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From the
Received for publication, June 18, 2002
The klotho mouse is an animal model
that prematurely shows phenotypes resembling human aging. Here we
report that in homozygotes for the klotho mutation
(kl The klotho (kl Non-erythroid spectrin is a heterodimeric actin-binding protein that
consists of Calpain, a calcium-dependent cytosolic cysteine protease,
is involved in many physiological and pathological processes (14-16). Calpain mediates proteolysis of various cellular proteins, including cytoskeletal proteins, and causes irreversible cell damage (10-13, 17,
18). Thus, calpain overactivation may contribute to the pathology of
cerebral and cardiac ischemia, Alzheimer's disease, arthritis, and
cataract formation (19, 20). Calpain has been shown to be regulated by
both calcium ion and calpastatin (16). Two types of isozymic calpain,
µ-calpain and m-calpain, are ubiquitously distributed in mammalian
cells. The former is activated by micromolar concentrations of calcium
and the latter is activated by millimolar concentrations of calcium.
Calpastatin is an endogenous inhibitor specific for calpain, but is
slowly degraded by calpain (21). Here, we report the cleavage of
Preparation of Mouse Tissue Extracts--
Kidneys were obtained
from 2- and 3-week-old kl+/+,
kl+/ Amino Acid Sequencing of 280-kDa Protein--
Kidney microsomal
fraction (250 µg) from 4-week-old kl+/+ and
kl Antibodies--
Rabbit antibodies specific to the pre- and
post-autolytic forms of µ-calpain (anti-pre-µ and anti-post-µ,
respectively) were raised against synthetic peptides as described
previously (23). Antibodies specific to the pre- and post-autolytic
forms of m-calpain (anti-pre-m and anti-post-m, respectively) were
produced using synthetic peptides corresponding to the N-terminal 21 residues (AGIAAKLAKDREAAEGLGSHE) of the intact form and the N-terminal 6 residues (KDREAA) of the autolytic form, respectively. A cysteine residue was added to the C terminus of each peptide so that the antigenic peptide could be conjugated to keyhole limpet
hemocyanin. The entire amino acid sequence and the autolytic
cleavage site of human m-calpain were obtained from previous reports
(24, 25). Antibodies specific to the calpain-generated N- and
C-terminal fragments of Western Blot Analysis--
The cytosolic and microsomal
fractions (15 µg each) were separated by SDS-PAGE (10 and 5% gel for
cytosolic and microsomal fractions, respectively), and proteins were
transferred to a polyvinylidene difluoride membrane. The membrane,
after blocking in phosphate-buffered saline containing 5% skim milk
and 0.5% Tween 20, was incubated with each antibody. Then the membrane
was treated with anti-goat (Santa Cruz Biotechnology), anti-mouse or
anti-rabbit (Amersham Biosciences) IgG conjugated with horseradish
peroxidase. Proteins bound to antibody were visualized with an ECL kit
(Amersham Biosciences).
Northern Blot Analysis--
Total RNA was isolated from
4-week-old kl+/+ and
kl Histological Examination--
Four-week-old and 3-week-old
(kl+/+ and kl Decrease of
A Western blot using an anti- Decrement of
To determine which of the calpain isozymes were activated in the
kl
The expression levels of mRNAs of calpastatin (Fig. 3H)
and Activation of µ-Calpain Depends on Klotho Protein Level--
To
elucidate the relation between the amount of Klotho protein and the
degree of µ-calpain activation, we examined the mouse heterozygotes
for the klotho mutation (kl+/
To elucidate the process of calpain activation, calpastatin decrement,
and Organ-specific Calpain Activation--
The susceptibility and
degree of proteolysis due to the klotho mutation varied
among different organs. Changes in the lung of 4-week-old
kl Calpain Activation in Aged Normal Mice--
Changes similar to
those observed in kl Our results show that the aberrant activation of µ-calpain and
the decrease of calpastatin in the kidney are caused by the klotho mutation, and such changes lead to the cleavage of
Our finding that µ-calpain, but not m-calpain, was activated in the
kidney of kl The degree of proteolysis and of activation of calpain caused by the
klotho mutation varied among different organs. Both
Our finding that normal aged mice show changes similar to those in
kl Based on the above results, we propose that tissue deterioration during
aging is caused by a decrease of Klotho protein, which leads to a
decrease of calpastatin and activation of µ-calpain, which leads to a
degradation of cytoskeletal components such as spectrin. The magnitude
of each of these effects correlates with the amount of Klotho
expression. A decrease of calpastatin accelerates the activation of
µ-calpain and vice versa. Such deterioration may trigger tissue
abnormalities in kl We thank Dr. Koichi Suzuki and Dr. Akira
Kobata for helpful discussions.
