Molecular and Cellular Biology, June 2000, p. 4474-4481, Vol. 20, No. 12
Department of
Biochemistry1 and Cancer Research
Laboratories, Department of Pathology,2 Queen's
University, Kingston, Ontario K7L 3N6, Canada
Received 10 August 1999/Returned for modification 28 September
1999/Accepted 16 March 2000
Calpains are a family of Ca2+-dependent intracellular
cysteine proteases, including the ubiquitously expressed µ- and
m-calpains. Both µ- and m-calpains are heterodimers, consisting of a
distinct large 80-kDa catalytic subunit, encoded by the genes
Capn1 and Capn2, and a common small 28-kDa
regulatory subunit (Capn4). The physiological roles and
possible functional distinctions of µ- and m-calpains remain unclear,
but suggested functions include participation in cell division and
migration, integrin-mediated signal transduction, apoptosis, and
regulation of cellular control proteins such as cyclin D1 and p53.
Homozygous disruption of murine Capn4 eliminated both µ-
and m-calpain activities, but this did not affect survival and
proliferation of cultured embryonic stem cells or embryonic
fibroblasts, or the early stages of organogenesis. However, mutant
embryos died at midgestation and displayed defects in the
cardiovascular system, hemorrhaging, and accumulation of erythroid progenitors.
The two forms of calpain found as
stable proteins in mammalian tissues are known as µ-calpain and
m-calpain (7, 39, 43, 53, 58). The enzymes are cytosolic
thiol proteases, absolutely dependent on Ca2+ for activity.
They both consist of an 80-kDa large subunit (from the genes
Capn1 and Capn2, respectively), each of which
forms a heterodimer with a common 28-kDa small subunit
(Capn4). The structure of these enzymes was first predicted
from their amino acid sequence (42) and has been established
very recently by X-ray crystallography (25, 56). The large
subunits contain a thiol protease region (domains I and II) related to
the papain and cathepsin families, a C2-like domain III, and a
C-terminal, calmodulin-like domain (domain IV), which binds
Ca2+ at several EF hands. The small subunit consists of an
N-terminal domain V, containing ~30% glycine residues, and a
C-terminal Ca2+-binding domain VI very similar to domain IV
(6, 31). The two subunits are bound to each other mainly
through contacts between domains IV and VI (25).
Historically it has been assumed that the calpain small subunit is
essential for µ- and m-calpain activity. The two enzymes are
invariably found as heterodimers in tissue extracts, and expression of
the m-calpain large subunit alone in Escherichia coli gave a
low yield of soluble protein with no activity (21).
Expression of the µ-calpain large subunit alone has, however, been
reported to yield some calpain activity, or to give an insoluble
product which was active following renaturation (37, 62),
suggesting that at least one function of the small subunit is to act as
a chaperone in assisting folding of the large subunit (58, 69, 70). It has also been reported that Ca2+ activation
of calpain involves subunit dissociation and that the large subunits
formed in this way can be active as monomers (58). There are
some reports which do not agree with this hypothesis (12,
71), but the question should be considered open.
In recent years, several other calpain-related cDNA sequences have been
described, but there is little information on the occurrence or
function of the corresponding proteins (5, 9, 20, 33).
Calpain 3 or p94 (Capn3) has attracted great interest since
deficiency of calpain 3 causes limb girdle muscular dystrophy 2A
(3, 18, 23, 28, 45). The natural substrates of calpain 3 are
not known, and the protein has an extremely short half-life in vivo,
making it very difficult to assay. In the context of this study on
disruption of the Capn4 gene, it may be noted that calpain 3 does not appear to require a calpain small subunit or to be
Ca2+ dependent. Calpain 3 mRNA is expressed briefly in
human fetal heart but later only in skeletal muscle, while in the mouse
embryo, it could not be detected before day 11.5 of gestation
(embryonic day 11.5 [E11.5]) (3, 18, 23). The conventional
calpains have not been extensively studied in the embryo, but m-calpain was detected in chick embryonic fibroblasts (30), and
expression in the mouse embryo of mRNAs for Capn5,
-6, and -11 has recently been described
(10).
The physiological roles of the calpains remain unclear (7,
43). The presence and conservation of µ- and m-calpains in almost all mammalian cells suggest that these enzymes are essential, but the absence of fully specific calpain inhibitors has so far prevented unambiguous proof of a particular physiological role. Many of
the calpain inhibitors commonly used in earlier work have been shown
also to inhibit the proteasome (36) or cathepsins or to
inhibit entirely different enzymes, for example, a protein tyrosine
phosphatase (49). The calpain genes have no major regulatory features in their promoter regions and are usually considered to be
housekeeping enzymes. The relative levels of µ- and m-calpains and of
their inhibitor, calpastatin, vary from tissue to tissue, again
suggesting some degree of regulation and importance to the cell
(59). Their Ca2+ dependence suggests a link to
signal transduction (19), and the common assumption that the
calpains become membrane bound on exposure to Ca2+
(35, 39) suggests that cytoskeletal proteins may be among the favored substrates. The list of proposed calpain substrates is very
long (19, 63), and many reports have suggested that calpain
may be involved in cell adhesion, spreading, and migration (26,
29, 49), myoblast fusion (4, 11), cell cycle control (36), and mitosis (50).
