Levels Of alphaB-crystallin Research Articles

Desmethoxycurcumin aids IFNα's anti-HBV activity by antagonizing CRYAB reduction and stabilizing IFNAR1 protein

The eradication of chronic hepatitis B (CHB) caused by hepatitis B virus (HBV) infection is a crucial goal in clinical practice. Enhancing the anti-HBV activity of interferon type I (IFNI) is a key strategy for achieving a functional cure for CHB. In this study, we investigated the effect of combined treatment with IFNα and Desmethoxycurcumin (DMC) on HBV replication in HepG2 cells and explored the underlying mechanism. Our results indicated IFNα alone was ineffective in completely inhibiting HBV replication, which was attributed to the virus-induced down-regulation of IFNI receptor 1 (IFNAR1) protein. However, the addition of a low dose of DMC significantly synergized with IFNα, leading to notable enhancement of IFNα anti-HBV activity. This effect was achieved by stabilizing the IFNAR1 protein. Further investigation revealed that low dose DMC effectively blocked the ubiquitination-mediated degradation of IFNAR1, which was accomplished by rescuing the protein levels of alphaB-crystallin (CRYAB) and orchestrating the interaction between CRYAB and the E3 ubiquitin ligase, β-Trcp. Importantly, over-expression of CRYAB was found to favor the antiviral activity of IFNα against HBV replication. In conclusion, our study demonstrates that low-dose DMC enhanced the anti-HBV activity of IFNα by counteracting the reduction of CRYAB and stabilizing the IFNAR1 protein.

Increased levels of alphaB-crystallin in vitreous fluid of patients with proliferative diabetic retinopathy and correlation with vascular endothelial growth factor.

AlphaB-crystallin has been shown to have angiogenic properties. The purpose of this study was to determine the levels of alphaB-crystallin in the vitreous fluid of patients with proliferative diabetic retinopathy and to confirm the association between the expression level of alphaB-crystallin and vascular endothelial growth factor. Vitreous samples were collected before vitrectomy from 46 eyes of 46 consecutive patients with proliferative diabetic retinopathy, and 19 patients without diabetes mellitus had vitrectomy for idiopathic macular hole. The concentrations of alphaB-crystallin and vascular endothelial growth factor were measured via enzyme-linked immunosorbent assay. The vitreous level (mean±SD) of alphaB-crystallin was significantly higher in patients with proliferative diabetic retinopathy (317.3±151.7ng/mL) than in control patients (idiopathic macular hole, 8.3±6.1ng/mL) (P<0.0001). The vitreous concentration of vascular endothelial growth factor was also significantly higher in patients with proliferative diabetic retinopathy (860.1±566.4pg/mL) than in control patients (9pg/mL) (P<0.0001). Meanwhile, both the expression levels of alphaB-crystallin and vascular endothelial growth factor were significantly higher in eyes with active proliferative diabetic retinopathy than in those with inactive proliferative diabetic retinopathy. Also, the vitreous concentration of alphaB-crystallin correlated significantly with that of vascular endothelial growth factor in vitreous fluid of proliferative diabetic retinopathy ([correlation coefficient], R=0.78, P<0.001). These results suggest a significant increase of alphaB-crystallin in the vitreous fluid of patients with proliferative diabetic retinopathy and present a crucial association between alphaB-crystallin and vascular endothelial growth factor with angiogenic activity in proliferative diabetic retinopathy.

