The basic helix-loop-helix transcription factor HESR1 regulates endothelial cell tube formation

Human endothelial cells can be induced to form capillary-like tubular networks in collagen gels. We have used this in vitro model and representational difference analysis to identify genes involved in the formation of new blood vessels. HESR1 (HEY-1/HRT-1/CHF-2/gridlock), a basic helix-loop-helix protein related to the hairy/enhancer of split/HES family, is absent in migrating and proliferating cultures of endothelial cells but is rapidly induced during capillary-like network formation. HESR1 is detectable in all adult tissues and at high levels in well vascularized organs such as heart and brain. Its expression is also enriched in aorta and purified capillaries. Overexpression of HESR1 in endothelial cells down-regulates vascular endothelial cell growth factor receptor-2 (VEGFR2) mRNA levels and blocks proliferation, migration, and network formation. Interestingly, reduction of expression of HESR1 by antisense oligonucleotides also blocks endothelial cell network formation in vitro. Finally, HESR1 expression is altered in several breast, lung, and kidney tumors. These data are consistent with a temporal model for HESR1 action where down-regulation at the initiation of new vessel budding is required to allow VEGFR2-mediated migration and proliferation, but re-expression of HESR1 is necessary for induction of tubular network formation and continued maintenance of the mature, quiescent vessel.     

AFAP120 regulates actin organization during neuronal differentiation

During development, dynamic changes in the actin cytoskeleton determine both cell motility and morphological differentiation. In most mature tissues, cells are generally minimally motile and have morphologies specialized to their functions. In metastatic cancer, cells generally lose their specialized morphology and become motile. Therefore, proteins that regulate the transition between the motile and morphologically differentiated states can play important roles in determining cancer outcomes. AFAP120 is a neuronal-specific protein that binds Src kinase and protein kinase C (PKC) and cross-links actin filaments. Here we report that expression and tyrosine phosphorylation of AFAP120 are developmentally regulated in the cerebellum. In cerebellar cultures, PKC activation induces Src kinase-dependent phosphorylation of AFAP120, indicating that AFAP120 may be a downstream effector of Src. In neuroblastoma cells induced to differentiate by treatment with a PKC activator, tyrosine phosphorylation of AFAP120 appears to regulate the formation of the lamellar actin structures and subsequent neurite initiation. Together, these results indicate that AFAP120 plays a role in organizing dynamic actin structures during neuronal differentiation and suggest that AFAP120 may help regulate the transition from motile precursor to morphologically differentiated neurons.     

MicroRNA miR-124 regulates neurite outgrowth during neuronal differentiation

MicroRNAs (miRNAs) are small RNAs with diverse regulatory roles. The miR-124 miRNA is expressed in neurons in the developing and adult nervous system. Here we show that overexpression of miR-124 in differentiating mouse P19 cells promotes neurite outgrowth, while blocking miR-124 function delays neurite outgrowth and decreases acetylated α-tubulin. Altered neurite outgrowth also was observed in mouse primary cortical neurons when miR-124 expression was increased, or when miR-124 function was blocked. In uncommitted P19 cells, miR-124 expression led to disruption of actin filaments and stabilization of microtubules. Expression of miR-124 also decreased Cdc42 protein and affected the subcellular localization of Rac1, suggesting that miR-124 may act in part via alterations to members of the Rho GTPase family. Furthermore, constitutively active Cdc42 or Rac1 attenuated neurite outgrowth promoted by miR-124. To obtain a broader perspective, we identified mRNAs downregulated by miR-124 in P19 cells using microarrays. mRNAs for proteins involved in cytoskeletal regulation were enriched among mRNAs downregulated by miR-124. A miR-124 variant with an additional 5′ base failed to promote neurite outgrowth and downregulated substantially different mRNAs. These results indicate that miR-124 contributes to the control of neurite outgrowth during neuronal differentiation, possibly by regulation of the cytoskeleton.