αB-crystallin In Cells Research Articles

Small Heat Shock Protein αB-Crystallin Controls Shape and Adhesion of Glioma and Myoblast Cells in the Absence of Stress.

Cell shape and adhesion and their proper controls are fundamental for all biological systems. Mesenchymal cells migrate at an average rate of 6 to 60 μm/hr, depending on the extracellular matrix environment and cell signaling. Myotubes, fully differentiated muscle cells, are specialized for power-generation and therefore lose motility. Cell spreading and stabilities of focal adhesion are regulated by the critical protein vinculin from immature myoblast to mature costamere of differentiated myotubes where myofibril Z-band linked to sarcolemma. The Z-band is constituted from microtubules, intermediate filaments, cell adhesion molecules and other adapter proteins that communicate with the outer environment. Mesenchymal cells, including myoblast cells, convert actomyosin contraction forces to tension through mechano-responsive adhesion assembly complexes as Z-band equivalents. There is growing evidence that microtubule dynamics are involved in the generation of contractile forces; however, the roles of microtubules in cell adhesion dynamics are not well determined. Here, we show for the first time that αB-crystallin, a molecular chaperon for tubulin/microtubules, is involved in cell shape determination. Moreover, knockdown of this molecule caused myoblasts and glioma cells to lose their ability for adhesion as they tended to behave like migratory cells. Surprisingly, αB-crystallin knockdown in both C6 glial cells and L6 myoblast permitted cells to migrate more rapidly (2.7 times faster for C6 and 1.3 times faster for L6 cells) than dermal fibroblast. On the other hand, overexpression of αB-crystallin in cells led to an immortal phenotype because of persistent adhesion. Position of matured focal adhesion as visualized by vinculin immuno-staining, stress fiber direction, length, and density were clearly αB-crystallin dependent. These results indicate that the small HSP αB-crystallin has important roles for cell adhesion, and thus microtubule dynamics are necessary for persistent adhesion.

Expression of αB-crystallin overrides the anti-apoptotic activity of XIAP.

Although crystallins are major structural proteins in the lens, α-crystallins perform non-lens functions, and αB-crystallin has been shown to act as an anti-apoptotic mediator in various cells. The present study was undertaken to examine whether αB-crystallin expressed in human malignant glioma cells exerts anti-apoptotic activity. In addition, we sought to elucidate the mechanism underlying any observed anti-apoptotic function of αB-crystallin in these cells. Three glioma cell lines, U373MG, U118MG, and T98G, were used. We observed that only the U373MG cell line expresses αB-crystallin, whereas the other 2 glioma cell lines, U118MG and T98G, demonstrated no endogenous expression of αB-crystallin. We next observed that the silencing of αB-crystallin sensitized U373MG cells to suberoylanilide hydroxamic acid (SAHA)-induced apoptosis and that αB-crystallin associates with caspase-3 and XIAP. Because XIAP is the most potent suppressor of mammalian apoptosis through the direct binding with caspases, we assessed whether XIAP also plays an anti-apoptotic role in SAHA-induced apoptosis in αB-crystallin-expressing U373MG cells. Of note, the silencing of XIAP did not alter the amount of cell death induced by SAHA, indicating that XIAP does not exert an anti-apoptotic activity in U373MG cells. We then determined whether the ectopic expression of αB-crystallin in glioma cells caused a loss of the anti-apoptotic activity of XIAP. Accordingly, we established 2 αB-crystallin over-expressing glioma cell lines, U118MG and T98G, and found that the silencing of XIAP did not sensitize these cells to SAHA-induced apoptosis. These findings suggest that αB-crystallin expressed in glioma cells overrides the anti-apoptotic activity exerted by XIAP.

