Hisaaki Kawakatsu

A Mechanism for Regulating Pulmonary Inflammation and Fibrosis: The Integrin αvβ6 Binds and Activates Latent TGF β1

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.ated, and in this configuration TGFb is unable to bind University of California, San Francisco to its receptors; that is, TGFb is latent. In most cases, San Francisco, California 94143-0854 the complex of LAP and TGFb (the small latent complex 5 Department of Medicine SLC) is joined by latent TGFb binding protein 1 (LTBP1), 6 Cell Biology and Kaplan Cancer Center a matrix protein with sequence similarity to the fibrillins, New York University School of Medicine and the complex of all three proteins is called the large New York, New York 10016-6402 latent complex (LLC). Latent TGFb can be linked by

The integrin αvβ8 mediates epithelial homeostasis through MT1-MMP–dependent activation of TGF-β1

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.

Transcriptional activation of integrin β6 during the epithelial-mesenchymal transition defines a novel prognostic indicator of aggressive colon carcinoma

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.

Transforming Growth Factor-β1 Decreases Expression of the Epithelial Sodium Channel αENaC and Alveolar Epithelial Vectorial Sodium and Fluid Transport via an ERK1/2-dependent Mechanism

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-β1 (TGF-β1) 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-β1 would also decrease vectorial ion and water transport across the distal lung epithelium. Therefore, we studied the effect of active TGF-β1 on 22Na+ uptake across monolayers of primary rat and human alveolar type II (ATII) cells. TGF-β1 significantly reduced the amiloride-sensitive fraction of 22Na+ uptake and fluid transport across monolayers of both rat and human ATII cells. TGF-β1 also significantly decreased αENaC mRNA and protein expression and inhibited expression of a luciferase reporter downstream of the αENaC promoter in lung epithelial cells. The inhibitory effect of TGF-β1 on sodium uptake and αENaC expression in ATII cells was mediated by activation of the MAPK, ERK1/2. Consistent with the in vitro results, TGF-β1 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-β1 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 αENaC expression mediated through an ERK1/2-dependent inhibition of the αENaC promoter activity.

Interleukin-1β Decreases Expression of the Epithelial Sodium Channel α-Subunit in Alveolar Epithelial Cells via a p38 MAPK-dependent Signaling Pathway

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-1β (IL-1β) is one of the most biologically active cytokines in the distal airspaces of patients with ALI. IL-1β has been shown to increase lung epithelial and endothelial permeability. In this study, we hypothesized that IL-1β would decrease vectorial ion and water transport across the distal lung epithelium. Therefore, we measured the effects of IL-1β on transepithelial current, resistance, and sodium transport in primary cultures of alveolar epithelial type II (ATII) cells. IL-1β significantly reduced the amiloride-sensitive fraction of the transepithelial current and sodium transport across rat ATII cell monolayers. Moreover, IL-1β decreased basal and dexamethasone-induced epithelial sodium channel α-subunit (αENaC) mRNA levels and total and cell-surface protein expression. The inhibitory effect of IL-1β on αENaC expression was mediated by the activation of p38 MAPK in both rat and human ATII cells and was independent of the activation of αvβ6 integrin and transforming growth factor-β. These results indicate that IL-1β 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 αENaC expression through p38 MAPK-dependent inhibition of αENaC promoter activity and to an alteration in ENaC trafficking to the apical membrane of ATII cells.

Follicle-stimulating Hormone Activates Extracellular Signal-regulated Kinase but Not Extracellular Signal-regulated Kinase Kinase through a 100-kDa Phosphotyrosine Phosphatase

