The small heat shock protein alpha B-crystallin is a novel inhibitor of TRAIL-induced apoptosis that suppresses the activation of caspase-3

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor alpha family of cytokines that preferentially induces apoptosis in transformed cells, making it a promising cancer therapy. However, many neoplasms are resistant to TRAIL-induced apoptosis by mechanisms that are poorly understood. We demonstrate that the expression of the small heat shock protein alpha B-crystallin (but not other heat shock proteins or apoptosis-regulating proteins) correlates with TRAIL resistance in a panel of human cancer cell lines. Stable expression of wild-type alpha B-crystallin, but not a pseudophosphorylation mutant impaired in its assembly and chaperone function, protects cancer cells from TRAIL-induced caspase-3 activation and apoptosis in vitro. Furthermore, selective inhibition of alpha B-crystallin expression by RNA interference sensitizes cancer cells to TRAIL. In addition, wild-type alpha B-crystallin promotes xenograft tumor growth and inhibits TRAIL-induced apoptosis in vivo in nude mice, whereas a pseudophosphorylation alpha B-crystallin mutant impaired in its anti-apoptotic function inhibits xenograft tumor growth. Collectively, these findings indicate that alpha B-crystallin is a novel regulator of TRAIL-induced apoptosis and tumor growth. Moreover, these results demonstrate that targeted inhibition of alpha B-crystallin promotes TRAIL-induced apoptosis, thereby suggesting a novel strategy to overcome TRAIL resistance in cancer.     

αB-crystallin, a small heat shock protein, modulates NF-κB activity in a phosphorylation-dependent manner and protects muscle myoblasts from TNF-α induced cytotoxicity

αB-crystallin, a member of the small heat shock protein family, has been implicated in various biological functions including response to heat shock, differentiation and apoptosis, the mechanisms of which have not been well understood. Myoblasts, the precursor cells in muscle regeneration, when subjected to growth factor deprivation differentiate to form myotubes or undergo apoptosis. During differentiation, myoblasts express elevated levels of αB-crystallin as well as TNF-α but the connecting link between these proteins in cell signaling is not clearly understood. We have therefore investigated the role of αB-crystallin in TNF-α induced regulation of NF-κB.We demonstrate that in response to TNF-α treatment, αB-crystallin associates with IKKβ and activate its kinase activity, facilitating the degradation of phosphorylated I-kBα, a prime step in NF-κB activation. Reducing the level of αB-crystallin using the RNAi approach reduces the translocation of p65, further confirming the role of αB-crystallin in NF-κB activation. Our study shows that the ability of αB-crystallin to activate NF-κB depends on its phosphorylation status. The present study shows that αB-crystallin-dependent NF-κB activation protects myoblasts from TNF-α induced cytoxicity by enhancing the expression of the anti-apoptotic protein, Bcl 2. Thus, our study identifies yet another mechanism by which αB-crystallin exerts its anti-apoptotic activity.     

BAG3 directly interacts with mutated alphaB-crystallin to suppress its aggregation and toxicity

A homozygous disruption or genetic mutation of the bag3 gene causes progressive myofibrillar myopathy in mouse and human skeletal and cardiac muscle disorder while mutations in the small heat shock protein αB-crystallin gene (CRYAB) are reported to be responsible for myofibrillar myopathy. Here, we demonstrate that BAG3 directly binds to wild-type αB-crystallin and the αB-crystallin mutant R120G, via the intermediate domain of BAG3. Peptides that inhibit this interaction in an in vitro binding assay indicate that two conserved Ile-Pro-Val regions of BAG3 are involved in the interaction with αB-crystallin, which is similar to results showing BAG3 binding to HspB8 and HspB6. BAG3 overexpression increased αB-crystallin R120G solubility and inhibited its intracellular aggregation in HEK293 cells. BAG3 suppressed cell death induced by αB-crystallin R120G overexpression in differentiating C2C12 mouse myoblast cells. Our findings indicate a novel function for BAG3 in inhibiting protein aggregation caused by the genetic mutation of CRYAB responsible for human myofibrillar myopathy.     

