Karen M. Kassel

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.

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 Gi/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.

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 μM 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). AG1478 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.

Modulation of epidermal growth factor receptor binding to human airway smooth muscle cells by glucocorticoids and β2-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.

Lipopolysaccharide enhances transforming growth factor β1-induced PDGF-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 β (TGFβ) and lipopolysaccharide (LPS) also contribute to the profibrogenic response after bile duct ligation. We tested the hypothesis that LPS and TGFβ1 synergistically induce PDGF‐B expression in BDECs.

Lipopolysaccharide enhances transforming growth factor β1-induced platelet-derived growth factor-B expression in bile duct epithelial cells: LPS enhances TGFβ1-induced PDGF-B mRNA

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 β (TGFβ) and lipopolysaccharide (LPS) also contribute to the profibrogenic response after bile duct ligation. We tested the hypothesis that LPS and TGFβ1 synergistically induce PDGF‐B expression in BDECs.