Apoptosis is another cell function for which there are conflicting
reports about the involvement of calpain. The existence of numerous
caspases (41), the problems of inhibitor specificity, and
the multiplicity of apoptosis pathways and experimental cell systems
have led to many conflicting reports about the role of calpain
(34, 46, 66, 68). Several members of the Bcl-2 and Bax
families, which are important in apoptosis, can be cleaved both by
caspases and by calpain (14). Calpain was reported to be
involved in radiation-induced apoptosis, upstream of caspase 3 (64), but caspase activation was found to be upstream of
calpain activation in drug-induced apoptosis (67). Calpain
activity was found to cause apoptosis of neutrophils, but Fas
antigen-induced apoptosis was independent of calpain (54,
55).
With the intention of resolving some of these questions, we have
therefore generated both heterozygous Capn4+/ The results have shown, as predicted, that homozygous
Capn4 Capn4+/ Disruption of the calpain small subunit gene.
Genomic and
cDNA clones of the mouse calpain small subunit (Capn4) gene
have been described elsewhere (1) (accession no. AF058297,
AF058298, and AF139373). The gene contains 11 exons. The left
(upstream) arm of the targeting vector consisted of a 2-kb genomic
PvuII fragment, extending from intron 6 to the middle of
exon 9. The right (downstream) arm was a 4-kb
BamHI-HindIII genomic DNA fragment extending
from intron 9 and including exons 10 and 11, to a point ~1.5 kb
downstream of the polyadenylation signal. These DNA segments were
inserted on either side of the PGK-neo cassette in pPNT
(Fig. 1) (61). This construct
replaces approximately 450 bp, including the 3'-terminal half of
Capn4 exon 9, with the PGK-neo cassette. The
thymidine kinase cassette was included in the vector to permit negative
selection of nonhomologous recombination events with ganciclovir
(GANC). This plasmid was linearized by NotI digestion for
electroporation into R1 ES cells, and transformed cells were selected
for G418 and GANC resistance by growth in the presence of 0.2 mg of
G418 (Gibco/BRL, Burlington, Ontario, Canada) per ml and 2 µM GANC
(Syntex, Inc.) for 8 to 9 days (24). Homozygous mutant ES
cells (Capn4
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Disruption of the Murine Calpain Small Subunit
Gene, Capn4: Calpain Is Essential for Embryonic Development
but Not for Cell Growth and Division
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
and homozygous Capn4/ embryonic stem (ES)
cells and have attempted to generate the corresponding mice. The work
was based on the assumption that loss of a functional Capn4
gene would abolish both µ- and m-calpain activity. The physiological
consequences of this mutation were impossible to predict, and it was
not known whether any other forms of calpain, or other protease systems
entirely, might compensate for the absence of the classical µ- and
m-calpains.
/ mouse cells lack µ- and m-calpain
activity as measured by casein zymography, but even in the absence of
detectable calpain activity, homozygous
Capn4/ ES cells and embryonic fibroblasts
grow and divide apparently normally. However, the large subunit genes
have not been altered and are still transcribed, although the resultant
large subunit proteins are unstable in the absence of the small
subunit. It is not impossible that these isolated large subunits may
still exert some calpain activity, even if transient and unregulated, but no assay to detect this activity unambiguously is available.
mice were viable, fertile, and
phenotypically normal, but the Capn4/
embryos died at midgestation, with indications of defects in both
vasculogenesis and erythropoiesis. Embryonic development is thus
absolutely dependent on the presence of a functional Capn4 gene, and we assume therefore on the presence of normal levels of at
least one of µ- and m-calpains in their normal heterodimeric form.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
/) were obtained by means of
further growth and selection of heterozygous (Capn4+/) ES cells in the presence of 1 mg of
G418 per ml.
View larger version (14K):
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FIG. 1.
Targeted disruption of the calpain small subunit gene.
Schematic representations show the murine Capn4 allele
(upper), the targeting vector (middle), and the targeted allele
(lower). The Capn4 gene was cloned from the 129SvJ mouse
strain (1). Positions of PstI (P),
PvuII (Pv), BamHI (B), and HindIII
(H) restriction endonuclease sites are indicated. Homologous
recombination replaces a 441-bp PvuII-BamHI
fragment containing the 3' end of Capn4 exon 9 with the
neo cassette. Tk, thymidine kinase.
Genotyping.
