Apoptotic Genes Induced by the Uveitogenic Bacterial Cell Wall Peptidoglycan, Muramyl Dipeptide.

Abstract

Abstract: : Purpose: Investigate the apoptotic pathway induced by the muramyl dipeptide (MDP) (smallest biologically active component of bacterial cell wall peptidoglycan that can induce anterior uveitis and conjunctivitis in rabbits); mediated via interaction of MDP/surface calreticulin/glucocorticosteroid receptor complex in rabbit kidney (RK₁₃) cells. Methods: PolyA–mRNA was isolated from control and MDP–treated RK₁₃ cells and the apoptotic genes expressed were determined in human and mouse apoptosis gene expression arrays (rabbit apoptosis gene expression arrays are not available). DNA ladder and caspase 3 assays were performed. Results: Caspase 3 activity and apoptotic DNA was detected in RK₁₃ cell cultures within 4 hr of MDP treatment. Four genes were induced in RK₁₃ cells by MDP–treatment; c–myc (inhibits NFΚß and promotes TNF–induced release of cytochrome c), cyclin A2 (promotes DNA synthesis), caspase recruiting domain –IL–1 activator enzyme (CARDIA K), and MAP kinase (ras) activator (CRAF1). Six apoptotic genes were expressed at higher levels in apoptotic MDP treated RK₁₃ cells than in control RK₁₃ cells. These included TNF receptor activator of NFΚß (TRANK), human apoptosis–related factor (TFAR15), cyclin dependent kinase (cdc–2), glyceraldehyde–3–phosphate dehydrogenase (GAPDH), protein inhibitor of NOS (PIN), and sentrin (inhibitor of Fas/APO–1 and TNF–induced cell death). Constitutive genes were detected in the control and MDP–treated RK₁₃ cells in human apoptosis genes arrays. Some of the were identified as caspase–6, DNA fragmentation factor (DFF 40), cyclophilin, cyclooxygenase 1 (COX–1), cytochrome P450 (metabolizes arachidonic acid), poly(ADP–ribose) polymerase (PARP), endogenous apoptosis suppressor (DAD–1), redox factor–1 (ref–1), retinoid X receptor (RxR–ß) and iNOS. Conclusions: Several apoptotic genes were detected in MDP–induced apoptotic RK₁₃ cells using human and mouse apoptosis gene arrays. Our results suggest that the apoptotic pathway induced by MDP in RK₁₃ cells differs from the apoptotic pathway induced by LPS in monocytes. While other apoptotic genes may have been detected using a rabbit apoptosis gene array (i.e., casapase–3), the data are compatible with activation of caspase 3 and the known effects of COX inhibitors on MDP induced apoptosis and uveitis.

Keywords: apoptosis/cell death • bacterial disease • uveitis–clinical/animal model