Thrombin inhibition with dabigatran protects against high-fat diet-induced fatty liver disease in mice.

Nonalcoholic fatty liver disease (NAFLD) is the hepatic manifestation of obesity and metabolic syndrome. Robust coagulation cascade activation is common in obese patients with NAFLD. We identified a critical temporal relationship between thrombin generation and the manifestation of hepatic steatosis, inflammation, and injury in C57BL/6J mice fed a high-fat diet (HFD) for 1, 2, and 3 months. Mice fed a HFD exhibited dramatic increases in hepatocellular injury and inflammation over time. Hepatic fibrin deposition preceded an increase in serum alanine aminotransferase, and the most dramatic changes in liver histopathology occurred in conjunction with a detectable increase in plasma thrombin-antithrombin levels at 3 months. To directly determine whether thrombin activity promotes NAFLD pathogenesis, mice were fed a HFD and simultaneously treated with the direct thrombin inhibitor dabigatran etexilate for 3 months. Notably, dabigatran treatment significantly reduced hepatic fibrin deposition, hepatic inflammation, hepatocellular injury, and steatosis in mice fed a HFD. Of interest, dabigatran treatment also significantly attenuated HFD-induced body weight gain. Gene expression analysis suggested that thrombin potentially drives NAFLD pathogenesis by altering the expression of genes associated with lipid metabolism and bile acid synthesis. Collectively, the results suggest that thrombin activity is central to HFD-induced body weight gain, liver injury, and inflammation and provide the proof-of-principle evidence that pharmacological thrombin inhibition could be effective in limiting NAFLD and associated pathologies.

Toxicogenomic analysis reveals profibrogenic effects of trichloroethylene in autoimmune-mediated cholangitis in mice.

Epidemiological studies suggest that exposure to environmental chemicals increases the risk of developing autoimmune liver disease. However, the identity of specific chemical perpetrators and the mechanisms whereby environmental chemicals modify liver disease is unclear. Previous studies link exposure to trichloroethylene (TCE) with the development of autoimmune liver disease and exacerbation of autoimmunity in lupus-prone MRL mice. In this study, we utilized NOD.c3c4 mice, which spontaneously develop autoimmune cholangitis bearing resemblance to some features of primary biliary cirrhosis. Nine-week-old female NOD.c3c4 mice were given TCE (0.5 mg/ml) or its vehicle (1% Cremophor-EL) in drinking water for 4 weeks. TCE had little effect on clinical chemistry, biliary cyst formation, or hepatic CD3+ T-cell accumulation. Hepatic microarray profiling revealed a dramatic suppression of early growth response 1 (EGR1) mRNA in livers of TCE-treated mice, which was verified by qPCR and immunohistochemical staining. Consistent with a reported link between reduced EGR1 expression and liver fibrosis, TCE increased hepatic type I collagen (COL1A1) mRNA and protein levels in livers of NOD.c3c4 mice. In contrast, TCE did not increase COL1A1 expression in NOD.ShiLtJ mice, which do not develop autoimmune cholangitis. These results suggest that in the context of concurrent autoimmune liver disease with a genetic basis, modification of hepatic gene expression by TCE may increase profibrogenic signaling in the liver. Moreover, these studies suggest that NOD.c3c4 mice may be a novel model to study gene-environment interactions critical for the development of autoimmune liver disease.

H1-antihistamines exacerbate high-fat diet-induced hepatic steatosis in wild-type but not in apolipoprotein E knockout mice.

