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. 2008 May 16;283(20):14012-21.
doi: 10.1074/jbc.M709776200. Epub 2008 Mar 3.

Coordination of growth and endoplasmic reticulum stress signaling by regulator of calcineurin 1 (RCAN1), a novel ATF6-inducible gene

Peter J Belmont  1 , Archana Tadimalla, Wenqiong J Chen, Joshua J Martindale, Donna J Thuerauf, Marie Marcinko, Natalie Gude, Mark A Sussman, Christopher C Glembotski
Affiliations

Coordination of growth and endoplasmic reticulum stress signaling by regulator of calcineurin 1 (RCAN1), a novel ATF6-inducible gene

Peter J Belmont et al. J Biol Chem. .

Abstract

Exposing cells to conditions that modulate growth can impair endoplasmic reticulum (ER) protein folding, leading to ER stress and activation of the transcription factor, ATF6. ATF6 binds to ER stress response elements in target genes, inducing expression of proteins that enhance the ER protein folding capacity, which helps overcome the stress and foster survival. To examine the mechanism of ATF6-mediated survival in vivo, we developed a transgenic mouse model that expresses a novel conditionally activated form of ATF6. We previously showed that activating ATF6 protected the hearts of ATF6 transgenic mice from ER stresses. In the present study, transcript profiling identified modulatory calcineurin interacting protein-1 (MCIP1), also known as regulator of calcineurin 1 (RCAN1), as a novel ATF6-inducible gene that encodes a known regulator of calcineurin/nuclear factor of activated T cells (NFAT)-mediated growth and development in many tissues. The ability of ATF6 to induce RCAN1 in vivo was replicated in cultured cardiac myocytes, where adenoviral (AdV)-mediated overexpression of activated ATF6 induced the RCAN1 promoter, up-regulated RCAN1 mRNA, inhibited calcineurin phosphatase activity, and exerted a striking growth modulating effect that was inhibited by RCAN1-targeted small interfering RNA. These results demonstrate that RCAN1 is a novel ATF6 target gene that may coordinate growth and ER stress signaling pathways. By modulating growth, RCAN1 may reduce the need for ER protein folding, thus helping to overcome the stress and enhance survival. Moreover, these results suggest that RCAN1 may also be a novel integrator of growth and ER stress signaling in many other tissues that depend on calcineurin/NFAT signaling for optimal growth and development.

