Small Heat Shock Protein alphaB-crystallin Research Articles

Activation of an immune-regulatory macrophage response and inhibition of lung inflammation in a mouse model of COPD using heat-shock protein alpha B-crystallin-loaded PLGA microparticles

As an extracellular protein, the small heat-shock protein alpha B-crystallin (HSPB5) has anti-inflammatory effects in several mouse models of inflammation. Here, we show that these effects are associated with the ability of HSPB5 to activate an immune-regulatory response in macrophages via endosomal/phagosomal CD14 and Toll-like receptors 1 and 2. Humans, however, possess natural antibodies against HSPB5 that block receptor binding. To protect it from these antibodies, we encapsulated HSPB5 in porous PLGA microparticles. We document here size, morphology, protein loading and release characteristics of such microparticles. Apart from effectively protecting HSPB5 from neutralization, PLGA microparticles also strongly promoted macrophage targeting of HSPB via phagocytosis. As a result, HSPB5 in porous PLGA microparticles was more than 100-fold more effective in activating macrophages than free soluble protein. Yet, the immune-regulatory nature of the macrophage response, as documented here by microarray transcript profiling, remained the same. In mice developing cigarette smoke-induced COPD, HSPB5-loaded PLGA microparticles were selectively taken up by alveolar macrophages upon intratracheal administration, and significantly suppressed lung infiltration by lymphocytes and neutrophils. In contrast, 30-fold higher doses of free soluble HSPB5 remained ineffective. Our data indicate that porous HSPB5-PLGA microparticles hold considerable promise as an anti-inflammatory biomaterial for humans.

Solid-state NMR and SAXS studies provide a structural basis for the activation of αB-crystallin oligomers

The small heat shock protein alphaB-crystallin (alphaB) contributes to cellular protection against stress. For decades, high-resolution structural studies on oligomeric alphaB have been confounded by its polydisperse nature. Here, we present a structural basis of oligomer assembly and activation of the chaperone using solid-state NMR and small-angle X-ray scattering (SAXS). The basic building block is a curved dimer, with an angle of approximately 121 degrees between the planes of the beta-sandwich formed by alpha-crystallin domains. The highly conserved IXI motif covers a substrate binding site at pH 7.5. We observe a pH-dependent modulation of the interaction of the IXI motif with beta4 and beta8, consistent with a pH-dependent regulation of the chaperone function. N-terminal region residues Ser59-Trp60-Phe61 are involved in intermolecular interaction with beta3. Intermolecular restraints from NMR and volumetric restraints from SAXS were combined to calculate a model of a 24-subunit alphaB oligomer with tetrahedral symmetry.

Transactivation Response DNA-Binding Protein 43 Microvasculopathy in Frontotemporal Degeneration and Familial Lewy Body Disease

We describe novel transactivation response DNA-binding protein of 43 kd (TDP-43)-positive structures in the brains of patients with frontotemporal lobar degeneration with ubiquitin-positive inclusions and familial Lewy body disease. The TDP-43 immunohistochemistry revealed small round structures closely associated with small blood vessels. By immunoelectron microscopy, these TDP-43-positive structures were unmyelinated cell processes located adjacent to and sometimes enclosed by the capillary basal lamina. Some processes protruded from outside of the vascular basal lamina to a position beneath the basal lamina. The processes contained 10- to 17-nm-diameter straight filaments or filaments coated with granular material similar to those described in neurites in frontotemporal lobar degeneration with ubiquitin-positive inclusions and other disorders. In some of the abnormal structures, electron-dense material formed paracrystalline arrays composed of TDP-43. The inclusions were variably positive by immunostaining for the small heat shock protein alphaB-crystallin and less often glial fibrillary acidic protein. Bundles of astrocytic glial fibrils characteristic of reactive astrocytes were often found in proximity, but glial fibrils were negative for TDP-43. These data suggest that these processes are astrocytic end-feet with abnormal TDP-43 fibrillary inclusions. The significance of this novel TDP-43 microvasculopathy on blood-brain barrier integrity warrants further investigation.