We examined the effects of two over-the-counter H1-antihistamines on the progression of fatty liver disease in male C57Bl/6 wild-type and apolipoprotein E (ApoE)-/- mice. Mice were fed a high-fat diet (HFD) for 3 mo, together with administration of either cetirizine (4 mg/kg body wt) or fexofenadine (40 mg/kg body wt) in drinking water. Antihistamine treatments increased body weight gain, gonadal fat deposition, liver weight, and hepatic steatosis in wild-type mice but not in ApoE-/- mice. Lobular inflammation, acute inflammation, and necrosis were not affected by H1-antihistamines in either genotype. Serum biomarkers of liver injury tended to increase in antihistamine-treated wild-type mice. Serum level of glucose was increased by fexofenadine, whereas lipase was increased by cetirizine. H1-antihistamines reduced the mRNA expression of ApoE and carbohydrate response element-binding protein in wild-type mice, without altering the mRNA expression of sterol regulatory element-binding protein 1c, fatty acid synthase, or ApoB100, in either genotype. Fexofenadine increased both triglycerides and cholesterol ester, whereas cetirizine increased only cholesterol ester in liver, with a concomitant decrease in serum triglycerides by both antihistamines in wild-type mice. Antihistamines increased hepatic levels of conjugated bile acids in wild-type mice, with the effect being significant in fexofenadine-treated animals. The increase was associated with changes in the expression of organic anion transport polypeptide 1b2 and bile salt export pump. These results suggest that H1-antihistamines increase the progression of fatty liver disease in wild-type mice, and there seems to be an association between the severity of disease, presence of ApoE, and increase in hepatic bile acid levels.

Mechanisms of STAT3 activation in the liver of FXR knockout mice.

Farnesoid X receptor (FXR, Nr1h4) is a ligand-activated transcription factor belonging to the nuclear receptor superfamily. FXR is essential in maintaining bile acid (BA) homeostasis, and FXR(-/-) mice develop cholestasis, inflammation, and spontaneous liver tumors. The signal transducer and activator of transcription 3 (STAT3) is well known to regulate liver growth, and STAT3 is feedback inhibited by its target gene, the suppressor of cytokine signaling 3 (SOCS3). Strong activation of STAT3 was detected in FXR(-/-) mouse livers. However, the mechanism of STAT3 activation with FXR deficiency remains elusive. Wild-type (WT) and FXR(-/-) mice were used to detect STAT3 pathway activation in the liver. In vivo BA feeding or deprivation was used to determine the role of BAs in STAT3 activation, and in vitro molecular approaches were used to determine the direct transcriptional regulation of SOCS3 by FXR. STAT3 was activated in FXR(-/-) but not WT mice. BA feeding increased, but deprivation by cholestyramine reduced, serum inflammatory markers and STAT3 activation. Furthermore, the Socs3 gene was determined as a direct FXR target gene. The elevated BAs and inflammation, along with reduced SOCS3, collectively contribute to the activation of the STAT3 signaling pathway in the liver of FXR(-/-) mice. This study suggests that the constitutive activation of STAT3 may be a mechanism of liver carcinogenesis in FXR(-/-) mice.

Small heterodimer partner overexpression partially protects against liver tumor development in farnesoid X receptor knockout mice.

Farnesoid X receptor (FXR, Nr1h4) and small heterodimer partner (SHP, Nr0b2) are nuclear receptors that are critical to liver homeostasis. Induction of SHP serves as a major mechanism of FXR in suppressing gene expression. Both FXR(-/-) and SHP(-/-) mice develop spontaneous hepatocellular carcinoma (HCC). SHP is one of the most strongly induced genes by FXR in the liver and is a tumor suppressor, therefore, we hypothesized that deficiency of SHP contributes to HCC development in the livers of FXR(-/-) mice and therefore, increased SHP expression in FXR(-/-) mice reduces liver tumorigenesis. To test this hypothesis, we generated FXR(-/-) mice with overexpression of SHP in hepatocytes (FXR(-/-)/SHP(Tg)) and determined the contribution of SHP in HCC development in FXR(-/-) mice. Hepatocyte-specific SHP overexpression did not affect liver tumor incidence or size in FXR(-/-) mice. However, SHP overexpression led to a lower grade of dysplasia, reduced indicator cell proliferation and increased apoptosis. All tumor-bearing mice had increased serum bile acid levels and IL-6 levels, which was associated with activation of hepatic STAT3. In conclusion, SHP partially protects FXR(-/-) mice from HCC formation by reducing tumor malignancy. However, disrupted bile acid homeostasis by FXR deficiency leads to inflammation and injury, which ultimately results in uncontrolled cell proliferation and tumorigenesis in the liver.

Hepatocyte tissue factor activates the coagulation cascade in mice.

