Subtoxic oxidative stress induces senescence in retinal pigment epithelial cells via TGF-beta release.

PURPOSE: The goal of the present study was to determine whether oxidative stress and transforming growth factor (TGF)-beta induce cellular senescence in human retinal pigment epithelial (RPE) cells. METHODS: Cultured human RPE cells were exposed to 50 to 150 microM hydrogen peroxide (H(2)O(2)) for 1 and 2 hours or treated with 1.0 ng/mL TGF-beta1 or -beta2 for 12, 24, and 48 hours. Senescence-associated beta-galactosidase (SA-beta-Gal) activity was detected by histochemical staining. Expression of senescence-associated genes (apolipoprotein J [Apo J], connective tissue growth factor [CTGF], fibronectin, and SM22) was examined by real-time PCR and induction of signal transduction proteins (p21, p16, and pRb) by Western blot analysis. The effects of TGF-beta blocking on the oxidative stress-induced expression of senescence-associated biomarkers were investigated by simultaneous incubation with neutralizing antibodies against the TGF-beta1, -beta2, and -beta3 isoforms and the TGF-betaII receptor. RESULTS: H(2)O(2) markedly increased the number of SA-beta-Gal-positive cells to up to 89% and the expression of Apo J, CTGF, fibronectin, and SM22 by approximately three to fourfold. Treatment with TGF-beta1 and -beta2 showed similar changes. H(2)O(2)and TGF-beta1 and -beta2 markedly enhanced the expression of p21 but downregulated pRb. In contrast, they had no effect on p16 expression. Simultaneous treatment with neutralizing antibodies against the TGF-beta1, -beta2, and -beta3 isoforms and the TGF-betaII receptor prevented the oxidative stress-mediated elevation of senescence-associated biomarkers. CONCLUSIONS: Oxidative stress, TGF-beta1, and TGF-beta2 are capable of inducing cellular senescence in cultured human RPE cells. Therefore, reduction of oxidative stress and minimizing TGF-beta may help to prevent senescence-associated changes in the RPE as seen in early age-related macular degeneration.

Reactivation of optic nerve head astrocytes by TGF-beta2 and H2O2 is accompanied by increased Hsp32 and Hsp47 expression.

PURPOSE: Histologic studies have previously demonstrated increased expression of small heat shock proteins (Hsps) in reactive optic nerve head (ONH) astrocytes of patients with glaucoma. Transforming growth factor (TGF)-beta2 and hydrogen peroxide (H(2)O(2)) are known to induce ONH astrocyte reactivation. The goal of the present study was to determine whether new potentially involved Hsps, such as Hsp32, -47, -60, and -70, are expressed in the reactivation process of ONH astrocytes mediated by TGF-beta2 and H(2)O(2). METHODS: Cultured human ONH astrocytes were treated with 1.0 ng/mL TGF-beta2 for up to 48 hours. In addition, the cells were exposed to 100, 200, or 400 microM H(2)O(2) for 1 hour. Expression of Hsp32, -47, -60, and -70 was examined by immunohistochemistry, real-time PCR, and Western blot analyses. RESULTS: Treatment with TGF-beta2 increased Hsp32 after 4 and 6 hours, whereas Hsp47 was upregulated after treatment with TGF-beta2 for 12, 24, and 48 hours. Exposure of the cells to H(2)O(2) could increase both Hsp32 and -47. No significant effects on the expression of Hsp60 and -70 were observed after treatment of the cells with TGF-beta2 or H(2)O(2). CONCLUSIONS: TGF-beta2 increased Hsp32 after short-term treatment and Hsp47 after longer periods in cultured human ONH astrocytes. H(2)O(2) increased both Hsp32 and -47 levels. No effects on Hsp60 and -70 levels were induced by TGF-beta2 and H(2)O(2). These results may provide further insights into the cellular stress responses of reactive human ONH astrocytes. Further extensive studies are needed to examine the potential roles of Hsps in the ONH of glaucomatous eyes.

Oxidative stress and TGF-beta2 increase heat shock protein 27 expression in human optic nerve head astrocytes.

