1 Pathology and Physiology Research Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Morgantown, West Virginia 26505; and 2 Department of Pathology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033
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ABSTRACT |
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To address the involvement of the calpain system
in both basal and silica-induced nuclear factor (NF)-B activation,
several human bronchial epithelial cell lines were established in which an intracellular inhibitor of calpain, calpastatin, was stably expressed. Reduced basal and silica-induced inhibitor (I
B
)
degradation and NF-
B activation were observed in cells stably
overexpressing calpastatin. In addition, the cells in which calpain was
constitutively inhibited by the overexpression of calpastatin exhibited
a notable morphological change. Whereas empty vector-transfected cells
displayed a morphology indistinguishable from that of parental cells,
cells overexpressing calpastatin exhibited a mosaic morphological
change with reduced formation of lamella 30 min after the cells were seeded. Genefilter microarray experiments, in which 3,965 human genes
can be evaluated for their expression at the same time, showed that
calpastatin downregulated genes encoding several membrane-associated proteins or nuclear proteins and upregulated genes of collagen
2,
DAZ, and mitochondrial capsule selenoprotein. These results suggest
that, in addition to their proteolytic activities on cytoskeletal proteins and other cellular regulatory proteins, calpain-calpastatin systems can also affect the expression levels of genes encoding structural or regulatory proteins.
nuclear factor-B; calpain; silica; epithelial cells
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INTRODUCTION |
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NUCLEAR
FACTOR (NF)-B proteins regulate transcription of a number of
cellular genes that are critical for the maintenance of normal immune
functions as well as the initiation or progression of human diseases
(3, 11, 28). This transcription factor can also govern the
expression of several viral genes, such as genes of human
immunodeficiency virus and human T-lymphocyte leukemia virus (1,
6, 15). Unlike many other transcription factors that are
constitutively localized in the nucleus, NF-
B is mainly sequestered
in the cytoplasm by the binding of its inhibitor, i.e., I
B family
members, in unstimulated cells. The most abundant and
best-characterized inhibitor of NF-
B is I
B
, which prevents activation, translocation, and DNA binding of NF-
B transcription factor. Upon activation of cells with a variety of agents, such as the
inflammatory cytokines interleukin-1 and tumor necrosis factor (TNF),
bacterial endotoxins, and environmental or occupational particles,
I
B
is phosphorylated by activated I
B kinases in collaboration
with mitogen-activated protein kinases. The phosphorylated I
B is
then ubiquitinated by an F-box/WD40 protein called
-TrCP (
-transducin repeat-containing protein) and rapidly degraded by a
variety of proteolytic enzymes (7, 30). The degradation of
I
B leads to the activation and translocation of NF-
B into the
nucleus to regulate the transcription of target genes.
Two major proteolytic pathways have been studied with regard to their
role in signal-induced NF-B activation: proteasomes and calpains.
Accumulating evidence suggests that the ubiquitin-proteasome pathway
plays a major role in the degradation of I
B
protein and,
consequently, in the activation of NF-
B transcription factor (2, 10, 22). Several recent studies have shown that the calpain system might be involved also under certain circumstances in
basal or signal-induced degradation of I
B
and the activation of
NF-
B (4, 8, 14, 20, 29). Whereas a potent proteasome inhibitor, MG132, failed to abrogate silica-induced I
B
degradation in macrophages, transient overexpression of calpastatin, a
specific endogenous inhibitor for calpain, resulted in an appreciable
inhibition of I
B
degradation induced by silica (8).
In vitro digestion of recombinant I
B
by purified calpain or
cytosolic extracts from silica-stimulated cells demonstrated further
that calpain was capable of degrading I
B
protein by the cleavage
of several leucine-rich domains. In an independent study, Han et al.
