Cell signaling pathways to alphaB-crystallin following stresses of the cytoskeleton

Small heat shock proteins (sHSPs) act as chaperone, but also in protecting the different cytoskeletal components. Recent results suggest that αB-crystallin, a member of sHSPs family, might regulate actin filament dynamics, stabilize them in a phosphorylation dependent manner, and protect the integrity of intermediate filaments (IF) against extracellular stress. We demonstrate that vinblastin and cytochalasin D, which respectively disorganize microtubules and actin microfilaments, trigger the activation of the p38/MAPKAP2 kinase pathway and lead to the specific αB-crystallin phosphorylation at serine 59. Upstream of p38, we found that RhoK, PKC and PKA are selectively involved in the activation of p38 and phosphorylation of αB-crystallin, depending on the cytoskeletal network disorganized. Moreover, we demonstrate that chronic perturbations of IF network result in the same activation of p38 MAPK and αB-crystallin phosphorylation, as with severe disorganization of other cytoskeletal networks. Finally, we also show that Ser 59 phosphorylated αB-crystallin colocalizes with cytoskeletal components. Thus, disturbance of cytoskeleton leads by converging signaling pathways to the phosphorylation of αB-crystallin, which probably acts as a protective effector of the cytoskeleton.     

alphaB-crystallin is phosphorylated during myocardial infarction: involvement of platelet-derived growth factor-BB

alphaB-crystallin is the most abundant low-molecular-weight heat shock protein in heart and recent studies have demonstrated that it plays a cardioprotective role during myocardial infarction both in vivo and in vitro. On the other hand, platelet-derived growth factor (PDGF), a potent serum mitogen, has been reported to improve cardiac function after myocardial infarction. In the present study, using a mouse myocardial infarction model, we investigated whether alphaB-crystallin is phosphorylated during myocardial infarction and the implication of PDGF-BB. Phosphorylation of alphaB-crystallin at Ser-59 was time dependently induced and plasma PDGF-BB levels were concomitantly increased. Moreover, PDGF-BB-stimulated phosphorylation of alphaB-crystallin was suppressed by SB203580, a specific inhibitor of p38 mitogen-activated protein (MAP) kinase, in primary cultured cardiac myocytes. Our results indicate that PDGF-BB induces phosphorylation of alphaB-crystallin via p38 MAP kinase during myocardial infarction.     

Rho-kinase induces association of adducin with the cytoskeleton in platelet activation

We examined whether adducin function is regulated through Rho-kinase after agonist stimulation in platelets. A variety of stimuli such as thrombin, STA(2) (a stable analog of TXA(2)), Ca(2+) ionophore, phorbol diester, and shear stress induced phosphorylation of alpha-adducin at Thr445. Preincubation with the Rho-kinase inhibitor Y-27632 in platelets inhibited agonist-induced phosphorylation of alpha-adducin. STA(2) stimulation led to a redistribution of adducin from Triton-insoluble (high speed) fraction (membrane skeleton) to Triton-insoluble (low speed) fraction (cytoskeleton) and detergent-soluble fraction. Phosphoadducin at Thr445 was selectively isolated in the cytoskeletal fraction, whereas phosphoadducin at Ser726 was mainly present in the Triton-soluble fraction. Y-27632 inhibition of STA(2)-induced alpha-adducin phosphorylation at Thr445 inhibited incorporation of alpha-adducin and spectrin into the platelet cytoskeleton, although Y-27632 did not affect phosphorylation of alpha-adducin at Ser726. These results suggest that Rho-kinase regulates the association of alpha-adducin and spectrin with the actin cytoskeleton in platelet activation.     

Interactions between lens epithelial and fiber cells reveal an intrinsic self-assembly mechanism