Consumption of fumonisin B1 for 9 days induces stress proteins along the gastrointestinal tract of pigs

Fumonisin B(1) (FB1) is a mycotoxin which alters intestinal epithelial cell physiology and barrier properties, and accumulates in the colon. Data on effects of FB1 on stress proteins in the gastrointestinal tract (GIT) are lacking. Therefore, we hypothesized that repeated consumption of FB1 alters GIT tissue levels of stress proteins. This was tested using 36 weaned pigs fed a FB1 solution (n=18) or the vehicle (control; n=18) for 9 days. The pigs were then slaughtered, the organs were weighed and GIT tissues were collected for assessing GIT integrity, and for analysing stress proteins by Western blotting and densitometry (n=7 in each group). FB1 had little effects on growth rate but the liver was heavier (P<0.01) in FB1-fed pigs. alphaB crystallin and COX-1 concentrations were eight-fold and 12-fold higher in the colon of FB1-fed pigs than in the controls (P<0.0001). Concentrations of COX-1 and nNOS in the stomach, HSP 70 in the jejunum and HO-2 in the colon were also higher in FB1-fed pigs (P<0.05 to P<0.001). In conclusion, the FB1 extract drastically enhanced colonic levels of alphaB crystallin and COX-1, with milder increases in other stress proteins along the GIT of pigs. The data suggest that the colon is an important target for FB1-induced stress responses.

Heat shock factor 1 deficiency via its downstream target gene αB‐crystallin (Hspb5) impairs p53 degradation

Heat shock factor Hsf1 regulates the stress-inducibility of heat shock proteins (Hsps) or molecular chaperones. One of the functions attributed to Hsps is their participation in folding and degradation of proteins. We recently showed that hsf1(-/-) cells accumulate ubiquitinated proteins. However, a direct role for Hsf1 in stability of specific proteins such as p53 has not been elucidated. We present evidence that cells deficient in hsf1 accumulate wild-type p53 protein. We further show that hsf1(-/-) cells express lower levels of alphaB-crystallin and cells deficient in alphaB-crystallin also accumulate p53 protein. Reports indicate that alphaB-crystallin binds to Fbx4 ubiquitin ligase, and they target cyclin D1 for degradation through a pathway involving the SCF (Skp1-Cul1-F-box) complex. Towards determining a mechanism for p53 degradation involving alphaB-crystallin and Hsf1, we have found that ectopic expression of Fbx4 in wild-type mouse embryo fibroblasts (MEFs) expressing mutant p53 (p53R175H) leads to increase in its degradation, while MEFs deficient in hsf1 or alphaBcry are defective in degradation of this p53 protein. In addition, immunoprecipitated p53R175H from wild-type MEFs is able to pull-down both alphaB-crystallin and Fbx4. Finally, immunoprecipitated wild-type p53 from doxorubicin treated U2OS cells can pull-down endogenous alphaB-crystallin and Fbx4. These results indicate that hsf1- and alphaBcry-deficient cells accumulate p53 due to reduced levels of alphaB-crystallin in these cells. Elevated levels of p53 in hsf1- and alphaBcry-deficient cells lead to their increased sensitivity to DNA damaging agents. These data reveal a novel mechanism for protein degradation through Hsf1 and alphaB-crystallin.

Differential expression of αB‐crystallin and evidence of its role as a mediator of matrix gene expression in osteoarthritis

Alpha B-crystallin belongs to the family of small heat-shock proteins (HSPs). The role of this protein family in chondrocytes is not well understood. The present study was undertaken to investigate expression levels of alphaB-crystallin in chondrocytes isolated from healthy subjects and patients with osteoarthritis (OA), and to explore the functional role of this potentially interesting protein in chondrocyte metabolism. Western blot and real-time reverse transcriptase-polymerase chain reaction (RT-PCR) analyses were performed to determine expression levels of alphaB-crystallin in healthy and OA chondrocytes cultured in alginate beads. RNA interference-mediated gene knockdown was used to explore the role of this small HSP in chondrocyte biology, by transfecting low concentrations of small interfering RNA (siRNA) in cultured chondrocytes. We initially identified alphaB-crystallin as a small HSP that was differentially expressed between healthy and OA-affected chondrocytes. The decreased abundance of this protein in OA chondrocytes was confirmed by Western blotting. Moreover, real-time RT-PCR confirmed the differential expression between chondrocytes isolated from visibly intact and visibly damaged zones of OA cartilage. The proinflammatory cytokines interleukin-1beta and tumor necrosis factor alpha both down-regulated alphaB-crystallin expression. Transfection of low concentrations of siRNA in cultured chondrocytes resulted in efficient knockdown of alphaB-crystallin gene expression. This was accompanied by altered expression of the chondrocyte-specific bone morphogenetic protein 2, aggrecan, and type II collagen genes. The present findings identify the small HSP alphaB-crystallin as a novel mediator of chondrocyte matrix gene expression that may contribute to altered chondrocyte metabolism during the development of OA.