Initial screening for
Capn4+/ ES cell clones was carried out by PCR,
using a sense-strand primer located just upstream of the 2-kb left arm
of homology and an antisense-strand primer located within the
PGK-neo cassette. Tentatively identified
Capn4+/ ES cell clones were characterized by
Southern blot hybridization of PstI-digested genomic DNA
with the 487-bp PvuII fragment located upstream of the 2-kb
left arm of homology. This probe recognized a 5.5-kb PstI
fragment from the wild-type Capn4 locus and a 2.5-kb PstI fragment from the targeted Capn4 locus (Fig.
1).
Germ line transmission. Germ line transmission of the Capn4 mutant allele was achieved by the "darning needle" aggregation chimera method (40). Briefly, eight-cell embryos were recovered from CD1 matings at a point prior to blastomere compaction. Acid Tyrode's buffer was used to remove the zona pellucida, and the embryos were placed in smooth depressions made with a darning needle in 35-mm-diameter culture dishes. Clumps of 8 to 12 ES cells were placed adjacent to each embryo within the depressions. During overnight culture, the ES cells were absorbed into the compacting morula, and those embryos which developed further to the blastocyst stage were transferred into the uteri of surrogate mothers. At term, chimeric animals were identified first by black eye pigmentation and subsequently by patches of agouti coat color. Chimeric males were initially bred with CD1 females to identify males capable of germ line transmission of the mutant allele. These males were subsequently bred with 129SvJ females to establish the mutant allele in an inbred state.
Northern blotting.
Total RNA was isolated from ES cells
using TRIzol reagent (Gibco/BRL) according to the manufacturer's
instructions, and poly(A)+ RNA was fractionated using the
polyATtract system (Promega, Madison, Wis.). Samples of
poly(A)+ RNA were resolved on 1.2% formaldehyde agarose
gels and transferred to nylon membranes (65). The membranes
were probed with [
-32P]dATP-labeled cDNA fragments
(15) for calpain subunits as follows: for the mouse
m-calpain large subunit, EST ms22d05.ri (mouse calpain 2; accession no.
AA168283), obtained from IMAGE Consortium, Lawrence Livermore National
Laboratory; for the mouse µ-calpain large subunit, a rat µ-calpain
large subunit cDNA cloned in this laboratory with kind assistance from
H. Sorimachi (52); and for the mouse calpain small subunit,
a cDNA coding for the C-terminal domain VI of the mouse calpain small
subunit and the 3' untranslated region (1). Equal gel
loading was confirmed both by ethidium bromide staining and by
stripping the blots and reprobing with an actin cDNA.
Separation and analysis of µ- and m-calpain activities. Calpain activities in cell and tissue extracts were detected by casein zymography (2, 44). Cultured cells were scraped into lysis buffer (50 mM HEPES [pH 7.6], 150 mM NaCl, 1% Triton X-100, 5 mM EDTA, 10 mM 2-mercaptoethanol, 0.1 mM phenylmethylsulfonyl fluoride, 10 µg of leupeptin per ml) without trypsin treatment. E9.5-E10.5 embryos were homogenized by pipetting up and down in this buffer. After centrifugation to remove particulate debris, aliquots representing 30 µg of protein from cell extracts, or 20 to 40% of each soluble embryo extract, were mixed with nondenaturing gel loading buffer and electrophoresed in nondenaturing polyacrylamide gels containing casein. Calpain activity was detected by incubating the gels overnight with Ca2+, followed by fixation and staining with Coomassie brilliant blue. Calpain activities were observed as colorless regions cleared of casein in a blue background.
Gel electrophoresis, immunoblotting, and antibodies. Protein samples denatured in sodium dodecyl sulfate (SDS) sample buffer were run on SDS-polyacrylamide gels (9%) in Tris-Tricine buffer (48) and blotted onto Immobilon membranes as previously described (60). A polyclonal antibody which binds the large (rat Capn2) subunit of rat and mouse m-calpain has been described elsewhere (47). Antibodies to the mitogen-activated protein kinase ERK1 (K-23) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, Calif.).
ES cell culture. Fetal bovine serum was from HyClone Laboratories Inc. (Logan, Utah) and was tested for its ability to support ES cell growth. Gelatin was from Sigma-Aldrich Canada (Oakville, Ontario, Canada). All other reagents for tissue culture were from Gibco/BRL. Murine ES cells were maintained on gelatinized plates at 37°C under 5% CO2 in ES medium (Dulbecco modified Eagle medium [high glucose] supplemented with 15% fetal bovine serum, 0.1 mM nonessential amino acids, antibiotics [penicillin, 100 U/ml; streptomycin, 100 µg/ml; amphotericin, 25 µg/ml], 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM 2-mercaptoethanol, and 1,000 U of recombinant leukemia inhibitory factor per ml).
Histological analysis.
