Levels Of αB-crystallin Research Articles

Effects of a 10-d Military Field Exercise on Body Composition, Physical Performance, and Muscle Cells in Men and Women.

This study aimed to investigate the effects of a demanding military field exercise on physical performance, body composition, and muscle cellular outcomes in men and women. Ten men (20.5 ± 0.5 yr) and 8 women (21.4 ± 1.4 yr) completed a 10-d field exercise consisting of extensive physical activity with food and sleep restriction. Acquisition of body composition, physical performance, blood, and muscle biopsies samples were done before and 1, 7, and 14 d after the exercise. There were no sex differences in the response to the exercise. Body mass was decreased with 5.6% ± 1.8% and fat mass with 31% ± 11% during the exercise. Both were still reduced after 14 d (2.5% ± 2.3%, P < 0.001, and 12.5% ± 7.7%, P < 0.001, respectively). Isometric leg strength did not change. Peak leg extension torque at 240°·s -1 and counter movement jump height were reduced with 4.6% ± 4.8% ( P = 0.012) and 6.7% ± 6.2% ( P < 0.001), respectively, and was still reduced after 14 d (4.3% ± 4.2%, P = 0.002, and 4.1% ± 4.7%, P = 0.030). No changes occurred in fiber CSA, fiber types, proteins involved in calcium handling, or HSP70. During the exercise, αB-crystallin levels decreased by 14% ± 19% ( P = 0.024) in the cytosolic fraction and staining intensity on muscle sections tended to increase (17% ± 25%, P = 0.076). MuRF1 levels in the cytosolic fraction tended to decrease (19% ± 35%) and increased with 85% ± 105% ( P = 0.003) in the cytoskeletal fraction 1 wk after the exercise. The field exercise resulted in reduced body mass and physical performance in both sexes. The ability to produce force at high contraction velocities and explosive strength was more affected than isometric strength, but this was not related to any changes in fiber type composition, fiber area, Ca 2+ handling, or fiber type-specific muscle damage.

Serum advanced glycation end-products and αB-crystallin in diabetic retinopathy patients.

αB-crystallin, one of the small heat shock proteins, which is also known as HSPB5, has cytoprotective effects under inflammatory conditions. Advanced glycation end-products (AGE) are produced through non-enzymatic glycation under conditions of hyperglycemia and they contribute to angiogenesis and inflammation. The aim of this study was to examine the levels of serum αB-crystallin and AGE concentrations in blood samples collected from proliferative diabetic retinopathy (PDR) patients. Blood samples were collected from seven diabetic patients with PDR and eight patients without diabetes mellitus who underwent vitrectomy due to PDR and idiopathic macular diseases, respectively, in a single center. The levels of serum αB-crystallin and AGE were measured by ELISA and correlations were assessed statistically. The serum levels (mean ± SEM) of AGE were significantly higher in PDR patients (28.41±0.46 µg/ml) than in patients with non-diabetic macular diseases (25.76±0.60 µg/ml; P=0.015), whereas there was no significant difference in serum αB-crystallin levels. There was one patient with an extremely high level of αB-crystallin, who was treated with systemic corticosteroid due to chronic autoimmune inflammatory diseases. The current prospective study showed that serum AGE levels were significantly higher in PDR patients; however, no correlations between serum AGE and αB-crystallin levels were identified.

Elevated vitreous αB-crystallin in patients with rhegmatogenous retinal detachment and association with proliferative vitreoretinopathy and retinal detachment area.

To investigate the relationship between αB-crystallin levels in the vitreous of patients with rhegmatogenous retinal detachment (RRD) and correlate these levels with the presence of proliferative vitreoretinopathy (PVR) and to the extent of RRD. Prospective, cross-sectional study. Vitreous samples were collected from twenty-six patients with RRD at the surgical repair. PVR was evaluated using the Updated Proliferative Vitreoretinopathy Grade Classification system. RRD area was measured using ultra-widefield retinal photographs. The concentration of αB-crystallin in the vitreous was measured using an enzyme-linked immunosorbent assay kit. The average concentration of αB-crystallin was significantly higher in the PVR Grade B and C group (97.83 ± 69.47) than in the PVR (-) and Grade A group (39.43 ± 13.53ng/mL, P = 0.005). The level of αB-crystallin was also significantly higher in patients with large RD area (RD area > 1/4, 85.52 ± 66.07ng/mL) than in patients with small RD area (RD area ≤ 1/4, 39.20 ± 13.02ng/mL, P = 0.016). The level of αB-crystallin was significantly increased in the vitreous of RRD patients with high-grade PVR and large RD area. This finding suggests that αB-crystallin may function as an endogenous mediator in fibrotic processes in RRD patients.

