The work of Dr Masaya Baba, at the National Cancer Institute, was highlighted in last week’s blog. Dr Baba was part of the team who identified Folliculin-interacting protein 1 (FNIP1) and a recent paper by this group now suggests a function for the protein (Baba et al., 2012). This new paper ties in well with the study by Park et al. (2012) which was discussed in a previous blog post. Both papers independently identified a similar function for FNIP1 in B cell development.
Baba and authors generated Fnip1 knockout mice and upon inspection of the mice, found them to have a reduced spleen size as compared to heterozygous or wild type controls. After further investigation, the thymocyte numbers and peripheral T cells were found to be normal, but there was seen to be a reduced number of pre-B cells and an accumulation of pro-B cells. These results suggest there is a developmental block at the pro-B cell to pre-B cell stage.
The FLCN-FNIP1 complex is involved in mTOR signalling (Baba et al., 2006) and it was considered whether defects in this pathway were causing the B cell developmental arrest. Unlike Flcn-/- mice, Fnip1-/- mice had no cysts in their kidneys, suggesting the mTOR pathway was unaffected. The phosphorylation of mTOR and its downstream target, ribosomal protein S6, were unchanged between wild type and mutant mice, further suggesting that mTOR signalling is not affected by Fnip1 deletion. Finally, treatment with rapamycin did not bypass the B cell developmental arrest, confirming that the block in B cell development is independent of mTOR signalling.
Subsequent experiments investigated whether the lack of pre-B cells resulted from increased cell death. Fnip1-/- pre-B cells had enhanced caspase activity relative to wild type cells, and also an increase in the number of dead cells as seen by DAPI staining. This suggests that Fnip1 is required for B cell survival and that deletion of Fnip1 leads to apoptosis of precursor B cells. This was confirmed by introducing an anti-apoptotic Bcl2 transgene into Fnip1-/- cells, which bypassed the B cell block and rescued the B cell population.
This study also investigated the effect of Flcn knockout on B cell development. Interestingly, Flcn-/- mice had the same B cell phenotype as the Fnip1-/- mice, suggesting that both Flcn and Fnip1 are required for B cell development. BHD syndrome and FLCN mutations have not previously been associated with B cell defects; however patients with BHD have monoallelic FLCN mutations and so perhaps loss of the second FLCN allele in B cells is required for the developmental defect (as is thought to be the case for the development of RCC in the kidney).
Although the studies by Park et al. and Baba et al. both show B cell developmental defects caused by Fnip1 deletion, the proposed reason for these defects differs. Park et al. suggest Fnip1 deletion causes energy stress which leads to B cell developmental arrest, whereas Baba et al. suggest Fnip1 deletion causes increased apoptosis of pre-B cells. Given that FLCN and FNIP2 have been implicated in MNU-induced apoptosis (Lim et al., 2012 – discussed in this blog post), it would be interesting to investigate this link with apoptosis further. Future work investigating if FNIP2 also has a role in B cell development will be interesting and will shed more light on the FLCN-FNIP complex.
- Baba M, Keller JR, Sun HW, Resch W, Kuchen S, Suh HC, Hasumi H, Hasumi Y, Kieffer-Kwon KR, Gonzalez CG, Hughes RM, Klein ME, Oh HF, Bible P, Southon E, Tessarollo L, Schmidt LS, Linehan WM, Casellas R. The Folliculin-FNIP1 pathway deleted in human Birt-Hogg-Dube syndrome is required for mouse B cell development. Blood. 2012 Jun 18. [Epub ahead of print]. PMID: 22709692
- Park H, Staehling K, Tsang M, Appleby MW, Brunkow ME, Margineantu D, Hockenbery DM, Habib T, Liggitt HD, Carlson G, Iritani BM. Disruption of fnip1 reveals a metabolic checkpoint controlling B lymphocyte development. Immunity. 2012 May 25;36(5):769-81. Epub 2012 May 17. PMID: 22608497
- Baba M, Hong SB, Sharma N, Warren MB, Nickerson ML, Iwamatsu A, Esposito D, Gillette WK, Hopkins RF 3rd, Hartley JL, Furihata M, Oishi S, Zhen W, Burke TR Jr, Linehan WM, Schmidt LS, Zbar B. Folliculin encoded by the BHD gene interacts with a binding protein, FNIP1, and AMPK, and is involved in AMPK and mTOR signaling. Proc Natl Acad Sci U S A. 2006 Oct 17;103(42):15552-7. PMID: 17028174
- Lim TH, Fujikane R, Sano S, Sakagami R, Nakatsu Y, Tsuzuki T, Sekiguchi M, Hidaka M. Activation of AMP-activated protein kinase by MAPO1 and FLCN induces apoptosis triggered by alkylated base mismatch in DNA. DNA Repair (Amst). 2012 Mar 1;11(3):259-66. PMID: 22209521
www.bhdsyndrome.org – the primary online resource for anyone interested in BHD Syndrome.