Cleavage of focal adhesion proteins and PKCd during lovastatin-induced apoptosis in spontaneously immortalized rat brain neuroblasts ´ ´ Lauro Gonzalez-Fernandez1,*, Maria Isabel Cerezo-Guisado2,*, Sonja Langmesser1, ´ ´ ´ ´ Maria Julia Bragado2, Maria Jesus Lorenzo2 and Luis Jesus Garcıa-Marın1 ´ ´ ´ ´ Departamento de Fisiologıa and Departamento de Bioquımica y Biologıa Molecular y Genetica, Facultad de Veterinaria, Universidad de ´ Extremadura, Caceres, Spain Keywords apoptosis; caspases; lovastatin; neuroblasts; proteolysis Correspondence ´ ´ L J Garcıa-Marın, Departamento de ´ Fisiologıa, Facultad de Veterinaria, Avda ´ Universidad s ⁄ n, E-10071 Caceres, Spain Fax: +34 927 257110 Tel: +34 927 257000 Ext 1327 E-mail: lgarcia@unex.es *Note These authors contributed equally to this work (Received 13 July 2005, revised 27 September 2005, accepted 18 October 2005) doi:10.1111/j.1742-4658.2005.05023.x We have previously shown that lovastatin induces apoptosis in spontaneously immortalized rat brain neuroblasts Focal adhesion proteins and protein kinase Cd (PKCd) have been implicated in the regulation of apoptosis We found that lovastatin exposure induced focal adhesion kinase, Crk-associated substrate (p130Cas), PKCd cleavage and caspase-3 activation in a concentration-dependent manner Lovastatin effects were fully prevented by mevalonate The cleavage of p130Cas was almost completely inhibited by z-DEVD-fmk, a specific caspase-3 inhibitor, and z-VAD-fmk, a broad spectrum caspase inhibitor, indicating that cleavage is mediated by caspase-3 In contrast, the lovastatin-induced cleavage of PKCd was only blocked by z-VAD-fmk suggesting that PKCd cleavage is caspase-dependent but caspase-3-independent Additionally, z-VAD-fmk partially prevented lovastatin-induced neuroblast apoptosis The present data show that lovastatin may induce neuroblast apoptosis by both caspase-dependent and independent pathways These findings may suggest that the caspase-dependent component leading to the neuroblast cell death is likely to involve the cleavage of focal adhesion proteins and PKCd, which may be partially responsible for some biochemical features of neuroblast apoptosis induced by lovastatin The mevalonate pathway plays an important role in cell growth and survival [1,2] Mevalonate is intracellularly synthesized from 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA), and this process is catalysed by HMG-CoA reductase, the rate-limiting enzyme in this pathway [2] Mevalonate metabolism yields a series of isoprenoid compounds which are incorporated into cholesterol, isopentenyl adenine, prenylated proteins and other end products essential for cell growth [2,3] Competitive inhibitors of HMGCoA reductase (statins), such as lovastatin, compactin, simvastatin and pravastatin, not only block the biosyn- thesis of mevalonate but, in addition, inhibit the proliferation and induce apoptosis of both normal and tumor cells [4–8] HMG-CoA reductase activity [9] and cholesterol biosynthesis [10,11] are very high in the early phase of ontogenetic development of the brain These data indicate that the mevalonate pathway is essential to ensure normal growth, differentiation and maintenance of neuronal tissues In accordance with this, we have shown that lovastatin induces the apoptosis of spontaneously immortalized rat brain neuroblasts Lovastatin effects were associated with both cell morphological Abbreviations Ac-DEVD-AMC, Acetyl-Asp-Glu-Val-Asp-7-amido-4methyl coumarin; FAK, focal adhesion kinase; HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide; PKCd, protein kinase Cd; p130Cas, Crk-associated substrate; z-DEVD-fmk, benzyloxycarbonyl-Asp-Glu-Val-Asp fluoromethyl ketone; z-VAD-fmk, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone FEBS Journal 273 (2006) 1–13 ª 2005 The Authors Journal Compilation ª 2005 FEBS ´ ´ndez et al L Gonzalez-Ferna Caspase-dependent effects in neuroblast apoptosis changes and a decrease in the level of prenylation of Ras and RhoA proteins [12] Rho small GTPases are important regulators of the actin cytoskeleton and the morphological heterogeneity of vertebrate cells [13,14] Among the family members of the Rho GTPases, RhoA is one of the most studied and has been implicated in the formation of stress fibres and focal adhesion complexes [15] Focal adhesion complexes are protein aggregates linking actin filaments to the cytoplasmic domain of integrins Maintenance of cell–matrix contact is an important cell survival factor and the loss of cell–matrix and cell– cell contact is a characteristic feature of apoptosis [16] Focal adhesion complexes consist of integrin proteins, the tyrosine kinases Src and focal adhesion kinase (FAK), actin-binding structural proteins, and the adaptor proteins paxillin and Crk-associated