Abstract B035: The role of αB-crystallin in chemotherapy-induced migration of triple negative breast cancer

Abstract Background: Triple negative breast cancer (TNBC) is an aggressive subtype of breast cancer marked by higher rate of metastasis and poorer overall survival. Although chemotherapy is the standard care of TNBC, clinical reports have shown that neoadjuvant chemotherapy treatment promoted metastasis in TNBC patients. Neoadjuvant chemotherapy-induced pro-metastasis in breast cancer cells was well explained by tumor microenvironment of metastasis, while its intracellular process has not been clarified yet. One of the small heat shock proteins, αB-crystallin, showed its high expression in metastatic sites of TNBC comparing to primary site, suggesting that αB-crystallin might be a potential target of metastasis. Even through researchers have also showed that αB-crystallin promoted tube formation, the relationship between αB-crystallin and chemotherapy-induced metastasis is not clear and the intracellular role of αB-crystallin needs to be explored. Purpose: To identify whether αB-crystallin involves in chemotherapy treatment and how αB-crystallin works in induced metastasis in this study. The expression of αB-crystallin in chemotherapy-induced TNBC migration were examined and its influence in Epithelial-Mesenchymal Transition (EMT) markers was confirmed. Methods: Cell viability of chemotherapy (Cisplatin, Fluorouracil, or Paclitaxel) in HCC1806 were tested by wst-8 assay. Preformed wound healing assay to measure migration ratio after treating HCC1806 cells with chemotherapy of three concentrations and confirming cell confluence. Expression level of αB-crystallin was examined after the same treatment, and the migration ability was measured when silencing αB-crystallin with small interfering RNA. Expression level of αB-crystallin and EMT markers were examined by western blotting after the same chemotherapy treatment. Results: The results of wst-8 assay showed that low concentrations of chemotherapy (0.01 μM and 0.1 μM of cisplatin, 0.01 μM and 0.1 μM of fluorouracil, 0.5 nM and 1 nM of paclitaxel) have no impact on cell viability of HCC1806, and high concentration of chemotherapy (1 μM of cisplatin, 1 μM of fluorouracil, 2 nM of paclitaxel) led to 80% alive cells comparing to non-treatment group. In addition, to identify the role of αB-crystallin in chemotherapy-induced migration, wound healing assay and western blotting were performed, showing that low concentration of chemotherapy treatment induced migration and promoted αB-crystallin expression, and that high concentration of chemotherapy blocked cell migration. Moreover, EMT markers were examined by western blotting after chemotherapy treatment, the increase of N-cadherin and decrease of E-cadherin were showed in HCC1806 after low concentration of chemotherapy treatment. Finally, to confirmed the relationship between αB-crystallin and chemotherapy-induced migration, wound healing assay was performed after silencing αB-crystallin and treating with chemotherapy. Silencing αB-crystallin reversed migration of HCC1806 cells and the expression of N-cadherin and E-cadherin induced by low concentrations of chemotherapy. Conclusion: These results suggest that the treatment of low concentrations of chemotherapy promoted epithelial to mesenchymal transition through increasing αB-crystallin expression, and induced migration in HCC1806. Citation Format: Lili YANG, Kazuma HIGASHISAKA, Yuya HAGA, Naoki SEKINE, Kotoe KIYOMOTO, Ying LIN, Hirofumi TSUJINO, Kazuya NAGANO, Yasuo TSUTSUMI. The role of αB-crystallin in chemotherapy-induced migration of triple negative breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B035. doi:10.1158/1535-7163.TARG-19-B035