Time of fertilization was determined
by observation of copulation plugs, and noon of that day was defined as
E0.5. Embryos were dissected from pregnant
Capn4+/ females that had been bred with
Capn4+/ males, and the yolk sacs were
separated and used for genotype analysis. Embryos were fixed overnight
in 4% paraformaldehyde in phosphate-buffered saline (pH 7.2),
dehydrated through graded ethanol, and embedded in paraffin wax. Serial
cross or sagittal 7-µm sections were dewaxed in xylene, rehydrated
through graded ethanol, and stained with hematoxylin and eosin. TUNEL
(terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end
labeling) assays were performed on dewaxed sections using the DeadEnd
colorimetric apoptosis detection system from Promega according to the
manufacturer's instructions. Photographs were taken with a Kodak DC120
digital camera mounted on a Nikon inverted microscope using an MDS 120 Universal C-mount adapter.
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RESULTS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The murine Capn4 gene (1) was disrupted by replacement of the 3'-terminal half of exon 9 with a PGK-neo cassette (61) (Fig. 1). This construct may give rise by alternative splicing of the RNA transcript to several possible forms of the small subunit, all having deletions or disruptions of the 30 to 40 C-terminal amino acids. These altered subunits are unlikely to be functional, since the C-terminal residues are known to be essential for activity and for heterodimer formation with the large subunit (13, 25).
ES cells.
Two independent targeted
Capn4+/ ES cell clones were generated, and one
of these was converted to the homozygous mutant state (Capn4/) by further selection of cells in
elevated G418 concentrations. The ES cell genotypes were characterized
by Southern blotting, showing the predicted wild-type 5.5-kb and
targeted 2.5-kb PstI bands (Fig.
2A). Northern blot analysis (Fig. 2B)
showed that the levels of µ-calpain 80-kDa subunit (Capn1)
mRNA were equal in all three lines. The levels of m-calpain 80-kDa
subunit (Capn2) mRNA were equal in
Capn4+/+ and Capn4/
ES cells but were slightly reduced in some
Capn4+/ ES cell lines. The level of the
calpain small subunit (Capn4) mRNA was significantly lower
in Capn4+/ ES cells than in
Capn4+/+ ES cells, consistent with the presence
of only one correct allele. In Capn4/ ES
cells, a trace amount of an mRNA of approximately the same size was
detected, which is assumed to represent an unstable or low abundance
mRNA arising from the disrupted Capn4 gene.
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, or cells, but neither activity was
detectable in Capn4/ cells (Fig. 2C).
Immunoblotting analysis showed that the m-calpain large subunit protein
was present in equal amounts in Capn4+/+ and
Capn4+/ ES cells but was either absent or
detectable only in trace amounts in Capn4/
ES cells (Fig. 2D). Reduced levels of m-calpain large subunit protein
were also seen in Capn4/ embryos (Fig. 4C).
No satisfactory immunoblots have been obtained for the µ-calpain
large subunit or for the small subunit, owing to the lack of
appropriate antibodies. Commercial antibodies claiming specificity for
these mouse proteins were purchased but were found to bind only to
irrelevant proteins in wild-type mouse tissue extracts. As a control,
the expression of Erk1/2 kinase was also assessed by immunoblotting
(Fig. 2E). Approximately equal protein loading in the immunoblot
analysis was also demonstrated by Coomassie blue staining (data not shown).
Growth rate.
The growth rates of the different ES cell lines
in ES medium were measured over 4 days, and the results are shown in
Fig. 3. A simple exponential fit of the
data to an equation of the form y = y0.eat gave population doubling times as
follows: Capn4+/+ ES cells, 13.1 ± 0.7 h; Capn4+/ ES cells, 14.0 ± 0.2 h; and Capn4/ ES cells, 12.7 ± 0.4 h.
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), Capn4+/
), and Capn4
) ES cells.
Embryonic fibroblasts.
Primary fibroblast cultures were
established from E9.5 embryos, and the growth characteristics of
Capn4/ embryonic fibroblasts were
indistinguishable from those of Capn4+/+ or
Capn4+/ genotypes. Casein zymography of
fibroblast extracts to detect calpain activity, and immunoblotting to
detect the m-calpain large subunit, all gave results entirely
consistent with those for the ES cells and embryos of each genotype
(data not shown).
Transgenic mice.
Capn4+/ ES cell clones
were used to produce chimeric animals by the aggregation method
(40), and germ line transmission of the targeted
Capn4 allele was achieved with two independent ES cell
clones. The Capn4+/ F1 animals
showed no apparent defects either in gross anatomy or by histological
analysis. The F2 weanlings (n = 225) from
heterozygous intercrosses consisted of 67%
Capn4+/, 33% Capn4+/+,
and no Capn4/ animals; in contrast, embryos
(n = 252) isolated from heterozygous intercrosses at E9.5
and E10.5 showed the expected Mendelian ratio of
Capn4/ (24%),
Capn4+/ (48%), and
Capn4+/+ (28%) (Fig.