substrate (p130Cas) [17] Among these, FAK, p130Cas and paxillin tyrosine phosphorylation are closely associated with the reorganization of the actin cytoskeleton [18,19] In addition, the transition from flat to round cell morphology, which is a characteristic feature in cells undergoing apoptosis, is accompanied by cytoskeletal rearrangement and changes in focal adhesion proteins Indeed, proteolytic cleavage of FAK [20–25] and p130Cas [22,26,27] by caspases in many cell types undergoing apoptosis further suggests the important role played by the association of focal adhesion proteins, the cytoskeleton and the extracellular matrix in the maintenance of cell morphology and survival Previous studies have shown that several signalling molecules including protein kinases are also cleaved by caspases during apoptosis, examples include p21 (CDKN1A)-activated kinase (PAK2) [28], MAPK kinase kinase (MEKK-1) [29], MAPK kinase kinase kinase (Raf-1), protein kinase B (Akt) [30,31] and protein kinase C (PKC) [32] PKC is a family of serine ⁄ threonine protein kinases that has been recently implicated in apoptosis in a variety of cell types [33– 35] At least 11 isoforms of PKC have been identified and can be classified into conventional (a, bI, bII and c), novel (d, e, g, and h), atypical (f and k ⁄ i) and novel ⁄ atypical (l) based upon their cofactor requirements PKC isozymes are known to be activated by proteolytic separation of the regulatory domain from the catalytic domain While the classical and atypical PKC isozymes are associated with cell survival, the novel PKC isozymes are proapoptotic in function [33–37] Notably, proteolytic cleavage and activation of PKCd by caspases have been shown to represent an important step in apoptosis induced by many stimuli [32,36–43] Furthermore, expression of the catalytic domain of PKCd is sufficient to induce apoptosis in various cell types, suggesting that PKCd may be an important effector of apoptosis [32] As it has been previously mentioned, lovastatin promotes growth suppression and apoptosis in rat brain neuroblasts [12] The involvement of caspase activity in the neuronal apoptosis induced by lovastatin as well as the relevance of the structural integrity of kinases and focal adhesion proteins after lovastatin treatment in neuroblasts have not been studied Therefore, the main objective of this work was to investigate the intracellular effects of lovastatin treatment in rat brain neuroblasts, i.e., activation of caspases and the cleavage of focal adhesion proteins and PKCd The elucidation of intracellular mechanisms affected by lovastatin will contribute to gaining insight in the growth inhibition and apoptosis induced by this statin in neuroblasts, and additionally it will contribute to establish the role of the mevalonate pathway in the physiological growth and development of cells from the central nervous system Results Effect of lovastatin on the cleavage of focal adhesion proteins We have previously shown that lovastatin induces apoptosis of rat brain neuroblasts; its effect being associated with a change in cell morphology from a flat to a round shape [12] Recent findings on apoptosis indicate that this morphological change is accompanied by proteolytic cleavage of focal adhesion proteins Therefore, in the present work, we first examined the integrity of FAK and p130Cas proteins by western blot in our experimental conditions Incubation of neuroblasts with different lovastatin concentrations for 24 h caused the cleavage of both FAK (Fig 1A) and p130Cas (Fig 1B) proteins in a concentration-dependent manner The cleavage induced by lovastatin was significant at 10 lm for both proteins, with 58 ± 12% of intact FAK and 36 ± 11% of intact p130Cas, respectively, remaining at this concentration (expressed as percentage of noncleavaged proteins in untreated cells) Proteolysis of FAK generated two main cleavage products of about 100 and 80 kDa, whereas proteolysis of p130Cas generated three cleavage products of about 80, 60 and 31 kDa The appearance of FAK and p130Cas proteolytic products correlated well with the decrease in the level of the respective full-length proteins Under the same experimental conditions, the level of the cytoskeletal protein actin remained constant (Fig 1C), suggesting that the effect of lovastatin on FAK and p130Cas cleavage was specific for these proteins and FEBS Journal 273 (2006) 1–13 ª 2005 The Authors Journal Compilation ª 2005 FEBS ´ ´ L Gonzalez-Fernandez et al A B Caspase-dependent effects in neuroblast apoptosis apoptosis induced by lovastatin [12] Therefore, to test the ability of mevalonate at preventing the effect of lovastatin on FAK and p130Cas cleavage, neuroblasts were incubated with 10 lm lovastatin in the absence or presence of mevalonate (100 lm) for 