p-Benzoquinone-induced aggregation and perturbation of structure and chaperone function of α-crystallin is a causative factor of cigarette smoke-related cataractogenesis.

Cigarette smoking is a significant risk factor for cataract. However, the mechanism by which cigarette smoke (CS) causes cataract remains poorly understood. We had earlier shown that in CS-exposed guinea pig, p-benzoquinone (p-BQ) derived from CS in the lungs is carried by the circulatory system to distant organs and induces various smoke-related pathogeneses. Here, we observed that CS exposure caused accumulation of the p-BQ-protein adduct in the eye lens of guinea pigs. We also observed accumulation of the p-BQ-protein adduct in resected lens from human smokers with cataract. No such accumulation was observed in the lens of never smokers. p-BQ is a strong arylating agent that forms Michael adducts with serum albumin and haemoglobin resulting in alterations of structure and function. A major protein in the mammalian eye lens is αA-crystallin, which is a potent molecular chaperone. αA-crystallin plays a key role in maintaining the integrity and transparency of the lens. SDS-PAGE indicated that p-BQ induced aggregation of αA-crystallin. Various biophysical techniques including UV-vis spectroscopy, fluorescence spectroscopy, FT-IR, bis-ANS titration suggested a perturbation of structure and chaperone function of αA-crystallin upon p-BQ modification. Our results indicate that p-BQ is a causative agent involved in the modification of αA-crystallin and pathogenesis of CS-induced cataract. Our findings would educate public about the impacts of smoking on eye health and help to discourage them from smoking. The study might also help scientists to develop new drugs for the intervention of CS-induced cataract at an early stage.

Alpha basic crystallin expression in canine mammary tumors.

The aim of this study was to evaluate prognostic and/or diagnostic factors of canine mammary tumors by immunohistochemically analyzing the expression of alpha basic crystallin (αB-c). For this, formalin-fixed, paraffin-embedded blocks of 51 naturally-occurring canine mammary tumors (11 benign and 40 malignant) were used. Tissue from eight normal canine mammary glands were served as a control. Immunohistochemically, in the control mammary tissues, a few luminal epithelial cells were αB-c positive but myoepithelial cells were negative. In benign or simple type malignant tumors, αB-c expression was observed in luminal epithelial cells while the myoepithelial basal cells were negative. In benign or complex type malign tumors, positive staining was predominantly found in the cytoplasm of epithelial cells. Immunoreactivity of αB-c was also observed in neoplastic myoepithelial cells. Statistically, the number of cells immunolabeled with αB-c was found to be significantly different among tissues from normal canine mammary glands, benign lesions, and malignant tumors (p < 0.05). αB-c immunoreactivity was higher in malignant tumors than the control mammary tissues (p < 0.001). Data obtained in the current study revealed a strong association between high expression levels of αB-c and primary mammary gland tumors in canines.

Inactivation of Smad4 leads to impaired ocular development and cataract formation.

PURPOSE: Signaling by members of the TGFß superfamily of molecules is essential for embryonic development and homeostasis. Smad4, a key intracellular mediator in TGFß signaling, forms transcriptional activator complexes with Activin-, BMP-, and TGFß-restricted Smad proteins. However, the functional role of Smad4 in controlling different visual system compartments has not been fully investigated. METHODS: Using the Pax6 promoter-driven Cre transgenic, smad4 was conditionally inactivated in the lens, cornea and ectoderm of the eyelids. Standard histological and molecular analytical approaches were employed to reveal morphological and cellular changes. RESULTS: Inactivation of Smad4 in the lens led to microphthalmia and cataract formation in addition to the persistent adhesion of the retina to the lens and the iris to the cornea. Inactivation of Smad4 from the ectoderm of the eyelid and cornea caused disruption to eyelid fusion and proper development of the corneal epithelium and corneal stroma. CONCLUSIONS: Smad4 is required for the development and maintenance of the lens in addition to the proper development of the cornea, eyelids, and retina.

Contribution of IL-6 to the Hsp72, Hsp25, and alphaB-crystallin [corrected] responses to inflammation and exercise training in mouse skeletal and cardiac muscle.

The heat shock proteins (Hsps) Hsp72, Hsp25, and alphaB-crystallin (alphaB C) [corrected]may protect tissues during exercise and/or inflammatory insults; however, no studies have investigated whether exercise training increases both basal and inflammation-induced expression of these Hsps in skeletal or cardiac muscle. IL-6 is produced by muscle during both exercise and inflammation and has been shown to modulate Hsp expression. These studies tested the hypothesis that voluntary wheel running (RW) increases basal and inflammation-induced Hsp72, Hsp25, and alphaB C [corrected] protein through an IL-6-dependent mechanism. We compared Hsp72, Hsp25, alphaB C, [corrected] and IL-6 protein levels 4 h after systemic inflammation induced by lipopolysaccharide (LPS) in skeletal and cardiac muscles of wild-type (IL-6(+/+)) and IL-6 deficient (IL-6(-/-)) mice after 2 wk of RW or normal cage activity (Sed). LPS significantly increased skeletal Hsp72 and Hsp25 relative to saline in Sed IL-6(+/+), but not IL-6(-/-) mice. LPS increased Hsp72 relative to saline in Sed IL-6(+/+) cardiac muscle. RW increased basal Hsp72, Hsp25, and alphaB C [corrected] in skeletal muscle in IL-6(+/+) and IL-6(-/-) mice. However, LPS was not associated with increases in any Hsp in RW IL-6(+/+) or IL-6(-/-) mice. LPS increased IL-6 protein in skeletal muscle and plasma in Sed and RW groups, with a significantly greater response in RW. The major results provide the first in vivo evidence that the absence of IL-6 is associated with reduced skeletal muscle Hsp72 and Hsp25 responses to LPS, but that IL-6 is not required for exercise-induced Hsp upregulation in skeletal or cardiac muscle.