4A). This suggested that the
Capn4/ animals might have died during
embryonic development or perinatally. Furthermore, the average litter
size at weaning age was 8.3, compared to an average of 9.6 embryos
isolated from pregnant mice. Indeed, all
Capn4/ embryos isolated after E11.5 were
dead or moribund. Analysis of calpain activity in E10.5 embryos by
means of casein zymography revealed µ- and m-calpain activities in
Capn4+/+ and Capn4+/
embryos but not in Capn4/ embryos (Fig. 4B).
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Histological analysis of embryos.
Examination of embryos
isolated at different stages of development revealed that all
Capn4/ embryos older than E11.5 were dead or
dying. However at earlier times, live Capn4/
embryos were found in the expected Mendelian ratios, and E9.5 Capn4/ embryos were indistinguishable from
their Capn4+/ and
Capn4+/+ littermates (data not shown). At E10.5,
although Capn4/ embryos were still alive,
the degree of yolk sac vasculature was reduced, and there was
hemorrhaging into the space between the embryo and the amnion.
Histological analysis of serial sections prepared from E10.5
Capn4/ embryos revealed that the
cardiovascular system appeared to have developed normally up to this
stage, but that the extensive morphogenic events in the heart chambers
and vessels leading to and from the heart did not appear to be
proceeding normally (Fig. 5). In
particular, the common atrial chamber had begun to collapse, and the
asymmetric restructuring of the sinus venosus did not appear to be
occurring. Endothelial cells in the atrial walls appeared to be
rounding up at E10.5, and by E11.5 they had delaminated (Fig. 5I to L). Another striking observation was the large accumulation of nucleated erythroid cells within the heart chambers, blood vessels, and developing liver (Fig. 5D, H, L, and P). This accumulation of erythroid
cells correlated with the death of Capn4/
embryos at or around E11.5. We also observed other, more subtle defects
which invite further study. For example, histological analysis
suggested the presence of more apoptotic cells among migrating neural
crest population from the trigeminal ganglion and within the adjacent
fourth ventricle (Fig. 5Q to T). By E11.5 the neural crest no longer
displayed a morphology typical of migrating cells, and there appeared
to be a higher proportion of apoptotic cells. However, in situ TUNEL
analysis at earlier embryonic stages (E9.5 to E10.5) failed to detect
significant differences in the number of apoptotic cells (data not
shown).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The Capn4 gene was disrupted on the basis of the
assumption that absence of a functional calpain small subunit would
abolish the activity of the classical µ- and m-calpains. This
assumption was found to be essentially correct. Casein zymography
showed that Capn4+/+ and
Capn4+/ cells and embryos contained normal
levels of calpain activity, but no calpain activity could be detected
in extracts of Capn4/ ES cells, embryonic
fibroblasts, or embryos. The casein zymogram method is not
quantitative, but it represents the only feasible means to separate and
detect the activities of both µ- and m-calpains in very small
samples, such as ES cell or fibroblast cultures, or single E9.5-E10.5
mouse embryos. Methods for small-scale separation and quantitative
assay of µ- and m-calpains have been described, but they require two
column steps (hydrophobic and ion exchange) for each sample, to
separate the enzymes from each other and from calpastatin, and require
at least 0.5 g of tissue or cultured cells (27, 30).
The embryonic lethality at E11.5 also indicates clearly that at least
one of the two µ- and m-calpain activities is absolutely required for
the later stages of fetal development. However,
Capn4/ embryos developed normally until
midgestation, and cells lacking calpain activity still proliferated at
normal rates and remained attached to the tissue culture substrate.
This suggests either that calpain is not required for cell growth and
division and for some forms of cell adhesion, a conclusion which does
not agree with some previous work, or that these cells still contained
some residual calpain activity.
Calpain activity could persist in Capn4/
cells by two mechanisms: by an unexpected activity of the disrupted
small subunit, which is highly improbable; or by a residual activity of
the isolated large subunits, which cannot presently be excluded. With
respect to the small subunit, Northern blotting showed that a trace
amount of a Capn4-derived transcript was still present in
Capn4/ ES cells. The sequence of this
putative residual Capn4 transcript has not been determined,
but the three most likely possibilities are: (i) transcription
termination in the PGK-neo cassette; (ii) splicing from exon
8 to cryptic splice sites in the beginning of the neo coding
sequence (38) followed by termination in the PGK-neo cassette; or (iii) splicing around the
PGK-neo cassette from exon 8 to exon 10. In all three cases,
the resulting transcripts would encode small subunits that lacked
critical portions of the C terminus. The disrupting PGK-neo
cassette is located within exon 9 in the codon for residue 230 of the
natural 268-amino-acid small subunit. Translational readthrough from
exon 9 into the PGK-neo cassette would result in replacement
of the natural 38 C-terminal residues with 98 heterologous amino acid
residues encoded by sequences in the PGK promoter.
