Knock-down of plasminogen-activator inhibitor-1 enhances expression of E-cadherin and promotes epithelial differentiation of human pancreatic adenocarcinoma cells.

High levels of plasminogen activator inhibitor-1 (PAI-1), which is produced by stromal, endothelial, and cancer cells and has multiple complex effects on cancers, correlate with poor cancer prognosis. To more definitively study the role of endogenously produced PAI-1 in human pancreatic adenocarcinoma (PAC) PANC-1 cell line biology, we used anti-PAI-1 shRNA to create stable PAI-1 deficient cells (PD-PANC-1s). PD-PANC-1s exhibited a heterogeneous morphology. While the majority of cells exhibited a cuboidal shape similar to the parental PANC-1 or the vector-infected control cells, numerous large cells with long filopodia and a neuronal-like appearance were observed. Although both Vector-control cells and PD-PANC-1s expressed mRNAs that are characteristic of mesenchymal, neural, and epithelial phenotypes, epithelial marker RNAs were up-regulated (e.g., E-cadherin, 32-fold) whereas mesenchymal marker RNAs were down-regulated (e.g., Thy1, ninefold) in PD-PANC-1s, suggesting mesenchymal-to-epithelial transition. Neural markers exhibited both up- and down-regulation. Immunocytochemistry indicated that epithelial-like PD-PANC-1s expressed E-cadherin and ß-catenin in significantly more cells, while neural-like cells exhibited robust expression of organized ß-3-tubulin. PAI-1 and E-cadherin were rarely co-expressed in the same cells. Indeed, examination of PAI-1 and E-cadherin mRNAs expression in additional cell lines yielded clear inverse correlation. Indeed, infection of Colo357 PAC cells (that exhibit high expression of E-cadherin) with PAI-1-expressing adenovirus led to a marked decrease in E-cadherin expression and to enhanced migration of cells from clusters. Our results suggest that endogenous PAI-1 suppresses expression of E-cadherin and differentiation in PAC cells in vitro, supporting its negative impact on tumor prognosis.

Kaposi's sarcoma-associated herpesvirus-G protein-coupled receptor-expressing endothelial cells exhibit reduced migration and stimulated chemotaxis by chemokine inverse agonists.

A constitutively active G protein-coupled receptor (GPCR) encoded by Kaposi's sarcoma-associated herpesvirus (human herpesvirus-8) (KSHV) is expressed in endothelial (spindle) cells of Kaposi's sarcoma lesions. In this study, we report novel effects of basal signaling by this receptor and of inverse agonist chemokines on migration of KSHV-GPCR-expressing mouse lung endothelial cells. We show that basal signaling by KSHV-GPCR inhibits migration of endothelial cells in two systems, movement through porous filters and in vitro wound closure. Naturally occurring chemokines, interferon gamma-inducible protein-10 and stromal-derived factor-1, which act as inverse agonists at KSHV-GPCR, abrogate the inhibition of migration and stimulate directed migration (or chemotaxis) of these cells. Thus, the expression of KSHV-GPCR may allow infected endothelial cells in situ to remain in a localized environment or to directionally migrate along a gradient of specific chemokines that are inverse agonists at KSHV-GPCR.

Plasminogen-induced aggregation of PANC-1 cells requires conversion to plasmin and is inhibited by endogenous plasminogen activator inhibitor-1.

PANC-1 cells express proteinase-activated receptors (PARs)-1, -2, and respond to their activation by transient elevation of cytosolic [Ca(2+)] and accelerated aggregation (Wei et al., 2006, J Cell Physiol 206:322-328). We studied the effect of plasminogen (PGN), an inactive precursor of the PAR-1-activating protease, plasmin (PN) on aggregation of pancreatic adenocarcinoma (PDAC) cells. A single dose of PGN time- and dose-dependently promoted PANC-1 cells aggregation in serum-free medium, while PN did not. PANC-1 cells express urokinase plasminogen activator (uPA), which continuously converted PGN to PN. This activity and PGN-induced aggregation were inhibited by the uPA inhibitor amiloride. PGN-induced aggregation was also inhibited by alpha-antiplasmin and by the PN inhibitor epsilon-aminocaproic acid (EACA). Direct assay of uPA activity revealed very low rate, markedly enhanced in the presence of PGN. Moreover, in PGN activator inhibitor 1-deficient PANC-1 cells, uPA activity and PGN-induced aggregation were markedly potentiated. Two additional human PDAC cell lines, MiaPaCa and Colo347, were assayed for PGN-induced aggregation. Both cell lines responded by aggregation and exhibited PGN-enhanced uPA activity. We hypothesized that the continuous conversion of PGN to PN by endogenous uPA is limited by PN's degradation and negatively controlled by endogenously produced PAI-1. Indeed, we found that PANC-1 cells inactivate PN with t1/2 of approximately 7 h, while the continuous addition of PN promoted aggregation. Our data suggest that PANC-1 cells possess intrinsic, PAI-1-sensitive mechanism for promotion of aggregation and differentiation by prolonged exposure to PGN and, possibly, additional precursors of PARs agonists.