P.80Imbalances in protein homeostasis caused by mutant desmin

Mutations of the human desmin gene cause myopathies and cardiomyopathies. We investigated immortalized R349P desmin knock-in myoblasts and myotubes in conjunction with the corresponding desminopathy mice as models for desminopathies to analyse major protein quality control processes in response to the presence of R349P mutant desmin. Desmin immunoblots and immunostains of differentiated myotubes revealed that these desminopathy cell models closely mirror central aspects of the desmin pathology observed in our desminopathy mice. Proteasomal activity was increased in muscle tissue lysates derived from hetero- and homozygous R349P desmin knock-in mice. Notably, treatment of the desminopathy myoblasts with the proteasome inhibitor MG132 did not alter the levels of both wild-type and R349P mutant desmin protein. Immunoblotting of tissue and myotube lysates demonstrated markedly increased levels of the autophagy-related markers serin/threonin protein kinase Ulk-1 and sequestosome 1 (p62/SQSTM1) as well as of p62/SQSTM1 and LC3-II. Furthermore, we detected increased protein levels of filamin-C and BAG-3. Finally, we found increased protein levels of αB-crystallin, Hsp27, and Hsp90 in skeletal muscle homogenates from homozygous mice, while only αB-crystallin and Hsp27, but not Hsp90, were increased in both desminopathy myotube genotypes. Both αB-crystallin and Hsp27 as well as Hsp90 displayed translocation patterns from Z-discs as well as Z-I junctions, respectively, to the level of sarcomeric I-bands. Our findings demonstrate that the presence of R349P mutant desmin causes a general imbalance in skeletal muscle protein homeostasis via aberrant activity of all major protein quality control systems. The augmented activity of these systems and the subcellular shift of essential heat shock proteins may deleteriously contribute to the previously observed increased turnover of desmin itself and desmin-binding partners, which triggers progressive dysfunction of the extrasarcomeric cytoskeleton and the myofibrillar apparatus in the course of the development of desminopathies.

Eccentric training enhances the αB-crystallin binding to the myofibrils and prevents skeletal muscle weakness in adjuvant-induced arthritis rat.

Patients with rheumatoid arthritis (RA) frequently suffer from muscle weakness. We examined whether eccentric training prevents skeletal muscle weakness in adjuvant-induced arthritis (AIA) rat, a widely used animal model for RA. AIA was induced in the knees of Wistar rats by injection of complete Freund's adjuvant. To induce eccentric contractions (ECCs), neuromuscular electrical stimulation (45 V) was applied to the plantar flexor muscles simultaneously with forced dorsiflexion of the ankle joint (0-40°) and was given every 6 s. ECC exercise was applied every other day for a total of 11 sessions and consisted of 4 sets of 5 contractions. There was a significant reduction in in vitro maximum Ca2+-activated force in skinned fibers in gastrocnemius muscle from AIA rats. These changes were associated with reduced expression levels of contractile proteins (i.e., myosin and actin), increased levels of inflammation redox stress-related biomarkers (i.e., TNF-α, malondialdehyde-protein adducts, NADPH oxidase 2, and neuronal nitric oxide synthase), and autolyzed active calpain-1 in AIA muscles. ECC training markedly enhanced the steady-state levels of αB-crystallin, a small heat shock protein, and its binding to the myofibrils and prevented the AIA-induced myofibrillar dysfunction, reduction in contractile proteins, and inflammation-oxidative stress insults. Our findings demonstrate that ECC training preserves myofibrillar function without muscle damage in AIA rats, which is at least partially attributable to the protective effect of αB-crystallin on the myofibrils against oxidative stress-mediated protein degeneration. Thus ECC training can be a safe and effective intervention, counteracting the loss of muscle strength in RA patients. NEW & NOTEWORTHY Eccentric contractions (ECCs) are regarded as an effective way to increase muscle strength. No studies, however, assess safety and effectiveness of ECC training on muscle weakness associated with rheumatoid arthritis. Here, we used adjuvant-induced arthritis (AIA) rats to demonstrate that ECC training prevents intrinsic contractile dysfunction without muscle damage in AIA rats, which may be attributed to the protective effect of αB-crystallin on the myofibrils against inflammation-oxidative stress insults.

