The activation of the atypical PKC zeta in light-induced retinal degeneration and its involvement in L-DNase II control

Abstract

Light-induced retinal degeneration is characterized by photoreceptor cell death. Many studies showed that photoreceptor demise is caspase-independent. In our laboratory we showed that leucocyte elastase inhibitor/LEI-derived DNase II (LEI/L-DNase II), a caspase-independent apoptotic pathway, is responsible for photoreceptor death. In this work, we investigated the activation of a pro-survival kinase, the protein kinase C (PKC) zeta. We show that light exposure induced PKC zeta activation. PKC zeta interacts with LEI/L-DNase II and controls its DNase activity by impairing its nuclear translocation. These results highlight the role of PKC zeta in retinal physiology and show that this kinase can control caspase-independent pathways.

Keywords: PKC zeta; light; retinal apoptosis; retinal degeneration; serpin B1.

Figure 1

PKC zeta in retina during LIRD. (A) Rats were exposed to a continuous white light for 1–7 days, and then killed; eyes were mounted in optimal cutting-temperature (OCT). Cryosections were stained with anti-PKC zeta (green) and with propidium iodide (DNA, red). Arrow head shows translocation of PKC zeta to the plasma membrane. ONL: outer nuclear layer; INL: inner nuclear layer; GCL: ganglion cell layer. Scale bar represents 20 μm. (B) Western blot analysis of PKC zeta expression at different times of light exposure. Specificity of the antibody was verified by competition with the specific peptide (sc-216P). Lower panel shows the quantification; means statistically different with respect to control (P < 0.01 (anova test)) are indicated by an ‘*’. (C) same experiment as in B using anti-PAR-4 antibody. Specificity of the antibody was verified by competition with the specific peptide (sc-4216). ‘*’ indicates significant differences with respect to the control, as before. Actin was used as a charge control.

Figure 2

Physical interaction between LEI/L-DNase II and PKC zeta. (A) Pull down of PKC zeta using a LEI affinity column. An His-cartridge resin was loaded with an His-tagged LEI. Crude extract of 100 μg from HeLa cells was loaded on the column that was then washed and eluted. The LEI line represents the material eluted from this column. As a negative control an his-tagged calmodulin was used as naïve protein. Ctl line represents 10 μg of HeLa crude extract. (B) Crude extract of 100 μg from HeLa cells was immunoprecipitated with anti-PKC zeta (PKC z line) or anti-LEI (LEI line). A column without IgG was used as control (Ctl). Western blots were developed with anti-PKC zeta (upper panel) or anti-LEI (lower panel). These are representative experiments out of three.

Figure 3

Putative phosphorylation site of LEI. (A) Fragment of the sequence of LEI presenting the consensus site for PKC zeta. LEI from several species are aligned showing that the sequence is well conserved among species. (B) GFP-LEI (WT) was transfected to BHK cells together with pDsRed-T195E-LEI (mutant protein). Cells were left untreated (Ctl panel) or treated with HMA to induce apoptosis. Nuclear translocation of the mutant protein is impaired. Scale bar represents 10 μm.

Figure 4

L-DNase II control by PKC zeta in HeLa cells. HeLa cells were induced in apoptosis by TNFα/cycloheximide. (A) Western blot analysis of PKC zeta in total extract of HeLa cells treated with TNFα/CHX (TNF) or left untreated (Ctl). Arrow heads indicate the fragments of PKC zeta. (B) Western blot analysis of serine 311 phosphorylated PKC zeta in TNFα/CHX treated or untreated cells (Ctl) The specificity of the antibody was performed by digesting the protein extract with 1 IU of calf intestinal alkaline phosphatase, invitrogen 18009-019. (C) Western blot analysis of LEI/L-DNase II in total extract of HeLa cells treated with TNFα/CHX (TNF) or left untreated (Ctl). Arrow head indicates L-DNase II. (D) Immunocytochemistry analysis of LEI/L-DNase II (green), PKC zeta (red) and NFkB (green) activation. Nuclei were counterstained with DAPI (blue). Arrow heads indicate intensely stained nuclei. Scale bar represents 10 μm. (E) HeLa cells were transfected with a siRNA against PKC zeta (siRNA) or with a control siRNA (Ctl). The decrease in PKC expression was controlled by Western blot (data not shown). After induction of apoptosis by TNFα/CHX treatment, the rate of cell death was evaluated using the MTT method. The means are significantly different P < 0.05, as determined by the test (*). (F) Same experiment as in E but nuclear and cytoplasmic fractions were performed and studied by Western blot for LEI/L-DNase II expression. The presented results correspond to cells transfected with a siRNA against PKC zeta (+) or a control siRNA (−). Arrow head indicates L-DNase II.

Figure 5

L-DNase II expression and activity in light-exposed rat retinas. (A) Rats were exposed to continuous white light for 2 days (2d) or kept in a normal, cycling light (Ctl). Previous to illumination the inhibitor of PKC zeta was intravitreally injected in the right eye. The left eye was injected with the vehicle. After killing, the retinas were dissected, extracted and analysed for L-DNase II activity using a supercoiled plasmid in ionic DNase II activating conditions. The right panel is a quantification of the lower band (product of degradation). This is a representative experiment out of three. (B) Same experiment as in A but in this case LEI/L-DNase II was analysed by Western blot right panel represent a representative experiment out of four. Right panel represents the quantification of the L-DNase II band; P < 0.05 (*).

Figure 6

Schematic model of PKC zeta and LEI/L-DNase II in LIRD. Light induces intracellular increase in Ca²⁺ concentration that activates calpains. In turn, calpain 1 permeabilizes the lysosomal membrane. This releases cathepsin D that activates L-DNase II which is translocated to the nucleus to induce photoreceptor cell death. This nuclear translocation is inhibited by PKC zeta to protect the cell. PKC zeta can also activate other anti-apoptotic proteins. Its protective action can be inhibited by its specific inhibitor PAR-4 (*).