Childrens Hospital Research Foundation

Cincinnati, OH, United States

Childrens Hospital Research Foundation

Cincinnati, OH, United States

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Godin J.,University of Liège | Thomas N.,University of Liège | Laguesse S.,University of Liège | Malinouskaya L.,University of Liège | And 13 more authors.
Developmental Cell | Year: 2012

The migration of cortical interneurons is characterized by extensive morphological changes that result from successive cycles of nucleokinesis and neurite branching. Their molecular bases remain elusive, and the present work describes how p27Kip1 controls cell-cycle-unrelated signaling pathways to regulate these morphological remodelings. Live imaging reveals that interneurons lacking p27Kip1 show delayed tangential migration resulting from defects in both nucleokinesis and dynamic branching of the leading process. At the molecular level, p27Kip1 is a microtubule-associated protein that promotes polymerization of microtubules in extending neurites, thereby contributing to tangential migration. Furthermore, we show that p27Kip1 controls actomyosin contractions that drive both forward translocation of the nucleus and growth cone splitting. Thus, p27Kip1 cell-autonomously controls nucleokinesis and neurite branching by regulating both actin and microtubule cytoskeletons.

Plageman Jr. T.F.,United Medical Systems | Plageman Jr. T.F.,Childrens Hospital Research Foundation | Chung M.-I.,University of Texas at Austin | Lou M.,Lamar University | And 8 more authors.
Development | Year: 2010

Embryonic development requires a complex series of relative cellular movements and shape changes that are generally referred to as morphogenesis. Although some of the mechanisms underlying morphogenesis have been identified, the process is still poorly understood. Here, we address mechanisms of epithelial morphogenesis using the vertebrate lens as a model system. We show that the apical constriction of lens epithelial cells that accompanies invagination of the lens placode is dependent on Shroom3, a molecule previously associated with apical constriction during morphogenesis of the neural plate. We show that Shroom3 is required for the apical localization of F-actin and myosin II, both crucial components of the contractile complexes required for apical constriction, and for the apical localization of Vasp, a Mena family protein with F-actin anti-capping function that is also required for morphogenesis. Finally, we show that the expression of Shroom3 is dependent on the crucial lens-induction transcription factor Pax6. This provides a previously missing link between lens-induction pathways and the morphogenesis machinery and partly explains the absence of lens morphogenesis in Pax6-deficient mutants. © 2010. Published by The Company of Biologists Ltd.

Raghu H.,Childrens Hospital Research Foundation | Cruz C.,Childrens Hospital Research Foundation | Rewerts C.L.,Childrens Hospital Research Foundation | Frederick M.D.,Childrens Hospital Research Foundation | And 5 more authors.
Blood | Year: 2015

Rheumatoid arthritis is a chronic inflammatory disease characterized by synovial hyperplasia, inflammatory cell infiltration, irreversible cartilage and bone destruction, and exuberant coagulation system activity within joint tissue. Here, we demonstrate that the coagulation transglutaminase, factor XIII (fXIII), drives arthritis pathogenesis by promoting local inflammatory and tissue degradative and remodeling events. All pathological features of collagen-induced arthritis (CIA) were significantly reduced in fXIII-deficient mice. However, the most striking difference in outcome was the preservation of cartilage and bone in fXIIIA-/- mice concurrent with reduced osteoclast numbers and activity. The local expression of osteoclast effectors receptor activator ofnuclear factor-κB ligand (RANKL) and tartrate resistant acid phosphatase were significantly diminished in CIA-challenged and even unchallenged fXIIIA-/- mice relative to wild-type animals, but were similar in wild-typeandfibrinogen-deficientmice. Impairedosteoclast formationin fXIIIA-/- mice was not due to an inherent deficiency of monocyte precursors, but it was linked to reduced RANKL-driven osteoclast formation. Furthermore, treatment ofmice with the pan-transglutaminase inhibitor cystamine resulted in significantly diminishedCIA pathology and local markers of osteoclastogenesis. Thus, eliminating fXIIIA limits inflammatory arthritis and protects from cartilage and bone destruction in part through mechanisms linked to reduced RANKL-mediated osteoclastogenesis. In summary, therapeutic strategies targeting fXIII activity may prove beneficial in limiting arthropathies and other degenerative bone diseases. © 2015 by The American Society of Hematology.

