Dynamic mitochondrial-nuclear redistribution of the immunophilin FKBP51 is regulated by the PKA signaling pathway to control gene expression during adipocyte differentiation

Abstract

Glucocorticoids play an important role in adipogenesis through the glucocorticoid receptor (GR) that forms a heterocomplex with Hsp90•Hsp70 and one high molecular weight immunophilin, either FKBP51 or FKBP52. When 3T3-L1 preadipocytes are induced to differentiate, FKBP51 expression progressively increases, whereas FKBP52 decreases, and Hsp90, Hsp70, p23 and Cyp40 remain unchanged. Interestingly, FKBP51 rapidly translocates from mitochondria to the nucleus where it is retained upon its interaction with chromatin and the nuclear matrix. FKBP51 nuclear localization is transient, and after 48 hours it cycles back to mitochondria. Importantly, this dynamic FKBP51 mitochondrial-nuclear shuttling depends on PKA signaling, because its inhibition by PKI or knockdown of PKA-cα by siRNA, prevented FKBP51 nuclear translocation induced by IBMX. In addition, the electrophoretic pattern of migration of FKBP51 is altered by treatment of cells with PKI or knockdown of PKA-cα, suggesting that FKBP51 is a PKA substrate. In preadipocytes, FKBP51 colocalizes with PKA-cα in mitochondria. When adipogenesis is triggered, PKA-cα also moves to the nucleus colocalizing with FKBP51 mainly in the nuclear lamina. Moreover, FKBP51 and GR interaction increases when preadipocytes are induced to differentiate. GR transcriptional capacity is reduced when cells are incubated in the presence of IBMX, forskolin or dibutyryl-cAMP, compounds that induced FKBP51 nuclear translocation, but not by a specific activator of EPAC. FKBP51 knockdown facilitates adipogenesis, whereas ectopic expression of FKBP51 blocks adipogenesis. These findings indicate that the dynamic mitochondrial-nuclear shuttling of FKBP51 regulated by PKA may be key in fine-tuning the transcriptional control of GR target genes required for the acquisition of adipocyte phenotype.

Keywords: Adipogenesis; FKBP51; Glucocorticoid receptor; PKA.

Fig. 1.

FKBP51 expression increases during the process of 3T3-L1 preadipocyte differentiation. (A) 3T3-L1 cells were differentiated for the indicated periods of time, cell lysates were obtained and proteins were separated by SDS-PAGE. Immunoblotting was performed with the indicated antibodies. (B) mRNA was isolated from 3T3-L1 at the indicated times post-induction of adipogenesis and analyzed as described in Materials and Methods. Similar results were obtained in three different experiments.

Fig. 2.

FKBP51 rapidly shuttles from mitochondria to the nucleus at the onset of adipogenesis. (A–P) 3T3-L1 cells were grown on coverslips and induced to differentiate for 1 (E–L) or 8 days (M–P), subjected to IIF with the indicated antibodies, and images were analyzed by confocal microscopy. In O and P lipid vesicles in 3T3-L1 adipocytes were stained with LipidTOX. Nuclei were counterstained with DAPI. Results are representative of five independent experiments. Panel p shows a magnification of the boxed area in P. Arrows indicate nucleoli and arrowheads indicate chromocenters. Scale bar: 5 µm. (Q) Total (T), cytosolic (C) and nuclear (N) fractions from 3T3-L1 cells prior (day 0) and 1 or 4 days post-induction of differentiation were resolved by SDS-PAGE, and analyzed by immunoblotting with the indicated antibodies. Bands representing FKBP51 with different electrophoretic migration (bands a–c) are indicated. (R) Total (T), cytosolic (C) and mitochondrial (M) fractions from 3T3-L1 cells prior (day 0) and 1 day post-induction of differentiation were resolved by SDS-PAGE, and analyzed by immunoblotting with the antibodies indicated in Q, and anti-Tom20 as marker for mitochondria. (S) The nuclear fraction from 3T3-L1 cells induced to differentiate for 1 day was incubated in the absence (N) or the presence of alkaline phosphatase (N+AP) as described in the Materials and Methods. The indicated samples and total 3T3-L1 cell lysates (T) were then subjected to western blot analysis. Blots are representative of at least three independent experiments.

Fig. 3.

FKBP51 interacts with chromatin and the nuclear matrix. 3T3-L1 cells grown on coverslips were induced to differentiate with MDI for 24 hours and then fixed (A–D) or subjected to in situ extraction (E–H), in situ extraction followed by RNase (I–L) or DNase treatment (M–P). Cells were then fixed, subjected to IIF with the indicated antibodies and samples analyzed by confocal microscopy. Results are representative of four independent experiments, with an average of 50 cells imaged per experiment. Scale bar: 5 µm.

Fig. 4.

FKBP51 mitochondrial–nuclear shuttling is regulated by cAMP-dependent and steroid hormone signaling. 3T3-L1 cells were grown on coverslips and incubated in the absence (A,B) or the presence of 520 µM IBMX (C,D,G,H) or 1 µM DEXA (E,F,I,J) for the indicated periods of time, and then subjected to IIF with anti-FKBP51. Nuclei were counterstained with DAPI. (K,L) After IBMX treatment cells were grown in the absence of IBMX for another 24 hours prior to IIF. Results are representative of four independent experiments. Scale bar: 5 µm.

Fig. 5.