*
This work was supported by Health and Labor Sciences
Research Grants for Comprehensive Research on Aging and Health from the Ministry of Health, Labor and Welfare, Japan (to T. E.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Published, JBC Papers in Press, July 15, 2002, DOI 10.1074/jbc.M206033200
1
T. Fujimori, H. Tsujikawa, and Y. Nabeshima, unpublished results.
Klotho Protein Deficiency Leads to Overactivation of
µ-Calpain*
,
,,,
Glycobiology and § Biomembrane
Research Groups, Tokyo Metropolitan Institute of Gerontology,
Foundation for Research on Aging and Promotion of Human Welfare, Tokyo
173-0015, Japan, the ¶ Department of Pathology and Tumor Biology,
Kyoto University Graduate School of Medicine, Kyoto 606-8501, Japan,
the Division of Biomolecular Characterization, RIKEN
(Institute of Physical and Chemical Research), Saitama 351-0198, Japan, and the ** Core Research for Evolutional Science & Technology (CREST), Saitama 332-0012, Japan
ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
/), 
II-spectrin is highly
cleaved, even before the occurrence of aging symptoms such as
calcification and arteriosclerosis. Because II-spectrin
is susceptible to proteolysis by calpain, we examined the activation of
calpain in kl/ mice. m-Calpain was not
activated, but µ-calpain was activated at an abnormally high level,
and an endogenous inhibitor of calpain, calpastatin, was significantly
decreased. Proteolysis of II-spectrin increased with
decreasing level of Klotho protein. Similar phenomena were observed in
normal aged mice. Our results indicate that the abnormal activation of
calpain due to the decrease of Klotho protein leads to degradation of
cytoskeletal elements such as II-spectrin. Such
deterioration may trigger renal abnormalities in
kl/ mice and aged mice, but Klotho protein
may suppress these processes.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/) mouse shows
multiple phenotypes resembling human aging caused by the mutation of a
single gene (1). This mutation is caused by the insertion of ectopic
DNA into the regulatory region of the klotho gene. The
klotho gene encodes a type I membrane protein that is
expressed predominantly in the kidney and brain. The extracellular
domain of Klotho protein consists of two internal repeats that share
sequence similarity to the 
-glucosidases of both bacteria and plants
(1, 2). As a result of a defect in klotho gene expression,
the kl/ mouse exhibits multiple
age-associated disorders, such as arteriosclerosis, osteoporosis, skin
atrophy, pulmonary emphysema, short life span, and infertility.
However, the mechanism by which the klotho gene product
suppresses the aging phenomena has not been identified. Analysis of the
pathophysiology of kl/ mice is expected to
give clues not only to understanding the mechanisms of individual
diseases associated with aging but also the relationship between these
mechanisms during human aging.
II- and II-spectrin and is
usually found on the cytoplasmic side of the plasma membrane (3, 4). It is thought to participate in the establishment and maintenance of cell
polarity, shape, and receptor distribution (5). Recently, it was
proposed that spectrin retained and stabilized various proteins at
specific regions on the cell surface (6-9). II-Spectrin has been shown to be cleaved by calpain and/or caspase during apoptosis
and necrosis (10-13).
II-spectrin due to the continuous activation of
µ-calpain in kl/ mice. Furthermore, we
also observe similar phenomena in normal aged mice.
EXPERIMENTAL PROCEDURES
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INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
, and kl/ mice
and from 4-week-old and 29-month-old C57BL/6 mice. Brain, lung, heart,
liver, and kidney were obtained from 4-week-old and 8-week-old
kl+/+ and kl/ mice.
