Excessive accumulation of extracellular matrix (ECM) in the kidneys and epithelial-to-mesenchymal transition (EMT) of renal tubular epithelial cells contributes to the renal fibrosis that is associated with diabetic nephropathy. Histone deacetylase (HDAC) determines the acetylation status of histones and thereby controls the regulation of gene expression. This study examined the effect of HDAC inhibition on renal fibrosis induced by diabetes or transforming growth factor (TGF)-beta1 and determined the role of reactive oxygen species (ROS) as mediators of HDAC activation. In streptozotocin (STZ)-induced diabetic kidneys and TGF-beta1-treated normal rat kidney tubular epithelial cells (NRK52-E), we found that trichostatin A, a nonselective HDAC inhibitor, decreased mRNA and protein expressions of ECM components and prevented EMT. Valproic acid and class I-selective HDAC inhibitor SK-7041 also showed similar effects in NRK52-E cells. Among the six HDACs tested (HDAC-1 through -5 and HDAC-8), HDAC-2 activity significantly increased in the kidneys of STZ-induced diabetic rats and db/db mice and TGF-beta1-treated NRK52-E cells. Levels of mRNA expression of fibronectin and alpha-smooth muscle actin were decreased, whereas E-cadherin mRNA was increased when HDAC-2 was knocked down using RNA interference in NRK52-E cells. Interestingly, hydrogen peroxide increased HDAC-2 activity, and the treatment with an antioxidant, N-acetylcysteine, almost completely reduced TGF-beta1-induced activation of HDAC-2. These findings suggest that HDAC-2 plays an important role in the development of ECM accumulation and EMT in diabetic kidney and that ROS mediate TGF-beta1-induced activation of HDAC-2.

Rho GTPases (Rac, Rho, and Cdc42) play important roles in regulating cell function through their ability to coordinate the actin cytoskeleton, modulate the formation of signaling reactive oxidant species, and control gene transcription. Activation of Rho GTPase signaling pathways requires the regulated release of Rho GTPases from RhoGDI complexes, followed by their reuptake after membrane cycling. We show here that Src kinase binds and phosphorylates RhoGDI both in vitro and in vivo at Tyr156. Analysis of Rho GTPase-RhoGDI complexes using in vitro assays of complexation and in vivo by coimmunoprecipitation analysis indicates that Src-mediated phosphorylation of Tyr156 causes a dramatic decrease in the ability of RhoGDI to form a complex with RhoA, Rac1, or Cdc42. Phosphomimetic mutation of Tyr1563 Glu results in the constitutive association of RhoGDI Y156E with the plasma membrane and/or associated cortical actin. Substantial cortical localization of tyrosine-phosphorylated RhoGDI is also observed in fibroblasts expressing active Src, where it is most evident in podosomes and regions of membrane ruffling. Expression of membrane-localized RhoGDI Y156E mutant is associated with enhanced cell spreading and membrane ruffling. These results suggest that Src-mediated RhoGDI phosphorylation is a novel physiological mechanism for regulating Rho GTPase cytosol membrane-cycling and activity. INTRODUCTIONThe Rho GTPases regulate cellular activities that include growth and differentiation, vesicular transport, production of reactive oxygen species (ROS), apoptosis, cell motility, and various other aspects of cytoskeletal dynamics and cell polarity (Van Aelst and Souza-Schorey, 1997;Bishop and Hall, 2000). Rho GTPases function as molecular switches in cell signaling, alternating between inactive GDP-bound states maintained as cytosolic complexes with GDP dissociation inhibitors (GDIs), and active GTP-bound states usually associated with membranes where effector targets reside. Complexation of Rho, Rac, or Cdc42 with GDIs inhibits GDP dissociation and localizes GTPases to the cytosol in inert forms unable to interact with GEFs (guanine nucleotide exchange factors), GAPs (GTPase activating proteins), or effector targets (Van Aelst and Souza-Schorey, 1997;DerMardirossian and Bokoch, 2005). GDIs maintain Rho GTPases as soluble cytosolic proteins by forming high-affinity complexes that mask the C-terminal geranylgeranyl membrane-targeting moiety within a hydrophobic pocket formed by the immunoglobulin-like domain of RhoGDI (Gosser et al., 1997;Keep et al., 1997;Hoffman et al., 2000). When Rho proteins are released from GDIs, they insert into the membrane lipid bilayer through their isoprenylated and polybasic C-terminal domains to be activated by membrane-associated GEFs, initiating the association with effector targets at the membrane. A reassociation with GDI, possibly associated with GTP hydrolysis, is postulated to induce recycling of the GTPase to the cytosol. Regulation of the cytosol-membrane cycling of the Rho GTPase by G...