Translational readthrough from the end of exon 8 into the
neo coding region would result in replacement of the natural
68 C-terminal residues with an undetermined heterologous sequence
derived from the neo coding region. Splicing of exon 8 to
exon 10 would generate a protein lacking residues 204 to 242. These
hypothetical small subunit peptides are most unlikely to combine with
the calpain large subunit to form active enzyme, since the natural
C-terminal portion of the small subunit is essential for heterodimer
formation and activity (13). The instability of the large
subunits, discussed below, is also a proof that no functional small
subunit was present.
With respect to the large subunits, their mRNAs were present at normal
levels in cells of all genotypes, but only trace levels of the
m-calpain large subunit could be detected by immunoblotting. The same
is almost certainly true for the µ-calpain large subunit, although
only poor immunoblot evidence could be obtained. It is clear that the
calpain large subunits in Capn4/ cells are
unstable in the absence of functional small units and are rapidly
turned over. There is no unambiguous assay available to measure the
possible in vivo activity of these large subunits (particularly at
trace levels); thus, it remains unresolved whether the isolated large
subunits simply fail to fold correctly in the absence of the small
subunit and are rapidly degraded, or whether they exert some transient
and unregulated activity prior to (auto)degradation. The latter case
would be similar to that of calpain 3, which has a very short half-life
and can be assayed only by observation of characteristic autolysis
products (23). If it is assumed that the isolated calpain
large subunits in Capn4/ cells possess some
short-lived activity, it is also necessary to assume that this activity
is sufficient to support normal growth and adhesion of
Capn4/ ES cells and fibroblasts but not
sufficient for full development of the fetus. The idea of a dose effect
is rather unsatisfactory, and it is attractive to speculate that the
difference between cell survival and tissue survival reflects some more
specific calpain-dependent cell-cell interaction.
It was also not clear at the outset whether the activities of other
calpain-related genes, or of other protease systems such as caspases
and the proteasome, might compensate for the absence of µ- and
m-calpains, but compensation was not observed. The death at
midgestation of all Capn4/ embryos shows
clearly that the presence of the calpain small subunit is essential for
normal fetal development. We assume that the absence of the small
subunit exerts its effect by causing the loss of normal µ- and
m-calpain activities.
Embryonic lethality at or around E10 has become a common observation in
mouse knockout studies, and defects in the cardiovascular system are
frequently observed in these cases (8, 16, 17, 22, 32, 51,
57). In the Capn4/ mice, embryonic
death coincided with a critical stage in heart development when
septation of both heart chambers is occurring as well as formation of
the atrioventricular canal. Histological analysis of
Capn4/ embryos showed that the cell linings
of both atrium and ventricle were losing their integrity by E10.5 and
were delaminated by E11.5 (Fig. 5J and L). As the dual-chambered atrium
was not observed in E10.5 Capn4/ embryos, we
have tentatively concluded that embryonic death may have resulted from
a failure in heart morphogenesis. We cannot rule out other
possibilities, including hemorrhaging and the apparent accumulation of
erythroid cells that is observed at the time of embryonic death. These
erythroid cells appear to be arising in the primitive liver because we
observed large islands of erythropoiesis in the E11.5 liver (Fig. 5P).
However, the erythroid cells accumulating in the vasculature and
throughout the embryo retain their nuclei. This raises the interesting
possibility of a blockage in erythroid development as a result of loss
of calpain activity, which will be investigated further. By E11.5 there
were more dead cells in the brain ventricles and neural crest of
Capn4/ embryos than in the controls, and
these latter cells did not appear to be migrating normally (Fig. 5T).
However, this may not be directly related to a role for calpain
activity in programmed cell death because in situ TUNEL analysis of
embryos failed to reveal differences in the proportion of apoptotic
cells at slightly earlier stages (E9.5 to E10.5).
While the essential functions that are disrupted in the absence of normal calpain activity remain unknown, it is important to note that the presence of the calpain small subunit is not required for the growth of ES cells or of embryonic fibroblasts. This appears to rule out a strict requirement for µ- or m-calpain in general cell functions including cell cycle regulation, survival signaling pathways, adhesion, and migration. Such a conclusion would disagree with previous work from several laboratories, and further study of these cell lines is required to establish the importance of calpain in these phenomena and in processing many of the reported calpain substrates. The activity of at least one of the two µ- or m-calpain activities is, however, clearly essential to survival and development of the embryo beyond midgestation. This observation increases the importance of experiments designed to disrupt each of the Capn1 and Capn2 genes alone. Our results do not provide the hoped-for demonstration of a physiological function for calpain, but they have provided homozygous cells for further work in tissue culture and point the way to alternative mouse models of calpain function. It has been established very clearly that calpain is essential to life.
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ACKNOWLEDGMENTS |
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We thank Andras Nagy for providing the R1 ES cell line, Janet Rossant for the 129SvJ mouse genomic DNA library, Ralph Zirngibl for modification of the pPNT vector and for helpful discussions, and Marion Arnold for histology.