In this study, we characterized tissue factor (TF) expression in mouse hepatocytes (HPCs) and evaluated its role in mouse models of HPC transplantation and acetaminophen (APAP) overdose. TF expression was significantly reduced in isolated HPCs and liver homogenates from TF(flox/flox)/albumin-Cre mice (HPC(ΔTF) mice) compared with TF(flox/flox) mice (control mice). Isolated mouse HPCs expressed low levels of TF that clotted factor VII-deficient human plasma. In addition, HPC TF initiated factor Xa generation without exogenous factor VIIa, and TF activity was increased dramatically after cell lysis. Treatment of HPCs with an inhibitory TF antibody or a cell-impermeable lysine-conjugating reagent prior to lysis substantially reduced TF activity, suggesting that TF was mainly present on the cell surface. Thrombin generation was dramatically reduced in APAP-treated HPC(ΔTF) mice compared with APAP-treated control mice. In addition, thrombin generation was dependent on donor HPC TF expression in a model of HPC transplantation. These results suggest that mouse HPCs constitutively express cell surface TF that mediates activation of coagulation during hepatocellular injury.

Therapeutic administration of the direct thrombin inhibitor argatroban reduces hepatic inflammation in mice with established fatty liver disease.

Thrombin generation is increased in patients with nonalcoholic fatty liver disease (NAFLD) and in mouse models of diet-induced obesity. Deficiency in the thrombin receptor protease activated receptor-1 reduces hepatic inflammation and steatosis in mice fed a Western diet. However, it is currently unclear whether thrombin inhibitors can modify the pathogenesis of established NAFLD. We tested the hypothesis that thrombin inhibition could reverse hepatic steatosis and inflammation in mice with established diet-induced NAFLD. Low-density lipoprotein receptor-deficient LDLr(-/-) mice were fed a control diet or a Western diet for 19 weeks. Mice were given the direct thrombin inhibitor argatroban ∼15 mg/kg/day or its vehicle via a miniosmotic pump for the final 4 weeks of the study. Argatroban administration significantly reduced hepatic proinflammatory cytokine expression and reduced macrophage and neutrophil accumulation in livers of mice fed a Western diet. Argatroban did not significantly impact hepatic steatosis, as indicated by histopathology, Oil Red O staining, and hepatic triglyceride levels. Argatroban reduced serum triglyceride and cholesterol levels in mice fed a Western diet. Argatroban reduced both α-smooth muscle actin expression and Type 1 collagen mRNA levels in livers of mice fed a Western diet, indicating reduced activation of hepatic stellate cells. This study indicates that therapeutic intervention with a thrombin inhibitor attenuates hepatic inflammation and several profibrogenic changes in mice fed a Western diet.

Fibrin(ogen)-independent role of plasminogen activators in acetaminophen-induced liver injury.

Hepatic fibrin(ogen) has been noted to occur after acetaminophen (APAP)-induced liver injury in mice. Deficiency in plasminogen activator inhibitor-1 (PAI-1), an endogenous inhibitor of fibrinolysis, increases APAP-induced liver injury in mice. However, the roles of fibrinogen and fibrinolysis in APAP-induced liver injury are not known. We tested the hypothesis that hepatic fibrin(ogen) deposition reduces severity of APAP-induced liver injury. APAP-induced (300 mg/kg) liver injury in mice was accompanied by thrombin generation, consumption of plasma fibrinogen, and deposition of hepatic fibrin. Neither fibrinogen depletion with ancrod nor complete fibrinogen deficiency [via knockout of the fibrinogen alpha chain gene (Fbg(-/-))] affected APAP-induced liver injury. PAI-1 deficiency (PAI-1(-/-)) increased APAP-induced liver injury and hepatic fibrin deposition 6 hours after APAP administration, which was followed by marked hemorrhage at 24 hours. As in PAI-1(-/-) mice, administration of recombinant tissue plasminogen activator (tenecteplase, 5 mg/kg) worsened APAP-induced liver injury and hemorrhage in wild-type mice. In contrast, APAP-induced liver injury was reduced in both plasminogen-deficient mice and in wild-type mice treated with tranexamic acid, an inhibitor of plasminogen activation. Activation of matrix metalloproteinase 9 (MMP-9) paralleled injury, but MMP-9 deficiency did not affect APAP-induced liver injury. The results indicate that fibrin(ogen) does not contribute to development of APAP-induced liver injury and suggest rather that plasminogen activation contributes to APAP-induced liver injury.

Lipopolysaccharide enhances transforming growth factor ß1-induced platelet-derived growth factor-B expression in bile duct epithelial cells.

