Small GTP-binding protein Rho-mediated signaling promotes proliferation of rheumatoid synovial fibroblasts

Rho is a major small GTP-binding protein that is involved in the regulation of various cell functions, including proliferation and cell migration, through activation of multiple signaling molecules in various types of cells. We studied its roles in synovial fibroblasts (SFs) in patients with rheumatoid arthritis (RA) and clarified its relevance to RA synovitis, with the following results. 1)We found that the thrombin receptor was overexpressed on RA synovial fibroblasts (RA SFs) and that thrombin induced a marked proliferation and progression of the cell cycle to the S phase in these cells. 2)We also found that thrombin efficiently activated Rho. 3)Rho activation and proliferation and the progression of the cell cycle to the S phase were completely blocked by p115RGS (an N-terminal regulator of the G-protein signaling domain of p115RhoGEF) and by the C-terminal fragments of Gα13 (an inhibitor of the interaction of receptors with G13). 4)Thrombin induced the secretion of IL-6 by RA SFs, but this action was blocked by p115RGS or Gα13. Our findings show that the actions of thrombin on the proliferation of RA SFs, cell-cycle progression to the S phase, and IL-6 secretion were mainly mediated by the G13 and RhoGEF pathways. These results suggest that p115RGS and Gα13 could be potent inhibitors of such functions. A rational design of future therapeutic strategies for RA synovitis could perhaps include the exploitation of the Rho pathway to directly reduce the growth of synovial cells

Pepducin-mediated cardioprotection via β-arrestin-biased β2-adrenergic receptor-specific signaling

Reperfusion as a therapeutic intervention for acute myocardial infarction-induced cardiac injury itself induces further cardiomyocyte death. β-arrestin (βarr)-biased β-adrenergic receptor (βAR) activation promotes survival signaling responses in vitro; thus, we hypothesize that this pathway can mitigate cardiomyocyte death at the time of reperfusion to better preserve function. However, a lack of efficacious βarr-biased orthosteric small molecules has prevented investigation into whether this pathway relays protection against ischemic injury in vivo. We recently demonstrated that the pepducin ICL1-9, a small lipidated peptide fragment designed from the first intracellular loop of β2AR, allosterically engaged pro-survival signaling cascades in a βarr-dependent manner in vitro. Thus, in this study we tested whether ICL1-9 relays cardioprotection against ischemia/reperfusion (I/R)-induced injury in vivo. Methods: Wild-type (WT) C57BL/6, β2AR knockout (KO), βarr1KO and βarr2KO mice received intracardiac injections of either ICL1-9 or a scrambled control pepducin (Scr) at the time of ischemia (30 min) followed by reperfusion for either 24 h, to assess infarct size and cardiomyocyte death, or 4 weeks, to monitor the impact of ICL1-9 on long-term cardiac structure and function. Neonatal rat ventricular myocytes (NRVM) were used to assess the impact of ICL1-9 versus Scr pepducin on cardiomyocyte survival and mitochondrial superoxide formation in response to either serum deprivation or hypoxia/reoxygenation (H/R) in vitro and to investigate the associated mechanism(s). Results: Intramyocardial injection of ICL1-9 at the time of I/R reduced infarct size, cardiomyocyte death and improved cardiac function in a β2AR- and βarr-dependent manner, which led to improved contractile function early and less fibrotic remodeling over time. Mechanistically, ICL1-9 attenuated mitochondrial superoxide production and promoted cardiomyocyte survival in a RhoA/ROCK-dependent manner. RhoA activation could be detected in cardiomyocytes and whole heart up to 24 h post-treatment, demonstrating the stability of ICL1-9 effects on βarr-dependent β2AR signaling. Conclusion: Pepducin-based allosteric modulation of βarr-dependent β2AR signaling represents a novel therapeutic approach to reduce reperfusion-induced cardiac injury and relay long-term cardiac remodeling benefits

Leukemia-associated RhoGEF (LARG) is a novel RhoGEF in cytokinesis and required for the proper completion of abscission.

