Birt-Hogg-Dube syndrome is a novel ciliopathy

Abstract

Birt-Hogg-Dubé (BHD) syndrome is an autosomal dominant disorder where patients are predisposed to kidney cancer, lung and kidney cysts and benign skin tumors. BHD is caused by heterozygous mutations affecting folliculin (FLCN), a conserved protein that is considered a tumor suppressor. Previous research has uncovered multiple roles for FLCN in cellular physiology, yet it remains unclear how these translate to BHD lesions. Since BHD manifests hallmark characteristics of ciliopathies, we speculated that FLCN might also have a ciliary role. Our data indicate that FLCN localizes to motile and non-motile cilia, centrosomes and the mitotic spindle. Alteration of FLCN levels can cause changes to the onset of ciliogenesis, without abrogating it. In three-dimensional culture, abnormal expression of FLCN disrupts polarized growth of kidney cells and deregulates canonical Wnt signalling. Our findings further suggest that BHD-causing FLCN mutants may retain partial functionality. Thus, several BHD symptoms may be due to abnormal levels of FLCN rather than its complete loss and accordingly, we show expression of mutant FLCN in a BHD-associated renal carcinoma. We propose that BHD is a novel ciliopathy, its symptoms at least partly due to abnormal ciliogenesis and canonical Wnt signalling.

Figure 1.

BHD syndrome is associated with development of renal cysts. (A) CT scan of a BHD patient. Coronal plane. Arrows indicate cysts in liver and kidney. (B) Paraffin-embedded samples were obtained from a renal carcinoma from a BHD patient with a c.499C>T mutation (encoding pGln167X). Immunohistochemical staining with custom-made C terminal FLCN antibody revealed FLCN around kidney tubules and within the tumor. Highlighted area around the kidney cyst is shown in (C). Magnification ×50. Scale bar is 400 µm. (C) Magnification highlighted area B. Magnification ×400. (D) H&E stain of highlighted area B. Magnification 400×. (E) H&E stain (composite of three images) of a cyst from Nihon Rat kidney tissue. H&E, magnification ×50. Scale bar is 100 µm. (F) H&E stain (composite of four images) of a tumor from Nihon Rat kidney tissue. Magnification ×50. Scale bar is 100 µm. (G) H&E stain (composite of six images) of a cyst from Nihon Rat kidney tissue. There are clear cysts containing two distinct populations of cells. The first population with cuboidal morphology (arrow), and the second with an eosinophilic cytoplasm that protruded to into the cyst lumen (arrowhead). Magnification ×50. Scale bar is 100 µm.

Figure 2.

FLCN is a constitutive ciliary, basal body and centrosomal protein. In this figure, all endogenous FLCN stains were obtained using the FLCN AP antibody. (A) FLCN localizes to primary cilia. HK-2 cells serum starved for 48 h were fixed and stained for endogenous FLCN (green) and acetylated alpha tubulin (red). Nuclei were stained with DAPI (blue). Cilium in the white box is shown in orthogonal view below. Scale bar is 10 µm. (B) Endogenous FLCN localizes to the centrosome. IMCD3 cells were fixed and stained for gamma-tubulin (red) and endogenous FLCN (green). Nuclei are stained with DAPI (blue). Scale bar is 10 µm. (C) EGFP-FLCN WT localizes to the centrosome. MDCK cells stably expressing EGFP-tagged FLCN WT were fixed and counterstained with anti-gamma-tubulin (red) to mark the centrosomes (indicated by arrows). EGFP-FLCN WT localizes at centrosome before (B i, scale bar is 20 µm) and after centrosome duplication (B ii, scale bar is 10 µm). Nuclei are stained with DAPI (blue). (D) FLCN localizes to motile cilia. (i) Single stain. Primary HNE cells were fixed and stained for endogenous FLCN (green). Middle panel is DIC image for visualization of cilia (indicated by the arrow). Scale bar is 20 µm. (ii) Double stained. HNE cells were fixed and stained for endogenous FLCN (red) and acetylated alpha tubulin (green). Scale bar is 10 µm. (E) FLCN localizes to the basal body. HNE cells were fixed and stained for endogenous FLCN (red) and pericentrin (green) to mark the base plate. Scale bar is 20 µm. (F) In human sperm endogenous FLCN localizes to the centriole and annulus. Human sperm cells were fixed and stained for endogenous FLCN (red). Note the staining at the centriole (arrowhead) and annulus (arrow). Scale bar is 10 µm. (G) FLCN localizes to the mitotic spindle. HK-2 cells overexpressing EGFP-FLCN WT were fixed and counterstained with acetylated alpha tubulin (red) to mark the mitotic spindles (indicated by arrows) and DAPI (blue). Scale bar is 20 µm.