Alpha-crystallin is a target gene of the farnesoid X-activated receptor in human livers.

Alpha-crystallins comprise 35% of soluble proteins in the ocular lens and possess chaperone-like functions. Furthermore, the alphaA subunit (alphaA-crystallin) of alpha crystallin is thought to be "lens-specific" as only very low levels of expression were detected in a few non-lenticular tissues. Here we report that human alphaA-crystallin is expressed in human livers and is regulated by farnesoid X-activated receptor (FXR) in response to FXR agonists. AlphaA-crystallin was identified in a microarray screen as one of the most highly induced genes after treatment of HepG2 cells with the synthetic FXR ligand GW4064. Northern blot and quantitative real-time PCR analyses confirmed that alphaA-crystallin expression was induced in HepG2-derived cell lines and human primary hepatocytes and hepatic stellate cells in response to either natural or synthetic FXR ligands. Transient transfection studies and electrophoretic mobility shift assays revealed a functional FXR response element located in intron 1 of the human alphaA-crystallin gene. Importantly, immunohistochemical staining of human liver sections showed increased alphaA-crystallin expression in cholangiocytes and hepatocytes. As a member of the small heat shock protein family possessing chaperone-like activity, alphaA-crystallin may be involved in protection of hepatocytes from the toxic effects of high concentrations of bile acids, as would occur in disease states such as cholestasis.

Influence of hormones and growth factors on lens protein composition: the effect of dexamethasone and PDGF-AA.

PURPOSE: To investigate the effect of hormones and ocular growth factors on the expression of alpha-, beta-, and gamma-crystallins in rat lens epithelial and fiber cells. METHODS: PDGF-AA, EGF, NGF, M-CSF, BMP-2, BMP-4, dexamethasone, and estrogen were tested for their ability to alter the spectrum of crystallins in explanted newborn rat lens epithelial cells or in vitro differentiating newborn rat lens fiber cells. The accumulation of alphaA-, aB-, betaA3/1-, betaB2-, and gamma-crystallin was measured by western blot and dot blot analysis. The morphology of the rat lens explants after culture was examined by hematoxylin-eosin staining, while crystallins were localized by immunofluorescence. RESULTS: Only dexamethasone and PDGF-AA showed an effect on relative crystallin levels. In the presence of dexamethasone the amount of alphaB-crystallin was increased in lens epithelial cells, but dexamethasone did not affect the crystallin spectrum in fiber cells. In rat lens epithelial explants cultured with PDGF-AA an increase in beta- and gamma-crystallin expression was seen. The spectrum of beta- and gamma-crystallins synthesized differed from that present in lens fiber cells. The cells expressing beta- and gamma-crystallin after culture with PDGF-AA were scattered in the epithelial cell layer and retained an epithelial morphology. PDGF-AA did not change the spectrum of crystallins synthesized in lens fiber cells but did enhance the rate of fiber cell differentiation, in agreement with results of others. CONCLUSIONS: Both dexamethasone and PDGF-AA influence crystallin gene expression in cultured rat lens epithelial cells. Dexamethasone enhances the expression of alphaB-crystallin while culturing in the presence of PDGF-AA caused an increase in beta- as well as gamma-crystallin synthesis. Since at least the gamma-crystallin genes are known to be silenced in epithelial cells by DNA methylation, PDGF-AA may be able to induce one of the steps towards fiber cell differentiation in some epithelial cells.

Activation of metallothioneins and alpha-crystallin/sHSPs in human lens epithelial cells by specific metals and the metal content of aging clear human lenses.

PURPOSE: To identify those metallothionein and alpha-crystallin/small heat-shock genes induced by toxic metals in human lens cells and to evaluate the levels of these metals between young and aged human lenses. METHODS: Human SRA01/04 and primary human lens epithelial cells were cultured and exposed to Cd(2+), Cu(2+), and Zn(2+). The levels of lens metallothioneins (Ig, If, Ih, Ie, and IIa) and alpha-crystallin/small heat-shock (alphaA-crystallin, alphaB-crystallin, and HSP27) genes were analyzed by semiquantitative and quantitative competitive RT-PCR. The content of aluminum, cadmium, calcium, chromium, copper, iron, lead, magnesium, manganese, nickel, potassium, sodium, and zinc in young (mean, 32.8 years), middle-aged (mean, 52.3 years), and old (mean, 70.5 years) human lenses was analyzed by inductively coupled plasma-emission spectroscopy. RESULTS: Lens metallothioneins (Ig, If, Ih, Ie, and IIa) and alpha-crystallin/small heat-shock genes (alphaA-crystallin, alphaB-crystallin, and HSP27) were differentially induced by specific metals in SRA01/04 human lens epithelial cells. Cd(2+) and Zn(2+), but not Cu(2+), induced the metallothioneins, whereas Cd(2+) and Cu(2+), but not Zn(2+), induced alphaB-crystallin and HSP27. alphaA-crystallin was induced by Cu(2+) only. Similar responses of the metallothionein IIa gene were detected in identically treated primary human lens epithelial cells. Cd(2+) and Zn(2+) induced metallothionein IIa to five times higher levels than metallothionein Ig. Of 13 different metals, only iron was altered, exhibiting an 81% decrease in old versus young lenses. CONCLUSIONS: Induction of metallothioneins and alpha-crystallin/small heat shock proteins by different metals indicates the presence of metal-specific lens regulatory pathways that are likely to be involved in protection against metal-associated stresses.