Alpha2beta1 integrin signalling enhances cyclooxygenase-2 expression in intestinal epithelial cells

Inflammatory bowel diseases (IBD) are linked to an increased risk of developing colon cancer, by inflammatory mediators and alterations to the extracellular matrix (ECM). The events induced by inflammatory mediators lead to dysregulated activation and induction of inflammatory genes such as cyclooxygenase-2 (COX-2). COX-2 is involved in the conversion of arachidonic acid to biologically active prostanoids and is highly upregulated in colon cancer. Since inflammation-induced changes to the extracellular matrix could affect integrin activities, we here investigated the effect of integrin signalling on the level of COX-2 expression in the non-transformed intestinal epithelial cell lines, Int 407 and IEC-6. Adhesion of these cells to a collagen I- or IV-coated surface, increased surface expression of alpha2beta1 integrin. Activation of integrins with collagen caused an increased cox-2 promoter activity, with a subsequent increase in COX-2 expression. The signalling cascade leading to this increased expression and promoter activity of cox-2, involves PKCalpha, the small GTPase Ras and NFkappaB but not Erk1/2 or Src activity. The integrin-induced increase in cellular COX-2 activity is responsible for an elevated generation of reactive oxygen species (ROS) and increased cell migration. This signalling pathway suggests a mechanism whereby inflammation-induced modulations of the ECM, can promote cancer transformation in the intestinal epithelial cells.     

Transforming growth factor-beta enhances secretory component and major histocompatibility complex class I antigen expression on rat IEC-6 intestinal epithelial cells.

Transforming growth factor-beta (TGF-beta) has been implicated as having a role in inflammatory responses by inducing cellular infiltration and the release of inflammatory cytokines. In this study, the IEC-6 rat intestinal epithelial cell line was used as a model to assess the effect of TGF-beta 1 on the expression of various plasma membrane determinants. TGF-beta 1 induced a dose-dependent increase in the percentage of cells expressing surface secretory component (SC) and class I major histocompatibility (MHC) antigens. However, the expression of class II MHC was unaffected. In contrast, epidermal growth factor had no effect on any of the surface proteins studied. The TGF-beta 1-enhanced expression of SC was accompanied by an enhanced binding of polymeric, but not monomeric, immunoglobulin A (IgA). Preincubation of the TGF-beta 1-treated cells with an anti-human beta-galactosyltransferase (beta-GT) antiserum did not block the binding of the anti-SC antibody, indicating that the TGF-beta-induced increase in SC staining was due to SC expression and not the polymeric immunoglobulin-binding enzyme, beta-GT. These results indicate that TGF-beta 1 may be important in immune functions involving intestinal epithelial cells by enhancing the expression of surface class I MHC antigens and SC, a protein responsible for the transport of polymeric IgA into the intestinal lumen.     

Transforming growth factor-beta stimulates the expression of endothelin mRNA by vascular endothelial cells

Endothelin is a potent vasoconstrictor peptide produced by vascular endothelial cells. Incubation of the serum-deprived confluent porcine aortic endothelial cells with 10-300 pM TGF-beta 1, resulted in a several fold increase in endothelin mRNA levels with a peak time of 2 h. An enzyme-linked immunosorbent assay revealed that the levels of endothelin in endothelial cell conditioned media was also increased by TGF-beta 1. These results suggest that TGF-beta 1, secreted by activated platelets, is involved not only in wound healing, but in the regulation of local vascular tone by stimulating endothelin production in the endothelial cells.     

Transforming growth factor-beta 1 expression in irradiated liver

The expression of transforming growth factor-beta 1 (TGF-beta 1) in the liver of irradiated rats was increased in a dose-dependent fashion 9 months after irradiation. Expression of TGF-beta 1 was confined primarily to hepatocytes in the pericentral region of the liver, and the percentage of hepatocytes strongly positive for TGF-beta 1 was significantly correlated with the extent of fibrosis. We further showed that a localized injection of TGF-beta 1 into normal rat liver elicited a strong fibrotic reaction at the injection site. These results suggest that the increased hepatic concentration of TGF-beta 1 in response to radiation injury may be important in the pathogenesis of radiation hepatitis. TGF-beta 1 was also found to be present at a significantly higher concentration in unirradiated human hepatocytes than in normal rat hepatocytes, implying that the propensity for humans to develop radiation hepatitis may result in part from the elevated levels of TGF-beta 1 normally found in human liver.     

