Birt-Hogg-Dubé (BHD) syndrome is caused by mutations of the folliculin (FLCN) gene. FLCN is a tumour-suppressor protein which associates with two homologous proteins, folliculin-interacting protein (FNIP)1 and FNIP2. Previous studies have reported that the FLCN/FNIP complex acts as a positive regulator of the AMP-activated protein kinase (AMPK) complex (Hasumi et al., 2015), while other studies suggest the opposite (Reyes et al., 2015). To address this issue, a new report from Siggs et al. (2016) uses a new mouse model of FNIP1 deficiency to find that Fnip1 mutation leads to B-cell deficiency and development of cardiomyopathy with gain-of-function mutations in AMPK supporting the idea that FNIP acts as a negative regulator of AMPK.
Using whole-genome sequencing in mouse mutants with an autosomal recessive B-cell deficiency phenotype the authors identified that this was associated with a splice donor variant mutation in Fnip1. By flow cytometry they showed that B cells were absent from the peritoneum and spleen of Fnip1 homozygous mutants. In the bone marrow (BM) there was an absence of IgM+ or IgD+ cells, although B-cell precursors were still present. Heterozygous mutants revealed a reduced frequency of the marginal zone B-cell population. Because the antiapoptotic protein BCL2 is important for B-cell survival the effects of its overexpression on B-cell development in Fnip1 mutants were measured. Unlike previously reported (Baba et al., 2012), BCL2 overexpression only partially restored B-cell values in the BM, peritoneum and spleen. Examination of the hearts of homozygous Fnip1 mutants revealed that they were enlarged due to hypertrophy and that skeletal muscle was darker than heterozygous or wild-type mice. Histology and biochemical analysis revealed that the mutant myocardium had a marked accumulation of glycogen. Homozygous Fnip1 mutant mice also showed high expression of Nppa, Nppb and Acta1, markers of cardiac stress. Due to the known interaction between FNIP1 and AMPK (Baba et al., 2006) and the cardiac phenotype observed, authors assessed the effect of the Fnip1 mutation on cardiac AMPK activity. A basal activation of the γ2 subunit-containing AMPK complexes but reduced AMP responsiveness, in homozygous Fnip1 mutants, was observed in cardiac tissue but not in the liver. This suggests that Fnip1 and Fnip2 may be functionally redundant in the liver as they are in the kidney (Hasumi et al., 2015). One of the roles of AMPK is to promote autophagy via different pathways including the unc-51-like autophagy activating kinase 1 (ULK1) (Gwinn et al., 2008; Egan et al., 2011) which is activated via phosphorylation at serine 555. In the present study, S555 p-ULK1 is higher in Fnip1 mutant BM cells suggesting that autophagy is enhanced. This was supported by the staining of LC3, an autophagosome marker, in Fnip1 mutant B cells. Overexpression of BLC2 reduced LC3 staining in mutant and wild type cells indicating that BLC2 could oppose the enhanced autophagy in Fnip1 mutant B cells while increasing survival (Cash et al., 2011). AMPK promotes cell-cycle arrest by stabilizing p53 (Jones et al., 2005), p53 is known to enhance autophagy and apoptosis in response to cellular stress (Crighton et al., 2006). However, no differences in B-cell numbers were observed between p53-sufficient or deficient Fnip1 mutants in this study. Lack of Fnip1 caused a block in B-cell development and although autophagy was increased, its inhibition, via deletion of the autophagy protein Atg3, failed to rescue the phenotype, suggesting that enhanced autophagy alone is not sufficient to block B-cell development.
In summary, the study supports other reports showing a non-redundant role for FNIP1 in B-cell development (Baba et al., 2012), skeletal muscle position (Reyes et al., 2015) and cardiac function (Hasumi et al., 2015) and it provides evidence that FNIP1 is an endogenous negative regulator of AMPK. Supporting this idea, previous studies have reported that FLCN repressed AMPK in C. elegans and mammalian cells (Possik et al., 2014; Yan et al., 2014). In these models, absence of FLCN resulted in AMPK activation, which induced autophagy. However, these models oppose the suggestion that autophagy is reduced in BHD-associated tumour tissue (Dunlop et al., 2014).
Authors are unsure how the lack of Fnip1 causes a block in B-cell development and it remains to be investigated if another AMPK-regulated pathway or a different pathway is responsible for the phenotype. It is therefore crucial to pursue future investigations on the role of AMPK and the FLCN/FNIP complex in BHD.
- Siggs OM, Stockenhuber A, Deobagkar-Lele M, Bull KR, Crockford TL, Kingston BL, Cornall RJ. (2016). Mutation of Fnip1 is associated with B-cell deficiency, cardiomyopathy, and elevated AMPK activity. Proceedings of the National Academy of Sciences of the United States of America. PMID: 27303042