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Proc Natl Acad Sci U S A. May 9, 2006; 103(19): 7327–7332.
Published online Apr 28, 2006. doi:  10.1073/pnas.0509340103
PMCID: PMC1464340
Cell Biology

Modulatory calcineurin-interacting proteins 1 and 2 function as calcineurin facilitators in vivo

Abstract

The calcium-activated phosphatase calcineurin is regulated by a binding cofactor known as modulatory calcineurin-interacting protein (MCIP) in yeast up through mammals. The physiologic function of MCIP remains an area of ongoing investigation, because both positive and negative calcineurin regulatory effects have been reported. Here we disrupted the mcip1 and mcip2 genes in the mouse and provide multiple lines of evidence that endogenous MCIP functions as a calcineurin facilitator in vivo. Mouse embryonic fibroblasts deficient in both mcip1/2 showed impaired activation of nuclear factor of activated T cells (NFAT), suggesting that MCIP is required for efficient calcineurin–NFAT coupling. Mice deficient in mcip1/2 showed a dramatic impairment in cardiac hypertrophy induced by pressure overload, neuroendocrine stimulation, or exercise, similar to mice lacking calcineurin Aβ. Moreover, simultaneous deletion of calcineurin Aβ in the mcip1/2-null background did not rescue impaired hypertrophic growth after pressure overload. Slow/oxidative fiber-type switching in skeletal muscle after exercise stimulation was also impaired in mcip1/2 mice, similar to calcineurin Aβ-null mice. Moreover, CD4+ T cells from mcip1/2-null mice showed enhanced apoptosis that was further increased by loss of calcineurin Aβ. Finally, mcip1/2-null mice displayed a neurologic phenotype that was similar to calcineurin Aβ-null mice, such as increased locomotor activity and impaired working memory. Thus, a loss-of-function analysis suggests that MCIPs serve either a permissive or facilitative function for calcineurin–NFAT signaling in vivo.

Keywords: heart, PP2B, signal transduction

Calcineurin is a serine/threonine-specific protein phosphatase that is uniquely activated by sustained elevations in intracellular calcium (1, 2). Calcineurin is composed of a 59- to 63-kDa catalytic subunit [calcineurin A (CnA)] and a 19-kDa calcium-binding subunit [calcineurin B (CnB)]. Three mammalian CnA catalytic genes (α, β, and γ) and two CnB regulatory genes (B1 and B2) have been identified in vertebrates. Once activated, calcineurin directly dephosphorylates nuclear factor of activated T cells (NFAT) transcription factors within the cytoplasm, promoting their translocation into the nucleus (1). Calcineurin–NFAT signaling is critically involved in regulating the cardiac hypertrophic growth response and cellular protection after ischemia in the heart (35). In T cells, calcineurin–NFAT signaling regulates cytokine gene expression and cellular activation after antigen challenge (1, 6). Indeed, genetic deletion of CnAβ leads to severe defects in T cell maturation and activation (7). In skeletal muscle, calcineurin promotes cellular differentiation and controls fiber-type switching between a slow/oxidative and a fast/glycolytic program (810). In the central nervous system, calcineurin–NFAT signaling regulates different aspects of brain function such as synaptic plasticity, neurotransmission, activity-dependent gene expression, and memory (1114). For example, forebrain-specific deletion of calcineurin increased locomotor activity and reduced specific aspects of memory function (11, 12).

Although calmodulin complexed with Ca2+ is the only known activator of calcineurin, a wide range of inhibitory proteins has been described. One such inhibitor, known as modulatory calcineurin-interacting protein (MCIP), or Down Syndrome Critical Region 1 (DSCR1) and DSCR1-like proteins/calcipressins, is composed of a gene family that includes mcip1, mcip2, and mcip3 (15). When overexpressed, MCIPs inhibit calcineurin activity both in vitro and in vivo by direct binding to the active site, hence blocking NFAT activation (16). However, in yeast and Cryptococcus neoformans, the MCIP homologue, RCN1/CBP1, has functional and phenotypic characteristics of a calcineurin activator (1719). Consistent with this observation, mcip1−/− mice showed an impaired cardiac hypertrophic response to pressure overload, suggesting that it may also function as a calcineurin facilitator in vivo (20). However, under other conditions or in other mammalian cell types, endogenous MCIP can mediate an inhibitory function (21).