24 h As shown in Fig 2, mevalonate completely prevented the cleavage of both FAK (106 ± 12% of intact FAK) and p130Cas (91 ± 11% of intact p130Cas) proteins Mevalonate alone had no effect on the integrity of focal adhesion proteins (101 ± 11% of intact FAK and 96 ± 5% of intact p130Cas) Actin levels remained constant (Fig 2C) Effect of lovastatin on the cleavage of PKCd C Fig Effect of lovastatin on FAK and p130Cas proteins in immortalized rat brain neuroblasts Cells initially cultured for 24 h in Ham’s F12 ⁄ 10% fetal bovine serum were incubated for an additional 24 h in medium alone (0) or in the presence of different concentrations of lovastatin At the end of the experiment, cells were lysed and total proteins (20 lg per lane) were subjected to SDS ⁄ PAGE and western blotting using specific antibodies against FAK (A), p130Cas (B) and actin (C) as described under Experimental procedures Molecular mass (kDa) is indicated by lines on the left Full-length proteins and cleavage fragments of proteins are indicated by arrows on the right Each blot is representative of three independent experiments that an equal amount of proteins were loaded in each lane We have shown that mevalonate prevents the morphological and biochemical features of neuroblast Previous studies have shown that PKCd is cleaved into a 41 kDa catalytically active and a 38 kDa regulatory fragment in cells undergoing apoptosis Furthermore, these studies indicated that proteolytic activation of PKCd contributes to phenotypic changes associated with apoptosis [32] Therefore, we next studied if PKCd cleavage also occurs during lovastatin-induced neuroblast apoptosis As shown in Fig 3A (upper panel), lovastatin did not appear to modify the expression of full-length PKCd but induced the generation of a cleavage product of about 42 kDa in a concentration-dependent manner Actin levels did not change after lovastatin treatment (Fig 3A, lower panel) In order to know whether this effect was specific of HMG-CoA reductase inhibition, cells were incubated with lovastatin (10 lm) in the presence or absence of mevalonate (100 lm) for 24 h Cotreatment with mevalonate prevented the cleavage of PKCd induced by lovastatin (Fig 3B, upper panel) Treatment of cells with mevalonate alone did not modify PKCd expression Actin levels did not change under the same experimental conditions (Fig 3B, lower panel) Effect of lovastatin on caspase-3 activation Previous studies have shown that proteolytic cleavage of focal adhesion proteins and PKCd is mediated by caspases, mainly by caspase-3 [20,25,32,37,44] To determine whether these proteins are cleaved by caspase-3 under our experimental conditions, we first examined the effect of lovastatin on caspase-3 activation Caspase-3 is expressed as a 32 kDa proenzyme, which is activated by proteolytic cleavage into an active 12–17 kDa form To determine whether lovastatin induced the activation of caspase-3, neuroblasts were exposed to different amounts of lovastatin for 24 h and the appearance of the activated form of caspase-3 FEBS Journal 273 (2006) 1–13 ª 2005 The Authors Journal Compilation ª 2005 FEBS ´ ´ndez et al L Gonzalez-Ferna Caspase-dependent effects in neuroblast apoptosis A A B B C Fig Mevalonate prevents FAK and p130Cas cleavage induced by lovastatin in immortalized rat brain neuroblasts Cells initially cultured for 24 h in Ham’s F12 ⁄ 10% fetal bovine serum were incubated with lovastatin (10 lM) in the absence or presence of mevalonate (100 lM) for an additional 24 h Control cells were incubated in presence of medium or mevalonate alone At the end of the experiment, cells were lysed and total proteins (20 lg per lane) were subjected to SDS ⁄ PAGE and western blotting using specific antibodies against FAK (A), p130Cas (B) and actin (C) as described Molecular mass (kDa) is indicated by lines on the left Full-length proteins and cleavage fragments of proteins are indicated by arrows on the right Each blot is representative of three independent experiments Fig Effect of lovastatin alone or in combination with mevalonate on PKCd in immortalized rat brain neuroblasts Cells previously cultured for 24 h in growth medium were incubated for an additional 24 h with different concentrations of lovastatin (A) or with 10 lM lovastatin in the absence or presence of 100 lM mevalonate (B) Control cells received medium or mevalonate alone Proteins from cell lysates (20 lg) were separated by SDS ⁄ PAGE and analyzed by western Blotting using anti-PKCd (upper panel) and anti-actin (lower panel) as described Molecular mass (kDa) is indicated by lines on the left Full-length PKCd and a cleavage fragment are indicated by arrows on the right Each blot is representative of three independent experiments was evaluated by western blot As seen in Fig 4A, lovastatin