Brief treatment with transforming growth factor (TGF)-␤1 stimulated the migration of macrophages, whereas long-term exposure decreased their migration. Cell migration stimulated by TGF-␤1 was markedly inhibited by 10 g/mL Tat-C3 exoenzyme. TGF-␤1 increased mRNA and protein levels of macrophage inflammatory protein (MIP)-1␣ in the initial period, and these effects also were inhibited by 10 g/mL Tat IntroductionTransforming growth factor (TGF)-␤ regulates diverse cellular functions, including tissue differentiation, cell proliferation, and cell migration. Monocytes/macrophages, in particular, secrete TGF-␤, which in turn stimulates numerous responses: production of a variety of cytokines, including interleukin-1␣ (IL-1␣) and -␤ (IL-1␤), tumor necrosis factor (TNF)-␣, platelet-derived growth factor (PDGF)-BB, and basic fibroblast growth factor (bFGF); recruitment of monocytes to sites of injury or inflammation; phagocytic activity (by up-regulating the expression of cell-surface Fc␥RIII); and the expression of several integrin receptors on monocytes, including leukocyte function-associated antigen-1 (LFA-1: integrin ␣L␤2), ␣3␤1, and ␣5␤1, thereby increasing their cell-cell and cell-matrix interactions. 1 These observation indicate a proinflammatory function for TGF-␤ on monocytes. 2 In contrast to its activating effects on peripheral blood monocytes, TGF-␤ reduces the host response to a variety of inflammatory stimuli and is a potent immunosuppressive, anti-inflammatory, and macrophage deactivating agent. 3 Resting monocytes express high levels of TGF-␤ type 1 and 2 receptors, whereas receptor levels decline as cells mature and are then activated by agents such as lipopolysaccharide (LPS) and interferon-␥ (IFN-␥). 1 The functional complex of TGF-␤1 receptors at the cell surface is composed of 2 type 2 (T␤RII) and 2 type 1 (T␤RI) transmembrane Ser/Thr kinase receptors. 4 Receptor-activated Smads (Rsmads: Smad1, Smad2, Smad3, Smad5, and Smad 8), which are phosphorylated by type 1 receptors, are released from the receptor complex to form a heterotrimeric complex of 2 R-Smads and a common Smad4 (Co-Smad); the complex then translocates to the nucleus, where it regulates transcription. The structurally distinct Smads, Smad6 and Smad7, act as inhibitory Smads (I-Smads) by competing with R-Smads for receptors. 5 The expression of I-Smads is strongly regulated by extracellular signals, and the induction of Smad6 and Smad7 expression by TGF-␤1 reveals an inhibitory feedback mechanism for ligand-induced signaling. 6 In addition to the R-Smad/Co-Smad activation pathway, TGF-␤ can activate the extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38MAPK pathways, the last 2 of which are activated via TGF-␤-activated kinase 1 (TAK1). 4 Rho GTPases regulate the actin cytoskeleton, cell polarity, gene expression, microtubule dynamics, and vesicular trafficking. 7 Regulation of the nucleotide-bound state of RhoGTPases, alternative cycling between active GTP-and inactive GDP-bound states, is accomplished ...

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