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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.
– 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 FGF23-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 FGF23 signaling by
Klotho
• The common phenotypes of Klotho and
FGF23 overexpression and deletion,
respectively, led to the postulate of a
– Common signal transduction pathway
– Kuro et al.* showed that
– Klotho protein directly bound to multiple
FGFRs
– The Klotho–FGFR complex bound to FGF23
with higher affinity than FGFR or Klotho alone
*J Biol Chem 2006; 281: 6120–6123
Regulation of FGF23 signaling by
Klotho
• Conversion by Klotho of canonical FGF
receptor into FGF23-specific receptor
Nephrol Dial Transplant 2007; 22: 1524–1526
Regulation of FGF23 signaling by
Klotho
• The fact that FGF23 requires Klotho as a
co-receptor explains
– Why Klotho-deficient mice develop
phenotypes identical with those observed in
FGF23-deficient mice and
– Why Klotho-deficient mice had extremely high
serum FGF23 levels
Nephrol Dial Transplant 2009;24: 1705–1708
Function of the
transmembrane form of
Klotho
1- Regulation of phosphate metabolism
• Recent studies have identified FGF23 as a novel
endocrine factor that lowers blood phosphate
and vitamin D levels .
• FGF23 is secreted from osteocytes in response
to high blood phosphate and vitamin D levels .
FGF23 acts on kidney to induce phosphaturia .
• FGF23 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 insulinlike 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 (SOD2) .
• Catalase and SOD2 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 FGF23 and accumulation of
phosphates .Accumulated phosphates can
accelerate aging .Phosphate retention can lead
to an aging phenotype .
FGF23 and its relationship to Klotho are linked to
a number of bone and joint diseases .
Klotho is a regulator of oxidative stress and cell
senescence .
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
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
more-recent publications
• 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 TWEAK and
TNFα, downregulate Klotho expression through
an NFκB-dependent 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
Age-related decline in Klotho
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 FGF23 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.