BACKGROUND AND AIM: Platelet-derived growth factor (PDGF)-B is a potent profibrogenic mediator expressed by bile duct epithelial cells (BDECs) that contributes to liver fibrosis after bile duct ligation. However, the mechanism of PDGF-B induction in BDECs during cholestasis is not known. Transforming growth factor ß (TGFß) and lipopolysaccharide (LPS) also contribute to the profibrogenic response after bile duct ligation. We tested the hypothesis that LPS and TGFß1 synergistically induce PDGF-B expression in BDECs. METHODS: Transformed human BDECs (MMNK-1 cells) and primary rat BDECs were stimulated with LPS and/or TGFß1, and signaling pathways through which LPS potentiates TGFß1-induced PDGF-B mRNA expression were investigated. RESULTS: Stimulation of MMNK-1 cells with LPS alone did not significantly induce PDGF-B mRNA expression. However, LPS co-treatment enhanced TGFß1 induction of PDGF-B mRNA in MMNK-1 cells and also in primary rat BDECs. Importantly, co-treatment of MMNK-1 cells with LPS and TGFß1 also significantly increased PDGF-BB protein expression. Interestingly, LPS did not affect TGFß1 activation of a SMAD-dependent reporter construct. Rather, stimulation of MMNK-1 cells with LPS, but not TGFß1, increased JNK1/2 phosphorylation. Expression of dominant negative JNK2, but not dominant negative JNK1, inhibited the LPS potentiation of TGFß1-induced PDGF-B mRNA expression in MMNK-1 cells. In addition, LPS treatment caused IκBα degradation and activation of a nuclear factor κB (NFκB)-dependent reporter construct. Expression of an IκBα super repressor inhibited activation of NFκB and attenuated LPS potentiation of TGFß1-induced PDGF-B mRNA. CONCLUSIONS: The results indicate that LPS activation of NFκB and JNK2 enhances TGFß1-induced PDGF-B expression in BDECs.

Protease-activated receptor 1 and hematopoietic cell tissue factor are required for hepatic steatosis in mice fed a Western diet.

Nonalcoholic fatty liver disease (NAFLD) is the hepatic manifestation of obesity and metabolic syndrome and contributes to increased risk of cardiovascular disease and liver-related morbidity and mortality. Indeed, obese patients with metabolic syndrome generate greater amounts of thrombin, an indication of coagulation cascade activation. However, the role of the coagulation cascade in Western diet-induced NAFLD has not been investigated. Using an established mouse model of Western diet-induced NAFLD, we tested whether the thrombin receptor protease-activated receptor 1 (PAR-1) and hematopoietic cell-derived tissue factor (TF) contribute to hepatic steatosis. In association with hepatic steatosis, plasma thrombin-antithrombin levels and hepatic fibrin deposition increased significantly in C57Bl/6J mice fed a Western diet for 3 months. PAR-1 deficiency reduced hepatic inflammation, particularly monocyte chemoattractant protein-1 expression and macrophage accumulation. In addition, PAR-1 deficiency was associated with reduced steatosis in mice fed a Western diet, including reduced liver triglyceride accumulation and CD36 expression. Similar to PAR-1 deficiency, hematopoietic cell TF deficiency was associated with reduced inflammation and reduced steatosis in livers of low-density lipoprotein receptor-deficient mice fed a Western diet. Moreover, hematopoietic cell TF deficiency reduced hepatic fibrin deposition. These studies indicate that PAR-1 and hematopoietic cell TF are required for liver inflammation and steatosis in mice fed a Western diet.

Differential roles of unsaturated and saturated fatty acids on autophagy and apoptosis in hepatocytes.

