The role of the Birt-Hogg-Dubé protein in mTOR activation and renal tumorigenesis

Abstract

Birt-Hogg-Dubé (BHD) syndrome is a tumor-suppressor gene disorder characterized by skin tumors, cystic lung disease and renal cell carcinoma. Very little is known about the molecular pathogenesis of BHD. Clinical similarities between BHD and tuberous sclerosis complex (TSC) suggest that the BHD and TSC proteins may function within a common pathway. The TSC proteins inhibit the activity of the mammalian target of rapamycin complex 1 (TORC1), and in Schizosaccharomyces pombe, Bhd and Tsc1/Tsc2 have opposing roles in the regulation of amino-acid homeostasis. We report here that in mammalian cells, downregulation of BHD reduces the phosphorylation of ribosomal protein S6, an indicator of TORC1 activity. To determine whether folliculin, the product of the BHD gene, regulates mammalian target of rapamycin activity in vivo, we generated a mouse with targeted inactivation of the Bhd gene. The mice developed spontaneous oncocytic cysts and tumors composed of cells that resemble the renal cell carcinomas in BHD patients. The cysts and tumors had low levels of phospho-S6. Taken together, these data indicate that folliculin regulates the activity of TORC1, and suggest a new paradigm in which both inappropriately high and inappropriately low levels of TORC1 activity can be associated with renal tumorigenesis.

Figure 1

Endogenous BHD mRNA levels in tissues and cell lines. (a) Quantitative real-time RT-PCR was used to identify tissues with high levels of BHD mRNA. A panel of normal human tissue RNA (Ambion) was utilized, with each sample being a pool from at least three donors. (b) RNA was prepared from 20 human cancer cell lines derived from kidney, ovary, brain and lung. Quantitative real-time RT-PCR was used to detect BHD mRNA levels. Relative mRNA levels are normalized to a renal cell line, CAKI-1.

Figure 2

BHD downregulation causes a reduction in phospho-S6 levels. (a) U251 cells were treated with TSC2 siRNA, BHD siRNA or control siRNA in 10% FBS for 24 hours, then serum starved for 24 hours (serum -) or grown in 10% FBS for 24 hours (serum +). Blot was probed with anti-TSC2 (tuberin), anti-folliculin mouse mAb, anti-phospho-S6 (Ser235/236), and anti-β-actin for a loading control. (b) HEK293 cells were treated with indicated siRNA in 10% FBS for 48 hours. Cells were either lysed (serum +) or serum starved for 24 additional hours (serum -). Both long and short exposures are shown for phospho-S6. (c) Human kidney proximal tubule epithelial cells (HK-2) were treated with indicated siRNA in 10% FBS for 24 hours. Cells were either lysed (serum 24 hr) or serum starved for 24 additional hours and lysed (serum -) or incubated for 15 minutes with 20% FBS (serum 15 m) to stimulate the mTOR pathway. Both long and short exposures are shown for folliculin and phospho-S6. (d) U251 cells were treated with BHD or control siRNA in 10% FBS for 24 hours. Cells were either lysed (serum 24 hr) or serum starved for 24 additional hours and lysed (serum -) or incubated for 15 minutes of 20% FBS (serum 15 m) to stimulate the mTOR pathway, as indicated. Blot is probed with anti-folliculin rabbit polyclonal and anti-β-actin. Similar results were seen in at least two independent experiments for each cell line shown.

Figure 3

Gene trap disruption of the Bhd gene in mouse embryonic stem (ES) cells. (a) ES cell line RRX115 was produced by Baygenomics. The βgeo cassette (green box) is inserted into intron 8 of the Bhd gene and causes a truncated folliculin-βgalactosidase/neomycin fusion protein. (b) Primers were designed to confirm the presence of the βgeo cassette in processed Bhd mRNA. (c) RT-PCR confirmation of Bhd-βgeo spliced mRNA in the ES cells. Lane 1, Water control (forward primer exon 7, reverse primer βgeo); Lane 2, gene trap ES cell RNA (forward primer exon 7, reverse primer βgeo); Lane 3, normal mouse fibroblast RNA (forward primer exon 7, reverse primer exon 9); Lane 4, myc-BHD plasmid cDNA wild type positive control (forward primer exon 7, reverse primer exon 9).

Figure 4

Bhd heterozygotes develop spontaneous oncocytic cysts and tumors. (a) A typical kidney with small visible cysts (arrows) from a 14-month-old Bhd^+/- mouse is shown next to a kidney from an age matched wild type mouse. (b) An oncocytic tumor and (c) an oncocytic cyst from a 5-month-old Bhd^+/- mouse, 40X magnification. (d,e) A large tumor associated with a cyst from a 13-month-old Bhd^+/- mouse, 10X (d) and 40X (e) magnification. Normal kidney tubules can be seen adjacent to altered tissue in b-d. All panels are hematoxylin and eosin (H&E) stained.

Figure 5

ENU accelerates development of oncocytic cysts and tumors in BHD heterozygotes. Pregnant Bhd^+/- mice were injected with ENU at day E14. Both wild type and Bhd^+/- littermates were sacrificed between 3 and 5 months of age. (a) An example of a non-oncocytic cyst that developed in an ENU injected wild type mouse. (b-e) Examples of oncocytic cysts from 4 different ENU-treated Bhd^+/- mice. (f) A small oncocytic tumor associated with a small cyst from an ENU-treated Bhd^+/- mouse. (g) An oncocytic tumor from an ENU-treated Bhd^+/- mouse. (h-i) Oncocytic cysts from the same mouse shown in (g). All images are H&E stained and at 40X magnification.

Figure 6

Oncocytic cysts and tumors from Bhd^+/- mice have no or weak phospho-S6 levels. (a) Oncocytic cyst lining epithelial cells (white arrow) from a 13-month-old untreated Bhd^+/- mouse were negative for anti-phospho-S6 ribosomal protein (Ser 235/236) staining. (b) Oncocytic cyst lining cells (white arrow) from a 3.5-month-old ENU-treated Bhd^+/- mouse were negative for phospho-S6 staining. (c) Oncocytic cyst lining cells (white arrow) from a second 3.5-month-old ENU-treated Bhd^+/- mouse were negative for phospho-S6 staining. (d,e) Oncocytic tumor cells (black asterisks) from a 13-month-old untreated Bhd^+/- mouse were negative for phospho-S6 staining. (f) Oncocytic tumor cells (black asterisk) from an ENU-treated 3.5-month-old Bhd^+/- mouse were primarily negative for phospho-S6 staining, with a few weakly positive (1+) staining cells. (g,h) Normal kidney tissue from ENU treated Bhd^+/+ mice stained with anti-phospho-S6 ribosomal protein (Ser 235/236). (i) Normal kidney tissue from an ENU treated Bhd^+/+ mouse using phosphate buffered saline in place of phospho-S6 primary antibody. In panels a-d black arrowheads indicate moderately (2+, 3+) phospho-S6 positive normal tubules adjacent to the cysts and tumors in Bhd^+/- mice. Slides were counterstained with hematoxylin. All images shown at 40X magnification.