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The effect of PKC activation on the survival of rat retinal ganglion cells in culture
Brain Research 853 Ž2000. 338–343 www.elsevier.comrlocaterbres
Research report
The effect of PKC activation on the survival of rat retinal ganglion cells in culture Aline Araujo dos Santos, Elizabeth Giestal de Araujo
)
Departamento de Neurobiologia, Instituto de Biologia, Centro de Estudos Gerais, UniÕersidade Federal Fluminense, Caixa Postal a100180, Niteroi, ´ Rio de Janeiro CEP 24001-970, Brazil Accepted 2 November 1999
Abstract Natural cell death is a degenerative phenomenon occurring during the development of the nervous system. Approximately half the neurons initially generated during this period die. The role of trophic molecules produced by target and afferent neurons as well as by glial cells controlling this regressive event has been extensively demonstrated. The aim of this work was to study the role of activated protein kinase C ŽPKC., an enzyme involved in apoptosis regulation, on the survival of retinal ganglion cells kept ‘‘in vitro’’ for 48 h. For this purpose, we used the phorbol 12-myristate 13-acetate ŽPMA., a tumor promoter agent that activates PKC. Our results showed that PMA increases the survival of ganglion cells. The effect was dose-dependent and PMA concentrations of 10 or 100 ngrml produced the maximal effect Ža two-fold increase on ganglion cells survival compared with 48 h control.. This effect was totally abolished by 1.25 mM chelerythrine chloride Žan inhibitor of PKC. and 30 mM genistein Žan inhibitor of tyrosine kinase enzymes.. Otherwise, PMA was effective only when it was chronically present in the cultures. On the other hand, treatment with 20 mM 5-fluoro-2X-deoxyuridine, an inhibitor of cell proliferation, or 25 mM BAPTA-AM, an intracellular calcium chelator, did not block PMA effect. Our results suggest that the survival of retinal ganglion cells ‘‘in vitro’’ may be mediated by a mechanism that involves PKC activation. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Retinal ganglion cell; PMA; Protein kinase C; Natural cell death; Retina; Neuronal survival
1. Introduction Since the pioneer study of Hamburger and LeviMontalcini w8x, natural cell death within nervous system has been extensively studied. It is known that during the development of the nervous system approximately half the neurons initially generated die. It has been assumed that an excessive number of neuronal cells are generated in order to promote the appropriate neuronal connections that fit neuronal projections to the size and function of target cells w20x. Natural cell death is concomitant to synaptogenesis and neurotrophins released by target cells play a fundamental role in this regressive phenomenon w20x. Neurotrophins are a family of small polypeptides including nerve growth factor ŽNGF. w13x, brain-derived neurotrophic factor ŽBDNF. w2x, neurotrophin 3 ŽNT-3. w9,14,23x, neurotrophin
) Corresponding author. [email protected]
Fax:
q 55-21-719-5934;
e-mail:
4r5 ŽNT-4r5. w3,7x, and neurotrophin 6 ŽNT-6. w6x. Recently, a new member, neurotrophin 7 ŽNT-7., was purified w12,18x. All neurotrophins bind to the low affinity receptor p75 w4x. They also bind to high affinity receptors represented by the Trk family that includes TrkA, TrkB and TrkC receptors. NGF binds to TrkA, BDNF and NT-4r5 bind to Trk-B, NT-3 binds to TrkC and NT6rNT-7 also bind to TrkA w4x. Afferent cells also play an important role in controlling natural cell death. It has been extensively demonstrated that the blockade of electrical activity of afferent cells induces neuronal degeneration either during early stages of development or in the adult phase w20x. The importance of electrical activity as well as the release of trophic factors by afferent and glial cells on the control of neuronal cell death is quite evident w27x. However, until now, some mechanisms involved in the afferent control of cell death are not clearly understood. Several morphological changes are observed during natural cell death including cell shrinkage, nuclear chromatin condensation, organized DNA fragmentation by endonu-
0006-8993r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 2 3 1 9 - 7
A.A. dos Santos, E.G. de Araujor Brain Research 853 (2000) 338–343
cleases, among others. Cell fragments are promptly phagocyted by macrophages and neighbouring cells, avoiding inflammatory response. For these reasons, the natural cell death has been identified, in many neuronal populations, as a death by apoptosis w1x. Several developmental events depend on the apoptotic process, including hormonal tissue atrophy, cell-mediated immunity, tumoral regression and the selection of a cellular population w15x. Intracellular calcium mobilization plays an important role in regulating apoptosis. Previous evidences showed that an increase in the cytoplasmic calcium concentration can either prevent or trigger apoptosis, an observation that varied with the cell type studied. In thymocytes and T-cells lines, it was shown that when the concentration of intracellular calcium decreased, apoptosis was stimulated. On the other hand, the apoptosis of IL-3 dependent hematopoietic cells would be blocked when cells were treated with a calcium ionophore w15x. Apoptosis can be triggered by different stimuli such as TNF, radiation and oncogenes. On the other hand, Bcl-2 and other viral proteins can inhibit apoptosis in different cell types w29x. Prevention of apoptosis is an important aspect of viral infection since apoptosis of infected cells prevents the replication of the virus. Viral anti-apoptotic genes inhibit apoptosis and are responsible for the increasing of virulence w30x. The study of