N-cadherin activation substitutes for the cell contact control in cell cycle arrest and myogenic differentiation: involvement of p120 and beta-catenin

N-cadherin is expressed throughout skeletal myogenesis and has been proposed to be involved in the differentiation program of myogenic precursors. Here, we further characterize the N-cadherin involvement and its mechanism of action at the onset of differentiation, through controlled N-cadherin activation by plating isolated C2 myoblasts on surfaces coated with a chimeric Ncad-Fc homophilic ligand (N-cadherin ectodomain fused to the immunoglobulin G Fc fragment). We show that N-cadherin activation substitutes for the cell density in myogenic differentiation by promoting myogenin and troponin T expression. In addition, N-cadherin adhesion participates to the associated cell cycle arrest through the nuclear accumulation of cyclin-dependent kinase inhibitors p21 and p27. Mouse primary myoblast cultures exhibited similar responses to N-cadherin as C2 cells. RNA interference knockdowns of the N-cadherin-associated cytoplasmic proteins p120 and beta-catenin produced opposite effects on the differentiation pathway. p120 silencing resulted in a decreased myogenic differentiation, associated with a reduction in cadherin-catenin content, which may explain its action on myogenic differentiation. beta-Catenin silencing led to a stimulatory effect on myogenin expression, without any effect on cell cycle. Our results demonstrate that N-cadherin adhesion may account for cell-cell contact-dependent cell cycle arrest and differentiation of myogenic cells, involving regulation through p120 and beta-catenins.     

Tumor necrosis factor-like weak inducer of apoptosis inhibits skeletal myogenesis through sustained activation of nuclear factor-kappaB and degradation of MyoD protein

In this study we have investigated the effect and the mechanisms by which tumor necrosis factor-like weak inducer of apoptosis (TWEAK) modulates myogenic differentiation. Treatment of C2C12 myoblasts with TWEAK inhibited their differentiation evident by a decrease in the expression of creatine kinase, myosin heavy chain-fast twitch, myogenin, and the formation of multinucleated myotubes. TWEAK also inhibited the differentiation of mouse primary myoblasts. Conversely, the proliferation of C2C12 myoblasts and the expression of a cell-cycle regulator cyclin D1 were increased in response to TWEAK treatment. Inhibition of cellular proliferation using hydroxyurea only partially reversed the inhibitory effect of TWEAK on myogenic differentiation. Treatment of C2C12 myoblasts with TWEAK resulted in the activation of nuclear factor-kappaB (NF-kappaB), the (IkappaB) IkappaB kinase (IKK) complex, and the phosphorylation and degradation of IkappaBalpha protein. Inhibition of NF-kappaB activity by overexpression of a dominant negative mutant of IkappaBalpha (IkappaBalphaDeltaN) significantly increased the myogenic differentiation in TWEAK-treated C2C12 cultures. Furthermore, overexpression of a dominant negative mutant of IKKbeta (IKKbetaK44A) but not IKKalpha (IKKalphaK44M) reversed the inhibitory effect of TWEAK on myogenesis. TWEAK inhibited the expression of myogenic regulatory factors MyoD and myogenin and also induced the degradation of MyoD protein. Finally, inhibition of NF-kappaB activation through overexpression of IKKbetaK44A prevented the degradation of MyoD protein. Overall, our data suggest that TWEAK inhibits myogenesis through the activation of NF-kappaB signaling pathway and degradation of MyoD protein.     

Mirk/Dyrk1B mediates survival during the differentiation of C2C12 myoblasts

The kinase Mirk/dyrk1B is essential for the differentiation of C2C12 myoblasts. Mirk reinforces the G0/G1 arrest state in which differentiation occurs by directly phosphorylating and stabilizing p27(Kip1) and destabilizing cyclin D1. We now demonstrate that Mirk is anti-apoptotic in myoblasts. Knockdown of endogenous Mirk by RNA interference activated caspase 3 and decreased myoblast survival by 75%, whereas transient overexpression of Mirk increased cell survival. Mirk exerts its anti-apoptotic effects during muscle differentiation at least in part through effects on the cell cycle inhibitor and pro-survival molecule p21(Cip1). Overexpression and RNA interference experiments demonstrated that Mirk phosphorylates p21 within its nuclear localization domain at Ser-153 causing a portion of the typically nuclear p21 to localize in the cytoplasm. Phosphomimetic GFP-p21-S153D was pancellular in both cycling C2C12 myoblasts and NIH3T3 cells. Endogenous Mirk in myotubes and overexpressed Mirk in NIH3T3 cells were able to cause the pancellular localization of wild-type GFP-p21 but not the nonphosphorylatable mutant GFP-p21-S153A. Translocation to the cytoplasm enables p21 to block apoptosis through inhibitory interaction with pro-apoptotic molecules. Phosphomimetic p21-S153D was more effective than wild-type p21 in blocking the activation of caspase 3. Transient expression of p21-S153D also increased myoblast viability in colony forming assays, whereas the p21-S153A mutant had no effect. This Mirk-dependent change in p21 intracellular localization is a natural part of myoblast differentiation. Endogenous p21 localized exclusively to the nuclei of proliferating myoblasts but was also found in the cytoplasm of post-mitotic multinucleated myotubes and adult human skeletal myofibers.     