Tissue samples were homogenized with 9 volumes (weight/volume) of 10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 250 mM sucrose. After centrifugation at 900 × g for 10 min, the supernatant was subjected to ultra centrifugation at 100,000 × g for 1 h. The
supernatants and precipitates were used as the cytosolic fraction and
microsomal membrane fraction, respectively. Protein concentration was
determined by BCA assay (Pierce). All experimental procedures using
laboratory animals were approved by the Animal Care and Use Committee
of Tokyo Metropolitan Institute of Gerontology. All efforts were made
to minimize the number of animals used and their suffering.
/ mice was subjected to SDS-PAGE under
reducing conditions followed by staining with Coomassie Brilliant Blue
R-250. A protein band of ~280 kDa was excised and treated with 0.1 µg of Achromobacter protease I (lysylendopeptidase) at
37 °C for 12 h in 0.1 M Tris-HCl, pH 9.0, containing 0.1% SDS and 1 mM EDTA (22). The peptides were
separated on columns of DEAE-5PW (1 × 20 mm; Tosoh, Tokyo, Japan)
and CAPCELL PAK C18 UG120 (1 × 100 mm; Shiseido, Tokyo, Japan).
Solvent A was 0.085% (v/v) trifluoroacetic acid in distilled water,
and solvent B was 0.075% (v/v) trifluoroacetic acid in 80% (v/v)
acetonitrile. The peptides were eluted at a flow rate of 30 µl/min
using a linear gradient of 1-60% solvent B. Selected peptides were
subjected to Edman degradation using a Procise 494 cLC protein
sequencer (Applied Biosystems, Foster City, CA) and to matrix-assisted
laser desorption ionization time-of-flight mass spectrometry on a
Reflex MALDI-TOF (Bruker Daltonics, Billerica, MA) in linear mode using
2-mercaptobenzothiazole as a matrix.
II-spectrin (136 and 148 kDa,
respectively) were produced by the peptide antigens QQQEVY
(anti-BDP-136) and GAMPRD (anti-BDP-148), respectively (see Fig. 2). A
cysteine residue was added to the N terminus of QQQEVY peptide or was
added to the C terminus of GAMPRD peptide. The amino acid sequence and the cleavage site in mouse II-spectrin by calpain were
as determined by others previously (22, 26). An antibody against domain IV of human calpastatin was produced using a synthetic peptide corresponding to residues 601-630 (AEHRDKLGERDDTIPPEYRHLLDDNGQDKP) (27) with a cysteine residue added to the C terminus. Rabbits were
immunized with the antigenic peptide-keyhole limpet hemocyanin conjugates. Affinity purification of polyclonal antibodies was carried
out using antigenic peptides immobilized on epoxy-activated Sepharose
6B (Amersham Biosciences, Buckinghamshire, UK).
Anti-human-II-spectrin polyclonal antibody C-20 from
goat was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
Anti-Klotho monoclonal antibody (KM2076) from rat was a generous gift
from Kyowa Hakko Kogyo Co., Ltd (28).
/ mice kidney by the guanidinium
thiocyanate method using Isogen (Nippon Gene, Toyama, Japan). Total RNA
(15 µg) was electrophoresed through a 1% agarose-formaldehyde
denaturing gel and transferred to a nylon membrane,
Hybond-N+ (Amersham Biosciences). Northern blot analysis
was performed using The Gene images AlkPhos Direct (Amersham
Biosciences) according to the manufacturer's instruction. Probe DNA
fragments for mouse II-spectrin, calpastatin, and
glyceraldehyde-3-phosphate dehydrogenase were prepared by reverse
transcriptase-PCR and nested PCR using total RNA from the
kl+/+ mouse kidney. Outer primers of
II-spectrin were 5'-AACAGCACAAACAAGGATTGGTGG-3' and
5'-TGCAGATCATGGGAGTCACCCAAT-3'; inner primers were
5'-GGTTTCGTGCCAGCTGCATA-3' and 5'-ACCCAATTTGGCCTTGCGCT-3'. Outer
primers of calpastatin were 5'-TTTCGCTGCGTTTTCCCGGA-3' and
5'-TTCTCCTTGGGGGGAACAGA-3'; inner primers are
5'-TTCACCGAAAAATGTCCCAGCCCGG-3' and 5'-GGTCACTCCTGCAGACTGAGCTTTG-3'. Primers of glyceraldehyde-3-phosphate dehydrogenase were
5'-TGAAGGTCGGTGTGAACGGATTTGGC-3' and
5'-CATGTAGGCCATGAGGTCCACCAC-3'.