Shang X.,Childrens Hospital Research Foundation | Marchioni F.,Childrens Hospital Research Foundation | Sipes N.,Childrens Hospital Research Foundation | Evelyn C.R.,Childrens Hospital Research Foundation | And 5 more authors.
Chemistry and Biology | Year: 2012

Rho GTPases have been implicated in diverse cellular functions and are potential therapeutic targets. By virtual screening, we have identified a Rho-specific inhibitor, Rhosin. Rhosin contains two aromatic rings tethered by a linker, and it binds to the surface area sandwiching Trp58 of RhoA with a submicromolar Kd and effectively inhibits GEF-catalyzed RhoA activation. In cells, Rhosin specifically inhibited RhoA activity and RhoA-mediated cellular function without affecting Cdc42 or Rac1 signaling activities. By suppressing RhoA or RhoC activity, Rhosin could inhibit mammary sphere formation by breast cancer cells, suppress invasion of mammary epithelial cells, and induce neurite outgrowth of PC12 cells in synergy with NGF. Thus, the rational designed RhoA subfamily-specific small molecule inhibitor is useful for studying the physiological and pathologic roles of Rho GTPase. © 2012 Elsevier Ltd All rights reserved.

Plageman T.F.,Childrens Hospital Research Foundation | Zacharias A.L.,University of Michigan | Gage P.J.,University of Michigan | Lang R.A.,Childrens Hospital Research Foundation | Lang R.A.,University of Cincinnati
Developmental Biology | Year: 2011

The cytoskeletal protein Shroom3 is a potent inducer of epithelial cell shape change and is required for lens and neural plate morphogenesis. Analysis of gut morphogenesis in Shroom3 deficient mouse embryos revealed that the direction of gut rotation is also disrupted. It was recently established that Pitx2-dependent, asymmetrical cellular behaviors in the dorsal mesentery (DM) of the early mid-gut, a structure connecting the gut-tube to the rest of the embryo, contribute to the direction of gut rotation in chicken embryos by influencing the direction of the dorsal mesenteric tilt. Asymmetric cell shapes in the DM epithelium are hypothesized to contribute to the tilt, however, it is unclear what lies downstream of Pitx2 to alter epithelial cell shape. The cells of the left DM epithelium in either Pitx2 or Shroom3 deficient embryos are shorter and wider than those in control embryos and resemble the shape of those on the right, demonstrating that like Pitx2, Shroom3 is required for cell shape asymmetry and the leftward DM tilt. Because N-cadherin expression is specific to the left side and is Pitx2 dependent, we determined whether Shroom3 and N-cadherin function together to regulate cell shape in the left DM epithelium. Analysis of mouse embryos lacking one allele of both Shroom3 and N-cadherin revealed that they possess shorter and wider left epithelial DM cells when compared with Shroom3 or N-cadherin heterozygous embryos. This indicates a genetic interaction. Together these data provide evidence that Shroom3 and N-cadherin function cooperatively downstream of Pitx2 to directly regulate cell shape changes necessary for early gut tube morphogenesis. © 2011 Elsevier Inc.

Evelyn C.R.,Childrens Hospital Research Foundation | Duan X.,Childrens Hospital Research Foundation | Biesiada J.,Childrens Hospital Research Foundation | Seibel W.L.,Childrens Hospital Research Foundation | And 3 more authors.
Chemistry and Biology | Year: 2014

Summary Ras GTPases regulate intracellular signaling involved in cell proliferation. Elevated Ras signaling activity has been associated with human cancers. Ras activation is catalyzed by guanine nucleotide exchange factors (GEFs), of which SOS1 is a major member that transduces receptor tyrosine kinase signaling to Ras. We have developed a rational approach coupling virtual screening with experimental screening in identifying small-molecule inhibitors targeting the catalytic site of SOS1 and SOS1-regulated Ras activity. A lead inhibitor, NSC-658497, was found to bind to SOS1, competitively suppress SOS1-Ras interaction, and dose-dependently inhibit SOS1 GEF activity. Mutagenesis and structure-activity relationship studies map the NSC-658497 site of action to the SOS1 catalytic site, and define the chemical moieties in the inhibitor essential for the activity. NSC-658497 showed dose-dependent efficacy in inhibiting Ras, downstream signaling activities, and associated cell proliferation. These studies establish a proof of principle for rational design of small-molecule inhibitors targeting Ras GEF enzymatic activity. ©2014 Elsevier Ltd. All rights reserved.

Plageman Jr. T.F.,United Medical Systems | Plageman Jr. T.F.,Childrens Hospital Research Foundation | Lang R.A.,United Medical Systems | Lang R.A.,Childrens Hospital Research Foundation | Lang R.A.,University of Cincinnati
PLoS ONE | Year: 2012

Genetic deletion of mouse genes has played a crucial role in our understanding of embryonic eye development. Transgenic, tissue specific Cre recombinase expression in various eye structures has facilitated these experiments. However, an early expressing, temporally-regulated, optic vesicle-specific Cre line has not been available. In this report, we detail the generation and analysis of a knock-in, inducible Cre line designed to drive recombination specifically within the Rx expression domain. Crossing this line with a reporter line demonstrates that recombination can be mediated within the early optic vesicle and throughout retinal development. Recombination can also be mediated in the Rx-expressing, ventral diencephalon and future posterior pituitary gland. Furthermore, it was demonstrated that dietary doxycycline could effectively modulate Cre activity. This line has the potential to facilitate conditional knock-out experimentation to study early retina and/or posterior pituitary development. © 2012 Plageman, Lang.

