RhoJ/TCL Regulates Endothelial Motility and Tube Formation and Modulates Actomyosin Contractility and Focal Adhesion Numbers

Objective—RhoJ/TCL was identified by our group as an endothelial-expressed Rho GTPase. The aim of this study was to determine its tissue distribution, subcellular localization, and function in endothelial migration and tube formation. Methods and Results—Using in situ hybridization, RhoJ was localized to endothelial cells in a set of normal and cancerous tissues and in the vasculature of mouse embryos; endogenous RhoJ was localized to focal adhesions by immunofluorescence. The proangiogenic factor vascular endothelial growth factor activated RhoJ in endothelial cells. Using either small interfering (si)RNA-mediated knockdown of RhoJ expression or overexpression of constitutively active RhoJ (daRhoJ), RhoJ was found to positively regulate endothelial motility and tubule formation. Downregulating RhoJ expression increased focal adhesions and stress fibers in migrating cells, whereas daRhoJ overexpression resulted in the converse. RhoJ downregulation resulted in increased contraction of a collagen gel and increased phospho–myosin light chain, indicative of increased actomyosin contractility. Pharmacological inhibition of Rho-kinase (which phosphorylates myosin light chain) or nonmuscle myosin II reversed the defective tube formation and migration of RhoJ knockdown cells. Conclusion—RhoJ is endothelial-expressed in vivo, activated by vascular endothelial growth factor, localizes to focal adhesions, regulates endothelial cell migration and tube formation, and modulates actomyosin contractility and focal adhesion numbers

Chemically-induced Neurite-like Outgrowth Reveals Multicellular Network Function in Patient-derived Glioblastoma Cells

Tumor stem cells and malignant multicellular networks have been separately implicated in the therapeutic resistance of Glioblastoma Multiforme (GBM), the most aggressive type of brain cancer in adults. We show that small molecule inhibition of RHO-associated serine/threonine kinase (ROCKi) significantly promoted the outgrowth of neurite-like cell projections in cultures of heterogeneous patient-derived GBM stem-like cells. These projections formed de novo -induced cellular network (iNet) ‘webs’, which regressed after withdrawal of ROCKi. Connected cells within the iNet web exhibited long range calcium signal transmission, and significant lysosomal and mitochondrial trafficking. In contrast to their less-connected vehicle control counterparts, iNet cells remained viable and proliferative after high-dose radiation. These findings demonstrate a link between ROCKi-regulated cell projection dynamics and the formation of radiation-resistant multicellular networks. Our study identifies means to reversibly induce iNet webs ex vivo , and may thereby accelerate future studies into the biology of GBM cellular networks

Scleroderma fibroblasts suppress angiogenesis via TGF-β/caveolin-1 dependent secretion of pigment epithelium-derived factor

Objectives: Systemic sclerosis (SSc) is characterised by tissue fibrosis and vasculopathy with defective angiogenesis. Transforming growth factor beta (TGF-β) plays a major role in tissue fibrosis, including downregulation of caveolin-1 (Cav-1); however, its role in defective angiogenesis is less clear. Pigment epithelium-derived factor (PEDF), a major antiangiogenic factor, is abundantly secreted by SSc fibroblasts. Here, we investigated the effect of TGF-β and Cav-1 on PEDF expression and the role of PEDF in the ability of SSc fibroblasts to modulate angiogenesis. Methods: PEDF and Cav-1 expression in fibroblasts and endothelial cells were evaluated by means of immunohistochemistry on human and mouse skin biopsies. PEDF and Cav-1 were silenced in cultured SSc and control fibroblasts using lentiviral short-hairpin RNAs. Organotypic fibroblast–endothelial cell co-cultures and matrigel assays were employed to assess angiogenesis. Results: PEDF is highly expressed in myofibroblasts and reticular fibroblasts with low Cav-1 expression in SSc skin biopsies, and it is induced by TGF-β in vitro. SSc fibroblasts suppress angiogenesis in an organotypic model. This model is reproduced by silencing Cav-1 in normal dermal fibroblasts. Conversely, silencing PEDF in SSc fibroblasts rescues their antiangiogenic phenotype. Consistently, transgenic mice with TGF-β receptor hyperactivation show lower Cav-1 and higher PEDF expression levels in skin biopsies accompanied by reduced blood vessel density. Conclusions: Our data reveal a new pathway by which TGF-β suppresses angiogenesis in SSc, through decreased fibroblast Cav-1 expression and subsequent PEDF secretion. This pathway may present a promising target for new therapeutic interventions in SSc

