The Ras-dependent activation of mitogen-activated protein (MAP) kinase pathways by many receptors coupled to heterotrimeric guanine nucleotide binding proteins (G proteins) requires the activation of Src family tyrosine kinases. Stimulation of beta2 adrenergic receptors resulted in the assembly of a protein complex containing activated c-Src and the receptor. Src recruitment was mediated by beta-arrestin, which functions as an adapter protein, binding both c-Src and the agonist-occupied receptor. beta-Arrestin 1 mutants, impaired either in c-Src binding or in the ability to target receptors to clathrin-coated pits, acted as dominant negative inhibitors of beta2 adrenergic receptor-mediated activation of the MAP kinases Erk1 and Erk2. These data suggest that beta-arrestin binding, which terminates receptor-G protein coupling, also initiates a second wave of signal transduction in which the "desensitized" receptor functions as a critical structural component of a mitogenic signaling complex.

Întegrins, matrix metalloproteases (MMPs), and the cytokine TGF-β have each been implicated in homeostatic cell behaviors such as cell growth and matrix remodeling. TGF-β exists mainly in a latent state, and a major point of homeostatic control is the activation of TGF-β. Because the latent domain of TGF-β1 possesses an integrin binding motif (RGD), integrins have the potential to sequester latent TGF-β (SLC) to the cell surface where TGF-β activation could be locally controlled. Here, we show that SLC binds to αvβ8, an integrin expressed by normal epithelial and neuronal cells in vivo. This binding results in the membrane type 1 (MT1)-MMP–dependent release of active TGF-β, which leads to autocrine and paracrine effects on cell growth and matrix production. These data elucidate a novel mechanism of cellular homeostasis achieved through the coordination of the activities of members of three major gene families involved in cell–matrix interactions.

Transforming growth factor beta (TGF beta) family members are secreted in inactive complexes with a latency-associated peptide (LAP), a protein derived from the N-terminal region of the TGF beta gene product. Extracellular activation of these complexes is a critical but incompletely understood step in regulation of TGF beta function in vivo. We show that TGF beta 1 LAP is a ligand for the integrin alpha v beta 6 and that alpha v beta 6-expressing cells induce spatially restricted activation of TGF beta 1. This finding explains why mice lacking this integrin develop exaggerated inflammation and, as we show, are protected from pulmonary fibrosis. These data identify a novel mechanism for locally regulating TGF beta 1 function in vivo by regulating expression of the alpha v beta 6 integrin.

We have recently reported that angiotensin II (Ang II)-induced mitogen-activated protein kinase (MAPK) activation is mainly mediated by Ca 2؉ -dependent activation of a protein tyrosine kinase through G q -coupled Ang II type 1 receptor in cultured rat vascular smooth muscle cells (VSMC). In the present study, we found Ang II rapidly induced the tyrosine phosphorylation of the epidermal growth factor (EGF) receptor and its association with Shc and Grb2. These reactions were inhibited by the EGF receptor kinase inhibitor, AG1478. The Ang II-induced phosphorylation of the EGF receptor was mimicked by a Ca 2؉ ionophore and completely inhibited by an intracellular Ca 2؉ chelator. Thus, AG1478 abolished the MAPK activation induced by Ang II, a Ca 2؉ ionophore as well as EGF but not by a phorbol ester or platelet-derived growth factor-BB in the VSMC. Moreover, Ang II induced association of EGF receptor with catalytically active c-Src. This reaction was not affected by AG1478. These data indicate that Ang II induces Ca 2؉ -dependent transactivation of the EGF receptor which serves as a scaffold for pre-activated c-Src and for downstream adaptors, leading to MAPK activation in VSMC.

We used a spheroid model of colon carcinoma to analyze integrin dynamics as a function of the epithelial-mesenchymal transition (EMT), a process that provides a paradigm for understanding how carcinoma cells acquire a more aggressive phenotype. This EMT involves transcriptional activation of the beta6 integrin subunit and a consequent induction of alphavbeta6 expression. This integrin enhances the tumorigenic properties of colon carcinoma, including activation of autocrine TGF-beta and migration on interstitial fibronectin. Importantly, this study validates the clinical relevance of the EMT. Kaplan-Meier analysis of beta6 expression in 488 colorectal carcinomas revealed a striking reduction in median survival time of patients with high beta6 expression. Elevated receptor expression did not simply reflect increasing tumor stage, since log-rank analysis showed a more significant impact on the survival of patients with early-stage, as opposed to late-stage, disease. Cox regression analysis confirmed that this integrin is an independent variable for these tumors. These findings define the alphavbeta6 integrin as an important risk factor for early-stage disease and a novel therapeutic candidate for colorectal cancer.

We used a spheroid model of colon carcinoma to analyze integrin dynamics as a function of the epithelialmesenchymal transition (EMT), a process that provides a paradigm for understanding how carcinoma cells acquire a more aggressive phenotype. This EMT involves transcriptional activation of the β6 integrin subunit and a consequent induction of αvβ6 expression. This integrin enhances the tumorigenic properties of colon carcinoma, including activation of autocrine TGF-β and migration on interstitial fibronectin. Importantly, this study validates the clinical relevance of the EMT. Kaplan-Meier analysis of β6 expression in 488 colorectal carcinomas revealed a striking reduction in median survival time of patients with high β6 expression. Elevated receptor expression did not simply reflect increasing tumor stage, since log-rank analysis showed a more significant impact on the survival of patients with early-stage, as opposed to late-stage, disease. Cox regression analysis confirmed that this integrin is an independent variable for these tumors. These findings define the αvβ6 integrin as an important risk factor for early-stage disease and a novel therapeutic candidate for colorectal cancer.