Induction of the small heat shock protein αB-crystallin by genotoxic stress is mediated by p53 and p73

The small heat shock protein alphaB-crystallin is a molecular chaperone that is induced by stress and protects cells by inhibiting protein aggregation and apoptosis. To identify novel transcriptional regulators of the alphaB-crystallin gene, we examined the alphaB-crystallin promoter for conserved transcription factor DNA-binding elements and identified a putative response element for the p53 tumor suppressor protein. Ectopic expression of wild-type p53 induced alphaB-crystallin mRNA and protein with delayed kinetics compared to p21. Additionally, the induction of alphaB-crystallin by genotoxic stress was inhibited by siRNAs targeting p53. Although the p53-dependent transactivation of an alphaB-crystallin promoter luciferase reporter required the putative p53RE, chromatin immunoprecipitation failed to detect p53 binding to the alphaB-crystallin promoter. These results suggested an indirect mechanism of transactivation involving p53 family members p63 or p73. DeltaNp73 was dramatically induced by p53 in a TAp73-dependent manner, and silencing p73 suppressed the transcriptional activation of alphaB-crystallin by p53. Moreover, ectopic expression of DeltaNp73alpha (but not other p73 isoforms) increased alphaB-crystallin mRNA levels in the absence of p53. Collectively, our results link the molecular chaperone alphaB-crystallin to the cellular genotoxic stress response via a novel mechanism of transcriptional regulation by p53 and p73.

Single Molecule Force Spectroscopy of the Cardiac Titin N2B Element: EFFECTS OF THE MOLECULAR CHAPERONE αB-CRYSTALLIN WITH DISEASE-CAUSING MUTATIONS

The small heat shock protein alphaB-crystallin interacts with N2B-Us, a large unique sequence found in the N2B element of cardiac titin. Using single molecule force spectroscopy, we studied the effect of alphaB-crystallin on the N2B-Us and its flanking Ig-like domains. Ig domains from the proximal tandem Ig segment of titin were also studied. The effect of wild type alphaB-crystallin on the single molecule force-extension curve was determined as well as that of mutant alphaB-crystallins harboring the dilated cardiomyopathy missense mutation, R157H, or the desmin-related myopathy mutation, R120G. Results revealed that wild type alphaB-crystallin decreased the persistence length of the N2B-Us (from approximately 0.7 to approximately 0.2 nm) but did not alter its contour length. alphaB-crystallin also increased the unfolding force of the Ig domains that flank the N2B-Us (by 51 +/- 3 piconewtons); the rate constant of unfolding at zero force was estimated to be approximately 17-fold lower in the presence of alphaB-crystallin (1.4 x 10(-4) s(-1) versus 2.4 x 10(-3) s(-1)). We also found that alphaB-crystallin increased the unfolding force of Ig domains from the proximal tandem Ig segment by 28 +/- 6 piconewtons. The effects of alphaB-crystallin were attenuated by the R157H mutation (but were still significant) and were absent when using the R120G mutant. We conclude that alphaB-crystallin protects titin from damage by lowering the persistence length of the N2B-Us and reducing the Ig domain unfolding probability. Our finding that this effect is either attenuated (R157H) or lost (R120G) in disease causing alphaB-crystallin mutations suggests that the interaction between alphaB-crystallin and titin is important for normal heart function.

α-Synuclein Promotes the Recruitment of Tau to Protein Inclusions in Oligodendroglial Cells: Effects of Oxidative and Proteolytic Stress

alpha-Synuclein is the major building block of cytoplasmic inclusions in neurodegenerative disorders named synucleinopathies. These inclusion bodies often contain the small heat shock protein alphaB-crystallin and the microtubule-associated protein tau. Oxidative modification of alpha-synuclein has been linked to fibril formation, and alpha-synuclein aggregation may induce the fibrillization of tau. To study alpha-synuclein aggregate formation, we have engineered oligodendroglial cells (OLN-93 cells) to stably express the longest human isoform of tau and wild-type alpha-synuclein or the A53T alpha-synuclein mutation. Under normal growth conditions, small punctuated alpha-synuclein aggregates were formed, which were more abundant in cells expressing the A53T mutation. After exposure to oxidative stress, protein inclusions were enlarged and were positive for thioflavin S, but the solubility of alpha-synuclein was not altered. Oxidative stress followed by proteasomal inhibition caused the occurrence of larger thioflavin S-positive inclusions, immunoreactive for tau and alphaB-crystallin, thus resembling glial cell inclusion bodies. Furthermore, this double stress situation led to a decrease in alpha-synuclein solubility, and alphaB-crystallin and HSP90 were present in the insoluble fraction. The formation and recruitment of tau to thioflavin S-positive protein aggregates in OLN-93 cells only expressing tau in the absence of alpha-synuclein, either after oxidative or proteasomal stress or both, was not observable. The data indicate that oxidatively modified alpha-synuclein is degraded by the proteasome and that it plays a pro-aggregatory role for tau in this cell culture model system.