Serine 59 Phosphorylation of αB-Crystallin Down-regulates Its Anti-apoptotic Function by Binding and Sequestering Bcl-2 in Breast Cancer Cells

The small heat shock protein (sHSP) αB-crystallin is a new oncoprotein in breast carcinoma that predicts poor clinical outcome in breast cancer. However, although several reports have demonstrated that phosphorylation of sHSPs modify their structural and functional properties, the significance of αB-crystallin phosphorylation in cancer cells has not yet been investigated. In this study, we have characterized the phosphorylation status of αB-crystallin in breast epithelial carcinoma cells line MCF7 submitted to anti-cancer agents like vinblastine. We have showed that the main phosphorylation site of αB-crystallin in response to vinblastine is serine 59 and determined a correlation between this post-translational modification and higher apoptosis level. The overexpression of the serine 59 “pseudophosphorylated” mutant (S59E) induces a significant increase in the apoptosis level of vinblastine-treated MCF7 cells. In contrast, overexpression of wild-type αB-crystallin or “nonphosphorylatable” mutant (S59A) result in a resistance to this microtubule-depolymerizing agent, while inhibition of endogenous levels of αB-crystallin by expression of shRNA lowers it. Analyzing further the molecular mechanism of this phenomenon, we report for the first time that phosphorylated αB-crystallin preferentially interacts with Bcl-2, an anti-apoptotic protein, and this interaction prevents the translocation of Bcl-2 to mitochondria. Hence, this study identifies serine 59 phosphorylation as an important key in the down-regulation of αB-crystallin anti-apoptotic function in breast cancer and suggests new strategies to improve anti-cancer treatments.     

Alpha B-crystallin expression in mouse NIH 3T3 fibroblasts: glucocorticoid responsiveness and involvement in thermal protection.

alpha B-crystallin, a major soluble protein of vertebrate eye lenses, is a small heat shock protein which transiently accumulates in response to heat shock and other kinds of stress in mouse NIH 3T3 fibroblasts. Ectopic expression of an alpha B-crystallin cDNA clone renders NIH 3T3 cells thermoresistant. alpha B-crystallin accumulates in response to the synthetic glucocorticoid hormone dexamethasone. Dexamethasone-treated NIH 3T3 cells become thermoresistant to the same extent as they accumulate alpha B-crystallin. A cell clone in which alpha B-crystallin is superinduced upon heat shock acquires augmented thermotolerance. Expression of the ras oncogene causes a rapid but transient accumulation of alpha B-crystallin within 1 day. Later, sustained ras oncogene expression suppresses the dexamethasone-mediated alpha B-crystallin accumulation. Thus, oncogenic transformation triggered by the ras oncogene interferes with hormone-mediated accumulation of alpha B-crystallin and concomitant acquisition of thermoresistance. Other known heat shock proteins do not accumulate in response to ectopic alpha B-crystallin expression or to dexamethasone treatment. These results indicate that alpha B-crystallin can protect NIH 3T3 fibroblasts from thermal shock.     

The roles of integrin-linked kinase in the regulation of myogenic differentiation

Myogenic differentiation is a highly orchestrated, multistep process that is coordinately regulated by growth factors and cell adhesion. We show here that integrin-linked kinase (ILK), an intracellular integrin- and PINCH-binding serine/threonine protein kinase, is an important regulator of myogenic differentiation. ILK is abundantly expressed in C2C12 myoblasts, both before and after induction of terminal myogenic differentiation. However, a noticeable amount of ILK in the Triton X-100-soluble cellular fractions is significantly reduced during terminal myogenic differentiation, suggesting that ILK is involved in cellular control of myogenic differentiation. To further investigate this, we have overexpressed the wild-type and mutant forms of ILK in C2C12 myoblasts. Overexpression of ILK in the myoblasts inhibited the expression of myogenic proteins (myogenin, MyoD, and myosin heavy chain) and the subsequent formation of multinucleated myotubes. Furthermore, mutations that eliminate either the PINCH-binding or the kinase activity of ILK abolished its ability to inhibit myogenic protein expression and allowed myotube formation. Although overexpression of the ILK mutants is permissive for the initiation of terminal myogenic differentiation, the myotubes derived from myoblasts overexpressing the ILK mutants frequently exhibited an abnormal morphology (giant myotubes containing clustered nuclei), suggesting that ILK functions not only in the initial decision making process, but also in later stages (fusion or maintaining myotube integrity) of myogenic differentiation. Additionally, we show that overexpression of ILK, but not that of the PINCH-binding defective or the kinase-deficient ILK mutants, prevents inactivation of MAP kinase, which is obligatory for the initiation of myogenic differentiation. Finally, inhibition of MAP kinase activation reversed the ILK-induced suppression of myogenic protein expression. Thus, ILK likely influences the initial decision making process of myogenic differentiation by regulation of MAP kinase activation.     