Fatty acid-induced lipotoxicity plays a critical role in the pathogenesis of nonalcoholic liver disease. Saturated fatty acids and unsaturated fatty acids have differential effects on cell death and steatosis, but the mechanisms responsible for these differences are not known. Using cultured HepG2 cells and primary mouse hepatocytes, we found that unsaturated and saturated fatty acids differentially regulate autophagy and apoptosis. The unsaturated fatty acid, oleic acid, promoted the formation of triglyceride-enriched lipid droplets and induced autophagy but had a minimal effect on apoptosis. In contrast, the saturated fatty acid, palmitic acid, was poorly converted into triglyceride-enriched lipid droplets, suppressed autophagy, and significantly induced apoptosis. Subsequent studies revealed that palmitic acid-induced apoptosis suppressed autophagy by inducing caspase-dependent Beclin 1 cleavage, indicating cross-talk between apoptosis and autophagy. Moreover, our data suggest that the formation of triglyceride-enriched lipid droplets and induction of autophagy are protective mechanisms against fatty acid-induced lipotoxicity. In line with our in vitro findings, we found that high-fat diet-induced hepatic steatosis was associated with autophagy in the mouse liver. Potential modulation of autophagy may be a novel approach that has therapeutic benefits for obesity-induced steatosis and liver injury.

Fibrinogen deficiency increases liver injury and early growth response-1 (Egr-1) expression in a model of chronic xenobiotic-induced cholestasis.

Chronic cholestatic liver injury induced by cholestasis in rodents is associated with hepatic fibrin deposition, and we found evidence of fibrin deposition in livers of patients with cholestasis. Key components of the fibrinolytic pathway modulate cholestatic liver injury by regulating activation of hepatocyte growth factor. However, the exact role of hepatic fibrin deposition in chronic cholestasis is not known. We tested the hypothesis that fibrinogen (Fbg) deficiency worsens liver injury induced by cholestasis. Fbg-deficient mice (Fbgα(-/-) mice) and heterozygous control mice (Fbgα(+/-) mice) were fed either the control diet or a diet containing 0.025% α-naphthylisothiocyanate (ANIT), which selectively injures bile duct epithelial cells in the liver, for 2 weeks. Hepatic fibrin and collagen deposits were evident in livers of heterozygous control mice fed the ANIT diet. Complete Fbg deficiency was associated with elevated serum bile acids, periportal necrosis, and increased serum alanine aminotransferase activity in mice fed the ANIT diet. Fbg deficiency was associated with enhanced hepatic expression of the transcription factor early growth response-1 (Egr-1) and enhanced induction of genes encoding the Egr-1-regulated proinflammatory chemokines monocyte chemotactic protein-1, KC growth-regulated protein, and macrophage inflammatory protein-2. Interestingly, peribiliary collagen deposition was not evident near necrotic areas in Fbg-deficient mice. The results suggest that in this model of chronic cholestasis, fibrin constrains the release of bile constituents from injured intrahepatic bile ducts, thereby limiting the progression of hepatic inflammation and hepatocellular injury.

Regulation of transforming growth factor-ß1-dependent integrin ß6 expression by p38 mitogen-activated protein kinase in bile duct epithelial cells.

Bile duct epithelial cells (BDECs) contribute to liver fibrosis by expressing αVß6 integrin, a critical activator of latent transforming growth factor ß (TGF-ß). ß6 integrin (Itgß6) mRNA induction and αVß6 integrin expression in BDECs are partially TGF-ß-dependent. However, the signaling pathways required for TGF-ß-dependent Itgß6 mRNA induction in BDECs are not known. We tested the hypothesis that the p38 mitogen-activated protein kinase (MAPK) signaling pathway contributes to TGF-ß1 induction of Itgß6 mRNA by activating SMAD and activator protein 1 (AP-1) transcription factors. Pretreatment of transformed human BDECs (MMNK-1 cells) with two different p38 MAPK inhibitors, but not a control compound, inhibited TGF-ß1 induction of Itgß6 mRNA. Inhibition of p38 also reduced TGF-ß1 activation of a SMAD-dependent reporter construct. Expression of a dominant-negative SMAD3 (SMAD3ΔC) significantly reduced TGF-ß1-induced Itgß6 mRNA expression. Expression of JunB mRNA, but not other AP-1 proteins, increased in TGF-ß1-treated MMNK-1 cells, and induction of JunB expression was p38-dependent. Consistent with a requirement for de novo induction of JunB protein, cycloheximide pretreatment inhibited TGF-ß1 induction of Itgß6 mRNA. Expression of a dominant-negative AP-1 mutant (TAM67) also inhibited TGF-ß1 induction of Itgß6 mRNA. Overall, the results suggest that p38 contributes to TGF-ß1-induced Itgß6 mRNA expression in MMNK-1 cells by regulating activation of both SMAD and AP-1 transcription factors.