apoptosis is responsible for many medical advances in cancer therapy. Treatment with chemotherapic drugs elicits apoptosis as well as radiotherapy that induces DNA damage leading to apoptosis. It is also important to notice that certain cytokines and hormones can induce apoptosis and this finding led to the advent of hormone therapy w30x. Cell death can be modulated by electrical activity. It has been shown that chronic depolarization enhances the survival of neuronal cells in the absence of trophic factors. Either high Kq concentration or treatment with veratridine, a drug that keeps Naq channels opened, are able to increase the survival of neuronal cells w5,21x. The effect of high Kq concentration on neuronal survival is mediated by the activation of voltage dependent L-type Caq2 channels. The increase on cytoplasmic calcium levels mediates several events on neurons such as neurotransmitter release, ionic channels activation or inactivation and gene inductions w5x. Another second messenger involved in the control of cell death is cAMP. Some neuronal populations increase their survival when cAMP concentration is increased, although in other populations such as thymocytes and human mammary cells, the increase of cellular cAMP levels stimulates apoptosis w15x. Protein kinase C ŽPKC. is an enzyme involved in many cellular events such as proliferation and differentiation, and is also involved in apoptosis regulation. Its effect in apoptosis depends on the cell type and on the isoenzymes activated. There are 11 isoenzymes of PKC with different functions depending on the cell type. These isoenzymes
339
are classified in three different classes of enzymes according to their calcium dependence and phorbol ester activation. The C2 section of the regulatory domain is responsible for calcium binding and C1 section for phorbol ester binding. Phorbol esters are natural products from Croton tiglium and other plants of Euphorbiacea family w11x. We use phorbol 12-myristate 13-acetate ŽPMA., a tumor promoter phorbol ester that crosses plasma cell membrane and directly activates PKC. It is a long lasting activation since it is not metabolized by the cells w22x. The conÕentional type of PKC isozyme is calcium dependent and is stimulated by phorbol ester. It is represented by the isoforms a , bI, bII and g. The noÕel class Žisoforms d, ´, h and u. is also activated by phorbol ester but is calcium independent. The atypical class Žisoforms z and l . is also calcium independent and cannot be activated by phorbol esters w26x. An interesting finding was reported by Mosior and Epand w17x, who showed that even in the absence of the C2 section of its regulatory domain, PKC remains selective for calcium. However, in this situation, other divalent cations are also able to stimulate this kinase. In this study, we examined the effect of PKC activation by PMA on the survival of neonatal rat retinal ganglion cells ‘‘in vitro’’. Our aim was to investigate if activation of PKC was involved on the control of neuronal degeneration. Our results indicate that PKC plays an important role on retinal ganglion cells degeneration since treatment with PMA was able to keep these neurons alive for 48 h. The effect was dose-dependent and inhibited by chelerythrine chloride and genistein but not affected by BAPTA-AM and fluorodeoxyuridine treatments.
2. Materials and methods 2.1. Materials Medium 199 was purchased from Gibco ŽGaithersburg, USA . . Genistein, 1.2-bis Ž 2-aminophenexy .ethaneN, N, N X , N X-tetraacetic acid ŽBAPTA., PMA, 5-fluoro-2Xdeoxyuridine, glutamine, penicillin and streptomycin were obtained from Sigma ŽSt. Louis, USA.. 2.2. Retrograde labelling of retinal ganglion cells Within the first 24 h after birth, Lister Hooded rats were anaesthetized by hypothermia. One microliter of a solution of 30% horseradish peroxidase ŽHRP. in 2% dimethyl sulphoxide ŽDMSO. was injected in each superior colliculus. The animals were returned to their mothers and survived for ; 16 h before the procedures used for cell culture. 2.3. Preparation of monolayer cultures of retinal cells The rat pups were killed by decapitation and the eyes were rapidly removed and immersed in a calcium- and
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A.A. dos Santos, E.G. de Araujor Brain Research 853 (2000) 338–343
magnesium-free ŽCMF. salt solution. The retinae were gently isolated and incubated at 378C for 20 min in CMF containing 0.2% trypsin ŽWorthington 38a797.. Following, the tissue was resuspended in complete culture medium and triturated through a Pasteur pipette. After complete dissociation of the retinae, 1 ml of the cell suspension was placed on glass coverslips previously coated with 50 mgrml poly-L-ornithine placed in 35 mm Petri dishes. We used 199 medium supplemented with 2 mM glutamine, 100 mgrml streptomycin, 100 Urml penicillin and 5% fetal calf serum. The cultures were usually incubated for 4 h to allow the cells to attach to the coverslips. Then, culture medium or culture medium with drugs to be tested were added to each Petri dish. Plating density was 650 000 cells per Petri dish. The cultures were maintained in a humidified atmosphere of 5% CO 2 and 95% air at 378C.
Fig. 2. Time-dependent effect of PMA on retinal ganglion cells survival. Retinal cells were treated with PMA after the initial 4 h in cultures and the drug was maintained for 4, 16 or 48 h. In some experiments, PMA was added after the initial 16 h in culture and the survival of retinal ganglion cells was evaluated after 48 h. CT, control; PMA, phorbol myristate acetate. Data are reported as the mean"S.E.M. of seven different experiments each performed with at least two different Petri dishes. U P - 0.001 compared to the 48 h control.
