Epithelial Naϩ channels (ENaCs) mediate sodium reabsorption in the cortical collecting duct (CCD), but the regulatory pathways that modulate the activity of these channels are incompletely understood. Here, we observed that endothelin-1 (ET-1) attenuates ENaC activity acutely by reducing the channel's open probability and chronically by decreasing the number of channels in the plasma membrane. To investigate whether ␤ 1 Pix, a signaling protein activated by ET-1, mediates ENaC activity, we reconstituted ENaC in CHO cells with or without coexpressed ␤ 1 Pix and found that ␤ 1 Pix negatively regulates ENaC. Knockdown of ␤Pix in native principal cells abolished the ET-1-induced decrease in ENaC channel number. Furthermore, we found that ␤Pix does not decrease ENaC activity through its guanine nucleotide exchange factor (GEF) activity for Rac1 and Cdc42. Instead, coexpression of ␤ 1 Pix mutant constructs revealed that ␤ 1 Pix affects ENaC activity through binding 14-3-3 proteins. Coimmunoprecipitation experiments supported a physical interaction between ␤ 1 Pix and 14-3-3␤ in cultured principal cells. Coexpression of 14-3-3␤ increased ENaC activity in CHO cells, but concomitant expression of ␤ 1 Pix attenuated this increase. Recruitment of 14-3-3␤ by ␤ 1 Pix impaired the interaction of 14-3-3␤ with the ubiquitin ligase Nedd4-2, thereby promoting ubiquitination and degradation of ENaC. Taken together, these results suggest that the inhibitory effects of chronic ET-1 on ENaC result from ␤Pix interacting with the 14-3-3/Nedd4-2 pathway.

Muscarinic receptors (CHRM) are overexpressed in colon cancer. To explore a role for muscarinic receptor signaling in colon cancer metastasis, we used human H508 and HT29 colon cancer cells that coexpress epidermal growth factor (ERBB) and CHRM3 receptors. In a wound closure model, following 8-h incubation of H508 cells with 100 μM ACh we observed a threefold increase in cell migration indistinguishable from the actions of epidermal growth factor (EGF). Atropine blocked the actions of ACh but not of EGF. In SNU-C4 colon cancer cells that express ERBB but not CHRM, EGF caused a threefold increase in migration; ACh had no effect. ACh-induced cell migration was attenuated by chemical inhibitors of ERBB1 activation, by anti-ERBB1 antibody, and by inhibitors of ERK and phosphatidylinositol 3-kinase (PI3K) signaling. Consistent with matrix metalloproteinase-7 (MMP7)-mediated release of an ERBB1 ligand, heparin binding epidermal growth factor-like growth factor (HBEGF), ACh-induced migration was inhibited by an MMP inhibitor and by anti-MMP7 and -HBEGF antibodies. ACh-induced cell migration was blocked by inhibiting RhoA and ROCK, key proteins that interact with the actin cytoskeleton. ACh-induced RhoA activation was attenuated by agents that inhibit ERBB1, ERK, and PI3K activation. Collectively, these findings indicate that ACh-induced cell migration is mediated by MMP7-mediated release of HBEGF, an ERBB ligand that activates ERBB1 and downstream ERK and PI3K signaling. In a cell invasion model, ACh-induced HT29 cell invasion was blocked by atropine. In concert with previous observations, these findings indicate that muscarinic receptor signaling plays a key role in colon cancer cell proliferation, survival, migration, and invasion.

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,...

The phosphorylation of forkhead transcription factor FOXO3a by Akt is critical regulator of cell proliferation induced by serum. We show that endothelin-1 (ET-1) stimulation of primary human mesangial cells (HMCs) induces betaPix and p66Shc up-regulation, resulting in the formation of the betaPix/p66Shc complex. In transformed HMCs, ET-1 induces a biphasic phosphorylation of p66Shc and FOXO3a. The second phase leads to p27(kip1) down-regulation independently of Akt. Depletion of betaPix blocks the second phase of p66Shc and FOXO3a phosphorylation and prevents p27(kip1) down-regulation induced by ET-1. Depletion of either betaPix or p66Shc inhibits ET-1-induced cell proliferation. The expression of beta(1)Pix induces FOXO3a phosphorylation through activation of Rac1, ERK1/2, and p66Shc. Using either p66Shc- or Akt-depleted cells; we show that beta(1)Pix-induced FOXO3a phosphorylation requires p66Shc but not Akt. beta(1)Pix-induced p27(kip1) down-regulation was blocked by U0126 but not by wortmannin. Endogenous betaPix and FOXO3a are constitutively associated with endogenous p66Shc. FOXO3a and p66Shc binding requires beta(1)Pix homodimerization. Expression of beta(1)Pix homodimerization deficient mutant abrogates beta(1)Pix-induced p27(kip1) down-regulation and cell proliferation. Our results identify p66Shc and FOXO3a as novel partners of beta(1)Pix and represent the first direct evidence of beta(1)Pix in cell proliferation via Erk/p66Shc-dependent and Akt-independent mechanisms.