Regulation of αB-crystallin gene expression by the transcription factor Ets1 in breast cancer

Recent studies indicate that the small heat shock protein alphaB-crystallin is expressed in poor prognosis basal-like breast tumors and likely contributes to their aggressive phenotype. However, the mechanisms underlying the deregulated expression of alphaB-crystallin in basal-like tumors are poorly understood. Using a bioinformatics approach, we identified a putative DNA binding motif in the human alphaB-crystallin promoter for the proto-oncogene Ets1, a member of the ETS transcription factor family that bind to DNA at palindromic ETS-binding sites (EBS). Here we demonstrate that ectopic expression of Ets1 activates the alphaB-crystallin promoter by an EBS-dependent mechanism and increases alphaB-crystallin protein levels, while silencing Ets1 reduces alphaB-crystallin promoter activity and protein levels. Chromatin immunoprecipitation analyses showed that endogenous Ets1 binds to the alphaB-crystallin promoter in basal-like breast cancer cells in vivo. Interrogation of publically available gene expression data revealed that Ets1 is expressed in human basal-like breast tumors and is associated with poor survival. Collectively, our results point to a previously unrecognized link between the oncogenic transcription factor Ets1 and alphaB-crystallin in basal-like breast cancer.

αB-crystallin: A novel marker of invasive basal-like and metaplastic breast carcinomas

Basal-like tumors are a newly recognized estrogen receptor (ER) negative and HER2 negative breast cancer subtype that express basal epithelial genes and are associated with poor survival. Metaplastic carcinomas are thought to belong within the basal-like group. We have recently demonstrated that the small heat shock protein alphaB-crystallin is commonly expressed in basal-like tumors and contributes to their aggressive phenotype. The current study examined the rates and patterns of alphaB-crystallin expression in whole tissue sections of human breast, including normal tissue, proliferative lesions, in situ and invasive carcinomas (ER positive, HER2 positive, basal-like, and metaplastic cancers). In normal breast tissue, proliferative lesions and in situ carcinomas, alphaB-crystallin expression was restricted to the myoepithelial cell compartment of ductal and lobular units. Most basal-like and metaplastic carcinomas demonstrated cytoplasmic expression of alphaB-crystallin (81% and 86%, respectively). Conversely, no staining for alphaB-crystallin was observed in nonbasal-like (ie, ER positive or HER2 positive) breast carcinomas. Taken together, our results indicate that alphaB-crystallin is a sensitive (81%) and specific (100%) marker for basal-like breast carcinomas. Moreover, the high rates of expression of alphaB-crystallin in metaplastic breast carcinomas (86%) suggest that these tumors may represent a histologically distinctive subset of basal-like breast tumors with a similar underlying molecular etiology.

Region-specific alterations of global protein expression in the remodelled rat myocardium

We applied the novel ProteinChip technology (SELDI-MS) to investigate and identify differentially regulated proteins upon myocardial remodelling in different heart regions. Tissue samples were isolated from the atria, the interventricular septum, and the right and left ventricles three months after surgically-induced myocardial infarction (MI) in rats. Corresponding protein extracts from control versus MI hearts were analysed on two different ProteinChip surfaces. In each of the functionally distinct cardiac regions, we obtained specific protein profile alterations upon myocardial remodelling. Most alterations were observed in the non-infarcted right ventricle, where down-regulation occurred more frequently than up-regulation of protein expression. Three of the differentially regulated proteins were identified: the metabolic enzyme triosephosphate isomerase (TIM), the cell signalling protein Raf-1 kinase inhibitory protein (RKIP), also known as phosphatidylethanolamine binding protein (PEBP), and the small heat shock protein alphaB-crystallin. These proteins showed a pronounced tissue-dependent regulation. TIM was down-regulated only in the atrium and in the left ventricle, RKIP/PEBP was down-regulated only in the right ventricle and in the interventricular septum, and alphaB-crystallin was up-regulated only in the right and in the left ventricle. A simple correlation of peak intensity changes using two of the identified peaks demonstrated the diagnostic potential of SELDI-MS. We conclude that this novel proteomic method is a powerful high-throughput tool for the fast detection of region-specific cardiac protein profiles in small biopsy samples, and that SELDI-MS may become a useful complementary technique for the diagnosis and prognosis of cardiac diseases.

