Serine/threonine protein phosphatase 5 (PP5) participates in the regulation of glucocorticoid receptor nucleocytoplasmic shuttling

Abstract

Background: In most cells glucocorticoid receptors (GR) reside predominantly in the cytoplasm. Upon hormone binding, the GR translocates into the nucleus, where the hormone-activated GR-complex regulates the transcription of GR-responsive genes. Serine/threonine protein phosphatase type 5 (PP5) associates with the GR-heat-shock protein-90 complex, and the suppression of PP5 expression with ISIS 15534 stimulates the activity of GR-responsive reporter plasmids, without affecting the binding of hormone to the GR.

Results: To further characterize the mechanism by which PP5 affects GR-induced gene expression, we employed immunofluorescence microscopy to track the movement of a GR-green fluorescent fusion protein (GR-GFP) that retained hormone binding, nuclear translocation activity and specific DNA binding activity, but is incapable of transactivation. In the absence of glucocorticoids, GR-GFP localized mainly in the cytoplasm. Treatment with dexamethasone results in the efficient translocation of GR-GFPs into the nucleus. The nuclear accumulation of GR-GFP, without the addition of glucocorticoids, was also observed when the expression of PP5 was suppressed by treatment with ISIS 15534. In contrast, ISIS 15534 treatment had no apparent effect on calcium induced nuclear translocation of NFAT-GFP.

Conclusion: These studies suggest that PP5 participates in the regulation of glucocorticoid receptor nucleocytoplasmic shuttling, and that the GR-induced transcriptional activity observed when the expression of PP5 is suppressed by treatment with ISIS 15534 results from the nuclear accumulation of GR in a form that is capable of binding DNA yet still requires agonist to elicit maximal transcriptional activation.

Figure 1

Inhibition of PP5 by okadaic acid. The purified catalytic subunit of PP5 was assayed, using [³²P]labeled phosphohistone as a substrate as described in Materials and Methods. The data is expressed as % of controls, with control activity being 4.2 ± 0.25 μmole/min/mg protein. Okadaic acid was mixed with the enzymes for 10 min at 23°C prior to the initiation of the reaction with the addition of substrate. Inhibition assays contained ~ 200 pM PP5. The data represent the mean SD (n = 4).

Figure 2

Effects of ISIS 15534 and dexamethasone on the subcellular distribution of GR-GFP.A. Representative subcellular distribution of GR-GFP. A549 cells were grown and treated with ISIS 15521 (a mismatched control antisense oligonucleotide for ISIS 1535), ISIS 15534 (antisense targeting PP5), dexamethasone (500 nM), ISIS 15534 and 500 nM dexamethasone, or vehicle alone (control) as described in Material and Methods. Eighteen hours later, GR-GFP expressing plasmids were microinjected into the nuclei of cells, incubated for 6 hours in serum-containing medium, and then serum-starved for an additional 18 hours. Between 150 and 200 cells were injected for each condition. The cells were either left untreated (control, ISIS 15521, ISIS 15534) or incubated with 500 nM dexamethasone (dexamethasone, ISIS + dexamethasone) for 30 minutes, prior to fixing and observation by fluorescence microscopy. Representative cells from 4 independent experiments are shown. B. Percentage of cells displaying nuclear GR-GFP. Cells from 4 independent experiments were scored for their intracellular distribution of GR-GFP and the mean percentage of cells having greater fluorescence in their nuclei than cytoplasm are shown (± SD). ^*p ≤ 0.0001 by ANOVA vs untreated or mismatch-treated cells.

Figure 3

Nucleo-cytoplasmic shuttling of GFP-NFAT.A) GFP-NFAT expressing HeLa cells were grown on glass coverslips and either left untreated (control) or treated with ISIS 15534 or the mismatched control (ISIS 15521) as described in Materials and Methods. Twenty-four hours after oligonucleotide treatment, the cells were either directly fixed with paraformaldehyde (No ionomycin), or ionomycin was added to 1 μM for 30 minutes. After treatment with ionomycin, the cells were either fixed (+ ionomycin) or rinsed in PBS and grown for an additional 6 hours in medium in the absence of drug (washout) before fixing. Representative fields of cells were viewed by fluorescence microscopy and photographed. B). Northern analysis of HeLa cells following treatment with 500 nM ISIS 15534, 500 nM ISIS 155521 or lipofectin alone (control), probing for PP5 and GAPDH confirms that the antisense oligonucleotides are also effective HeLa cells.