© 2000 American Heart Association, Inc.
UltraRapid Communication |
Independent Signals Control Expression of the Calcineurin Inhibitory Proteins MCIP1 and MCIP2 in Striated Muscles
From the Departments of Internal Medicine and Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Tex.
Correspondence to R. Sanders Williams, University of Texas Southwestern Medical Center, 6000 Harry Hines Blvd, NB11.200, Dallas, TX 75390-8573. E-mail williams{at}ryburn.swmed.edu
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Abstract |
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Abstract—Calcineurin, a calcium/calmodulin-regulated protein phosphatase, modulates gene expression in cardiac and skeletal muscles during development and in remodeling responses such as cardiac hypertrophy that are evoked by environmental stresses or disease. Recently, we identified two genes encoding proteins (MCIP1 and MCIP2) that are enriched in striated muscles and that interact with calcineurin to inhibit its enzymatic activity. In the present study, we show that expression of MCIP1 is regulated by calcineurin activity in hearts of mice with cardiac hypertrophy, as well as in cultured skeletal myotubes. In contrast, expression of MCIP2 in the heart is not altered by activated calcineurin but responds to thyroid hormone, which has no effect on MCIP1. A
900-bp intragenic segment located between exons 3 and 4 of
the MCIP1 gene functions as an alternative promoter that responds to
calcineurin. This region includes a dense cluster of 15 consensus
binding sites for NF-AT transcription factors. Because MCIP proteins
can inhibit calcineurin, these results suggest that MCIP1 participates
in a negative feedback circuit to diminish potentially deleterious
effects of unrestrained calcineurin activity in cardiac and skeletal
myocytes. Inhibitory effects of MCIP2 on calcineurin activity may be
pertinent to gene switching events driven by thyroid hormone in
striated muscles. The full text of this article is available at
http://www.circresaha.org.
Key Words: calcineurin • hypertrophy • gene transcription • thyroid hormone
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Introduction |
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Changes in intracellular calcium concentrations control gene expression in many cell types by calmodulin-dependent activation of calcineurin, a serine-threonine protein phosphatase.1 2 Signaling pathways controlled by calcineurin have been most intensively characterized in lymphocytes, where binding of antigen to cell surface receptors triggers calcium entry in a pattern that activates calcineurin, which then removes phosphate groups from transcriptional regulatory proteins of the NF-AT family. Dephosphorylated NF-AT proteins translocate from the cytoplasm to the nucleus, where they bind cognate recognition elements within target genes, in association with other transcription factors such as AP-1. Downstream genes required for T-cell activation (eg, interleukin-2) are induced in this manner. The pharmacological actions of immunosuppressive drugs such as cyclosporin A and FK-506 are based on inhibition of calcineurin activity in immune effector cells.
Recent studies also have revealed roles for calcineurin-dependent signaling pathways in cardiac and skeletal muscles. Transgenic mice that express constitutively active forms of calcineurin or NF-AT3 in the heart develop massive cardiac hypertrophy that progresses to dilated cardiomyopathy.3 Administration of calcineurin antagonist drugs prevents cardiac hypertrophy induced by a calcineurin transgene and also blocks hypertrophic responses to other stimuli in some, but not all, models.4 5 6 7 In skeletal muscles, calcineurin signaling has been implicated in both the hypertrophic response to insulin-like growth factor-18 9 and the remodeling of myofiber phenotypes in response to motor neuron activity.10 11 12 13 Calcineurin also stimulates differentiation of myogenic precursor cells,14 and animals lacking NF-AT2 fail to develop normal cardiac valves,15 indicating that calcineurin signaling is pertinent to development of heart and skeletal muscles as well.
