Opposite, binary regulatory pathways involved in IL-1-mediated stromelysin gene expression in rat mesangial cells

Abstract

Glomerular mesangial cells express matrix metalloproteinase sromelysin in response to the proinflammatory cytokine IL-1 beta. The present study was conducted to identify intracellular machinery involved in this IL-1 action, especially focusing on the role of the TPA response element (TRE) located in the 5'-flanking region of the stromelysin gene. Using transient transfection with a pTRE-LacZ reporter plasmid, we detected no obvious up-regulation of TRE activity in rat mesangial cells following the IL-1 stimulation. However, the basal activity of TRE was found to be essential to the stromelysin induction, since (i) mesangial cells stably expressing a transdominant negative mutant of c-Jun, which effectively suppressed both basal and inducible TRE activity, exhibited the blunted expression of stromelysin in response to IL-1 beta, whereas (ii) transfection with a c-fos antisense gene, which suppressed only the inducible TRE activity, did not affect the stromelysin induction. To seek cooperative pathways required for the IL-1 action, we next focused on protein kinases, the potential regulators of the stromelysin gene. Stimulation of mesangial cells with a protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), induced the stromelysin transcript without affecting TRE activity. Depletion of intracellular PKC by high-dose PMA or inhibition of PKC activity with calphostin C suppressed the stromelysin induction by IL-1 beta, suggesting the crucial contribution of a PKC-mediated, but TRE-independent pathway. In contrast, either cAMP inducer forskolin or dibutyryl cAMP suppressed the IL-1-mediated stromelysin expression. An inhibitor of cAMP-dependent protein kinase A (PKA), HA1004, enhanced the IL-1 effect in a dose-dependent manner. Unexpectedly, the inhibitory action of PKA was not through cAMP response element (CRE) but through TRE, because (i) activation of CRE was not induced by IL-1 beta, and (ii) cAMP-mediated activation of PKA suppressed the basal TRE activity. These findings elucidated the unique, binary regulation of stromelysin by IL-1 beta; that is, IL-1 up-regulated the transcript via the PKC-dependent pathway under the cooperation with constitutively active TRE, and this stimulatory effect was in part counterbalanced by the IL-1-inducible PKA which down-regulated the basal TRE activity.