Results

Generation and Characterization of mcip1−/− and mcip2−/− Mice.

Many studies conducted in mammalian model systems have concluded that MCIPs function as negative regulators of calcineurin activity. However, some experiments in lower organisms have suggested that these proteins might also selectively augment calcineurin signaling (15, 17, 18). To elucidate the function of MCIP proteins in vivo the genes encoding mcip1 and mcip2 were targeted by homologous recombination in embryonic stem cells. Exons 5 and 6 were deleted from mcip1, whereas exons 2 and 3 were deleted from mcip2 (Fig. 1A and B). mcip1−/− and mcip2−/−, as well as mcip1/2 double-null mice (2KO) mice, were viable and able to breed as adults. However, mcip1−/− mice demonstrated a ≈10% reduction in body weight as adults and a reduction in fertility as they aged (data not shown). Protein extracts were generated from heart, skeletal muscle, thymus, and brain for Western blotting with MCIP1-, MCIP2-, and MCIP3-specific polyclonal antisera from WT and 2KO mice (Fig. 1C). The data demonstrate that MCIP1 and MCIP2 protein is present in heart, skeletal muscle, and brain of WT mice, but not in 2KO mice. MCIP3 protein was not detected in heart and skeletal muscle. Surprisingly, the brain expresses the most MCIP1/2 protein, in contrast to reported levels of mRNA distribution (22, 23). There was no compensatory alteration in CnA or CnB protein levels in the absence of mcip1 and mcip2 (Fig. 1C and data not shown).

Fig. 1.
Generation of mcip1 and mcip2 gene-targeted mice. (A and B) Schematic representation of the mcip1 and mcip2 genetic loci and the exons that were targeted for replacement with neomycin (Neo) in embryonic stem cells. The position of the relevant restriction ...

Loss of MCIP1/2 Attenuates Calcineurin–NFAT Signaling in 2KO Mouse Embryonic Fibroblasts (MEFs).

As described, even subtle overexpression of MCIP1 can inhibit calcineurin–NFAT signaling. Here we used an NFAT-dependent luciferase reporter and thapsigargin stimulation to increase intracellular Ca2+ in WT MEFs (Fig. 2A). However, to evaluate the effect associated with loss of endogenous MCIPs on calcineurin–NFAT signaling, MEFs were generated from mcip1−/−, mcip2−/−, and 2KO embryos and compared with identically generated WT MEFs (Fig. 2B). The data demonstrate a significant reduction in NFAT transcriptional activity at 4 h in mcip1−/−, mcip2−/−, and 2KO MEFs, which was also observed at 2 and 6 h (Fig. 2B and data not shown). However, by 8 and 12 h, only mcip1−/− and 2KO MEFs showed reduced NFAT transcriptional activity to increased Ca2+ compared with WT MEFs (Fig. 2B). Similarly, an adenovirus encoding NFATc1-GFP was infected in WT and 2KO MEFs for analysis of acute nuclear translocation after thapsigargin and angiotensin II (Ang II) stimulation. Once again, 2KO MEFs demonstrated reduced calcineurin–NFAT signaling characterized by less efficient nuclear occupancy/translocation in 2KO MEFs versus WT MEFs at 30 min and 2 h (Fig. 2 C and D). Consistent with these results, Western blotting for the migration rates of total NFATc1 protein showed less dephosphorylation (dephosphorylation increases migration) after thapsigargin stimulation in 2KO compared with WT MEFs (Fig. 2E). There was no compensatory alteration in CnA or CnB proteins levels in 2KO MEFs versus WT MEFs (Fig. 2F). Assessment of calcineurin activation through its association with calmodulin after thapsigargin stimulation was not altered in 2KO MEFs versus WT MEFs (Fig. 2G). Thus, loss of mcip1/2 in fibroblasts only compromises NFAT activation.

Fig. 2.
Assessment of calcineurin function in 2KO MEFs. (A) NFAT transcriptional activity in WT MEFs infected with the NFAT-dependent luciferase reporter adenovirus and increasing concentration of AdMCIP1 at baseline or in response to thapsigargin (2 μM) ...

MCIP1/2 Facilitate Calcineurin in the Heart.

