Protein kinase activity-dependent inhibition of urokinase-type plasminogen activator gene transcription by cyclic AMP in human pre-B lymphoma cell line RC-K8

Abstract

We investigated the effects of cAMP on the urokinase-type plasminogen activator (uPA) production in human pre-B lymphoma cell line RC-K8 that is consistently secreting uPA in the conditioned medium. Both Bt2cAMP and PGE1 inhibited the uPA accumulation in a dose-dependent manner. Northern blot analysis and nuclear run-on assay revealed that uPA gene transcription was repressed by Bt2cAMP and the repression was negated by inhibition of de novo protein synthesis by cycloheximide. Pretreatment with H89 (N-[2-(p-bromocinnamyl-amino) ethyl]-5-isoquinoline sulfonamide), a specific cAMP-dependent protein kinase (PKA) inhibitor, strongly inhibited both the PKA activation and the supression of uPA mRNA accumulation induced by cAMP. H85 (N-[2-(N-formyl-p-chlorocinnamyl-amino) ethyl]-5-isoquinoline sulfonamide), which closely resembles H89 in its chemical structure but is not a selective inhibitor of PKA, showed little effect on the regulation of uPA gene regulation by Bt2cAMP. These results suggest that cAMP represses uPA gene transcription in human pre-B lymphoma cells through PKA pathway and in which de novo protein synthesis is required.

Urokinase (u‐PA) is produced by collecting duct principal cells and is post‐transcriptionally regulated by SV40 large‐T, arginine vasopressin, and epidermal growth factor

“…These discrepant results may be attributed to LLC-PK1 unusual properties since those cells respond to AVP although they derive from the proximal tubule, a tubule segment devoid of AVP receptors. In other cell types including human pre-B lymphoma cells (Shinbo et al, 1995), Sertoli cells (Tolli et al, 1995), astrocytes (Tranque et al, 1992), and glomerular epithelial cells (Rondeau et al, 1989), u-PA synthesis is decreased by cAMP. Stimulation of u-PA by EGF has also been reported in a wide variety of cell types (Rorth et al, 1990) (Watabe et al, 1998) including mouse renal mIMCD3 cells that originate from the collecting duct.…”

Regulation of plasminogen activator inhibitor‐1 secretion by urokinase and tissue plasminogen activator in rat epithelioid‐type smooth muscle cells

“…The concentrations of inhibitors were chosen to optimally inhibit the selected targets. These included the broadly specific protein tyrosine kinase inhibitor genistein (final concentration 0·14 mmol/l) (Akiyama & Ogawara, 1991; Lazo et al , 1997), protein kinase A (PKA) inhibitors H‐89 (15 μmol/l) (Shinbo et al , 1995) and KT‐5720 (2 μmol/l) (Shinbo et al , 1995), PKA activators 8‐bromo‐cAMP (1 mmol/l), dibutyryl cAMP (1 mmol/l), forskolin (0·1 mmol/l) and isobutyl methylxanthane (0·1 mmol/l), protein kinase C (PKC) inhibitor GF109208X (4 μg/ml) (Shraga‐Levine et al , 1994; Chang et al , 1998), Mek 1 inhibitor PD98059 (50 μmol/l) (Lynch et al , 1999; Burns et al , 2000), phosphoinositol‐3‐kinase (PI3‐K) inhibitor LY‐294 002 (25 μmol/l) (Duan et al , 2000; Kim et al , 2000), ras isoprenylation inhibitor (–)‐perillic acid (1 mmol/l) (Schulz et al , 1997; Borsellino et al , 1999), syk inhibitor piceatannol (10 μg/ml) (Oliver et al , 1994; Tkaczyk et al , 1999), and src inhibitor PP1 (10 μmol/l) (Igishi & Gutkind, 1998; Waltenberger et al , 1999). The PKC activator, PMA, was added at a final concentration of 50 ng/ml for 5 min and removed before adding any stimulating agonist, in order to maximally activate MAPK, according to Jones et al (1994).…”

Microlaryngoscopic repair of iatrogenic pharyngeal pouch perforations: Treatment of choice?

“…These discrepant results may be attributed to LLC‐PK1 unusual properties since those cells respond to AVP although they derive from the proximal tubule, a tubule segment devoid of AVP receptors. In other cell types including human pre‐B lymphoma cells (Shinbo et al, 1995), Sertoli cells (Tolli et al, 1995), astrocytes (Tranque et al, 1992), and glomerular epithelial cells (Rondeau et al, 1989), u‐PA synthesis is decreased by cAMP. Stimulation of u‐PA by EGF has also been reported in a wide variety of cell types (Rorth et al, 1990) (Watabe et al, 1998) including mouse renal mIMCD3 cells that originate from the collecting duct.…”