Pathway of urokinase-type plasminogen activator induction in the T47D and LLC-PK1 cell lines

Induction of urokinase-type plasminogen activator (uPA) in response to either reagents activating cAMP-dependent protein kinase (cAMP-PK) or the calcium ion phospholipid-dependent kinase (C-kinase) was compared in the LLC-PK1 and T47D cell lines. The two cell lines exhibited quantitatively different responses to calcitonin, to the phosphodiesterase inhibitor isobutylmethylxanthine, and to the adenylate cyclase activator forskolin. Both showed activation of cAMP-PK in response to all these reagents, with T47D cells displaying a greater extent of activation. T47D cells, however, failed to produce uPA in response to calcitonin, forskolin, or the cAMP analog 8-bromo-cAMP, whereas LLC-PK1 cells produced high levels of uPA in response to all these agents. Both cell lines responded to phorbol esters in terms of uPA induction, though to differing extents. Phorbol myristate acetate (PMA) was shown conclusively not to activate cAMP-PK in either cell line, even at concentrations 10-fold higher than those promoting maximal uPA induction. It was concluded that phorbol ester-mediated induction of uPA does not involve cAMP or cAMP-PK activation. These results are discussed in relation to proposed models concerning the role of cAMP-PK in uPA induction.     

Mechanisms of cAMP-mediated gene induction: examination of renal epithelial cell mutants affected in the catalytic subunit of the cAMP-dependent protein kinase

The precise mechanistic role of the cAMP-dependent protein kinase (cAMP-PK) in cAMP-mediated gene induction remains unclear. Renal epithelial cell mutants were compared to the LLC-PK1 parental cell line for induction of the cAMP-responsive urokinase-type plasminogen activator (uPA) gene, as quantitated by the technique of mRNA solution hybridization. The FIB4 and FIB6 mutants, which possess less than 10% parental cAMP-PK catalytic (C) subunit activity, showed markedly diminished uPA mRNA induction in response to agents elevating intracellular cAMP such as the cAMP analogue 8-bromo-cAMP and the adenylate cyclase-stimulating hormones vasopressin and calcitonin. In contrast, the mutant cells responded to a similar or greater extent than the parental cells in terms of uPA mRNA induction following treatment with the Ca2+/phospholipid-dependent protein kinase activator phorbol 12-myristate 13-acetate (PMA). Elevation of intracellular cAMP was found to induce a translocation of the cAMP-PK C subunit from the perinuclear Golgi region to the nucleus in both parental and mutant cell lines, as shown by immunocytochemical techniques. Results argue for the role of the cAMP-PK C subunit activity and possibly nuclear translocation of the C subunit in cAMP-mediated uPA induction, which is mechanistically distinct from the PMA-stimulated response.     

A novel LLC-PK1 renal epithelial cell mutant impaired in in vivo down-regulation of cAMP-mediated hormonal response.

A novel "cAMP-resistant" variant of LLC-PK1 renal epithelial cells which is impaired in in vivo down-regulation of response following hormonal stimulation of adenylate cyclase (AC) is described. Compared to parental cells, the BIB27 mutant exhibited markedly higher in vivo activation of cAMP-dependent protein kinase (cAMP-PK) in response to the hormones salmon calcitonin (SCT) or [Arg8]-vasopressin (AVP) or the AC activator forskolin. The activation of cAMP-PK subsequent to agonist stimulation also persisted much longer in the mutant than in LLC-PK1 cells, although the cAMP-PK of BIB27 cells was normal in terms of both absolute levels and regulation by cAMP in vitro. Intracellular cAMP accumulation was also much higher in BIB27 than in LLC-PK1 cells following agonist stimulation. Production of cAMP could be detected in BIB27 cells even 12 h after treatment with AVP or SCT, whereas cAMP production in LLC-PK1 had returned to basal within 1 and 8 h, respectively. High levels of free cAMP-PK catalytic (C) subunit in BIB27 persisted even 12 h after hormone addition, meaning that the higher cAMP production in BIB27 did not result in the normal down-regulation of cAMP-PK C subunit levels. In vitro AC activity in BIB27 cell homogenates could be stimulated by hormones or receptor-independent agonists, but to a lesser extent than in LLC-PK1 cell homogenates. The SCT and AVP concentrations promoting half-maximal AC activation in BIB27 cells were about 10- and 3-fold higher than parental, respectively. BIB27 accordingly appeared to possess a mutation in AC responsible for the impairment of both in vitro response to agonists and the normal in vivo down-regulation processes following hormonal stimulation.     