Clinical and Neuropathologic Findings in a Woman With the FMR1 Premutation and Multiple Sclerosis

Multiple sclerosis (MS) and fragile X-associated tremor/ataxia syndrome (FXTAS) have overlapping clinical signs and symptoms. To present a case with evidence of both MS and FXTAS and to discuss the relationship of both disorders. Case report. Fragile X Research and Treatment Center at the University of California, Davis, Medical Center. Patient Woman with the FMR1 premutation who died of MS at the age of 52 years. Magnetic resonance imaging, physical examination, and neuropathologic examination results. Magnetic resonance imaging, physical examination, and autopsy neuropathologic examination revealed diagnostic features of MS and FXTAS. The molecular mechanism of RNA toxicity, including the elevation of alphaB-crystallin levels observed in FXTAS, may lead to enhanced predisposition to autoimmune diseases.

Alpha B-Crystallin, a New Independent Marker for Poor Prognosis in Head and Neck Cancer

Gene expression profiling has provided many insights into tumor progression but translation to clinical practice has been limited. We have previously identified a list of potential markers by the differences of expression profiling of seven matched head and neck cancer (HNSCC) tumors with autologous normal oral mucosa (NOM). Alpha B-crystallin (CRYAB) was in the top 5% of genes identified with statistically significant differences in expression between tumor and NOM at the mRNA level. The objective was to confirm this in routine paraffin sections at the protein level. The level of alpha B-crystallin was determined in tumors of 62 HNSCC patients whose prognosis was known for 5 years. Immunohistochemical detection of alpha B-crystallin expression was performed on HNSCC paraffin sections. Univariate survival analysis identified lack of alpha B-crystallin staining as an independent prognostic marker for disease-free interval (P < 0.001) and overall survival (P < 0.002) of HNSCC patients over the 5-year observation period. Notably, all 13 patients (100%), including 5 patients with nodal disease whose tumors lacked alpha B-crystallin had no recurrences (P < 0.001). Nineteen of 27 node-negative patients stained positive for alpha B-crystallin and seven of the 19 (36.8%) had recurrences. Presence or absence of expression of alpha B-crystallin was a powerful marker for prognosis in this series of patients.

Small Heat Shock Protein αB-Crystallin Is Part of Cell Cycle-dependent Golgi Reorganization

AlphaB-crystallin is a developmentally regulated small heat shock protein known for its binding to a variety of denatured polypeptides and suppression of protein aggregation in vitro. Elevated levels of alphaB-crystallin are known to be associated with a number of neurodegenerative pathologies such as Alzheimer disease and multiple sclerosis. Mutations in alphaB-crystallin gene have been linked to desmin related cardiomyopathy and cataractogenesis. The physiological function of this protein, however, is unknown. Using discontinuous sucrose density gradient fractionation of post-nuclear supernatants, prepared from rat tissues and human glioblastoma cell line U373MG, we have identified discrete membrane-bound fractions of alphaB-crystallin, which co-sediment with the Golgi matrix protein, GM130. Confocal microscopy reveals co-localization of alphaB-crystallin with BODIPY TR ceramide and the Golgi matrix protein, GM130, in the perinuclear Golgi in human glioblastoma U373MG cells. Examination of synchronized cultures indicated that alphaB-crystallin follows disassembly of the Golgi at prometaphase and its reassembly at the completion of cytokinesis, suggesting that this small heat shock protein, with its chaperone-like activity, may have an important role in the Golgi reorganization during cell division.