This work was supported by grants from the Medical Research Council of Canada, the Heart and Stroke Foundation of Canada, and the National Cancer Institute of Canada. P.A.G. is an MRC scholar.
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FOOTNOTES |
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* Corresponding author. Mailing address: Cancer Research Laboratories, Department of Pathology, Queen's University, Kingston, Ontario K7L 3N6, Canada. Phone: (613) 533-2813. Fax: (613) 533-6830. E-mail: greerp{at}post.queensu.ca.
Present address: MRC Protein Phosphorylation Unit, University of
Dundee, Dundee DD1 5EH, United Kingdom.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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129: 435-442
[Abstract] [Full Text] -
Kramerova, I., Kudryashova, E., Tidball, J.G., Spencer, M. J.
(2004). Null mutation of calpain 3 (p94) in mice causes abnormal sarcomere formation in vivo and in vitro. Hum Mol Genet
13: 1373-1388
[Abstract] [Full Text] -
Yadavalli, R., Guttmann, R. P., Seward, T., Centers, A. P., Williamson, R. A., Telling, G. C.
(2004). Calpain-dependent Endoproteolytic Cleavage of PrPSc Modulates Scrapie Prion Propagation. J. Biol. Chem.
279: 21948-21956
[Abstract] [Full Text] -
Honda, S., Marumoto, T., Hirota, T., Nitta, M., Arima, Y., Ogawa, M., Saya, H.
(2004). Activation of m-Calpain Is Required for Chromosome Alignment on the Metaphase Plate during Mitosis. J. Biol. Chem.
279: 10615-10623
[Abstract] [Full Text] -
Franz, T., Winckler, L., Boehm, T., Dear, T. N.
(2004). Capn5 Is Expressed in a Subset of T Cells and Is Dispensable for Development. Mol. Cell. Biol.
24: 1649-1654
[Abstract] [Full Text] -
Altznauer, F., Conus, S., Cavalli, A., Folkers, G., Simon, H.-U.
(2004). Calpain-1 Regulates Bax and Subsequent Smac-dependent Caspase-3 Activation in Neutrophil Apoptosis. J. Biol. Chem.
279: 5947-5957
[Abstract] [Full Text] -
Moldoveanu, T., Jia, Z., Davies, P. L.
(2004). Calpain Activation by Cooperative Ca2+ Binding at Two Non-EF-hand Sites. J. Biol. Chem.
279: 6106-6114
[Abstract] [Full Text] -
Suzuki, K., Hata, S., Kawabata, Y., Sorimachi, H.
(2004). Structure, Activation, and Biology of Calpain. Diabetes
53: S12-18
[Abstract] [Full Text] -
Zhou, J., Ryan, A. J., Medh, J., Mallampalli, R. K.
(2003). Oxidized Lipoproteins Inhibit Surfactant Phosphatidylcholine Synthesis via Calpain-mediated Cleavage of CTP:Phosphocholine Cytidylyltransferase. J. Biol. Chem.
278: 37032-37040
[Abstract] [Full Text] -
Kifor, O., Kifor, I., Moore, F. D. Jr., Butters, R. R. Jr., Brown, E. M.
(2003). m-Calpain Colocalizes with the Calcium-sensing Receptor (CaR) in Caveolae in Parathyroid Cells and Participates in Degradation of the CaR. J. Biol. Chem.
278: 31167-31176
[Abstract] [Full Text] -
Sedarous, M., Keramaris, E., O'Hare, M., Melloni, E., Slack, R. S., Elce, J. S., Greer, P. A., Park, D. S.
(2003). Calpains Mediate p53 Activation and Neuronal Death Evoked by DNA Damage. J. Biol. Chem.
278: 26031-26038
[Abstract] [Full Text] -
GOLL, D. E., THOMPSON, V. F., LI, H., WEI, W., CONG, J.
(2003). The Calpain System. Physiol. Rev.
83: 731-801
[Abstract] [Full Text] -
Crocker, S. J., Smith, P. D., Jackson-Lewis, V., Lamba, W. R., Hayley, S. P., Grimm, E., Callaghan, S. M., Slack, R. S., Melloni, E., Przedborski, S., Robertson, G. S., Anisman, H., Merali, Z., Park, D. S.
(2003). Inhibition of Calpains Prevents Neuronal and Behavioral Deficits in an MPTP Mouse Model of Parkinson's Disease. J. Neurosci.
23: 4081-4091
[Abstract] [Full Text] -
Welm, A. L., Timchenko, N. A., Ono, Y., Sorimachi, H., Radomska, H. S., Tenen, D. G., Lekstrom-Himes, J., Darlington, G. J.
(2002). C/EBPalpha Is Required for Proteolytic Cleavage of Cyclin A by Calpain 3 in Myeloid Precursor Cells. J. Biol. Chem.
