Rational identification of a Cdc42 inhibitor presents a new regimen for long-term hematopoietic stem cell mobilization.

Mobilization of hematopoietic stem cells (HSCs) from bone marrow (BM) to peripheral blood (PB) by cytokine granulocyte colony-stimulating factor (G-CSF) or the chemical antagonist of CXCR4, AMD3100, is important in the treatment of blood diseases. Due to clinical conditions of each application, there is a need for continued improvement of HSC mobilization regimens. Previous studies have shown that genetic ablation of the Rho GTPase Cdc42 in HSCs results in their mobilization without affecting survival.

Combined rational design and a high throughput screening platform for identifying chemical inhibitors of a Ras-activating enzyme.

The Ras family small GTPases regulate multiple cellular processes, including cell growth, survival, movement, and gene expression, and are intimately involved in cancer pathogenesis. Activation of these small GTPases is catalyzed by a special class of enzymes, termed guanine nucleotide exchange factors (GEFs). Herein, we developed a small molecule screening platform for identifying lead hits targeting a Ras GEF enzyme, SOS1.

Small-molecule inhibitors targeting G-protein-coupled Rho guanine nucleotide exchange factors.

The G-protein-mediated Rho guanine nucleotide exchange factor (GEF)-Rho GTPase signaling axis has been implicated in human pathophysiology and is a potential therapeutic target. By virtual screening of chemicals that fit into a surface groove of the DH-PH domain of LARG, a G-protein-regulated Rho GEF involved in RhoA activation, and subsequent validations in biochemical assays, we have identified a class of chemical inhibitors represented by Y16 that are active in specifically inhibiting LARG binding to RhoA. Y16 binds to the junction site of the DH-PH domains of LARG with a ∼80 nM K(d) and suppresses LARG catalyzed RhoA activation dose dependently.

Rational design of Rho GTPase-targeting inhibitors.

Rho GTPases have been implicated in diverse cellular functions and are potential therapeutic targets in inflammation, cancer, and neurologic diseases. Virtual screening of compounds that fit into surface grooves of RhoA known to be critical for guanine nucleotide exchange factor (GEF) interaction produced chemical candidates with minimized docking energy. Subsequent screening for inhibitory activity of RhoA binding to the Rho-GEF, LARG, identified a Rho-specific inhibitor as a lead compound capable of blocking RhoA-LARG interaction and RhoA activation by LARG specifically and dose dependently.

Rational design of small molecule inhibitors targeting RhoA subfamily Rho GTPases.

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.

A Trio-RhoA-Shroom3 pathway is required for apical constriction and epithelial invagination.

Epithelial invagination is a common feature of embryogenesis. An example of invagination morphogenesis occurs during development of the early eye when the lens placode forms the lens pit. This morphogenesis is accompanied by a columnar-to-conical cell shape change (apical constriction or AC) and is known to be dependent on the cytoskeletal protein Shroom3.

Gene targeting implicates Cdc42 GTPase in GPVI and non-GPVI mediated platelet filopodia formation, secretion and aggregation.

Background: Cdc42 and Rac1, members of the Rho family of small GTPases, play critical roles in actin cytoskeleton regulation. We have shown previously that Rac1 is involved in regulation of platelet secretion and aggregation. However, the role of Cdc42 in platelet activation remains controversial.

Rho GTPases in hematopoietic stem/progenitor cell migration.

Rho GTPases including RhoA, Rac1, and Cdc42 are a class of intracellular signaling proteins critical for the regulation of cytoskeleton organization, adhesion, and migration. Molecular mechanisms of mammalian cell migration were first revealed in fibroblasts where RhoA, Rac1, and Cdc42 facilitate in the multistep process including establishment and maintenance of polarity, formation of actin-rich protrusions, remodeling of adhesive contacts, and generation of force. In hematopoietic stem/progenitor cells, Rho GTPases relay signals from chemokines and cytokines such as SDF-1α and SCF to the actin and microtubule cytoskeleton through effector kinases and/or adaptor molecules that affect adhesion or transcription.

R-Ras and Rac GTPase cross-talk regulates hematopoietic progenitor cell migration, homing, and mobilization.