2.4. Identification of retinal ganglion cells in culture Presence of the enzyme peroxidase in the cytoplasm of retinal ganglion cells was revealed according to the protocol of Mesulan w16x. Briefly, the monolayers were fixed after 2 days in culture with a mixture of 1% paraformaldehyde and 2% glutaraldehyde in sodium phosphate buffer Ž0.1 M for 5 min. washed in phosphate buffer and reacted with tetramethylbenzidine. 2.5. Quantification of the results Retinal ganglion cells were counted using a Zeiss microscope at a magnification of 400 = , under bright-field. As an internal control for the variable percentage of ganglion cells labelled with HRP in distinct experiments, the number of labelled cells at 4 h in culture was taken as 100%. All data are expressed as mean " S.E.M. from experiments performed at least in duplicate and each experiment was repeated at least three times. Statistical anal-
Fig. 1. Dose-dependent effect of PMA on retinal ganglion cells survival after 48 h in culture. Cultures were treated with PMA Ž0.01–100 ngrml.. CT, control; PMA, phorbol myristate acetate. Data are reported as the mean"S.E.M. of four different experiments each performed with at least two different Petri dishes. U P - 0.001 compared to the 48-h control.
ysis was done using analysis of variance ŽANOVA. followed by Newman–Keuls’ test.
3. Results We first analyzed the effect of PMA on retinal ganglion cells survival after 48 h ‘‘in vitro’’. Fig. 1 shows that PMA increased ganglion cell survival and the effect was dose-dependent. After 48 h, the number of retinal ganglion cells in control cultures was reduced to approximately 50% of the initial number. However, treatment with PMA at 0.1, 1.0, 10.0 and 100 ngrml increased ganglion cells survival. The greatest effect was observed with 10 and 100 ngrml of PMA. In these conditions all the ganglion cells initially plated were alive after 48 h. According to these results, we decided to use 10 ngrml of PMA in all further experiments.
Fig. 3. Chelerythrine chloride 1.25 mM inhibited the PMA effect on retinal ganglion cells survival after 48 h in culture. CT, control; PMA, phorbol myristate acetate; CC, chelerythrine chloride. Data are reported as the mean"S.E.M. of two different experiments each performed with at least three different Petri dishes. U P - 0.001 compared to the 48-h control.
A.A. dos Santos, E.G. de Araujor Brain Research 853 (2000) 338–343
Fig. 4. An intracellular Ca2q chelator ŽBAPTA-AM 25 mM. did not inhibit the PMA effect on retinal ganglion cells survival. CT, control; PMA, phorbol myristate acetate. Data are reported as the mean"S.E.M. of three different experiments each performed with at least two different Petri dishes. U P - 0.001 compared to the 48-h control.
To understand the effect of PMA better, we investigated if only chronic treatment would increase the survival of the ganglion cells ŽFig. 2.. We began testing if a 4-h pulse of PMA would be able to rescue ganglion cells after 48 h. The result of this experiment showed that a 4-h pulse of PMA did not enhance the ganglion cell survival. Then, we studied if a 16-h treatment with PMA would increase ganglion cells survival. As Fig. 2 shows, no effect of PMA on survival of the cells was observed in this condition. Moreover, treatment of the cultures with PMA after the first 16 h also did not elicit a statistically significant increase on ganglion cells survival ŽFig. 2.. Data from literature show that treatment of cells with PMA can lead to the down regulation of the enzyme PKC w22x. To verify if the effect of PMA involved the down regulation of PKC in our cultures, we investigated the effect of chelerythrine chloride Ž1.25 mM., a specific inhibitor of PKC. As Fig. 3 shows, when PKC was inhibited no increase on ganglion cell survival was observed. These results suggest that the effect of PMA be mediated by the activation of PKC, and not by the downregulation
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Fig. 6. An inhibitor of cell division Žfluorodeoxyuridine, 20 mM. did not abolished the PMA effect on retinal ganglion cells survival. CT, control; PMA, phorbol myristate acetate; FDUR, fluorodeoxyuridine. Data are reported as the mean"S.E.M. of two different experiments each performed with at least two different Petri dishes. U P - 0.001 compared to the 48-h control.
of this enzyme. In the next experiments, we examined if the effect of PMA was mediated by an increase in the intracellular calcium levels. Fig. 4 shows that when cells were treated with PMA, in the presence of an intracellular calcium chelator ŽBAPTA 25 mM., the increase on retinal ganglion cells survival was abolished. We also did experiments with higher concentrations of BAPTA-AM. However, a toxic effect of this drug was observed Ždata not shown.. To investigate if tyrosine kinase enzymes were involved in the effect of PMA, we studied the survival of ganglion cells treated with PMA and genistein 10, 15, 25 and 30 mM Žan inhibitor of tyrosine kinase.. Fig. 5 shows that genistein inhibited the effect of PMA in a dose-dependent manner. A complete blockade of PMA effect was obtained when 30 mM genistein was used. This result suggests that tyrosine kinase pathways are involved on the increased survival of ganglion cells induced by PMA. Morphological analysis of retinal cells in culture shows that glial cells keep their proliferative capacity all over the time ‘‘in vitro’’. To study if the effect of PMA was mediated by an increase in the glial cell population, we tested its effect in the presence of an inhibitor of cell division Ž20 mM fluorodeoxyuridine.. Fig. 6 shows that the anti-mitotic drug did not interfere with the PMA effect. This result indicates that the increase on ganglion cells survival induced by PMA is not due to an increase in glial cells population.