Monocyte chemoattractant protein-1 deficiency does not affect steatosis or inflammation in livers of mice fed a methionine-choline-deficient diet.

Monocyte chemoattractant protein-1 (MCP-1, Ccl2) expression is increased in livers of patients with nonalcoholic steatohepatitis and in murine models of steatohepatitis. Several studies in rodents indicate that MCP-1 contributes to liver steatosis induced by feeding a high-fat diet. However, the extent of MCP-1 involvement in the widely utilized methionine-choline-deficient (MCD) diet model of steatohepatitis has not been determined. We tested the hypothesis that MCP-1 contributes to steatohepatitis in mice fed the MCD diet. MCP-1-deficient mice on a C57Bl/6J background and age-matched C57Bl/6J mice were fed either MCD diet or control diet for 4 weeks. MCP-1 deficiency did not affect steatohepatitis, as indicated by liver histopathology, nor did it affect serum alanine aminotransferase activity, hepatic triglyceride levels, hepatic inflammatory gene induction, or macrophage accumulation in mice fed the MCD diet. MCP-1 deficiency reduced the expression of the profibrogenic genes, pro-collagen 1a1, connective tissue growth factor, and transforming growth factor-ß, in mice fed the MCD diet. MCP-1 deficiency significantly reduced collagen deposition and α-smooth muscle actin protein levels in the livers of mice fed the MCD diet. The results indicate that MCP-1 does not contribute to liver steatosis or inflammation in the MCD diet model of steatohepatitis. Rather, the data suggest that MCP-1 contributes to fibrosis in mice fed the MCD diet, independent of effects on steatosis and inflammation.

Regulation of human cytidine triphosphate synthetase 2 by phosphorylation.

Cytidine triphosphate synthetase (CTPS) is the rate-limiting enzyme in de novo CTP synthesis and is required for the formation of RNA, DNA, and phospholipids. This study determined the kinetic properties of the individual human CTPS isozymes (hCTPS1 and hCTPS2) and regulation through substrate concentration, oligomerization, and phosphorylation. Kinetic analysis demonstrated that both hCTPS1 and hCTPS2 were maximally active at physiological concentrations of ATP, GTP, and glutamine, whereas the K(m) and IC(50) values for the substrate UTP and the product CTP, respectively, were close to their physiological concentrations, indicating that the intracellular concentrations of UTP and CTP may precisely regulate hCTPS activity. Low serum treatment increased hCTPS2 phosphorylation, and five probable phosphorylation sites were identified in the hCTPS2 C-terminal domain. Metabolic labeling of hCTPS2 with [(32)P]H(3)PO(4) demonstrated that Ser(568) and Ser(571) were two major phosphorylation sites, and additional studies demonstrated that Ser(568) was phosphorylated by casein kinase 1 both in vitro and in vivo. Interestingly, mutation of Ser(568) (S568A) but not Ser(571) significantly increased hCTPS2 activity, demonstrating that Ser(568) is a major inhibitory phosphorylation site. The S568A mutation had a greater effect on the glutamine than ammonia-dependent activity, indicating that phosphorylation of this site may influence the glutaminase domain of hCTPS2. Deletion of the C-terminal regulatory domain of hCTPS1 also greatly increased the V(max) of this enzyme. In summary, this is the first study to characterize the kinetic properties of hCTPS1 and hCTPS2 and to identify Ser(568) as a major site of CTPS2 regulation by phosphorylation.

Modulation of epidermal growth factor receptor binding to human airway smooth muscle cells by glucocorticoids and beta2-adrenergic receptor agonists.