Astrocytic α7 Nicotinic Receptor Activation Inhibits Amyloid-β Aggregation by Upregulating Endogenous αB-crystallin through the PI3K/Akt Signaling Pathway.

β-amyloid (Aβ) aggregation plays an important role in the pathogenesis of Alzheimer's disease (AD), and astrocytes can significantly inhibit Aβ aggregation. Astrocytic α7 Neuronal Nicotinic Acetylcholine Receptor (nAChR) upregulation detected in the AD brains is closely associated with Aβ deposits. However, the relationships between the astrocytic α7 nAChRs and Aβ aggregation remain unclear. The Aβ oligomers levels in astrocytic cell lysates and culture medium were measured after treatment with nicotine or co-treatment with a Phosphatidylinositol 3-Kinase (PI3K)-protein kinase B (Akt) inhibitor. The level of αB-Crystallin (Cryab) in astrocytes treated with nicotine for different times or co-treated with α7 nAChR antagonists as well as co-incubated with a PI3K or mitogen-activated protein kinase kinase 1/2 (MEK1/2) inhibitor was determined by western blotting. In this study, nicotine pre-treatment in primary astrocytes markedly inhibited Aβ aggregation and upregulated endogenous astrocytic Cryab, while the nicotine-mediated neuroprotective effect was reversed by pre-treatment with a selective α7 nAChR antagonist. Furthermore, this neuroprotection against Aβ aggregation was suppressed by LY294002, a PI3K inhibitor. Pre-treatment with nicotine significantly increased the levels of phosphorylated Akt, an effector of PI3K in astrocytes. α7 nAChR activation and PI3K/Akt signaling transduction contributed to nicotinemediated neuroprotection against Aβ aggregation by modulating endogenous astrocytic Cryab.

Imbalances in protein homeostasis caused by mutant desmin.

We investigated newly generated immortalized heterozygous and homozygous R349P desmin knock-in myoblasts in conjunction with the corresponding desminopathy mice as models for desminopathies to analyse major protein quality control processes in response to the presence of R349P mutant desmin. We used hetero- and homozygous R349P desmin knock-in mice for analyses and for crossbreeding with p53 knock-out mice to generate immortalized R349P desmin knock-in skeletal muscle myoblasts and myotubes. Skeletal muscle sections and cultured muscle cells were investigated by indirect immunofluorescence microscopy, proteasomal activity measurements and immunoblotting addressing autophagy rate, chaperone-assisted selective autophagy and heat shock protein levels. Muscle sections were further analysed by transmission and immunogold electron microscopy. We demonstrate that mutant desmin (i) increases proteasomal activity, (ii) stimulates macroautophagy, (iii) dysregulates the chaperone assisted selective autophagy and (iv) elevates the protein levels of αB-crystallin and Hsp27. Both αB-crystallin and Hsp27 as well as Hsp90 displayed translocation patterns from Z-discs as well as Z-I junctions, respectively, to the level of sarcomeric I-bands in dominant and recessive desminopathies. Our findings demonstrate that the presence of R349P mutant desmin causes a general imbalance in skeletal muscle protein homeostasis via aberrant activity of all major protein quality control systems. The augmented activity of these systems and the subcellular shift of essential heat shock proteins may deleteriously contribute to the previously observed increased turnover of desmin itself and desmin-binding partners, which triggers progressive dysfunction of the extrasarcomeric cytoskeleton and the myofibrillar apparatus in the course of the development of desminopathies.

Acute and long-term effects of blood flow restricted training on heat shock proteins and endogenous antioxidant systems.

Blood flow restricted exercise (BFRE) with low loads has been demonstrated to induce considerable stress to exercising muscles. Muscle cells have developed a series of defensive systems against exercise-induced stress. However, little is known about acute and long-term effects of BFRE training on these systems. Nine previously untrained females trained low-load BFRE and heavy load strength training (HLS) on separate legs and on separate days to investigate acute and long-term effects on heat shock proteins (HSP) and endogenous antioxidant systems in skeletal muscles. BFRE and HLS increased muscle strength similarly by 12±7% and 12±6%, respectively, after 12weeks of training. Acutely after the first BFRE and HLS exercise session, αB-crystallin and HSP27 content increased in cytoskeletal structures, accompanied by increased expression of several HSP genes. After 12weeks of training, this acute HSP response was absent. Basal levels of αB-crystallin, HSP27, HSP70, mnSOD, or GPx1 remained unchanged after 12weeks of training, but HSP27 levels increased in the cytoskeleton. Marked translocation of HSP to cytoskeletal structures at the commencement of training indicates that these structures are highly stressed from BFRE and HLS. However, as the muscle gets used to this type of exercise, this response is abolished.

