FKBP12, the 12-kDa FK506-binding protein, is a physiologic regulator of the cell cycle

Abstract

FKBP12, the 12-kDa FK506-binding protein, is a ubiquitous abundant protein that acts as a receptor for the immunosuppressant drug FK506, binds tightly to intracellular calcium release channels and to the transforming growth factor beta (TGF-beta) type I receptor. We now demonstrate that cells from FKBP12-deficient (FKBP12(-/-)) mice manifest cell cycle arrest in G(1) phase and that these cells can be rescued by FKBP12 transfection. This arrest is mediated by marked augmentation of p21(WAF1/CIP1) levels, which cannot be further augmented by TGF-beta1. The p21 up-regulation and cell cycle arrest derive from the overactivity of TGF-beta receptor signaling, which is normally inhibited by FKBP12. Cell cycle arrest is prevented by transfection with a dominant-negative TGF-beta receptor construct. TGF-beta receptor signaling to gene expression can be mediated by SMAD, p38, and ERK/MAP kinase (extracellular signal-regulated kinase/mitogen-activated protein kinase) pathways. SMAD signaling is down-regulated in FKBP12(-/-) cells. Inhibition of ERK/MAP kinase fails to affect p21 up-regulation. By contrast, activated phosphorylated p38 is markedly augmented in FKBP12(-/-) cells and the p21 up-regulation is prevented by an inhibitor of p38. Thus, FKBP12 is a physiologic regulator of cell cycle acting by normally down-regulating TGF-beta receptor signaling.

Figure 1

FKBP12^−/− fibroblasts are arrested in G₁ phase of the cell cycle. (A) Cell growth. For FKBP12^+/+ or FKBP12^−/− cells, 5 × 10⁴ cells were plated in six-well dishes and counted in 24-h intervals by hematocytometry. Trypan blue dye was used to count viable cells. Data points are the mean ± SEM from three experiments. (B) Cyclin D1 staining. (Left) Nuclear localization of cyclin D1 was observed in more than 80% of FKBP12^−/− cells. (Right) The bar graph presents the mean ± SEM from three experiments, with at least 200 cells counted in 10 fields for each experiment (*, P < 0.001). (C) Cell cycle progression in FKBP12^−/− cells stimulated with bFGF (25 ng/ml) was monitored by using cyclin D1 (G₁ phase) and PCNA (S phase) as markers. In FKBP12^+/+ cells, a 24-h bFGF treatment stimulates expression of cyclin D1 4-fold (bar b is different from bar a, P < 0.001) and expression of PCNA 3.5-fold (bar d is different from bar c, P < 0.001), indicating an increase in cells in S phase. In FKBP12^−/− cells, cyclin D1 is stimulated 5-fold (bar d different from bar c, P < 0.001) with only a modest increase in PCNA, indicating G₁-phase blockade and failure to progress to S phase. Values are the mean ± SEM of five determinations.

Figure 2

FKBP12 transfection reverses the G₁ arrest of FKBP12^−/− fibroblasts. Hemagglutinin (HA)-tagged FKBP12 was transfected into FKBP12^−/− cells and nuclear localization of cyclin D1 was monitored. At least 200 transfected or untransfected cells in 10 fields were counted in three experiments with mean ± SEM reported in the bar graph. As a control green fluorescent protein (GFP) was transfected in FKBP12^−/− cells and the transfected GFP cells were also counted.