AlphaB-crystallin as a marker of lymph node involvement in breast carcinoma.

It was found previously that alphaB-crystallin, a small heat-shock protein, was overexpressed in a metastatic variant of the GI101A human breast carcinoma cell line. The objective of the current study was to determine whether the expression of alphaB-crystallin in primary breast carcinomas was associated with lymph node metastasis and survival. Expression of alphaB-crystallin was measured in human breast carcinoma cell lines by immunoblotting. Expression in human breast carcinomas was evaluated by immunohistochemical staining of tissue microarrays that contained samples from 317 patients with lymph node-negative breast carcinoma and 291 patients with lymph node-positive breast carcinoma. It was found that alphaB-crystallin was expressed constitutively in certain breast carcinoma cell lines, including those that were capable of metastasizing in immunodeficient mice. Expression of alphaB-crystallin in human tissue samples was associated strongly with lymph node involvement (P < 0.0001; chi-square test) and, to a lesser degree, with high nuclear grade (P = 0.05). Increased intensity of expression was correlated with shorter survival (P = 0.0091; log-rank test). Multivariate analysis indicated that alphaB-crystallin expression was not independent of lymph node status as a predictor of survival. The data obtained in the current study revealed a strong association between high expression levels of alphaB-crystallin in primary breast carcinoma specimens and lymph node involvement. Further studies will be needed to prospectively elucidate the role of this novel tumor marker as a clinical prognostic marker in local and locally advanced breast carcinoma as well as its potential status as a new target for therapy in patients with breast carcinoma.

Effect of dicarbonyl modification of fibronectin on retinal capillary pericytes.

To determine effects of alpha-dicarbonyl modification of an extracellular matrix protein on retinal capillary pericyte attachment and viability. Primary cultures of bovine retinal pericytes (BRPs) were seeded on either normal fibronectin (FN) or FN modified by methylglyoxal (MGO) and glyoxal (GO). Apoptosis was measured by flow cytometry along with caspase-3 activity. Phosphorylation of p38 mitogen-activated protein kinase (MAPK) and Akt/PKB were evaluated by Western blot analysis. Cellular glutathione and reactive oxygen species were measured. alphaB-crystallin was measured by Western blot analysis and, to determine its role in apoptosis, experiments were conducted using BRPs that were transiently transfected with alphaB-crystallin. Cultures seeded on MGO- or GO-modified FN showed a significant reduction in the number of viable cells, an increase in the number of apoptotic cells, and increased caspase-3 activity, which correlated with the extent of FN modification. Pericytes seeded on either type of modified FN showed phosphorylation of p38 MAPK and dephosphorylation of Akt/PKB. Cultures seeded on dicarbonyl-modified FN had reduced glutathione and increased levels of reactive oxygen species compared with those on a normal matrix. Cells on the altered matrices had reduced alphaB-crystallin levels as well. Transient transfection of rat alphaB-crystallin into BRPs significantly reduced the apoptosis triggered by alpha-dicarbonyl-modified FN. These observations indicate that modification of FN by alpha-dicarbonyl compounds triggers apoptosis through a combination of increased oxidative stress and reduction of alphaB-crystallin. This mechanism may contribute to loss of pericytes in diabetic retinopathy and contribute to the resultant vascular lesions.

Use of Proteomics to Discover Novel Markers of Cardiac Allograft Rejection

Endomyocardial biopsy remains the most reliable method of detecting rejection following cardiac transplantation. Despite numerous attempts to detect rejection using a blood assay, none have proved reliable enough to replace the biopsy. Here, we have investigated the hypothesis that proteomics has the potential to reveal many molecules which are upregulated in the heart during rejection, some of which may serve as novel blood markers of rejection. Initially, sequential cardiac biopsies (33 in total) from 4 patients were analysed by two-dimensional gel electrophoresis according to whether they showed rejection (n = 16) or no rejection (n = 17); over 100 proteins were found to be upregulated by between 2- and 50-fold during rejection. Of these, 13 were identified and were found to be cardiac specific or heat shock proteins. Two of these (alphaB-crystallin, tropomyosin) were measured by ELISA in the sera of 17 patients followed for 3 months after their transplants. Mean levels of alphaB-crystallin and tropomyosin were significantly higher in sera associated with biopsies showing 1A (p = 0.007) or all grades of rejection (p = 0.022) compared to no rejection. These studies demonstrate that proteomics is a powerful method that can be used to identify novel serum markers of human cardiac allograft rejection.