PURPOSE: Reactive astrocytes of glaucomatous optic nerve heads (ONHs) are characterized by an increased expression of transforming growth factor (TGF)-beta2 and heat shock proteins (Hsps) such as Hsp27. The goal of the present study was to determine the effect of oxidative stress on TGF-beta2 and Hsp27 expression in human ONH astrocytes. METHODS: Cultured ONH astrocytes were exposed to 100, 200, and 400 muM hydrogen peroxide (H(2)O(2)) for 1 hour. Levels of TGF-beta2 were analyzed by ELISA. Additionally, cells were treated with 1.0 ng/mL TGF-beta2 for 12, 24, and 48 hours, respectively. Expression of Hsp27 was detected by immunohistochemistry, real-time PCR, and Western blot analyses. To investigate the role of signal transduction proteins, cells were pretreated with the specific inhibitors SB203580, PD98059, U0126, and SP600125. Furthermore, induction of p38MAP kinase and its phosphorylated form were determined by Western blot analyses. RESULTS: H(2)O(2) exposure increased the secretion of TGF-beta2. Both H(2)O(2) and TGF-beta2 increased the Hsp27 expression. Pretreatment of cells with SB203580, an inhibitor for p38MAP kinase, reduced the H(2)O(2)- and TGF-beta2-stimulated Hsp27 expression, whereas pretreatment with PD98059 and U0126, specific inhibitors of ERK1/2, and SP600125, a specific c-Jun N-terminal kinase inhibitor, had no effects. H(2)O(2) and TGF-beta2 upregulated the phosphorylated p38MAP kinase. CONCLUSIONS: Oxidative stress increased TGF-beta2 secretion in cultured human ONH astrocytes. Both H(2)O(2) and TGF-beta2 increased Hsp27 expression via p38MAP kinase activation. Therefore, it is tempting to speculate that reduction of oxidative stress and TGF-beta2 may help to minimize the incidence of characteristic changes in the ONH of patients with glaucoma.

Tissue transglutaminase catalyzes the deamidation of glutamines in lens betaB(2)- and betaB(3)-crystallins.

Tissue transglutaminase (tTG) is a Ca(2+)-dependent enzyme catalyzing the formation of covalent crosslinks between peptide-bound glutamine and lysine residues. Lens crystallins, including alphaB-crystallin and several beta-crystallins, are in vitro substrates for tTG. In both human and bovine fetal lens extracts treated with commercially available guinea pig liver tTG we detected the formation of high molecular weight (HMW) aggregates containing crosslinked betaB(2)- and betaA(3)-crystallin. More interestingly, 2D-gel electrophoresis combined with mass spectrometry analysis revealed that glutamines present in the N-terminal arms of betaB(2)- and betaB(3)-crystallins deamidate readily in the presence of tTG. We found that both tTG-catalyzed crosslinking and deamidation disrupt the beta-crystallin complex, suggesting that these tTG-catalyzed modifications can influence the macromolecular assembly of lens crystallins. These data together suggest that tTG can contribute to the age-related deamidation of glutamine residues of lens crystallins.

Hypoxia/reoxygenation and TGF-beta increase alphaB-crystallin expression in human optic nerve head astrocytes.

Reactive astrocytes in glaucomatous optic nerve changes are characterized by an increased expression of alphaB-crystallin and transforming growth factor-beta (TGF-beta). In the pathogenesis of glaucomatous optic nerve damage, ischemia/reperfusion injury may play an important role. The goal of the present study was to determine the influence of hypoxia/reoxygenation and TGF-beta on the expression of alphaB-crystallin in cultured human astrocytes of the optic nerve head (ONH). Cultured human astrocytes were incubated under hypoxic conditions (1% O2 for 4-12 h) with subsequent reoxygenation (12-24 h). Additionally, cells were treated with 1.0 ng/ml TGF-beta1 and TGF-beta2 for 12-48 h. Expression of alphaB-crystallin was examined by Northern- and Western-blotting. Levels of TGF-beta1 and TGF-beta2 were analyzed by RT-PCR analysis and ELISA. The effect of TGF-beta blocking on the hypoxia/reoxygenation modulated expression of alphaB-crystallin was investigated by simultaneous incubation with neutralizing antibodies against TGF-beta during the reoxygenation phase. Hypoxia/reoxygenation increased the expression of alphaB-crystallin at the mRNA (2.8- to 3.1-fold) and protein level (1.8- to 2.1-fold). Treatment with 1.0 ng/ml TGF-beta1 and TGF-beta2 for 12-48 h markedly enhanced alphaB-crystallin mRNA expression approximately three- to fourfold. Using Western blot analysis, this increase ranged from 2 to 3 times. Both cytokines showed a twofold increase after 12 and 24 h of reoxygenation at the mRNA and a two- to threefold increase at the protein level. Simultaneous treatment with neutralizing antibodies against both TGF-beta isoforms prevented the hypoxia/reoxygenation-mediated elevation of alphaB-crystallin. The process of hypoxia/reoxygenation is capable of inducing the expression of alphaB-crystallin and TGF-ss in cultured ONH astrocytes. Therefore, optimization of conditions leading to hypoxia/reoxygenation in the ONH of glaucomatous patients may help to lower the incidence of characteristic changes in the optic nerve.

