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John Montalvo,
Carrie Spencer,
Alexander Hackathorn,
Katherine Masterjohn,
Ashley Perkins,
Christopher Doty,
Arunkumar Arumugam,
Pat P Ongusaha,
Rajkumar Lakshmanaswamy,
James K Liao,
Dianne C Mitchell, Brad A Bryan
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ABSTRACT: The serine/threonine protein kinase paralogs ROCK1 & 2 have been implicated as essential modulators of angiogenesis; however their paralog-specific roles in endothelial function are unknown. shRNA knockdown of ROCK1 or 2 in endothelial cells resulted in a significant disruption of in vitro capillary network formation, cell polarization, and cell migration compared to cells harboring non-targeting control shRNA plasmids. Knockdowns led to alterations in cytoskeletal dynamics due to ROCK1 & 2-mediated reductions in actin isoform expression, and ROCK2-specific reduction in myosin phosphatase and cofilin phosphorylation. Knockdowns enhanced cell survival and led to ROCK1 & 2-mediated reduction in caspase 6 and 9 cleavage, and a ROCK2-specific reduction in caspase 3 cleavage. Microarray analysis of ROCK knockdown lines revealed overlapping and unique control of global transcription by the paralogs, and a reduction in the transcriptional regulation of just under 50% of VEGF responsive genes. Finally, paralog knockdown in xenograft angiosarcoma tumors resulted in a significant reduction in tumor formation. Our data reveals that ROCK1 & 2 exhibit overlapping and unique roles in normal and dysfunctional endothelial cells, that alterations in cytoskeletal dynamics are capable of overriding mitogen activated transcription, and that therapeutic targeting of ROCK signaling may have profound impacts for targeting angiogenesis.
Current Molecular Medicine 08/2012; · 5.10 Impact Factor
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ABSTRACT: Rho kinase (ROCK) proteins are Rho-GTPase activated serine/threonine kinases that function as modulators of actin-myosin cytoskeletal dynamics via regulation of Lin11, Isl-1 & Mec-3 domain (LIM) kinase, myosin light chain (MLC), and MLC phosphatase. A strong correlation between cytoskeletal rearrangements and tumor cell invasion, metastasis, and deregulated microenvironment interaction has been reported in the literature, and the utilization of pharmacological inhibitors of ROCK signaling for the treatment of cancer is actively being pursued by a number of pharmaceutical companies. Indeed, in many preclinical models ROCK inhibitors have shown remarkable efficacy in reducing tumor growth and metastasis. Interestingly, ROCK signaling has been shown to be either pro-apoptotic or pro-survival in a cell type and context dependent manner, though the molecular mechanisms controlling ROCK-mediated cell fate decisions are unknown. This review summarizes the many pleiotropic roles of ROCK signaling in survival and apoptosis, and suggests that controlled modulation of ROCK activity in tumor cells has the potential to significantly affect tumor survival and patient outcome.
Anticancer research 11/2011; 31(11):3645-57. · 1.73 Impact Factor
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ABSTRACT: The role of the RhoA/Rho kinase (ROCK) signaling pathway in cell survival remains a very controversial issue, with its activation being pro-apoptotic in many cell types and anti-apoptotic in others. To test if ROCK inhibition contributes to tumor cell survival or death following chemotherapy, we treated cisplatin damaged neuroblastoma cells with a pharmacological ROCK inhibitor (Y27632) or sham, and monitored cell survival, accumulation of a chemoresistant phenotype, and in vivo tumor formation. Additionally, we assayed if ROCK inhibition altered the expression of genes known to be involved in cisplatin resistance. Our studies indicate that ROCK inhibition results in increased cell survival, acquired chemoresistance, and enhanced tumor survival following cisplatin cytotoxicity, due in part to altered expression of cisplatin resistance genes. These findings suggest that ROCK inhibition in combination with cisplatin chemotherapy may lead to enhanced tumor chemoresistance in neuroblastoma.
International Journal of Oncology 11/2010; 37(5):1297-305. · 2.40 Impact Factor
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ABSTRACT: Healthy cells, as well as benign and malignant tumors, depend upon the body's blood supply to bring in oxygen and nutrients and carry away waste products. Using this property against tumors, anti-angiogenic therapy targets the tumor vasculature with the aim of starving the tumor, and has demonstrated exceptional clinical efficacy against a number of tumors. This review discusses the current state of knowledge regarding anti-angiogenic therapies presently available to patients, and garners from both preclinical and clinical literature the benefits and side effects associated with anti-angiogenic therapies, the unfortunate mechanisms of acquired resistance to these novel therapeutics, and highlights promising next generation anti-angiogenics that may overcome the limitations encountered with first generation therapies.