Redistribution of GFAP and αB-crystallin after thermal stress in C6 glioma cell line

Some intermediate filament (IF) proteins expressed in the development of glia include nestin, vimentin, and glial fibrillary acidic protein (GFAP). However, GFAP is the major intermediate filament protein of mature astrocytes. To determine the organization of GFAP in glial cells, rat GFAP cDNA tagged with enhanced green fluorescent protein (EGFP) was transfected into the rat C6 glioma cell line. After selection, two stable C6-EGFP-GFAP cell lines were established. Stable C6-EGFP-GFAP cell lines with or without heat shock treatment were analyzed by immunocytochemistry, electron microscopy, and Western blot analysis. In the transient transfection study, EGFP-GFAP transiently expressed in C6 cells formed punctate aggregations in the cytoplasm right after transfection, but gradually a filamentous structure of EGFP-GFAP was observed. The protein level of nestin in the C6-EGFP-GFAP stable clone was similar to that in the pEGFP-C1 transfected C6 stable clones and non-transfected C6 cells, whereas the level of vimentin was reduced in Western blotting. Interestingly, the expression level of small heat shock protein alphaB-crystallin in C6-EGFP-GFAP cells was also enhanced after transfection. Immunostaining patterns of C6-EGFP-GFAP cells showed that GFAP was dispersed as a fine filamentous structure. However, after heat shock treatment, GFAP formed IF bundles in C6-EGFP-GFAP cells. In the meantime, alphaB-crystallin also colocalized with IF bundles of GFAP in C6-EGFP-GFAP cells. The heat-induced GFAP reorganization we found suggested that small heat shock protein alphaB-crystallin may play a functional role regulating the cytoarchitecture of GFAP.

The effect of alphaB-crystallin and Hsp27 on the availability of translation initiation factors in heat-shocked cells.

The mechanism of the translational thermotolerance provided by the small heat shock proteins (sHsps) alphaB-crystallin or Hsp27 is unknown. We show here that Hsp27, but not alphaB-crystallin, increased the pool of mobile stress granule-associated enhanced green fluorescent protein (EGFP)-eukaryotic translation initiation factor (eIF)4E in heat-shocked cells, as determined by fluorescence recovery after photobleaching. Hsp27 also partially prevented the sharp decrease in the pool of mobile cytoplasmic EGFP-eIF4G. sHsps did not prevent the phosphorylation of eIF2alpha by a heat shock, but promoted dephosphorylation during recovery. Expression of the C-terminal fragment of GADD34, which causes constitutive dephosphorylation of eIF2alpha, fully compensated for the stimulatory effect of alphaB-crystallin on protein synthesis in heat-shocked cells, but only partially for that of Hsp27. Our data show that sHsps do not prevent the inhibition of protein synthesis upon heat shock, but restore translation more rapidly by promoting the dephosphorylation of eIF2alpha and, in the case of Hsp27, the availability of eIF4E and eIF4G.

Differential expression of alphaB-crystallin and Hsp27-1 in anaplastic thyroid carcinomas because of tumor-specific alphaB-crystallin gene (CRYAB) silencing

Expression of the small heat shock protein alphaB-crystallin in differentiated thyroid tumors has been described recently. In this study, we investigated the molecular mechanisms that affect the expression of alphaB-crystallin in benign goiters (n = 7) and highly malignant anaplastic thyroid carcinomas (ATCs) (n = 3). AlphaB-crystallin expression was compared with that of Hsp27-1. Immunoblot and quantitative real-time (RT) polymerase chain reaction revealed marked downregulation of alphaB-crystallin in all the tested ATCs and the ATC-derived cell line C-643 . In contrast, considerable expression of Hsp27-1 in benign and malignant thyroid tissue was demonstrated. Immunofluorescence analysis revealed no relevant topological differences between benign and malignant thyrocytes in the cytoplasmic staining of both proteins. Consistent and marked downregulation of TFCP2L1 was identified as one of the main mechanisms contributing to CRYAB gene silencing in ATCs. In addition, CRYAB gene promoter methylation seems to occur in distinct ATCs. In silico analysis revealed that the differential expression of alphaB-crystallin and Hsp27-1 results from differences between the alphaB-crystallin and Hsp27-1 promoter fragments (712 bp upstream from the transcriptional start site). Biological activity of the analyzed promoter element is confirmed by its heat shock inducibility. In conclusion, we demonstrate downregulation of alphaB-crystallin expression in highly dedifferentiated ATCs because of a tumor-specific transcription factor pattern. The differential expression of alphaB-crystallin and Hsp27-1 indicates functional differences between both proteins.

Mimicking phosphorylation of the small heat‐shock protein αB‐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.

