be modulated by dehydrationmodulated by dehydration..

• Some observations disclose a Some observations disclose a powerful effect of dehydration on powerful effect of dehydration on decrease Klotho expression, an effect decrease Klotho expression, an effect at least partially mediated by at least partially mediated by enhanced release of ADH and enhanced release of ADH and aldosteronealdosterone..”   ”  

Klotho as a tumor suppressor Klotho as a tumor suppressor genegene

• Epigenetic silencing of the tumor supEpigenetic silencing of the tumor suppressor pressor klothoklotho in human breast cancer in human breast cancer , , Cancer colon and gastric cancerCancer colon and gastric cancer

AgeAge--related decline in related decline in Klotho may be related to Klotho may be related to promoter methylationpromoter methylation..

• methylation of the promoter can methylation of the promoter can decrease gene transcriptiondecrease gene transcription. . These These results provide evidence that results provide evidence that changes in KL gene expression with changes in KL gene expression with age may, at least in part, be the age may, at least in part, be the result of epigenetic changes to the 5′ result of epigenetic changes to the 5′ regulatory regionregulatory region..“ “

ConclusionsConclusions

• The discovery of the The discovery of the klothoklotho gene has led to identification of gene has led to identification of multiple novel endocrine axes mediated by endocrine FGFs multiple novel endocrine axes mediated by endocrine FGFs and Klothos that regulates various metabolic processes. and Klothos that regulates various metabolic processes.

• The transmembrane form of Klotho protein functions as a The transmembrane form of Klotho protein functions as a co-receptor for FGF23 and regulates phosphate, calcium, co-receptor for FGF23 and regulates phosphate, calcium, and vitamin D metabolism.and vitamin D metabolism.

• The extracellular domain of Klotho protein is shed and The extracellular domain of Klotho protein is shed and

secreted into the systemic circulation where it functions as secreted into the systemic circulation where it functions as an endocrine factor. The secreted Klotho protein controls an endocrine factor. The secreted Klotho protein controls multiple ion channels and growth factor signaling pathways multiple ion channels and growth factor signaling pathways including insulin, IGF-1.including insulin, IGF-1.

ConclusionsConclusions

• potentially participating in biology of potentially participating in biology of cancer and stem cells. Identification cancer and stem cells. Identification of the common molecular basis for of the common molecular basis for these multiple function of Klotho will these multiple function of Klotho will be of particular importance to be of particular importance to understand the anti-aging properties understand the anti-aging properties of Klotho. of Klotho.