Journal of Cellular Biochemistry 10/2010; 111(3):543-53. · 2.87 Impact Factor
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ABSTRACT: The small GTPase RhoA and its downstream effectors, ROCK1 and ROCK2, regulate a number of cellular processes, including cell motility, proliferation, survival, and permeability. Pharmacological inhibitors of the Rho pathway reportedly block angiogenesis; however, the molecular details of this inhibition are largely unknown. We demonstrate that vascular endothelial growth factor-A (VEGF) rapidly induces RhoA activation in endothelial cells (ECs). Moreover, the pharmacological inhibition of ROCK1/2 using 10 microM Y-27632 (the IC(50) for this compound in ECs) strongly disrupts vasculogenesis in pluripotent embryonic stem cell cultures, VEGF-mediated regenerative angiogenesis in ex vivo retinal explants, and VEGF-mediated in vitro EC tube formation. Furthermore, using small interfering RNA knockdown and mouse heterozygote knockouts of ROCK1 and ROCK2, we provide data indicating that VEGF-driven angiogenesis is largely mediated through ROCK2. These data demonstrate that Rho/ROCK signaling is an important mediator in a number of angiogenic processes, including EC migration, survival, and cell permeability, and suggest that Rho/ROCK inhibition may prove useful for the treatment of angiogenesis-related disorders.
The FASEB Journal 09/2010; 24(9):3186-95. · 5.71 Impact Factor
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ABSTRACT: Vascular endothelial growth factor (VEGF) is considered the master regulator of angiogenesis during growth and development, as well as in disease states such as cancer, diabetes, and macular degeneration. This review details our current understanding of VEGF signaling and discusses the benefits and unexpected side effects of promising anti-angiogenic therapeutics that are currently being used to inhibit neovacularization in tumors.
BioFactors 06/2009; 35(4):332-7. · 4.93 Impact Factor
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ABSTRACT: Angiogenesis is largely controlled by hypoxia-driven transcriptional up-regulation and secretion of vascular endothelial growth factor (VEGF) and its binding to the endothelial cell tyrosine receptor kinases, VEGFR1 and VEGFR2. Recent expression analysis suggests that VEGF is expressed in a cell-specific manner in normoxic adult tissue; however, the transcriptional regulation and role of VEGF in these tissues remains fundamentally unknown. In this report we demonstrate that VEGF is coordinately up-regulated during terminal skeletal muscle differentiation. We reveal that this regulation is mediated in part by MyoD homo- and hetero-dimeric transcriptional mechanisms. Serial deletions of the VEGF promoter elucidated a region containing three tandem CANNTG consensus MyoD sites serving as essential sites of direct interaction for MyoD-mediated up-regulation of VEGF transcription. VEGF-null embryonic stem (ES) cells exhibited reduced myogenic differentiation compared with wild-type ES cells, suggesting that VEGF may serve a role in skeletal muscle differentiation. We demonstrate that VEGFR1 and VEGFR2 are expressed at low levels in myogenic precursor cells and are robustly activated upon VEGF stimulation and that their expression is coordinately regulated during skeletal muscle differentiation. VEGF stimulation of differentiating C2C12 cells promoted myotube hypertrophy and increased myogenic differentiation, whereas addition of sFlt1, a VEGF inhibitor, resulted in myotube hypotrophy and inhibited myogenic differentiation. We further provide evidence indicating VEGF-mediated myogenic marker expression, mitogenic activity, migration, and prosurvival functions may contribute to increased myogenesis. These data suggest a novel mechanism whereby VEGF is coordinately regulated as part of the myogenic differentiation program and serves an autocrine function regulating skeletal myogenesis.
Molecular biology of the cell 04/2008; 19(3):994-1006. · 5.98 Impact Factor
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ABSTRACT: Vascular assembly, patterning, and maintenance is a complex and highly regulated process that begins with the formation of a primary capillary plexus by means of angiogenesis or vasculogenesis and ends when the primitive vessels have been remodeled into quiescent, differentiated vessels. Differentiated or "mature" microvessels are characterized in large part by their association with pericytes, and failure of these interactions results in severe, and often lethal, defects that have been implicated in a number of human pathologic conditions, including tumor angiogenesis, diabetic microangiopathy, ectopic tissue calcification, stroke, and dementia. This chapter describes methods that can be used to isolate and culture primary pericytes, as well as to study pericyte-endothelial cell interactions with in vitro cell culture systems.
