To investigate CD40 signaling complex formation in living cells, we used green fluorescent protein (GFP)-tagged CD40 signaling intermediates and confocal life imaging. The majority of cytoplasmic TRAF2-GFP and, to a lesser extent, TRAF3-GFP, but not TRAF1-GFP or TRAF4-GFP, translocated into CD40 signaling complexes within a few minutes after CD40 triggering with the CD40 ligand. The inhibitor of apoptosis proteins cIAP1 and cIAP2 were also recruited by TRAF2 to sites of CD40 signaling. An excess of TRAF2 allowed recruitment of TRAF1-GFP to sites of CD40 signaling, whereas an excess of TRAF1 abrogated the interaction of TRAF2 and CD40. Overexpression of TRAF1, however, had no effect on the interaction of TRADD and TRAF2, known to be important for tumor necrosis factor receptor 1 (TNF-R1)-mediated NF-B activation. Accordingly, TRAF1 inhibited CD40-dependent but not TNF-R1-dependent NF-B activation. Moreover, down-regulation of TRAF1 with small interfering RNAs enhanced CD40/ CD40 ligand-induced NF-B activation but showed no effect on TNF signaling. Because of the trimeric organization of TRAF proteins, we propose that the stoichiometry of TRAF1-TRAF2 heteromeric complexes ((TRAF2) 2 -TRAF1 versus TRAF2-(TRAF1) 2 ) determines their capability to mediate CD40 signaling but has no major effect on TNF signaling. CD40 and its ligand CD40L1 /CD154 are members of the tumor necrosis factor (TNF) receptor and TNF ligand family and represent major regulators of lymphocyte function (1). Aside from T-and B-cells, CD40 and CD40L are expressed in a variety of non-lymphocytic cell types including monocytes, dendritic cells, fibroblasts, smooth muscle, and endothelial cells (1). The CD40/CD40L system plays a critical role in the regulation of thymus-dependent humoral immune responses but also contributes to chronic inflammatory processes in autoimmune diseases, neurodegenerative disorders, graft-versus-host disease, cancer, and atherosclerosis (1).Engagement of CD40 results in the recruitment of members of the TNF receptor-associated factor (TRAF) adaptor protein family (1, 2). In addition, triggering of CD40 leads to Janus family kinase 3 (Jak3)-dependent activation of signal transducers and activators of transcription (STAT) proteins and to activation of the Src-related tyrosine kinase Lyn (3-6). TRAF proteins couple TNF receptors and Toll/interleukin-1 receptor family members to pathways leading to the activation of the inhibitor of I-B kinases and kinases of the mitogen-activated protein kinase (MAPK) family (2). All members of the TRAF family share a conserved C-terminal homology domain of ϳ180 amino acids (TRAF domain), which mediates interactions with the above mentioned receptors and the majority of cytosolic factors known for their TRAF binding capacity, including kinases, inhibitor of apoptosis proteins, and death domain adaptor proteins (2). With the exception of TRAF1, the N-terminal domain of all six known mammalian TRAFs comprise a single RING finger followed by several zinc finger motifs (2) that are important for ...

A process termed "restitution" enables rapid repair of the respiratory epithelium by migration of neighbouring cells. Mucin-associated TFF-peptides (formerly P-domain peptides or trefoil factors) are typical motogens enhancing migration of cells in various in vitro models mimicking restitution of the intestine. The human bronchial epithelial cell line BEAS-2B was used as a model system of airway restitution. The motogenic activities of recombinant human TFF2 as well as porcine TFF2 were demonstrated by in vitro wound healing assays of BEAS-2B cells. TFF2 did not induce phosphorylation of the epidermal growth factor (EGF) receptor. EGF was capable of enhancing the motogenic effect of human TFF2 at a concentration of 3 x 10(-10) M whereas EGF itself (i.e., in the absence of TFF2) did not stimulate migration at this low concentration. Furthermore, TFF2 as well as monomeric and dimeric forms of TFF3 enhanced migration of BEAS-2B cells in Boyden chambers. Motogenic activity of TFF2 was also shown for normal human bronchial epithelial (NHBE) cells in Boyden chambers. These results suggest that TFF-peptides act as motogens in the human respiratory epithelium triggering rapid repair of damaged mucosa in the course of airway diseases such as asthma.