Proteolytic stress causes heat shock protein induction, tau ubiquitination, and the recruitment of ubiquitin to tau-positive aggregates in oligodendrocytes in culture.

Molecular chaperones and the ubiquitin-proteasome system are participants in the defense against unfolded proteins and provide an effective protein quality control system that is essential for cellular functions and survival. Ubiquitinated tau-positive inclusion bodies containing the small heat shock protein alphaB-crystallin in oligodendrocytes are consistent features of a variety of neurodegenerative diseases, and defects in the proteasome system might contribute to the aggregation process. Oligodendrocytes, the myelin-forming cells of the CNS, are specifically sensitive to stress situations. Here we can show that in cultured rat brain oligodendrocytes proteasomal inhibition by MG-132 or lactacystin caused apoptotic cell death and the induction of heat shock proteins in a time- and concentration-dependent manner. Specifically, alphaB-crystallin was upregulated, and ubiquitinated proteins accumulated. After incubation with MG-132 the tau was dephosphorylated, which enhanced its microtubule-binding capacity. Proteasomal inhibition led to ubiquitination of tau and its association with alphaB-crystallin and to the occurrence of thioflavine S-positive aggregates in the oligodendroglial cytoplasm. These aggregates were positive for tau and also contained ubiquitin and alphaB-crystallin; hence they resembled the glial cytoplasmic inclusions observed in white matter disease and frontotemporal dementias with parkinsonism linked to chromosome 17 (FTDP-17). In summary, the data underscore the specific sensitivity of oligodendrocytes to stress situations and point to a causal relationship of proteasomal impairment and inclusion body formation.

Activation of PP2A-like phosphatase and modulation of tau phosphorylation accompany stress-induced apoptosis in cultured oligodendrocytes.

In a number of neurodegenerative diseases, tau-positive glial cytoplasmic inclusions (GCIs), immunochemically labeled with antibodies to the small heat shock protein (HSP) alphaB-crystallin, occur in oligodendrocytes. The microtubule-associated protein tau is functionally modulated by phosphorylation. We have shown previously that oxidative stress (OS) and heat shock (HS) induce apoptotic cell death in oligodendrocytes. The present study was undertaken to test whether stress responses in oligodendrocytes cause abnormalities in the expression and posttranslational modification of tau proteins, and whether the dynamic phosphorylation and dephosphorylation of tau are involved in the pathogenesis of glial cells. Cultured rat brain oligodendrocytes were subjected to OS, exerted by hydrogen peroxide, or HS (44 degrees C, 30 min). Immunoblot analysis with a panel of phosphorylation-dependent antibodies shows that OS and HS caused the rapid dephosphorylation of tau proteins at multiple sites, before characteristic features of apoptosis were observed. Concomitantly, ERK1,2 (extracellular signal-regulated kinase) was activated. Tau phosphorylation and rephosphorylation after stress was mediated by glycogen synthase kinase 3beta (GSK-3beta), and not by ERK1,2 and could be suppressed by lithium chloride, a specific inhibitor of GSK-3beta. Stress-induced dephosphorylation could be mimicked by alkaline phosphatase and suppressed by the protein phosphatase inhibitor okadaic acid (OA), indicating that PP2A in oligodendrocytes is activated by stress. OA at low concentrations could prevent stress-induced DNA fragmentation, but eventually exerted cytotoxic effects. Hence, stress-induced activation of PP2A in oligodendrocytes and tau dephosphorylation constitute a major feature of the response to injury in these cells, which eventually undergo apoptotic cell death.

The Small Heat Shock Protein αB-Crystallin Negatively Regulates Cytochrome c- and Caspase-8-dependent Activation of Caspase-3 by Inhibiting Its Autoproteolytic Maturation

Caspases are universal effectors of apoptosis. The mitochondrial and death receptor pathways activate distinct apical caspases (caspase-9 and -8, respectively) that converge on the proteolytic activation of the downstream executioner caspase-3. Caspase-9 and -8 cleave procaspase-3 to produce a p24 processing intermediate (composed of its prodomain and large subunit), which then undergoes autoproteolytic cleavage to remove the prodomain from the active protease. Recently, several heat shock proteins have been shown to selectively inhibit the mitochondrial apoptotic pathway by disrupting the activation of caspase-9 downstream of cytochrome c release. We report here that the small heat shock protein alphaB-crystallin inhibits both the mitochondrial and death receptor pathways. In S-100 cytosolic extracts treated with cytochrome c/dATP or caspase-8, alphaB-crystallin inhibits the autoproteolytic maturation of the p24 partially processed caspase-3 intermediate. In contrast, neither the closely related small heat shock protein family member Hsp27 nor Hsp70 inhibited the maturation of the p24 intermediate. We also demonstrate that alphaB-crystallin co-immunoprecipitates with the p24 partially processed caspase-3 in vivo. Taken together, our results demonstrate that alphaB-crystallin is a novel negative regulator of apoptosis that acts distally in the conserved cell death machinery by inhibiting the autocatalytic maturation of caspase-3.