Methods in enzymology 02/2008; 443:315-31. · 1.90 Impact Factor
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ABSTRACT: The small GTPases function as "molecular switches" by binding and releasing GTP to mediate downstream signaling effects. The Rho-family of GTPases is central in modulating cell differentiation and cytoskeletal changes. Since eye development requires comprehensive morphogenetic movements and extensive cellular differentiation, we hypothesize that different small GTPases may play important roles during morphogenesis of eye development. To explore this possibility, we examined the expression patterns of three major Rho-GTPases: RhoA, Rac1, and Cdc42 in embryonic, postnatal (one day after birth), and adult (two-month old) mouse eye.
Various ocular tissues were collected from embryonic, postnatal, and adult C57BL/6 mice. Western blots were conducted using total proteins extracted from cornea, retina, lens epithelial cells, and lens fiber cells of the adult mice or different fractions of rat lenses. Immunohistochemistry (IHC) was performed with 6 mum thick sections cut through the eye ball region of 11.5 pc, 14.5 pc, 17.5 pc, postnatal, and adult mice. Parallel controls were run using the rabbit preimmune and GTPase-specific antibodies blocked with saturating levels of corresponding peptide antigen.
In the embryonic mouse eye, RhoA and Cdc42 expressions were initially detectable in all three compartments at 11.5 pc. However, Rac1 became easily detectable in these compartments at 14.5 pc. Increased levels of RhoA, Rac1, and Cdc42 were detected in the three compartments at 17.5 pc and the strongest signals for RhoA, Rac1, and Cdc42 were observed in the primary lens fiber cells at 17.5 pc. In the postnatal mouse eye, the three small GTPases were significantly expressed in both endothelial and epithelial cells of mouse cornea, epithelial cells of the ocular lens, photoreceptors, horizontal/amacrine/Muller's cells, and some ganglian cells of the retina. Much lower level of expression was observed in the corneal stroma fibroblasts, lens fiber cells, and the inner and outer plexiform layers of the mouse retina. In the adult mouse eye, all three Rho-GTPases were expressed in corneal epithelial cells and retina. However, only RhoA protein was detected in corneal endothelial cells and Rac1 protein detected in the ocular lens.
The strong expression of the three small GTPases in the cornea, lens, and retina of mouse eye at embryonic 17.5 pc and postnatal stage suggests their important functions for the morphogenesis of the different compartments of the mouse eye. Particularly, high levels of expression of RhoA, Rac1, and Cdc42 in embryonic lens fiber cells suggest their involvement in differentiation of primary lens fiber cells. In the adult mouse eye, all three Rho-GTPases seem to be involved in differentiation of corneal epithelial cells and retina, however, RhoA alone may be required for endothelial cell differentiation and Rac1 likely plays an important role in supporting continuous lens growth and maintenance of lens transparency.
Molecular vision 02/2007; 13:1144-53. · 2.20 Impact Factor
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ABSTRACT: The Rho GTPases are important regulators of neurite outgrowth and pathfinding. We have recently reported that a Rho-family guanine nucleotide exchange factor, GEFT, modulates dendrite spine morphology and basal neurite outgrowth in primary hippocampal neurons and Neuro2A cells, respectively. Here we demonstrate that GEFT protein is highly expressed in all regions of the brain and is highly up-regulated upon treatment of Neuro2A cells with retinoic acid and dibutyric cAMP, which promote dendrite and axon-like neurite extensions, respectively. Within retinoic acid-induced neurite extensions, GEFT is localized to actin-enriched regions in the primary neurites, with little or no expression from secondary branches. Dibutyric cAMP-induced neurite extensions are highly concentrated for GEFT at the actin-rich distal tip of the growth cone. Additionally, we demonstrate that GEFT promotes neurite outgrowth in undifferentiated as well as differentiated Neuro2A cells. Together, our data provide new evidence suggesting that GEFT is an important regulator of multiple processes involved in axon and dendrite formation.