Human bcl-2 gene attenuates the ability of rabbit lens epithelial cells against H2O2-induced apoptosis through down-regulation of the alpha B-crystallin gene.

It is well established that the proto-oncogene, bcl-2, can prevent apoptosis induced by a variety of factors. Regarding the mechanism by which BCL-2 prevents cell death, one theory suggests that it acts by protecting cells from oxidative stress. In the lens system, oxidative stress-induced apoptosis is implicated in cataractogenesis. To explore the possibility of anti-apoptotic gene therapy development for cataract prevention and also to further test the anti-oxidative stress theory of BCL-2 action, we have introduced the human bcl-2 gene into an immortalized rabbit lens epithelial cell line, N/N1003A. The stable expression clones of both vector- and bcl-2-transfected cells have been established. Treatment of the two cell lines with H(2)O(2) revealed that bcl-2-transfected cells were less capable of detoxifying H(2)O(2) than the control cells. Moreover, bcl-2-transfected cells are more susceptible to H(2)O(2)-induced apoptosis. To explore why bcl-2-transfected cells have reduced resistance to H(2)O(2)-induced apoptosis, we examined the expression patterns of several relevant genes and found that expression of the alphaB-crystallin gene was distinctly down-regulated in bcl-2-transfected cells compared with that in vector-transfected cells. This down-regulation was specific because a substantial inhibition of BCL-2 expression through antisense bcl-2 RNA significantly restored the level of alphaB-crystallin and, moreover, enhanced the ability of the bcl-2-transfected cells against H(2)O(2)-induced apoptosis. Introduction of a mouse alphaB-crystallin gene into bcl-2-transfected cells also counteracted the BCL-2 effects. Down-regulation of alphaB-crystallin gene was largely derived from changed lens epithelial cell-derived growth factor activity. Besides, alphaB-crystallin prevents apoptosis through interaction with procaspase-3 and partially processed procaspase-3 to prevent caspase-3 activation. Together, our results reveal that BCL-2 can regulate gene expression in rabbit lens epithelial cells. Through down-regulation of the alphaB-crystallin gene, BCL-2 attenuates the ability of rabbit lens epithelial cells against H(2)O(2)-induced apoptosis.

AlphaB-crystallin, a low-molecular-weight heat shock protein, acts as a regulator of platelet function.

It has recently been reported that alphaB-crystallin, a low-molecular-weight heat shock protein, may be released from cells by mechanical stretch. We investigated a physiological role of alphaB-crystallin in platelet function. AlphaB-crystallin inhibited platelet aggregation induced by thrombin or botrocetin in hamsters and humans. These platelets had specific binding sites for alphaB-crystallin. Moreover, alphaB-crystallin significantly reduced thrombin-induced Ca2+ influx and phosphoinositide hydrolysis by phospholipase C in human platelets. Additionally, plasma levels of alphaB-crystallin were markedly elevated in cardiomyopathic hamsters. Levels of alphaB-crystallin in vessel walls after endothelial injury were markedly reduced. Therefore, our results suggest that alphaB-crystallin, which is discharged from vessel walls in response to endothelial injury, acts intercellularly as a regulator of platelet function.

