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. 2004 Apr;15(4):1833-42.
doi: 10.1091/mbc.e03-10-0730. Epub 2004 Feb 6.

Calcineurin regulates cyclin D1 accumulation in growth-stimulated fibroblasts

Christina R Kahl  1 , Anthony R Means
Affiliations

Calcineurin regulates cyclin D1 accumulation in growth-stimulated fibroblasts

Christina R Kahl et al. Mol Biol Cell. 2004 Apr.

Abstract

Calcium (Ca(2+)) and calmodulin (CaM) are required for progression of mammalian cells from quiescence into S phase. In multiple cell types, cyclosporin A causes a G(1) cell cycle arrest, implicating the serine/threonine phosphatase calcineurin as one Ca(2+)/CaM-dependent enzyme required for G(1) transit. Here, we show, in diploid human fibroblasts, that cyclosporin A arrested cells in G(1) before cyclin D/cdk4 complex activation and retinoblastoma hyperphosphorylation. This arrest occurred in early G(1) with low levels of cyclin D1 protein. Because cyclin D1 mRNA was induced normally in the cyclosporin A-treated cells, we analyzed the half-life of cyclin D1 in the presence of cyclosporin A and found no difference from control cells. However, cyclosporin A treatment dramatically reduced cyclin D1 protein synthesis. Although these pharmacological experiments suggested that calcineurin regulates cyclin D1 synthesis, we evaluated the effects of overexpression of activated calcineurin on cyclin D1 synthesis. In contrast to the reduction of cyclin D1 with cyclosporin A, ectopic expression of calcium/calmodulin-independent calcineurin promoted synthesis of cyclin D1 during G(1) progression. Therefore, calcineurin is a Ca(2+)/CaM-dependent target that regulates cyclin D1 accumulation in G(1).