McLendon P.M.,Childrens Hospital Research Foundation | Robbins J.,Childrens Hospital Research Foundation
Circulation Research | Year: 2015

Baseline physiological function of The mammalian heart is under The constant threat of environmental or intrinsic pathological insults. Cardiomyocyte proteins are thus subject to unremitting pressure to function optimally, and this depends on them assuming and maintaining proper conformation. This review explores The multiple defenses a cell may use for its proteins to assume and maintain correct protein folding and conformation. There are multiple quality control mechanisms to ensure that nascent polypeptides are properly folded and mature proteins maintain their functional conformation. When proteins do misfold, either in The face of normal or pathological stimuli or because of intrinsic mutations or post-translational modifications, they must either be refolded correctly or recycled. In The absence of these corrective processes, they may become toxic to The cell. Herein, we explore some of The underlying mechanisms that lead to proteotoxicity. The continued presence and chronic accumulation of misfolded or unfolded proteins can be disastrous in cardiomyocytes because these misfolded proteins can lead to aggregation or The formation of soluble peptides that are proteotoxic. This in turn leads to compromised protein quality control and precipitating a downward spiral of The cell's ability to maintain protein homeostasis. Some underlying mechanisms are discussed and The therapeutic potential of interfering with proteotoxicity in The heart is explored. © 2015 American Heart Association, Inc.

Zhang S.,Childrens Hospital Research Foundation | Zhou X.,Childrens Hospital Research Foundation | Lang R.A.,Childrens Hospital Research Foundation | Guo F.,Childrens Hospital Research Foundation
PLoS ONE | Year: 2012

RhoA is a member of the Rho family small GTPases that are implicated in various cell functions including proliferation and survival. However, the physiological role of RhoA in vivo remains largely unknown. Here, we deleted RhoA in the B cell and hematopoietic stem cell (HSC) populations in RhoA flox/flox mice with CD19 and Mx promoter-driven Cre expression, respectively. Deletion of RhoA by CD19 Cre/+ significantly blocked B cell development in spleen, leading to a marked reduction in the number of transitional, marginal zone, and follicular B cells. Surprisingly, neither B cell proliferation in response to either LPS or B cell receptor (BCR) engagement nor B cell survival rate in vivo was affected by RhoA deletion. Furthermore, RhoA -/- B cells, like control cells, were rescued from apoptosis by BCR crosslinking in vitro. In contrast, RhoA deficiency led to a defect in B cell activating factor (BAFF)-mediated B cell survival that was associated with a dampened expression of BAFF receptor and a loss of BAFF-mediated Akt activation. Finally, HSC deletion of RhoA by Mx-Cre severely reduced proB/preB and immature B cell populations in bone marrow while common lymphoid progenitors were increased, indicating that RhoA is also required for B cell progenitor/precursor differentiation. Taken together, our results uncover an important role for RhoA at multiple stages of B cell development. © 2012 Zhang et al.

Raghu H.,Childrens Hospital Research Foundation | Flick M.J.,Childrens Hospital Research Foundation
Current Pharmaceutical Biotechnology | Year: 2011

Fibrinogen is a provisional matrix protein of the coagulation system that following proteolytic cleavage by the protease thrombin polymerizes to form fibrin, the structural basis of the blood clot. Fibrin polymer formation at sites of vessel injury is critical to normal hemostasis. However, fibrin deposition within damaged tissues is also a common pathological feature of inflammatory diseases, including rheumatoid arthritis. Fibrin deposition has been readily detected along articular surfaces, within inflamed hyperplastic synovial tissue, and as a component of insoluble "rice bodies" within the synovial fluid of arthritic joints. Recent data has suggested that fibrin deposition within inflamed tissues is not simply a reflection of a disease process but rather actively contributes to disease pathogenesis. One mechanism that has been demonstrated to directly link fibrin(ogen) to the regulation of inflammation is the ability of fibrin(ogen) to serve as a ligand for cell-surface receptors, particularly integrins. Indeed, engagement of fibrin(ogen) by the leukocyte integrin receptor α Mβ 2 appears to be a common and fundamental event driving local inflammation. Recent studies have demonstrated that eliminating fibrin(ogen)- α Mβ 2 interactions can significantly limit the progression of multiple inflammatory diseases, including arthritis, without compromising the ability of fibrinogen to function in coagulation. These exciting findings have opened the door to new opportunities for targeting fibrinogen as an inflammatory mediator while leaving intact its hemostatic properties. © 2011 Bentham Science Publishers.

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