4. Discussion
Fig. 5. A tyrosine kinase inhibitor Žgenistein. abolished the PMA effect on retinal ganglion cells survival after 48 h in culture. The blockage of genistein was dose-dependent and the maximum effect was obtained with 30 mM. CT, control; PMA, phorbol myristate acetate; Gen, genistein. Data are reported as the mean"S.E.M. of five different experiments each performed with at least two different Petri dishes. U P - 0.001 compared to the 48-h control.
Our results show that treatment of retinal cultures with PMA induced the survival of retinal ganglion cells after 48 h ‘‘in vitro’’. The effect was dose-dependent and 10 ngrml PMA rescued all retinal ganglion cells initially plated. Data from the literature show conflicting results concerning the role of PKC on apoptosis. Treatment with PMA activates PKC-b1 and induces apoptosis in
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A.A. dos Santos, E.G. de Araujor Brain Research 853 (2000) 338–343
myelomonocytic cell line U937, as well as in thymocytes. However, when hepatocytes are stimulated with PMA, apoptosis is inhibited w22x. These results could be explained by the finding that, during the development, PKC isoenzymes are expressed independently, accordingly to the cell type. Thus, it is reasonable to suppose that different PKC isoenzymes could be related to the suppression or to the activation of apoptosis. In agreement with this hypothesis, it has been shown that, in cell line U937, apoptosis is associated with an increase in PKC-b isoenzyme and a decrease in PKC-z isoenzyme. Furthermore, in thymocytes, the triggering of apoptosis elicited by glucocorticoids involves the translocation and activation of PKC-e w15x. The presence of PKC isoenzymes has been demonstrated in rat retinae. Using polyclonal antibodies, it was shown the presence of immunoreactivity on both plexiform layers and on the photoreceptor layer. It was also observed by electronic microscopy, the presence of PKC immunoreactivity in the nuclei of ganglion cells w31x. Moreover, using monoclonal antibodies, a isoenzymes were detected on both bipolar and ganglion cells w19x. The presence of the calcium independent isoenzymes z, e and d was also demonstrated by Western blotting w10x. Therefore, treatment with PMA could increase ganglion cell survival by direct stimulation of PKC enzymes present in this neuronal population. Previous data show that chronic treatment with PMA can induce the downregulation of PKC isoenzymes w22x. As downregulation decreases PKC activity, we decided to test if the inhibition of PKC could increase the survival of retinal ganglion cells. Our results clearly show that treatment with an inhibitor of PKC Žchelerythrine chloride. did not increase ganglion cells survival but abolished the effect of PMA. These results suggest that the effect mediated by PMA in our cultures involves the activation of PKC. However, only when PMA was present in cultures for 48 h the survival of ganglion cells was observed, suggesting that chronic stimulation of PKC is important for the rescue of retinal ganglion cells. Intracellular calcium is involved in apoptosis regulation. In some cells, intracellular calcium chelators can inhibit apoptosis. However, in IL-3 dependent hematopoietic cells, the increase in intracellular calcium can block apoptosis w15x. In our retinal cultures, we observed that an increase on intracellular calcium levels was not required for the effect of PMA since the treatment with BAPTA-AM did not inhibit the increase on the survival of ganglion cells stimulated by PMA. Since calcium independent isoenzymes of PKC were identified in rat retinae, one possibility to explain our results is the activation of these specific isoenzymes by PMA. However, further experiments will be necessary to identify which PKC isoenzymes are involved in the control of ganglion cell survival. It has been shown that PKC is able to downregulate the signaling of tyrosine kinase receptors such as the insulin
receptor and epidermal growth factor receptor w25x. Our results showing that genistein inhibited the PMA effect, suggest that PKC did not induce the downregulation of tyrosine kinase enzymes in our cultures. The effect of PMA on the survival of retinal ganglion cells can be explained either by a direct activation of PKC isoenzymes identified in ganglion cells w19x or by a release of trophic factors mediated by PKC activation in our cultures. Since many trophic molecules signal through activation of tyrosine kinase enzymes and since we observed an inhibition of PMA effect with genistein treatment, it is reasonable to assume that trophic molecules, released by PMA activation of PKC, mediate the survival of ganglion cells in our cultures. One possible source of trophic molecules could be glial cells. It has been demonstrated that glial cells produce and release trophic molecules after appropriate stimulation w24,28x. Since these cells, in our cultures, maintain the proliferative capacity and PKC activation can stimulate cellular division, we investigated if the PMA effect was mediated by an increase in glial cells population. Our results demonstrated that the blockade of cell division with fluorodeoxyuridine did not abolish the effect of PMA. Therefore, although we cannot exclude the release of trophic factors by glial cells in the cultures, our data shows that an increase in the population of glial cells is not related to the effect of PMA on the survival of ganglion cells.
Acknowledgements We would like to thank Alexandre Jose´ Fernandes and Bernardino Matheus dos Santos for technical assistance. We also thank Dr. Ana Lucia ´ Marques Ventura for helpful discussions. Aline Santos is the recipient of a FAPERJ fellowship. This work was supported by grants from FAPERJ and FINEP-UFF.