Structural and immunocytochemical alterations in eye lens fiber cells from Cx46 and Cx50 knockout mice.

In the current study we describe the changes of overall organization of lens fiber cells in connexin 46 (Cx46) and connexin 50 (Cx50) knockout mice. Morphometric analyses and the application of immunocytochemical techniques revealed that in Cx46 knockout lens (Cx46 -/-), where Cx50 is expressed alone, the postnatal differentiation of secondary fiber cells proceeds faster and is characterized by an increased number of smaller fiber cells. Conversely, in Cx50 knockout mice (Cx50 -/-), the lenticular mass is considerably reduced and characterized by a small number of fiber cells added during the postnatal period. The process of terminal differentiation was impaired and generated larger fiber cells still possessing cytoplasmic organelles. Freeze-fracture and fracture labeling revealed that the junctional assembly, packing organization and topographic interactions between connexons and MP26 differed when Cx46 and Cx50 were co-assembled in the wild-type or expressed separately in the two distinct knockout phenotypes. Filipin cytochemistry provided indirect evidence that Cx46 and Cx50 expressed alone are recruited into different lipid environments. Our results represent the structural proof that interaction of connexins and MP26 contributes to the overall organization of the fiber cells.

Ageing and vision: structure, stability and function of lens crystallins.

The alpha-, beta- and gamma-crystallins are the major protein components of the vertebrate eye lens, alpha-crystallin as a molecular chaperone as well as a structural protein, beta- and gamma-crystallins as structural proteins. For the lens to be able to retain life-long transparency in the absence of protein turnover, the crystallins must meet not only the requirement of solubility associated with high cellular concentration but that of longevity as well. For proteins, longevity is commonly assumed to be correlated with long-term retention of native structure, which in turn can be due to inherent thermodynamic stability, efficient capture and refolding of non-native protein by chaperones, or a combination of both. Understanding how the specific interactions that confer intrinsic stability of the protein fold are combined with the stabilizing effect of protein assembly, and how the non-specific interactions and associations of the assemblies enable the generation of highly concentrated solutions, is thus of importance to understand the loss of transparency of the lens with age. Post-translational modification can have a major effect on protein stability but an emerging theme of the few studies of the effect of post-translational modification of the crystallins is one of solubility and assembly. Here we review the structure, assembly, interactions, stability and post-translational modifications of the crystallins, not only in isolation but also as part of a multi-component system. The available data are discussed in the context of the establishment, the maintenance and finally, with age, the loss of transparency of the lens. Understanding the structural basis of protein stability and interactions in the healthy eye lens is the route to solve the enormous medical and economical problem of cataract.

Nuclear staining for the small heat shock protein alphaB-crystallin colocalizes with splicing factor SC35.

