Phosphoinositol 3 kinase inhibitor, LY294002 increases bcl-2 protein and inhibits okadaic acid-induced apoptosis in Bcl-2 expressing renal epithelial cells.

Protein phosphorylation plays an indispensable role in cellular regulation of mitosis, metabolism, differentiation, and death. We previously reported that the protein phosphatase inhibitor okadaic acid (OKA) induces apoptosis in renal epithelial cells in culture. In the present study, we examined the role of phosphotidylinositol 3 (PI3) kinase signaling in okadaic acid-induced apoptosis by pre-treating normal rat kidney renal epithelial cells expressing human bcl-2 with the PI3 kinase inhibitors, LY294002 and wortmannin, followed by apoptosis-inducing concentrations of okadaic acid. Given the reported cell survival activity of PI3 kinase signaling mostly attributed to Akt kinase activation, we hypothesized that inhibition of PI3 kinase would enhance okadaic-induced apoptosis. Surprisingly, our data show that pretreatment with LY294002, but not wortmannin, attenuated okadaic acid-induced apoptosis. In contrast, to LY294002, wortmannin enhanced apoptosis. Interestingly, we also found that LY294002 treatment increased bcl-2 protein levels in normal rat kidney epithelial cells expressing bcl-2 (NRK-bcl-2). In untreated cells, bcl-2 appeared to be mainly perinuclear, coincident with the nuclear membrane, or in the cytosol. In OKA treated cells that were pre-treated with Ly294002, bcl-2 was highly co-localized with mitochondria, but in cells treated with okadaic acid alone, bcl-2 was associated with fragmented chromatin. In this model, it appears that LY294002 may exert anti-apoptotic effects by a previously unreported treatment related increase in bcl-2. Although it is widely accepted that bcl-2 protein can inhibit apoptosis, we propose that the subcellular location of bcl-2 is an important determinant in whether bcl-2 effectively inhibits apoptosis.

Herbimycin A and geldanamycin inhibit okadaic acid-induced apoptosis and p38 activation in NRK-52E renal epithelial cells.

It is important to understand the mechanisms by which phosphorylation-dependent events play a role in regulation of apoptosis in toxicant-metabolizing organs such as the kidney. Our previous work demonstrated that the toxicant and phosphatase inhibitor okadaic acid induces apoptosis of renal epithelial cells via a mechanism that appears to involve the modulation of c-raf-1, p38 kinase, and extracellular regulatory kinase (ERK) cascades. Using the benzoquinone ansamycins and tyrosine kinase inhibitors geldanamycin and herbimycin A, we examined the contribution of tyrosine phosphorylation and c-raf-1 activities to okadaic acid-induced apoptosis. In this report we show that both geldanamycin and herbimycin A protected NRK-52E cells from okadaic acid-induced apoptosis, abrogated the overall okadaic acid-induced kinase activation, and specifically inhibited activation of p38 kinase by okadaic acid. Herbimycin A and geldanamycin also abrogated okadaic-acid induced morphologic changes such as cell rounding and cell membrane blebbing. Herbimycin A and geldanamycin caused pronounced cell spreading, cell flattening, and a decrease in okadaic acid-induced loss of actin filaments. Interestingly, herbimycin A showed more potent inhibitory effect than geldanamycin, and herbimycin A alone inhibited okadaic acid-induced movement of p38 kinase into the cytosol. These results imply that decreased p38 activity and its cytosolic translocation together with cellular resistance to cytoskeletal disorganization may play a significant role in resistance to phosphorylation-dependent apoptosis. Furthermore, the results imply that changes in cell shape may partially modulate the observed alterations in signal transduction induced by okadaic acid.