(14) showed that calpain provides a parallel proteolytic
pathway to the ubiquitin-proteasome pathway for TNF-
-induced
I
B
degradation in human HepG2 cells (14). In WEHI231
immature B cells, a rapid degradation of I
B
was insensitive to
proteasome inhibitors but was substantially inhibited by calpain
inhibitors (20). More recently, Baghdiguian and associates
(4) provided direct evidence demonstrating that patients
with an autosomal muscular dystrophy caused by calpain 3 deficiency
exhibited a substantial impairment of I
B
degradation and NF-
B
activation in muscular cells (4).
Calpains are calcium-dependent cysteine proteinases present in a variety of cells (31, 33). Two major groups of calpains, termed ubiquitous calpains and tissue-specific calpains, have been identified. The ubiquitous calpains include calpain 1 (µ-calpain) and calpain 2 (m-calpain), which require micromolar and millimolar concentrations of calcium for their activation, respectively. Calpain 3 (n-calpain-1), requiring nanomolar concentrations of calcium for its activation, is skeletal muscle specific, whereas calpain 4 (n-calpain-2) was mainly found in smooth muscle. Experiments using synthetic inhibitors of calpains have shown them to be pivotal proteases participating in a limited proteolytic reaction of a number of cellular structural or regulatory proteins, such as cytoskeletal proteins (24), kinases (32), cytokines (17), and the tumor-suppressing protein p53 (12, 18, 23, 34). However, the reliability of the use of synthetic pharmacological inhibitors to delineate the role of calpains has been compromised because of the low cellular permeability and poor substrate specificity of these inhibitors. These inhibitors include calpain inhibitor I/II, E64 family compounds, and leupeptin. Recently, an intracellular calpain inhibitor, calpastatin, has been identified as a specific inhibitor of calpains, having no direct effect on other cellular proteases such as proteasome and lysosome systems (5, 33).
Silica is one of the earliest recognized inhaled occupational dusts,
inducing severe and debilitating lung diseases among miners and
constructional workers through damaging of bronchial epithelial cells
and alveolar cells (19). The aim of the present study was
to determine whether NF-B activation by silica in human bronchial
epithelial cells is mediated by a similar mechanism found in mouse
macrophages cell lines as described previously. Using several cell
lines derived from human bronchial epithelial cells, we investigated
the effect of calpastatin, an intracellular inhibitor of calpains, on
silica-induced NF-
B activation. We demonstrated that the activation
of NF-
B transcription factor and the expression of a variety of
cellular genes, such as cytoskeletal protein genes, are impaired or
altered by the constitutive inhibition of calpains.
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MATERIALS AND METHODS |
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Cells. Human bronchial epithelial cells, BEAS-2B, were purchased from American Type Culture Collection (ATCC, Rockville, MD). The cells were cultured in DMEM (Mediatech, Washington, DC) supplemented with 10% fetal bovine serum, 2 mM glutamine, and 1,000 U/ml penicillin-streptomycin (Sigma, St. Louis, MO).
Expressing plasmids. Human calpastatin cDNA was a gift from Dr. Masatoshi Maki (School of Agricultural Sciences, Nagoya University, Japan). An expression vector for calpastatin was constructed by inserting a full-length human calpastatin cDNA into the EcoR V and Xba I sites on the pcDNAI/Neo vector (Invitrogen, Carlsbad, CA).
Stable transfection. Lipofectin (GIBCO BRL) was used for transfection of BEAS-2B cells with an empty vector (pcDNA I/Neo) or an expression vector for full-length human calpastatin. The cells were seeded at a density of 2 × 105 cells/well in six-well tissue culture plates and cultured for 2 days before the performance of transfection. The cells, containing the empty vector or expression vector for calpastatin, were screened in geneticin (700 µg/ml, GIBCO) for 3 wk.
Electrophoretic mobility shift assay.