Inflammatory cytokines inhibit myogenic differentiation through activation of nuclear factor-kappaB.

Muscle wasting is often associated with chronic inflammation. Because tumor necrosis factor alpha (TNF-alpha) has been implicated as a major mediator of cachexia, its effects on C2C12 myocytes were examined. TNF-alpha activated nuclear factor-kappaB (NF-kappaB) and interfered with the expression of muscle proteins in differentiating myoblasts. Introduction of a mutant form of inhibitory protein kappaBalpha (IkappaBalpha) restored myogenic differentiation in myoblasts treated with TNF-alpha or interleukin 1beta. Conversely, activation of NF-kappaB by overexpression of IkappaB kinase was sufficient to block myogenesis, illustrating the causal link between NF-kappaB activation and inhibition of myogenic differentiation. The inhibitory effects of TNF-alpha on myogenic differentiation were reversible, indicating that the effects of the cytokine were not due to nonspecific toxicity. Treatment of differentiated myotubes with TNF-alpha did not result in a striking loss of muscle-specific proteins, which shows that myogenesis was selectively affected in the myoblast stage by TNF-alpha. An important finding was that NF-kappaB was activated to the same extent in differentiating and differentiated cells, illustrating that once myocytes have differentiated they become refractory to the effects of NF-kappaB activation. These results demonstrate that inflammatory cytokines may contribute to muscle wasting through the inhibition of myogenic differentiation via a NF-kappaB-dependent pathway.     

Mutation of COOH-terminal lysines in overexpressed alpha B-crystallin abrogates ischemic protection in cardiomyocytes

High levels of alpha B-crystallin are present in the cardiomyocyte, yet little is understood about the function and importance of this protein. Like many other small heat shock proteins, alpha B-crystallin forms large oligomeric complexes whose size can be regulated by posttranslational modifications. The size of these complexes can modify the function of the protein. A naturally occurring COOH-terminal mutant has many detrimental effects in the lens of the eye and altered oligomerization. Therefore, we mutated the two COOH-terminal lysines of alpha B-crystallin to glycines (K174/175G) and adenovirally mounted them to transduce cardiomyocytes. We analyzed the effect of this mutation on oligomerization, microtubular stabilization, and ischemic outcome. A nearly 45% downward shift in complex size was observed with the mutant by native PAGE followed by immunoblotting. The overexpressed protein no longer protected the tubulin cytoskeleton against ischemic stress by confocal analysis. The mutant caused a 30% increase in cytosolic enzyme release with ischemia compared with control, whereas a 33% decrease was associated with wild-type alpha B-crystallin overexpression. We conclude that the COOH terminus of alpha B-crystallin is crucial to its proper function.     

αB-Crystallin Phosphorylation: Advances and Problems

The review is dedicated to phosphorylation of αB-crystallin (HspB5), one of ubiquitously expressed small heat shock proteins. We describe the structure and properties of αB-crystallin and protein kinases involved in its phosphorylation in different cells and tissues, advantages and drawbacks of pseudophosphorylation mutants in elucidation of the mechanism of αB-crystallin functioning, effects of phosphorylation on the quaternary structure and intracellular location of αB-crystallin, interactions of αB-crystallin with different elements of the cytoskeleton, and effect of phosphorylation on the chap-erone-like activity of αB-crystallin. We also discuss the validity of experimental data obtained by overexpression of pseudophosphorylation mutants for understanding the effect of phosphorylation on physiologically important properties of αB-crystallin, as well as the question why multiple attempts to phosphorylate αB-crystallin in vitro have been unsuccessful so far.     

Mimicking phosphorylation of the small heat-shock protein alphaB-crystallin recruits the F-box protein FBX4 to nuclear SC35 speckles.