Figure 3.

The BHD-derived renal cancer cell line UOK257 shows abnormal ciliation. (A) UOK257 and UOK257-2 cells were maintained at confluence for indicated amount of days, fixed and stained with acetylated alpha tubulin to mark the ciliary axoneme and pericentrin to mark the ciliary base. Nuclei were stained with DAPI and the percentage of ciliated cells was counted using ImageJ. After 6 days of confluence, UOK257 had a significantly reduced number of cilia compared with UOK257-2 (P = 0.023). After 10 days of confluence, both cell lines have a similar number of cilia (P = 0.422). Error bars represent standard error of the mean. A minimum of 365 cells were counted per cell line per time point. *P < 0.05. (B) Sequencing of genomic DNA isolated from HK-2 and UOK257 cells showing the presence of the wild-type allele (starting sequence indicated by the arrow).

Figure 4.

Knockdown and exogenous expression of FLCN affects ciliogenesis in kidney cells. (A) Western blot of knockdown and control cells. Western blot on 30 µg of WCL showing stable knockdown of FLCN using shRNA in HK-2 clone FLCN KD compared with wild-type HK-2 and HK-2 with scrambled control shRNA clone NT (also see Supplementary Material, Fig. S6). Membrane was probed with FLCN AP antibody. Actin was used as a loading control. (B) Measurement of ciliogenesis in FLCN knockdown versus non target control. HK-2 FLCN KD and NT cells were serum starved for the indicated amount of hours, fixed and stained with acetylated alpha tubulin to mark the ciliary axoneme and pericentrin to mark the ciliary base. Nuclei were stained with DAPI and the percentage of ciliated cells and cilium length was measured using ImageJ. After 48 h (P < 0.001) and 72 h (P < 0.001) of serum starvation, HK-2 FLCN KD cells showed a statistically significant reduction in the percentage of ciliated cells compared with NT cells. After 96 h (P = 0.915) and 120 h (P = 0.067) of serum starvation, there was no difference in the percentage of ciliated cells between the two cell lines. Error bars represent standard error of the mean. Average of 350 cells counted per cell line per time point. *P < 0.05. (C) Measurement of cilia length in FLCN knockdown versus non-target control. Average cilium length of HK-2 FLCN KD cells (n = 40) was significantly decreased compared with cilia of NT cells (n = 124) after 48 h of serum starvation (P = 0.001). After 72 h of serum starvation, no difference in cilia length was observed between HK-2 FLCN KD (n = 24) and NT cells (n = 55) (P = 0.55). Error bars represent standard error of the mean. *P < 0.05. (D) Western blot of FLCN expressing cell lines. Western blot probed with EGFP antibody showing MDCK cells with stable expression of EGFP alone, EGFP-FLCN WT and the disease-causing FLCN missense variant EGFP-FLCN K508R. Actin was used as a loading control. Actin is not detected in the EGFP-only MDCK cells, as a result of 10-fold dilution of this sample due to very high EGFP expression in this cell line. (E) Growth curve in FLCN expressing cell lines. At a starting density of 10⁵ cells per well, untransfected MDCK cells and MDCK cells with stable expression of EGFP, EGFP-FLCN WT or EGFP-FLCN K508R were plated. Cell number was determined at each time point. Each data point is the average of six independent counts. Error bars represent standard error of the mean. (F) Measurement of ciliogenesis in FLCN expressing cell lines. Untransfected MDCK (MDCK) and MDCK cells stably expression EGFP, EGFP-FLCN WT (WT) or EGFP-FLCN K508R (K508R) were maintained at confluence for 5 days, fixed and stained with acetylated alpha tubulin to mark the ciliary axoneme and with DAPI to stain nuclei. Cells expressing EGFP-FLCN WT showed a statistically significant reduction in the percentage of ciliated cells compared with cells expressing EGFP alone (P < 0.001) or EGFP-FLCN K508R (P < 0.001), as counted using ImageJ. Error bars represent standard error of the mean. *P < 0.05.