The extraction of nuclear proteins and preparation of a
32P-labeled double-stranded oligonucleotide containing the
consensus B-site was performed as described previously
(9). For electrophoretic mobility shift assay (EMSA), 4 µg of nuclear protein were mixed with the labeled double-stranded
probe and incubated at room temperature for 30 min. The reaction
solution was subjected to electrophoresis on a native 5%
polyacrylamide gel in 0.25× Tris base EDTA buffer for 2-3
h. The protein-DNA complexes, indicating NF-
B binding, and
free probe were visualized in dried gels by autoradiography.
Proteolysis reaction.
In vitro degradation of recombinant IB
protein (Santa Cruz
Biotechnology, Santa Cruz, CA) was performed as described previously with minor modification (8). Briefly, total cytoplasmic
extracts were prepared by lysing the cells in a hypotonic buffer
containing 10 mM HEPES (pH 7.4), 1 mM EDTA, 10 mM KCl, 1 mM
dithiothreitol, 50 mM NaF, 1 mM sodium orthovanadate, and 0.2 mM
phenylmethylsulfonyl fluoride. After being incubated on ice for 30 min,
the cells were vortexed for 10 s and centrifuged at 2,000 g for 5 min. The supernatant was collected and further
centrifuged at 80,000 g for 1 h at 4°C. For the
proteolysis assay, the glutathione-S-transferase (GST) tag
in the GST-I
B
fusion protein was enzymatically removed by thrombin digestion. The resulting recombinant I
B
was
incubated with various concentrations of µ-calpain or cytoplasmic
extracts from the cells transfected with empty vector or a vector for
calpastatin in 20 µl of proteolysis reaction buffer [30 mM
Tris · HCl (pH 7.4), 30 mM NaCl, and 10 mM CaCl2]
at 30°C for 10 min. The proteolytic reaction was terminated by adding
8 µl of 3× SDS reducing buffer [350 mM Tris · HCl (pH 6.8),
15% SDS, 10% glycerol, 3.6 M
-mercaptoethanol, and 0.01%
bromphenol blue] and subjected to SDS-polyacrylamide gel
electrophoresis in 12% gels. Western blot analysis was performed to
determine the degradation of I
B
protein, using
anti-NH2-terminal and anti-COOH-terminal I
B
antibodies (C-15 and C-21, respectively; Santa Cruz) and enhanced
chemiluminescence immunoblot detection reagents (ECL; Amersham Life
Science, Arlington Heights, IL).
Genefilter microarray. The gene expression pattern in both vector- and calpastatin-transfected cells was determined by microarray experiments using Genefilter membrane gf211 (Research Genetics, Huntsville, AL). Briefly, 1 µg of total RNA extracted from cells transfected with empty vector or expression vector for calpastatin was incubated with 2 µg of oligo(dT), 1.5 µl of reverse transcriptase, 20 mM of dATP, dGTP, and dTTP, and 100 µCi [33P]dCTP in 30 µl of diethyl pyrocarbonate-treated water for 90 min at 37°C. After purification through a Bio-Spin 6 Chromatography Column, labeled probe was mixed with prehybridization solution and incubated with Genefilter membranes overnight at 42°C. To minimize possible manufacturer's variations among individual membranes, the same membrane was stripped and rehybridized with the second probe after the first round of hybridization.
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RESULTS |
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Impaired NF-B activation in calpastatin-transfected cells.
Even though the ubiquitin-proteasome system is considered to be a
universal pathway mediating the activation of NF-
B transcription factor, a possible alternative pathway under certain circumstances cannot be excluded (8, 14, 16). Previous studies using a
murine macrophage cell line, RAW264.7, demonstrated that inhibition of
calpain by transient transfection of calpastatin resulted in a
reduction of NF-
B activation in response to silica particles (8). To examine the possibility that a calpain-calpastatin system may also be involved in the activation of NF-
B in human bronchial epithelial cells, stable transfected cell lines were established in which calpastatin was constitutively expressed. EMSA
showed that, in contrast to the cells transfected with an empty vector
in which a time-dependent NF-
B induction by silica could be
identified (Fig. 1A,
lanes 5-8), calpastatin-expressing cells exhibited no
or only marginal activation of NF-
B in response to silica
(lanes 1-4). Likewise, immunoblot experiments indicated that the degradation of intracellular I
B
protein induced by silica was impaired in the cells in which calpains were constitutively inhibited by stable overexpression of calpastatin (Fig. 1B,
lanes 1-4).