Prostaglandins stimulate the stress‐induced synthesis of hsp27 and αB crystallin

The effects were examined of various prostaglandins (prostaglandin A1, A2, J2, E2, and D2) on the stress-induced accumulation of hsp27 and αB crystallin in C6 rat glioma cells. The levels of hsp27 and αB crystallin, which were determined by specific immunoassays, were low in cells in confluent cultures. The levels of the two proteins increased after exposure of cells to heat (42°C for 30 min) or arsenite (50 μM for 1 h). Cells exposed to 10 μM each of prostaglandin A1, A2, or J2 for 1 h resulted in stimulation of the binding to the heat shock element (HSE) of heat shock transcription factor (HSF). However, there was no phosphorylation-dependent mobility shift of HSF1 and no subsequent increase in the transcription and translation for hsp27, αB crystallin, and hsp70. When cells were exposed to arsenite in the presence of 10-40 μM prostaglandin, the accumulation of hsp27 and αB crystallin in cells was enhanced markedly. The levels of hsp70 also increased in cells that had been treated with arsenite in the presence of a prostaglandin, as estimated by Western blot analysis. Northern blot analysis revealed that the expression of messenger RNAs (mRNAs) for hsp27, αB crystallin, and hsp70 was enhanced in cells that had been exposed to arsenite in the presence of each prostaglandin. Similar stimulatory effects of prostaglandins also were observed in the case of the heat-induced responses of hsp27, αB crystallin, and hsp70. Gel mobility shift assays revealed that each prostaglandin prolonged the arsenite-induced binding of HSF to HSE. These results suggest that the pharmacological dose of prostaglandins stimulates the stress-induced synthesis of stress proteins via activation of the HSF. J. Cell. Physiol. 170:255–262, 1997. © 1997 Wiley-Liss, Inc.

Prostaglandins stimulate the stress-induced synthesis of hsp27 and αB crystallin

The effects were examined of various prostaglandins (prostaglandin A1, A2, J2, E2, and D2) on the stress-induced accumulation of hsp27 and alpha B crystallin in C6 rat glioma cells. The levels of hsp27 and alpha B crystallin, which were determined by specific immunoassays, were low in cells in confluent cultures. The levels of the two proteins increased after exposure of cells to heat (42 degrees C for 30 min) or arsenite (50 microM for 1 h). Cells exposed to 10 microM each of prostaglandin A1, A2, or J2 for 1 h resulted in stimulation of the binding to the heat shock element (HSE) of heat shock transcription factor (HSF). However, there was no phosphorylation-dependent mobility shift of HSF1 and no subsequent increase in the transcription and translation for hsp27, alpha B crystallin, and hsp70. When cells were exposed to arsenite in the presence of 10-40 microM prostaglandin, the accumulation of hsp27 and alpha B crystallin in cells was enhanced markedly. The levels of hsp70 also increased in cells that had been treated with arsenite in the presence of a prostaglandin, as estimated by Western blot analysis. Northern blot analysis revealed that the expression of messenger RNAs (mRNAs) for hsp27, alpha B crystallin, and hsp70 was enhanced in cells that had been exposed to arsenite in the presence of each prostaglandin. Similar stimulatory effects of prostaglandins also were observed in the case of the heat-induced responses of hsp27, alpha B crystallin, and hsp70. Gel mobility shift assays revealed that each prostaglandin prolonged the arsenite-induced binding of HSF to HSE. These results suggest that the pharmacological dose of prostaglandins stimulates the stress-induced synthesis of stress proteins via activation of the HSF.

Ubiquitin—protein conjugates and αB crystallin are selectively present in cells undergoing major cytomorphological reorganisation in early chicken embryos

Ubiquitin—protein conjugates and αB crystallin are detected immunohistochemically in cells undergoing extensive morphological reorganisation in early chicken embryos. Cytoplasmic ubiquitinated proteins and αB crystallin are coordinately found in cells of the lens, notochord and myotome. The antigens appear in the myotome cells precisely, at the point at which the cells begin to migrate from the dorsomedial lip of the dermamyotome. The findings indicate that ubiquitin and αB crystallin may have a coordinate role in the extensive architectural remodeling which occurs in these developing tissues in the early chick embryo, Some form of functional association between protein ubiquitination and αB crystallin in cells may explain why αB crystallin is found with ubiquitin—protein deposits in some neurodegenerative diseases.