EGF receptors (EGFRs) are increased in airway smooth muscle in asthma, which may contribute to both their hyperproliferation and hypercontractility. Lysophosphatidic acid (LPA) is a candidate pathological agent in asthma and other airway diseases, and LPA upregulates EGFRs in human airway smooth muscle (HASM) cells. We tested whether therapeutic glucocorticoids and/or beta(2)-adrenergic receptor (beta(2)AR) agonists also alter EGFR binding in HASM cells. Exposure to glucocorticoids for 24 h induced a twofold increase in EGFR binding similar to that with LPA; fluticasone was markedly more potent than dexamethasone. The increase in EGFR binding by glucocorticoids required 24-h exposure, consistent with transcription-mediated effects. Although the increase in EGFR binding was blocked by the protein synthesis inhibitor cycloheximide for LPA, fluticasone, and dexamethasone, only LPA induced a significant increase in EGFR protein expression detected by immunoblotting. In contrast to the increased binding induced by the glucocorticoids, the beta(2)AR agonists isoproterenol, albuterol, and salmeterol all induced a decrease in EGFR binding. beta(2)AR agonist effects were multiphasic, with an initial decline at 2-4 h that reversed by 6 h and a second, somewhat greater decrease by 18-24 h. In cells pretreated with glucocorticoids, the decreases in EGFR binding by subsequent beta(2)AR treatment were not statistically significant; glucocorticoid upregulation of EGFRs also prevented further increases by LPA. Similar increases by glucocorticoids and decreases by beta(2)AR agonists were found in HFL-1 human lung fibroblasts. These complex and opposing effects of clinically relevant glucocorticoids and beta(2)AR agonists on airway mesenchymal cell EGFRs likely contribute to their overall therapeutic profile in the diseased airway.

Lysophosphatidic acid induces rapid and sustained decreases in epidermal growth factor receptor binding via different signaling pathways in BEAS-2B airway epithelial cells.

Lysophosphatidic acid (LPA) and epidermal growth factor (EGF) are important mediators of lung cell function and lung diseases. We showed previously that LPA decreases epidermal growth factor receptor (EGFR) binding rapidly in BEAS-2B airway epithelial cells, and this decrease is sustained to at least 18 h. The current studies investigate which LPA signaling pathways mediate the rapid versus sustained decreases in EGFR binding in BEAS-2B cells. The G(i/o) inhibitor pertussis toxin and the Rho kinase inhibitor Y-27632 [(R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide] had no effect on the rapid or sustained decreases. However, the mitogen-activated protein kinase kinase (MEK) inhibitor U0126 [1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto)-butadiene ethanolate] decreased extracellular signal-regulated kinase (ERK) 1/2 phosphorylation, completely inhibited the rapid decrease in binding, and partially inhibited the sustained decrease. The direct Ca2+- and phospholipid-dependent protein kinase (PKC) activator phorbol-12-myristate-13-acetate stimulated ERK1/2 phosphorylation and decreased EGFR binding at both 15 min and 18 h. Furthermore, inhibitors of PKC partially inhibited ERK1/2 phosphorylation and the 15-min decrease but completely inhibited the 18-h decrease. Inhibitor time course studies showed that PKC induction of the 18-h decrease occurred during the first 3 h of treatment. We showed previously that LPA-stimulated EGFR transactivation contributes to the rapid decrease. Two transactivation inhibitors partially inhibited ERK1/2 phosphorylation, and U0126 partially inhibited EGFR transactivation, indicating that MEK may be involved both upstream and downstream of EGFR activation. Together, the data presented here indicate that LPA mediates the rapid decrease in EGFR binding via EGFR transactivation, MEK/ERK, and PKC, whereas the sustained decrease is regulated primarily by PKC.

Inhibition of human airway smooth muscle cell proliferation by beta 2-adrenergic receptors and cAMP is PKA independent: evidence for EPAC involvement.

Mechanisms by which beta-adrenergic receptor (beta AR) agonists inhibit proliferation of human airway smooth muscle (HASM) cells were investigated because of their potential relevance to smooth muscle hyperplasia in asthma. We hypothesized that beta AR agonists would inhibit mitogenesis in HASM cells via the beta 2AR, an increase in cAMP, and PKA activation. HASM cells were treated for 24 h with various agents and then analyzed for [3H]thymidine incorporation as a measure of cell proliferation. EGF stimulated proliferation by approximately 10-fold. The nonselective beta AR agonist isoproterenol and the beta 2AR-selective agonists albuterol and salmeterol inhibited EGF-stimulated proliferation by more than 50%, with half-maximal effects at 4.8 nM, 110 nM, and 6.7 nM, respectively. A beta 2AR-selective antagonist inhibited the isoproterenol effect with 100-fold greater potency than a beta 1AR-selective antagonist, confirming beta 2AR involvement in the inhibition of proliferation. The cAMP-elevating agents PGE2 and forskolin decreased EGF-induced proliferation, suggesting cAMP as the mediator. beta 2AR agonists and forskolin also inhibited proliferation stimulated by lysophosphatidic acid (LPA) as well as the synergistic proliferation stimulated by LPA+EGF. Importantly, PKA-selective cAMP analogs did not inhibit proliferation at concentrations that maximally activated PKA (10-100 microM), whereas a cAMP analog selective for the exchange protein directly activated by cAMP (EPAC), 8-(4-chlorophenylthio)-2'-O-methyl-cAMP, maximally inhibited proliferation at a concentration that did not activate PKA (10 microM). These data show that beta 2AR agonists and other cAMP-elevating agents decrease proliferation in HASM cells via a PKA-independent mechanism, and they provide pharmacological evidence for involvement of EPAC or an EPAC-like cAMP effector protein instead.