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IB
is a substrate for calpain both in vitro and in vivo.
The ability of calpain to regulate protein turnover has suggested an
important role of this protease in cellular functions. To investigate
the digestion of I
B
protein by calpain in vitro, purified
µ-calpain was incubated with recombinant I
B
for 10 min. As
depicted in Fig. 2, A and
B, digestion of I
B
by calpain was dose dependent.
Antibody C-15, raised against amino acids from positions 6 to 20 at the
NH2 terminus of I
B
, recognizes three closely migrated
fragments with approximate molecular weights between 20 and 25 kDa
(Fig. 2A, lane 2, fragments b-d).
When an antibody directed to the amino acids from positions 297 to 317 at the COOH terminus of I
B
protein was used (antibody C-21), an
additional immunoreactive fragment with an approximate molecular weight
of 28 kDa was also detected (Fig. 2B, lanes 2 and
3, fragment a). On the basis of their apparent
molecular masses and their patterns of recognition by NH2-
and COOH-terminal I
B
antibodies, the major calpain cleavage sites
of I
B
should be within the NH2 terminus and the
middle of I
B
. It is also important to note that further
proteolytic processing of fragments b-d occurs when the
ratio of calpain to I
B
is increased, e.g., 1 µg vs. 0.25 µg
of calpain (Fig. 2, A and B, lanes 3).
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Morphological changes of calpain inhibition.
The most prominent activity of calpain is its limited proteolytic
effect on proteins that are closely associated with membranes, such as
cytoskeletal proteins and membrane proteins (27).
Consequently, structural alteration of the plasma membrane and
morphological changes of cells can be anticipated when the activity of
calpains is inhibited. In our initial experiments using murine
macrophage cells, RAW264.7, we found that the cells transfected with
calpastatin exhibited a cubelike morphology rather than a spherical
shape as exhibited by the parental cells or the cells transfected with an empty vector (data not shown). In human bronchial epithelial cells,
although a dramatic morphological change could not be identified by the
transfection of calpastatin, the cells expressing calpastatin did
exhibit an impaired spreading and an enhanced early adhesion onto
tissue culture flasks. More than 50% of empty vector-transfected cells
displayed a thin and smooth-edged circumferential lamella 30 min after
seeding into the cell culture flasks (Fig.
3A, arrows). Instead of the
lamella formation, cells overexpressing calpastatin showed a triangular
morphological characteristic and formed a mosaic monolayer among
individual cells (Fig. 3B). Moreover, although the lamella
formation and cell spreading were absent among the cells overexpressing
calpastatin, these cells exhibited a faster adhesion to the tissue
culture flasks. No morphological differences could be distinguished
between the cells transfected with an empty vector and the cells
transfected with calpastatin 4 h after seeding.
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Altered gene expression in the cells expressing calpastatin.
The proteolytic activity of calpains is well established and considered
as a major contributor to the turnover of proteins involved in the
morphological changes, spreading, and adhesion of cells (24,
27). Little is known, however, concerning the role calpains play
in the regulation of cellular gene expression. The fact that the
activation or functional maintenance of many transcription factors is
dependent on proteolytic processes suggests that it is possible that
calpains participate in the regulation of gene expression. To determine
what set of genes or how many genes were affected by the inhibition of
calpains, we carried out Genefilter microarray experiments in which the
expression of 3,965 randomly selected human genes was compared between
the cells transfected with an empty vector and the cells expressing calpastatin. We arbitrarily set a criterion of a twofold difference to
judge substantial inhibition or induction of gene expression in the
cells stably expressing calpastatin. A total of 12 genes satisfied this
criterion. As presented in Table 1, a
constitutive inhibition of calpain by overexpressing calpastatin in the
cells resulted in a decreased expression of nine genes. It is
intriguing to note that most of the decreased genes were those genes
encoding membrane-associated proteins or ion channel-related proteins
(protein phosphatase 2B and the sodium-/chloride-dependent betaine
transporter). The expression of three genes was upregulated by the
inhibition of calpains. These genes were collagen type I2, DAZ, and
mitochondrial capsule selenoprotein (MCS). Both DAZ and MCS are
important for the spermatogenesis and fertilization of male
reproductive cells.