Figure 3

Increased p21(WAF1/CIP1) expression in FKBP12^−/− cells. (A) TGF-β1 and bFGF treatments for 24 h induce p21 protein in FKBP12^+/+ cells (bars b–d are different from bar a, P < 0.01) and their effects are additive (bar d is different from bars b and c, P < 0.01). In FKBP12^−/− cells, p21 protein is greatly augmented (bar e is different from bar a, P < 0.001), and TGF-β1 does not elicit any further increase although bFGF does augment p21 (bar g is different from bar e, P < 0.05). Data are the mean ± SEM of five experiments. (B) p21 mRNA is augmented in FKBP12^−/− fibroblasts. p21 mRNA was monitored by real-time PCR and RT-PCR; RNA was extracted from FKBP12^+/+ and FKBP12^−/− cells. p21 mRNA was 8-fold higher in FKBP12^−/− cells. TGF-β1 treatment (3 h) induced p21 in FKBP12^+/+ cells but not in FKBP12^−/− cells. Data are representative of data from three experiments, whose results varied less than 15%. (C) PAI-1 mRNA is augmented in FKBP12^−/− fibroblasts. PAI-1 mRNA was monitored by real-time PCR and RT-PCR; RNA was extracted from FKBP12^+/+ and FKBP12^−/− cells. PAI-1 mRNA was 2.5-fold higher in FKBP12^−/− cells than in FKBP12^+/+ cells. However, consistent with previous reports (12, 23), TGF-β1 treatment (3 h) induced PAI-1 in both FKBP12^+/+ cells and FKBP12^−/− cells by 5- and 3-fold, respectively. Data are representative of data from three experiments, whose results varied less than 15%.

Figure 4

Perturbation of TGF-β signaling pathway rescues cells from G₁-phase arrest. FKBP12^−/− cells were transfected with hemagglutinin (HA)-tagged kinase-deficient (K232R) TGF-β RI, which decreased cells in G₁ phase as monitored by nuclear cyclin D1. Values in the bar graph are the mean ± SEM for three experiments, each conducted with at least 200 cells counted in 10 fields.

Figure 5

Overactivation of TGF-β signaling is due to increased p38 MAP kinase signaling. (A) TGF-β1 treatment increases the phosphorylation of SMAD2/3 in FKBP12^+/+ but not FKBP12^−/− cells. FKBP12^+/+ and FKBP12^−/− cells were incubated with [³²P]orthophosphate. Immunoprecipitation with anti-SMAD2/3 was used to determine the amount of ³²P incorporated into SMAD2/3. There was no significant difference in basal SMAD2/3 phosphorylation in the FKBP12^+/+ or FKBP12^−/− cells. TGF-β1 treatment (30 min) significantly increased SMAD2/3 phosphorylation in FKBP12^+/+ but not in FKBP12^−/− cells. (B) TGF-β1 treatment increases nuclear localization of SMAD4 in FKBP12^+/+ but not in FKBP12^−/− cells. After TGF-β1 treatment, nuclear localization of SMAD4 was observed in more than 80% of FKBP12^+/+ cells but no significant increase was seen in FKBP12^−/− cells. (C) SMAD7 mRNA is significantly higher in FKBP12^−/− than FKBP12^+/+ cells. TGF-β1 treatment increased SMAD7 mRNA in FKBP12^+/+ cells almost to the levels of the FKBP12^−/− cells but did not augment the SMAD7 mRNA in the FKBP12^−/− cells. (D) p38 MAP kinase is constitutively phosphorylated in FKBP12^−/− fibroblasts. TGF-β1 treatment (30 min) increased phosphorylation of p38 in FKBP12^+/+ cells; however, p38, in FKBP12^−/− cells, was already extensively phosphorylated and TGF-β1 treatment did not increase phosphorylation. No change in total p38 expression was observed relative to actin or total protein. The experiment was replicated three times. (E) Inhibition of p38 MAP kinase blocks p21 induction in FKBP12^−/− fibroblasts. SB203580 (20 μM, 24 h), an inhibitor of p38 MAP kinase, blocked the TGF-β1 induction of p21 in FKBP12^+/+ cells and reduced p21 in the FKBP12^−/− cells. PD98059 (20 μM, 24 h), an inhibitor working upstream of MAP kinase/Erk kinase, did not affect p21 in FKBP12^−/− cells.

Figure 6

Model depicting FKBP12 regulation of type I TGF-β receptor signaling. (Left) In FKBP12^+/+ cells, TGF-β1 induces gene transcription by SMAD2/3, p38, and ERK MAP kinase leading to induction of p21, SMAD7, PAI-1, and other genes under the influence of TGF-β receptor signaling. (Upper Right) FKBP12^−/− cells are partially activated in a ligand-independent manner leading to augmentation of p21, SMAD7, and p38. (Lower Right) TGF-β1 treatment of the FKBP12^−/− cells cannot further induce or activate these pathways due to feedback influenced by SMAD7. PAI-1 is basally activated in FKBP12^−/− cells and can be further activated by TGF-β1.