Angiotensin II bi-directionally regulates cyclooxygenase-2 expression in intestinal epithelial cells

We previously demonstrated that angiotensin II (Ang II) receptor signaling is involved in azoxymethane-induced mouse colon tumorigenesis. In order to clarify the role of Ang II in COX-2 expression in the intestinal epithelium, the receptor subtype-specific effect on COX-2 expression in a rat intestinal epithelial cell line (RIE-1) has been investigated. Ang II dose- and time-dependently increased the expression of COX-2, but not COX-1 mRNA and protein. This stimulation was completely blocked by the AT(1) receptor antagonist but not the AT(2) receptor antagonist. Ang II and lipopolysaccharide (LPS) additively induced COX-2 protein in RIE-1 cells, whereas the LPS-induced COX-2 expression was significantly attenuated by low concentrations of Ang II or the AT(2) agonistic peptide CGP-42112A only in AT(2) over-expressed cells. These data indicate that Ang II bi-directionally regulates COX-2 expression via both AT(1) and AT(2) receptors. Control of COX-2 expression through Ang II signaling may have significance in cytokine-induced COX-2 induction and colon tumorigenesis.     

Transforming growth factor-beta2 enhances differentiation of cardiac myocytes from embryonic stem cells

Stem cell therapy holds great promise for the treatment of injured myocardium, but is challenged by a limited supply of appropriate cells. Three different isoforms of transforming growth factor-beta (TGF-beta) -beta1, -beta2, and -beta3 exhibit distinct regulatory effects on cell growth, differentiation, and migration during embryonic development. We compared the effects of these three different isoforms on cardiomyocyte differentiation from embryonic stem (ES) cells. In contrast to TGF-beta1, or -beta3, treatment of mouse ES cells with TGF-beta2 isoform significantly increased embryoid body (EB) proliferation as well as the extent of the EB outgrowth that beat rhythmically. At 17 days, 49% of the EBs treated with TGF-beta2 exhibited spontaneous beating compared with 15% in controls. Cardiac myocyte specific protein markers sarcomeric myosin and alpha-actin were demonstrated in beating EBs and cells isolated from EBs. In conclusion, TGF-beta2 but not TGF-beta1, or -beta3 promotes cardiac myocyte differentiation from ES cells.     

Transforming growth factor-beta1 regulates basement membrane formation by alveolar epithelial cells in vitro

Immortalized alveolar type II epithelial (SV40-T2) cells formed a continuous, thin lamina densa when they were cultured on collagen fibrils with the supplement of 1.0 ng/ml TGF-beta1. Corresponding to lamina densa formation, immunohistochemical analysis of laminin, type IV collagen, perlecan, and entactin (nidogen) indicated integration of these components in a linear array beneath the SV40-T2 cells. Synthesis of these basement membrane constituents was significantly enhanced by TGF-beta1 in a dose-dependent manner. On the other hand, TGF-beta1 did not affect the synthesis of extracellular matrix-regulatory enzymes and their inhibitors, such as type II transglutaminase, matrix metalloproteinase-2, plasminogen activator inhibitor-1, or tissue inhibitor of matrix metalloproteinase-1. These results indicate that basement membrane formation in the presence of 1.0 ng/ml TGF-beta1 is attributable to enhanced synthesis of basement membrane constituents. However, formation of a continuous basement membrane was inhibited at a TGF-beta1 concentration of 5.0 ng/ml. Synthesis of the basement membrane constituents was further enhanced at this concentration and the extracellular matrix-regulatory enzymes remained unchanged. The deposits of cellular fibronectin and type I collagen beneath SV40-T2 cells were significantly augmented. Thus excessive production of interstitial extracellular matrix components appears to obstruct the integration of basement membrane constituents into a continuous architecture. These results indicate that the basement membrane formation by SV40-T2 cells is achieved at the optimal TGF-beta1 concentration.     

Transforming growth factor-beta1 downregulation of Smad1 gene expression in rat hepatic stellate cells

Smads are intracellular signaling molecules of the transforming growth factor-beta (TGF-beta) superfamily that play an important role in the activation of hepatic stellate cells (HSCs) and hepatic fibrosis. Excepting the regulation of Smad7, receptor-regulated Smad gene expression is still unclear. We employed rat HSCs to investigate the expression and regulation of the Smad1 gene, which is a bone morphogenetic protein (BMP) receptor-regulated Smad. We found that the expression and phosphorylation of Smad1 are increased during the activation of HSCs. Moreover, TGF-beta significantly inhibits Smad1 gene expression in HSCs in a time- and dose-dependent manner. Furthermore, although both TGF-beta1 and BMP2 stimulate the activation of HSCs, they have different effects on HSC proliferation. In conclusion, Smad1 expression and phosphorylation are increased during the activation of HSCs and TGF-beta1 significantly inhibits the expression of the Smad1 gene.     

