Regulation of gene activation by the estrogen receptor (ER) is complex and involves co-regulatory proteins, oncoproteins (such as Fos and Jun), and phosphorylation signaling pathways. Here we report the cloning and initial characterization of a novel protein, Brx, that contains a region of identity to the oncogenic Rhoguanine nucleotide exchange (Rho-GEF) protein Lbc, and a unique region capable of binding to nuclear hormone receptors, including the ER. Western and immunohistochemistry studies showed Brx to be expressed in estrogen-responsive reproductive tissues, including breast ductal epithelium. Brx bound speci®cally to the ER via an interaction that required distinct regions of ER and Brx. Furthermore, overexpression of Brx in transfection experiments using an estrogenresponsive reporter revealed that Brx augmented gene activation by the ER in an element-speci®c and liganddependent manner. Moreover, activation of ER by Brx could be speci®cally inhibited by a dominant-negative mutant of Cdc42Hs, but not by dominant negative mutants of RhoA or Rac1. Taken together, these data suggest that Brx represents a novel modular protein that may integrate cytoplasmic signaling pathways involving Rho family GTPases and nuclear hormone receptors.

p21-activated kinase (Pak)-interacting exchange factor (Pix), a Rho family guanine nucleotide exchange factor (GEF), has been shown to co-localize with Pak and form activated Cdc42-and Rac1-driven focal complexes. In this study we have presented evidence that treatment of human mesangial cells (HMC) with endothelin 1 (ET-1) and stimulation of adenylate cyclase with either forskolin or with the cAMP analog 8-Br-cAMP activated the GTP loading of Cdc42. Transient expression of constitutively active G␣ s also stimulated Cdc42. In addition, overexpression of ␤ 1 Pix enhanced ET-1-induced Cdc42 activation, whereas the expression of ␤ 1 Pix SH3m(W43K), which lacks the ability to bind Pak, and ␤ 1 PixDHm(L238R/L239S), which lacks GEF activity, decreased ET-1-induced Cdc42 activation. Mesangial cells are smooth muscle-like cells situated within the renal glomerulus that play an important role in regulating glomerular filtration and function, both by contraction and release of proinflammatory substances. Endothelin (ET), 1 a potent vasoconstrictor peptide implicated in chronic renal diseases, plays a crucial role in the physiology and pathology of glomerular cells. ET release is increased in response to inflammatory cytokines, suggesting that ET-1 synthesis might increase in glomerulonephritis by intrinsic glomerular cells such as glomerular endothelial, mesangial, and epithelial cells (1). Two receptors for ET isopeptides, ET A and ET B , are G proteincoupled receptors with seven transmembrane domains (2, 3). ET not only stimulates mesangial cell proliferation (4) but also increases the expression of extracellular matrix proteins such as collagen and fibronectin (5) and induces active cytoskeletal rearrangement. This process is governed largely by the precise temporal and spatial modulation of small GTPase proteins of the Rho family, Cdc42, Rac, and RhoA.Cdc42 and Rac1 function as molecular switches (6, 7). They are converted from the GDP-bound inactive form to a GTPbound active state by a reaction catalyzed by guanine nucleotide exchange factors (GEFs) (8). Since their identification, GEFs have become increasingly involved in mediating the effects of G protein-coupled receptor agonists. Recently, a Cdc42/ Rac-GEF termed Pix (Pak-interacting exchange factor) was identified (9). Pix has a diffuse B cell lymphoma homology (DH) domain and a flanking pleckstrin homology domain, which are conserved in all of the GEFs for Rho GTPases. Pix family proteins consist of two isoforms, ␣Pix and ␤Pix, and recently a new splice variant of ␤Pix designated ␤ 2 Pix has been identified (10). The human Pix family bind tightly through an N-terminal SH3 domain to a conserved proline-rich Pak sequence located at the C terminus and are colocalized with Pak to form activated Cdc42-and Rac1-driven focal complexes (9). Recently, Pix has been shown to form a trimolecular complex with Pak1 and p95PKL (also known as G protein-coupled receptor kinaseinteracting target, GIT1) (11). Furthermore, tyrosine-phosphorylated p95PKL can also bind paxillin (12,...

In microvessels of patients with coronary artery disease (CAD), flow-mediated dilation (FMD) is largely dependent upon the endothelium-derived hyperpolarizing factor HO. The goal of this study is to examine the influence of age and presence or absence of disease on the mechanism of FMD. Human coronary or adipose arterioles (~150 µm diameter) were prepared for videomicroscopy. The effect of inhibiting COX [indomethacin (Indo) or NOS (L-NAME), eliminating HO (polyethylene glycol-catalase (PEG-CAT)] or targeting a reduction in mitochondrial ROS with scavengers/inhibitors [Vitamin E (Vitamin E); phenylboronic acid (PBA)] was determined in children aged 0-18 years; young adults 19-55 years; older adults >55 years without CAD, and similarly aged adults with CAD. Indo eliminated FMD in children and reduced FMD in younger adults. This response was mediated mainly by PGI, as the prostacyclin-synthase-inhibitor trans-2-phenyl cyclopropylamine reduced FMD in children and young adults. L-NAME attenuated dilation in children and younger adults and eliminated FMD in older adults without CAD, but had no effect on vessels from those with CAD, where mitochondria-derived HO was the primary mediator. The magnitude of dilation was reduced in older compared to younger adults independent of CAD. Exogenous treatment with a sub-dilator dose of NO blocked FMD in vessels from subjects with CAD, while prolonged inhibition of NOS in young adults resulted in a phenotype similar to that observed in disease. The mediator of coronary arteriolar FMD evolves throughout life from prostacyclin in youth, to NO in adulthood. With the onset of CAD, NO-inhibitable release of HO emerges as the exclusive mediator of FMD. These findings have implications for use of pharmacological agents, such as nonsteroidal anti-inflammatory agents in children and the role of microvascular endothelium in cardiovascular health.

No abstract

Renal preglomerular arterioles regulate vascular tone to ensure a large pressure gradient over short distances, a function that is extremely important for maintaining renal microcirculation. Regulation of renal microvascular tone is impaired in salt-sensitive (SS) hypertension-induced nephropathy, but the molecular mechanisms contributing to this impairment remain elusive. Here, we assessed the contribution of the SH2 adaptor protein p66Shc (encoded by Shc1) in regulating renal vascular tone and the development of renal vascular dysfunction associated with hypertension-induced nephropathy. We generated a panel of mutant rat strains in which specific modifications of Shc1 were introduced into the Dahl SS rats. In SS rats, overexpression of p66Shc was linked to increased renal damage. Conversely, deletion of p66Shc from these rats restored the myogenic responsiveness of renal preglomerular arterioles ex vivo and promoted cellular contraction in primary vascular smooth muscle cells (SMCs) that were isolated from renal vessels. In primary SMCs, p66Shc restricted the activation of transient receptor potential cation channels to attenuate cytosolic Ca2+ influx, implicating a mechanism by which overexpression of p66Shc impairs renal vascular reactivity. These results establish the adaptor protein p66Shc as a regulator of renal vascular tone and a driver of impaired renal vascular function in hypertension-induced nephropathy.