How tissues and organs develop and maintain their characteristic three-dimensional cellular architecture is often a poorly understood part of their developmental program; yet, as is clearly the case for the eye lens, precise regulation of these features can be critical for function. During lens morphogenesis cells become organized into a polarized, spheroidal structure with a monolayer of epithelial cells overlying the apical tips of elongated fiber cells. Epithelial cells proliferate and progeny that shift below the lens equator differentiate into new fibers that are progressively added to the fiber mass. It is now known that FGF induces epithelial to fiber differentiation; however, it is not fully understood how these two forms of cells assemble into their characteristic polarized arrangement. Here we show that in FGF-treated epithelial explants, elongating fibers become polarized/oriented towards islands of epithelial cells and mimic their polarized arrangement in vivo. Epithelial explants secrete Wnt5 into the culture medium and we show that Wnt5 can promote directed behavior of lens cells. We also show that these explants replicate aspects of the Notch/Jagged signaling activity that has been shown to regulate proliferation of epithelial cells in vivo. Thus, our in vitro study identifies a novel mechanism, intrinsic to the two forms of lens cells, that facilitates self-assembly into the polarized arrangement characteristic of the lens in vivo. In this way the lens, with its relatively simple cellular composition, serves as a useful model to highlight the importance of such intrinsic self-assembly mechanisms in tissue developmental and regenerative processes.     

alpha-Crystallin localizes to the leading edges of migrating lens epithelial cells

alpha-crystallin (alphaA and alphaB) is a major lens protein, which belongs to the small heat-shock family of proteins and binds to various cytoskeletal proteins including actin, vimentin and desmin. In this study, we investigated the cellular localization of alphaA and alphaB-crystallins in migrating epithelial cells isolated from porcine lens. Immunofluorescence localization and confocal imaging of alphaB-crystallin in confluent and in migrating subconfluent cell cultures revealed a distinct pattern of subcellular distribution. While alphaB-crystallin localization was predominantly cytoplasmic in confluent cultures, it was strongly localized to the leading edges of cell membrane or the lamellipodia in migrating cells. In accordance with this pattern, we found abundant levels of alphaB-crystallin in membrane fractions compared to cytosolic and nuclear fractions in migrating lens epithelial cells. alphaA-crystallin, which has 60% sequence identity to alphaB-crystallin, also exhibited a distribution profile localizing to the leading edge of the cell membrane in migrating lens epithelial cells. Localization of alphaB-crystallin to the lamellipodia appears to be dependent on phosphorylation of residue serine-59. An inhibitor of p38 MAP kinase (SB202190), but not the ERK kinase inhibitor PD98059, was found to diminish localization of alphaB-crystallin to the lamellipodia, and this effect was found to be associated with reduced levels of Serine-59 phosphorylated alphaB-crystallin in SB202190-treated migrating lens epithelial cells. alphaB-crystallin localization to the lamellipodia was also altered by the treatment with RGD (Arg-Ala-Asp) peptide, dominant negative N17 Rac1 GTPase, cytochalasin D and Src kinase inhibitor (PP2), but not by the Rho kinase inhibitor Y-27632 or the myosin II inhibitor, blebbistatin. Additionally, in migrating lens epithelial cells, alphaB-crystallin exhibited a clear co-localization with the actin meshwork, beta-catenin, WAVE-1, a promoter of actin nucleation, Abi-2, a component of WAVE-1 protein complex and Arp3, a protein of the actin nucleation complex, suggesting potential interactions between alphaB-crystallin and regulatory proteins involved in actin dynamics and cell adhesion. This is the first report demonstrating specific localization of alphaA and alphaB-crystallins to the lamellipodia in migrating lens epithelial cells and our findings indicate a potential role for alpha-crystallin in actin dynamics during cell migration.     

Methylglyoxal induces cellular damage by increasing argpyrimidine accumulation and oxidative DNA damage in human lens epithelial cells

Methylglyoxal (MGO) is a cytotoxic metabolite and modifies tissue proteins through the Maillard reaction, resulting in advanced glycation end products (AGEs), which can alter protein structure and functions. Several MGO-derived AGEs have been described, including argpyrimidine, a fluorescent product of the MGO reaction with arginine residues. Herein, we evaluated the cytotoxic role of MGO in human lens epithelial cell line (HLE-B3). HLE-B3 cells were exposed to 400 μM MGO in the present or absence of pyridoxamine for 24 h. We then examined the formation of argpyrimidine, apoptosis and oxidative stress in HLE-B3 cells. In MGO-treated HLE-B3 cells, the accumulation of argpyrimidine was markedly increased, and caspase-3 and 8-hydroxydeoxyguanosine (8-OHdG) were highly expressed, which paralleled apoptotic cell death. However, pyridoxamine (AGEs inhibitor) prevented the argpyrimidine formation and apoptosis of MGO-treated HLE-B3 cells. These results suggested that the accumulation of argpyrimidine and oxidative DNA damage caused by MGO are involved in apoptosis of HLE-B3 cells.     