increased the amount of the activated form of caspase-3 in a concentration-dependent manner FEBS Journal 273 (2006) 1–13 ª 2005 The Authors Journal Compilation ª 2005 FEBS ´ ´ L Gonzalez-Fernandez et al A B Caspase-dependent effects in neuroblast apoptosis Mevalonate (100 lm) abolished caspase-3 processing induced by lovastatin, indicating that lovastatin effect was specific (Fig 4B) Actin levels did not change under the same experimental conditions (Fig 4A,B) To verify the results obtained above, we measured caspase-3 activity using the synthetic substrate Ac-DEVD-AMC Lysates containing active caspase-3 cleave the substrate releasing the fluorescent molecule AMC, which is detected in a spectrofluorimeter with excitation wavelength of 380 nm and emission wavelength of 440 nm As shown in Fig 4C, lovastatin (10 lm) markedly enhanced the activity of caspase-3 Again, cotreatment with mevalonate prevented the effect of lovastatin and completely restored caspase-3 activity to control levels Mevalonate alone did not affect caspase-3 activation (Fig 4B,C) Effect of caspase inhibition on lovastatin-induced protein cleavage and neuronal apoptosis C Fig Effect of lovastatin alone or in combination with mevalonate on caspase-3 activation in immortalized rat brain neuroblasts Cells previously cultured for 24 h in growth medium were incubated for an additional 24 h with different concentrations of lovastatin (A) or with 10 lM lovastatin in the absence or presence of 100 lM mevalonate (B and C) Control cells received medium or mevalonate alone At the end of the experiment, cell extracts were obtained and used either to analyze, by western blot, the proteolytic activation of pro-caspase-3 (A and B, upper panel) or actin levels (A and B, lower panel) or the activity of caspase-3 (C) as described Each blot is representative of three independent experiments (A and B) Data are represented as mean ± SEM and representative of three independent experiments performed in duplicate (C) Once we showed that lovastatin induced caspase-3 activation, we next investigated whether lovastatin-induced proteolytic cleavage of focal adhesion proteins and PKCd was mediated by caspase-3 Cells were incubated with lovastatin in the absence or presence of the specific inhibitor of caspase-3, z-DEVD-fmk (50 lm) or the broad spectrum caspase inhibitor, z-VAD-fmk (50 lm) We previously confirmed that the specific inhibitor z-DEVD-fmk completely blocked caspase-3 activity induced by lovastatin at 30 (0.1 ± vs 2.1 ± 0.1 nmol product per 100 lg protein, respectively) On the other hand z-VAD-fmk is able to block the appearance of the active fragment of caspase-3 induced by lovastatin (data not shown) Both inhibitors z-DEVD-fmk (Fig 5A) and z-VAD-fmk (Fig 5B) partially prevented p130Cas cleavage induced by lovastatin by 67% and 74%, respectively Regarding PKCd, the specific caspase-3 inhibitor z-DEVD-fmk only prevented its proteolytic cleavage by 10% (Fig 5C), but surprisingly z-VAD-fmk prevented it by 100% (Fig 5D) Both caspase inhibitors partially prevented the cleavage of FAK induced by lovastatin (data not shown) Because z-VAD-fmk could inhibit lovastatin-induced protein cleavage, the ability of this peptide to prevent lovastatin-evoked apoptosis was assessed by three independent assays, neuroblast viability, internucleosomal DNA fragmentation, and quantification of the percentage of neuroblasts undergoing apoptosis, by flow cytometry Morphology of the cells was also studied by phase contrast microscopy As we have shown previously, the treatment of neuroblasts with 10 lm lovastatin during 24 h caused a significant decrease in FEBS Journal 273 (2006) 1–13 ª 2005 The Authors Journal Compilation ª 2005 FEBS ´ ´ndez et al L Gonzalez-Ferna Caspase-dependent effects in neuroblast apoptosis A C B D Fig Effect of caspase inhibitors on lovastatin-induced p130Cas and PKCd cleavage in immortalized rat brain neuroblasts Cells previously cultured for 24 h in growth medium were incubated with 10 lM lovastatin in the absence or presence of 50 lM z-DEVD-fmk (A and C) or 50 lM z-VAD-fmk (B and D) for an additional 24 h Control cells received media, z-DEVD-fmk or z-VAD-fmk alone At the end of the experiment, cells were lysed and total proteins (20 lg per lane) were subjected to SDS ⁄ PAGE and western blotting using specific antibodies against p130Cas (A and B), PKCd (C and D, upper panel) and actin (C and D, lower panel) as described Molecular mass (kDa) is indicated by lines on the left Full-length proteins and cleavage fragments of proteins are indicated by arrows on the right Each blot is representative of three independent experiments cell viability (Fig 6A), the appearance of internucleosomal DNA fragmentation (Fig 6B) and an increase in the percentage of apoptotic neuroblasts (Fig 6C) These lovastatin effects were partially blocked when z-VAD-fmk (50 lm) was present in the medium However, this general caspase inhibitor failed to prevent lovastatin-induced cell shape changes (Fig 6D–G), and z-VAD-fmk alone had no effect, either on cell survival or on morphology (Fig 6) Discussion We have recently shown that lovastatin, a competitive HMG-CoA reductase inhibitor, induces apoptosis of spontaneously immortalized rat brain neuroblasts, its effect being associated with cell morphological changes and a decrease in the level of prenylation of RhoA protein [12] These findings suggest that lovastatin may trigger apoptosis of neuroblasts by inducing cytoskeletal FEBS Journal 273 (2006) 1–13 ª 2005 The Authors Journal Compilation ª 2005 FEBS ´ ´ L Gonzalez-Fernandez et al Caspase-dependent effects in neuroblast apoptosis A D B E F C G Fig Effect of lovastatin alone or in combination with z-VAD-fmk on cell viability (A), internucleosomal DNA fragmentation (B), percentage of apoptotic cells (C), and cell morphology (D–G) in immortalized rat brain neuroblasts Cells previously cultured for 24 h in growth medium were incubated with 10 lM lovastatin in the absence or presence of 50 lM z-VAD-fmk for an additional 24 h Control cells were kept in medium alone or supplemented with z-VAD-fmk only At the end of the experiment, cell viability was determined by the MTT assay (A); internucleosomal DNA degradation was analyzed by using electrophoresis on 2% agarose gel (B); the percentage of cells with hypodiploid DNA content was evaluated by flow cytometry (C) Each value represents the mean ± standard error of three independent experiments made in triplicate ns, Not significant; *, P < 0.05; ***, P < 0.001; compared to untreated cells (A and C) A representative photograph of three independent experiments is shown in (B) where M represents 100 bp molecular mass markers Morphological changes (D–G) were determined by using phase contrast microscopy and photographs were taken (Magnification: 200·) (D) medium alone; (E) 10 lM lovastatin; (F) 10 lM lovastatin + 50 lM z-VAD-fmk; (G) 50 lM z-VAD-fmk alone FEBS Journal 273 (2006) 1–13 ª 2005 The Authors Journal Compilation ª 2005 FEBS ´ ´ndez et al L Gonzalez-Ferna Caspase-dependent effects in neuroblast apoptosis rearrangement as a consequence of changes in the expression and ⁄ or activation of proteins that control the organization of the cytoskeleton, such as focal adhesion proteins and PKCd [20,23,26,33,35] However, the effect of lovastatin on the integrity of focal adhesion proteins and PKCd has not been reported previously In the present work, we show for the first time that lovastatin induces the cleavage of FAK and p130Cas in spontaneously immortalized rat brain neuroblasts undergoing apoptosis Lovastatin effects were concentration dependent and completely prevented by simultaneous exposure of cells to exogenous mevalonate, demonstrating that the effect of lovastatin on FAK and p130Cas cleavage was due to its specific inhibitory action on intracellular mevalonate synthesis The cleavage of FAK and p130Cas in response to various apoptotic inducers in different normal and tumour cell lines has been reported previously [20,21,23–26,44–49], which indicates that cleavage of these proteins may play an important role in the execution of apoptosis FAK and p130Cas cleavage patterns induced by lovastatin are very similar to those seen in cells exposed to other apoptotic stimuli, however, we could not detect FAK cleavage products of about 40 kDa and 30 kDa that have been described in some reports [21,44,46], possibly due to the different cell type or antibody used We have also shown for the first time that lovastatin specifically induces the cleavage of PKCd in a concentration-dependent manner, producing a fragment of about 42 kDa that corresponds in size to the catalytic domain of the kinase Our result is in good agreement with previous works that show that PKCd is cleaved in various cell types undergoing apoptosis, including neuronal cells [32,36,37,40,43], and suggests that lovastatin may induce the activation of PKCd in rat brain neuroblasts by its proteolytic cleavage In difference to other reports, we show that the appearance of the PKCd cleavage product is not accompanied by a decrease in the full-length protein levels These data suggest that lovastatin may be inducing PKCd expression in neuroblasts In agreement with this, Kaasinen et al [40] have shown that the expression of PKCd mRNA is strikingly up-regulated during kainiteinduced apoptosis in the cortex and the CA1 and CA3 hippocampal regions Previous works indicate that the proteolytic cleavage of focal adhesion proteins and PKCd is mediated by members of the caspase family of cysteine proteases, particularly by caspase-3 [20,25,32,37,44] Therefore, we first studied whether caspase-3 is activated under our experimental conditions We show that lovastatin induces proteolytic activation of caspase-3 in a concen8 tration-dependent manner, and that mevalonate prevents this effect The ability of lovastatin to induce caspase-3 activation has recently been documented in various non-neuronal cell lines [50–53], but, to our knowledge, this is the first report that demonstrates that lovastatin is a potent inducer of caspase-3 activity in neuronal cells Focking et al [54] previously showed ă that lovastatin was not able to activate caspase-3 in a murine hippocampal cell line Taken together, these results suggest that lovastatin-induced caspase-3 activation in neuronal cells may be cell type specific To examine the contribution of caspases to cleavage of focal adhesion proteins and PKCd induced by lovastatin, pharmacological caspase inhibitors were employed Our results suggest that the cleavage of FAK (data not shown) and p130Cas in rat brain neuroblasts is likely to be mediated partially by caspase-3, other caspases and also by other proteases In this regard, calpain-induced FAK and p130Cas proteolysis has been reported in cells undergoing apoptosis [27,46] Whether or not lovastatin induces the activation of calpain in these cells is currently under study On the other hand, we also show that the cleavage of PKCd during lovastatin-induced neuroblast apoptosis is completely dependent of caspase activity However, our results suggest that activation of other caspases different than caspase-3 may be involved in this event This finding differs from those reported in other apoptotic models, in which z-DEVD-fmk completely prevented PKCd cleavage [36,37,39,55,56], but is in agreement with the fact that caspase-3 did not cleave PKCd in the colon cancer line, COLO205 [57] At present, we not know which caspase may be mediating PKCd cleavage in our experimental conditions However, the fact that lovastatin induces caspase-7 activation in the prostate cancer cell line, LNCaP [58], and that the fluorogenic peptide substrate Ac-DEVD-AMC is also a substrate for caspase-7 [59] suggests that the cleavage of PKCd induced by lovastatin in rat brain neuroblasts undergoing apoptosis may be mediated by caspase-7 Because caspase inhibition could block lovastatininduced cleavage of focal adhesion proteins and PKCd, we investigated whether it also blocks lovastatininduced neuroblast morphological changes and apoptosis Our results show that z-VAD-fmk partially prevent the biochemical features of apoptosis but fail to block the morphological changes induced by lovastatin at the same concentration that blocked PKCd cleavage and almost completely inhibited both FAK and p130Cas degradation Our findings differ from those of other studies in which the pan-caspase inhibitor prevents not only these caspase-mediated cleavage events but also the morphological changes and FEBS Journal 273 (2006) 1–13 ª 2005 The Authors Journal Compilation ª 2005 FEBS ´ ´ L Gonzalez-Fernandez et al apoptosis induced by different agents in various cell types [21,22,24,36,37,43,45] Taken together, our data allow us to speculate that the cleavage of FAK, p130Cas and PKCd could be, at least partially, mediating the biochemical features of apoptosis induced by lovastatin Therefore, our work suggests that other proteins have to mediate the morphological changes observed On the other hand, the fact that z-VAD-fmk partially prevents the apoptosis suggests that lovastatin may induce neuroblast death by both caspase-dependent and -independent pathways Caspase-independent apoptosis has been extensively described during normal cell physiology [60,61] In summary, we have demonstrated for the first time that HMG-CoA reductase inhibition by lovastatin stimulates caspase activity in rat brain neuroblasts, which may help explain its apoptotic effect In a parallel way, our data showed that lovastatin induces proteolysis of FAK, p130Cas and PKCd in these cells Lovastatin effects were concentration-dependent and prevented by the HMG-CoA reductase product, which indicates that lovastatin effects were due to an inhibition of mevalonate synthesis While a general caspase inhibition partially prevented both proteolytic processes and the biochemical parameters of apoptosis, inhibition of caspase activity seems unrelated to the morphological changes associated to this type of cell death Therefore, our results suggest that lovastatin may induce neuroblast apoptosis by both caspase-dependent and -independent pathways In addition, our data allow us to speculate that the caspase-dependent component leading to neuroblast cell death may involve the cleavage of focal adhesion proteins and PKCd, which may be partially responsible for some