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DISCUSSION |
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Proteolysis is a pivotal physiological event required for the control of fundamental cellular processes including cytoskeletal remodeling, cell migration, cell cycle progression, transcription factor activation or inactivation, antigen presentation, and programmed cell death (21). Not surprisingly, proteolytic systems are also crucial for the initiation of disease processes. Eukaryotic cells contain two major nonlysosomal proteolytic pathways, the ubiquitin-proteasome pathway and the calpain pathway, that participate in the signal-induced proteolysis.
A vast majority of studies have indicated that ubiquitin-proteasome
plays a major role in signal-induced IB
degradation and
consequent activation of NF-
B transcription factor (10, 22). However, several lines of evidence suggest the presence of
alternative degradation pathways for I
B
proteins. First, in the
murine macrophage cell line (RAW264.6), the inhibition of proteasome
was unable to block I
B
degradation induced by the occupational
dust, silica (8). Second, a proteasome-independent proteolytic pathway mediating the degradation of I
B
was
considered responsible for the constitutive nuclear localization of
NF-
B c-Rel/p50 complex in pre-B lymphocytes (20).
Third, a pathway parallel to proteasome, calpain degradation pathway,
has been identified in TNF-
- and respiratory syncytial virus-induced
I
B
degradation in human liver cells and airway epithelial cells
(14). Finally, calpain 3 deficiency in patients with
limb-girdle muscular dystrophy type 2A showed a substantial inhibition
of I
B
degradation, which was accountable for the cytoplasmic
sequestration of NF-
B (4).
The findings in the present report provide further supportive and
confirmational evidence to these observations. We have shown here that
the inhibition of calpains by stable transfection of calpastatin in
human bronchial epithelial cells led to a significant reduction of
NF-B DNA binding and I
B
degradation induced by silica.
Moreover, in vitro digestion analysis revealed preferential cleavage
site(s) of µ-calpain on the NH2 terminus of the
recombinant I
B
molecule (Fig. 2, A and B),
which is consistent with previous reports by us and others (8,
14, 29). This implies that despite the existence of a
COOH-terminal PEST domain [a sequence rich in proline (P), glutamic
and aspartic acid (E), serine (S), and threonine (T)] assumed as a
target region of calpain cleavage (25), the
NH2 terminus is more susceptible to calpain than the COOH
terminus of I
B
. A most recent study by Reuther and Baldwin (26) indicated that I
B
truncated at the amino
terminus by caspase-3 might function as a stable inhibitor of NF-
B.
This appears not to be the case in calpain-mediated degradation of I
B
because the fragments of NH2-terminal truncated
I
B
are still susceptible to further degradation by calpains.
Another important observation from the present study is the alteration
of the gene expression profile due to the constitutive inhibition of
calpain. In agreement with others, our finding that calpastatin-overexpressing cells exhibited impaired migration and
altered morphological characteristics implies a crucial role of
calpains in the regulation of cell mobility and morphology by cleaving
and remodeling cytoskeletal proteins in a site-specific manner.
However, there was limited information on calpain regulation of gene
expression. On the basis of the involvement of calpain in the
activation or functional maintenance of transcription factors including
NF-B- and c-Fos-containing complexes, it should be predictable that
the expression of some housekeeping and inducible genes will be
affected by the inhibition of calpains. An unanticipated result from
the Genefilter microarray experiments is that only a few sets of genes
were downregulated substantially in calpastatin-overexpressing cells.