Somatostatin induces rapid contraction of neuroendocrine cells

The peptide hormone somatostatin, as well as the somatostatin analog octreotide, induces rapid morphological changes in neuroendocrine cells. The effect can be detected in less than 2 min: retraction fibers are formed, cells round up and cell-cell contacts are broken. Somatostatin-dependent cell contraction is inhibited by Y-27632, indicating that this effect is dependent on Rho kinase. In BON1 cells, the somatostatin-induced inhibition of forskolin-induced secretion of chromogranin A is not blocked by Y-27632. It is therefore concluded that the inhibitory effect of somatostatin in forskolin-stimulated cells is not dependent on cell contraction.     

Nonchromatin nuclear proteins of mammalian lens epithelial cells

The nuclear matrix (NM) proteins of six tissue cultured lens epithelial cell lines and one embryonic rabbit epidermal cell line were analyzed to determine possible tissue and species specificity of these proteins. The NM proteins were isolated by the modified Penman technique. The tissue cultured cells were pulsed with [³⁵S] methionine and nuclear matrix proteins were fractionated by two-dimensional (2-D) gel electrophoresis. The 2-D gels were dried and autoradiographed. The relative abundance of spot patterns of nuclear matrix proteins of different cells were compared. The data from these experiments revealed that all the examined cell lines have distinct spot patterns, however, all of NM profile showed a spot pattern in the 45 kDa region with acidic pH. Some of these spots cross-reacted with anti-vimentin antibodies, whereas a prominent protein spot in this region did not cross react with either vimentin or actin antibodies. The observed variations in the NM protein patterns of lens epithelial cells may reflect tissue and species specificity and also a role in the regulatory properties of these nuclear proteins in the eye tissue development. J. Cell. Biochem. 64:644–650. © 1997 Wiley-Liss, Inc.     

Rho GDP dissociation inhibitor-mediated disruption of Rho GTPase activity impairs lens fiber cell migration, elongation and survival

To explore the role of the Rho GTPases in lens morphogenesis, we overexpressed bovine Rho GDP dissociation inhibitor (RhoGDIα), which serves as a negative regulator of Rho, Rac and Cdc42 GTPase activity, in a lens-specific manner in transgenic mice. This was achieved using a chimeric promoter of δ-crystallin enhancer and αA-crystallin, which is active at embryonic day 12. Several individual transgenic (Tg) lines were obtained, and exhibited ocular specific phenotype comprised of microphthalmic eyes with lens opacity. The overexpression of bovine RhoGDIα disrupted membrane translocation of Rho, Rac and Cdc42 GTPases in Tg lenses. Transgenic lenses also revealed abnormalities in the migration pattern, elongation and organization of lens fibers. These changes appeared to be associated with impaired organization of the actin cytoskeleton and cell-cell adhesions. At E14.5, the size of the RhoGDIα Tg lenses was larger compared to wild type (WT) and the central lens epithelium and differentiating fibers exhibited an abnormal increase of bromo-deoxy-uridine incorporation. Postnatal Tg eyes, however, were much smaller in size compared to WT eyes, revealing increased apoptosis in the disrupted lens fibers. Taken together, these data demonstrate a critical role for Rho GTPase-dependent signaling pathways in processes underlying morphogenesis, fiber cell migration, elongation and survival in the developing lens.     

Rat lens epithelial cells in vitro

Summary Lens epithelial cells from rats aged 5 days were grown in long-term cultures. These cells age, differentiate and transform spontaneously. Morphological observations indicate five different stages (A-E). The epithelial character is lost after the first two passages. Elongated cells appearing afterwards are considered as cells that have started differentiation to fiberlike cells. Big flattened cells are considered as senescent cells that have lost their proliferative capacity. Data from population kinetics also reflect these five stages. Chromosome analysis shows that three of the five stages are no longer diploid. Two alternative modes of spontaneous transformation are possible. The proliferative capacity of rat lens epithelial cells is higher than that of rat embryonic fibroblast systems. The investigations were supported by the Deutsche Forschungsgemeinschaft (Biology of Aging, Grants Ri 285/2 and Ri 285/3).     