277: 33848-33856
[Abstract] [Full Text] -
Kobayashi, S., Yamashita, K., Takeoka, T., Ohtsuki, T., Suzuki, Y., Takahashi, R., Yamamoto, K., Kaufmann, S. H., Uchiyama, T., Sasada, M., Takahashi, A.
(2002). Calpain-mediated X-linked Inhibitor of Apoptosis Degradation in Neutrophil Apoptosis and Its Impairment in Chronic Neutrophilic Leukemia. J. Biol. Chem.
277: 33968-33977
[Abstract] [Full Text] -
Gil-Parrado, S., Fernandez-Montalvan, A., Assfalg-Machleidt, I., Popp, O., Bestvater, F., Holloschi, A., Knoch, T. A., Auerswald, E. A., Welsh, K., Reed, J. C., Fritz, H., Fuentes-Prior, P., Spiess, E., Salvesen, G. S., Machleidt, W.
(2002). Ionomycin-activated Calpain Triggers Apoptosis. A PROBABLE ROLE FOR Bcl-2 FAMILY MEMBERS. J. Biol. Chem.
277: 27217-27226
[Abstract] [Full Text] -
Xu, Y., Mellgren, R. L.
(2002). Calpain Inhibition Decreases the Growth Rate of Mammalian Cell Colonies. J. Biol. Chem.
277: 21474-21479
[Abstract] [Full Text] -
Lid, S. E., Gruis, D., Jung, R., Lorentzen, J. A., Ananiev, E., Chamberlin, M., Niu, X., Meeley, R., Nichols, S., Olsen, O.-A.
(2002). The defective kernel 1 (dek1) gene required for aleurone cell development in the endosperm of maize grains encodes a membrane protein of the calpain gene superfamily. Proc. Natl. Acad. Sci. USA
99: 5460-5465
[Abstract] [Full Text] -
Wiggin, G. R., Soloaga, A., Foster, J. M., Murray-Tait, V., Cohen, P., Arthur, J. S. C.
(2002). MSK1 and MSK2 Are Required for the Mitogen- and Stress-Induced Phosphorylation of CREB and ATF1 in Fibroblasts. Mol. Cell. Biol.
22: 2871-2881
[Abstract] [Full Text] -
Tompa, P., Mucsi, Z., Orosz, G., Friedrich, P.
(2002). Calpastatin Subdomains A and C Are Activators of Calpain. J. Biol. Chem.
277: 9022-9026
[Abstract] [Full Text] -
Bhatt, A., Kaverina, I., Otey, C., Huttenlocher, A.
(2002). Regulation of focal complex composition and disassembly by the calcium-dependent protease calpain. J. Cell Sci.
115: 3415-3425
[Abstract] [Full Text] -
Carragher, N. O., Westhoff, M. A., Riley, D., Potter, D. A., Dutt, P., Elce, J. S., Greer, P. A., Frame, M. C.
(2002). v-Src-Induced Modulation of the Calpain-Calpastatin Proteolytic System Regulates Transformation. Mol. Cell. Biol.
22: 257-269
[Abstract] [Full Text] -
Dourdin, N., Bhatt, A. K., Dutt, P., Greer, P. A., Arthur, J. S. C., Elce, J. S., Huttenlocher, A.
(2001). Reduced Cell Migration and Disruption of the Actin Cytoskeleton in Calpain-deficient Embryonic Fibroblasts. J. Biol. Chem.
276: 48382-48388
[Abstract] [Full Text] -
Azam, M., Andrabi, S. S., Sahr, K. E., Kamath, L., Kuliopulos, A., Chishti, A. H.
(2001). Disruption of the Mouse {micro}-Calpain Gene Reveals an Essential Role in Platelet Function. Mol. Cell. Biol.
21: 2213-2220
[Abstract] [Full Text] -
Hosfield, C. M., Moldoveanu, T., Davies, P. L., Elce, J. S., Jia, Z.
(2001). Calpain Mutants with Increased Ca2+ Sensitivity and Implications for the Role of the C2-like Domain. J. Biol. Chem.
276: 7404-7407
[Abstract] [Full Text] -
Glading, A., Uberall, F., Keyse, S. M., Lauffenburger, D. A., Wells, A.
(2001). Membrane Proximal ERK Signaling Is Required for M-calpain Activation Downstream of Epidermal Growth Factor Receptor Signaling. J. Biol. Chem.
276: 23341-23348
[Abstract] [Full Text] -
Lid, S. E., Gruis, D., Jung, R., Lorentzen, J. A., Ananiev, E., Chamberlin, M., Niu, X., Meeley, R., Nichols, S., Olsen, O.-A.
(2002). The defective kernel 1 (dek1) gene required for aleurone cell development in the endosperm of maize grains encodes a membrane protein of the calpain gene superfamily. Proc. Natl. Acad. Sci. USA
99: 5460-5465
[Abstract] [Full Text]
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