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Figures

FIGURE 1.
FIGURE 1.
Effect of tamoxifen on the ATF6-inducible gene, GRP78, in ATF6-MER transgenic mouse hearts. A, the mechanism of tamoxifen-mediated ATF6 activation and induction of ATF6-inducible ER stress response genes, such as GRP78, in ATF6-MER TG mice is shown. The transcriptional activation domain of ATF6, shown in red, is masked by proteins, such as Hsp90, which bind to the MER fragment in the absence of tamoxifen, rendering the ATF6-MER fusion protein inactive. Tamoxifen displaces these proteins, unmasking the transcriptional activation domain, conferring transcriptional activation to ATF6-MER. B and C, TG mice were treated with vehicle (panel B) or tamoxifen (panel C), as previously described (16), n = 3 mice per treatment, one heart/treatment is shown in this figure. Heart sections were stained for GRP78 protein (green) (1), tropomyosin (red) (2), or treated with TOPRO-3 to identify nuclei (blue) (3). Samples were viewed by laser scanning confocal fluorescence microscopy. Images 1-3 show each stain separately, image 4 shows an enlarged overlay of images 1-3. Image 5 in panel C was acquired from a different section and is shown at higher magnification. All immunofluorescence and confocal microscopy procedures have been previously described (6).
FIGURE 2.
FIGURE 2.
Validation of microarray results using real time quantitative PCR. A, the RNA samples that were used for the microarray analysis were subjected to RT-qPCR to examine the levels of the mRNAs encoded by the GRP78, Erp72, DnaJB11, Hyou1, XBP1, and RCAN1 genes. Shown are the mean ± S.E. for each target gene (n = 3 mouse hearts per treatment). Veh, vehicle; Tx, tamoxifen. B, NRVMC were infected with either AdV-Con or AdV-ATF6 (n = 3 cultures per treatment). 48 h after infection, cultures were extracted and the RNA was subjected to RT-qPCR to examine the levels of mRNA for the same target genes described in panel A. Shown are the mean ± S.E. for each target gene (n = 3 cultures per treatment). * = p ≤ 0.05 different from all other values for each target gene.
FIGURE 3.
FIGURE 3.
Effect of various treatments on RCAN1 mRNA and RCAN1 promoter activity in cultured cardiac myocytes. A, NRVMC were treated ± tunicamycin (TM, 10 μg/ml, bar 2) for 16 h, or subjected to sI for 24 h (bar 3), or sI for 20 h followed by simulated reperfusion for 20 h (sI/R, bar 4); n = 3 cultures for each treatment. sI and sI/R were carried out as previously described (6). RNA was isolated from culture extracts and subjected to RT-qPCR to determine the levels of mRNAs encoded by the GAPDH and RCAN1 genes. The primers used for RCAN1 were designed to amplify a region of exon 4. Shown are the mean values of RCAN1/GAPDH mRNA, expressed as the -fold of control (bar 1) ± S.E. for each treatment (n = 3 cultures per treatment). B, the human RCAN1 gene promoter (-984 to +30 of the region located to the 5′ of exon 4) and a version harboring a mutation in a putative ERSR element located at -329 to -311 (RCAN1-M), were cloned into a luciferase expression construct. NRVMC were transfected with RCAN1-luciferase or RCAN1-M-luciferase and CMV-β-galactosidase and then infected with AdV-Con, AdV-ATF6, or AdV-DN-ATF6, as previously described (6). 24 h after infection, cultures were treated ± TM, as shown and as described in panel A. 16 h later, cultures were extracted and luciferase and β-galactosidase reporter enzyme activities were determined, as previously described (6). AdV-DN-ATF6 alone had no effect on RCAN1-luciferase, or RCAN1-M-luciferase activation (not shown). Shown are the mean relative luciferase (luciferase/β-galactosidase), expressed as the -fold of control (bar 1) ± S.E. for each treatment (n = 3 cultures per treatment). * and §, p ≤ 0.05 different from all other values.
FIGURE 4.
FIGURE 4.
Effect of phenylephrine on calcineurin phosphatase activity, and on ANP and BNP induction. NRVMC were infected with AdV-Con or AdV-ATF6, and 24 h later, cultures were treated ± PE (50 μm) for 48 h. Cultures were then examined for calcineurin activity (panel A), ANP mRNA (panel B), or BNP mRNA (panel C). Shown are the mean values of calcineurin phosphatase activity, or for ANP or BNP mRNA/GAPDH mRNA expressed as the -fold of control (bar 1) ± S.E. for each treatment (n = 3 cultures per treatment). * and §, p ≤ 0.05 different from all other values.
FIGURE 5.
FIGURE 5.
Effect of RCAN1 siRNA and activated ATF6 on ANP and BNP gene induction by phenylephrine. NRVMC were transfected with either control siRNA (Con siRNA) or RCAN1 siRNA, and then infected with AdV-Con or AdV-ATF6. 24 h later, cultures were treated ± phenylephrine (50 μm) for 24 h. Cultures were then extracted and ANP (panel A), or BNP (panel B) mRNA levels were determined by RT-qPCR. Shown are mean values of each target gene/GAPDH mRNA, expressed as the -fold of control (bar 1) ± S.E. for each treatment (n = 3 cultures per treatment). * and §, p ≤ 0.05 different from all other values.
FIGURE 6.
FIGURE 6.
Effect of activated ATF6, RCAN1 siRNA, and phenylephrine on cardiomyocyte area and [3H]leucine incorporation into protein. A-D, NRVMC were transfected with control siRNA or RCAN1 siRNA, infected with AdV-Con or AdV-ATF6, and treated ± PE, as described in the legend to Fig. 5. Cultures were then fixed and immunostained for α-actinin protein (red) and the nuclei were identified using TOPRO-3 (blue), as previously described (6). Fluorescence confocal micrographs show samples of cells after exposure to each treatment. Bar in panel A = 40 μm. E, cell images obtained from micrographs similar to those shown in panels A-C were quantified densitometrically for area, which provides an estimate of cell size. n = at least 250 cells counted for each treatment; each treatment was carried out on 3 different cultures. Shown is the mean of the relative cell area ± S.E., normalized to the control, which was set at 100% (n = 3 cultures per treatment). *, §, and #, p ≤ 0.05 different from all other values. F, cells were cultured and treated as described in panels A-D, except they were incubated with [3H]leucine and the amount of radiolabel incorporated into trichloroacetic acid-precipitable protein was examined, as described under “Experimental Procedures.” Shown is the mean ± S.E. of labeled protein, normalized to the control. n = 3 cultures/treatment/experiment. *, §, and #, p ≤ 0.05 different from all other values.
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