Klotho The AgeingSuppressor Gene By Naglaa Fathy Alhusseini

Klotho: The Ageing-Suppressor Gene By Naglaa Fathy Alhusseini Assistant prof. of Medical Biochemistry Faculty of Medicine Benha University 2013

Overview • Introduction • Klotho-deficient mice • Klotho-overexpressing transgenic mice • Klotho protein functions • Conclusions

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

Introduction – Control the life (spins the thread of life) and destiny of everyone – She is the goddess who helps life to unfold, in contrast to The (apoptotic) Atropos, who cuts the thread of life

Introduction Gene • Identified app. 16 years ago, by the Japanese group of Kuro-o et al. * • Named after Greek goddess, As prolonging lifespan is probably the most important role of this ageing-suppressor gene • The klotho gene is composed of 5 exons • The klotho gene is expressed in limited tissues and cell types. The highest expression is observed in distal convoluted tubules in the kidney and choroid plexus in the brain. • Lower expression is detected in pituitary gland, brain, parathyroid gland pancreas, ovary, testis, placenta, skeletal muscle, urinary bladder, colon , inner ear , and breast epithelial cells *Nature 1997; 390: 45– 51

Introduction • Japanese researchers reported – Defect in Klotho gene expression in the mouse resulted in a syndrome that resembled • Human ageing, including – Short lifespan, infertility, arteriosclerosis, skin atrophy, osteoporosis and emphysema • The gene encoded a membrane protein that Shared sequence similarity with the beta-glucosidase enzymes Nature 1997; 390: 45– 51

Klotho- 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

Introduction • The Klotho gene encodes – Single-pass transmembrane protein • Belongs to a family 1 glycosidase • Expressed primarily in renal tubules (distal tubules) – Present in the circulation and urine Blood Cells Mol Dis 1998; 24: 83– 100

• Klotho-deficient mice and FGF 23 -deficient mice • have an identical phenotype including – Hyperphosphataemia, hypercalcaemia, elevated plasma calcitriol and vascular calcification, in addition to premature ageing In contrast, over-expression of the Klotho gene – Extends the lifespan and increases resistance to oxidative stress Nephrol Dial Transplant 2007; 22: 1524– 1526

Regulation of FGF 23 signaling by Klotho • The common phenotypes of Klotho and FGF 23 overexpression and deletion, respectively, led to the postulate of a – Common signal transduction pathway – Klotho protein directly bound to multiple FGFRs – The Klotho–FGFR complex bound to FGF 23 with higher affinity than FGFR or Klotho alone *J Biol Chem 2006; 281: 6120– 6123

Regulation of FGF 23 signaling by Klotho • Conversion by Klotho of canonical FGF receptor into FGF 23 -specific receptor Nephrol Dial Transplant 2007; 22: 1524– 1526

Regulation of FGF 23 signaling by Klotho • The fact that FGF 23 requires Klotho as a co-receptor explains – Why Klotho-deficient mice develop phenotypes identical with those observed in FGF 23 -deficient mice and – Why Klotho-deficient mice had extremely high serum FGF 23 levels Nephrol Dial Transplant 2009; 24: 1705– 1708

Function of the transmembrane form of Klotho 1 - Regulation of phosphate metabolism • Recent studies have identified FGF 23 as a novel endocrine factor that lowers blood phosphate and vitamin D levels. • FGF 23 is secreted from osteocytes in response to high blood phosphate and vitamin D levels. FGF 23 acts on kidney to induce phosphaturia. • FGF 23 induces a negative phosphate balance by functioning as a phosphaturic hormone as well as a counter-regulatory hormone for vitamin D Kuro-o 2009, Klotho and aging NIH

Function of the secreted form of Klotho 1 - Suppression of insulin/IGF-1 signaling • Klotho-deficient mice are hypoglycemic and extremely sensitive to insulin. In contrast, Klotho-overexpressing transgenic mice are moderately resistant to insulin and IGF-1, although they maintain normal fasting blood glucose levels and are not diabetic. • These observations suggest that Klotho may inhibit the insulin/IGF-1 signaling pathway. • The ability of Klotho to inhibit insulin/IGF-1 signaling may be associated with anti-aging properties of Klotho, since numerous lines of genetic evidence indicate that moderate inhibition of insulin-like signaling pathway is one of the evolutionarily conserved mechanisms for suppressing aging Kuro-o 2009, Klotho and aging NIH

2 - Suppression of oxidative stress • Activation of insulin/IGF-1 signaling increases activity of • • a serine-threonine kinase, which inactivate the transcription factors FOXOs (up-regulate expression of multiple target genes, including antioxidant enzymes such as catalase and mitochondrial manganesesuperoxide dismutase (SOD 2). Catalase and SOD 2 detoxify harmful reactive oxygen species (ROS) hydrogen peroxide and superoxide, respectively, and reduce oxidative stress. Thus, activation of FOXOs by inhibiting insulin/IGF-1 signaling can increase cellular protection against oxidative stress and may contribute to the suppression of aging.