Lysophosphatidic acid decreases epidermal growth factor receptor binding in airway epithelial cells.

We showed previously that treatment of human airway smooth muscle cells and lung fibroblasts with lysophosphatidic acid (LPA) increases the binding of epidermal growth factor (EGF) to EGF receptors (EGFRs). The purpose of this study was to determine whether LPA also regulates EGFR binding in airway epithelial cells. Airway epithelial cells were incubated in the absence or presence of 10 microM LPA for increasing times, and binding of 125I-EGF to intact cells on ice was measured. Exposure to LPA for only 15 min caused a 30 to 70% decrease in EGFR binding in a dose-dependent manner, depending on the cell line. This decrease in binding was sustained to at least 18 h in BEAS-2B and primary human bronchial epithelial cells. In contrast, the LPA-induced decrease in binding reversed rapidly in two lung cancer epithelial cell lines, H292 and A549, returning to control levels within 3 h. LPA increased phosphorylation of the EGFR in BEAS-2B cells, and this phosphorylation was inhibited by both 4-(3'-chloroanilino)-6,7-dimethoxy-quinazoline (AG1478; EGFR tyrosine kinase inhibitor) and N-[(2R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl]-l-tryptophan methylamide (GM6001; matrix metalloproteinase inhibitor) but not by CRM197 (heparin-binding EGF inhibitor). AG-1478 and GM6001 also inhibited the LPA-induced decrease in EGFR binding but only by 50%, suggesting only partial involvement of EGFR transactivation in the decrease in EGFR binding. In summary, LPA stimulates a decrease in EGFR binding in airway epithelial cells that is sustained in normal cells but that rapidly reverses in cancer cells. LPA-induced transactivation of EGFRs occurs and contributes to the decrease in EGFR binding, but additional pathway(s) may also be involved.

Impaired receptor-mediated endocytosis by the asialoglycoprotein receptor in ethanol-fed mice: implications for studying the role of this receptor in alcoholic apoptosis.

During receptor-mediated endocytosis (RME), extracellular molecules are internalized after being recognized and bound to specific cell surface receptors. In previous studies of the asialoglycoprotein receptor (ASGPR) in rats, we showed that ethanol impairs RME at multiple ASGPR sites. Ethanol administration has been shown to increase apoptosis, and we demonstrated increased sensitization to apoptotic induction in hepatocytes from ethanol-fed rats. Although a physiological role for the ASGPR has not been identified, investigators have shown its involvement in the uptake/clearance of apoptotic cells in vitro. This suggests a potential role for the ASGPR in the removal of apoptotic cells, and the recent availability of an ASGPR-deficient mouse strain provides an excellent opportunity to examine the role of the ASGPR during ethanol impairment. In this study, we examined ethanol-impaired RME in mice and began the characterization of ASGPR-deficient mice for use in ethanol studies. Similar to our findings with rats, ligand binding, internalization, and degradation were decreased 45-50% in hepatocytes from ethanol-fed wild-type mice. In ASGPR-deficient mice, these parameters did not vary among the chow-fed, pair-fed control, or ethanol groups and were negligible compared with those of wild-type mice. TUNEL analysis of liver sections showed an ethanol-induced increase in apoptotic bodies in all mouse strains with a significant difference in the receptor-deficient mice. Further, the livers of ASGPR-deficient mice had three times more apoptotic bodies, in all feeding groups, compared with wild-type mice. These results support the use of the ASGPR-deficient mouse model for studying ethanol-induced liver injury, specifically ethanol-induced apoptosis.