Proper completion of mitosis requires the concerted effort of multiple RhoGEFs. Here we show that leukemia-associated RhoGEF (LARG), a RhoA-specific RGS-RhoGEF, is required for abscission, the final stage of cytokinesis, in which the intercellular membrane is cleaved between daughter cells. LARG colocalizes with α-tubulin at the spindle poles before localizing to the central spindle. During cytokinesis, LARG is condensed in the midbody, where it colocalizes with RhoA. HeLa cells depleted of LARG display apoptosis during cytokinesis with unresolved intercellular bridges, and rescue experiments show that expression of small interfering RNA-resistant LARG prevents this apoptosis. Moreover, live cell imaging of LARG-depleted cells reveals greatly delayed fission kinetics in abscission in which a population of cells with persistent bridges undergoes apoptosis; however, the delayed fission kinetics is rescued by Aurora-B inhibition. The formation of a Flemming body and thinning of microtubules in the intercellular bridge of cells depleted of LARG is consistent with a defect in late cytokinesis, just before the abscission event. In contrast to studies of other RhoGEFs, particularly Ect2 and GEF-H1, LARG depletion does not result in cytokinetic furrow regression nor does it affect internal mitotic timing. These results show that LARG is a novel and temporally distinct RhoGEF required for completion of abscission

Plasma Membrane Association but Not Midzone Recruitment of RhoGEF ECT2 Is Essential for Cytokinesis

Cytokinesis, the final step of cell division, begins with the formation of a cleavage furrow. How the mitotic spindle specifies the furrow at the equator in animal cells remains unknown. Current models propose that the concentration of the RhoGEF ECT2 at the spindle midzone and the equatorial plasma membrane directs furrow formation. Using chemical genetic and optogenetic tools, we demonstrate that the association of ECT2 with the plasma membrane during anaphase is required and sufficient for cytokinesis. Local membrane targeting of ECT2 leads to unilateral furrowing, highlighting the importance of local ECT2 activity. ECT2 mutations that prevent centralspindlin binding compromise concentration of ECT2 at the midzone and equatorial membrane but sustain cytokinesis. While the association of ECT2 with the plasma membrane is essential for cytokinesis, our data suggest that ECT2 recruitment to the spindle midzone is insufficient to account for equatorial furrowing and may act redundantly with yet-uncharacterized signals.Cancer Research U

Calcium-Sensing Receptor Regulates Cytosolic [Ca 2+ ] and Plays a Major Role in the Development of Pulmonary Hypertension.

Pulmonary arterial hypertension (PAH) is a progressive disease characterized by elevated pulmonary vascular resistance (PVR) leading to right heart failure and premature death. The increased PVR results in part from pulmonary vascular remodeling and sustained pulmonary vasoconstriction. Excessive pulmonary vascular remodeling stems from increased pulmonary arterial smooth muscle cell (PASMC) proliferation and decreased PASMC apoptosis. A rise in cytosolic free Ca2+ concentration ([Ca2+]cyt) in PASMC is a major trigger for pulmonary vasoconstriction and a key stimulus for PASMC proliferation and migration, both contributing to the development of pulmonary vascular remodeling. PASMC from patients with idiopathic PAH (IPAH) have increased resting [Ca2+]cyt and enhanced Ca2+ influx. Enhanced Ca2+ entry into PASMC due to upregulation of membrane receptors and/or Ca2+ channels may contribute to PASMC contraction and proliferation and to pulmonary vasoconstriction and pulmonary vascular remodeling. We have shown that the extracellular Ca2+-sensing receptor (CaSR), which is a member of G protein-coupled receptor (GPCR) subfamily C, is upregulated, and the extracellular Ca2+-induced increase in [Ca2+]cyt is enhanced in PASMC from patients with IPAH in comparison to PASMC from normal subjects. Pharmacologically blockade of CaSR significantly attenuate the development and progression of experimental pulmonary hypertension in animals. Additionally, we have demonstrated that dihydropyridine Ca2+ channel blockers (e.g., nifedipine), which are used to treat PAH patients but are only effective in 15-20% of patients, activate CaSR resulting in an increase in [Ca2+]cyt in IPAH-PASMC, but not normal PASMC. Our data indicate that CaSR functionally couples with transient receptor potential canonical (TRPC) channels to mediate extracellular Ca2+-induced Ca2+ influx and increase in [Ca2+]cyt in IPAH-PASMC. Upregulated CaSR is necessary for the enhanced extracellular Ca2+-induced increase in [Ca2+]cyt and the augmented proliferation of PASMC in patients with IPAH. This review will highlight the pathogenic role of CaSR in the development and progression of PAH

Wnt5a induces ROR1 to recruit cortactin to promote breast-cancer migration and metastasis.