Knockdown of PKA-cα prevents phosphorylation and nuclear translocation of FKBP51. (A–P) HEK293T cells were transfected with a scrambled siRNA (A–L) or a specific siRNA for PKA-cα (M–P). Two days later cells were subjected to the indicated treatments, and subcellular distribution of FKBP51 and PKA-cα were evaluated by IIF and confocal microscopy. Scale bar: 5 µm. (Q) HEK293T cells transfected with scrambled or specific siRNA for PKA-cα were treated with 520 µM IBMX in the absence or presence of 0.5 µM PKI for 24 hours, and cell lysates were analyzed by immunoblotting with the indicated antibodies. AP: cell lysates from HEK293T cells incubated 24 hours with IBMX were treated with alkaline phosphatase before applying them to the gel. Bands a and b correspond to FKBP51 with different electrophoretic migration. Results are representative of at least three independent experiments.

Fig. 6.

FKBP51 and PKA-cα transiently colocalize in the nuclear lamina at the onset of adipocyte differentiation. (A–T) 3T3-L1 preadipocytes grown on coverslips were induced to differentiate for the indicated periods of time and subcellular localization of PKA-cα and FKBP51 was assessed by IIF and confocal microscopy. Nuclei were counterstained with DAPI. For M, O and P, IIF was performed using anti-lamin B to label the nuclear lamina. Arrowheads in M and N indicate loss of lamin B or FKBP51 in the nuclear rim, respectively. (Q–T) PKA-cα is shown in light blue (Q) to distinguish it from the nucleus stained with DAPI (blue) and the lipid vesicles were stained with LipidTOX (red; S). Scale bars: 2 µm (A–P); 5 µm (Q–T). (U) FKBP51 was immunoprecipitated from 3T3-L1 cells prior to (day 0) and 2 days after induction of differentiation, and immunoprecipitated complexes were resolved by SDS-PAGE and analyzed by immunoblotting with the indicated antibodies. NI, non-immune antibody; I, immune antibody. Results are representative of four independent experiments.

Fig. 7.

PKA-dependent FKBP51 nuclear translocation restrains GR transcriptional capacity. (A–F) 3T3-L1 preadipocytes grown on coverslips were not induced (A; day 0) or induced to differentiate (B: day 1, C–F: day 8), and then subjected to IIF using anti-FKBP51 and anti-GR. Nuclei were counterstained with DAPI. A, B, E and F are merged confocal images of FKBP51 and GR. Scale bar: 10 µm. (G) GR was immunoprecipitated from 3T3-L1 preadipocytes prior and at the indicated time post-induction of adipogenesis. Immunoprecipitated complexes were resolved by SDS-PAGE and analyzed by immunoblotting using the indicated antibodies. (H) HEK293T cells were transiently transfected with MMTV-Luc and RSV-β-galactosidase plasmids in the absence or the presence of 0.01, 0.1 or 1 µg of pCI-Neo-hFKBP51 and the amount of transfected DNA was normalized to that of the empty vector. After transfection cells were cultured for 24 hours in steroid-free medium, and then incubated for 18 hours with 1 µM DEXA in the absence or the presence of 50 and 520 µM IBMX, 15 and 30 µM forskolin (FK), 50 and 500 µM dibutyryl-cAMP (DB) or 5 and 100 µM Me-cAMP (Me). Luciferase activity was measured and normalized to β-galactosidase activity. Each bar represents the mean ± s.e.m. (P<0.01) for five independent experiments. (I–X) 3T3-L1 cells grown on coverslips were treated with vehicle (I–L) or 1 µM DEXA in the absence (M–P) or presence of 520 µM IBMX (Q–T) or 100 µM Me-cAMP (U–X) for 1 hour, and then subjected to IIF with the indicated antibodies. Nuclei were counterstained with DAPI. Results are representative of three independent experiments. Scale bar: 10 µm.

Fig. 8.

FKBP51 restrains differentiation of 3T3-L1 preadipocytes. Plasmids with mock shRNA or shRNA specific for FKBP51 were transfected in 3T3-L1 cells, and 48 hours later cells were induced to differentiate. Adipogenesis was evaluated by IIF using LipidTOX to stain vesicles containing lipids (A,B), and by evaluating mRNA for FKBP51, adiponectin and resistin (C). 3T3-L1 cells were transfected with empty vector or FLAG-FKBP51, and 24 hours later cells were induced to differentiate and adipocyte differentiation was evaluated by Oil Red O staining (D,E), and by detecting mRNA for FKBP51, adiponectin and resistin (F). β-Actin was used as a control of loading in C and F. (G) Fold change in the percentage of cells that differentiated into adipocytes for control cells, and 3T3-L1 cells in which FKBP51 was knocked down (shRNA FKBP51) or overexpressed (Flag-FKBP51), *P<0.01. (H) Model of FKBP51 activity in adipocyte differentiation. In the adipogenic cocktail MDI, IBMX and DEXA are responsible for FKBP51 nuclear translocation. IBMX inhibits the phosphodiesterase (PDE) increasing intracellular cAMP that activates PKA, and induces PKA-cα and mitochondrial FKBP51 nuclear translocation, where they mainly colocalize in the nuclear lamina (NL). In the nucleus, FKBP51 is retained by its interaction with the nuclear matrix (NM) and chromatin, regulating GR-target genes, and possibly other targets.