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First published online 29 April 2008
doi: 10.1242/jcs.022640
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Research Article |
-induced apoptosisDepartment of Pathology and Cell Biology, Université de Montréal, 2900 Edouard-Montpetit, Montréal, Québec, H3T 1J4, Canada
* Author for correspondence (e-mail: maria.leiza.vitale{at}umontreal.ca)
Accepted 19 February 2008
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Summary |
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or short-term with TGF together with cycloheximide (CHX) undergo apoptosis. Cell shrinkage and detachment during apoptosis is dependent on actomyosin contractility. Myosin II heavy chain (MHCII) isoforms have shared and distinct functions. Here, we investigated whether the involvement of MHCII isoforms A and B (MHCIIA and MHCIIB, respectively) in cell shrinkage and detachment differs during apoptosis. We show that TNF induces caspase-dependent MHCIIA degradation, whereas MHCIIB levels and association with the cytoskeleton remained virtually unchanged in TtT/GF cells and NIH 3T3 fibroblasts. MHCIIA proteolysis also occurred in fibroblasts that lack MHCIIB when treated with TNF and CHX together. The absence of MHCIIB did not affect cell death rate. However, MHCIIB–/– cells showed more resistance to TNF–induced actin disassembly, cell shrinkage and detachment than wild-type fibroblasts, indicating the participation of MHCIIB in these events. Moreover, inhibition of atypical PKC
, which targets MHCIIB but not MHCIIA, blocked TNF-induced shrinkage and detachment in TtT/GF cells and wild-type fibroblasts, but the inhibitory effect was significantly reduced in MHCIIB–/– fibroblasts. TNF treatment increased cytoskeleton-associated myosin light chain (MLC) phosphorylation but did not induce actin cleavage. In conclusion, our results demonstrate that MHCIIB, together with MLC phosphorylation and actin, constitute the actomyosin cytoskeleton that mediates contractility during apoptosis.
Key words: Myosin, Apoptosis, Actin
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Introduction |
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(TNF) is a pro-inflammatory cytokine involved in distinct bioregulatory functions such as cell proliferation, differentiation and programmed cell death or apoptosis (Leong and Kaplan, 2000
). During apoptosis, TNF signalling triggers the trimerisation of TNF-receptor-1 (TNFR1) leading to the sequential recruitment of a series of adaptor proteins to constitute the death-inducing signalling complex (DISC) (Baud and Karin, 2001). DISC assembly induces the cleavage and activation of the initiator procaspase 8 (Baud and Karin, 2001), which triggers the `execution phase' of apoptosis during which several effector caspases are activated that cause the cleavage of target proteins (Mills et al., 1999) – amongst them several cytoskeletal proteins (Chen et al., 2001; Jänicke et al., 1998; Kook et al., 2003). The execution phase of the apoptotic process is characterised by specific changes in cell morphology, such as chromatin condensation and fragmentation, cell shrinkage, membrane blebbing, and apoptotic-body formation (Earnshaw, 1995; Mills et al., 1999). Actin-myosin interaction is required to generate the contractile force necessary for most of these morphological responses (Croft et al., 2005; Mills et al., 1999; Suarez-Huerta et al., 2000b).
Myosins are motor proteins that bind to and move on actin filaments. Myosins are oligomers formed by one or two myosin heavy chains (MHCs) and one or more myosin light chains (MLCs). Several classes of myosins have been described and classified on the basis of the MHC isoform in the complex (Korn, 2000). Contractile actomyosin fibers are mainly formed by the association of actin and myosin II. Class II conventional myosins are hexamers constituted through the noncovalent association of a pair of MHCs (MHCII; 200 kDa) and two pairs of MLCs (17 and 20 kDa) one of which is `regulatory' and the other `constitutive' (Bresnick, 1999).
Actomyosin fiber contraction in nonmuscle cells is regulated by the phosphorylation state of the regulatory MLC, which is dependent on MLC kinase (MLCK) (Gallagher et al., 1997), the Rho-regulated kinases (ROCK1 and ROCK2) (Amano et al., 1996; Maekawa et al., 1999) and on myosin phosphatase (Kimura et al., 1996; Noda et al., 1995). All these enzymes are necessary for cell contraction, shrinkage, membrane bleb formation and relocalisation of fragmented DNA into apoptotic bodies (Coleman et al., 2001; Croft et al., 2005; Mills et al., 1998; Orlando et al., 2006; Song et al., 2002).