Acute lung injury (ALI) is characterized by the flooding of the alveolar airspaces with protein-rich edema fluid and diffuse alveolar damage. We have previously reported that transforming growth factor-␤1 (TGF-␤1) is a critical mediator of ALI after intratracheal administration of bleomycin or Escherichia coli endotoxin, at least in part due to effects on lung endothelial and alveolar epithelial permeability. In the present study, we hypothesized that TGF-␤1 would also decrease vectorial ion and water transport across the distal lung epithelium. Therefore, we studied the effect of active TGF-␤1 on 22 Na ؉ uptake across monolayers of primary rat and human alveolar type II (ATII) cells. TGF-␤1 significantly reduced the amiloride-sensitive fraction of 22 Na ؉ uptake and fluid transport across monolayers of both rat and human ATII cells. TGF-␤1 also significantly decreased ␣ENaC mRNA and protein expression and inhibited expression of a luciferase reporter downstream of the ␣ENaC promoter in lung epithelial cells. The inhibitory effect of TGF-␤1 on sodium uptake and ␣ENaC expression in ATII cells was mediated by activation of the MAPK, ERK1/2. Consistent with the in vitro results, TGF-␤1 inhibited the amiloride-sensitive fraction of the distal airway epithelial fluid transport in an in vivo rat model at a dose that was not associated with any change in epithelial protein permeability. These data indicate that increased TGF-␤1 activity in the distal airspaces during ALI promotes alveolar edema by reducing distal airway epithelial sodium and fluid clearance. This reduction in sodium and fluid transport is attributable in large part to a reduction in apical membrane ␣ENaC expression mediated through an ERK1/2-dependent inhibition of the ␣ENaC promoter activity. Acute lung injury (ALI)1 is a devastating syndrome characterized by flooding of alveolar spaces with a protein-rich exudate that impairs pulmonary gas exchange, leading to arterial hypoxemia and respiratory failure (1). Epithelial injury can contribute to alveolar flooding, because the epithelial barrier is much less permeable under normal conditions than the endothelial barrier. Injury to alveolar epithelial cells can also disrupt normal epithelial fluid transport, impairing the removal of edema fluid from the alveolar space. Clinical studies have demonstrated that impaired alveolar fluid clearance is a characteristic feature of clinical lung injury (2, 3), but the mechanisms for this decrease in epithelial fluid transport have not been well worked out. The removal of edema fluid from the airspaces occurs via an active transport-dependent sodium concentration gradient across the distal lung epithelium. The ratelimiting step in the transport of fluid across the lung epithelium is the movement of sodium and chloride across the apical plasma membrane, specifically the movement of sodium through amiloride-sensitive and -insensitive channels (4). Among the sodium channels at the apical membrane of lung epithelial cells, amiloride-sensitive channels represent 50 -60% ...

Acute lung injury (ALI) is a devastating syndrome characterized by diffuse alveolar damage, elevated airspace levels of pro-inflammatory cytokines, and flooding of the alveolar spaces with protein-rich edema fluid. Interleukin-1␤ (IL-1␤) is one of the most biologically active cytokines in the distal airspaces of patients with ALI. IL-1␤ has been shown to increase lung epithelial and endothelial permeability. In this study, we hypothesized that IL-1␤ would decrease vectorial ion and water transport across the distal lung epithelium. Therefore, we measured the effects of IL-1␤ on transepithelial current, resistance, and sodium transport in primary cultures of alveolar epithelial type II (ATII) cells. IL-1␤ significantly reduced the amiloride-sensitive fraction of the transepithelial current and sodium transport across rat ATII cell monolayers. Moreover, IL-1␤ decreased basal and dexamethasone-induced epithelial sodium channel ␣-subunit (␣ENaC) mRNA levels and total and cellsurface protein expression. The inhibitory effect of IL-1␤ on ␣ENaC expression was mediated by the activation of p38 MAPK in both rat and human ATII cells and was independent of the activation of ␣v␤6 integrin and transforming growth factor-␤. These results indicate that IL-1␤ may contribute to alveolar edema in ALI by reducing distal lung epithelial sodium absorption. This reduction in ion and water transport across the lung epithelium is in large part due to a decrease in ␣ENaC expression through p38 MAPK-dependent inhibition of ␣ENaC promoter activity and to an alteration in ENaC trafficking to the apical membrane of ATII cells. Acute lung injury (ALI)1 is a devastating clinical syndrome manifested by diffuse alveolar damage, capillary injury, and disruption of the alveolar epithelium. The acute phase of ALI is characterized by the influx of protein-rich edema fluid that impairs gas exchange, causing arterial hypoxemia and respiratory failure with an overall mortality rate of 30 -40% (1). Along with an increase in lung endothelial and epithelial permeability to protein, this syndrome is associated with abnormal surfactant production and decreased vectorial fluid transport across the lung epithelial barrier (2, 3). A number of inflammatory mediators have been found to be elevated in the alveolar space during the early phase of ALI, including interleukin (IL)-1␤, tumor necrosis factor-␣, IL-6, and IL-8 (4). IL-1␤ is one of the most biologically active cytokines in pulmonary edema and bronchoalveolar lavage fluids of patients with ALI (4 -6). Indeed, IL-1␤ increases microvascular lung epithelial permeability in in vitro and in vivo models of ALI (7). IL-1␤ also enhances alveolar epithelial repair by increasing cell spreading (8) and fibroblast activation and proliferation (5). In contrast, the role of IL-1␤ in distal lung epithelial ion transport remains unclear. In other epithelia such as the colonic epithelium, IL-1␤ inhibits aldosterone-induced electrogenic sodium absorption and attenuates aldosterone-induced up-regulation of ␤-and ␥-subunit ...