In this report we sought to elucidate the mechanism by which the follicle-stimulating hormone (FSH) receptor signals to promote activation of the p42/p44 extracellular signal-regulated protein kinases (ERKs) in granulosa cells. Results show that the ERK kinase MEK and upstream intermediates Raf-1, Ras, Src, and L-type Ca2+ channels are already partially activated in vehicle-treated cells and that FSH does not further activate them. This tonic stimulatory pathway appears to be restrained at the level of ERK by a 100-kDa phosphotyrosine phosphatase that associates with ERK in vehicle-treated cells and promotes dephosphorylation of its regulatory Tyr residue, resulting in ERK inactivation. FSH promotes the phosphorylation of this phosphotyrosine phosphatase and its dissociation from ERK, relieving ERK from inhibition and resulting in its activation by the tonic stimulatory pathway and consequent translocation to the nucleus. Consistent with this premise, FSH-stimulated ERK activation is inhibited by the cell-permeable protein kinase A-specific inhibitor peptide Myr-PKI as well as by inhibitors of MEK, Src, a Ca2+ channel blocker, and chelation of extracellular Ca2+. These results suggest that FSH stimulates ERK activity in immature granulosa cells by relieving an inhibition imposed by a 100-kDa phosphotyrosine phosphatase.

Shear Stress Stimulation of p130 cas Tyrosine Phosphorylation Requires Calcium-dependent c-Src Activation

Fluid shear stress (flow) modulates endothelial cell function via specific intracellular signaling events. Previously we showed that flow activated ERK1/2 in an integrin-dependent manner (Takahashi, M., and Berk, B. C. (1996) J. Clin. Invest. 98, 2623–2631). p130 Crk-associated substrate (Cas), a putative c-Src substrate, was originally identified as a highly phosphorylated protein that is localized to focal adhesions and acts as an adapter protein. Recent reports have shown that Cas is important in cardiovascular development and actin filament assembly. Flow (shear stress = 12 dynes/cm2) stimulated Cas tyrosine phosphorylation within 1 min in human umbilical vein endothelial cells. Phosphorylation peaked at 5 min (3.5 ± 0.7-fold) and was sustained to 20 min. Tyrosine phosphorylation of Cas was functionally important because flow stimulated association of Cas with Crk in a time- and force-dependent manner. Flow-mediated activation of c-Src, phosphorylation of Cas, and association of Cas with Crk were all inhibited by calcium chelation and pretreatment with the Src family-specific tyrosine kinase inhibitor PP1. To determine the role of c-Src in flow-stimulated phosphorylation of Cas, we transduced cells with adenovirus encoding kinase-inactive Src. Expression of kinase-inactive Src prevented flow-induced Cas tyrosine phosphorylation but not ERK1/2 activation. Calcium-dependent activation of c-Src and tyrosine phosphorylation of Cas defines a new flow-stimulated signal pathway, different from ERK1/2 activation. This pathway may be involved in focal adhesion remodeling and actin filament assembly.

A New Monoclonal Antibody Which Selectively Recognizes the Active Form of Src Tyrosine Kinase

Phosphorylation and dephosphorylation of Tyr-530 in human c-Src (Tyr-527 in avian c-Src) is critical in regulating c-Src kinase activity. So far, it has not been possible to distinguish the active and inactive forms in vivo. We now report a new monoclonal antibody that selectively recognizes the active form of c-Src. This antibody, termed clone 28, recognized a region adjacent to Tyr-530 (QYQP) in the C-terminal regulatory domain of c-Src, and its binding was hindered by phosphorylation of this tyrosine as determined by peptide competition assay. Combined immunoprecipitation/Western blotting revealed that clone 28 reacted with a 60-kDa protein that was precipitated by mAb 327, a well known monoclonal antibody against v-Src and c-Src. Cyanogen bromide cleavage and two-dimensional tryptic maps confirmed that clone 28 was specific for the active form (Tyr-530 not phosphorylated), whereas mAb 327 recognized the inactive form (Tyr-530 phosphorylated) as well as the active form. Clone 28 selectively immunoprecipitated the active form and augmented its kinase activity. Preabsorption experiments revealed that clone 28 could not completely immunoprecipitate the mAb 327 binding 60-kDa protein in either an in vitro or an in vivo phosphorylation system. These observations, taken together, strongly suggest the existence of multiple forms of c-Src as proposed by Cooper and Howell(1993) (Cooper, J. A., and Howell, B.(1993) Cell 73, 1051-1054). Using clone 28, we demonstrated a distinct localization of the active form of c-Src within cultured normal fibrobast cells. In liver tissue sections, we also examined the distribution of the active form in embryonic mice. Megakaryocytes were strongly stained, in contrast to completely negative immunoreactivity in hepatocytes, reticulocytes, and granulocytes. This result provides the first direct evidence that c-Src is highly activated in platelets.

