FLCN regulates transferrin receptor 1 transport and iron homeostasis

Abstract

Birt-Hogg-Dubé (BHD) syndrome is a multiorgan disorder caused by inactivation of the folliculin (FLCN) protein. Previously, we identified FLCN as a binding protein of Rab11A, a key regulator of the endocytic recycling pathway. This finding implies that the abnormal localization of specific proteins whose transport requires the FLCN-Rab11A complex may contribute to BHD. Here, we used human kidney-derived HEK293 cells as a model, and we report that FLCN promotes the binding of Rab11A with transferrin receptor 1 (TfR1), which is required for iron uptake through continuous trafficking between the cell surface and the cytoplasm. Loss of FLCN attenuated the Rab11A-TfR1 interaction, resulting in delayed recycling transport of TfR1. This delay caused an iron deficiency condition that induced hypoxia-inducible factor (HIF) activity, which was reversed by iron supplementation. In a Drosophila model of BHD syndrome, we further demonstrated that the phenotype of BHD mutant larvae was substantially rescued by an iron-rich diet. These findings reveal a conserved function of FLCN in iron metabolism and may help to elucidate the mechanisms driving BHD syndrome.

Keywords: BHD; FLCN; HIF; iron.

Figure 1

Co-IP assays of the protein–protein interactions.A, cells cultured under normal conditions were lysed. The cell lysates were precipitated with monoclonal antibodies against either LAMP1 (negative control) or TfR1, followed by western blotting with the indicated antibodies. B and C, similar assays as described in A, except that cells were deprived of iron by incubation with 100 μM DFO for 12 h. In C, plasmids containing different FLCN-HA constructs were transfected into FLCN^−/− cells to exclude the influence of endogenous FLCN.

Figure 2

FLCN regulates the recycling transport of TfR1 and the uptake of Tf-iron.A, wild-type (WT) and FLCN^−/− cells were labeled with FITC-Tf, and confocal images are displayed. The arrows indicate the putative PRC sites at which Rab11A normally accumulates. Scale bars: 20 μm. B, the intensity of the FITC-Tf signal as shown in A was measured with Nikon confocal software. At least 30 cells from each panel were evaluated. C, cells were stained with calcein-AM and were then incubated with holo-Tf for 3 h. Calcein fluorescence was measured by flow cytometry. Uptake of Tf-iron was indicated by the QIP (ΔMFI). MFI, median fluorescence intensity. ∗p < 0.05; ∗∗p < 0.01.

Figure 3

FLCN regulates the cellular iron pool.A, ferrozine assay of the total cellular iron concentration (n = 3 repeated experiments). B, assay of the labile iron pool by calcein-AM staining. Calcein fluorescence was measured by flow cytometry. FMI, median fluorescence intensity. C, WB showing that FTH expression was decreased by FLCN loss. D, E, and G; RT-PCR analysis of mRNA levels. In E, shN indicates the nonsense shRNA (negative control). In G, FLCN OE indicates FLCN overexpression. F, a brief summary of the published data showing the expression of TfR1 and DMT1 in different types of FLCN-deficient cells. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.

Figure 4

FLCN and Rab11A cooperatively regulate iron homeostasis (RT-PCR analysis).A, Rab11A knockdown (shRab11A) increased the expression of both TfR1 and DMT1, and this effect was reversed by FAC supplementation. B, increasing Rab11A activity by Rab11A-act expression counteracted the effect of FLCN knockdown. The stable cell lines of shFLCN (9) and shRab11A (25), and the Rab11A-act plasmid (active form of Rab11A) (25) have been described before.

Figure 5

The increase in HIF activity upon FLCN loss is due to the iron deficiency condition (RT-PCR analysis).A, the activity of PGC-1α, TFEB, and HIFs was increased under both iron depletion (DFO chelation) and FLCN loss (FLCN−/−) conditions. Note that DFO chelation increased the expression of both iron metabolism genes (TfR1 and DMT1) and HIF target genes more strongly than FLCN deficiency. B and C, iron supplementation (30 μM FAC for 4 h) had no effect on PGC-1α expression (B) or TFEB (C) activity in FLCN^−/− cells. D, The increase in HIF activity in FLCN^−/− cells was reversed by FAC supplementation. E, HIF activity in FLCN^−/− cells was increased further by DFO chelation.

Figure 6

A, representative images of two male Drosophila pupae collected from the same tube of food supplemented with FAC. The mutant (DBHD^−/−) can be distinguished from the heterozygote (+/−) by the white eyes and long bristles (arrows). Note that the mutant has completed metamorphosis. B, quantification of the adults (+/−) and the rescued mutant pupae (−/−) from the same food tubes. The rescue efficiency was calculated as the ratio of the number of DBHD^−/− pupae to 1/2 the number of (+/−) adults. Note that high concentrations of FAC (>0.4 mM) were toxic to the mutants.