Muscle plasticity related to changes in tubulin and αB-crystallin levels induced by eccentric contraction in rat skeletal muscles.

We used the model of eccentric contraction of the hindlimb muscle by Ochi etal. to examine the role of eccentric contraction in muscle plasticity. This model aims to focus on stimulated skeletal muscle responses by measuring tissue weights and tracing the quantities of αB-crystallin and tubulin. The medial gastrocnemius muscle (GCM) responded to electrically induced eccentric contraction (EIEC) with significant increases in tissue weight (p < 0.01) and the ratio of tissue weight to body weight (p < 0.05); however, there was a decrease in soleus muscle weight after EIEC. EIEC in the GCM caused contractile-induced sustenance of the traced proteins, but the soleus muscle exhibited a remarkable decrease in α-tubulin and a 19% decrease in αB-crystallin. EIEC caused fast-to-slow myosin heavy chain (MHC) isoform type-oriented shift within both the GCM and soleus muscle. These results have shown that different MHC isoform type-expressing slow and fast muscles commonly undergo fast-to-slow type MHC isoform transformation. This suggests that different levels of EIEC affected each of the slow and fast muscles to induce different quantitative changes in the expression of αB-crystallin and α-tubulin.

Different alpha crystallin expression in human age-related and congenital cataract lens epithelium.

BackgroundThe purpose of this study was to investigate the different expressions of αA-crystallin and αB-crystallin in human lens epithelium of age-related and congenital cataracts.MethodsThe central part of the human anterior lens capsule approximately 5 mm in diameter together with the adhering epithelial cells, were harvested and processed within 6 hours after cataract surgery from age-related and congenital cataract patients or from normal eyes of fresh cadavers. The mRNA and soluble protein levels of αA-crystallin and αB-crystallin in the human lens epithelium were detected by real-time PCR and western blots, respectively.ResultsThe mRNA and soluble protein expressions of αA-crystallin and αB-crystallin in the lens epithelium were both reduced in age-related and congenital cataract groups when compared with the normal control group. However, the degree of α-crystallin loss in the lens epithelium was highly correlated with different cataract types. The α-crystallin expression of the lens epithelium was greatly reduced in the congenital cataract group but only moderately decreased in the age-related cataract group. The reduction of αA-crystallin soluble protein levels in the congenital cataract group was approximately 2.4 fold decrease compared with that of the age-related cataract group, while an mRNA fold change of 1.67 decrease was observed for the age-related cataract group. Similarly, the reduction of soluble protein levels of αB-crystallin in the congenital cataract group was approximately a 1.57 fold change compared with that of the age-related cataract group. A 1.75 fold change for mRNA levels compared with that of the age-related cataract group was observed.ConclusionsThe results suggest that the differential loss of α-crystallin in the human lens epithelium could be associated with the different mechanisms of cataractogenesis in age-related versus congenital cataracts, subsequently resulting in different clinical presentations.

High level of αB-crystallin contributes to the progression of osteosarcoma.

Accumulating evidences indicate the elevated expression of αB-Crystallin (Cryab) is implicated in tumorigenesis. However, the expression and biologic role of Cryab in osteosarcoma (OS) are still unknown. In this study, we showed that Cryab expression was elevated in OS tissues and cell lines, and down-regulation of Cryab in MG-63 and U-2OS cells led to a decline in the cells’ aggressiveness, and reduced secretion of matrix metalloproteinase-9 (MMP-9) in vitro, and lower metastasis potential in vivo. Further study indicated that the Cryab expression was positively associated with the activity of ERK1/2 which is responsible for the cells’ aggressiveness and MMP-9 secretion. Clinically, our data confirmed that the high level of Cryab was associated with shorten survival and tumor recurrence for the postoperative OS patients. Together, our results indicate that high level of Cryab is a new adverse outcomes marker for OS patients and may be used as a new therapeutic target.