Experimental evidence has demonstrated a role for MLC phosphorylation and for the corresponding kinases in generating contractile forces within cells that undergo apoptosis, but the potential role of MHC has received little attention. Interaction between actin and myosin II and, thus, the formation of actomyosin contractile elements, also depend on the heavy chains that possess the actin-binding site and the actin-activated ATPase activity (Bresnick, 1999). The C-terminal region of the MHC binds to cellular structures and acts as a crosslinker that stabilises the actomyosin complex (Bresnick, 1999). The class II nonmuscle MHC consists of three isoforms, MHCIIA, MHCIIB (Sellers and Goodson, 1995) and the recently discovered MHCIIC (Golomb et al., 2004). Whereas MHCIIA and MHCIIB share a high degree of homology in the amino-acid sequence (Takahashi et al., 1992), their catalytic activity and intracellular distribution differ (Kawamoto and Adelstein, 1991; Kelley et al., 1996; Lo et al., 2004). This suggests that, although some of the activities of the isoforms overlap (Wylie and Chantler, 2001), intrinsic differences also occur (Kolega, 2003; Lo et al., 2004; Straussman et al., 2001; Swailes et al., 2006; Togo and Steinhardt, 2004; Vicente-Manzanares et al., 2007; Wei and Adelstein, 2000; Wilson et al., 2001; Wylie and Chantler, 2003). In addition, differential phosphorylation of MHCIIs has been shown to be implicated in regulating myosin fiber assembly (Redowicz, 2001). Numerous studies have focussed on the role of MHCII isoforms in cell locomotion and cytokinesis but few have attempted to elucidate the role of the isoforms in apoptosis. MHCIIA has been reported to be a caspase 3 target during apoptosis (Gerner et al., 2000; Kato et al., 2005; Suarez-Huerta et al., 2000a).
Our study addresses the contribution of MHCII isoforms A and B in actin cytoskeleton remodelling during TNF-induced apoptosis. The study used the folliculostellate (FS) cell line TtT/GF, a stable mouse cell line characterised by morphological and biochemical features of normal FS cells (Allaerts and Vankelecom, 2005; Inoue et al., 1992). TtT/GF cells express the TNFR1 receptor (Kobayashi et al., 1997) and are responsive to TNF (Fortin et al., 2006; Kobayashi et al., 1997; Meilleur et al., 2007). Here, we report a distinctive participation of MHCIIA and MHCIIB in the execution phase of apoptosis. MHCIIB, together with the phosphorylated MLCs, is required for the formation of a functional contractile cytoskeleton that is needed for shrinkage and detachment of apoptotic cells. In addition, our results show that PKC facilitates this process.
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treatment induces apoptosis of TtT/GF cells). Treatment of TtT/GF cells with TNF for increasing periods of time (0-96 hours) induced the cells to acquire a rounded shape and to detach from the culture support (Fig. 1A, TNF). Detached cells showed abundant membrane blebbing (Fig. 1A, TNF, inset). These morphological responses of TtT/GF cells to TNF were similar to those seen in cells undergoing apoptosis (Earnshaw, 1995; Mills et al., 1999). To confirm or infirm whether long-term TNF treatment alone caused apoptosis of TtT/GF cells, we searched for the presence of distinct apoptotic hallmarks.
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. Caspase 8 activation was observed as early as 48 hours after addition of TNF- (Fig. 1B), whereas caspase 3 activation was observed from 72 hours onwards (Fig. 1C). Caspase 3 cleavage was blocked by the wide-range caspase inhibitor Z-VAD-fmk (Fig. 1D). Interestingly, the caspase 3 fragment corresponding to the active enzyme was recovered in the cytoskeleton-enriched-fraction of TNF-treated cells (Fig. 1D), suggesting a close interaction between cytoskeletal proteins and activated caspase 3 during TNF treatment.
Second, three different methods were used to quantify and determine whether TNF-treated TtT/GF cells undergo apoptosis: (1) detection and counting of cell nuclei containing condensed chromatin using an assay in which healthy cells show low levels of blue fluorescence, apoptotic cells show high levels of blue fluorescence and necrotic cells show a combination of blue and red fluorescence; (2) measurement of cytoplasmic nucleosome release induced by endonuclease activation using a photometric enzyme-immunoassay that detects cytoplasmic histone-associated-DNA fragments (mono and oligonucleosomes) and; (3) counting of detached cells. Our time-course studies showed that there was an increasing trend in the number of TNF-treated cells displaying these apoptotic markers from 48 hours onwards (Fig. 1E). TNF-induced TtT/GF cell contraction and detachment (Fig. 1F), and nucleosome release (Fig. 1G) were blocked by the caspase inhibitor Z-VAD-fmk.
Third, in accordance with the orderly dismantling of the apoptotic cells due to the proteolysis of key structural proteins, exposure of TtT/GF cells to TNF for more than 48 hours caused the downregulation of the focal-adhesion-associated protein -actinin and the cleavage of the actin-anchoring protein spectrin into two fragments (Fig. 1H). The 150 kDa spectrin fragment corresponds to the calpain-catalysed cleavage of spectrin, whereas the 120 kDa fragment corresponds to the caspase-3-mediated cleavage of the protein (Boivin et al., 1990; Jänicke et al., 1998). The TNF-induced proteolysis of these actin-binding proteins was coincident with the activation of caspase 8 and caspase 3, and was blocked by Z-VAD-fmk (not shown).