/) mice
were examined. The kidneys were excised, fixed with 10% formaldehyde,
embedded in paraffin, sectioned in 4-µm slices and stained with
hematoxylin-eosin and von Kossa staining.
RESULTS
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II-Spectrin in the Kidney of
kl/ Mice--
To determine whether mouse homozygotes
of the klotho gene mutation (kl/)
have a different pattern of proteins in the kidney, we examined the
kidney microsomal fractions from 4-week-old mice by SDS-PAGE. A band of
about 280 kDa was found to be significantly weaker in kl/ mice than in
kl+/+ mice (Fig.
1A). Similar results
were obtained with five other kl/ mice. The
280-kDa protein band was subjected to in-gel lysylendopeptidase digestion (22), and the sequences of two of the resulting peptides were
determined to be LQTASDESYK and KHEAFETDFTVHK by a combination of
Edman degradation and mass spectrometry. A data base search of protein
sequences revealed that these peptide sequences were homologous to
those of human II-spectrin (GenBankTM
accession number AAB41498).
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Fig. 1.
Decrease of
II-spectrin in the
kl/
kidney before morphological change. A, SDS-PAGE
of kidney microsomal fractions of 4-week-old male mice, stained with
Coomassie Blue. B, Western blot analysis of gel assessed
with anti-II-spectrin (C-20) antibody. Lane
1, kl+/+; lane 2,
kl/. Arrowheads indicate the
position of II-spectrin. Open triangle in
B indicates the position of the degraded product of
II-spectrin. Molecular mass markers are indicated on the
left. C-F, histological examination of kidney sections.
C, 4-week-old kl+/+ mice,
hematoxylin-eosin. D, kl/ mice,
hematoxylin-eosin. E, 4-week-old
kl+/+ mice, von Kossa staining. F,
kl/ mice, von Kossa staining.
Arrowhead in F indicates calcium (stained by von
Kossa staining). Bar, 20 µm.
II-spectrin antibody (C-20)
confirmed that the 280-kDa protein is II-spectrin and
that the reactivity of the antibody was drastically decreased in
kl/ mice (Fig. 1B). The antibody
also stained a 145-kDa band in kl/ mice, but
this band was below the detectable level in
kl+/+ mice (Fig. 1B). Because the
anti-II-spectrin antibody recognizes the C terminus of
II-spectrin, it is likely that the 145-kDa band is a
C-terminal fragment of II-spectrin. Although the kidney of 4-week-old kl/ mice was not
morphologically different from that of kl+/+
mice, it did show a small amount of calcification (Fig. 1,
C-F).
II-Spectrin and Calpastatin with
Increased Activation of µ-Calpain--
II-Spectrin
was previously shown to be cleaved at a particular site by calpain
(29), yielding 136- and 148-kDa fragments. To determine whether calpain
is involved in proteolysis of II-spectrin in the kidney
of kl/ mice, we prepared specific antibodies
to sequences on either side of the cleavage site (Fig.
2). The anti-BDP-136 antibody, which was
produced against a sequence (QQQEVY) in the C-terminal region of
BDP-136, recognized only the 136-kDa fragment of
II-spectrin. BDP-136 was detected only in
kl/ mice (Fig.
3A). On the other hand, the
anti-BDP-148 antibody, which was produced against a sequence (GAMPRD)
in the N-terminal region of BDP-148, recognized not only the 148 kDa
fragment but also full-length II-spectrin. BDP-148 was
detected only in kl/ mice (Fig.
3B). These results indicated that II-spectrin was degraded by calpain in the kidney of kl/
mice.
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Fig. 2.
Design of synthetic peptides for antibodies
to the 136- and 148-kDa fragments of
II-spectrin cleaved by calpain.
Antigenic peptides were designed to correspond the C-terminal sequence
(QQQEVY) of the 136-kDa fragment and the N-terminal sequence (GAMPRD)
of the 148-kDa fragment of II-spectrin cleaved by
calpain.