Expression profiles of heat shock protein 27 and αB-crystallin and their effects on heat-stressed rat myocardial cells in vitro and in vivo.

The present study established a heat-stressed rat heart model, and used an H9c2 myocardial cell line to investigate the expression profiles of heat shock protein (Hsp)27 and αB-crystallin, both in vivo and in vitro. Rats and myocardial cells were subjected to 42 ˚C for 0, 20, 40, 60, 80 or 100 min, following which the mRNA and protein expression levels of Hsp27 and αB-crystallin were measured. Following heat shock, the protein expression levels of Hsp27 and αB-crystallin were significantly decreased in the rat heart cells in vivo, whereas their mRNA levels were significantly increased. The opposing association between the protein and mRNA expression levels of Hsp27 and αB-crystallin suggests that the progression from mRNA into proteins via translation may delayed, or proteins may exist as either oligomers or in the phosphorylated form under heat stress. In vitro, Hsp27 and αB-crystallin exhibited similar reductions in the protein levels at 40 and 60 min, then increased to normal values following 80 min of heat stress. However, the mRNA levels were not consistent with the protein levels. The mRNA levels of Hsp27 and αB-crystallin did however exhibit similar tendencies following 60 min of heat stress. The present study investigated these apparently conflicting results between the in vitro cell line and the in vivo body system. The results demonstrated that the protein and mRNA expression levels of Hsp27 and αB-crystallin exhibited similar trends in vivo and in vitro, respectively. These results were confirmed by analysis with STRING 9.1 software, which indicated that Hsp27 and αB-crystallin are co-expressed in rat myocardial cells. However, the individual cell lines and whole body system exhibited different trends in Hsp27 and αB-crystallin levels prior to and following heat stress, thus require further investigation.

Gravitational Effects on Human Physiology.

Physical working capacity decreases with age and also in microgravity. Regardless of age, increased physical activity can always improve the physical adaptability of the body, although the mechanisms of this adaptability are unknown. Physical exercise produces various mechanical stimuli in the body, and these stimuli may be essential for cell survival in organisms. The cytoskeleton plays an important role in maintaining cell shape and tension development, and in various molecular and/or cellular organelles involved in cellular trafficking. Both intra and extracellular stimuli send signals through the cytoskeleton to the nucleus and modulate gene expression via an intrinsic property, namely the "dynamic instability" of cytoskeletal proteins. αB-crystallin is an important chaperone for cytoskeletal proteins in muscle cells. Decreases in the levels of αB-crystallin are specifically associated with a marked decrease in muscle mass (atrophy) in a rat hindlimb suspension model that mimics muscle and bone atrophy that occurs in space and increases with passive stretch. Moreover, immunofluorescence data show complete co-localization of αB-crystallin and the tubulin/microtubule system in myoblast cells. This association was further confirmed in biochemical experiments carried out in vitro showing that αB-crystallin acts as a chaperone for heat-denatured tubulin and prevents microtubule disassembly induced by calcium. Physical activity induces the constitutive expression of αB-crystallin, which helps to maintain the homeostasis of cytoskeleton dynamics in response to gravitational forces. This relationship between chaperone expression levels and regulation of cytoskeletal dynamics observed in slow anti-gravitational muscles as well as in mammalian striated muscles, such as those in the heart, diaphragm and tongue, may have been especially essential for human evolution in particular. Elucidation of the intrinsic properties of the tubulin/microtubule and chaperone αB-crystallin protein complex systems is expected to provide valuable information for high-pressure bioscience and gravity health science.

Level of vitreous alpha-B crystallin in eyes with rhegmatogenous retinal detachment.