Fourth, changes in the phosphorylation state of MLC during apoptosis are required for cell retraction, membrane blebbing, nuclear disintegration and the formation of apoptotic bodies (Croft et al., 2005; Mills et al., 1998). We found that MLC phosphorylated at serine residue 19 (MLC-Ser19-P) was mainly associated with the cytoskeleton-enriched fraction of control and TNF-treated cells. Long-term exposure of TtT/GF cells to TNF increased levels of MLC-Ser19-P, which were reduced by caspase inhibition (Fig. 2A). Most MLC was recovered in the cytoskeleton fraction of control and TNF-treated cells (Fig. 2A). The cytoskeleton-associated MLC levels slightly increased after TNF treatment and this was independent of caspase activation (Fig. 2A). By contrast, the TNF-induced MLC decreased in the non-cytoskeleton-enriched fraction was partially blocked by Z-VAD-fmk (Fig. 2A). Long-term incubation with TNF led to ROCK1 translocation to the cytoskeleton and to its cleavage into a 122 kDa fragment that was also recovered in the cytoskeleton fraction (Fig. 2B). ROCK1 cleavage, but not its translocation to the cytoskeleton, was blocked by caspase inhibition (Fig. 2B). ROCK2 was not cleaved by TNF treatment (data not shown). The ROCK inhibitor Y-27632 blocked TNF-induced MLC-Ser19-P increase (not shown). ROCK inhibition induced broad lamellipodia and thin cytoplasmic extensions in untreated cells, and blocked TNF-induced cell contraction and detachment (Fig. 2C). Together, the results prove that long-term TNF treatment induced the apoptosis of TtT/GF cells. Moreover, the findings demonstrate that there is a time-related activation of caspase 3 and cytoskeleton remodelling in TNF-induced TtT/GF cell apoptosis.
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-induced TtT/GF cell apoptosis. TtT/GF cells were incubated 96 hours with either with culture medium only or with culture medium containing TNF. Next, MHCIIA and MHCIIB expression was analysed by western blotting in the non-cytoskeleton (N)- and cytoskeleton (C)-enriched subcellular fractions. MHCIIA levels were reduced in the non-cytoskeleton fraction in TNF-treated cells and the levels recovered after blocking caspase activity (Fig. 3A, N-labelled lanes in MHCIIA blot). MHCIIA content in the cytoskeleton-enriched fraction was decreased separately by Z-VAD-fmk and TNF when compared with control levels (Fig. 3A, C-labelled lanes in MHCIIA blot). Treatment of TtT/GF cells with TNF in combination with Z-VAD-fmk increased MHCIIA levels in the cytoskeleton fractions with respect to those measured in cells treated with TNF alone. However, MHCIIA levels were lower than those found in Z-VAD-fmk-treated or control cells (Fig. 3A, C-labelled lanes in MHCIIA blot). Two different polyclonal MHCIIA antibodies yielded the same result. No MHCIIA fragments were detected by these antibodies in any of the subcellular factions. The effect of TNF on MHCIIB was very different. Cytoskeleton-associated MHCIIB was not affected by TNF treatment, whereas non-cytoskeleton-MHCIIB was slightly reduced by the treatment and this reduction was abolished by the caspase inhibitor (Fig. 3A, MHCIIB). Similar results were obtained with three different antibodies against MHCIIB.
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These results demonstrate that TNF alone is capable of inducing TtT/GF cell apoptosis when the cells are incubated for long periods of time. Probably, the delay in inducing apoptosis is because TNF elicits both anti-apoptotic and pro-apoptotic signals, but with time the apoptotic signals prevail in TtT/GF cells. To block the TNF-induced anti-apoptotic signals, we inhibited the survival pathways using the eukaryotic protein synthesis inhibitor cycloheximide (CHX, 5 µg/ml). In addition, to assess whether the different behaviour of the two myosin II isoforms also takes place in other cells, we also used NIH 3T3 fibroblasts. A 24 hours-incubation with either TNF or CHX did not affect MHCIIA levels; however, TNF together with CHX reduced MHCIIA levels which were restored by Z-VADfmk in both cell types (Fig. 3B). On the contrary, MHCIIB levels in TtT/GF cells were not affected by neither TNF nor CHX nor a combination of the compounds (Fig. 3B). TNF alone did not affect MHCIIB but together with CHX slightly decreased the levels of the protein in NIH 3T3 fibroblasts (Fig. 3B).
To characterise the differential stability of MHCII isoforms A and B, TtT/GF cells were incubated with the protein synthesis inhibitor CHX (50 µg/ml; Fig. 3C) or with the transcription inhibitor actinomycin D (ActD, 1 µg/ml; Fig. 3D) for increasing periods of time. The expression levels of each myosin II isoform was assessed by western blotting. The MHCIIA protein and mRNA showed a faster turnover than those of MHCIIB.
MHCIIB–/– embryonic fibroblasts
Our results show that MHCIIB was less affected than MHCIIA during cytoskeleton reorganisation in cells undergoing apoptosis. Perhaps, MHCIIB is the isoform that remains longer in the cells, to form the active actomyosin fibers that mediate some morphological responses during the execution phase of the apoptotic process. This was investigated by using a stable mouse embryonic cell line lacking MHCIIB (MHCIIB–/– fibroblasts) with the corresponding wild-type cells (Tullio et al., 1997). The MHCIIB–/– fibroblasts express neither MHCIIB nor MHCIIC but only MHCIIA (Lo et al., 2004), which is therefore the only MHCII isoform to interact with MLC and to be incorporated in the actomyosin contractile fibers in these cells.