Differential Protective Activity of αA- and αB-crystallin in Lens Epithelial Cells

alphaA- and alphaB-crystallins are molecular chaperones expressed at low levels in lens epithelial cells, and their expression increases dramatically during differentiation to lens fibers. However, the functions of alphaA- and alphaB-crystallins in lens epithelial cells have not been studied in detail. In this study, the relative ability of alphaA- and alphaB-crystallin, in protecting lens epithelial cells from apoptotic cell death was determined. The introduction of alphaA-crystallin in the transformed human lens epithelial (HLE) B-3 lens epithelial cell line (which expresses low endogenous levels of alphaB-crystallin) led to a nearly complete protection of cell death induced by staurosporine, Fas monoclonal antibody, or the cytokine tumor necrosis factor alpha. To further study the relative protective activities of alphaA- and alphaB-crystallins, we created a cell line derived from alphaA-/-alphaB-/- double knockout mouse lens epithelia by infecting primary cells with Ad12-SV40 hybrid virus. The transformed cell line alphaAalphaBKO1 derived from alphaA/alphaB double knockout cells was transfected with alphaA- or alphaB-crystallin cDNA contained in pCIneo mammalian expression vector. Cells expressing different amounts of either alphaA-crystallin or alphaB-crystallin were isolated. The ability of alphaA- or alphaB-crystallin to confer protection from apoptotic cell death was determined by annexin labeling and flow cytometry of staurosporine- or UVA- treated cells. The results indicate that the anti-apoptotic activity of alphaA-crystallin was two to three-fold higher than that of alphaB-crystallin. Our work suggests that comparing the in vitro annexin labeling of lens epithelial cells is an effective way to measure the protective activity of alphaA- and alphaB-crystallin. Since the expression of alphaA-crystallin is largely restricted to the lens, its greater protective effect against apoptosis suggests that it may play a significant role in protecting lens epithelial cells from stress.

Ischemic acute renal failure induces differential expression of small heat shock proteins.

AlphaB-crystallin and heat shock protein (hsp) 25 are structurally and functionally related small stress proteins induced by a variety of insults, including heat and ischemia. Cytoprotection by these two hsp is thought to result from molecular chaperoning and/or cytoskeletal stabilization. Because renal ischemia is characterized by disruption of the renal tubular cell actin cytoskeleton, this study was conducted to determine the localization and quantify the expression and phosphorylation of both hsp in renal cortex, isolated glomeruli, outer medulla, and inner medulla of rats after bilateral renal ischemia. Sham-operated kidneys had similarly small amounts of hsp25 and alphaB-crystallin in cortex and glomeruli, with substantially greater amounts of alphaB-crystallin versus hsp25 in outer and inner medulla. Ischemia resulted in significantly increased hsp25 (and hsp70i) but variable alphaB-crystallin levels in cortex and outer medulla, and progressively decreased glomerular hsp25 phosphorylation. In sham-operated kidneys, hsp25 localized to glomeruli, vessels, and collecting ducts, with alphaB-crystallin primarily in medullary thin limbs and collecting ducts. After ischemia, hsp25 accumulated in proximal tubules in cortex and outer medulla, while alphaB-crystallin labeling became nonhomogeneous in outer medulla, and increased in Bowman's capsule. It is concluded that: (1) There is striking differential expression of hsp25 and alphaB-crystallin in various renal compartments; and (2) Renal ischemia results in differential accumulation of hsp25 and alphaB-crystallin, with hsp25 part of a generalized stress response in renal proximal tubular cells, which may play a role in recovery from ischemia-induced actin filament disruption.

Responses of Heat Shock Proteins hsp27, αB‐Crystallin, and hsp70 in Rat Brain After Kainic Acid‐Induced Seizure Activity