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Research report
The effect of PKC activation on the survival of rat retinal ganglion cells in culture Aline Araujo dos Santos, Elizabeth Giestal de Araujo
)
Departamento de Neurobiologia, Instituto de Biologia, Centro de Estudos Gerais, UniÕersidade Federal Fluminense, Caixa Postal a100180, Niteroi, ´ Rio de Janeiro CEP 24001-970, Brazil Accepted 2 November 1999
Abstract Natural cell death is a degenerative phenomenon occurring during the development of the nervous system. Approximately half the neurons initially generated during this period die. The role of trophic molecules produced by target and afferent neurons as well as by glial cells controlling this regressive event has been extensively demonstrated. The aim of this work was to study the role of activated protein kinase C ŽPKC., an enzyme involved in apoptosis regulation, on the survival of retinal ganglion cells kept ‘‘in vitro’’ for 48 h. For this purpose, we used the phorbol 12-myristate 13-acetate ŽPMA., a tumor promoter agent that activates PKC. Our results showed that PMA increases the survival of ganglion cells. The effect was dose-dependent and PMA concentrations of 10 or 100 ngrml produced the maximal effect Ža two-fold increase on ganglion cells survival compared with 48 h control.. This effect was totally abolished by 1.25 mM chelerythrine chloride Žan inhibitor of PKC. and 30 mM genistein Žan inhibitor of tyrosine kinase enzymes.. Otherwise, PMA was effective only when it was chronically present in the cultures. On the other hand, treatment with 20 mM 5-fluoro-2X-deoxyuridine, an inhibitor of cell proliferation, or 25 mM BAPTA-AM, an intracellular calcium chelator, did not block PMA effect. Our results suggest that the survival of retinal ganglion cells ‘‘in vitro’’ may be mediated by a mechanism that involves PKC activation. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Retinal ganglion cell; PMA; Protein kinase C; Natural cell death; Retina; Neuronal survival
1. Introduction Since the pioneer study of Hamburger and LeviMontalcini w8x, natural cell death within nervous system has been extensively studied. It is known that during the development of the nervous system approximately half the neurons initially generated die. It has been assumed that an excessive number of neuronal cells are generated in order to promote the appropriate neuronal connections that fit neuronal projections to the size and function of target cells w20x. Natural cell death is concomitant to synaptogenesis and neurotrophins released by target cells play a fundamental role in this regressive phenomenon w20x. Neurotrophins are a family of small polypeptides including nerve growth factor ŽNGF. w13x, brain-derived neurotrophic factor ŽBDNF. w2x, neurotrophin 3 ŽNT-3. w9,14,23x, neurotrophin
) Corresponding author. [email protected]
Fax:
q 55-21-719-5934;
e-mail:
4r5 ŽNT-4r5. w3,7x, and neurotrophin 6 ŽNT-6. w6x. Recently, a new member, neurotrophin 7 ŽNT-7., was purified w12,18x. All neurotrophins bind to the low affinity receptor p75 w4x. They also bind to high affinity receptors represented by the Trk family that includes TrkA, TrkB and TrkC receptors. NGF binds to TrkA, BDNF and NT-4r5 bind to Trk-B, NT-3 binds to TrkC and NT6rNT-7 also bind to TrkA w4x. Afferent cells also play an important role in controlling natural cell death. It has been extensively demonstrated that the blockade of electrical activity of afferent cells induces neuronal degeneration either during early stages of development or in the adult phase w20x. The importance of electrical activity as well as the release of trophic factors by afferent and glial cells on the control of neuronal cell death is quite evident w27x. However, until now, some mechanisms involved in the afferent control of cell death are not clearly understood. Several morphological changes are observed during natural cell death including cell shrinkage, nuclear chromatin condensation, organized DNA fragmentation by endonu-
0006-8993r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 2 3 1 9 - 7
A.A. dos Santos, E.G. de Araujor Brain Research 853 (2000) 338–343
cleases, among others. Cell fragments are promptly phagocyted by macrophages and neighbouring cells, avoiding inflammatory response. For these reasons, the natural cell death has been identified, in many neuronal populations, as a death by apoptosis w1x. Several developmental events depend on the apoptotic process, including hormonal tissue atrophy, cell-mediated immunity, tumoral regression and the selection of a cellular population w15x. Intracellular calcium mobilization plays an important role in regulating apoptosis. Previous evidences showed that an increase in the cytoplasmic calcium concentration can either prevent or trigger apoptosis, an observation that varied with the cell type studied. In thymocytes and T-cells lines, it was shown that when the concentration of intracellular calcium decreased, apoptosis was stimulated. On the other hand, the apoptosis of IL-3 dependent hematopoietic cells would be blocked when cells were treated with a calcium ionophore w15x. Apoptosis can be triggered by different stimuli such as TNF, radiation and oncogenes. On the other hand, Bcl-2 and other viral proteins can inhibit apoptosis in different cell types w29x. Prevention of apoptosis is an important aspect of viral infection since apoptosis of infected cells prevents the replication of the virus. Viral anti-apoptotic genes inhibit apoptosis and are responsible for the increasing of virulence w30x. The study of apoptosis is responsible for many medical advances in cancer therapy. Treatment with chemotherapic drugs