Adult hematopoietic progenitor cells (HPCs) are maintained by highly coordinated signals in the bone marrow. The molecular mechanisms linking intracellular signaling network of HPCs with their microenvironment remain poorly defined. The Rho family GTPase Rac1/Rac2 has previously been implicated in cell functions involved in HPC maintenance, including adhesion, migration, homing, and mobilization.

Increased susceptibility of 129SvEvBrd mice to IgE-Mast cell mediated anaphylaxis.

Background: Experimental analyses have identified strain-dependent factors that regulate susceptibility to anaphylaxis in mice. We assessed the susceptibility of the widely used 129SvEvBrd (also known as 129S5) mouse strain to IgE/mast cell-mediated anaphylaxis as compared to BALB/c. Mice were subjected to passive and oral Ovalbumin [OVA]-induced active anaphylaxis.

Defective homing is associated with altered Cdc42 activity in cells from patients with Fanconi anemia group A.

Previous studies showed that Fanconi anemia (FA) murine stem cells have defective reconstitution after bone marrow (BM) transplantation. The mechanism underlying this defect is not known. Here, we report defective homing of FA patient BM progenitors transplanted into mouse models.

Cdc42 critically regulates the balance between myelopoiesis and erythropoiesis.

The Rho GTPase Cdc42 regulates adhesion, migration, and homing, as well as cell cycle progression, of hematopoietic stem cells, but its role in multilineage blood development remains unclear. We report here that inducible deletion of cdc42 in cdc42-floxed mouse bone marrow by the interferon-responsive, Mx1-Cre-mediated excision led to myeloid and erythroid developmental defects. Cdc42 deletion affected the number of early myeloid progenitors while suppressing erythroid differentiation.

p200 RhoGAP promotes cell proliferation by mediating cross-talk between Ras and Rho signaling pathways.

p200 RhoGAP, a member of the Rho GTPase-activating protein (RhoGAP) family, was previously implicated in the regulation of neurite outgrowth through its RhoGAP activity. Here we show that ectopic expression of p200 RhoGAP stimulates fibroblast cell proliferation and cell cycle progression, leading to transformation. The morphology of the foci induced by p200 RhoGAP is distinct from that formed by Rac or Rho activation but similar to that induced by oncogenic Ras, raising the possibility that p200 RhoGAP may engage Ras signaling.

Repetitive deformation activates focal adhesion kinase and ERK mitogenic signals in human Caco-2 intestinal epithelial cells through Src and Rac1.

Intestinal epithelial cells are subject to repetitive deformation during peristalsis and villous motility, whereas the mucosa atrophies during sepsis or ileus when such stimuli are abnormal. Such repetitive deformation stimulates intestinal epithelial proliferation via focal adhesion kinase (FAK) and extracellular signal-regulated kinases (ERK). However, the upstream mediators of these effects are unknown.

Concerted regulation of cell dynamics by BNIP-2 and Cdc42GAP homology/Sec14p-like, proline-rich, and GTPase-activating protein domains of a novel Rho GTPase-activating protein, BPGAP1.

RhoA, Cdc42, and Rac1 are small GTPases that regulate cytoskeletal reorganization leading to changes in cell morphology and cell motility. Their signaling pathways are activated by guanine nucleotide exchange factors and inactivated by GTPase-activating proteins (GAPs). We have identified a novel RhoGAP, BPGAP1 (for BNIP-2 and Cdc42GAP Homology (BCH) domain-containing, Proline-rich and Cdc42GAP-like protein subtype-1), that is ubiquitously expressed and shares 54% sequence identity to Cdc42GAP/p50RhoGAP.

The BNIP-2 and Cdc42GAP homology/Sec14p-like domain of BNIP-Salpha is a novel apoptosis-inducing sequence.

We have cloned the cDNAs for two novel human proteins, designated BNIP-Salpha and beta (for BNIP-2 Similar) that are homologous to BNIP-2, a previously known Bcl-2 and E1B-associated protein. The BNIP-S gene encodes two protein isoforms; the longer protein (BNIP-Salpha) contains a complete BNIP-2 and Cdc42GAP Homology (BCH) domain, a novel protein domain that we recently identified, whereas its shorter variant (BNIP-Sbeta) lacks the full BCH domain as a result of an alternative RNA splicing that introduces a nonsense intron. Primer-specific reverse-transcription PCR revealed that both BNIP-Salpha and BNIP-Sbeta mRNA are differentially expressed in various cells and tissues.