We determined the changes in the levels of the mammalian small heat shock protein of 25-28 kDa (hsp27) and the hsp alphaB-crystallin in various regions of rat brain after kainic acid-induced seizure activity by means of specific immunoassays. The levels of hsp27 in the hippocampus and entorhinal cortex were markedly increased and reached a maximum (1.5-2 microg/mg of protein) 2-4 days after the seizure. The levels of hsp27 in these regions were considerably high even 10 days after the seizure. A marked increase in levels of mRNA for hsp27 was also observed in the hippocampus of rats 1-2 days after the seizure. A severalfold increase in the levels of alphaB-crystallin was observed in the hippocampus and entorhinal cortex of rats 2 days after the seizure. However, the maximum levels were <50 ng/mg of protein. The levels of protein sulfhydryl group and glutathione were significantly reduced in the hippocampus of rats at 24 h after the seizure, which might have enhanced the expressions of hsp27 and alphaB-crystallin. The expression of inducible mammalian hsp of 70 kDa (hsp70) was also enhanced in the hippocampus of rats after the seizure, as detected by western and northern blotting analyses. Immunohistochemically, an intensive staining of hsp27 was observed in both glial cells and neurons in the hippocampus, piriform cortex, and entorhinal cortex of rats with kainic acid-induced seizure. However, in the cerebellum, where the receptors for kainic acid are also rich, hsp27 was barely induced in the same rats. This might be due to high levels of the cerebellar calcium-binding proteins parvalbumin and 28-kDa calbindin-D, which might have a protective effect against the kainic acid-inducible damage.

Formation of GFAP Cytoplasmic Inclusions in Astrocytes and Their Disaggregation by αB-Crystallin

In several neuropathological conditions, alphaB-crystallin and glial fibrillary acidic protein (GFAP) accumulate and form cytoplasmic inclusions in astrocytes. To explore the pathogenesis of the inclusions and the possible functions of the accumulated alphaB-crystallin, GFAP and alphaB-crystallin were overexpressed in cultured astrocytes by transient transfection. Human GFAP formed filamentous, cytoplasmic inclusions in mouse astrocytes, NIH3T3 cells, rat C6 glioma cells, and human U251 glioma cells. These human GFAP inclusions did not contain the endogenous vimentin or beta-tubulin, and the intermediate filament and microtubular networks of the transfected cells appeared normal. alphaB-crystallin and hsp25 were associated with the GFAP inclusions. Increasing intracellular alphaB-crystallin levels using recombinant adenoviruses, either before or after GFAP inclusions were formed, decreased the number of inclusion-bearing astrocytes and converted the human GFAP from an inclusion to a spread, filamentous form. These results suggest that alphaB-crystallin reorganizes abnormal intermediate filament aggregates into the normal filamentous network.

Selective stimulation of Hsp27 and αB-crystallin but not Hsp70 expression by p38 MAP kinase activation

The levels of Hsp27 and alphaB-crystallin in C6 rat glioma cells, that had been heated at 43 degrees C for 30 min with a subsequent culture for 16 h at 37 degrees C, were markedly increased. The exposure of the cells to a low concentration (0.1-3 microg/ml) of anisomycin for a few hours after heat stress stimulated the accumulation of the small stress proteins Hsp27 and alphaB-crystallin, but not that of Hsp70. The levels of mRNAs for Hsp27 and alphaB-crystallin but not that for Hsp70 increased in cells that had been exposed to heat and subsequently for 2 h to 0.1-3 microg/ml anisomycin. The results of a reporter assay, using an alphaB-crystallin promotor fused to a luciferase reporter gene, suggested that the increase in level of alphaB-crystallin mRNA was due to the production of new mRNA. The activation of the binding of heat shock factors to heat shock elements induced in cells that had been heat stressed was barely affected by subsequent exposure to anisomycin at 0.3 microg/ml. The stimulatory effects of anisomycin were also observed in cells that had been exposed to NaAsO2 or CdCl2. The active form of p38 mitogen activated protein (MAP) kinase was increased in cell that had been subjected to heat shock and subsequent exposure to 0.3 microg/ml of anisomycin. The heat-induced accumulations of Hsp27 and alphaB-crystallin were also stimulated by cycloheximide, another stimulator of p38 MAP kinase. SB202190, a specific inhibitor of p38 MAP kinase, suppressed the stimulation by anisomycin of the heat stress-induced expressions of Hsp27 and alphaB-crystallin. These results suggest that the signal transduction pathway of the stress-induced expressions of Hsp27 and alphaB-crystallin in C6 glioma cells includes a process that is sensitive to p38 MAP kinase.