Activation of mitogen-activated protein kinases and p90 ribosomal S6 kinase in failing human hearts with dilated cardiomyopathy.

OBJECTIVE A new member of the MAP kinase family, big MAP kinase-1 (BMK1), has been recently identified to promote cell growth and attenuate apoptosis. P90 ribosomal S6 kinase (p90RSK), one of the potentially important substrates of extracellular signal regulated kinase (ERK), regulates gene expression in part via phosphorylation of CREB and the Na(+)/H(+) exchanger. Recently, we have demonstrated that the activity of BMK1, Src (the upstream regulator of BMK1) and p90RSK was increased in hypertrophied myocardium induced by pressure-overload in the guinea pig. However, the abundance and activity of these kinases in human hearts are unknown. METHODS In addition to the three classical MAP kinases (ERK, p38 kinase, and c-Jun NH(2)-terminal kinase (JNK)), we examined the protein expression and activity of Src, BMK1, and p90RSK in explanted hearts from patients with dilated cardiomyopathy (n=9). Normal donor hearts, which were not suitable for transplant for technical reasons, were used as controls (n=5). RESULTS There were no significant differences in the levels of protein expression of these kinases between normal and failing hearts. ERK1/2 and p90RSK were activated in heart failure compared to control (P<0.01 and P<0.03, respectively), while the activity of p38 kinase was decreased (P<0.05) and the activity of JNK was unchanged in heart failure. By contrast, the activities of Src and BMK1 were significantly reduced in end-stage heart failure compared to normal donor hearts (P<0.05). CONCLUSION These data suggest that multiple MAP kinases, p90RSK, and Src are differentially regulated in human failing myocardium of patients with idiopathic dilated cardiomyopathy and may be involved in the pathogenesis of this complex disease.

Differential Regulation of p90 Ribosomal S6 Kinase and Big Mitogen–Activated Protein Kinase 1 by Ischemia/Reperfusion and Oxidative Stress in Perfused Guinea Pig Hearts

Reactive oxygen species (ROS) activate members of the Src kinase and mitogen-activated protein kinase superfamily, including big mitogen-activated protein kinase 1 (BMK1) and extracellular signal-regulated kinases (ERK1/2). A potentially important downstream effector of ERK1/2 is p90 ribosomal S6 kinase (p90RSK), which plays an important role in cell growth through the activation of several transcription factors, as well as the Na(+)/H(+) exchanger. Previously, we showed that Src regulates BMK1 via a redox-sensitive signaling pathway. Because ROS are generated during ischemia and reperfusion after ischemia, we assessed the effects of these stimuli (H(2)O(2), ischemia, and reperfusion) in the activation of ERK1/2, p90RSK, Src, and BMK1 in perfused guinea pig hearts. H(2)O(2) (100 micromol/L) significantly activated all kinases. Ischemia alone stimulated p90RSK, Src, and BMK1 but not ERK1/2. These results suggest that p90RSK activation through ischemia occurs via a pathway other than ERK1/2. A role of Src in ischemia-mediated BMK1 activation was demonstrated through inhibition with the Src inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine. Reperfusion after ischemia stimulated both p90RSK and ERK1/2. In contrast, although ROS increase during reperfusion after ischemia, the activities of both BMK1 and its upstream regulator, Src, were markedly attenuated by reperfusion after ischemia. The activation of C-terminal Src kinase during ischemia but not during reperfusion suggests that the attenuation of Src and BMK1 activity by reperfusion was not regulated by C-terminal Src kinase activity. The antioxidant N-2-mercaptopropionylglycine completely inhibited ERK1/2 and p90RSK activation by reperfusion but only partially inhibited ischemia-induced Src and BMK1 activation. The present study is the first to show the coregulation of Src and BMK1 by reperfusion after ischemia, which we propose to occur via a novel, ROS-independent pathway.