Journal of Neuroscience Research 06/2006; 83(7):1151-9. · 2.74 Impact Factor
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ABSTRACT: Rho family guanine nucleotide exchange factors (GEFs) regulate diverse cellular processes including cytoskeletal reorganization, cell adhesion, and differentiation via activation of the Rho GTPases. However, no studies have yet implicated Rho-GEFs as molecular regulators of the mesenchymal cell fate decisions which occur during development and repair of tissue damage. In this study, we demonstrate that the steady-state protein level of the Rho-specific GEF GEFT is modulated during skeletal muscle regeneration and that gene transfer of GEFT into cardiotoxin-injured mouse tibialis anterior muscle exerts a powerful promotion of skeletal muscle regeneration in vivo. In order to molecularly characterize this regenerative effect, we extrapolate the mechanism of action by examining the consequence of GEFT expression in multipotent cell lines capable of differentiating into a number of cell types, including muscle and adipocyte lineages. Our data demonstrate that endogenous GEFT is transcriptionally upregulated during myogenic differentiation and downregulated during adipogenic differentiation. Exogenous expression of GEFT promotes myogenesis of C2C12 cells via activation of RhoA, Rac1, and Cdc42 and their downstream effector proteins, while a dominant-negative mutant of GEFT inhibits this process. Moreover, we show that GEFT inhibits insulin-induced adipogenesis in 3T3L1 preadipocytes. In summary, we provide the first evidence that the Rho family signaling pathways act as potential regulators of skeletal muscle regeneration and provide the first reported molecular mechanism illustrating how a mammalian Rho family GEF controls this process by modulating mesenchymal cell fate decisions.
Molecular and Cellular Biology 01/2006; 25(24):11089-101. · 5.53 Impact Factor
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ABSTRACT: Cell polarity and asymmetric cell division are fundamental traits of all living cells and play an essential role in embryonic development, neuronal cell chirality formation, and maintenance of mammalian epithelial cell morphology. Heterotrimeric GTP-binding proteins (G proteins) are involved in directing cell polarity and asymmetric cell division in different organisms. However, the mechanism for G-protein-mediated cell polarity and asymmetric cell division is poorly understood. In this study, we have demonstrated that G-protein-activated phospholipase C-beta (PLC-beta) interacts with cell polarity proteins Par3 and Par6 (Par: partition-defective) to form protein complexes and to mediate downstream signal transduction. The interactions between PLC-beta and Par proteins are direct and require the extreme C-terminal-specific sequence motifs of PLC-beta and the PDZ (PSD95/Dlg/ZO-1) domains of Par proteins. Binding of Par proteins with PLC-beta stimulates PLC-beta enzymatic activity, leading to the hydrolysis of phosphatidylinositol-4,5-bisphosphate, and the production of diacylglycerol and inositol 1,4,5-triphosphate, important mediators in cell polarity and cell asymmetric division processes. Furthermore, we have shown that coexpression of PLC-beta with Par proteins induces transcriptional activation coupled to intracellular Ca2+ and the Wnt signaling pathway. Therefore, our data suggest that the interaction of PLC-beta with cell polarity Par proteins may serve as a nexus to transduce extracellular signals to transcriptional regulation through G-protein-mediated signaling pathway in cell polarity and cell asymmetric division.
Oncogene 07/2005; 24(26):4293-300. · 6.37 Impact Factor
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ABSTRACT: Cell cycle progression of Saccharomyces cerevisiae cells was monitored in continuous cultures limited for glucose or nitrogen. The G1 cell cycle phase, before initiation of DNA replication, did not exclusively expand when growth rate decreased. Especially during nitrogen limitation, non-G1 phases expanded almost as much as G1. In addition, cell size remained constant as a function of growth rate. These results contrast with current views that growth requirements are met before initiation of DNA replication, and suggest that distinct nutrient limitations differentially impinge on cell cycle progression.
Archives of Microbiology 11/2004; 182(4):326-30. · 1.43 Impact Factor
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ABSTRACT: Exposure to UV causes a response in yeast and mammalian cells, which is distinct from the response to DNA damage. We report that the mitogen-activated protein kinase Slt2p is involved in this response in Saccharomyces cerevisiae. Thus, budding yeast and mammalian cells respond to UV by using very similar signal transduction pathways.
Current Microbiology 08/2004; 49(1):32-4. · 1.82 Impact Factor
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ABSTRACT: Rho kinase signaling plays an important role in the oncogenic process largely through its regulation of F-actin dynamics, and inhibition of this pathway results in reduction in tumor volume and metastasis across a number of tumor types. While the cytoskeletal-regulatory role of Rho kinase has been a topic of in-depth study, the mechanisms linking Rho kinase to altered gene expression are largely unknown.
Global gene expression analysis was performed on melanoma tumors treated with sham or the small molecule inhibitor Y27632.
Inhibition of Rho kinase activity in melanoma tumors results in a statistically significant change in gene transcription of 94 genes, many of which are critically involved in tumor initiation and progression.
In addition to regulating tumorigenesis through modulation of the phosphoproteome, Rho kinase signaling also contributes to the regulation of the tumor transcriptome.
Cancer genomics & proteomics. 8(2):77-85.