elicits apoptosis as well as radiotherapy that induces DNA damage leading to apoptosis. It is also important to notice that certain cytokines and hormones can induce apoptosis and this finding led to the advent of hormone therapy w30x. Cell death can be modulated by electrical activity. It has been shown that chronic depolarization enhances the survival of neuronal cells in the absence of trophic factors. Either high Kq concentration or treatment with veratridine, a drug that keeps Naq channels opened, are able to increase the survival of neuronal cells w5,21x. The effect of high Kq concentration on neuronal survival is mediated by the activation of voltage dependent L-type Caq2 channels. The increase on cytoplasmic calcium levels mediates several events on neurons such as neurotransmitter release, ionic channels activation or inactivation and gene inductions w5x. Another second messenger involved in the control of cell death is cAMP. Some neuronal populations increase their survival when cAMP concentration is increased, although in other populations such as thymocytes and human mammary cells, the increase of cellular cAMP levels stimulates apoptosis w15x. Protein kinase C ŽPKC. is an enzyme involved in many cellular events such as proliferation and differentiation, and is also involved in apoptosis regulation. Its effect in apoptosis depends on the cell type and on the isoenzymes activated. There are 11 isoenzymes of PKC with different functions depending on the cell type. These isoenzymes
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are classified in three different classes of enzymes according to their calcium dependence and phorbol ester activation. The C2 section of the regulatory domain is responsible for calcium binding and C1 section for phorbol ester binding. Phorbol esters are natural products from Croton tiglium and other plants of Euphorbiacea family w11x. We use phorbol 12-myristate 13-acetate ŽPMA., a tumor promoter phorbol ester that crosses plasma cell membrane and directly activates PKC. It is a long lasting activation since it is not metabolized by the cells w22x. The conÕentional type of PKC isozyme is calcium dependent and is stimulated by phorbol ester. It is represented by the isoforms a , bI, bII and g. The noÕel class Žisoforms d, ´, h and u. is also activated by phorbol ester but is calcium independent. The atypical class Žisoforms z and l . is also calcium independent and cannot be activated by phorbol esters w26x. An interesting finding was reported by Mosior and Epand w17x, who showed that even in the absence of the C2 section of its regulatory domain, PKC remains selective for calcium. However, in this situation, other divalent cations are also able to stimulate this kinase. In this study, we examined the effect of PKC activation by PMA on the survival of neonatal rat retinal ganglion cells ‘‘in vitro’’. Our aim was to investigate if activation of PKC was involved on the control of neuronal degeneration. Our results indicate that PKC plays an important role on retinal ganglion cells degeneration since treatment with PMA was able to keep these neurons alive for 48 h. The effect was dose-dependent and inhibited by chelerythrine chloride and genistein but not affected by BAPTA-AM and fluorodeoxyuridine treatments.
2. Materials and methods 2.1. Materials Medium 199 was purchased from Gibco ŽGaithersburg, USA . . Genistein, 1.2-bis Ž 2-aminophenexy .ethaneN, N, N X , N X-tetraacetic acid ŽBAPTA., PMA, 5-fluoro-2Xdeoxyuridine, glutamine, penicillin and streptomycin were obtained from Sigma ŽSt. Louis, USA.. 2.2. Retrograde labelling of retinal ganglion cells Within the first 24 h after birth, Lister Hooded rats were anaesthetized by hypothermia. One microliter of a solution of 30% horseradish peroxidase ŽHRP. in 2% dimethyl sulphoxide ŽDMSO. was injected in each superior colliculus. The animals were returned to their mothers and survived for ; 16 h before the procedures used for cell culture. 2.3. Preparation of monolayer cultures of retinal cells The rat pups were killed by decapitation and the eyes were rapidly removed and immersed in a calcium- and
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A.A. dos Santos, E.G. de Araujor Brain Research 853 (2000) 338–343
magnesium-free ŽCMF. salt solution. The retinae were gently isolated and incubated at 378C for 20 min in CMF containing 0.2% trypsin ŽWorthington 38a797.. Following, the tissue was resuspended in complete culture medium and triturated through a Pasteur pipette. After complete dissociation of the retinae, 1 ml of the cell suspension was placed on glass coverslips previously coated with 50 mgrml poly-L-ornithine placed in 35 mm Petri dishes. We used 199 medium supplemented with 2 mM glutamine, 100 mgrml streptomycin, 100 Urml penicillin and 5% fetal calf serum. The cultures were usually incubated for 4 h to allow the cells to attach to the coverslips. Then, culture medium or culture medium with drugs to be tested were added to each Petri dish. Plating density was 650 000 cells per Petri dish. The cultures were maintained in a humidified atmosphere of 5% CO 2 and 95% air at 378C.
Fig. 2. Time-dependent effect of PMA on retinal ganglion cells survival. Retinal cells were treated with PMA after the initial 4 h in cultures and the drug was maintained for 4, 16 or 48 h. In some experiments, PMA was added after the initial 16 h in culture and the survival of retinal ganglion cells was evaluated after 48 h. CT, control; PMA, phorbol myristate acetate. Data are reported as the mean"S.E.M. of seven different experiments each performed with at least two different Petri dishes. U P - 0.001 compared to the 48 h control.