Rat lens epithelial cells in vitro

Summary Serially subcultured rat lens epithelial cells grow in different stages, which can be classified according to morphology, chromosome numbers and population kinetics. A lensspecific γ-crystallin appears in the diploid stage, when elongated cell types are observed. One of the β-crystallin bands (pH 5.7) disappears during aging in higher passage numbers of the diploid stage B. A weak band in the β-crystallin region (pH 6.4), which is present in all stages, becomes very intensive in aneuploid cells of stage D, which exhibit a fibroblast-like morphology. The work was supported by Deutsche Forschungsgemeinschaft, Grant Ri 285/3.     

AMPK induces MUC5B expression via p38 MAPK in NCI-H292 airway epithelial cells

Adenosine monophosphate-activated protein kinase (AMPK) is a well-known serine/threonine kinase that has been implicated in modulation of glucose and fatty acid metabolism. Recent reports have also implicated AMPK in modulation of mucin secretion. In this study, the effects and signaling pathways of AMPK on MUC5B expression were investigated in human NCI-H292 airway epithelial cells. Metformin, as an activator of AMPK, induced MUC5B expression in a dose-dependent manner. Compound C, as an inhibitor of AMPK, inhibited metformin-induced MUC5B expression in a dose-dependent manner. Metformin significantly activated phosphorylation of AMPK; compound C inhibited metformin-activated phosphorylation of AMPK. Without treatment with metformin, there was no difference in MUC5B mRNA expression between Ad-dnAMPK transfected and wild-type adenovirus transfected NCI-H292 cells. However, after treatment with metformin, MUC5B mRNA expression was increased in wild-type adenovirus transfected NCI-H292 cells; MUC5B mRNA expression was significantly decreased in Ad-dnAMPK transfected NCI-H292 cells. Metformin activated phosphorylation of p38 mitogen-activated protein kinase (MAPK); compound C inhibited metformin-activated phosphorylation of p38 MAPK. SB203580, as an inhibitor of p38 MAPK, significantly inhibited metformin-induced MUC5B mRNA expression, while U0126, as an inhibitor of ERK1/2 MAPK, had no effect. In addition, knockdown of p38 MAPK by p38 MAPK siRNA significantly blocked metformin-induced MUC5B mRNA expression. In conclusion, results of this study show that AMPK induces MUC5B expression through the p38 MAPK signaling pathway in airway epithelial cells.     

Post-translational modifications in host cells during bacterial infection

Post-translational modification of proteins is a widespread mechanism used by both prokaryotic and eukaryotic cells to modify the activity of key factors that plays fundamental roles in cellular physiology. This review focuses on how bacterial pathogens can interfere with host post-translational modifications to promote their own survival and replication.     

Intracellular protein degradation in cultured bovine lens epithelial cells

Summary Although several proteases have been identified in homogenates of cultured epithelial cells of the eye lens and in lens tissues, there is little information regarding intracellular protein degradation in intact lens cells in vitro. Cultured lens cells may be useful in the study of intracellular protein degradation in the lens, a tissue with a wide range of protein half-lives. This is of interest because alterations in protein turnover in the lens have been implicated in cataract formation. This study examines intracellular protein degradation in cultured bovine lens epithelial cells (BLEC). Cell cultures were incubated with radiolabeled leucine to label intracellular proteins. Protein degradation was measured by monitoring the release of trichloroacetic-acid-soluble radioactivity into the culture medium. The average half-life of long-lived proteins (half-life >50 h) was typically about 57 h in serum-supplemented medium. Average rates of degradation of long-lived proteins increased by up to 73% when fetal bovine serum was withdrawn from the culture medium. Serum had no effect on the degradation of short-lived proteins (half-life <10 h). Degradation of long-lived proteins in the presence and absence of serum was further studied in cultured BLEC from population doubling level (PDL) 2 to 43. Average half-life of proteins in serum-supplemented medium was 52 to 58 h and did not vary significantly as a function of PDL. Degradation rates in serum-free medium increased approximately twofold up to PDL 7, but returned by PDL 25 to original levels, which were maintained through PDL 43. This work was supported in part by grants from U. S. Department of Agriculture contract 53-3K06-5-10, Massachusetts Lions Eye Research Fund, Inc., and the Daniel and Florence Guggenheim Foundation. D. A. E. is a recipient of a National Eye Institute postdoctoral fellowship.     