A number of different proteins have been shown to bind to the catalytic subunit of calcineurin (calcineurin A) and to regulate its enzymatic activity. The holoenzyme includes calcineurin A and a regulatory subunit, calcineurin B. This complex is activated upon binding of calcium/calmodulin.16 CHP is a calcineurin B homologue that inhibits calcineurin activity by hindering the formation of the calcineurin/calmodulin/calcineurin B heterotrimer.17 Cyclosporin A and FK506 when bound to their respective binding proteins, cyclophilin A and FKBP12, form oligomeric complexes with calcineurin and inhibit its activity.18 Other proteins act to localize calcineurin within the cell. FKBP12, in the absence of FK506 ligand, anchors calcineurin to IP3 and ryanodine receptors,19 and AKAP79 is a scaffolding protein that binds calcineurin, protein kinase A, and protein kinase C.20 Forced expression of a ubiquitously expressed calcineurin-binding protein called Cabin/Cain inhibits calcineurin signaling in cultured cells and can prevent hypertrophic responses in rat cardiomyocytes.21 22 23
We have recently described a family of proteins—MCIP1 and MCIP2—that are highly expressed in striated muscles and that inhibit calcineurin through a direct physical interaction.24 The human gene encoding MCIP1 resides on chromosome 21 within the Down syndrome critical region (termed DSCR1) and was shown independently by other laboratories to function as an inhibitor of calcineurin.25 26 Two other human genes annotated as ZAKI-4/DSCR1L1 and DSCR1L2 encode closely related proteins that we term MCIP2 and MCIP3, respectively.27 28 The MCIP gene family includes a yeast protein Rcn1p capable of inhibiting calcineurin.25 MCIP proteins differ from previously described inhibitors of calcineurin in several important respects. Unlike immunophilin and FKBP, no exogenous molecules are required for the ability of MCIPs to inhibit calcineurin. MCIP proteins bind directly to calcineurin A using different binding surfaces compared with the larger AKAP79 or Cabin/Cain proteins. Finally, MCIP1 and MCIP2 are expressed most abundantly in striated muscles, compared with the ubiquitous expression of AKAP79, Cabin/Cain, and CHP.
In the present study, we report that the genes encoding
MCIP1 and MCIP2 are subject to distinctive mechanisms of regulation.
Specifically, expression of MCIP1 is induced by calcineurin activity,
whereas the MCIP2 gene fails to respond to this stimulus. Conversely,
MCIP2 expression is regulated by thyroid hormone, which has no
discernible effects on MCIP1. An intragenic region of the MCIP1 gene
located 5' to exon 4 contains a dense cluster of 15 NF-AT binding
motifs within a 900-bp segment and functions as an alternative
calcineurin-responsive promoter. These results identify independent
mechanisms by which different MCIP proteins are induced, presumably to
protect the cell from otherwise deleterious effects of unrestrained
calcineurin activity in different contexts.
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Materials and Methods |
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Plasmid Constructions
The segment of intron 3 from the human MCIP1 (DSCR1) gene was isolated by polymerase chain reaction (PCR) using human genomic DNA as template and primers based on sequence information from the human chromosome 21 databank.29 This
900-bp
fragment was subcloned into a pGL3 luciferase reporter vector
(Promega). Other plasmids were previously
described.10 24
Tissue Culture, Cell Transfection or Infection,
and Reporter Gene Assays
C2C12 myoblasts and myotubes were cultured as
previously described.30
Ionomycin (2 µmol/L) and cyclosporin A (50 to 200 nmol/L) were added
4 hours before harvesting the cells. When included,
cycloheximide (25 µmol/L) was added 15 minutes before
ionomycin. Transient transfection with plasmids or infection with
recombinant, replication-defective adenoviruses and luciferase assays
were performed as previously
reported.10 24 31
Animal Experiments
Lines of transgenic mice in which the 
-myosin
heavy chain promoter is used to drive expression of a constitutively
active form of calcineurin selectively in the heart were generated and
described previously.3
Wild-type male C57Bl/6 mice were injected intraperitoneally with
3,5,3'-triiodothyromine (T3) (0.1 µg/g body weight) or an equal
volume of 0.9% saline once a day for 10
days.32 All experiments
involving animals were conducted using IACRAC-approved
protocols.