AlphaB-crystallin interacts with intermediate filaments in response to stress.

The small heat shock protein alphaB-crystallin interacts with intermediate filament proteins. Using a co-sedimentation assay, we showed that in vitro binding of alphaB-crystallin to peripherin and vimentin was temperature-dependent. Specifically, a synthetic peptide representing the first ten residues of alphaB-crystallin was involved in this interaction. When cells were submitted to different stress conditions such as serum starvation, hypertonic stress, or heat shock, we observed a dynamic reorganisation of the intermediate filament network, and concomitant recruitment of alphaB-crystallins on intermediate filament proteins. Under normal conditions alphaB-crystallin was extracted from cells by detergent. In stressed cells, alphaB-crystallin colocalised with intermediate filament proteins, and became resistant to detergent extraction. The intracellular state of alphaB-crystallin seemed to correlate directly with the remodelling of the intermediate filament network in response to stress. This suggested that alphaB-crystallin functions as a molecular chaperone for intermediate filament proteins.

Examination of the molecular basis for the lack of alphaB-crystallin expression in L929 cells.

We have previously shown that murine L929 cells do not express the small heat shock protein alphaB-crystallin upon exposure to thermal stress (Mol Cell Biochem 155: 51-60, 1996). In these studies, we demonstrate that L929 cells also fail to express alphaB-crystallin upon exposure dexamethasone, whereas NIH 3T3 and Swiss 3T3 murine cells exhibit alphaB-crystallin expression under identical conditions. Mobility shift assays demonstrated heat-inducible binding, presumably by heat shock factor(s), to an alphaB-crystallin heat shock element (HSE) oligomeric sequence in total cellular extracts from L929 cells. Transient transfection of a plasmid containing the alphaB-crystallin promoter linked to a CAT reporter gene exhibited heat-inducible expression in L929 cells. In addition, L929 cells stably transfected with a plasmid containing the complete alphaB-crystallin gene showed expression of this gene following heat shock. The presence of the endogenous alphaB-crystallin gene was detected by Southern blot hybridization of genomic L929 DNA, and sequence analysis revealed identical nucleotide structure to published murine sequences throughout the entire promoter. Treatment of L929 cells with 5-azacytidine enabled heat-inducible expression of alphaB-crystallin from the endogenous gene, however, methylation of the putative heat shock element (HSE) and flanking promoter sequences of L929 cell genomic DNA was not detected. In vivo genomic footprinting demonstrated constitutive binding to the endogenous HSE of the alphaB-crystallin promoter in L929, L929/alphaB-crystallin transfectant cells, and Swiss 3T3 cells during unstressed and heat stressed conditions. Therefore, the genomic alphaB-crystallin HSE region in L929 cells appears to be available for binding of putative transcription factors, but methylation in other regions of the gene or genome repress the expression of alphaB-crystallin in L929 cells. In vitro culture of L929 cells appears to have rendered the alphaB-crystallin gene loci inactive through methylation, thus providing a unique system by which to study the function of transfected small heat shock proteins.

Multiple sclerosis: an altered immune response or an altered stress response?

The pathogenesis of multiple sclerosis (MS), the major neurological disease of young adults in the Western world, is still poorly understood, and no effective therapy to block MS is available as yet. The clinical symptoms of MS result from inflammatory damage to the insulating myelin sheath of axons in the CNS and-at later stages-to axons themselves. A local autoimmune process involving activation of helper T cells against CNS protein components is likely to be crucial in this development. Especially at the first stages of MS, therapies aimed at the selective downregulation of MS-specific autoimmune responses may contribute to controlling the disease. Key to the success of such approaches is the identification of CNS proteins that are the target of local T cell responses. We recently identified the small heat-shock protein alpha B-crystallin as the single immunodominant myelin antigen in MS-affected myelin. This review discusses the functional and therapeutic implications of this finding along with other data on MS, and hypothesizes that an inappropriate stress response within the CNS itself is crucial as an initiating event in disease development.