The wild-type and MHCIIB–/– fibroblasts were treated with culture medium alone, or with culture medium containing either TNF or TNF plus CHX for 24 hours. Short-term incubation with TNF did not affect MHCIIA levels in either wild-type or MHCIIB–/– fibroblasts (Fig. 4A,B). However, TNF together with CHX decreased MHCIIA levels in both cell lines (Fig. 4A,B). The effect was blocked by the caspase inhibitor Z-VAD but not by ROCK inhibitor Y-27632 (Fig. 4A,B). MHCIIB levels were not affected by any of those treatments in the wild-type fibroblasts (Fig. 4A). Spectrin cleavage was similar in both wild-type and MHCIIB–/– embryonic fibroblasts (Fig. 4A), indicating that other members of the actin cytoskeleton were similarly affected in wild-type and MHCIIB–/– cells. Indeed, caspase 3 was processed similarly in wild-type and MHCIIB–/– fibroblasts (Fig. 4A). Furthermore, a time-course study revealed that wild-type and MHCIIB–/– embryonic fibroblasts responded to TNF+CHX treatment by activating caspase 3 and by degrading MHCIIA with similar kinetics (Fig. 5A). In addition, there was no difference in the percentage of active caspase-3-positive cells during TNF+CHX treatment in both cell lines, indicating that the absence of MHCIIB did not affect the rate of cell death (Fig. 5B).
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+CHX retracted and detached (Fig. 6A). By contrast, most MHCIIB–/– fibroblasts subjected to the same treatment remained flat and adherent (Fig. 6A). Quantification of cell attachment demonstrated a tenfold increase in the number of attached cells in the MHCIIB–/– cell line with respect to wild-type fibroblasts after TNF+CHX treatment (Fig. 6B). The percentage of apoptotic cells, i.e. cells labelled with active caspase 3 that remained flat and adherent during TNF+CHX treatment, was also higher within the MHCIIB–/– than within the wild-type fibroblasts from 16 hours onwards (Fig. 6C). Since we have shown that the percentage of apoptotic cells for each time point is similar in both cell lines (Fig. 5B), this result indicates that there is an accumulation of flat cells within the apoptotic MHCIIB–/– cell population.
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We further characterised this differential behaviour by double labelling the TtT/GF cells and the wild-type and MHCIIB–/– embryonic fibroblasts with Rhodamine-phalloidin to exhibit the filamentous actin (F-actin) distribution, and with antibody against active caspase 3 to label the apoptotic cells (Fig. 7). All control cells were flat and showed plasma membrane projections, and cortical and cytoplasmic F-actin (Fig. 7, control, F-actin). As expected, the control cells displayed a low level of caspase 3 activation (Fig. 7, control, active caspase 3). Following TNF+CHX treatment for 16 hours, most TtT/GF cells and wild-type fibroblasts that remained adherent and showed labelling for active caspase 3 were retracted (Fig. 7, TNF+CHX-treated TtT/GF and wild-type cells). Note that caspase 3 was not activated in TNF+CHX-treated TtT/GF cells that stayed flat (Fig. 7, TNF+CHX, TtT/GF cells, arrows). However, most TNF+CHX-treated MHCIIB–/– fibroblasts that remained flat and adherent showed actin filaments and activation of caspase 3 (Fig. 7, TNF+CHX, MHCIIB–/– fibroblasts, see also supplementary material Fig. S1). In these cells, active caspase 3 was distributed as cytoplasmic dots (Fig. 7, MHCIIB–/– fibroblasts).
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in TNF-induced morphological responses during apoptosis (Even-Faitelson and Ravid, 2006). PKC is activated by phosphorylation at threonine residue 140 (Thr410) (Smith et al., 2003) and by a caspase-mediated cleavage that releases the C-terminal catalytic domain from the N-terminal autoinhibitory domain (Smith et al., 2000). We investigated whether PKC is involved in MHIIB-mediated cell rounding up and detachment during apoptosis. TNF+CHX treatment of TtT/GF cells caused the cleavage of endogenous PKC with a time course similar to that of caspase 3 cleavage (Fig. 8A). The PKC fragment was recovered in the cytoskeleton-enriched fraction (Fig. 8B). TNF+CHX also induced the translocation of full-length PKC to the cytoskeleton fraction (Fig. 8B). PKC cleavage but not its translocation to the cytoskeleton was blocked by caspase inhibition (Fig. 8B). PKC cleavage following TNF+CHX treatment also took place in NIH 3T3 fibroblasts, wild-type and MHCIIB–/– embryonic fibroblasts although with different kinetics (Fig. 8C).