RNA Isolation and Northern Blot
Analysis
Total RNA was prepared from mouse tissues or C2C12
cells using Tripure (Boehringer Mannheim, Inc) following the
manufacturer’s protocol. Northern blot analysis was performed with 20
µg of total RNA in each lane and probed in Ultrahyb (Ambion) with
complementary sequences representing the 3' untranslated region (UTR)
of MCIP1 (common to all known splicing variants), exon 1 of MCIP1, exon
4 of MCIP1, or ORF segments of MCIP2 or GAPDH cDNA. Probes were
generated by PCR and labeled as described
previously.24 Signals from
Northern blots were detected on a Storm PhosphorImager (Molecular
Dynamics) and quantified using ImageQuant (version
1.2).
cDNA Microarray Analysis of
Calcineurin-Transgenic Mice
RNA was isolated from two calcineurin-transgenic
mice3 at 10 weeks of age and
from a wild-type littermate. One of the transgenic mice was determined
to be in heart failure, on the basis of anasarca with massive ascites,
whereas the other appeared grossly normal. Both transgenic mice showed
>100% increase in heart weight relative to the wild-type control (250
and 240 mg versus 96 mg, respectively). Total body weights of the
wild-type (26 g) and nonfailing calcineurin transgenic mice (27.5 g)
were comparable, whereas the calcineurin-transgenic mouse in heart
failure had a greater body weight (35.6 g), reflecting the edematous
state. Microarray analyses were conducted by Incyte Genomics as
described elsewhere.33
Briefly, polyA+ RNA was labeled with Cy3/Cy5 fluorescent dyes and
hybridized with a mouse cDNA microarray (mouse GEM 1.14) containing
8734 elements each (7832 unique genes: 3336 annotated/4496 unannotated)
genes. Differential expression was calculated as the ratio of
fluorescent signals after subtraction of
background.
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Results |
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Calcineurin Induces Expression of MCIP1 but Not MCIP2
Gene expression profiling by microarray analysis was conducted to identify genes that are differentially regulated in hearts of transgenic mice engineered to express a constitutively active form of calcineurin compared with normal controls. Calcineurin-transgenic animals (
MHC-CnA*) develop massive cardiac hypertrophy that
progresses to dilated
cardiomyopathy.3 This
analysis identified MCIP1 as a gene that is potently upregulated in
this model
(Figure 1A
). Other genes known to be controlled by
hypertrophic signals (eg, atrial natriuretic factor) also were
identified by this analysis and were induced to a comparable extent
(3-fold) as MCIP1 in hypertrophic, nonfailing hearts of animals at
10 weeks of age compared with a wild-type littermate
(Figure 1A, top). Both MCIP1 and calcineurin A transcripts
were elevated further in hearts of age-matched animals that had
progressed to overt heart failure compared with levels noted in
hypertrophic, nonfailing hearts
(Figure 1A, bottom). This induction of MCIP1 gene expression
within the intact myocardium by activated calcineurin was confirmed by
Northern blot analysis
(Figure 1B). Quantitative estimates of the extent of MCIP1
induction in MHC-CnA* hearts
(Table 1A) varied among individual animals of comparable age
(3- to 17-fold), perhaps reflecting different stages in disease
progression. Northern blots
(Figure 1B) also revealed that the MCIP2 gene was not
similarly regulated. In contrast to the upregulation of MCIP1 mRNA in
this model, there was no detectable change in MCIP2 mRNA
(Figure 1B and
Table 1A).
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We also assessed the ability of calcineurin to stimulate
expression of MCIP1 in C2C12 cells that differentiate into skeletal
myotubes in cell culture. Increased intracellular calcium
concentrations evoked by administration of the calcium ionophore
ionomycin led rapidly (
4 hours) to an increased abundance of MCIP1
mRNA in C2C12 cells
(Figure 2A). MCIP1 mRNA is increased during differentiation
of C2C12 cells (reported
previously,24 not shown
here), but a 2-fold induction of MCIP1 by ionomycin was evident
irrespective of the stage of differentiation (myoblasts or myotubes)
and the correspondingly lower or higher initial levels of MCIP1 mRNA.
In contrast, expression of MCIP2 mRNA was unaffected by ionomycin
(Figure 2A and
Table 1A
). To determine whether the induction of MCIP1 gene
expression by calcium ionophore was attributable to calcineurin
activity, as opposed to other calcium-regulated signaling events, we
assessed the effects of cyclosporin A on this response. In a
dose-dependent manner, cyclosporin A blocked the ability of ionomycin
to upregulate MCIP1 transcript levels in C2C12 cells
(Figure 2B) and even reduced MCIP1 transcripts below the
levels observed in cells untreated with either drug, presumably by
blocking basal as well as ionophore-stimulated calcineurin activity.