biochemical features of neuroblast apoptosis induced by lovastatin These findings might contribute to elucidation of the molecular mechanisms of some statin effects described in the central nervous system, such as growth suppression or induction of neuroblast apoptosis Experimental procedures Reagents Lovastatin (Mevinolin, MK-803) was from Calbiochem (La Jolla, CA, USA) Mevalonic acid, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide (MTT), propidium iodide, ribonuclease A, proteinase K and crystal violet were purchased from Sigma Chemical Company (Sigma-Aldrich, St Louis, MO, USA) The caspase-3 substrate (Ac-DEVD-AMC), the caspase-3 inhibitor (z-DEVD-fmk) and the general caspase inhibitor (z-VAD-fmk) were from PharMingen (BD Biosciences Europe, Brussels, Belgium) Caspase-dependent effects in neuroblast apoptosis Complete protease inhibitor cocktail tablets were from Roche Molecular Biochemicals (Indianapolis, IN, USA) Ham’s F-12 medium, fetal bovine serum, l-glutamine, streptomycin, penicillin and trypsin ⁄ EDTA solution were from PAN Biotech (Aidenbach, Germany) Tissue culture flasks and dishes were from TPP (Trasadingen, Switzerland) Other reagents were obtained from different commercial sources and were of the highest purity available Cell line and culture Spontaneously immortalized rat brain neuroblasts were used in this study The cell line was obtained by spontaneous immortalization from cultures of 18 day old fetal rat cerebral cortices and was kindly provided by A Munoz ˜ ´ (Instituto de Investigaciones Biomedicas, CSIC, Madrid, Spain) Cells were grown in Ham’s F-12 supplemented with 10% fetal bovine serum, l-glutamine (2 mm), streptomycin (100 lgỈmL)1) and penicillin (100 mL)1) Cells were seeded at · 105 in 75 cm2 tissue culture flask in 10 mL of culture medium and incubated at 37 °C under a 5% CO2 ⁄ 95% air atmosphere Cultures were passaged twice weekly by trypsinization using a trypsin ⁄ EDTA solution Cell treatments Confluent cells in 75 cm2 tissue culture flasks were trypsinized and seeded in tissue culture dishes at a concentration of · 104 cells per cm2 Twenty-four hours later, the medium was aspirated and replaced with fresh medium alone or containing the indicated concentrations of lovastatin, mevalonate and caspase inhibitors and the incubation was continued for a further 24 h Cell morphology was analyzed by phase contrast microscopy in a Leica DMIL inverted microscope (Wetzlar, Germany) Photographs were taken at the end of each experiment Western blot analysis Cells cultured under different experimental conditions were washed with NaCl ⁄ Pi and lysed in Hepes buffer [50 mm Hepes pH 7.4, 150 mm NaCl, mm MgCl2, 25 mm NaF, ` 10 mm Na4P2O7, 10% glycerol, 1% TritonX-100, 0.5 mm Na3VO4 and tablet (50 mL) of complete protease inhibitor cocktail] After centrifugation at 10 000 g for 15 at °C, protein concentration in each sample was determined by using the Bio-Rad Protein Assay (Munich, Germany), according to the instructions of the manufacturer Equal amounts of protein (20 lg) were subjected to electrophoretic separation on denaturing 10% polyacrylamide gels, by SDS ⁄ PAGE, and transferred to nitrocellulose membranes (Protran, Schleicher and Schuell, Dassel, Germany) Membranes were blocked in Blotto [50 mm Tris ⁄ HCl pH 8.0, mm CaCl2, 80 mm NaCl, 0.05% (v ⁄ v) Tween 20 and 5% FEBS Journal 273 (2006) 1–13 ª 2005 The Authors Journal Compilation ª 2005 FEBS ´ ´ndez et al L Gonzalez-Ferna Caspase-dependent effects in neuroblast apoptosis (w ⁄ v) nonfat dry milk] and incubated with primary antibodies Incubation conditions were: mouse monoclonal anti-FAK (Transduction Laboratories, BD Bioscience Europe) : 1000 for 90 at room temperature, mouse monoclonal anti-p130Cas (Transduction Laboratories, BD Bioscience Europe) : 750 for 90 at room temperature, rabbit polyclonal anti-PKCd (Santa Cruz Biotechnology, Santa Cruz, CA, USA) 0.25 lgỈmL)1 for 120 at room temperature, and mouse monoclonal anti-caspase-3, active form (17 kDa) (Cell Signalling, Beverly, MA, USA) : 1000 for 90 at room temperature Membranes were washed twice with Blotto and incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (anti-mouse IgG : 6000 or anti-rabbit IgG : 10000, Pierce, Rockford, Il, USA) for 45 at room temperature After three washes (10 each) with 50 mm Tris ⁄ HCl pH 8.0, mm CaCl2, 80 mm NaCl, membranes were incubated with Super SignalÒ West Pico Chemiluminescent Substrate (Pierce) for at room temperature and exposed to Hyperfilm ECL (Amersham, Piscataway, NJ, USA) Blots were stripped and reprobed with rabbit anti-actin (Sigma) : 5000 for 90 at room temperature, to verify equal loading of protein in all lanes Analysis of caspase-3 activity Caspase-3 activity was measured using the synthetic substrate Ac-DEVD-AMC Cells exposed to