LLC-PK1 cell mutants in cAMP metabolism respond normally to phorbol esters

Mutants of the pig kidney cell line, LLC-PK1, affected in cAMP metabolism, were examined for cAMP-dependent protein kinase (cAMP-PK) activity and for cAMP-mediated induction of urokinase-type plasminogen activator (uPA). The FIB4 and FIB6 mutant cell lines possessed about 10% parental levels of cAMP-PK activity and concomitantly reduced uPA production (10-20% parental) in response to calcitonin, forskolin and 8-bromo cAMP. The FIB1, FIB2 and FIB5 mutant cell lines had about 70% parental levels of cAMP-PK and the synthesis of uPA was 40-60% parental. Thus, cAMP-mediated induction of uPA showed a dependence on the absolute levels of cAMP-PK. However, uPA synthesis in response to phorbol-12-myristate-13-acetate by all of the mutants was similar to parental, which indicates that enzyme induction mediated by phorbol esters does not involve cAMP or cAMP-PK.     

A Nerve Growth Factor-Sensitive S6 Kinase in Cell-Free Extracts from PC 12 Cells

Soluble extracts from nerve growth factor (NGF)-stimulated PC12 cells prepared by alkaline lysis show a two- to 10-fold greater ability to phosphorylate the 40S ribosomal protein S6 than do extracts from control cells. The alkaline lysis method yields a preparation of much higher specific activity than does sonication. Half-maximal incorporation of 32P from [32P]ATP into S6 occurred after 4-7 min of NGF treatment. The partially purified NGF-sensitive S6 kinase has a molecular weight of 45,000. It is not inhibited by NaCl, chlorpromazine, or the specific inhibitor of cyclic AMP (cAMP)-dependent protein kinase, nor is it activated by addition of diolein plus phosphatidylserine. Trypsin treatment of either crude extracts or partially purified S6 kinase from control or NGF-treated cells was without effect. These data suggest that the S6 kinase stimulated by NGF is neither cAMP-dependent protein kinase or protein kinase C nor the result of tryptic activation of an inactive proenzyme. Treatment of intact cells with dibutyryl cAMP or 5'-N-ethylcarboxamideadenosine also increases the subsequent cell-free phosphorylation of S6. This observation suggests that cAMP-dependent protein kinase may be involved in the phosphorylation of S6 kinase.     

Isolation of a mutant LLC-PK1 cell line defective in hormonal responsiveness. A pleiotropic lesion in receptor function

A mutant LLC-PK1 cell line, M18, was isolated after a single treatment of the parent culture with N-methyl-N'-nitro-N-nitroso-guanidine. In contrast to LLC-PK1 cells, the mutant did not exhibit production of urokinase-type plasminogen activator (uPA) in response to the hormones calcitonin and vasopressin, but produced the expected levels of uPA upon stimulation by the receptor-independent adenylate cyclase activators forskolin and cholera toxin, as well as by the phosphodiesterase inhibitor isobutylmethylxanthine and the 8-bromo analogue of adenosine cyclic monophosphate, Br8cAMP. The patterns of activation of cAMP-dependent protein kinase were identical to those of uPA induction: calcitonin and vasopressin were without effect, but the response to all other agents was normal. In similar fashion, mutant cell homogenates displayed normal activation of adenylate cyclase upon treatment with sodium fluoride, forskolin, or the non-hydrolyzable GTP analogue guanosine 5'-[beta, gamma-imino]triphosphate, but were unresponsive to calcitonin or vasopressin. The ability of M18 cells to bind radioactively labelled calcitonin and vasopressin was measured. The mutant possessed less than 4% of the normal levels of the receptor binding activity for both hormones. Somatic cell hybrids formed between M18 and LLC-PK1 cells were found to retain normal hormone binding activity and responsiveness to hormones, indicating that the defect in M18 cells was recessive. M18 was concluded most probably to contain a single mutation impairing the function of two distinct polypeptide hormone receptors.     

Activation of cAMP-dependent protein kinase alters the chromatin structure of the urokinase-type plasminogen activator gene promoter.

In LLC-PK1 cells, the urokinase-type plasminogen activator (uPA) gene is induced by two of the major signal transduction pathways, the protein kinase C (PKC) and the cAMP-dependent protein kinase (PKA) pathways. We have analyzed the chromatin structure of 26 kb of the uPA gene locus and have shown that PKA activation but not PKC activation induce major chromatin structural alterations in the uPA gene promoter. In uninduced cells, several DNase I hypersensitive (HS) sites were detected in the 5' and 3' flanking regions but not in the transcribed region. Two of the sites correspond to previously characterized regulatory sites: a cAMP responsive site at nucleotide position -3500 with respect to the initiation site, and the PEA3/AP1 site at -2100 that mediates PKC activation. After the activation of PKA but not PKC, a strong HS site was induced at -2600. Functional analysis of this region revealed cAMP responsive activity. Chromatin structural alterations again brought about specifically by PKA but not by PKC were were also detected in the upstream of the promoter by topoisomerase I cleavage site analysis, with two prominent sites appearing at -2800 and -3300. These results suggest that the strong cAMP induction of the uPA gene requires structural alterations that permit cooperative interactions between the multiple cAMP responsive sites.