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PKC and MHCIIB have been demonstrated to physically interact (Even-Faitelson and Ravid, 2006). Here, we show that PKC and MHCIIB co-immunoprecipitated in untreated TtT/GF cells (Fig. 9A,B) as well as in TtT/GF cells treated with TNF+CHX (Fig. 9C). To evaluate the involvement of PKC in TNF+CHX-induced cell retraction during apoptosis, TtT/GF cells and wild-type and MHCIIB–/– embryonic fibroblasts were treated with the specific PKC inhibitor Myr-PKC pseudosubstrate for 16 hours. Inhibition of PKC activity in control cells induced the formation of cytoplasmic projections but the cells remained adherent (Fig. 10). Myr-PKC pseudosubstrate neither induced the apoptosis of any of the cells examined (Fig. 10) nor inhibited TNF-induced cleavage of caspase 3 (supplementary material Fig. S2). However, PKC inactivation reduced TNF+CHX-induced cell retraction in TtT/GF cells and wild-type embryonic fibroblasts (Fig. 10) but was without effect on TNF+CHX-treated MHCIIB–/– cells (Fig. 10).
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has a facilitatory role in this process. Because TNF elicits both apoptotic and antiapoptotic signals (Baud and Karin, 2001), it is not considered a good apoptotic inducer and, therefore, it is used in combination with a protein synthesis inhibitor to selectively trigger the apoptotic pathway. However, we found that long-term TNF treatment induced biochemical and morphological features in TtT/GF cells that are typical to cells undergoing apoptosis (Earnshaw, 1995; Mills et al., 1999). Furthermore, the morphological and biochemical responses observed in cells treated with TNF either alone or in combination with CHX were similar, except that all events occurred faster in the presence of CHX.
The morphological responses taking place during the execution stage of the apoptotic process require the participation of the actin cytoskeleton (Coleman and Olson, 2002). Proteolysis of several actin-binding proteins contributes to cell shrinkage and detachment (Brancolini et al., 1997; Chen et al., 2001; Jänicke et al., 1998; Kook et al., 2003). At the same time, a contractile actomyosin system is necessary for cell contraction, apoptotic body formation and nuclear breakdown (Croft et al., 2005; Lane et al., 2005; Mills et al., 1998). A paradox seems to underlie this experimental evidence: a dismantling yet functional actin cytoskeleton is needed for a successful execution phase.
The involvement of MLC phosphorylation in the regulation of actomyosin contraction during apoptosis is well established; however, the implication of the MHCIIs in the apoptotic process is not. We show here, for the first time, a distinct contribution of MHCII isoforms A and B to the apoptotic process: MHCIIB levels remained unchanged, whereas those of MHCIIA decreased significantly during TNF or TNF+CHX treatment in TtT/GF cells, NIH 3T3 fibroblasts and embryonic fibroblasts. MHCIIA cleavage has already been recorded in TNF+CHX-treated aortic endothelial cells (Suarez-Huerta et al., 2000a) and anti-Fas-treated Jurkat cells (Gerner et al., 2000; Kato et al., 2005). Surprisingly, MHCIIA cleavage products were either not recognised by the antibodies used (Suarez-Huerta et al., 2000a) or their recognition was due to a crossreactivity with another antibody (Kato et al., 2005). The two different MHCIIA antibodies used in the present study also failed to detect any MHCIIA fragment.
Since we found that MHCIIA has a faster turnover than MHCIIB, low MHCIIA levels in TNF+CHX-treated cells may be the result of the rapid disappearance of MHCIIA following protein synthesis inhibition. However, the same downregulation of MHCIIA but not of MHCIIB was obtained in long-term TNF-treated cells in which protein synthesis was not exogenously inhibited. In addition, MHCIIA possesses consensus sequences for caspase 3 proteolysis (Kato et al., 2005); therefore, by default, it is broken down during the execution phase of the apoptotic process. In accordance with this, reduced MHCIIA levels in apoptotic cells were restored to control values by inhibiting caspase activity (Gerner et al., 2000; Suarez-Huerta et al., 2000a) (and this paper). Therefore, the experimental data indicate that the loss of MHCIIA and the conservation of MHCIIB are specific events of the apoptotic process not a consequence of a different stability of the isoforms.
But why then is the isoform A lost but the isoform B retained? On the one hand, experimental evidence has demonstrated the need of actin contractile fibers for cell contraction and detachment during apoptosis. Our results suggest that there is a specific dismantling of actomyosin fibers made of MHCIIA during apoptosis. The observation of MHCIIA in late blebs together with the phosphorylated MLCs (Lane et al., 2005) agrees with this idea. On the other hand, MHCIIB and actin were virtually not affected by TNF. MHCIIB was reported to bind to actin more slowly and weakly than MHCIIA (Bresnick, 1999; Kolega, 2003; Kolega, 2006). Thus, MHCIIA proteolysis could facilitate the access of MHCIIB molecules to the actin fibers due to a lack of competition with MHCIIA. In support of this view is the report that MHCIIB but not MHCIIC is assembled into cytoplasmic stress fibers in the absence of MHCIIA (Bao et al., 2005). Therefore, MHCIIB may be the main contributor to actomyosin fibers in apoptotic cells.