2.4. Identification of retinal ganglion cells in culture Presence of the enzyme peroxidase in the cytoplasm of retinal ganglion cells was revealed according to the protocol of Mesulan w16x. Briefly, the monolayers were fixed after 2 days in culture with a mixture of 1% paraformaldehyde and 2% glutaraldehyde in sodium phosphate buffer Ž0.1 M for 5 min. washed in phosphate buffer and reacted with tetramethylbenzidine. 2.5. Quantification of the results Retinal ganglion cells were counted using a Zeiss microscope at a magnification of 400 = , under bright-field. As an internal control for the variable percentage of ganglion cells labelled with HRP in distinct experiments, the number of labelled cells at 4 h in culture was taken as 100%. All data are expressed as mean " S.E.M. from experiments performed at least in duplicate and each experiment was repeated at least three times. Statistical anal-
Fig. 1. Dose-dependent effect of PMA on retinal ganglion cells survival after 48 h in culture. Cultures were treated with PMA Ž0.01–100 ngrml.. CT, control; PMA, phorbol myristate acetate. Data are reported as the mean"S.E.M. of four different experiments each performed with at least two different Petri dishes. U P - 0.001 compared to the 48-h control.
ysis was done using analysis of variance ŽANOVA. followed by Newman–Keuls’ test.
3. Results We first analyzed the effect of PMA on retinal ganglion cells survival after 48 h ‘‘in vitro’’. Fig. 1 shows that PMA increased ganglion cell survival and the effect was dose-dependent. After 48 h, the number of retinal ganglion cells in control cultures was reduced to approximately 50% of the initial number. However, treatment with PMA at 0.1, 1.0, 10.0 and 100 ngrml increased ganglion cells survival. The greatest effect was observed with 10 and 100 ngrml of PMA. In these conditions all the ganglion cells initially plated were alive after 48 h. According to these results, we decided to use 10 ngrml of PMA in all further experiments.
Fig. 3. Chelerythrine chloride 1.25 mM inhibited the PMA effect on retinal ganglion cells survival after 48 h in culture. CT, control; PMA, phorbol myristate acetate; CC, chelerythrine chloride. Data are reported as the mean"S.E.M. of two different experiments each performed with at least three different Petri dishes. U P - 0.001 compared to the 48-h control.
A.A. dos Santos, E.G. de Araujor Brain Research 853 (2000) 338–343
Fig. 4. An intracellular Ca2q chelator ŽBAPTA-AM 25 mM. did not inhibit the PMA effect on retinal ganglion cells survival. CT, control; PMA, phorbol myristate acetate. Data are reported as the mean"S.E.M. of three different experiments each performed with at least two different Petri dishes. U P - 0.001 compared to the 48-h control.
To understand the effect of PMA better, we investigated if only chronic treatment would increase the survival of the ganglion cells ŽFig. 2.. We began testing if a 4-h pulse of PMA would be able to rescue ganglion cells after 48 h. The result of this experiment showed that a 4-h pulse of PMA did not enhance the ganglion cell survival. Then, we studied if a 16-h treatment with PMA would increase ganglion cells survival. As Fig. 2 shows, no effect of PMA on survival of the cells was observed in this condition. Moreover, treatment of the cultures with PMA after the first 16 h also did not elicit a statistically significant increase on ganglion cells survival ŽFig. 2.. Data from literature show that treatment of cells with PMA can lead to the down regulation of the enzyme PKC w22x. To verify if the effect of PMA involved the down regulation of PKC in our cultures, we investigated the effect of chelerythrine chloride Ž1.25 mM., a specific inhibitor of PKC. As Fig. 3 shows, when PKC was inhibited no increase on ganglion cell survival was observed. These results suggest that the effect of PMA be mediated by the activation of PKC, and not by the downregulation
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Fig. 6. An inhibitor of cell division Žfluorodeoxyuridine, 20 mM. did not abolished the PMA effect on retinal ganglion cells survival. CT, control; PMA, phorbol myristate acetate; FDUR, fluorodeoxyuridine. Data are reported as the mean"S.E.M. of two different experiments each performed with at least two different Petri dishes. U P - 0.001 compared to the 48-h control.
of this enzyme. In the next experiments, we examined if the effect of PMA was mediated by an increase in the intracellular calcium levels. Fig. 4 shows that when cells were treated with PMA, in the presence of an intracellular calcium chelator ŽBAPTA 25 mM., the increase on retinal ganglion cells survival was abolished. We also did experiments with higher concentrations of BAPTA-AM. However, a toxic effect of this drug was observed Ždata not shown.. To investigate if tyrosine kinase enzymes were involved in the effect of PMA, we studied the survival of ganglion cells treated with PMA and genistein 10, 15, 25 and 30 mM Žan inhibitor of tyrosine kinase.. Fig. 5 shows that genistein inhibited the effect of PMA in a dose-dependent manner. A complete blockade of PMA effect was obtained when 30 mM genistein was used. This result suggests that tyrosine kinase pathways are involved on the increased survival of ganglion cells induced by PMA. Morphological analysis of retinal cells in culture shows that glial cells keep their proliferative capacity all over the time ‘‘in vitro’’. To study if the effect of PMA was mediated by an increase in the glial cell population, we tested its effect in the presence of an inhibitor of cell division Ž20 mM fluorodeoxyuridine.. Fig. 6 shows that the anti-mitotic drug did not interfere with the PMA effect. This result indicates that the increase on ganglion cells survival induced by PMA is not due to an increase in glial cells population.