Compared with cells treated with the highest dose of cyclosporin A,
MCIP1 mRNA was increased 4-fold by ionomycin in the absence of
cyclosporin A
(Figure 2B). In addition, MCIP1 mRNA was increased 2-fold in
cultured myocytes by infection with a recombinant adenoviral vector
encoding a constitutively activated form of calcineurin, compared with
the effects of infection by a control virus encoding green fluorescent
protein
(Figure 2C). Thus, experimental strategies based on
loss-of-function and gain-of-function approaches in cultured myocytes
support the conclusion that MCIP1 gene expression, but not that of
MCIP2, is regulated by calcineurin signaling.
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Induction of MCIP1 Expression by Calcineurin
Does Not Require New Protein Synthesis
In the presence of cycloheximide, an inhibitor of
protein synthesis, activation of calcineurin by ionomycin continued to
upregulate MCIP1 mRNA in C2C12 cells, indicating that this induction is
not dependent on the generation of new proteins
(Figure 2D). The magnitude of ionomycin-stimulated
upregulation of MCIP1 in the presence of cycloheximide (10-fold) was
greater than that observed when cycloheximide was absent (2-fold).
We interpret these data to support the hypothesis that calcineurin
stimulates MCIP1 gene transcription by posttranslational modification
of a preexisting pool of NF-AT proteins (and possibly other
transcription factors). The greater magnitude of MCIP1 induction by
calcineurin when new protein synthesis is blocked is potentially
attributable to abrogation of calcineurin-dependent induction of
endogenous MCIP1 synthesis, thereby eliminating negative feedback that
otherwise would restrain calcineurin activity.
Thyroid Hormone Induces Expression of MCIP2 but
Not MCIP1
The gene encoding MCIP2 was identified originally in a
subtractive cloning experiment designed to identify genes that are
upregulated by thyroid hormone in cultured human
fibroblasts.28 To determine
whether MCIP genes are regulated by thyroid hormone in hearts of intact
animals, hyperthyroidism was induced in wild-type mice by
intraperitoneal injection of T3 for 10 days. As noted
previously,32 T3-treated
hearts were uniformly hypertrophic (mean heart weight=180 mg versus 130
mg; mean heart weight/body weight ratio=7.2 mg/g versus 4.9 mg/g; n=4
animals in each group). In contrast to the effects of activated
calcineurin in the murine heart
(Figure 1), the expression of MCIP1 was unaltered in
hyperthyroid hearts
(Figure 3). However, MCIP2 transcript levels were increased
2-fold in both heart and soleus skeletal muscles of T3-treated mice
(Figure 3 and
Table 1B). It remains to be determined whether the effects
of T3 are a direct consequence of nuclear receptor binding to
regulatory elements of the MCIP2 gene or a result of indirect
mechanisms.
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An Intragenic Region Located 5' to Exon 4 of
the MCIP1 Gene Is Sufficient to Promote a Transcriptional Response to
Calcineurin
The human MCIP1 gene (annotated initially as DSCR1) was
reported to express four variant mRNAs with each of four alternative
exons incorporated selectively at the 5' terminus of the expressed
transcripts.34 The majority
of these transcripts were identified to represent isoforms that include
sequences encoded either by exon 1 or exon
4.34 These variants have
unique 5' UTR regions and encode proteins that differ within the first
29 amino acids. The remaining 168 residues of MCIP1, encoded by exons 5
to 7, are identical in all MCIP1 variants
(Figure 4A). In our experiments on hearts of transgenic mice,
we determined that expression of the exon 4 variant of MCIP1 mRNA was
particularly sensitive to calcineurin activity. The increased abundance
of MCIP1 mRNA detected by a probe complementary to the 3' UTR, which is
included within all variants of MCIP1
(Figure 4A), was mirrored by the increase detected with a
probe complementary only to unique exon 4 sequences
(Figure 4B). In contrast, MCIP1 transcripts that include exon
1 sequences were present only at the limit of detection in wild-type
murine hearts and were not induced by the activated calcineurin
transgene (not shown).
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The selectively increased expression of the exon 4 variant
of MCIP1 mRNA suggested the possibility of alternative promoter use as
a function of calcineurin activation, and we sought to determine
whether transcriptional regulatory elements involved in transducing
this signal reside in proximity to exon 4 of the MCIP1 gene.