Lamins of ocular lens epithelial cells

Experiments were performed to characterize a prominent nuclear matrix (NM) protein isolated from tissue cultured mouse lens epithelial cells. This NM protein was separated by SDS-PAGE and the stained gel band was analyzed by mass spectroscopy. Blast analysis of the amino acid sequence derived by mass spectroscopy revealed the presence of Lamin C in the NM of the mouse lens epithelial cells. We also examined nuclear proteins of adult and fetal human lenses. Data collected from these experiments showed the presence of Lamin C in both adult and fetal lens cells. However fetal lens cells only show Lamin C dimers, whereas adult human lens contained dimers, monomers and degraded Lamin C. Early and late passaged tissue cultured mouse lens epithelial cells also contained Lamin C in the nucleus with a preponderance of the dimer in the early passaged cells. The biological significance of the presence of dimers in human fetal lens cells and early passaged mouse lens cells is not known. However, it could suggest an enhanced docking capability of Lamin C dimers for other physiologically important nuclear proteins.     

Effect of acrylamide on the cytoskeleton and apoptosis of bovine lens epithelial cells

Acrylamide, a known disrupter of intermediate filaments, has been used to produce the collapse of vimentin filaments in bovine lens epithelial (BEL) cells, and its potential modulation of staurosporine-induced apoptosis has been investigated. In BEL cells, short treatments with acrylamide caused the collapse of vimentin filaments and microtubules and the almost complete disappearance of stress fibers, with thick f-actin bundles remaining in the cell periphery. Actin organization was less affected in cells pretreated with colchicine and in spreading cells, suggesting that extended microtubules and vimentin filaments are required for acrylamide to produce its maximal effects. Acrylamide alone slightly increased apoptosis compared to controls. However, simultaneous exposure to acrylamide and staurosporine for 8h produced significantly less apoptosis than staurosporine alone, and preincubation with acrylamide followed by staurosporine markedly reduced apoptosis at 8 and 24h of treatment. Acrylamide seems therefore to have a dual effect on BEL cell survival.     

Insulin utilizes the PI 3-kinase pathway to inhibit SP-A gene expression in lung epithelial cells

Background  

It has been proposed that high insulin levels may cause delayed lung development in the fetuses of diabetic mothers. A key event in lung development is the production of adequate amounts of pulmonary surfactant. Insulin inhibits the expression of surfactant protein A (SP-A), the major surfactant-associated protein, in lung epithelial cells. In the present study, we investigated the signal transduction pathways involved in insulin inhibition of SP-A gene expression.     

Aldosterone induces myofibroblastic transdifferentiation and collagen gene expression through the Rho-kinase dependent signaling pathway in rat mesangial cells

There is accumulating evidence indicating the role of aldosterone in the pathogenesis of hypertension and renal injury. In this study, we investigated the role of the Rho-kinase dependent signaling pathway in aldosterone-induced myofibroblastic transdifferentiation and collagen gene expression in rat mesangial cells (RMCs). Stimulation with aldosterone (1 nmol/L) significantly increased phosphorylation of myosin phosphatase target subunit-1 (MYPT-1), a marker of Rho-kinase activity, with a peak at 20 min in RMCs. Pre-incubation with a selective mineralocorticoid receptor antagonist, eplerenone (10 µmol/L), or a specific Rho-kinase inhibitor, Y27632 (10 µmol/L), attenuated the aldosterone-induced increase in MYPT-1 phosphorylation. Aldosterone also induced hypertrophy in RMCs, accompanied by an increase in actin polymerization and expression of α-smooth muscle actin (α-SMA), a myofibroblastic transdifferentiation marker. Collagen type I, III and IV mRNA levels were also increased with aldosterone stimulation. Pre-treatment with eplerenone or Y27632 prevented the aldosterone-induced cell hypertrophy, actin polymerization, the increase in α-SMA expression and the increases of collagen type I, III, IV mRNA levels in RMCs. These results suggest that aldosterone-induced mesangial cell hypertrophy is associated with cell transformation, leading to an increase in collagen gene expression via the Rho-kinase dependent signaling pathway.