The signaling routes linking G-protein-coupled receptors to mitogen-activated protein kinase (MAPK) may involve tyrosine kinases, phosphoinositide 3-kinase gamma (PI3Kgamma), and protein kinase C (PKC). To characterize the mitogenic pathway of bradykinin (BK), COS-7 cells were transiently cotransfected with the human bradykinin B(2) receptor and hemagglutinin-tagged MAPK. We demonstrate that BK-induced activation of MAPK is mediated via the alpha subunits of a G(q/11) protein. Both activation of Raf-1 and activation of MAPK in response to BK were blocked by inhibitors of PKC as well as of the epidermal growth factor (EGF) receptor. Furthermore, in PKC-depleted COS-7 cells, the effect of BK on MAPK was clearly reduced. Inhibition of PI3-Kgamma or Src kinase failed to diminish MAPK activation by BK. BK-induced translocation and overexpression of PKC isoforms as well as coexpression of inactive or constitutively active mutants of different PKC isozymes provided evidence for a role of the diacylglycerol-sensitive PKCs alpha and epsilon in BK signaling toward MAPK. In addition to PKC activation, BK also induced tyrosine phosphorylation of EGF receptor (transactivation) in COS-7 cells. Inhibition of PKC did not alter BK-induced transactivation, and blockade of EGF receptor did not affect BK-stimulated phosphatidylinositol turnover or BK-induced PKC translocation, suggesting that PKC acts neither upstream nor downstream of the EGF receptor. Comparison of the kinetics of PKC activation and EGF receptor transactivation in response to BK also suggests simultaneous rather than consecutive signaling. We conclude that in COS-7 cells, BK activates MAPK via a permanent dual signaling pathway involving the independent activation of the PKC isoforms alpha and epsilon and transactivation of the EGF receptor. The two branches of this pathway may converge at the level of the Ras-Raf complex.

The signaling routes connecting G protein-coupled receptors to the mitogen-activated protein kinase (MAPK) pathway reveal a high degree of complexity and cell specificity. In the human colon carcinoma cell line SW-480, we detected a mitogenic effect of bradykinin (BK) that is mediated via a pertussis toxin-insensitive G protein of the G q/11 family and that involves activation of MAPK. Both BK-induced stimulation of DNA synthesis and activation of MAPK in response to BK were abolished by two different inhibitors of phosphatidylinositol 3-kinase (PI3K), wortmannin and LY 294002, as well as by two different inhibitors of protein kinase C (PKC), bisindolylmaleimide and Ro 31-8220. Stimulation of SW-480 cells by BK led to increased formation of PI3K lipid products (phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,4-bisphosphate) and to enhanced translocation of the PKC⑀ isoform from the cytosol to the membrane. Both effects of BK were inhibited by wortmannin, too. Using subtype-specific antibodies, only the PI3K subunits p110␤ and p85, but not p110␣ and p110␥, were detected in SW-480 cells. Finally, p110␤ was found to be co-immunoprecipitated with PKC⑀. Our data suggest that in SW-480 cells, (i) dimeric PI3K␤ is activated via a G q/11 protein; (ii) PKC⑀ is a downstream target of PI3K␤ mediating the mitogenic signal to the MAPK pathway; and (iii) PKC⑀ associates with the p110 subunit of PI3K␤. Thus, these results add a novel possibility to the emerging picture of multiple pathways linking G protein-coupled receptors to MAPK.G protein-coupled receptors mediate effects of peptide hormones and neurotransmitters on intermediary metabolism as well as play an important role in the regulation of cell growth and differentiation. Similar to receptor tyrosine kinases, they initiate signaling pathways that finally activate members of the mitogen-activated protein kinase (MAPK) 1 family. One MAPK subfamily, which includes the extracellular signal-regulated kinases Erk1 and Erk2, is stimulated via a consecutive activation of the protein kinases Raf and MEK. The MAPK cascade is initially switched on via activation of the low molecular mass GTP-binding protein Ras. GTP-bound Ras associates the proximal kinase Raf to the plasma membrane, resulting in its activation. Several signal transduction pathways from G protein-coupled receptors to MAPK have been proposed that may be classified according to the type of G protein involved (for review, see Refs. 1 and 2). Thus, MAPK activation via pertussis toxin (PTX)-sensitive G i protein-coupled receptor, such as the m 2 muscarinic receptor, was found to be mediated by G ␤␥ subunits, phosphatidylinositol 3-kinase ␥ (PI3K␥), and Ras (3). In contrast, receptors coupled to G proteins of the PTX-insensitive G q/11 family, such as the m 1 muscarinic receptor, mediate MAPK activation via a G ␣ subunit that is Ras-independent and may involve PKC (4). Once activated, the different PKC isoforms, with the exception of PKC, activate the MAPK cascade at the level of Raf (5), but may al...

TFF-peptides (formerly P-

Cell membranes of the human epidermoid cell line A431 express classical bradykinin (BK) B2 receptors, as assessed by [3H]BK binding studies. Furthermore, stimulation by BK induced a time-dependent modulation of protein kinase C (PKC) activity in A431 cells: a rapid activation (t1/2 approximately 1 min) is followed by a slow inhibition (t1/2 approximately 20 min) of PKC translocation measured by [3H]phorbol 12,13-dibutyrate binding. In addition, BK stimulated both adenylate cyclase activity in A431 membranes and accumulation of intracellular cyclic AMP (cAMP) in intact cells in a retarded manner. A possible BK-induced activation of the cAMP pathway mediated via PKC, phospholipase D, prostaglandins or Ca2+/calmodulin was excluded. A 35 kDa protein was found in A431 membranes to be specifically phosphorylated in the presence of both BK and protein kinase A (PKA). An anti-alpha s-antibody, AS 348, abolished stimulation of adenylate cyclase activity in response to BK, cholera toxin and isoprenaline, strongly suggesting the involvement of Gs proteins in the BK action. The BK-activated cAMP signalling system might be important for the observed inactivation of PKC slowly evoked by BK: the BK-induced rapid activation of PKC is decreased by dibutyryl cAMP, and the slow inhibition of PKC is prevented by an inhibitor of PKA, adenosine 3':5'-monophosphothioate (cyclic, Rp isomer). The inhibition of PKC translocation might be exerted directly at the level of PKC activation, since stimulation of phosphoinositide hydrolysis by BK was affected by neither dibutyryl cAMP nor forskolin. Thus our results provide the first evidence that A431 cells BK is able to activate two independent signal-transduction pathways via a single class of B2 receptors but two different G proteins. The lagging stimulation of the cAMP signalling pathway via Gs might serve to switch off PKC, which is rapidly activated via Gq-mediated stimulation of phosphoinositide hydrolysis.

