Induction of cell migration by pro-urokinase binding to its receptor: possible mechanism for signal transduction in human epithelial cells.

A human epithelial cell line, WISH, and a mouse cell line, LB6-uPAR, transfected with the human urokinase receptor (uPAR), both expressed high affinity uPAR but undetectable levels of urokinase (uPA). In two independent assays, binding of exogenous pro-uPA produced an up to threefold enhancement of migration. The migration was time and concentration dependent and did not involve extracellular proteolysis. This biologic response suggested that uPAR can trigger an intracellular signal. Since this receptor is a glycosyl-phosphatidylinositol-linked protein, we postulated that it must do so by interacting with other proteins, among which, by analogy to other systems, would be a kinase. To test this hypothesis, we carried out a solid phase capture of uPAR from WISH cell lysates using either antibodies against uPAR or pro-uPA adsorbed to plastic wells, followed by in vitro phosphorylation of the immobilized proteins. SDS-PAGE and autoradiography revealed two phosphorylated protein bands of 47 and 55 kD. Both proteins were phosphorylated on serine residues. Partial sequence of the two proteins showed a 100% homology to cytokeratin 18 (CK18) and 8 (CK8), respectively. A similar pattern of phosphorylation was obtained with lysates from A459 cells, a lung carcinoma, but not HL60, LB6-uPAR or HEp3 cell lysates, suggesting that the identified multiprotein uPAR-complex may be specific for simple epithelia. Moreover, immunocapture with antibody to another glycosyl-phosphatidylinositol-linked protein, CD55, which is highly expressed in WISH cells, was ineffective. The kinase was tentatively identified as protein kinase C, because it was inhibited by an analogue of staurosporine more specific for PKC and not by a PKA or tyrosine kinase inhibitors. The kinase was tentatively identified as PKC epsilon because of its resistance to PMA down-modulation, independence of Ca2+ for activity, and reaction with a specific anti-PKC epsilon antibody in Western blots. Cell fractionation into cytosolic and particulate fractions revealed that all four proteins, the kinase, uPAR, CK18, and CK8, were present in the particulate fraction. In vivo, CK8, and to a lesser degree CK18, were found to be phosphorylated on serine residues. Occupation of uPAR elicited a time-dependent increase in the phosphorylation intensity of CK8, a cell shape change and a redistribution of the cytokeratin filaments. These results strongly suggest that uPAR serves not only as an anchor for uPA but participates in a signal transduction pathway resulting in a pronounced biological response.

Down-regulation of retinoic acid receptor activity associated with decreased alpha and gamma isoforms expression in F9 embryonal carcinoma cells differentiated by retinoic acid.

F9 embryonal carcinoma cells differentiate in response to retinoic acid (RA). To investigate the regulation of RA receptors (RARs) expression during this process, cDNA probes specific for the major RAR isoforms were used. In contrast to the level of RAR beta 2 mRNA which was high in cells treated 5 days with RA and below detection in untreated cells, as previously described, the steady state levels of RAR alpha 1, alpha 2, gamma 1, and gamma 2 mRNAs were markedly decreased in the RA-differentiated cells as compared to untreated cells. The down-regulation of the RA-responsive system in differentiated cells was also evident in gel shift assays as a marked decrease in binding capacity to a retinoid acid response element (beta 2RARE), as well as in chloramphenicol acetyltransferase (CAT) assays as a sixfold decrease in RA-mediated transacting activity via this element. The down-regulation of RAR DNA-binding and transacting activity coincided with the burst in tissue plasminogen activator secretion and thus, occurred at the hinge between early and late differentiation. The down-regulation of RA responsiveness may constitute an important event in the transition between early and late differentiation stage in F9 cells.

Recovery from opioid receptor alkylation: social conflict analgesia and brain [3H]etorphine binding in beta-chlornaltrexamine-treated mice.

The effects of beta-chlornaltrexamine (CNA, 5 mg/kg s.c.) on social conflict analgesia and brain opioid binding were investigated in mice at different times after the administration of the alkylating antagonist. The specific binding of [3H]etorphine to high-affinity binding sites and the stress-induced analgesia of attacked mice (50 bites) were prevented for 6 h after CNA administration. Stress-mediated inhibition of pain fully recovered within 3 days after CNA treatment. Brain opioid binding was still reduced to 45% at this time and reached control values 9 days after treatment.

Opiate receptor binding and behavioral effects of morphine in RHA/Verh and RLA/Verh rats.

Large baseline differences were found in open field activity and rearing behavior between 2 psychogenetically selected rat lines, as well as differences in the effects of peripherally administered morphine (2,4 and 10 mg/kg) on those behaviors and on body temperature. The 2 lines also showed different capacities of tritiated DAGO binding to preparations of striatal membranes.