␤ 1 Pix is a guanine nucleotide exchange factor (GEF) for the small GTPases Rac and Cdc42 which has been shown to mediate signaling pathways leading to cytoskeletal reorganization. In the present study, we show that the basal association between endogenous ␤Pix and endogenous 14-3-3␤ was increased after forskolin stimulation and significantly inhibited by protein kinase A inhibitor. However, forskolin stimulation failed to increase the interaction between 14-3-3␤ and a ␤ 1 Pix mutant that is insensitive to protein kinase A phosphorylation, ␤ 1 Pix(S516A, T526A). We present evidence indicating that forskolin-induced binding of 14-3-3␤ to ␤ 1 Pix results in inhibition of Rac1 GTP loading in 293 cells and in vitro. Furthermore, we show that deletion of 10 amino acid residues within the leucine zipper domain is sufficient to block ␤ 1 Pix homodimerization and 14-3-3␤ binding and modulates ␤ 1 Pix-GEF activity. These residues also play a crucial role in ␤ 1 Pix intracellular localization. These results indicate that 14-3-3␤ negatively affects the GEF activity of dimeric ␤ 1 Pix only. Altogether, these results provide a mechanistic insight into the role of 14-3-3␤ in modulating ␤ 1 Pix-GEF activity.

Mammalian matrix metalloproteinases (MMPs) which degrade extracellular matrix facilitate colon cancer cell invasion into the bloodstream and extra-colonic tissues; in particular, MMP1 expression correlates strongly with advanced colon cancer stage, hematogenous metastasis and poor prognosis. Likewise, muscarinic receptor signaling plays an important role in colon cancer; muscarinic receptors are over-expressed in colon cancer compared to normal colon epithelial cells. Muscarinic receptor activation stimulates proliferation, migration and invasion of human colon cancer cells. I n mouse intestinal neoplasia models genetic ablation of muscarinic receptors attenuates carcinogenesis. In the present work, we sought to link these observations by showing that MMP1 expression and activation plays a mechanistic role in muscarinic receptor agonist-induced colon cancer cell invasion. We show that acetylcholine, which robustly increases MMP1 expression, stimulates invasion of HT29 and H508 human colon cancer cells into human umbilical vein endothelial cell monolayers – this was abolished by pre-incubation with atropine, a non-selective muscarinic receptor inhibitor, and by pre-incubation with anti-MMP1 neutralizing antibody. Similar results were obtained using a Matrigel chamber assay and deoxycholyltaurine (DCT), an amidated dihydroxy bile acid associated with colon neoplasia in animal models and humans previously shown to interact functionally with muscarinic receptors. DCT treatment of human colon cancer cells resulted in time-dependent, 10-fold increased MMP1 expression, and DCT-induced cell invasion was also blocked by pre-treatment with anti-MMP1 antibody. This study contributes to understanding mechanisms underlying muscarinic receptor agonist-induced promotion of colon cancer and, more importantly, indicates that blocking MMP1 expression and activation has therapeutic promise to stop or retard colon cancer invasion and dissemination.

Both phospholipase D1 (PLD1) and PLD2 regulate degranulation when RBL-2H3 cells are stimulated via the immunoglobulin E receptor, FcRI. However, the activation mechanism for PLD2 is unclear. As reported here, PLD2 but not PLD1 is phosphorylated through the Src kinases, Fyn and Fgr, and this phosphorylation appears to regulate PLD2 activation and degranulation. For example, only hemagglutinin-tagged PLD2 was tyrosine phosphorylated in antigen-stimulated cells that had been made to express HA-PLD1 and HA-PLD2. This phosphorylation was blocked by a Src kinase inhibitor or by small interfering RNAs directed against Fyn and Fgr and was enhanced by overexpression of Fyn and Fgr but not by other Src kinases. The phosphorylation and activity of PLD2 were further enhanced by the tyrosine phosphatase inhibitor, Na 3 VO 4 . Mutation of PLD2 at tyrosines 11, 14, 165, or 470 partially impaired, and mutation of all tyrosines blocked, PLD2 phosphorylation and activation, although two of these mutations were detrimental to PLD2 function. PLD2 phosphorylation preceded degranulation, both events were equally sensitive to inhibition of Src kinase activity, and both were enhanced by coexpression of PLD2 and the Src kinases. The findings provide the first description of a mechanism for activation of PLD2 in a physiological setting and of a role for Fgr in FcRI-mediated signaling.

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