Different concentrations of two small stress proteins, alphaB crystallin and HSP27 in human urological tumor tissues.

Concentrations of two small stress proteins, alphaB crystallin and the 27-kDa heat shock protein (HSP27) were quantitated in tissues of the human normal genitourinary system and their tumors. Levels of HSP27 in renal cell carcinomas (mean +/- SE: 1450+/-262 ng/mg protein, n = 15) were significantly higher than in normal kidney (the cortex: 540+/-99 ng/ mg protein, n = 13; the medulla: 600+/-106 ng/mg protein, n = 13) while those of alphaB crystallin tended to be increased without statistical significance. These findings were similar to those previously reported for renal cell tumors chemically induced in rats. Concentrations of alphaB crystallin in prostatic carcinoma tissues (410+/-129 ng/mg protein, n = 10) were also significantly higher than in benign prostatic hyperplasia (54+/-12 ng/mg protein, n = 14), whereas alphaB crystallin levels in testicular tumors including seminomas (2.1+/-0.8 ng/mg protein, n = 11) and non-seminomas (5.2+/-2.3 ng/mg protein, n = 9) were significantly lower than in normal testicular tissues (29.7+/-6.2 ng/mg protein, n = 5). Both alphaB crystallin and HSP27 could be immunohistochemically localized in the normal kidney and renal cell carcinoma tissues.

Phosphorylation of αB-crystallin in Mitotic Cells and Identification of Enzymatic Activities Responsible for Phosphorylation

The immunofluorescence localization of alphaB-crystallin in U373 MG human glioma cells with an antibody specific for alphaB-crystallin that had been phosphorylated at Ser-45 revealed an intense staining of cells in the mitotic phase of the cell cycle. Phosphorylated forms of alphaB-crystallin in mitotic cells were detected in all cell lines examined and in tissue sections of mouse embryos. Increases in the levels of alphaB-crystallin that had been phosphorylated at Ser-45 and Ser-19, but not at Ser-59, were detected biochemically by isoelectric focusing or SDS-polyacrylamide gel electrophoresis and a subsequent Western blot analysis of extracts of cells collected at the mitotic phase. When we estimated the phosphorylation activity specific for alphaB-crystallin in extracts of mitotic U373 MG cells, using the amino-terminal 72-amino acid peptide derived from unphosphorylated alphaB2-crystallin as the substrate, we found that the activities responsible for the phosphorylation of Ser-45 and Ser-19 were markedly enhanced but that the activity responsible for the phosphorylation of Ser-59 was suppressed. The protein kinases responsible for the phosphorylation of Ser-45 and Ser-59 in the amino-terminal 72-amino acid peptide were partially purified from extracts of cells that had been stimulated by exposure to H2O2 in the presence of calyculin A. The activities responsible for the phosphorylation of Ser-45 and Ser-59 were eluted separately from a column of Superdex 200 at fractions corresponding to about 40 and 60 kDa, respectively, while the kinase for Ser-19 was unstable. p44/42 mitogen-activated protein (MAP) kinase and MAP kinase-activated protein (MAPKAP) kinase-2 were concentrated in the Ser-45 kinase fraction and Ser-59 kinase fraction, respectively. Recombinant human p44 MAP kinase and MAPKAP kinase-2 purified from rabbit muscle selectively phosphorylated Ser-45 and -59, respectively. The Ser-45 kinase fraction and Ser-59 kinase fraction phosphorylated myelin basic protein and hsp27, respectively. These results suggest that the phosphorylations of Ser-45 and Ser-59 in alphaB-crystallin are catalyzed by p44/42 MAP kinase and MAPKAP kinase-2, respectively, in cells and that the phosphorylation of Ser-45 by p44/42 MAP kinase is enhanced while the phosphorylation of Ser-59 by MAPKAP kinase-2 is suppressed during cell division.