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Fig. 3.
Changes of
II-spectrin,
µ-calpain, and calpastatin in the
kl/
kidney. A and B, Western blots of kidney
microsomal fractions of 4-week-old male mice using antibodies.
A, anti-BDP-136 antibody. B, anti-BDP-148
antibody. C-G, Western blots of kidney cytosolic fractions
of 4-week-old male mice using antibodies. C,
anti-pre-µ-calpain antibody; D, anti-post-µ-calpain
antibody; E, anti-pre-m-calpain antibody;
F, anti-post-m-calpain antibody; G,
anti-calpastatin antibody. H-J, Northern blot analyses of
4-week-old mice kidney. H, II-spectrin.
I, calpastatin. J, glyceraldehyde-3-phosphate
dehydrogenase. Lanes 1, kl+/+;
lanes 2, kl/.
Arrowheads indicate the positions of the corresponding
molecules. Molecular mass markers or ribosomal RNA bands are indicated
on the left.
/ kidney, we made a Western blot of kidney
cytosolic fractions using antibodies against four types of calpain: the
inactive and active forms of µ-calpain (pre- and post-µ-calpain)
and the inactive and active forms of m-calpain (pre- and
post-m-calpain). Pre-µ-calpain was detected in
kl+/+ mice but not in
kl/ mice (Fig. 3C).
Post-µ-calpain was detected in kl/ mice
but not in kl+/+ mice (Fig. 3D).
Pre-m-calpain was detected in both kl+/+ and
kl/ mice with no significant difference
between them (Fig. 3E). Post-m-calpain was barely detected in either
kl+/+ or kl/ mice
(Fig. 3F). These results indicate that µ-calpain, but not m-calpain, was specifically activated in the
kl/ kidney. Interestingly, calpastatin,
which is an endogenous inhibitor of calpain, was barely detected in
kl/ mice (Fig. 3G). The triplet
bands at about 122 kDa in Fig. 3G are probably alternative
splicing forms of calpastatin (30).
II-spectrin (Fig. 3I) were not different
between kl/ and kl+/+
mice, which suggests that the decreases of calpastatin and
II-spectrin in kl/ mice were
due to increased degradation rather than a down-regulation of transcription.
). The
expression level of Klotho protein in 2-week-old
kl+/ mice (Fig.
4A, lane 2), was
approximately half that in 2-week-old kl+/+ mice
(lane 1). A similar relation was found in 3-week-old mice (lanes 5 and 4, respectively). The levels of
expression of pre-µ-calpain, post-µ-calpain, and calpastatin in
2-week-old kl+/ mice (lane 2) were
intermediate between those of kl+/+ mice
(lane 1) and those of kl/ mice
(lane 3). Similar results were obtained in 3-week-old mice (lanes 5, 4, and 6, respectively).
These results showed that the expression level of Klotho protein
affected the activation of µ-calpain and the amount of calpastatin
(Fig. 4B).
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Fig. 4.
Dependence of calpain, calpastatin, and
II-spectrin on Klotho protein.
A, Western blots of kidney cytosolic and microsomal
fractions of 2- and 3-week-old male mice using anti-Klotho antibody and
other antibodies described in Fig. 3. Lanes 1 and
4, kl+/+; lanes 2 and
5, kl+/; lanes 3 and
6, kl/. Arrowheads
indicate the positions of each molecule. B, intensities of
bands in A were measured by densitometric scanning using a
densitometer and NIH Image 1.61/ppc software.