Accordingly, we isolated a 900-bp genomic segment from this position
(-874 to +30 relative to the first nucleotide of exon 4). This region
was found to contain a remarkably dense cluster of consensus NF-AT
binding motifs
(T/AGGAAANA/T/C)35
(Figure 4A). A reporter plasmid was constructed to link this
MCIP1 genomic region to a luciferase reporter gene
(Figure 4C), and this construct was tested for its ability to
respond to calcineurin after transfection into C2C12 cells. Like the
endogenous MCIP1 gene, expression of this transgene is increased by
activated calcineurin
(Figure 4D), which has no effect on a control plasmid
(minimal TATA plus luciferase; not shown). Inhibition of calcineurin
activity by concomitant overexpression of MCIP1 represses this response
(not shown). Luciferase reporter plasmids controlled by shorter
segments of this genomic region 5' to exon 4 (-231 to +30 or -163
to +30;
Figure 4C) retain basal activity equivalent to the -874 to
+30 segment but progressively lose calcineurin responsiveness as the
number of NF-AT binding sites is reduced
(Figure 4D).
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Discussion |
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A major finding of this study is that the genes encoding the calcineurin-interacting proteins MCIP1 and MCIP2 are regulated selectively in skeletal and cardiac myocytes by calcineurin and thyroid hormone, respectively. Both genes are expressed in striated myocytes, and both proteins are capable of inhibiting the enzymatic activity of calcineurin. However, only MCIP1 is induced by calcineurin and only MCIP2 by thyroid hormone. Regulatory responses of this nature have not been reported for genes encoding other proteins (Cabin/Cain, CHP) that function as endogenous inhibitors of calcineurin.
The induction of MCIP1 expression by calcineurin in striated
myocytes is rapid and robust. An increased abundance of MCIP1 mRNA is
detected within 4 hours after activation of calcineurin by calcium
influx into cultured C2C12 myoblasts or myotubes. A microarray analysis
capable of screening 7832 independent genes identified MCIP1 as one of
the genes most markedly upregulated in hearts of transgenic mice
engineered to express a constitutively active form of calcineurin.
Among several variants of MCIP1 mRNA that arise by alternative promoter
use and/or alternative splicing, transcripts including sequences
encoded by exon 4 were found to be induced by calcineurin. This
response was recapitulated by a plasmid construct that linked a
900-bp intragenic region located 5' to exon 4 to a luciferase
reporter gene. It is possible that other regions of the MCIP1 gene
contribute to its transcriptional regulation, but the exceptionally
dense clustering of NF-AT binding motifs upstream of exon 4 is likely
to mediate the potent response to calcineurin signaling. Serial
deletions of this promoter region lose responsiveness to calcineurin in
proportion to the number of NF-AT binding sites that are
removed.
These findings with respect to MCIP1 gene regulation by
calcineurin, in concert with the ability of MCIP1 to inhibit the
enzymatic activity of
calcineurin,24 support the
concept that MCIP1 functions in a negative feedback circuit
(Figure 5) to limit potentially deleterious consequences of
otherwise unhindered calcineurin signaling, such as
apoptosis.36 Our experiments
using cycloheximide show that new protein synthesis is not required for
induction of MCIP1 transcription by calcineurin, in keeping with a
mechanism based on posttranslational modification of NF-AT
(Figure 5). Moreover, the magnitude of MCIP1 induction by
calcineurin is increased in the presence of cycloheximide, as would be
predicted if newly synthesized MCIP1 negatively regulates calcineurin
activity. Findings recently reported by other
laboratories25 26
also are consistent with the notion of negative feedback on calcineurin
by MCIP proteins. It remains to be determined, however, whether
inhibition of calcineurin is the sole function of MCIP1 in mammalian
cells. The MCIP orthologue of
Saccharomyces cerevisiae
(Rcn1p) inhibits calcineurin signaling at high concentrations, but
calcineurin signaling is impaired if Rcn1p is absent. This latter
observation suggests that basal levels of Rcn1p function in some manner
to facilitate calcineurin signaling. Loss-of-function experiments based
on targeted disruption of each of the three MCIP genes will be critical
to determine whether this is also true for MCIP proteins in mammalian
cells.
