Folliculin regulates stem cells’ exit from pluripotency

A stem cell requires two properties: self-renewal and potency. Self-renewal describes stem cell division to generate more stem cells. Potency describes stem cell division where the new cell takes on different characteristics to the progenitor cell and ultimately becomes a different cell type; this process is called differentiation and is particularly important during embryonic development. Classical stem cells are pluripotent, meaning that they can differentiate into many different cell types to form specialised tissues and organs. Stem cell maintenance and differentiation is highly controlled, and a recent study by Austin Smith’s group at the University of Cambridge shows that the BHD gene, FLCN, promotes stem cells to exit from the pluripotent state and to become primed for differentiation.

Betschinger et al. identified FLCN as a regulator of mouse embryonic stem cell (ESC) exit from pluripotency using an RNAi screen, and further experiments showed that ESCs depleted of FLCN retained pluripotency and failed to differentiate upon exposure to the developmental factors Activin A, fibroblast growth factor 4 (FGF4) and bone morphogenetic protein 4 (BMP4). Depletion of FNIP1 and/or FNIP2 abrogated this phenotype, with the strongest effect being seen in the combined FNIP1/2 knock down, suggesting that FLCN, FNIP1 and FNIP2 act together to regulate pluripotency. Similar experiments showed that TSC2 – which causes the related syndrome Tuberous sclerosis complex (TSC) – knockdown cells also showed this phenotype.

As both FLCN and TSC2 have been linked to mTOR signalling (Baba et al., 2006; Inoki et al., 2002), the authors tested whether mTOR signalling was required for this effect and found that elevated mTOR signalling did indeed prevent stem cells from exiting pluripotency, but was not required for differentiation. While TSC2 seemed to exercise its effects on pluripotency via mTOR signalling, FLCN did not, suggesting that TSC2 acts upstream, and FLCN acts downstream or independently of mTOR signalling.

Furthermore, the authors showed that under normal conditions, the FLCN-FNIP1-FNIP2 complex acts to exclude the TFE3 protein from the nucleus, preventing the expression of Esrrb – a known pluripotency regulator (Martello et al., 2012) – and consequently stem cells to exit pluripotency. Interestingly, FLCN has been previously shown to inhibit TFE3’s activity (Hong et al., 2010) and recurrent Xp11.2 translocations involving TFE3 have been found in cases of papillary renal cell carcinoma (Kuroda et al., 2012).

The authors analysed the sub-cellular localisation of TFE3 throughout normal development in the mouse embryo and found that as development proceeds, TFE3 expression changes from being exclusively nuclear in pre-implantation embryos, to being predominantly cytoplasmic in post-implantation embryos. Given TFE3’s function in maintaining pluripotency, this result is to be expected as during development the proportion of stem cells in the embryo decreases as it grows and cells differentiate to form organs and tissues. Thus, it seems that FLCN, together with FNIP1 and FNIP2, may act as a developmental switch from the highly pluripotent state seen in the pre-implantation embryo, into the highly differentiating state seen in the post-implantation embryo, by controlling the sub-cellular localisation of TFE3. This could explain why FLCN-null mice show such early embryonic lethality (Hasumi et al., 2009).

FLCN has been previously linked to stem cell maintenance, although the mechanism through which it exerts this function has remained elusive (Singh et al., 2006). Betschinger et al., elegantly show that FLCN causes stem cells to exit pluripotency and become primed for differentiation by excluding TFE3 from the nucleus. However, given that the majority of stem cell differentiation occurs in the developing embryo, it is unclear why the symptoms of BHD syndrome do not manifest until adulthood. While stem cells do generally disappear during development to become differentiated tissues, several pools of stem cells are maintained in the adult to replace old and damaged cells. For example epidermal stem cells proliferate to heal cuts and wounds (Blanpain and Fuchs, 2006); hematopoetic stem cells produce new blood cells throughout life (Dzierzak and Speck, 2008); and kidney stem cells can repair damaged tissue upon renal injury (Reule and Gupta, 2011). Thus, it is possible that the hyperplastic lesions characteristic of BHD – fibrofolliculomas, lung cysts and kidney tumours – may arise from these adult stem cells.


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