Alpha-B crystallin (αB-crystallin) is a molecular chaperone, and one of the small heat shock proteins (sHSPs). αB-crystallin has been shown to have neuroprotective properties. The purpose of this study was to determine the level of αB-crystallin in eyes with a rhegmatogenous retinal detachment (RRD). Vitreous samples were collected before vitrectomy from 32 eyes of 32 consecutive patients with RRD. The concentration of αB-crystallin was measured by enzyme-linked immunosorbent assay (ELISA). Vitreous samples were also collected before vitrectomy from 16 patients with an idiopathic macular hole and epiretinal membrane, and used as controls. The mean age was 56 years (range, 31-67 years) in the RRD cases and 66 years (range, 49-77 years) in the controls. The mean level of αB-crystallin was significantly higher in the RRD cases (36.7 ± 37.4 ng/ml) than that in controls (8.5 ± 12.2 ng/ml; P = 0.0028). The increased level of αB-crystallin suggests that αB-crystallin may play a neuroprotective role by lessening retinal cell death after a retinal detachment.

OP1-3 - SFRR-E Young Investigator AwardeeαB-crystallin modulation after acute exercise in skeletal muscle: the role of oxidative stress and fiber composition

αB-crystallin (CRYAB) is a member of the small heat shock proteins implicated in various biological functions, particularly in skeletal muscle where it is involved in adaptive remodelling processes, activation of gene transcription and stabilization of nascent proteins.In this research we analysed αB-crystallin’ response in mouse gastrocnemius at 15’ and 30’ of recovery from an acute aerobic exercise (1hour), correlating its modulation with oxidative stress level and fiber composition, red (RG) and white gastrocnemius (WG).We found for the first time that the acute exercise lead to a short term, specific increase of phospho-αB-crystallin level (pCRYAB) in the RG, while no changes were observed in the WG. Moreover, this induction was correlated with increased level of 4-hydroxynonenal (HNE),suggesting a putative role for oxidative stress in driving CRYAB, but not hsp70 or hsp27, activity during exercise. Any increased level of αB-crystallin’ protein was observed neither in RG nor in WG. These data were also supported by our in vitro experiments showing a significant enhancement of pCRYAB in H2O2–treated C2C12 myotubes.Although our results seem suggest a fiber-dependent role of CRYAB, further experiments are in progress to clarify both the molecular pathway driving CRYAB phosphorylation and its fiber-specific induction after exercise –induced oxidative stress.This work was supported by MIUR - PRIN 2012 grant

Levels of oestrogen receptor, progesterone receptor and αB-crystallin in eutopic endometrium in relation to pregnancy in women with endometriosis

Endometriosis affects fertility in many women and may partly be due to decreased endometrial receptivity. Several mechanisms have been suggested, notably, progesterone resistance for which a number of candidate biomarkers have been suggested. Here we demonstrate aberrant levels of steroid hormone receptors and the small heat shock protein αB-crystallin in eutopic endometrial epithelium from 38 women with peritoneal endometriosis diagnosed during investigation for secondary infertility. Spontaneous pregnancies within 1 year after medical and surgical treatment for endometriosis were recorded and semi-quantitative immunohistochemistry data compared between women with endometriosis who did or did not become pregnant and healthy controls.Stronger immunostaining for ER-α was detected in luminal and glandular endometrial epithelium from women with endometriosis who did not become pregnant during the post-treatment observation period versus endometriosis patients who became pregnant and controls. Staining levels of PR and PR-B were lower in patients without subsequent pregnancies than in the two other groups. Endometrial levels of αB-crystallin in endometriosis patients similar to those in controls were strongly correlated with the chance of becoming pregnant, whereas higher or lower levels were not.

Acute response and subcellular movement of HSP27, αB-crystallin and HSP70 in human skeletal muscle after blood-flow-restricted low-load resistance exercise.

Heat-shock proteins (HSP) are important chaperones for stressed and damaged proteins. Low-load blood-flow-restricted resistance exercise (BFRE) is generally believed not to induce significant muscle damage, but is hitherto unverified with intracellular markers. Consequently, the aim of this study was to investigate the HSP response after BFRE in human skeletal muscle. Nine healthy volunteers performed five sets to failure of unilateral knee extension at 30% of 1RM with partial blood-flow restriction. The contralateral leg performed the same work with free blood flow. Muscle biopsies were collected before exercise, 1, 24 and 48 h after exercise and analysed for HSP27, αB-crystallin, HSP70, desmin, glycogen content and myosin heavy chain by immunohistochemistry, ELISA and western blotting. One hour after exercise, HSP27 and αB-crystallin levels were reduced in the cytosolic and increased in the cytoskeletal fraction in the BFRE leg. HSP70 showed a delayed response and was increased over 48 h in the BFRE leg. Immunohistochemical analyses showed higher staining intensity of HSP70 in type 1 fibres in the BFRE leg at 24 and 48 h post-exercise. PAS staining showed decreased glycogen levels after BFRE, and interestingly, glycogen was still depleted 48 h after exercise in the same fibres displaying high HSP70 staining (type 1 fibres). Translocation of HSP27 and αB-crystallin from cytosol to cytoskeletal structures indicates that cytoskeletal proteins are stressed during BFRE. However, overt signs of myofibrillar disruptions were not observed. Interestingly, the stress response was more pronounced in type 1 than in type 2 fibres and coincided with low glycogen levels.