Figure 5.

Knockdown or expression of exogenous FLCN impairs kidney cell spheroid growth in three-dimensional culture. (A) Knockdown of FLCN impairs kidney cell spheroid growth in three-dimensional culture. (i) IMCD3 cells were treated with non-targeting RNAi (siCON) or FLCN RNAi (siFlcn) for 48 h. The cells were subsequently split into two equal parts, with the first half cultured for 3 days in matrigel and the remainder in 2D culture for quantification of knockdown shown in (ii). Cultures were fixed and stained. Tight-junction marker ZO-1 (orange) localizes to the apical side, β-catenin (green) is enriched at lateral cell–cell contacts and acetylated alpha tubulin stains cilia (red) that are present protruding into the lumen. Nuclei are stained with DAPI. Representative spheroids are shown. SiCON cells form a typical spheroid that is evenly rounded and has a clear lumen. Cells treated with SiFlcn form spheroids with impaired ciliogenesis, reduced overall and luminal sizes. Arrows indicate cilia containing β-catenin. Scale bar is 10 µm. (ii) Quantification of Flcn levels using RT-PCR in IMCD3 cells treated with siCON or siFlcn (normalized to Rpl19). The RNA was extracted from cells at the same time point as the 3D spheroids shown in (i) were fixed. (iii) Quantification of the number of cilia observed in non-target siCON or the siFlcn spheroids. Spheroids with reduced Flcn levels exhibit significantly decreased numbers of cilia compared with the siCON control (P > 0.0001). (B) Knockdown of FLCN in IMCD3 decreases spheroid and lumen size in three-dimensional culture. IMCD3 cells with siRNA-mediated knockdown of Flcn form spheroids that consist of the same number of cells (i) but are significantly smaller (ii) in size (P = 0.0058) and have a smaller lumen (ii, P = 0.0003) compared with control spheroids. (C) Knockdown of FLCN impairs spindle orientation in three-dimensional culture. (i) Representative image of misoriented, dividing cell and a cell that ended up outside the normal plane (white arrows) in siFLCN spheroid versus control. Cells are stained with DAPI. Scale bar 20 µm. (ii) Quantification of cell division orientation in IMCD3 spheroids treated with SiFlcn, which show a significant increase in the number of misorientated spindles per spheroid compared with control spheroids (P < 0.0001). (D) Expression of wild-type but not K508R FLCN impairs kidney cell spheroid growth in three-dimensional culture. MDCK cells stably expression EGFP, EGFP-FLCN WT or EGFP-FLCN K508R were grown in collagen for 11 days. Cultures were fixed and stained with DAPI (blue). Note the lack of lumen formation in the cells expression EGFP-FLCN WT in the 2D and 3D projection. Scale bar is 20 µm. (E) Quantification of spheroid parameters with exogenous FLCN expression in IMCD3. (i) IMCD3 cells with stable expression of EGFP-FLCN WT form spheroids that are significantly smaller in size (P < 0.0001) and have a smaller lumen (P < 0.0001) compared with control spheroids expressing EGFP only. (ii) Expression of EGFP-FLCN K508R results in normal-sized spheroids (P = 0.2381) with a slight reduction in lumen size (P < 0.0001) compared with control spheroids expressing EGFP only.

Figure 6.

FLCN knockdown causes activation of canonical Wnt signalling. (A) Knockdown of FLCN results in activation of β-catenin. Western blot on 30 µg of WCL from treated IMCD3 cells in 2D culture. Cells were harvested at 60 h post transfection. Cells were serum starved for 18 h prior to harvesting. IMCD3 express equal amounts of total β-catenin (middle panel) upon treatment with non-targeting SiCON or SiFlcn or treatment with 20 mm KCl or LiCl for 16 h. Using an antibody to detect the unphosphorylated, active β-catenin, cells treated with LiCl (an activator of β-catenin through inhibition of GSK3β) showed increased active β-catenin as did SiFlcn treated cells (upper panel). The β-tubulin (lower panel) was used as a loading control. (B) Activation of downstream targets of the Wnt pathway upon FLCN knockdown. The activation of the Wnt pathway was further confirmed using quantitative RT-PCR of downstream target genes Axin2 and CyclinD1. Upon Flcn knockdown, mRNA levels of both genes exhibited up-regulation.