Modulation of the cytoskeletal architecture was shown to regulate the expression of CTGF (connective tissue growth factor, CCN2). The microtubule disrupting agents nocodazole and colchicine strongly up-regulated CTGF expression, which was prevented upon stabilization of the microtubules by paclitaxel. As a consequence of microtubule disruption, RhoA was activated and the actin stress fibers were stabilized. Both effects were related to CTGF induction. Overexpression of constitutively active RhoA induced CTGF synthesis. Interference with RhoA signaling by simvastatin, toxinB, C3 toxin, and Y27632 prevented up-regulation of CTGF. Likewise, direct disintegration of the actin cytoskeleton by latrunculin B interfered with nocodazolemediated up-regulation of CTGF expression. Disassembly of actin fibers by cytochalasin D, however, unexpectedly increased CTGF expression indicating that the content of F-actin per se was not the major determinant for CTGF gene expression. Given the fact that cytochalasin D sequesters G-actin, a decrease in G-actin increased CTGF, while increased levels of Gactin corresponded to reduced CTGF expression. These data link alterations in the microtubule and actin cytoskeleton to the expression of CTGF and provide a molecular basis for the observation that CTGF is up-regulated in cells exposed to mechanical stress.

The role of tumor necrosis factor (TNF) receptor-associated factor (TRAF)-1 in NF-B activation by various members of the TNF receptor family is not well understood, and conflicting data have been published. Here, we show that TRAF1 differentially affects TRAF2 recruitment and activation of NF-B by members of the TNF receptor family. Interestingly, a naturally occurring caspase-derived cleavage product of TRAF1 solely comprising its TRAF domain (TRAF1-(164 -416)) acted as a general inhibitor of NF-B activation. In contrast, a corresponding fragment generated by cleavage of TRAF3 showed no effect in this regard. In accordance with these functional data, TRAF1, but not TRAF3, interacted with the IKK complex via its N-TRAF domain. Endogenous TRAF1 and the overexpressed TRAF domain of TRAF1 were found to be constitutively associated with the IKK complex, whereas endogenous receptor interacting protein was only transiently associated with the IKK complex upon TNF stimulation. Importantly, the caspase-generated TRAF1-fragment, but not TRAF1 itself inhibited IKK activation. Our results suggest that TRAF1 and TRAF1-(164 -416) exert their regulatory effects on receptor-induced NF-B activation not only by modulation of TRAF2 receptor interaction but especially TRAF1-(164 -416) also by directly targeting the IKK complex.The tumor necrosis factor (TNF) 1 receptor-associated factor (TRAF) family of proteins has a pivotal role in signaling by members of the TNF receptor and the interleukin-1 receptor/ Toll-like receptor (IL1R/TLR) family (1-3). TRAF proteins are characterized by a carboxyl-terminal homology domain of about 180 amino acids, named the TRAF domain. Apart from TRAF1, all TRAF proteins show a similar overall architecture in their amino-terminal part: a single RING finger, which is followed by five or seven evenly separated zinc finger motifs (1-3). TRAF proteins have been recognized as mediators of NF-B activation and regulators of cell death, but also as activators of various kinases including ERK, JNK, and IRE1␣ (1-3). The TRAF proteins can interact with a plethora of proteins that play a role in the signaling pathways mentioned above via their TRAF domain. However, in a minority of cases associations can also occur via the amino-terminal ring/zinc finger domain. The carboxyl-terminal part of the TRAF domain allows direct binding to various TNF receptors (1-3). Thus, the TRAF molecules seem to act mainly as adaptor and scaffolding proteins. Several lines of evidence, especially analyses of knock-out mice, point to a critical role of TRAF2, TRAF5, and TRAF6 in TNF receptor and IL1R/Toll receptor-induced activation of JNK and the kinases of the IKK complex (1-3). Although the molecular mode of TRAF4 action is poorly understood, analyses of knock-out mice (4, 5) and the TRAF4 expression pattern (6, 7) argue for a role of this molecule in epithelialmesenchymal interactions and neurogenesis. The functions of TRAF1 and TRAF3 are rather unknown.With respect to its molecular architecture, TRAF1 is a unique member of the ...