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 ...

The GTPase Rab3A has been postulated to cycle on and off synaptic membranes during the course of neurotransmission. Moreover, a Rab guanine nucleotide dissociation inhibitor has been shown to cause Rab3A to dissociate from synaptic membranes in vitro. We demonstrate here that Ca 2؉ /calmodulin also can cause Rab3A to dissociate from synaptic membranes in vitro. Like Rab guanine nucleotide dissociation inhibitor, it forms a 1:1 complex with Rab3A that requires both the lipidated C terminus of Rab3A and the presence of bound guanine nucleotide. In addition, a synthetic peptide corresponding to the Lys The opening of voltage-gated Ca 2ϩ channels in active zones of nerve terminals causes a brief, localized influx of Ca 2ϩ followed by the secretion of neurotransmitters (1-3). The molecular basis of this effect is still unclear, but increased concentrations of intracellular Ca 2ϩ may act at several levels to trigger fast fusion of pre-docked synaptic vesicles with the synaptic plasma membrane, promote endocytosis of the vesicle membranes and subsequent vesicle reformation, and mobilize additional vesicles to release sites (1, 4). Proteins that bind Ca 2ϩ probably mediate many of these actions, and a number of candidate proteins have been identified. They include rabphilin (5, 6); the ␣-, ␤-II-, and ␥ isoforms of protein kinase C (7); and dynamin (8) (Table I) was from LC Laboratories. Stock solutions of peptides were prepared in Me 2 SO and then added to incubation mixtures at final Me 2 SO concentrations of Ͻ5%. GDP, GTP␥S, and unprenylated Rab3A were from Calbiochem. Rab GDI was purified from bovine brain as described (29), except that all buffers used after the ammonium sulfate precipitation step contained 10% glycerol, 0.25 mM phenylmethylsulfonyl fluoride, 2.5 g/ml each of aprotinin and leupeptin, and 1 g/ml pepstatin A. All other purchased chemicals were reagent grade from Sigma, and all procedures were performed at 4°C unless otherwise indicated.Preparation of Synaptosomes-Two different methods were used to prepare synaptosomes from cerebral cortex of nonhuman primates (Macaca nemestrina), obtained from the tissue distribution program of the Regional Primate Research Center at the University of Washington. In method 1, 50 g of cortex was sliced in ice-cold buffer A (320 mM sucrose; * This work was supported by the Howard Hughes Medical Institute and by National Institutes of Health Grant RR00166 to the Regional Primate Research Center at the University of Washington. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C.

Xvent homeobox genes encode transcription factors that repress organizer genes and are essential for dorsoventral specification during early embryogenesis in Xenopus. In contrast to the Xvent-2 gene subfamily, Xvent-1 subfamily members, including PV.1A, have been proposed as indirect targets of Bone Morphogenetic Protein-4 (BMP-4) signaling. Because PV.1A is a critical downstream mediator of, and tightly regulated by, BMP-4 signaling, we hypothesized that its promoter contains a direct BMP-4 response element to effect this transcriptional regulation. We demonstrate that direct regulation by BMP-4 is necessary for transcription of PV.1A: its proximal promoter contains cis-acting binding elements for Smads and Oaz crucial to induction in response to BMP-4 signaling. In addition to these direct cis-acting BMP-4 responsive elements, an indirect Xvent-2 response element and several repressive elements exist in the PV.1A promoter to regulate its transcription. In summary, PV.1A undergoes combinatorial regulation during early Xenopus development as both the direct target of BMP-4 signaling and as the direct and indirect target of positive and negative regulatory factors.