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The responsiveness of endogenous MCIP1 gene expression to calcineurin activity in cultured cells and in tissues of intact animals, and the ability of the exon 4 flanking region to recapitulate this response when placed into a reporter plasmid, potentially can be exploited as sensitive indicators of calcineurin activity. As noted in recent reviews,37 current methods used to assess the activation state of calcineurin in vivo are subject to problematical artifacts. It may be possible to redress this deficiency by designing assays based on the peculiar responsiveness of MCIP1 to calcineurin activation. Such assays could aid in the interpretation of experiments that seek to modify calcineurin signaling in animal models or be used in high-throughput screens for discovery of new chemical agents that alter calcineurin signaling.
The ability of T3 to induce expression of MCIP2 in mammalian striated muscles was anticipated on the basis of experiments in which a cDNA encoded by the human ZAKI-4 gene (here termed MCIP2) was identified in a screen for transcripts upregulated by treatment of human fibroblasts with thyroid hormone.28 In the present study, we confirm that this response occurs in skeletal and cardiac muscles, and we provide new information to show that such regulation by T3 does not extend to the MCIP1 gene. In addition, we demonstrate that, unlike MCIP1, MCIP2 is not subject to regulation by calcineurin signaling. It will be important in future studies to ascertain whether the induction of MCIP2 by thyroid hormone and the ensuing inhibition of calcineurin activity that should result from this response are pertinent to any of the consequences of hyperthyroidism that affect skeletal and cardiac muscles. For example, inhibition of calcineurin activity in skeletal muscles by cyclosporin A promotes transformation of slow myofibers to the fast fiber phenotype.10 13 Excess T3 can induce a similar transformation of myofiber subtypes, as well as myosin isoform switching in the heart.38 39 It is plausible to propose that T3-induced accumulation of the calcineurin inhibitory protein MCIP2 may contribute to these effects.
Our observation that, unlike MCIP2, expression of MCIP1 is not induced in hearts of thyrotoxic mice has additional implications. Unchanged levels of MCIP1 mRNA in hypertrophic hearts of T3-treated animals suggests that the induction of MCIP1 produced by expression of an activated calcineurin transgene is a direct consequence of calcineurin activity, rather than a uniform feature of all forms of cardiac hypertrophy. Moreover, on the premise that expression of MCIP1 provides an indicator of the state of activation of the calcineurin signaling pathway, normal levels of MCIP1 in hypertrophic hearts of T3-treated animals can be taken as evidence that calcineurin is less pertinent to the mechanisms of cardiac hypertrophy driven by T3, compared with other hypertrophic stimuli.40 In future studies, measurements of MCIP1 mRNA concentrations and studies of the effects of forced expression of MCIP1 in many different models of cardiac hypertrophy may help to distinguish calcineurin-dependent from calcineurin-independent pathways that control cardiac mass.
Calcineurin plays an important biological role in many different types of cells and tissues. Activating signals arising from calcineurin are directed to a large and diverse set of target genes, distinctive subsets of which are selected in different cell types and on the basis of the parallel activation of other signaling pathways. It is not surprising that such a potent signaling molecule as calcineurin, subject to activation by a great diversity of primary stimuli that generate appropriate waveforms of intracellular calcium, is also subject to negative regulation by a diversity of inhibitory processes. Because of their prominent expression in cardiac and skeletal muscles, MCIP proteins are of particular interest in this regard. The distinctive responses of genes encoding MCIP1 and MCIP2 to different regulatory stimuli reveal an additional level of complexity with respect to our understanding of calcineurin-dependent signaling in mammalian cells. Further studies of MCIP proteins may lead to the development of new measures to modulate calcineurin activity selectively in cardiac or skeletal muscles for experimental, and possibly clinical, purposes.
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Acknowledgments |
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This work was supported by grants from the National Institutes of Health (NIH) (to E.N.O and R.S.W.), the D.W. Reynolds Foundation (to E.N.O and R.S.W.), the Robert A. Welch Foundation (to E.N.O.), and Myogen, Inc (to E.N.O.). N.F. was supported by a fellowship of the Deutsche Forschungsgemeinschaft. T.A.M. is a Pfizer Fellow of the Life Sciences Research Foundation. J.Y. and R.V. were supported by an NIH training grant. We are grateful to John Shelton for preparing images of primary data for publication.
Received October 24, 2000; accepted November 9, 2000.
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