Proteinase-activated receptors differentially modulate in vitro invasion of human pancreatic adenocarcinoma PANC-1 cells in correlation with changes in the expression of CDC42 protein.

OBJECTIVES: Proteinase-activated receptor-1 (PAR-1) and PAR-2 have been associated with increased invasiveness and metastasis in human malignancies. The role of PAR-3 has been less investigated. We examined the role of PARs in a human pancreatic adenocarcinoma PANC-1 cell line phenotype in vitro. METHODS: We knocked down PAR-1, PAR-2, or PAR-3, whereas empty vector-infected cells served as controls. Specific peptide agonists of PARs were used to stimulate the receptors. In vitro assays of colony formation, migration, and invasion were used to characterize the phenotypes, and Western analysis was used to follow cell division control protein 42 homolog (CDC42) expression. RESULTS: PAR-1 and PAR-2 knockdowns (KDs) were markedly less, whereas PAR-3 KDs were robustly more migratory and invasive than the controls. Stimulation of PAR-1 or PAR-2 by their peptide agonists increased, whereas PAR-3 agonist reduced the invasion of the control cells. Knockdowns of all three PARs exhibited changes in the expression of CDC42, which correlated with the changes in their invasion. Conversely, stimulation of vector-control cells with PAR-1 or PAR-2 agonists enhanced, whereas PAR-3 agonist reduced the expression of CDC42. In the respective KD cells, the effects of the agonists were abrogated. CONCLUSION: The expression and/or activation of PARs is linked to the invasiveness of PANC-1 cells in vitro, probably via modulation of the expression of CDC42.

Carboxyl tail cysteine mutants of the thyrotropin-releasing hormone receptor type 1 exhibit constitutive signaling: role of palmitoylation.

We studied the role of carboxyl tail cysteine residues and their palmitoylation in constitutive signaling by the thyrotropin-releasing hormone (TRH) receptor type 1 (TRH-R1) in transfected mammalian cells and in Xenopus laevis oocytes. To study palmitoylation, we inserted a factor Xa cleavage site within the third extracellular loop of TRH-R1, added a carboxyl-terminal C9 immunotag and expressed the mutant receptor in Chinese hamster ovary cells. We identified TRH-R1-specific palmitoylation in the transmembrane helix-7/carboxyl-tail receptor fragment mainly at Cys-335 and Cys-337. In contrast to a mutant truncated at Cys-335 that was reported previously to be constitutively active, a receptor truncated at Lys-338 (K338Stop), which preserves Cys-335 and Cys-337, and C337Stop and N336Stop, which preserve Cys-335, did not exhibit increased constitutive signaling. TRH-R1 mutants substituted singly by Gly or Ser at Cys-335 or Cys-337 did not exhibit constitutive signaling. By contrast, substitution of both cysteines (C335G/C337G or C335S/C337S) yielded TRH-R1 mutants that exhibited marked constitutive signaling in mammalian cells. In the oocyte, constitutive signaling by C335G/C337G resulted in homologous (of C335G/C337G) and heterologous (of M1 muscarinic receptor) desensitization. Because both Cys-335 and Cys-337 have to be substituted or deleted for constitutive signaling, we propose that a single palmitoylation site in the proximal carboxyl tail is sufficient to constrain TRH-R1 in an inactive conformation.

Response to lysophosphatidic acid in Xenopus oocytes and its rapid desensitization: the role of Gq and Go G-protein families.

Native Xenopus oocytes exhibit dose-dependent depolarizing current responses to lysophosphatidic acid (LPA), with EC50 = 0.18 microM. Responses to LPA were subject to pronounced rapid desensitization. When oocytes were challenged with 5 nM LPA, the response was <10% of the maximal. Subsequent addition of 0.5 microM LPA resulted in 50-70% desensitization, when compared to naïve controls. Injection of antisense oligodeoxyoligonucleotides (ASODNs) targeted at either of the two endogenous LPA receptors inhibited the LPA response by approximately 50%, but did not alter the degree of rapid desensitization. To study the involvement of G-proteins in rapid homologous desensitization of responses to LPA, we selectively depleted native G-proteins by injection of specific ASDONs. Injection of ASDONs targeted at Galphaq family mRNAs (mainly Galpha11) reduced the response to 0.5 microM LPA by 50%. ASDONs targeted at either Galphao or Galphao1 caused a large decrease in the amount of their cognate mRNAs and the Galphao family proteins, while the response to LPA was inhibited by up to 30%. Injection of ASDONs targeted at Galphao1 mRNA decreased rapid desensitization from 69 to 23%, while pertussis toxin (PTX) completely abolished it. Expression of two dominant negative mutants of the human Galphao family homologs either decreased or virtually abolished rapid desensitization. Microinjection of CaCl(2) demonstrated that 50% of rapid desensitization could be attributed to inhibition of Ca(2+) activation of chloride channels. We propose that the apparent degenerate coupling of different G-proteins to LPA receptors in Xenopus oocytes actually serves both the generation of the response (by Gq and Go G-protein families) and its desensitization (mostly by Go G-protein family).