II-spectrin proteolysis, we examined mice that were
less than 4 weeks old. In kl/ mice,
pre-µ-calpain and calpastatin were present at low levels at 2 weeks
(Fig. 4A, lane 3) but were undetectable at 3 weeks (lane 6), while the amount of cleaved
II-spectrin was much higher at 3 weeks (lane
6) and 4 weeks than at 2 weeks (lane 3). In 2- and
3-week-old kl+/ mice (lanes 2 and
5), the amount of cleaved II-spectrin was much higher at 3 weeks (lane 5) than at 2 weeks (lane
2), while the levels of pre-µ-calpain and calpastatin at 3 weeks
were slightly less than those at 2 weeks. These findings suggest that:
1) pre-µ-calpain and calpastatin were originally expressed in
kl/ mouse kidney and that µ-calpain was
gradually activated and calpastatin was gradually decreased during
development, and 2) II-spectrin was hardly cleaved in
the presence of calpastatin, but intensive cleavage of
II-spectrin was observed after the complete
disappearance of calpastatin (Fig. 4B). No calcification was
observed in 3-week-old kl/ mice (data not
shown), indicating that degradation of II-spectrin in
the kidney of kl/ mice preceded the
occurrence of any tissue damage.
/ mice (Fig.
5) were similar to those observed in the
kidney. The intensity of intact II-spectrin drastically
decreased and lower molecular weight bands newly appeared. In addition,
post-µ-calpain, but not pre-µ-calpain, was detected, suggesting
that significant proteolysis occurred in the lung. It may be relevant
to that the first observation of the pulmonary emphysematous changes
occurs at 4 weeks of age in kl/ mice (31).
In the heart, partial activation of calpain was observed at 4 weeks,
and only post-µ-calpain was detected at 8 weeks. However,
II-spectrin was not cleaved in the heart. These results
suggest that the heart has a sufficient amount of calpastatin to
prevent II-spectrin degradation. On the other hand, no
II-spectrin degradation or calpain activation was
observed in the brain or liver at 8 weeks.
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Fig. 5.
Organ-specific differences in calpastatin,
calpain, and II-spectrin between
kl+/+ and
kl/
mice. Western blots of organ extracts of 4- and 8-week-old male
mice with antibodies. Lanes W, kl+/+;
lanes H, kl/.
/ mice occurred in aged
normal (C57BL/6) mice. As normal mice aged from 4 weeks to 29 months,
the expression of Klotho protein decreased, the activation of
µ-calpain increased, the level of calpastatin considerably decreased,
and the degradation of II-spectrin increased (Fig. 6). Similar changes were observed in five
other mice.
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Fig. 6.
Changes in the kidney of aged normal mice.
Western blots of 4-week-old (lane 1) and 29-month-old
(lane 2) C57BL/6 mice. Antibodies are described in the
legend to Fig. 4. Arrowheads indicate the positions of the
corresponding molecules.
DISCUSSION
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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II-spectrin. These phenomena are well correlated with
the expression level of Klotho protein. Our results also show that
similar changes in µ-calpain, calpastatin, and
II-spectrin occur in normal aged mice. The abnormal
activation of µ-calpain in the kidney occurs at an early age: in
kl/ mice, changes in µ-calpain activation
and II-spectrin degradation started to occur one to 2 weeks before the appearance of abnormal phenotypes, and in
kl+/ mice, µ-calpain was gradually activated
as they aged, even though these mice have a normal phenotypic appearance.
/ mice suggests that the
concentration of intracellular calcium ions in these mice is in the
micromolar range. Normally calpain is activated temporarily and
calpain-catalyzed proteolysis leads to modulation rather than
destruction of the substrate proteins. Therefore, continuous activation
of µ-calpain is unusual and elucidation of the mechanism is essential
to understanding its pathophysiological role. One possible mechanism is
that µ-calpain overactivation causes a deficiency of calpastatin, and
another is that a decrease in calpastatin causes an increase in
µ-calpain activation. Since the transcription levels of calpastatin
are the same in kl/ and
kl+/+ mice, the latter possibility is
unlikely, but we cannot completely rule it out. µ-Calpain activity,
in addition to being regulated by the calcium ion concentration, is
usually also regulated by the binding of calpastatin (16). Thus, a
deficiency of calpastatin may induce the overdestruction of substrates
such as II-spectrin by calpain. The mechanism by which
Klotho protein might regulate µ-calpain activity and calpastatin
level in the kidney is unknown. However, it is possible that this
regulation is mediated by nitric oxide (NO). NO has been shown to
inhibit calpain-mediated proteolysis (32), and systemic NO synthesis is
decreased in kl/ mice (33, 34). Furthermore,
adenovirus-mediated klotho gene delivery increased NO
production and restored vascular endothelial dysfunction (35). It is
noteworthy that calpain overactivation in
kl/ mice is not caused by ischemia due to
arteriosclerosis, while ischemia would cause overactivation of calpain
(13). A previous study revealed that, in kl/
mice, arteriosclerosis first appeared around 4 weeks after birth and
progressed gradually with age (1). However, in the lung and kidney in
kl/ mice, arteriosclerosis could not be the
cause of overactivation of µ-calpain, because the latter occurred as
early as 2~3 weeks.