Rho GTPases required for angiogenesis: role and regulation of RhoG

Angiogenesis is the formation of new blood vessels from pre-existing ones and it is necessary for physiological processes such as normal development, and pathological conditions like cancer growth and metastatic cell dissemination. In order to form new blood vessels, endothelial cells, the cells that line the blood vessels, must go through a number of distinct cellular processes. In order to sprout, cells have to initially detach from the vessel wall, migrate and divide. Endothelial cells must then establish new cell junctions and form functional lumens. During these processes the endothelial cell actin cytoskeleton and cell morphology change dramatically. The Rho family of small GTPases are key regulators of such morphological changes. Whilst the prototypical family members Rho, Rac and Cdc42 have been implicated in different aspects of endothelial cell behavior such as migration and cell assembly, other Rho GTPase family members are less characterized. The aim of this study has been to understand the role of the Rho GTPase RhoG in vessel formation and to identify its regulators and downstream effectors. For this purpose organotypic co-culture system was employed of endothelial cells and fibroblasts that gives rise to three-dimensional capillary-like tubes as an in vitro tool to model vessel development. Using RNAi-mediated knockdown in the endothelial cells in the in vitro coculture system, the role of putative regulators (guanine nucleotide exchange factors) and downstream effectors in angiogenesis was analysed and players that control lateral filopdia formation were identified. Pulldown assays that determine the activation status of Rho GTPases and gene overexpression and protein co-immunoprecipitation assays were then used to confirm phenotypic results seen in the co-culture assay. Using this system, a novel non-canonical SGEF-RhoG-DOCK4-Rac1-DOCK9-Cdc42 signaling module was identified that controls filopodia formation during endothelial cell assembly and sprouting. Knockdown of components of this signaling module block lateral filopodia formation, while tip filopodia persist suggesting that that different sub-populations of endothelial cell filopodia exist that are regulated independently during tubule morphogenesis. Mechanistic studies carried out in the course of this study showed that DOCK4 and DOCK9 interact with each other and with the RhoG effector ELMO. Complex formation of DOCK9 with ELMO is through DOCK4 whereas DOCK4 interacts directly with DOCK9. Domain deletion studies showed that DOCK4 interacts with DOCK9 via its SH3 domain. In summary, this work has identified a new signaling cascade that drives vessel formation via lateral filopodia formation and sprouting and demonstrated for the first time an interaction between a Rac1 exchange factor (DOCK4) and a Cdc42 exchange factor (DOCK9). In addition to providing new information about the process of angiogenic sprouting and functional interaction between different GEFs, the signaling components identified in this study may constitute useful therapeutic targets in pathological angiogenesis in cancer and other diseases

Peak BMP Responses in the Drosophila Embryo are Dependent on the Activation of Integrin Signalling.

SummaryWithin a 3D tissue, cells need to integrate signals from growth factors, such as BMPs, and the extracellular matrix (ECM) to coordinate growth and differentiation. Here, we use the Drosophila embryo as a model to investigate how BMP responses are influenced by a cell’s local ECM environment. We show that integrins, which are ECM receptors, are absolutely required for peak BMP signaling. This stimulatory effect of integrins requires their intracellular signaling function, which is activated by the ECM protein collagen IV. Mechanistically, integrins interact with the BMP receptor and stimulate phosphorylation of the downstream Mad transcription factor. The BMP-pathway-enhancing function of integrins is independent of focal adhesion kinase, but it requires conserved NPXY motifs in the β-integrin cytoplasmic tail. Furthermore, we show that an α-integrin subunit is a BMP target gene, identifying positive feedback between integrin signaling and BMP pathway activity that may contribute to robust cell fate decisions