AlphaB-Crystallin has for a long time been considered a specific eye lens protein. Later on it appeared that this protein belongs to the family of the small heat shock proteins and that it occurs also extra-lenticularly in many different cell types. AlphaB-Crystallin is mainly present in the cytoplasm, but there are some indications that it might have a function in the nucleus too. However, till now its presence in the nucleus is uncertain. We therefore compared the localization of alphaB-crystallin in nine cell lines cultured under normal conditions using four different antisera. All four antisera gave a diffuse staining for alphaB-crystallin in the cytoplasm, but one of the antibodies consistently showed nuclear staining in eight of the cell types, in the form of distinct speckles. These speckles are equally pronounced in the different cell types, whether or not cytoplasmic alphaB-crystallin is present. Preabsorption of the antiserum with alphaB-crystallin abolished the staining. Furthermore we demonstrate that if only minor amounts of alphaB-crystallin are present, the protein seems to be located exclusively in the nucleus. However, in case of higher amounts of protein, alphaB-crystallin is distributed between cytoplasm and nucleus. The nuclear alphaB-crystallin exists, like the cytoplasmic alphaB-crystallin, in non-phosphorylated and phosphorylated forms, is Triton-insoluble but can be extracted by 2 M NaCl. These data suggest that alphaB-crystallin might be bound to the nuclear matrix per se or to nuclear matrix proteins via other proteins. In agreement with other nuclear matrix proteins, nuclear alphaB-crystallin staining turns diffuse upon mitosis and leaves the chromosomes unstained. Double staining experiments revealed colocalization of alphaB-crystallin with the splicing factor SC35 in nuclear speckles, suggesting a role for alphaB-crystallin in splicing or protection of the splicing machinery.

Molecular mechanisms of exon shuffling: illegitimate recombination.

Illegitimate recombination (IR) is a process that takes place far more often than homologous recombination and is characterized by the recombination between non-homologous or short homologous sequences. The consequences of IR frequently emerge after the introduction of DNA in cell lines because it more frequently integrates in non-homologous than in homologous regions of the host genome. As a result, unexpected truncated or elongated products may be found. By not discarding those products as transfection artifacts, but by studying how they are generated, it might elucidate a possible molecular mechanism of IR. Here we review the current literature describing different mechanisms by which non-homologous DNA recombination can be induced.

Retinal pigment epithelium is protected against apoptosis by alphaB-crystallin.

PURPOSE: The degeneration of retinal pigment epithelial (RPE) cells is considered to be a crucial event in the pathophysiology of age-related macular degeneration (AMD). Cumulative oxidative damage has been implicated in the development of the changes seen in AMD. The present study was undertaken to evaluate the expression of the small heat shock protein alphaB-crystallin in the RPE in response to oxidative stress and to explore whether alphaB-crystallin expression confers an antiapoptotic cytoprotective effect on RPE cells. METHODS: Native human RPE cells from the macula and retinal periphery were analyzed by RT-PCR and Western blot analysis for expression of alphaB-crystallin. Monolayer cultures of human RPE cells were stressed by heat shock (42 degrees C for 20 minutes) or oxidant-mediated injury (50-300 micro M H(2)O(2) for 1 hour). Induction of alphaB-crystallin and the corresponding mRNA was assessed by Western and Northern blot analyses. To study the cytoprotective effect of alphaB-crystallin, human RPE cells were transfected with either a neomycin-selectable expression vector containing alphaB-crystallin cDNA or a control vector without alphaB-crystallin cDNA. Caspase-3 activity was determined by observing the cleavage of a colorimetric peptide substrate. Cell viability was quantified by combined propidium iodide and Hoechst 33342 staining. RESULTS: alphaB-crystallin is constitutively expressed in RPE under in vivo and in vitro conditions. Western blot analysis of freshly isolated RPE showed greater baseline expression levels in RPE derived from the macular area than in that from the more peripheral regions. Heat shock treatment and oxidative stress caused a significant increase in alphaB-crystallin mRNA and protein. Oxidant-mediated injury in RPE cells with baseline expression levels of alphaB-crystallin resulted in apoptotic cell death, as measured by caspase-3 activity, whereas RPE cells that had been stably transfected with alphaB-crystallin were more resistant to H(2)O(2)-induced cellular injury. CONCLUSIONS: alphaB-crystallin may function as a stress-inducible antiapoptotic protein in human RPE and is inducible by oxidative stress, a condition implicated in the pathogenesis of AMD. Overexpression of alphaB-crystallin may be an important mechanism for the RPE to prevent apoptotic cell death in response to cellular stress.