MHCIIA is responsible for the forward movement of the cell body during locomotion, whereas MHCIIB is responsible for cell retraction and remains associated with stable stress fibers (Kolega, 2003). We have shown that increased association of myosin II with cortical actin participates in the rounding-up of cells (Vitale and Carbajal, 2004). Disposal of MHCIIA and maintenance of MHCIIB in the apoptotic cells would shift the locomotion and contraction balance towards cell contraction. Our observation of a larger number of flat and adherent apoptotic MHCIIB–/– fibroblasts than apoptotic wild-type fibroblasts during TNF treatment, supports the view of a requirement of MHCIIB for actomyosin fiber assembly necessary for cell contraction during apoptosis. The fact that COS-7 cells, which lack MHCIIA, contract and detach during apoptosis adds supports to the notion (Orlando et al., 2006). However, the removal of MHCIIA and, therefore, the disassembly of actin-MHCIIA fibers probably contribute to the dismantling of focal adhesions that is also required for detachment. Instability of MHCIIA affects focal adhesions (Dulyaninova et al., 2007).
The contribution of MHCII phosphorylation in the stability of myosin fibers has become increasingly manifest (Even-Faitelson and Ravid, 2006; Murakami et al., 2000; Straussman et al., 2001). Once again, the kinetics of the phosphorylation and the kinases involved differ for the A and B isoforms (Dulyaninova et al., 2007; Kolega, 1999; Redowicz, 2001). Among the several kinases that phosphorylate the MHCIIs, the atypical PKC isoform is of particular significance because, on the one hand, this kinase interacts and phosphorylates MHCIIB not MHCIIA (Even-Faitelson and Ravid, 2006) and, on the other hand, PKC is itself a target of caspase 3 (Smith et al., 2003). PKC activity is stimulated by phosphorylation in Thr 410, and this phosphorylation normally occurs in serum cultured cells (Smith et al., 2003). However, full activation of the enzyme is achieved by the caspase-mediated cleavage that releases the C-terminal catalytic domain from the N-terminal autoinhibitory domain (Smith et al., 2000). Under our experimental conditions, only a small proportion of total PKC was found cleaved by TNF+CHX treatment. Nonetheless, because cleaved PKC has a shorter half-life than that of the full-length kinase (Smith et al., 2000; Smith et al., 2003), once the fragment is formed it is expected to be quickly degraded, hence reducing the intensity of the immunoreactive band.
Our findings that, (1) PKC and MHCIIB physically interacted not only in control cells but in TNF-treated cells as well, (2) TNF induced the translocation of PKC to the cytoskeleton and, (3) cleaved PKC was associated with the cytoskeleton fraction, provide strong support to the idea that MHCIIB is a target of PKC during TNF-induced apoptosis. Specific inhibition of PKC did not induce cell death and did not affect TNF-induced caspase 3 processing. However, PKC inactivation blocked TNF-induced retraction and detachment of TtT/GF cells and of wild-type embryonic fibroblasts, indicating that the activity of the kinase is necessary for actin cytoskeleton remodelling in apoptotic cells. The fact that TNF-induced morphological responses were less affected by PKC inhibition in MHCIIB–/– fibroblasts than in the cells expressing MHCIIB suggests that PKC-MHCIIB interaction plays an important role in TNF-induced cell contraction during apoptosis.
Besides MHCIIB contribution, our results confirm the implication of ROCK1-mediated MLC phosphorylation in cell contraction and detachment during the execution phase of apoptosis. Moreover, our findings extended previous observations by showing that cleaved, activated ROCK1 and phosphorylated MLC were both specifically associated with the cytoskeleton fraction. The results highlight the fact that TNF induced the translocation of some cytoskeleton-related proteins (such as MLC, ROCK1 and PKC) from the non-cytoskeleton to the cytoskeleton fraction, and that this effect was independent of caspase activation.
Together, our results indicate that, during the execution phase of apoptosis, there is an increased detachment of actin fibers to the cell periphery (downregulation or cleavage of MHCIIA, -actinin and spectrin) accompanied by a reinforcement of the actin-MHCIIB contractile fibers (increased cytoskeleton association or activation of ROCK1, phosphorylated MLC, MHCIIB and actin). These results may explain the apparent paradox of a dismantling yet functional actin cytoskeleton.
