Previous work has shown that FLCN appears to play a role in cellular metabolism (Klomp et al., 2010; Preston et al., 2011). In particular, Klomp et al. observed that human BHD tumours had a high expression of mitochondrial and oxidative phosphorylation-associated genes, such as Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PPARGC1A). A recent study by Hasumi et al. (2012) has now added to this work using a muscle-specific FLCN knockout mouse, showing that FLCN loss leads to an increase in both PPARGC1A expression and mitochondrial oxidative phosphorylation.
FLCN was knocked-out specifically in the muscle as it is an important metabolic organ, and this led to the tissue taking on a distinctive red colouring, which is indicative of an increase in the level of mitochondrial components (Lin et al., 2002). Further analysis of the FLCN-deficient muscle confirmed an increase in the expression of mitochondrial genes and proteins using real-time PCR and western blot. Moreover, a significant increase in mitochondrial area per muscle fibre area was detected using electron microscopy.
Mass spectroscopy was then used to investigate mitochondrial function in the FLCN-deficient muscle, and a significant increase in tricarboxylic acid cycle metabolites and coenzymes for the electron transport chain was observed. Additionally, there was a significant increase in both the respiratory capacity of isolated mitochondria and the levels of ATP, all of which suggests a metabolic shift to mitochondrial oxidative phosphorylation in the FLCN-null muscle tissue. Such a shift could provide the energy necessary for cell growth during BHD-associated tumourigenesis.
PPARGC1A is known to be involved in mitochondrial function, and accordingly there was an increase in PPARGC1A mRNA and protein in FLCN-deficient mouse muscle as shown by real-time PCR and western blot. In order to test whether the effects of FLCN loss were mediated through PPARGC1A, muscle-specific FLCN knockout mice were crossbred with muscle-specific PPARGC1A knockout mice. Consequently, the muscle tissue was less red and there was a decrease in the expression of mitochondrial genes and proteins in this double knockout. PPARGC1A is also thought to be involved in angiogenesis and ER stress response, and FLCN-deficient muscles had a higher mRNA expression of angiogenic factors and unfolded protein response genes. This increase was reversed in the muscle-specific double knockout, suggesting that these tumourigenic processes may also be regulated by PPARGC1A during FLCN deficiency.
To understand the role of PPARGC1A in BHD syndrome, it was noted that the expression of both PPARGC1A and COX4 (a downstream target of PPARGC1A) was upregulated in human BHD kidney tumours using real-time PCR and immunohistochemical analysis respectively. The number of mitochondria seen by electron microscopy was also increased in these tumours. Furthermore, using a kidney-specific FLCN knockout mouse model developed by Baba et al. (2008), it could be seen that the enlarged kidneys had both increased PPARGC1A protein levels and numbers of mitochondria. Subsequent crossbreeding with kidney-targeted PPARGC1A knockout mice led to a decrease in kidney size and the number of mitochondria in the double knockout, as well as a complete disappearance of oncocytic-like hyperplastic cells. These results highlight the importance of dysregulated mitochondrial function in BHD-associated tumourigenesis, and could suggest avenues for PPARGC1A-targeted therapies.
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