NODAL REGULATES P27 EXPRESSION AT MULTIPLE LEVELS
Lubna Nadeem, Frank Chen and Chun Peng
Department of Biology, York University, Toronto, Canada
Nodal, a member of transforming growth factor β (TGF-β) superfamily, is expressed in
human placenta throughout gestation (Roberts et al, 2003. Biol Reprod 68: 1719) and it
acts through activin receptor-like kinase 7 (ALK7) to inhibit proliferation and induce
apoptosis in human trophoblast cells (Munir et al., 2004. J Biol Chem 279: 31277). We
have recently found up-regulation of p27, a cyclin dependent kinase inhibitor, by
Nodal/ALK7 signaling in HTR8/SVneo cells in gene array analysis. Our data also
suggests that p27 is the key mediator of Nodal function and it mediates the inhibitory
effect of Nodal on trophoblast proliferation, migration and invasion. Therefore, in this
study we examined how Nodal regulates p27 expression. We found that Nodal upregulated p27 mRNA and protein levels. Regulation of p27 at transcription level was
confirmed by luciferase reporter assays. Further we tested whether Nodal regulates p27
mRNA stability using Actinomycin D, which blocks gene transcription. Through RT-PCR
analyses we found that Nodal increased the half-life of p27 mRNA. When protein
synthesis was blocked by cycloheximide, Nodal still increased p27 protein level.
Phosphorylation of p27 at T-187 is known to target p27 for ubiquitination and subsequent
degradation. We observed that Nodal inhibited p27 phosphorylation at T-187 and
decreased p27 ubiquitination. Taken together, our study revealed that Nodal regulates p27
synthesis and degradation at multiple levels to increase p27 levels, which in turn,
mediates the effects of Nodal on trophoblast cell proliferation, and migration.
[Supported by CIHR grants and OWHC/CIHR mid-career award to CP and OGS to LN]