Interestingly, five of nine underexpressed genes in
calpastatin-overexpressing cells are related to membrane-associated
proteins, such as surface proteins, ion channels, and Ca2+
channel-regulating proteins (Table 1). It is possible that the characteristic morphology of calpastatin-overexpressing cells may be
partially attributable to the downregulated expression of genes
encoding these membrane-associated proteins. Another four of nine
underexpressed genes are related to nuclear localized proteins with
transcription factor-like function, including hERR1, TRIP9, Y-linked
testis-specific protein, and cytokine-inducible nuclear protein. It is
recognized that a subtle difference of gene expression can have
dramatic biological consequences. Therefore, we also evaluated genes
whose repression or upregulation was between 1.4- and 2-fold of control
(Table 2) in cells overexpressing calpastatin, even though such changes
are usually considered insignificant in DNA microarray assays. It was
interesting to note that the expression of most of the genes encoding
ribosomal protein family members was inhibited.
It is unknown whether interconnected events are involved in NF-B
activation, altered gene expression, and cellular morphological changes
induced by the calpain-calpastatin system. There are no previous
reports indicating NF-
B-mediated expression of any of these genes
listed in Table 1. Whereas the gene structural data for most of these
genes are not available currently, DNA sequence analysis revealed
several highly conserved NF-
B binding sites, such as
499-GGGAATTTCA-508 and 549-GGGAATTTCCC-559, located within the promoter
region of the cartilage glycoprotein-39 gene (GenBank accession no.
Y08374; data not shown).
It would be premature to conclude that the observed alteration of gene
expression in calpastatin-overexpressing cells is the result of
impaired activation of NF-B. First, we compared the gene expression
pattern in the cells cultured under a basal condition without further
treatment. As shown in Fig. 1A, the basal level of NF-
B
activation could be notably inhibited by the inhibition of calpains
through constitutive overexpression of calpastatin. For the
determination of gene expression mediated by NF-
B, however, an
inducible condition is usually required. Second, it has been demonstrated that Cdc42, Rho, and Ras GTPases were associated with the
cytoskeleton (13). The effect of calpastatin on
calpain-mediated turnover and remodeling of the cytoskeleton system
could conceivably lead to alteration of signal transduction cascades
and gene expression, which bypasses regulation by NF-
B. Third,
besides NF-
B, other transcription factors may also serve as
targeting factors of calpains. Finally, certain secondary effects may
exist in this nonphysiological overexpression condition.
In summary, we explored two important functional aspects of calpains
that have not been well defined previously, i.e., involvement in the
activation of NF-B and regulation of gene expression. It is possible
that calpains may be involved in a number of physiological and
pathological processes. Several issues need to be addressed in future
studies, including the mechanisms of calpain activation, consequences
of calpain deficiency or overfunction, cross talk with proteasome and
other proteolytic systems, and the feasibility of targeting calpains to
interfere with disease processes.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. Jacques Corbeil (Director, Center for AIDS Research Genomics Core, University of California, San Diego) for the Genefilter microarray analysis. We also thank Dr. Masatoshi Maki (Nagoya University of Japan) for human calpastatin cDNA, Dr. Pinyi Du (University of California, San Diego) for participating in Genefilter microarray experiments, and Dr. Lyndell Millecchia for help with photography.
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FOOTNOTES |
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F. Chen was supported by a Career Development Award under a cooperative agreement from the Centers for Disease Control and Prevention through the Association of Teachers of Preventive Medicine.
Address for reprint requests and other correspondence: X. Shi, Pathology and Physiology Research Branch, National Institute for Occupational Safety and Health, 1095 Willowdale Rd., Morgantown, WV 26505 (E-mail: xas0{at}cdc.gov).
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. §1734 solely to indicate this fact.
Received 30 September 1999; accepted in final form 31 March 2000.
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