Mutation in the TSC2 tumor suppressor causes tuberous sclerosis complex, a disease characterized by hamartoma formation in multiple tissues. TSC2 inhibits cell growth by acting as a GTPase-activating protein toward Rheb, thereby inhibiting mTOR, a central controller of cell growth. Here, we show that Wnt activates mTOR via inhibiting GSK3 without involving beta-catenin-dependent transcription. GSK3 inhibits the mTOR pathway by phosphorylating TSC2 in a manner dependent on AMPK-priming phosphorylation. Inhibition of mTOR by rapamycin blocks Wnt-induced cell growth and tumor development, suggesting a potential therapeutic value of rapamycin for cancers with activated Wnt signaling. Our results show that, in addition to transcriptional activation, Wnt stimulates translation and cell growth by activating the TSC-mTOR pathway. Furthermore, the sequential phosphorylation of TSC2 by AMPK and GSK3 reveals a molecular mechanism of signal integration in cell growth regulation.

The molecular basis of learning and memory has been the object of several recent advances, which have focused attention on calcium-regulated pathways controlling transcription. One of the molecules implicated by pharmacological, biochemical and genetic approaches is the calcium/calmodulin-regulated phosphatase, calcineurin. In lymphocytes, calcineurin responds to specific calcium signals and regulates expression of several immediate early genes by controlling the nuclear import of the NF-ATc family of transcription factors. Here we show that NF-ATc4/NF-AT3 in hippocampal neurons can rapidly translocate from cytoplasm to nucleus and activate NF-AT-dependent transcription in response to electrical activity or potassium depolarization. The calcineurin-mediated translocation is critically dependent on calcium entry through L-type voltage-gated calcium channels. GSK-3 can phosphorylate NF-ATc4, promoting its export from the nucleus and antagonizing NF-ATc4-dependent transcription. Furthermore, we show that induction of the inositol 1,4,5-trisphosphate receptor type 1 is controlled by the calcium/calcineurin/NF-ATc pathway. This provides a new perspective on the function of calcineurin in the central nervous system and indicates that NF-AT-mediated gene expression may be involved in the induction of hippocampal synaptic plasticity and memory formation.

cells. Once transformed, the mesenchymal cells prolifer-1 Departments of Pathology and Developmental ate and invade the cardiac jelly, a basement membrane-Biology like substance elaborated by the myocardial cells. A Howard Hughes Medical Institute second process, which is even less well understood, 2 Cardiovascular Division begins after EMT. The endocardial cushion area elon-Department of Medicine gates and undergoes continuous remodeling that even-Stanford University Medical School tually refines the primitive cushion into thin elongated Stanford, California 94305 valve leaflets. Several signaling pathways have been implicated in heart valve morphogenesis. Studies with neutralizing Summary antibodies and antisense RNA indicate that TGF-␤ signaling is essential for EMT in the chick (Eisenberg and The delicate leaflets that make up vertebrate heart Markwald, 1995; Nakajima et al., 2000). However, probavalves are essential for our moment-to-moment exisbly due to functional redundancy in the mammalian systence. Abnormalities of valve formation are the most tem, mice with single mutations in the TGF-␤ family common serious human congenital defect. Despite members or their receptors do not have impaired EMT. their importance, relatively little is known about valve The Wnt/␤-catenin pathway is essential for EMT in zedevelopment. We show that the initiation of heart valve brafish (Hurlstone et al., 2003). In mice, however, Wnt morphogenesis in mice requires calcineurin/NFAT to plays an essential early role in cardiac myogenesis repress VEGF expression in the myocardium underly-(Olson and Schneider, 2003), thus obscuring any later ing the site of prospective valve formation. This represroles in heart valve formation. Also, Notch signaling has sion of VEGF at E9 is essential for endocardial cells recently been shown to promote EMT in mice (Timmerto transform into mesenchymal cells. Later, at E11, a man et al., 2004). EGF-related growth factors and their second wave of calcineurin/NFAT signaling is required receptors are important in regulating heart valve formain the endocardium, adjacent to the earlier myocardial tion in mice. Loss of endocardial HB-EGF or EGFR resite of NFAT action, to direct valvular elongation and sults in abnormal hyperplasia of cushion mesenchymal refinement. Thus, NFAT signaling functions sequencells (Chen et al., 2000; Iwamoto et al., 2003; Jackson tially from myocardium to endocardium within a valvuet al., 2003). Lack of endocardial neuregulin-1 or cushion lar morphogenetic field to initiate and perpetuate mesenchymal ErbB3 results in severe mesenchymal hyembryonic valve formation. This mechanism also oppoplasia in the cushion area (Camenisch et al., 2002; erates in zebrafish, indicating a conserved role for Erickson et al., 1997; Meyer and Birchmeier, 1995). Ras calcineurin/NFAT signaling in vertebrate heart valve signaling is also crucial for cushion mesenchymal develmorphogenesis. opment. Loss of endothelial neurofibromin-1, which inactivates Ras, results in hyperplasia of cushion mesen-

Intronic microRNAs have been proposed to complicate the design and interpretation of mouse knockout studies. The endothelialexpressed Egfl7/miR-126 locus contains miR-126 within Egfl7 intron 7, and angiogenesis deficits have been previously ascribed to Egfl7 gene-trap and lacZ knock-in mice. Surprisingly, selectively floxed Egfl7 ⌬ and miR-126 ⌬ alleles revealed that Egfl7 ⌬/⌬ mice were phenotypically normal, whereas miR-126 ⌬/⌬ mice bearing a 289-nt microdeletion recapitulated previously described Egfl7 embryonic and postnatal retinal vascular phenotypes. Regulation of angiogenesis by miR-126 was confirmed by endothelial-specific deletion and in the adult cornea micropocket assay. Furthermore, miR-126 deletion inhibited VEGF-dependent Akt and Erk signaling by derepression of the p85β subunit of PI3 kinase and of Spred1, respectively. These studies demonstrate the regulation of angiogenesis by an endothelial miRNA, attribute previously described Egfl7 vascular phenotypes to miR-126, and document inadvertent miRNA dysregulation as a complication of mouse knockout strategies.KEY WORDS: Angiogenesis, miRNA, miR-126 (Mirn126), Egfl7, p85β (Pik3r2)