Mutational inactivation of the tumor suppressor tuberous sclerosis complex 2 (TSC2) constitutively activates mTORC1, increases cell proliferation, and induces the pathological manifestations observed in tuberous sclerosis (TS) and in pulmonary lymphangioleiomyomatosis (LAM). While the role of mTORC1 in TSC2-dependent growth has been extensively characterized, little is known about the role of mTORC2. Our data demonstrate that mTORC2 modulates TSC2-null cell proliferation and survival through RhoA GTPase and Bcl2 proteins. TSC2-null cell proliferation was inhibited not only by reexpression of TSC2 or small interfering RNA (siRNA)-induced downregulation of Rheb, mTOR, or raptor, but also by siRNA for rictor. Increased RhoA GTPase activity and P-Ser473 Akt were inhibited by siRNA for rictor. Importantly, constitutively active V14RhoA reversed growth inhibition induced by siRNA for rictor, siRNA TSC1, reexpression of TSC2, or simvastatin. While siRNA for RhoA had a modest effect on growth inhibition, downregulation of RhoA markedly increased TSC2-null cell apoptosis. Inhibition of RhoA activity downregulated antiapoptotic Bcl2 and upregulated proapoptotic Bim, Bok, and Puma. In vitro and in vivo, simvastatin alone or in combination with rapamycin inhibited cell growth and induced TSC2-null cell apoptosis, abrogated TSC2-null tumor growth, improved animal survival, and prevented tumor recurrence by inhibiting cell growth and promoting apoptosis. Our data demonstrate that mTORC2-dependent activation of RhoA is required for TSC2-null cell growth and survival and suggest that targeting both mTORC2 and mTORC1 by a combination of proapoptotic simvastatin and cytostatic rapamycin shows promise for combinational therapeutic intervention in diseases with TSC2 dysfunction.

The ␣2␤1 integrin receptor plays a key role in angiogenesis. Here we investigated the effects of small molecule inhibitors (SMIs) designed to disrupt integrin ␣2 I or ␤1 I-like domain function on angiogenesis. In unchallenged endothelial cells, fibrillar collagen induced robust capillary morphogenesis. In contrast, tube formation was significantly reduced by SMI496, a ␤1 I-like domain inhibitor and by function-blocking anti-␣2␤1 but not -␣1␤1 antibodies. Endothelial cells bound fluorescein-labeled collagen I fibrils, an interaction specifically inhibited by SMI496. Moreover, SMI496 caused cell retraction and cytoskeletal collapse of endothelial cells as well as delayed endothelial cell wound healing. SMI activities were examined in vivo by supplementing the growth medium of zebrafish embryos expressing green fluorescent protein under the control of the vascular endothelial growth factor receptor-2 promoter. SMI496, but not a control compound, interfered with angiogenesis in vivo by reversibly inhibiting sprouting from the axial vessels. We further characterized zebrafish ␣2 integrin and discovered that this integrin is highly conserved, especially the I domain. Notably, a similar vascular phenotype was induced by morpholino-mediated knockdown of the integrin ␣2 subunit. By live videomicroscopy, we confirmed that the vessels were largely nonfunctional in the absence of ␣2␤1 integrin. Collectively , our results provide strong biochemical and genetic evidence of a central role for ␣2␤1 integrin in experimental and developmental angiogenesis.

Decorin, an archetypal member of the small leucine-rich proteoglycan gene family, regulates collagen fibrillogenesis and cell growth. To further explore its biological function, we examined the role of Decorin during zebrafish development. Zebrafish Decorin is a chondroitin sulfate proteoglycan that exhibits a high degree of conservation with its mammalian counterpart and displays a unique spatiotemporal expression pattern. Morpholino-mediated knockdown of zebrafish decorin identified a developmental role during medial-lateral convergence and anterior-posterior extension of the body plan, as well as in craniofacial cartilage formation. decorin morphants displayed a pronounced shortening of the head-to-tail axis as well as compression, flattening, and extension of the jaw cartilages. The morphant phenotype was efficiently rescued by zebrafish decorin mRNA. Unexpectedly, microinjection of excess zebrafish decorin mRNA or proteoglycan/protein core into onecell stage embryos caused cyclopia. The morphant and overexpression phenotype represent a convergent extension defect. Our results indicate a central function for Decorin during early embryogenesis.Proteoglycan-enriched extracellular matrices provide powerful messages to the cells via signaling events that vary from storing growth factors and morphogens to modulating their bioactivity and interactions with their cognate receptors (1). Decorin belongs to the family of the small leucine-rich proteoglycans (SLRPs) 3 (2-4). The Decorin protein core directly binds type I collagen, a key biological interaction that controls the pace and extent of collagen fibril formation both in vitro and in vivo (5). The attached glycosaminoglycan (GAG) chain also contributes by coordinating the proper spacing between the fibrils (6). The structural requirement of Decorin during these events was clearly manifested by the decorin-null mice. Gene targeting of murine decorin resulted in irregular collagen fibril morphology associated with fragility of the skin (7). In addition to various collagen types, Decorin binds to Zn 2ϩ , fibrin, fibronectin, C1q, thrombospondin, transforming growth factor ␤, LRP1, and EGFR (8 -17). Decorin is also involved in the pathogenesis of renal diseases (18 -20), angiogenesis (21), wound healing (22), myocardial infarction (23), lung mechanics (24), tooth development (25), and bone marrow stromal cell biology (26). The function of Decorin through the EGFR has been extensively linked to the pathobiology of cancer (27-33).Notably, double mutant mice lacking both decorin and the tumor suppressor p53 die early as a consequence of aggressive lymphomas (34), suggesting that Decorin is permissive for tumorigenesis. In line with this hypothesis, a recent study utilizing decorin-null animals, which were backcrossed into a different genetic background, has shown that the lack of decorin favors spontaneous occurrence of intestinal tumors in ϳ30% of the cases, and this tumor burden and frequency were exacerbated by subjecting the mutant mice to a high risk diet (35).In...

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