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 ...

J. Neurochem. (2012) 120, 684–698. Abstract cAMP induces neurite outgrowth in the rat pheochromocytoma cell line 12 (PC12). In particular, di‐butyric cAMP (db‐cAMP) induces a greater number of primary processes with shorter length than the number induced by nerve growth factor (NGF). db‐cAMP up‐ and down‐regulates GTP‐RhoA levels in PC12 cells in a time‐dependent manner. Tat‐C3 toxin stimulates neurite outgrowth, whereas lysophosphatidic acid (LPA) and constitutively active (CA)‐RhoA reduce neurite outgrowth, suggesting that RhoA inactivation is essential for the neurite outgrowth from PC12 cells stimulated by cAMP. In this study, the mechanism by which RhoA is inactivated in response to cAMP was examined. db‐cAMP induces phosphorylation of RhoA and augments the binding of RhoA with Rho guanine nucleotide dissociation inhibitor (GDI). Moreover, RhoA (S188D) mimicking phosphorylated RhoA induces greater neurite outgrowth than RhoA (S188A) mimicking dephosphorylated form does. Additionally, db‐cAMP increases GTP‐Rap1 levels, and dominant negative (DN)‐Rap1 and DN‐Rap‐dependent RhoGAP (ARAP3) block neurite outgrowth induced by db‐cAMP. DN‐p190RhoGAP and the Src inhibitor PP2 suppress neurite outgrowth, whereas transfection of c‐Src and p190RhoGAP cDNAs synergistically stimulate neurite outgrowth. Taken together, RhoA is inactivated by phosphorylation of itself, by p190RhoGAP which is activated by Src, and by ARAP3 which is activated by Rap1 during neurite outgrowth from PC12 cells in response to db‐cAMP.

Rat pheochromocytoma (PC12) cells have been used to investigate neurite outgrowth. Nerve growth factor (NGF) has been well known to induce neurite outgrowth from PC12 cells. RhoA belongs to Ras-related small GTP-binding proteins, which regulate a variety of cellular processes, including cell morphology alteration, actin dynamics, and cell migration. NGF suppressed GTP-RhoA levels after 12 h in PC12 cells and was consistently required for a long time to induce neurite outgrowth. Constitutively active (CA)-RhoA suppressed neurite outgrowth from PC12 cells in response to NGF, whereas dominant-negative (DN)-RhoA stimulated it, suggesting that RhoA inactivation is essential for neurite outgrowth. Here, we investigated the mechanism of RhoA inactivation. DN-p190RhoGAP abrogated neurite outgrowth, whereas wild-type (WT)-p190RhoGAP and WT-Src synergistically stimulated it along with accelerating RhoA inactivation, suggesting that p190RhoGAP, which can be activated by Src, is a major component in inhibiting RhoA in response to NGF in PC12 cells. Contrary to RhoA, Rap1 was activated by NGF, and DN-Rap1 suppressed neurite outgrowth, suggesting that Rap1 is also essential for neurite outgrowth. RhoA was co-immunoprecipitated with Rap1, suggesting that Rap1 interacts with RhoA. Furthermore, a DN-Rap-dependent RhoGAP (ARAP3) prevented RhoA inactivation, abolishing neurite formation from PC12 cells in response to NGF. These results suggest that NGF activates Rap1, which, in turn, up-regulates ARAP3 leading to RhoA inactivation and neurite outgrowth from PC12 cells. Taken together, p190RhoGAP and ARAP3 seem to be two main factors inhibiting RhoA activity during neurite outgrowth in PC12 cells in response to NGF.

Rac1 and Rac2 are essential for the control of oxidative burst catalyzed by NADPH oxidase. It was also documented that Rho is associated with the superoxide burst reaction during phagocytosis of serum-(SOZ) and IgG-opsonized zymosan particles (IOZ). In this study, we attempted to reveal the signal pathway components in the superoxide formation regulated by Rho GTPase. Tat PHOX may be subsequently activated, leading to activation of NADPH oxidase to produce superoxide.