Uloge signalnih puteva kinaze regulisane ekstracelularnim signalima, proteinskih kinaza PKB i PKC u razvoju i ekspresiji gena embriona ribe zebrice

Abstract

Uticaj ekstraćelijskim signalima regulisanih kinaza (ERK), proteinskih kinaza B (PKB; AKT) i proteinskih kinaza C (PKC) na adultne ćelija su dobro proučeni ali postoji relativno malo podataka o njihovim efektima tokom embriogeneze. U cilju definisanja uloga Erk, Akt i Pkc signalizacije tokom rane embriogeneze, embrioni riba zebrica su tretirani farmakološkim inhibitorima i stimulatorima ovih signalnih puteva. Rezultati su pokazali da Erk inhibiše eksprimiranje gena za glutation S-transferazu Pi (gstp1-2) i citohrom P450 1a (cyp1a) koji učestvuju u metabolizmu ksenobiotika i održavanju redoks ravnoteže tokom rane embriogeneze. Inhibitorni efekat Erk na eksprimiranje gstp1-2 se ostvaruje preko aktivacije transkripcionog faktora Creb (engl. cAMP response element-binding protein). Tokom oksidativnog stresa izazvanog terc-butilhidrokinonom (tBHQ), nije došlo do aktivacije Erk, te je i izostao efekat ove kinaze na tBHQ zavisnu indukciju eksprimiranja gstp1-2. Forbol 12-miristat 13-acetat (PMA) je doveo do povećanja fosforilacije Erk, ali nije menjao eksprimiranje gstp1-2. Inhibicija aktivnosti kinaza Erk sa U0126 je omogućila eksprimiranje gstp1-2 kod embriona tretiranih sa PMA. Globalna aktivacija Pkc izazvana delovanjem PMA je dovela do pojave razvojnih deformiteta, smanjenja stope preživljavanja, pojave apoptotičnih ćelija u regionu mozga, povećanja eksprimiranja proapoptotičnih gena casp9 (kaspaza 9) i puma (engl. p53 upregulated modulator of apoptosis) i gena za antioksidativne enzime superoksid dismutazu 2 (Sod2) i katalazu (Cat) kod embriona riba zebrica. Inhibicija Pkc je poboljšala stopu preživljavanja, smanjila apoptozu u mozgu i smanjila eksprimiranje casp9 i puma u embrionima izloženim PMA, ali nije sprečila pojavu deformacija niti oksidativni stres. Rezultati u ovoj disertaciji ukazuju da Erk deluje inhibitorno na eksprimiranje gena gstp1-2 i cyp1a u neometanim uslovima razvoja embriona, dok aktivacija ove kinaze sprečava povećanje gstp1-2 tokom oksidativnog stresa. Prekomerna aktivacija Pkc tokom rane embriogeneze zebrica dovodi do apoptoze i smanjene stope preživljavanja embriona riba zebrica.

Extracellular signal regulated kinase (ERK), protein kinase B (PKB, i.e. AKT) and protein kinase C (PKC) signaling pathways and their impact on the adult cell functions are well studied, but only few data exist regaridng their role during embryogenesis. In order to reveal the role of these signaling pathways during early embriogenesis, zebrafish embryos were treated with inhibitors or activators of the above mentioned signaling pathways. The results showed that Erk inhibited transcription of glutation S-transferase Pi (gstp1-2) and cytochrome P450 1a (cyp1a) genes, which are involved in metabolism of xenobiotics and redox regulation in embryos. The inhibitory effect of Erk is transmitted through the Creb (cAMP response element-binding protein) transcription factor. During oxidative stress provoked by tert-butylhydroquinone (tBHQ), the Erk activity was not enhanced and therefore it failed to prevent the stimulatory effect of tBHQ on gstp1-2 gene expression. On the other hand, inhibition of the Erk acitivity by U0126 allowed phorbol 12-myristate 13-acetate (PMA) to induce gstp1-2 during oxidative stress. Addition of PMA caused various developmental deformities, decreased survival rate, increased the number of apoptotic cells in the brain region, and enhanced the expression of proapoptotic casp9 (Caspase 9) and puma (p53 upregulated modulator of apoptosis, puma) and antioxidative sod2 (superoxide dismutase 2) and cat (catalase) genes in zebrafish embryos. The inhibition of Pkc by pharmalogical inhibitor improved overall survival rate, reduced apoptosis in the brain and decreased expression of casp9 and puma in the PMA-exposed embryos, but did not prevent deformities and oxidative stress. These data suggest that Erk inhibits gstp1-2 and cyp1a during normal embryogenesis. The activation of Erk during oxidative stress can also prevent the expression of gstp1-2. Direct over-activation of Pkc during early embryogenesis of zebrafish is associated with apoptosis and decreased survival rate of the embryos.