Effect of hypoxia on the expression of αB-crystallin in head and neck squamous cell carcinoma.

BackgroundThe presence of hypoxia in head and neck squamous cell carcinoma (HNSCC) is associated with therapeutic resistance and increased risk of metastasis formation. αB-crystallin (HspB5) is a small heat shock protein, which is also associated with metastasis formation in HNSCC. In this study, we investigated whether αB-crystallin protein expression is increased in hypoxic areas of HNSCC biopsies and analyzed whether hypoxia induces αB-crystallin expression in vitro and in this way may confer hypoxic cell survival.MethodsIn 38 HNSCC biopsies, the overlap between immunohistochemically stained αB-crystallin and pimonidazole-adducts (hypoxiamarker) was determined. Moreover, expression levels of αB-crystallin were analyzed in HNSCC cell lines under hypoxia and reoxygenation conditions and after exposure to reactive oxygen species (ROS) and the ROS scavenger N-acetylcysteine (NAC). siRNA-mediated knockdown was used to determine the influence of αB-crystallin on cell survival under hypoxic conditions.ResultsIn all biopsies αB-crystallin was more abundantly present in hypoxic areas than in normoxic areas. Remarkably, hypoxia decreased αB-crystallin mRNA expression in the HNSCC cell lines. Only after reoxygenation, a condition that stimulates ROS formation, αB-crystallin expression was increased. αB-crystallin mRNA levels were also increased by extracellular ROS, and NAC abolished the reoxygenation-induced αB-crystallin upregulation. Moreover, it was found that decreased αB-crystallin levels reduced cell survival under hypoxic conditions.ConclusionsWe provide the first evidence that hypoxia stimulates upregulation of αB-crystallin in HNSCC. This upregulation was not caused by the low oxygen pressure, but more likely by ROS formation. The higher expression of αB-crystallin may lead to prolonged survival of these cells under hypoxic conditions.

αB-Crystallin Expression is Correlated with Phospho-ERK1/2 Expression in Human Breast Cancer

αB-crystallin is regarded as a biomarker for triple-negative and/or basal-like breast cancer. In normal breast cells, overexpression of αB-crystallin leads to neoplastic-like changes, which likely relate to enhanced expression of phosphorylated ERK1/2 (pERK1/2). In this study, we investigated whether αB-crystallin expression is correlated to pERK1/2 expression in breast cancer. In a balanced tissue microarray the expression of αB-crystallin and pERK1/2 kinase were determined immunohistochemically, together with the triple-negativity and basal-like markers CK5/6 and SMA and the signaling molecules pAKT, pmTOR, EGFR, and IGF-1R. αB-crystallin expression significantly correlated with triple negativity and basal-like markers CK5/6 and SMA (Pearson Chi-square test p=0.004, p=0.001, and p&lt;0.001, respectively). A significant correlation was also observed with pERK1/2 expression (p=0.002). siRNA-mediated knockdown of αB-crystallin in the triple-negative breast cell line MDA-MB468 did not show an effect on pERK1/2 expression levels, indicating that lowering the level of αB-crystallin does not reduce pERK1/2 expression. Our results confirm that αB-crystallin can be used as a biomarker for triple-negative and/or basal-like breast cancer. The expression of αB-crystallin correlates with pERK1/2 expression in breast cancer tissue suggesting that therapies targeting αB-crystallin might be considered for treatment of triple-negative or basal-like breast cancer.