different treatments were washed with cold NaCl ⁄ Pi and lysed at °C in 10 mm Tris ⁄ HCl, 10 mm NaH2PO4, pH 7.5, 130 mm NaCl, 1% Triton X-100 and 10 mm NaPPi Lysates were clarified by centrifugation at 10 000 g for 10 at °C After measuring protein concentration, aliquots of 50 lg protein were diluted in reaction buffer (20 mm Hepes pH 7.5, 10% glycerol, mm dithiothreitol) and mixed with 20 lm caspase-3 substrate (Ac-DEVD-AMC) The reactions were incubated at 37 °C for the indicated period of time and product formation was monitored in a spectrofluorometer using excitation and emission wavelengths of 380 nm and 440 nm, respectively Fluorescence readings were calibrated using a solution of commercial AMC (Sigma) of known concentration allowed to dry, and 1% SDS was added to solubilize the dye The absorbance of dye was measured at a wavelength of 560 nm Viable cells were calculated as percent of absorbance with respect to untreated cells Results using both methods were similar DNA fragmentation assay At the end of each experiment, cells were washed twice in ice-cold NaCl ⁄ Pi without Ca2+ and Mg2+ and then scraped and pelleted at °C Cells were lysed in Tris buffer (10 mm Tris, pH 7.4, mm EDTA, and 0.5% Triton X-100) for 60 at °C After centrifugation at 10 000 g for 30 at °C, the supernatants were incubated with RNase A (0.1 mgỈmL)1) at 37 °C for 45 min, and then with proteinase K (0.2 mgỈmL)1) at 37 °C for 45 DNA was then extracted twice with phenol ⁄ chloroform (1 : 1) and precipitated with 0.1 volumes of sodium acetate (3 m) and 2.5 volumes of ice-cold ethanol at )80 °C overnight The precipitated DNA was collected by centrifugation at 10 000 g for 20 and resuspended in autoclaved water DNA was resolved on 2% agarose ⁄ 0.1 lgỈmL)1 ethidium bromide gels in TBE buffer (80 mmolỈL)1 Tris ⁄ borate, mmolỈL)1 EDTA, pH 8.0) After electrophoresis, gels were examined under ultraviolet light and photographed using a ChemiDoc System (Documentation and Analysis System, Bio-Rad, Hercules, CA, USA) Analysis of cell DNA content by flow cytometry The ploidy determination of neuroblasts was estimated by flow cytometry DNA analysis as described previously [12] After treatment, cells were trypsinized, washed with NaCl ⁄ Pi, fixed at °C in 70% ethanol and treated at 37 °C with RNase (10 lgỈmL)1) for 30 The DNA content per cell was evaluated in a Cyan flow cytometer (DAKO Cytomation, Glostrup, Denmark) after staining the cells with propidium iodide (50 lgỈmL)1) for 30 at room temperature in the dark For cell cycle analysis, only signals from single cells were considered (10 000 events per sample) Statistical analysis Cell viability assay Cell viability was determined by the colorimetric MTT assay as described previously [12] At the end of each treatment, cells were incubated for 15 at 37 °C with MTT (500 lgỈmL)1) and formazan precipitates were solubilized with acidic isopropanol (0.04–0.1 m HCl in absolute isopropanol) The absorbance of converted dye was measured at a wavelength of 570 nm In some experiments, cell viability was evaluated by the crystal violet method At the end of each experiment, medium was discarded and cells were stained with crystal violet (0.03% in 2% ethanol) for Subsequently, dishes were rinsed with tap water, 10 Each experiment was repeated at least three times, with good agreement among the results of individual experiments All data are expressed as the mean ± standard error of the mean Results were analyzed by one-way analysis of variance (anova) followed by Student’s t-test P-values of less than 0.05 were considered significant Acknowledgements The authors thank J Ricardo Argent for his excellent technical assistance and Dr Alberto Alvarez Barrientos FEBS Journal 273 (2006) 1–13 ª 2005 The Authors Journal Compilation ª 2005 FEBS ´ ´ L Gonzalez-Fernandez et al for his kind and valuable help on the flow cytometry experiments This work has been supported by Grants, SAF 2001-0154 from the Ministerio de Ciencia y Tecnologia, and 2PR01B007 from the Junta de Extrema´ ´ dura, Spain L Gonzalez-Fernandez was supported by ´ a fellowship from 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Oncogene 23, 2766–2773 13 ... lovastatin in rat brain neuroblasts undergoing apoptosis may be mediated by caspase-7 Because caspase inhibition could block lovastatininduced cleavage of focal adhesion proteins and PKCd, we investigated... Actin levels remained constant (Fig 2C) Effect of lovastatin on the cleavage of PKCd C Fig Effect of lovastatin on FAK and p130Cas proteins in immortalized rat brain neuroblasts Cells initially cultured... apoptosis in rat brain neuroblasts [12] The involvement of caspase activity in the neuronal apoptosis induced by lovastatin as well as the relevance of the structural integrity of kinases and focal adhesion