4. Discussion
Fig. 5. A tyrosine kinase inhibitor Žgenistein. abolished the PMA effect on retinal ganglion cells survival after 48 h in culture. The blockage of genistein was dose-dependent and the maximum effect was obtained with 30 mM. CT, control; PMA, phorbol myristate acetate; Gen, genistein. Data are reported as the mean"S.E.M. of five different experiments each performed with at least two different Petri dishes. U P - 0.001 compared to the 48-h control.
Our results show that treatment of retinal cultures with PMA induced the survival of retinal ganglion cells after 48 h ‘‘in vitro’’. The effect was dose-dependent and 10 ngrml PMA rescued all retinal ganglion cells initially plated. Data from the literature show conflicting results concerning the role of PKC on apoptosis. Treatment with PMA activates PKC-b1 and induces apoptosis in
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A.A. dos Santos, E.G. de Araujor Brain Research 853 (2000) 338–343
myelomonocytic cell line U937, as well as in thymocytes. However, when hepatocytes are stimulated with PMA, apoptosis is inhibited w22x. These results could be explained by the finding that, during the development, PKC isoenzymes are expressed independently, accordingly to the cell type. Thus, it is reasonable to suppose that different PKC isoenzymes could be related to the suppression or to the activation of apoptosis. In agreement with this hypothesis, it has been shown that, in cell line U937, apoptosis is associated with an increase in PKC-b isoenzyme and a decrease in PKC-z isoenzyme. Furthermore, in thymocytes, the triggering of apoptosis elicited by glucocorticoids involves the translocation and activation of PKC-e w15x. The presence of PKC isoenzymes has been demonstrated in rat retinae. Using polyclonal antibodies, it was shown the presence of immunoreactivity on both plexiform layers and on the photoreceptor layer. It was also observed by electronic microscopy, the presence of PKC immunoreactivity in the nuclei of ganglion cells w31x. Moreover, using monoclonal antibodies, a isoenzymes were detected on both bipolar and ganglion cells w19x. The presence of the calcium independent isoenzymes z, e and d was also demonstrated by Western blotting w10x. Therefore, treatment with PMA could increase ganglion cell survival by direct stimulation of PKC enzymes present in this neuronal population. Previous data show that chronic treatment with PMA can induce the downregulation of PKC isoenzymes w22x. As downregulation decreases PKC activity, we decided to test if the inhibition of PKC could increase the survival of retinal ganglion cells. Our results clearly show that treatment with an inhibitor of PKC Žchelerythrine chloride. did not increase ganglion cells survival but abolished the effect of PMA. These results suggest that the effect mediated by PMA in our cultures involves the activation of PKC. However, only when PMA was present in cultures for 48 h the survival of ganglion cells was observed, suggesting that chronic stimulation of PKC is important for the rescue of retinal ganglion cells. Intracellular calcium is involved in apoptosis regulation. In some cells, intracellular calcium chelators can inhibit apoptosis. However, in IL-3 dependent hematopoietic cells, the increase in intracellular calcium can block apoptosis w15x. In our retinal cultures, we observed that an increase on intracellular calcium levels was not required for the effect of PMA since the treatment with BAPTA-AM did not inhibit the increase on the survival of ganglion cells stimulated by PMA. Since calcium independent isoenzymes of PKC were identified in rat retinae, one possibility to explain our results is the activation of these specific isoenzymes by PMA. However, further experiments will be necessary to identify which PKC isoenzymes are involved in the control of ganglion cell survival. It has been shown that PKC is able to downregulate the signaling of tyrosine kinase receptors such as the insulin
receptor and epidermal growth factor receptor w25x. Our results showing that genistein inhibited the PMA effect, suggest that PKC did not induce the downregulation of tyrosine kinase enzymes in our cultures. The effect of PMA on the survival of retinal ganglion cells can be explained either by a direct activation of PKC isoenzymes identified in ganglion cells w19x or by a release of trophic factors mediated by PKC activation in our cultures. Since many trophic molecules signal through activation of tyrosine kinase enzymes and since we observed an inhibition of PMA effect with genistein treatment, it is reasonable to assume that trophic molecules, released by PMA activation of PKC, mediate the survival of ganglion cells in our cultures. One possible source of trophic molecules could be glial cells. It has been demonstrated that glial cells produce and release trophic molecules after appropriate stimulation w24,28x. Since these cells, in our cultures, maintain the proliferative capacity and PKC activation can stimulate cellular division, we investigated if the PMA effect was mediated by an increase in glial cells population. Our results demonstrated that the blockade of cell division with fluorodeoxyuridine did not abolish the effect of PMA. Therefore, although we cannot exclude the release of trophic factors by glial cells in the cultures, our data shows that an increase in the population of glial cells is not related to the effect of PMA on the survival of ganglion cells.
Acknowledgements We would like to thank Alexandre Jose´ Fernandes and Bernardino Matheus dos Santos for technical assistance. We also thank Dr. Ana Lucia ´ Marques Ventura for helpful discussions. Aline Santos is the recipient of a FAPERJ fellowship. This work was supported by grants from FAPERJ and FINEP-UFF.
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