Transforming growth factor-beta1 decreases expression of the epithelial sodium channel alphaENaC 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-beta1 (TGF-beta1) 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-beta1 would also decrease vectorial ion and water transport across the distal lung epithelium. Therefore, we studied the effect of active TGF-beta1 on 22Na+ uptake across monolayers of primary rat and human alveolar type II (ATII) cells. TGF-beta1 significantly reduced the amiloride-sensitive fraction of 22Na+ uptake and fluid transport across monolayers of both rat and human ATII cells. TGF-beta1 also significantly decreased alphaENaC mRNA and protein expression and inhibited expression of a luciferase reporter downstream of the alphaENaC promoter in lung epithelial cells. The inhibitory effect of TGF-beta1 on sodium uptake and alphaENaC expression in ATII cells was mediated by activation of the MAPK, ERK1/2. Consistent with the in vitro results, TGF-beta1 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-beta1 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 alphaENaC expression mediated through an ERK1/2-dependent inhibition of the alphaENaC promoter activity.     

Cigarette smoke enhances human rhinovirus-induced CXCL8 production via HuR-mediated mRNA stabilization in human airway epithelial cells

Background

Human rhinovirus (HRV) triggers exacerbations of asthma and chronic obstructive pulmonary disease (COPD). Cigarette smoking is the leading risk factor for the development of COPD and 25% of asthmatics smoke. Smoking asthmatics have worse symptoms and more frequent hospitalizations compared to non-smoking asthmatics. The degree of neutrophil recruitment to the airways correlates with disease severity in COPD and during viral exacerbations of asthma. We have previously shown that HRV and cigarette smoke, in the form of cigarette smoke extract (CSE), each induce expression of the neutrophil chemoattractant and activator, CXCL8, in human airway epithelial cells. Additionally, we demonstrated that the combination of HRV and CSE induces expression of levels of CXCL8 that are at least additive relative to induction by each stimulus alone, and that enhancement of CXCL8 expression by HRV+CSE is regulated, at least in part, via mRNA stabilization. Here we further investigate the mechanisms by which HRV+CSE enhances CXCL8 expression.

Methods

Primary human bronchial epithelial cells were cultured and treated with CSE alone, HRV alone or the combination of the two stimuli. Stabilizing/destabilizing proteins adenine/uridine-rich factor-1 (AUF-1), KH-type splicing regulatory protein (KHSRP) and human antigen R (HuR) were measured in cell lysates to determine expression levels following treatment. siRNA knockdown of each protein was used to assess their contribution to the induction of CXCL8 expression following treatment of cells with HRV and CSE.

Results

We show that total expression of stabilizing/de-stabilizing proteins linked to CXCL8 regulation, including AUF-1, KHSRP and HuR, are not altered by CSE, HRV or the combination of the two stimuli. Importantly, however, siRNA-mediated knock-down of HuR, but not AUF-1 or KHSRP, abolishes the enhancement of CXCL8 by HRV+CSE. Data were analyzed using one-way ANOVA with student Newman-Keuls post hoc analysis and values of p≤ 0.05 were considered significant.

Conclusions

Induction of CXCL8 by the combination of HRV and CSE is regulated by mRNA stabilization involving HuR. Thus, targeting the HuR pathway may be an effective method of dampening CXCL8 production during HRV-induced exacerbations of lower airway disease, particularly in COPD patients and asthmatic patients who smoke.     