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Figures

Figure 1.
Figure 1.
Synchronous cell cycle reentry of WI-38 fibroblasts. (A) DNA profiles of reentry. WI-38 fibroblasts were growth arrested in low serum and then, stimulated to reenter the cell cycle by the readdition of growth media. Cells were harvested at several times after serum addition (T = 0 to T = 24), fixed, stained with propidium iodide, and analyzed by fluorescence-activated cell sorting analysis. Each histogram plots cell count versus DNA content. In the histogram, the first peak represents cells in G0/G1 with 2N DNA content and the second peak represents cells in G2/M with 4N DNA content. Cells traversing S phase are between the two peaks with DNA content ranging from 2N to 4N. (B) Cyclin and calcineurin expression during reentry. Cell lysates were separated by SDS-PAGE and analyzed for the expression of the cyclins (D1, E, and A) and calcineurin A by Western blotting.
Figure 2.
Figure 2.
Cell cycle inhibition in G1 by cyclosporin A. (A) DNA profiles of reentry with W-13 and cyclosporin A. Serum-starved WI-38 cells were stimulated with growth media in the presence of the vehicle dimethyl sulfoxide, 15 μg/ml W-13, or 25 μM cyclosporin A. Cells were harvested at 18 h after serum addition and analyzed by fluorescence-activated cell sorting analysis (FACS), with each histogram plotting cell count versus DNA content. (B) DNA profiles of release from S phase. WI-38 cells were arrested in early S phase with hydroxyurea and then released into fresh media. Then, cells were harvested at 6 and 12 h for FACS analysis. (C) DNA profiles of release from M phase. WI-38 cells were arrested in M phase with nocodazole and then released into fresh media. Then, cells were harvested at increasing times after the removal of nocodazole for FACS analysis. (D) DNA profiles of release from the cyclosporin A G1 arrest. Serum-starved WI-38 cells were stimulated with growth media in the presence of cyclosporin A, followed by release into fresh media. Cells were harvested at 12 and 20 h for FACS analysis. (E) Time course of cyclosporin A addition. Serum-stimulated WI-38 cells, treated with cyclosporin A, were pulse labeled with BrdU for 30 min and harvested at 18 h after serum addition. Cells were counted at random for BrdU incorporation, and S phase percentage was determined by dividing the number of BrdU-positive cells by the total number of cells, as determined by DAPI nuclear staining.
Figure 3.
Figure 3.
Inhibition of pRb phosphorylation and cyclin D1 accumulation by cyclosporin A. (A) Cdk2 IP kinase assays. Serum-starved WI-38 cells were stimulated with growth media in the presence or absence of cyclosporin A and then harvested at 16 and 20 h. Cdk2 complexes were immunoprecipitated from cell lysates followed by an in vitro kinase assay with histone H1 as a substrate. (B) Cdk4 IP kinase assays. For cdk4 assays, cells were harvested at 18 h and then cdk4 complexes were immunoprecipitated and assayed for activity by using GST-pRb CT as a substrate. As a measure of nonspecific activity in the immunoprecipitation, extracts were immunoprecipitated in the presence of the cdk4 peptide (used to generate the immunoprecipitating cdk4 antibody) that prevents cdk4 immunoprecipitation and demonstrates minimal activity against the GST-pRb substrate. (C) pRb Western analysis. WI-38 lysates were separated by SDS-PAGE, and pRb was detected by Western blotting. Hypophosphorylated pRb migrates as a single band, whereas hyperphosphorylated pRb migrates with a reduced mobility shift. (D) Cyclin D1 and cdk4 Western analyses. The expression of cyclin D1 and cdk4 were determined at 18 h after serum addition by Western blotting. (E) Time course of cyclin D1 mRNA accumulation. Equal amounts of total RNA (15 μg) from cells at 1.5 and 4 h after serum addition were subject to Northern analysis by using a radiolabeled probe from mouse cyclin D1. (F) Cyclin D1 mRNA accumulation with cyclosporin A. Equal amounts of total RNA from cells at 4 h after serum stimulation, with and without cyclosporin A, were subject to Northern analysis for cyclin D1 expression.
Figure 4.
Figure 4.
Reduction of cyclin D1 synthesis by cyclosporin A. (A) Cyclin D1 labeling in the presence of cyclosporin A. Serum-stimulated WI-38 cells, in the presence or absence of cyclosporin A, were pulse labeled with 35S-EasyTag methionine/cystine (PerkinElmer Life Sciences) for 2 h between 6 and 8 h after serum addition. Endogenous cyclin D1 was immunoprecipitated, followed by separation of proteins by SDS-PAGE. (B) Half-life determination of cyclin D1. After the 2-h labeling, the media were removed and replaced with fresh media containing methionine/cystine for 40 min. PhosphorImager analysis was used to quantify the amount of 35S-labeled cyclin D1, and the results are graphed as percentage of remaining versus time. CsA represents cells in the presence of cyclosporin A, and No Tx represents cells in the absence of cyclosporin A. (C) Inhibition of cyclin D1 synthesis by cyclosporin A. The amount of labeled cyclin D1 was determined in three independent experiments, with the cyclosporin A-treated samples expressed as a percentage of untreated samples, which was set to 100%. (D) Cyclin D1 labeling in the presence of MG-132. WI-38 cells were pulse labeled as described above in the presence of 10 μM MG-132. Cyclin D1 immunoprecipitates were separated by SDS-PAGE.
Figure 5.
Figure 5.
Promotion of cyclin D1 synthesis by expression of Ca2+/CaM-independent calcineurin A. (A) Coexpression of calcineurin A and calcineurin B. Subconfluent WI-38 cells were infected with Ad-calcineurin A (wild-type, 1-397, and H151Q), with and without coinfection of Ad-calcineurin B, at multiplicities of infection of 100. Western analysis was performed using anti-calcineurin A and anti-calcineurin B. (B) Calcineurin A expression during reentry. Serum-starved WI-38 cells were infected with Ad-calcineurin A (1-397, H151Q) in the presence of Ad-calcineurin B. After 18 additional hours of serum starvation, cells were released into growth media and harvested at increasing times. Calcineurin A expression was determined by Western blotting. (C) Cyclin D1 labeling in the presence of calcineurin A overexpression. Serum-starved WI-38 cells were infected with either Ad-GFP or Ad-calcineurin A 1-397, both in the presence of Ad-calcineurin B. Cells were serum stimulated and pulse-labeled with 35S-EasyTag methionine/cystine as described in text. (D) Promotion of cyclin D1 synthesis by calcineurin A overexpression. The amount of 35S-labeled cyclin D1 was determined for three independent experiments.
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