Deletion of CnAβ in the mouse severely compromises the ability of the heart to hypertrophy in response to pressure overload and neuroendocrine stimulation (4). More recently, Olson and colleagues (20) deleted the mcip1 gene in the mouse, which also showed reduced hypertrophy after pressure overload stimulation, suggesting the possibility that endogenous levels of MCIP might have a stimulatory interaction with calcineurin. Here we analyzed mice null for mcip1 and mcip2 and 2KOs, as well as mice null for mcip1/2 × CnAβ (3KO). Baseline evaluation of 2KO mice at 2 months or 1 year of age showed no phenotypic manifestations of cardiac disease or dysfunction as assessed by echocardiography and histology (Table 1 and data not shown). Two-month-old mcip1−/−, mcip2−/−, and 2KO mice were subjected to pressure overload stimulation by transverse aortic constriction (TAC) for 2 weeks and compared with age- and strain-matched WT mice (Fig. 3A). Remarkably, each single null and the 2KO mouse showed significantly less increase in heart weight normalized to tibia length (HW/TL) in response to pressure overload stimulation (Fig. 3A). Analysis of myocyte area from histological sections revealed a similar profile of inhibited hypertrophy in the absence of mcip1/2 (Fig. 3B). Analysis of cardiac function by echocardiography over 4 weeks of pressure overload stimulation showed a partial protection from pressure overload-induced decompensation in the absence of mcip1/2 compared with WT mice (Fig. 3C), consistent with a similar profile of attenuated decompensation in the absence of CnAβ, WT mice treated with cyclosporine A, or MCIP1 overexpressing transgenic mice (5, 24, 25). Interestingly, 2KO mice also did not hypertrophy in response to 2 weeks of phenylephrine plus Ang II infusion by osmotic minipump or after 3 weeks of forced exercise by swimming (Fig. 3 D and E). Myocardial damage after ischemia-reperfusion injury was also increased in mcip1−/− and 2KO mice, similar to our previous observations in CnAβ−/− mice (Fig. 3F). However, loss of mcip2 did not increase the infarction area after ischemia-reperfusion injury, suggesting that MCIP1 and MCIP2 function differently in this stress response.

Fig. 3.
Assessment of MCIP1/2 function in the heart. (A) HW/TL in the indicated groups of mice 2 weeks after a sham or TAC surgical procedure. n = 6–16 mice per group; *, P < 0.05 versus WT sham. (B) Cardiomyocyte area in the indicated ...
Table 1.
Echocardiography in WT and 2KO mice at 2 months or 1 year

Although the individual or combined loss of mcip1/2 renders the heart largely resistant to cardiac hypertrophy, a slightly more complex regulatory paradigm was suggested by incorporation of the activated CnA transgene into the mcip1−/− and mcip2−/− backgrounds. At 3 weeks of age, the activated CnA transgene induced a nearly 2-fold increase in HW/TL in mcip1/2+/+ mice, a level of cardiac hypertrophy that was not altered by deletion of mcip2 (Fig. 3H). However, loss of mcip1 induced a significantly greater increase in cardiac hypertrophy associated with the activated CnA transgene, suggesting that mcip1 functioned as a repressor in the presence of the activated CnA transgene (Fig. 3H). This dichotomous result is likely due to the observation that endogenous levels of MCIP1 are massively augmented by the activated CnA transgene in the heart, rendering MCIP1 inhibitory toward calcineurin similar to MCIP1 overexpression by transgenesis (Fig. 3G). In addition, pressure overload and phenylephrine plus Ang II infusion did not detectably increase MCIP1 protein levels in the heart, suggesting that, although calcineurin is activated by these stimuli, it is not of sufficient magnitude to increase MCIP1 protein expression to inhibitory levels (data not shown). Finally, we also crossed both the mcip1- and mcip2-null alleles into the CnAβ-null background. If endogenous MCIP functions as a calcineurin inhibitor, crossing into the CnAβ-null background should partially rescue the attenuated profile of hypertrophy after pressure overload associated with the remaining CnAα gene. However, the data demonstrate that 3KO have a similar profile of inhibited hypertrophy compared with 2KO and CnAβ-null mice (Fig. 3I). Collectively, these results further suggest that MCIPs serve to facilitate calcineurin functionality in vivo.

MCIP1/2 Facilitate Calcineurin in Skeletal Muscle and T Cells.