ROR1 is a conserved oncoembryonic surface protein expressed in breast cancer. Here we report that ROR1 associates with cortactin in primary breast-cancer cells or in MCF7 transfected to express ROR1. Wnt5a also induced ROR1-dependent tyrosine phosphorylation of cortactin (Y421), which recruited ARHGEF1 to activate RhoA and promote breast-cancer-cell migration; such effects could be inhibited by cirmtuzumab, a humanized mAb specific for ROR1. Furthermore, treatment of mice bearing breast-cancer xenograft with cirmtuzumab inhibited cortactin phosphorylation in vivo and impaired metastatic development. We established that the proline at 841 of ROR1 was required for it to recruit cortactin and ARHGEF1, activate RhoA, and enhance breast-cancer-cell migration in vitro or development of metastases in vivo. Collectively, these studies demonstrate that the interaction of ROR1 with cortactin plays an important role in breast-cancer-cell migration and metastasis

The G protein-coupled receptor P2RY8 and follicular dendritic cells promote germinal center confinement of B cells, whereas S1PR3 can contribute to their dissemination.

The orphan Gα13-coupled receptor P2RY8 is mutated in human germinal center (GC)-derived lymphomas and was recently found to promote B cell association with GCs in a mouse model. Here we establish that P2RY8 promotes clustering of activated B cells within follicles in a follicular dendritic cell (FDC)-dependent manner. Although mice lack a P2RY8 orthologue, we show that mouse GC B cell clustering is also dependent on FDCs acting to support the function of a Gα13-coupled receptor. Mutations in GNA13 and its downstream effector ARHGEF1 are associated with the development of disseminated GC-derived lymphomas. We find that egress of Gna13 mutant GC B cells from lymph nodes in the mouse depends on sphingosine-1-phosphate receptor-3. These findings provide evidence that FDCs promote GC confinement of both human and mouse GC B cells via Gα13-dependent pathways, and they show that dissemination of Gα13-deficient GC B cells additionally requires an egress-promoting receptor

The epithelial-mesenchymal transition of the Drosophila mesoderm requires the Rho GTP exchange factor Pebble

Drosophila pebble (pbl) encodes a Rho-family GTP exchange factor (GEF) required for cytokinesis. The accumulation of high levels of PBL protein during interphase and the developmentally regulated expression of pbl in mesodermal tissues suggested that the primary cytokinetic mutant phenotype might be masking other roles. Using various muscle differentiation markers, we found that Even skipped (EVE) expression in the dorsal mesoderm is greatly reduced in pbl mutant embryos. EVE expression in the dorsalmost mesodermal cells is induced in response to DPP secreted by the dorsal epidermal cells. Further analysis revealed that this phenotype is likely to be a consequence of an earlier defect. pbl mutant mesodermal cells fail to undergo the normal epithelial-mesenchymal transition (EMT) and dorsal migration that follows ventral furrow formation. This phenotype is not a secondary consequence of failed cytokinesis, as it is rescued by a mutant form of pbl that does not rescue the cytokinetic defect. In wild-type embryos, newly invaginated cells at the lateral edges of the mesoderm extend numerous protrusions. In pbl mutant embryos, however, cells appear more tightly adhered to their neighbours and extend very few protrusions. Consistent with the dependence of the mesoderm EMT and cytokinesis on actin organisation, the GTP exchange function of the PBL RhoGEF is required for both processes. By contrast, the N-terminal BRCT domains of PBL are required only for the cytokinetic function of PBL. These studies reveal that a novel PBL-mediated intracellular signalling pathway operates in mesodermal cells during the transition from an epithelial to migratory mesenchymal morphology during gastrulation