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Source of antibodies and chemicals
Several antibodies against MHC were used: rabbit polyclonal anti-MHCIIA antibody raised against a synthetic peptide corresponding to amino acids 1950-1961 of human nonmuscle MHCIIA and rabbit polyclonal anti-MHCIIB antibody raised against synthetic peptide corresponding to amino acid residues 1965-1976 of human nonmuscle MHCIIB were obtained from Sigma Chemical Co. (St Louis, MO). Rabbit polyclonal anti-MHCIIA antibody raised against a peptide with the sequence GKADGAEAKPAE corresponding to the C-terminus of human nonmuscle MHCIIA and rabbit polyclonal anti-MHCIIB antibody generated against a peptide with the sequence SDVNETQPPQSE corresponding to the C-terminus of nonmuscle MHCIIB were purchased from Convance (Berkeley, CA). Mouse monoclonal nonmuscle anti-chicken MHCIIB antibody (CMII 23) was developed by G. W. Conrad and A. H Conrad (Division of Biology, Kansas State University, Manhattan, KS) and obtained from the Developmental Studies Hybridoma Bank University of Iowa, Dept Biological Sciences developed under the auspices of the NICHD. Rabbit polyclonal antibody against activated caspase 3, raised against a synthetic peptide corresponding to the cleavage site of human caspase 3 and that only recognises the 17 kDa fragment of active caspase 3, as well as mouse monoclonal anti-MLC, rabbit polyclonal anti-actin, horseradish peroxidase (HRP)-conjugated anti-mouse IgM antibodies, proteinA Sepharose-4B, cycloheximide and actinomycin were purchased from Sigma Chemical Co. HRP-conjugated anti-mouse IgG, HRP-conjugated anti-goat IgG and HRP-conjugated anti-rabbit IgG antibodies were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). Mouse monoclonal anti-ROCK1 antibody was purchased from BD Biosciences (Mississauga, ON, Canada). Mouse monoclonal antibodies against -actinin, non-erythroid -spectrin and goat polyclonal anti-vimentin antibody were from Chemicon International (Temecula, CA). The rabbit anti-caspase-8 p20 (H-134) antibody, that detects both the precursor and the activated fragment, rabbit anti-PKC (C-20) and goat anti-PKC (N-17) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Cleaved caspase 3 (Asp175) (5A1) rabbit monoclonal antibody from Cell Signaling Technology (Danvers, MA) detects endogenous levels of the large fragment (17/19 kDa) of activated caspase 3 resulting from cleavage adjacent to Asp175. This antibody does not recognise full-length caspase 3 or other cleaved caspases. It was raised against a synthetic peptide corresponding to N-terminal residues adjacent to Asp175 of human caspase-3. Antibody against MLC phosphorylated at serine residue 19 was also purchased from Cell Signaling Technology. The vybrantTM apoptosis assay kit no. 5 and Rhodamine-phalloidin were purchased from Molecular Probes (Eugene, OR). Human recombinant tumour TNF, the Cell Death Detection ELISAplus and the chemiluminescence substrate Lumilight were purchased from Roche (Laval, QC, Canada). The ROCK inhibitor Y27632 and the caspase inhibitor Z-VAD-fmk were purchased from Calbiochem (San Diego, CA). The specific PKC inhibitor, Myr-PKC pseudosubstrate was from BioSource (Camarillo, CA). Proteins in the samples were measured by the Bradford dye binding assay with materials provided by Bio Rad (Mississauga, ON, Canada).
Morphological analysis
Cells were treated with different compounds according to the specific protocol. The morphology of adherent control cells and treated cells was evaluated with an inverted microscope (Leica DMRIB, Wetzlar, Germany); the images were acquired with a Retiga Ex-I camera (Q-Imaging, Burnaby, Canada) and treated with the Northern Eclipse v6 program (Empix Imaging, Mississauga, Canada). Morphological analysis of detached cells, particularly the presence of membrane blebbing, was carried out with a Zeiss Axio-Imager Z1 microscope (Zeiss, Jena, Germany). Pictures were taken with a Zeiss HrM camera (Zeiss) and processed with the Axio-Vision v 4.5 program (Zeiss).
Fluorescence microscopy
Cells were grown on glass coverslips and incubated with different compounds according to the specific protocol. Next, cells were fixed with 3.7% formaldehyde and permeabilised with 0.5% Triton X-100 in PBS (137 mM NaCl, 3 mM KCl, 8 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.4). The cells were blocked with 5% skim milk in PBS. Cells were double labelled with Rhodamine-phalloidin (1:900 dilution) to show F-actin distribution and with antibody against active caspase 3 that only recognises the 17 kDa fragment active caspase 3 to mark apoptotic cells. Next, cells were incubated with anti-rabbit-IgG-FITC. Coverslips were mounted in PBS-glycerol (1:1) containing 5% 1,4 diazabiscyclo (2,2,2) octane (DABCO). Cells were viewed with a Carl Zeiss Axiophot II fluorescence microscope (Zeiss). Pictured were acquired using Northern Eclipse program.
Quantification of apoptosis
Chromatin condensation
We used the vibrant apoptosis assay kit no. 5, which detects the compacted state of the chromatin in apoptotic cells using Hoechst 33342 and propidium iodide. Hoechst 33342 is a DNA stain that possesses a blue fluorescence and is particularly bright in the condensed nuclei of apoptotic cells. Propidium iodide which is a DNA stain presenting a red fluorescence, only enters cells with compromised plasma membranes. Briefly, following treatments, cells were rinsed in PBS and incubated with a mixture of the dyes. Next, the cells were washed and fixed in 3.7% formaldehyde and observed with a Carl Zeiss Axiophot II fluorescence microscope. Nuclei of viable cells show a less bright blue fluorescence, apoptotic cells show a very bright blue fluorescence and necrotic cells show both blue and red fluorescence. The same procedure was applied to detached cells harvested in the culture medium. We did not observe necrotic cells under our experimental conditions.
