6533b872fe1ef96bd12d4378

RESEARCH PRODUCT

Inhibition of Rho modulates cytokine-induced prostaglandin E2 formation in renal mesangial cells.

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Stimulation of rat mesangial cells for 24 h with interleukin-1β(IL-1β) plus forskolin (Fk) leads to a marked increase in prostaglandin E 2 (PGE 2 ) synthesis. This effect is further enhanced by the small G-protein Rho inhibitor toxin A. A similar increase in PGE 2 formation is obtained with Y27632, a Rho-dependent kinase inhibitor, and with lovastatin, a hydroxymethylglutaryl-coenzyme A inhibitor which depletes cells from geranylgeranyl moieties and thus blocks Rho activation. In parallel to the increased PGE 2 synthesis, a potentiation of IL-1β-induced secretory group IIA phospholipases A 2 (sPLA 2 -IIA) protein expression also occurs by Rho inhibition. However, only toxin A triggers an increased sPLA 2 -IIA activity consistent with the elevated levels of protein expression, whereas Y27632 and lovastatin rather reduced IL-1β-induced sPLA 2 -IIA activity. In vitro activity studies reveal that Y27632 and lovastatin can directly block sPLA 2 -IIA enzyme activity in a concentration-dependent manner. Interestingly, in the absence of IL-1β/Fk stimulation and the lack of sPLA 2 -IIA protein expression, all Rho inhibitors exert a small but significant increase in PGE 2 formation suggesting that additional PLA 2 s or downstream enzymes like cyclooxygenases or prostaglandin synthases may be activated by Rho inhibitors. Western blot analyses of toxin A-, Y27632- and lovastatin-stimulated cells reveal that the cytosolic group IV PLA 2 (CPLA 2 ) and the cytosolic PGE 2 synthase (cPGES), but not the sPLA 2 -IIA, cyclooxygenase-2 or the microsomal PGE 2 synthase (mPGES), are upregulated compared to unstimulated cells. Furthermore, the Rho inhibitors induced arachidonic acid release from intact cells which is blocked by the cPLA 2 inhibitor methyl arachidonyl fluorophosphonate (MAFP). In summary, these data show that inhibition of the small G-protein Rho, either by toxin A, lovastatin, or Y27632, exert a dual effect on mesangial cells: (i) in the absence of an inflammatory stimulus it activates the constitutive cPLA 2 and CPGE 2 synthase and generates low amount of PGE 2 . (ii) In the presence of inflammatory cytokines it potentiates sPLA 2 -IIA expression and subsequent PGE 2 formation. In addition, we identified lovastatin and Y27632 as direct inhibitors of sPLA 2 -IIA in a cell-free system.