Transforming growth factor-beta down-regulates apolipoprotein M in HepG2 cells

Apolipoprotein M (apoM) is a novel apolipoprotein presented mostly in high-density lipoprotein (HDL) in human plasma, and is exclusively expressed in liver and in kidney. The pathophysiological function of apoM has not yet been elucidated. Apolipoprotein B (apoB), the characteristic apolipoprotein of low-density lipoprotein (LDL), is like apoM, a very hydrophobic protein, and thereafter they both must co-circulate with lipoprotein particles in plasma. The cytokine, transforming growth factor-beta (TGF-beta), has been shown to decreased apoB secretion in HepG2 cells, and we hypothesized that TGF-beta may have the same effects on apoM expression in HepG2 cells. In the present study, we used real-time RT-PCR to analyze apoM and apoB mRNA levels during administration of TGF-beta, as well as TGF-alpha, epidermal growth factor (EGF) and hepatic growth factor (HGF). TGF-beta significantly inhibited both apoM and apoB mRNA expression in HepG2 cells. The inhibitory effects of TGF-beta were dose-dependent, i.e. 1 ng/ml of TGF-beta decreased apoM mRNA levels by 30%, and 10 or 100 ng/ml of TGF-beta decreased apoM mRNA levels more than 65%. The effect of TGF-beta on apoB mRNA expression was slightly weaker than that of apoM, with a maximum effect at 10 or 100 ng/ml TGF-beta where apoB mRNA levels decreased about 55%. The inhibitory effects of TGF-beta on apoM and apoB mRNA levels also increased with increasing incubation time, where the maximum effect was obtained at 24 h. Moreover TGF-alpha, EGF and HGF all decreased both apoM and apoB mRNA levels, but to a less extent than TGF-beta. Further, all four cytokines had more pronounced effects on apoM mRNA expression than apoB mRNA expression. The present study suggested that apoM, like apoB, may be involved in the hepatic lipoprotein assembly in vivo.     

Apoptotic cell-derived sphingosine-1-phosphate promotes HuR-dependent cyclooxygenase-2 mRNA stabilization and protein expression

Removal of apoptotic cells by phagocytes is considered a pivotal immune regulatory process. Although considerable knowledge has been obtained on the postphagocytic macrophage phenotype, there is little information on molecular mechanisms, which provoke macrophage polarization. In this study, we show that human apoptotic Jurkat cells (AC) or AC-conditioned medium (CM) rapidly induces cyclooxygenase-2 (COX-2) expression in mouse RAW264.7 macrophages via sphingosine-1-phosphate (S1P). Pharmacological inhibition of S1P release from AC or using CM from cells with a knockdown of sphingosine kinase 2 in human MCF-7 cells abrogates this effect. Expression of COX-2 resulted from an increase in mRNA stability via its 3'-untranslated region (UTR), shown by COX-2-3'-UTR and AU-rich element-driven reporter assays. Western analysis corroborated increased nucleocytoplasmic shuttling of the RNA-binding protein HuR after CM treatment. RNA EMSA analysis revealed an S1P- and CM-mediated increase in HuR-RNA binding to a COX-2-specific UTR, whereas HuR knockdown pointed to its importance for S1P in CM-induced COX-2 expression. Immunofluorescence microscopy of phospholipase A2 (PLA2) and ELISA analysis of PGE2 revealed activation of PLA2 and production of PGE2 in response to CM but not S1P. S1P, released from AC, uses HuR to stabilize COX-2 mRNA and thus to increase COX-2 protein expression. However, only CM also activates PLA2 to provide the substrate for COX-2. Our data underscore the importance of S1P in AC-mediated immune regulation, by stabilizing COX-2 mRNA in macrophages, a prerequisite for PGE2 formation.     

Transforming growth factor-beta1 inhibits thrombin activation of endothelial cells

Transforming growth factor-beta1 (TGF-beta1) is reported to exert both pro- and anti-inflammatory effects on the chronic activation of endothelial cells (ECs) in vitro by cytokines such as tumour necrosis factor-alpha (TNF-alpha). However, the effects of TGF-beta1 on acute inflammatory responses of ECs in vitro (e.g. to thrombin) have not been characterised. Pretreatment with TGF-beta1 (10 ng/mL) effectively inhibited all the thrombin-stimulated responses in rat aortic endothelial cells (RAECs) examined: adhesion and migration of polymorphonuclear leukocytes, adhesion of platelets and lymphocytes. Substantial inhibition of thrombin stimulation occurred after 30 min of pretreatment with TGF-beta1 and maximal inhibition was obtained after 1-20 h of pretreatment. Inhibition by TGF-beta1 pretreatment for 30 min was not affected by cycloheximide and was therefore independent of protein synthesis. Treatment with TGF-beta1 for 20 h did not affect the total levels of P-selectin and von Willebrand factor (vWF) in RAECs, but reduced thrombin-stimulated recruitment of P-selectin and vWF to the cell surface. The data demonstrate that TGF-beta1 exerts a potent anti-thrombin effect on ECs, effective after long and short pretreatment times.