We have previously shown that loss of CnAβ or CnB from skeletal muscle severely reduces baseline and exercise-induced slow/oxidative fiber-type switching (10, 26). Given this known function of calcineurin in skeletal muscle, we compared the phenotype of 2KO mice for induction of the slow/oxidative program after 3 weeks of forced swimming. WT mice showed a significant increase in type I myosin heavy chain after exercise, a hallmark of the slow/oxidative fiber-type program, yet 2KO mice showed no switching (Fig. 4A). Quantitation of these results from six mice per group confirmed lack of fiber-type switching in 2KO mice (Fig. 4B). These results are consistent with the hypothesis that MCIPs can facilitate calcineurin function in skeletal muscle.

Fig. 4.
Assessment of MCIP function in skeletal muscle and T cells. (A) Western blotting for type I myosin heavy chain (MyHC I) and GAPDH in WT and 2KO mice at rest or after swimming. (B) Quantitation of MyHC I protein levels in the gastrocnemius in three independent ...

Loss of CnAβ and CnB in lymphocytes compromises their activation and/or development (6, 7). To further compare the phenotype of CnAβ−/− mice and mcip1/2-null mice, lymphocyte development and function were evaluated. FACS of splenocytes from 6-week-old mice demonstrated no reduction in B cells or CD3-, CD4-, or CD8-positive cells in mcip1−/−, mcip2−/− or 2KO mice (Fig. 4C and Table 2). These results are also consistent with previous observations made in mcip1−/− mice (21). However, consistent with our previous observations in CnAβ−/− mice, T cell development was compromised in the 3KO mice (Fig. 3C and Table 2). To evaluate T cell activation, CD4+ T cells were purified and stimulated with CD3/CD28 antibody for assessment of proliferation rates and cytokine production. Loss of mcip1−/−, mcip2−/−, or both genes together did not reduce CD4+ T cell activation, IL-4, or IFN-γ production (data not shown). However, phorbol 12-myristate 13-acetate/ionomycin stimulation of total splenocytes from 2KO, CnAβ−/−, and 3KO mice showed reduced proliferation and IL-2 production compared with WT (data not shown). Loss of mcip1 was previously shown to increase CD4+ T cell apoptosis (21). Similarly, here we identified increased CD4+ T cell annexin V labeling at baseline or after CD3/CD28 stimulation in mcip1−/−, mcip2−/−, 2KO, and 3KO mice (Fig. 4D). Importantly, loss of CnAβ did not rescue the increase in annexin V labeling associated with mcip1/2 deletion but instead significantly increased it (Fig. 4D). This latter result further supports the hypothesis that MCIPs are permissive for calcineurin signaling in vivo (see Discussion).

Table 2.
Quantitation of lymphocyte numbers from the spleens of the indicated genotypes

MCIP1/2 Facilitate Calcineurin in the Brain.

Targeted deletion of CnB1 in the brain resulted in a phenotype of hyperactivity and working memory (short-term memory) deficits in mice (11, 12). However, overexpression of activated calcineurin in the brain led to a distinct phenotype whereby only long-term memory was affected (27). To further associate the role of MCIPs in the brain with the known effects of calcineurin inhibition, behavioral and activity analyses were performed in 2KO mice. Consistent with CnB1-deleted mice, 2KO mice showed a significant increase in cumulative activity during light and dark conditions (Fig. 5A). CnAβ−/− mice showed an even greater increase in total locomotor activity, although this effect was not reduced in 3KO mice (Fig. 5A). Total contacts with an exercise wheel were also dramatically increased in 2KO mice compared with WT mice (Fig. 5B). Also of interest, 2KO mice showed faster overall movements in an open field test compared with WT mice (Fig. 5 C and D). As a control, 2KO mice did not show an alteration in their percentage of crossings in an open field test or time spent in the corners, suggesting that anxiety/stress levels were not altered in 2KO versus WT mice (Fig. 5E and data not shown). Finally, 2KO mice showed a subtle but significant impairment in working memory compared with WT mice, as assessed by novel and familiar object recognition analysis at 24 h (Fig. 5F). However, 2KO mice did not show a defect in long-term memory (data not shown). Thus, loss of mcip1/2 in the brain also appears to render a phenotype that resembles reduced calcineurin activation, further suggesting that MCIP proteins facilitate calcineurin signaling in vivo.