Cytoplasmic nucleosome release
The Cell Death Detection ELISAplus, a photometric enzyme-immunoassay that detects cytoplasmic histone-associated-DNA fragments (mono- and oligonucleosomes) was used. In short, after treatments, the cells were scraped off and recovered by centrifugation. The pellet was incubated with the lysis buffer. Twenty microlitres of cell lysate were transferred into streptavidin-coated microplates, and incubated with a mixture of anti-histone-biotin and anti-DNA-peroxidase (POD) antibodies for 2 hours. After the removal of unbound antibodies, nucleosomes were photometrically detected by measuring POD activity with ABTS [2,2-azino-di(3-ethyl-benzthiazolin-sulfonat)] as substrate. The absorbance corresponds to the enrichment of mono- and oligonucleosomes released in the cytoplasm in each experimental condition.
Counting of detached cells
Following treatments the detached cells were recovered from the medium surrounding the cultures and cells were counted using a hemocytometer.
Counting of active caspase3-positive cells
Cells cultured on glass coverslips were subjected to TNF+CHX treatment for increasing periods of time. At the end of each incubation period the cells were processed for immunofluorescence microscopy with active caspase 3 antibody and Rhodamine-phalloidin. Cells in the preparations were visualised under a fluorescence microscope and the number of (1) total, (2) active-caspase-3-positive and, (3) flat and active-caspase-3-positive cells was recorded. More than 300 cells were counted for each experimental condition.
Preparation of subcellular fractions
Following treatments, the cells were rinsed with cold PBS, scraped off and collected by centrifugation at 100 g (Beckman GS-6R, Beckman Canada, Mississauga, ON, Canada). Cells were resuspended and sonicated in PBS containing phosphatase and protease inhibitors {1 mM EDTA, 1 mM PMSF, 2 µg/ml leupeptin, 2 µg/ml aprotinin, 4 mM Na3VO4, 80 mM NaF, 20 mM Na4P2O7 and 10 µM bisperoxovanadium 1,10-phenanthroline [bpV(phen)]}. The total cell homogenate was centrifuged to remove the nuclei and cell debris. After centrifugation, the supernatant was incubated with 1% Triton X-100 (final concentration) in PBS that contained phosphatase and protease inhibitors for 30 minutes at room temperature, followed by a centrifugation at 15,000 g (Beckman microfuge E, Beckman Canada, Mississauga, ON, Canada). The pellet was considered the cytoskeleton-enriched fraction and the supernatant the non-cytoskeleton-enriched fraction. The cytoskeleton fractions were dissolved in RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% DOC, 0.1% SDS, 50 mM Tris pH 8). The characterisation of subcellular fractions had been be done and described before (Zheng et al., 2005).
Electrophoresis and immunoblotting
Fifteen micrograms of sample proteins were loaded onto 8-12% polyacrylamide gels. After electrophoresis, the proteins were transferred onto nitrocellulose membranes. Membranes were quickly stained with Ponceau Red to ensure equal loading, rinsed and blocked in PBS containing 5% milk and 0.1% Tween for 1 hour at room temperature. Membranes were incubated with the first antibody in PBS with 5% milk and 0.1% Tween, followed by incubation with the corresponding secondary antibody coupled to HRP. The antigen-antibody reactions were revealed by chemiluminescence. Bands on the films were scanned and the intensity of the bands was quantified by using the Scion Image Program (Scion Corporation, MD).
Co-immunoprecipitation studies
Co-immunoprecipitation studies were performed essentially as described before (Meilleur et al., 2007). Briefly, following treatments, cells were washed with cold PBS, scraped and collected by centrifugation at 100 g (Beckman GS-6R). Cells were incubated with lysis buffer [150 mM NaCl, 20 mM Tris-HCl pH 7.4, 1% NP-40, 100 µM PMSF, 5 mg/ml leupeptin, 5 µg/ml aprotinin, 10 µM bpV(pfen)] for 1 hour at 4°C. The cell lysate was centrifuged for 15 minutes at 15,000 g, and 1 ml of supernatant was incubated with 100 µl of proteinA-Sepharose bead slurry (50%) at 4°C for 10 minutes on a rocker to pre-clear the cell lysate. ProteinA-Sepharose beads were removed by centrifugation at 15,000 g at 4°C for 10 minutes. Protein concentration in the pre-cleared lysates was determined by the method of Bradford. Pre-cleared supernatants (1 mg protein) were incubated with 100 µl of proteinA-Sepharose beads pre-incubated with either goat or rabbit anti-PKC antibody or anti-MHCIIB at 4°C overnight on a rocker. Beads were rinsed several times in lysis buffer, and were finally resuspended in 60 µl electrophoresis sample buffer (2x) and boiled for 5 minutes. After centrifugation at 15,000 g at 4°C, the supernatant was subjected to SDS-PAGE. After electrophoresis and transfer, the membrane was incubated with anti-MHCIIB or anti-PKC antibodies. Controls for the immunoprecipitation studies included incubation of the protein lysate with ProteinA-Sepharose4B beads not pre-incubated with the PKC or MHCIIB antibodies, the use of two different PKC antibodies to co-immunoprecipitate MHCIIB and the use of an antibody that did not pull down PKC or MHCIIB, such as anti-vimentin.
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P<0.001, 72 hours vs 48 hours), nucleosome release (*P<0.0001, 48 hours vs 24 hours) and cell detachment (**P<0.0002, 48 hours vs 24 hours) in TNF
