http://www.bhdsyndrome.org/forum/bhd-research-blog/research-update-flcn-fnip2-ampk-and-mnu-induced-apoptosis/
Previous studies in mouse cell lines have suggested that FLCN, FNIP2 and AMPK are involved in N-methyl-N-nitrosourea (MNU)-induced apoptosis (Lim et al., 2011 – described here).
Using a human cell line, further work from the same group has now shown
that FNIP2 appears to be stabilised by MNU treatment, and that this
stability is modulated by FLCN and AMPK (Sano et al., 2012).
Sano et al. used a HeLa MR cell line which lacks the enzyme MGMT
that repairs MNU-induced DNA lesions, and confirmed that FNIP2 is
involved in the induction of apoptosis following MNU exposure (using
flow cytometry with miRNA-knockdown). Western blot analysis went on to
show that FNIP2 protein levels gradually increased after MNU treatment,
while those of FLCN and AMPKα remained unchanged. However, qRT-PCR
showed no change in the levels of FNIP2 mRNA after MNU treatment,
indicating that the observed increase in FNIP2 protein levels was not
due to transcriptional effects. Using HeLa MR cells which expressed
FLAG-tagged FNIP2, it could be seen that the proteosome inhibitor MG132
led to a significant increase in FNIP2 protein levels, while inhibiting
protein synthesis with cyclohexamide led to a significant decrease. In
both cases, the levels of FLCN and AMPKα remained constant.
Consequently, the authors suggested that FNIP2 may be regulated at the
post-transcriptional level following MNU exposure.
As FNIP2 is known to interact with FLCN and AMPK (Hasumi et al., 2008; Takagi et al., 2008; Lim et al.,
2011), the authors investigated whether these factors affected the
levels of FNIP2. siRNA knockdown of FLCN in HeLa MR cells led to a
decrease in FNIP2 protein levels, and treatment with MNU elicited no
significant changes. In contrast, siRNA knockdown of AMPKα led to an
increase in FNIP2 levels, which increased further after MNU treatment.
Together these results suggest that FLCN and AMPK may have opposing
effects on the stability of FNIP2. Moreover, the inhibition of AMPK
kinase activity with compound C led to an increase in the levels of
FNIP2, but no increase was observed after MNU exposure. This result
suggests that the kinase activity of AMPK is also linked to the
stability of FNIP2.
Finally, immunoprecipitation of
FLAG-tagged FNIP2 from HeLa MR cells treated with MNU over 72 hours
showed that FLCN interacted with FNIP2 throughout this period. However,
it could be seen that the interaction between FNIP2 and AMPKα gradually
decreased over the same timescale. Although the exact mechanistic
details appear to be quite complex, it is clear that FLCN, FNIP2 and
AMPK are associated with MNU-induced apoptosis in vitro.