Fig. 5.
Assessment of MCIP function in the brain. (A) Total cumulative motor activity during the dark and light phase over 24 h in the indicated genotypes of cage-housed mice (n = six mice per group). (B) Total number of running wheel contacts over 3–4 ...

Discussion

Here we presented multiple lines of evidence suggesting that the physiologic function of MCIP is to facilitate, or at least permit, calcineurin signaling in vivo. In heart, brain, skeletal muscle, and T cells, loss of mcip1/2 in mice led to a phenotype similar to mice lacking CnAβ, whereas the combined deletion of mcip1/2 with CnAβ did not suppress the 2KO phenotype in any of the tissues examined. MEFs null for mcip1, mcip2, or 2KO demonstrated a clear profile of reduced calcineurin–NFAT coupling. One unique caveat to these observations is that mcip2 deletion reduced calcineurin-NFAT signaling only at early time points after thapsigargin stimulation, whereas at later time points, MCIP1 appeared to be more important, suggesting some functional differences between MCIP1 and MCIP2. It is also important to note that loss of mcip1/2 did not alter calcineurin activity per se or its association with calmodulin, and in fact, overexpression of MCIP1 always served an inhibitory function in vivo and in vitro. Hence, it remains uncertain exactly how endogenous MCIPs facilitate calcineurin activity toward target proteins, although inhibitor-2 and DARPP-32 may play a similar role in permitting PP1 activity through an unknown mechanism (28, 29).

Many previous reports implicating MCIP as a calcineurin inhibitor have relied on overexpression approaches (17, 2123, 25, 3034). However, there is ample precedent in the literature that overexpression of a kinase or phosphatase docking/activating protein can actually have an inhibitory function based on a sequestration effect. For example, overexpression of the c-Jun N-terminal kinase (JNK)-interacting protein (JIP1) results in JNK inhibition, although endogenous levels of JIP1 normally function in specifically localizing and enhancing JNK activity within a cell (35). A kinase anchoring protein 79 (AKAP79), which docks protein kinase A, protein kinase C, and calcineurin to facilitate their selective and coordinated activity for localized substrates, can also function as a repressor when overexpressed (36, 37). Even more germane endogenous levels of inhibitor-2 protein (yeast homologue Glc8) were shown to function as facilitators of protein phosphatase 1 (PP1), although overexpression of inhibitor-2 blocks PP1 activity (28, 38). Phosphorylation of inhibitor-2 can alter its inhibitory characteristics toward PP1, not unlike the ability of specific kinases to alter MCIP1 function toward calcineurin by phosphorylation (19, 39). All these observations are particularly interesting given the relative conservation between the calcineurin docking domains in MCIP1 and NFAT proteins versus the docking domain in PP1 for inhibitor-2 and other interacting proteins (40). Deletion of rcn1 in yeast (MCIP homologue) produced a phenotype that was similar to Rcn1 overexpression, suggesting that both states inhibited calcineurin and that the true physiologic function of Rcn1 in vivo may be to facilitate calcineurin activity toward substrates (17). In support of this hypothesis, C. neoformans mutant for cbp1 (MCIP homologue) displays a phenotype that is similar to, albeit less severe than, calcineurin mutants (18). Finally, two separate reports have examined the effect of mcip1 deletion in the mouse. Olson and colleagues (20) observed depressed cardiac hypertrophy in mcip1-null mice after pressure overload stimulation or isoproterenol infusion, consistent with the hypothesis that MCIP1 is required to facilitate calcineurin signaling. McKeon and colleagues (21) reported that CD4+ T cells from mcip1-null mice had an increase in the ratio of dephosphorylated to phosphorylated NFATc2 and reduced IL-2 production after T cell receptor stimulation at specific dosages of CD3 antibody, suggesting that MCIP1 might serve an inhibitory role under certain conditions in these cells. In support of this result, partial reduction of MCIP1 protein using an antisense RNA approach increased NFAT activity and inflammatory response gene expression in endothelial cells (41).