II-spectrin degradation and calpain activation were
observed in the kidney and lung as early as 2~3 weeks. Spectrin was
not cleaved in the heart even at 8 weeks, while overactivation of
calpain was observed. The time course of activation of calpain in the
heart seemed to be proceeded slower than in the lung and kidney.
However, it is impossible to examine this possibility, because
kl/ mice die at ~8-9 weeks (1). On the
other hand, no II-spectrin degradation or calpain
activation was observed in the brain or liver at 8 weeks. In addition,
an organ's susceptibility to the klotho mutation did not
necessarily correspond to its expression of klotho mRNA.
Taken together, these results suggest that Klotho protein or its
metabolites may function as a humoral factor. In support of this
hypothesis, both mice and human have a secretory form of Klotho protein
(28, 36, 37), and the exogenous klotho gene expressed in the
brain and testis could improve systemic aging phenotypes in
kl/ mice (1, 38). It is important to
identify and characterize a target molecule (receptor) that is
responsive to Klotho protein or its metabolites. Thus, it may be that
the factor most responsible for an organ's sensitivity to the
klotho mutation is the density of such a receptor.
/ mice suggests that the decrease of
Klotho protein is closely related to aging processes. Recent studies
revealed that the expression of klotho gene was gradually
reduced in the rat kidney during long term hypertension (39) and that
calpastatin was gently degraded also in the kidney of hypertensive rats
(40). Furthermore, humans with chronic renal failure commonly develop
multiple complications resembling phenotypes observed in
kl/ mice (41-45), and the expression of
klotho mRNA and the production of Klotho protein were
severely reduced in these patients (46). Taken together, these results
suggest that Klotho protein in the kidney protects the progress of
age-related renal disorders.
/ mice and aged mice, but
Klotho protein may suppress these processes, while the detailed
mechanism is not clear yet. Very recently Yoshida et al.
(47) reported that calcium and phosphorus homeostasis could be
regulated through Klotho function via the action of
1,25-dihydroxyvitamin D due to the impaired regulation of
1-hydroxylase gene expression. This deterioration in the vitamin
D3 endocrine system may participate in many of the
phenotypes in kl/ mice via toxicity due to
increased levels of calcium, phosphorus, and 1,25-dihydroxyvitamin D. It should be noted that when serum concentrations of calcium,
phosphorus, and 1,25-dihydroxyvitamin D are restored to normal levels,
many of phenotypes are improved despite Klotho protein
deficiency.1 Thus, Klotho
protein may be a regulator of calcium homeostasis via the vitamin
D3 endocrine system. Alternatively, based on the homology
to -glucosidase (1, 2), Klotho protein may function as a
glycosidase-like enzyme and modify the glycan moieties of ion channels.
Since it is known that glycosylation appears important for the function
of ion channels (48-50), the change of glycosylation may affect
calcium homeostasis. In any case, the abnormal activation of calpain
due to the decrease of Klotho protein leads to degradation of
cytoskeletal elements such as II-spectrin is likely to
be integral to the pathogenic sequence in
kl/ mice and recapitulates effects seen in
normal aging. Future studies are needed to determine the definitive
role of Klotho protein in the regulation of calcium metabolism as well
as of intracellular calcium concentration. Such studies will also lead
to a better understand of age-related renal abnormalities and to
prevent renal diseases in the future.
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed: Glycobiology
Research Group, Tokyo Metropolitan Inst. of Gerontology, Foundation for
Research on Aging and Promotion of Human Welfare, 35-2 Sakaecho, Itabashi-ku, Tokyo 173-0015, Japan. Tel.: 81-3-3964-3241 (ext. 3080);
Fax: 81-3-3579-4776; E-mail: endo@tmig.or.jp.
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
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