The mammalian small heat shock protein alphaB-crystallin can be phosphorylated at three different sites, Ser19, Ser45 and Ser59. We compared the intracellular distribution of wild-type, nonphosphorylatable and all possible pseudophosphorylation mutants of alphaB-crystallin by immunoblot and immunocytochemical analyses of stable and transiently transfected cells. We observed that pseudophosphorylation at two (especially S19D/S45D) or all three (S19D/S45D/S59D) sites induced the partial translocation of alphaB-crystallin from the detergent-soluble to the detergent-insoluble fraction. Double immunofluorescence studies showed that the pseudophosphorylation mutants localized in nuclear speckles containing the splicing factor SC35. The alphaB-crystallin mutants in these speckles were resistant to mild detergent treatment, and also to DNase I or RNase A digestion, indicating a stable interaction with one or more speckle proteins, not dependent on intact DNA or RNA. We further found that FBX4, an adaptor protein of the ubiquitin-protein isopeptide ligase SKP1/CUL1/F-box known to interact with pseudophosphorylated alphaB-crystallin, was also recruited to SC35 speckles when cotransfected with the pseudophosphorylation mutants. Because SC35 speckles also react with an antibody against alphaB-crystallin endogenously phosphorylated at Ser45, our findings suggest that alphaB-crystallin has a phosphorylation-dependent role in the ubiquitination of a component of SC35 speckles.     

Apoptosis in differentiating C2C12 muscle cells selectively targets Bcl-2-deficient myotubes

Muscle cell apoptosis accompanies normal muscle development and regeneration, as well as degenerative diseases and aging. C2C12 murine myoblast cells represent a common model to study muscle differentiation. Though it was already shown that myogenic differentiation of C2C12 cells is accompanied by enhanced apoptosis in a fraction of cells, either the cell population sensitive to apoptosis or regulatory mechanisms for the apoptotic response are unclear so far. In the current study we characterize apoptotic phenotypes of different types of C2C12 cells at all stages of differentiation, and report here that myotubes of differentiated C2C12 cells with low levels of anti-apoptotic Bcl-2 expression are particularly vulnerable to apoptosis even though they are displaying low levels of pro-apoptotic proteins Bax, Bak and Bad. In contrast, reserve cells exhibit higher levels of Bcl-2 and high resistance to apoptosis. The transfection of proliferating myoblasts with Bcl-2 prior to differentiation did not protect against spontaneous apoptosis accompanying differentiation of C2C12 cells but led to Bcl-2 overexpression in myotubes and to significant protection from apoptotic cell loss caused by exposure to hydrogen peroxide. Overall, our data advocate for a Bcl-2-dependent mechanism of apoptosis in differentiated muscle cells. However, downstream processes for spontaneous and hydrogen peroxide induced apoptosis are not completely similar. Apoptosis in differentiating myoblasts and myotubes is regulated not through interaction of Bcl-2 with pro-apoptotic Bcl-2 family proteins such as Bax, Bak, and Bad.     

p130Cas-dependent actin remodelling regulates myogenic differentiation

Actin dynamics are implicated in various cellular processes, not only through the regulation of cytoskeletal organization, but also via the control of gene expression. In the present study we show that the Src family kinase substrate p130Cas (Cas is Crk-associated substrate) influences actin remodelling and concomitant muscle-specific gene expression, thereby regulating myogenic differentiation. In C2C12 myoblasts, silencing of p130Cas expression by RNA interference impaired F-actin (filamentous actin) formation and nuclear localization of the SRF (serum-response factor) co-activator MAL (megakaryocytic acute leukaemia) following the induction of myogenic differentiation. Consequently, formation of multinucleated myotubes was abolished. Re-introduction of wild-type p130Cas, but not its phosphorylation-defective mutant, into p130Cas-knockdown myoblasts restored F-actin assembly, MAL nuclear localization and myotube formation. Depletion of the adhesion molecule integrin β3, a key regulator of myogenic differentiation as well as actin cytoskeletal organization, attenuated p130Cas phosphorylation and MAL nuclear localization during C2C12 differentiation. Moreover, knockdown of p130Cas led to the activation of the F-actin-severing protein cofilin. The introduction of a dominant-negative mutant of cofilin into p130Cas-knockdown myoblasts restored muscle-specific gene expression and myotube formation. The results of the present study suggest that p130Cas phosphorylation, mediated by integrin β3, facilitates cofilin inactivation and promotes myogenic differentiation through modulating actin cytoskeleton remodelling.