Although the functional role that we have proposed for MCIPs would appear to contradict results from McKeon and colleagues (21), there are many points of similarity in the data. Similar to McKeon and colleagues, we also observed an increase in apoptosis rates in mcip1/2-null CD4+ T cells after CD3 stimulation and no obvious defects in T cell development. However, the most significant difference between our studies is related only to interpretation of data, because loss of CnAβ in the mcip1/2-null background did not rescue the phenotype, consistent with the proposal that MCIPs permit/facilitate calcineurin activity. By comparison, McKeon and colleagues (21) observed that cyclosporine A rescued the increase in cell death associated with mcip1 deletion, although cyclosporine A can also block cell death through a calcineurin-independent function involving inhibition of mitochondrial permeability transition.

Although CnAβ−/− mice had a phenotype similar to 2KO mice in the heart, skeletal muscle, brain, and T cells, there were some minor differences at baseline. 2KO mice showed no differences in CD4 or CD8 T cell numbers in the spleen or thymus (data not shown), yet CnAβ−/− mice were previously shown to have a substantial reduction in both T cell populations at baseline (7). These results indicate that loss of mcip1/2 does not impact T cell development the same as loss of CnAβ, possibly suggesting that MCIPs regulate at different times in T cell maturation or that MCIP facilitates only a select range of calcineurin targets. Similarly, loss of mcip1/2 did not affect basal type I myosin heavy chain protein expression in skeletal muscle (only exercise-induced expression), whereas CnAβ−/− and CnB1-null mice each showed dramatic reductions in basal expression (10, 26). With respect to the brain, loss of CnAβ−/− produced a more pronounced increase in cumulative locomotor activity compared with a significant, albeit more modest, effect in 2KO mice. This observation suggests that loss of mcip1/2 does not fully compromise calcineurin activation under all conditions or toward all targets. A similar conclusion was proposed based on the observations in C. neoformans whereby calcineurin mutants had a more severe phenotype than did cbp1 mutants (18). The results observed in mammals also need to be qualified by two additional considerations. Mammals contain a third mcip gene (mcip3) that is also expressed in brain and lymphocytes and could contribute to residual calcineurin target facilitation in the absence of mcip1/2 (42). MCIPs have also been reported to interact with proteins other than calcineurin, suggesting the possibility of calcineurin-independent effects. For example, MCIP1 was shown to immunoprecipitate with integrins and Raf-1 (43, 44). Thus, MCIPs are likely to function as calcineurin facilitators through direct and indirect mechanisms.

Methods

Generation of mcip1−/− and mcip2−/− Mice.

A more extensive description of the targeting strategy and strain background, as well as many other procedures, is given in Supporting Text, which is published as supporting information on the PNAS web site.

Western Blotting and Immunoprecipitation.

Generation of protein samples from tissue and cultured cells, along with Western blotting and chemifluorescent detection, was performed as described (4, 7, 45). Calmodulin immunoprecipitation for calcineurin binding in MEF protein extracts (500 μg) was performed as described (46).

Cell Culture.

MEFs were isolated at embryonic day 12.5 from 2KO or WT matings, as described (47). Cultures were harvested 24 h after recombinant adenoviral infection, and luciferase assays were performed as described (48).

Cardiac Hypertrophy Measurements in Vivo.

See Supporting Text.

Murine TAC, Agonist Infusion, Exercise, and Ischemia/Reperfusion Protocols.

Performed as described (48, 49) and see Supporting Text.

Preparation of Lymphocytes.

See Supporting Text.

Activity and Memory Assessment.

Home cage activity was measured via tse (TSE Systems, Midland, MI) dual array of infrared photo beams, using tse software to assess gross movement and rearing behaviors, as described (50). Additional tests are described in Supporting Text.

Supplementary Material

Supporting Text:

Acknowledgments

This work was supported by grants from the National Institutes of Health and by an American Heart Association Established Investigator grant (to J.D.M.).

Abbreviations

2KO
mcip1/2 double-null mice
3KO
mcip1/2 ×CnAβ triple-null mice
Ang II
angiotensin II
CnA
calcineurin A subunit
CnB
calcineurin B subunit
HW/TL
heart weight/tibia length
IL-2
interleukin-2
MCIP
modulatory calcineurin-interacting protein
MEF
mouse embryonic fibroblast
NFAT
nuclear factor of activated T cells
TAC
transverse aortic constriction
PP1
protein phosphatase 1

Footnotes

Conflict of interest statement: No conflicts declared.

This paper was submitted directly (Track II) to the PNAS office.

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