3 - Nitric oxide production • Klotho deficiency causes a reduction of NO synthesis in vascular endothelial cells. • Consistent with this finding, Klothodeficient mice exhibit impaired angiogenesis, which is dependent on endothelium-derived NO

4 - Klotho as a suppressor of breast cancer • clinical and laboratory data indicate a critical role for • • insulin/IGF-1 signaling in breast cancer. Notably: 1) increased serum insulin levels are associated with adverse prognosis in breast cancer 2) High circulating IGF-1 levels are associated with increased risk of pre-menopausal breast cancer and 3) Inhibition of insulin/IGF-1 signaling inhibits growth of breast cancer cells. Since secreted Klotho protein inhibits activation of insulin/IGF-1 receptors, Klotho may function as a suppressor of breast cancer. Kuro-o 2009, Klotho and aging NIH

• In addition, several lines of experimental • • • evidence support the notion that Klotho functions as a tumor suppressor. 1) Forced expression of Klotho in breast cancer cell lines reduces their proliferation. Inhibition of IGF-1 signaling by Klotho increased expression of CCAAT/enhancer-binding proteins (C/EBP) Klotho may be a suppressor of breast cancer. It remains to be determined whether the antibreast cancer activity of Klotho depends primarily on its ability to suppress IGF-1 signaling or on other unknown mechanisms.

• Defects in Klotho expression can lead to underexpression of FGF 23 and accumulation of • • phosphates . Accumulated phosphates can accelerate aging . Phosphate retention can lead to an aging phenotype. FGF 23 and its relationship to Klotho are linked to a number of bone and joint diseases. Klotho is a regulator of oxidative stress. Klotho inhibits growth and promotes apoptosis in some cancer lines. Klotho protein protects against endothelial dysfunction and hypertension.

• Control of Klotho expression comes about • • • through epigenetic mechanisms; some cancers can silence Klotho expression. Consuming cola drinks rich in phosphoric acid when coupled with Klotho insufficiency may exert a pro-aging effect. In humans, studies suggest that Klotho KL-VS gene polymorphisms may be associated with stroke, coronary artery disease and longevity. Klotho pathways might be targets for anti-aging interventions.

Human KLOTHO gene polymorphism and mutations • . A functional variant of human Klotho protein • contains six sequence variations. Interestingly, heterozygotes for this variant have advantage for longevity, while homozygotes are disadvantageous for survival, because homozygotes are significantly under-represented in the aged. In contrast, heterozygotes have longer life span than wild-types in multiple populations, suggesting that the KLOTHO gene variation affects human life span. Kuro-o 2009, Klotho and aging NIH

• In addition, homozygosity of this variant is • associated with traditional cardiovascular risk associated with traditional factors including low high-density lipoprotein cholesterol and high systolic blood pressure and newly identified as an independent risk factor for stroke and early-onset coronary artery disease. In contrast, heterozygosity is associated with lower risk for stroke and coronary artery disease, which is consistent with its association with long life span. In addition to this KLOTHO variant, several single-nucleotide polymorphisms (SNPs) are associated with bone mineral density glucose metabolism and cognitive function

Additional observations gleaned from recent publications-more • Low levels of Klotho may serve as an early • • warning biomarker for kidney disease and cardiovascular complications Klotho suppresses renal fibrosis. inflammatory cytokines, such as TNFα, down regulate Klotho expression through an NFκBdependent mechanism. These results may partially explain the relationship between inflammation and diseases characterized by accelerated aging of organs, including CKD.

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

Klotho as a tumor suppressor gene • Epigenetic silencing of the tumor suppressor klotho in human breast cancer , Cancer colon and gastric cancer

related decline in Klotho-Age may be related to promoter. methylation Methylation of the promoter can decrease gene transcription. These results provide evidence that changes in KL gene expression with age may, at least in part, be the result of epigenetic changes to the 5′ regulatory region “.

Conclusions • The discovery of the klotho gene has led to identification of multiple novel endocrine axes mediated by endocrine FGFs and Klothos that regulates various metabolic processes. • The transmembrane form of Klotho protein functions as a co receptor for FGF 23 and regulates phosphate, calcium, and vitamin D metabolism. • The extracellular domain of Klotho protein is shed and secreted into the systemic circulation where it functions as an endocrine factor. The secreted Klotho protein controls multiple ion channels and growth factor signaling pathways including insulin, IGF-1.

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