Organization of the cytokeratin network in an epithelial cell

The cytoskeleton is a dynamic three-dimensional structure mainly located in the cytoplasm. It is involved in many cell functions such as mechanical signal transduction and maintenance of cell integrity. Among the three cytoskeletal components, intermediate filaments (the cytokeratin in epithelial cells) are the best candidates for this mechanical role. A model of the establishment of the cytokeratin network of an epithelial cell is proposed to study the dependence of its structural organization on extracellular mechanical environment. To implicitly describe the latter and its effects on the intracellular domain, we use mechanically regulated protein synthesis. Our model is a hybrid of a partial differential equation of parabolic type, governing the evolution of the concentration of cytokeratin, and a set of stochastic differential equations describing the dynamics of filaments. Each filament is described by a stochastic differential equation that reflects both the local interactions with the environment and the non-local interactions via the past history of the filament. A three-dimensional simulation model is derived from this mathematical model. This simulation model is then used to obtain examples of cytokeratin network architectures under given mechanical conditions, and to study the influence of several parameters.     

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.     

Long-distance propagation of forces in a cell

A fundamental question in the field of mechanotransduction is how forces propagate inside a cell. Recent experiments have shown that a force of a physiological magnitude, applied via a focal adhesion, can propagate a long distance into the cell. This observation disagrees with existing models that regard the cell as a homogeneous body. We show that this "action at a distance" results from the inhomogeneity in the cell: a prestressed and stiff actin bundle guides the propagation of forces over long distances. Our models highlight the enormous ratios of the prestress and the modulus of the actin bundle to the modulus of the cytoskeleton network. For a normal cell, the models predict that forces propagate over characteristic lengths comparable to the size of the cell. The characteristic lengths can be altered, however, by treatments of the cell. We provide experimental evidence and discuss biological implications.     

Simultaneous labelling of microtubules and fibrillar bundles in tobacco BY-2 cells by the anti-intermediate filament antibody,ME 101

Summary In previous studies on plant cells, antibodies directed against intermediate filaments (IFs) have shown that IF antigens are distributed in one of two quite distinct forms. The first co-distributes with each of the four microtubule arrays (cortical, preprophase band, spindle and phragmoplast), while the second form is associated with cytoplasmic paracrystalline fibrillar bundles (FBs) of 10 nm filaments. Conditions allowing one form to be labelled with antibody have generally proved unsuitable for labelling of the other; this has prevented the simultaneous visualization of the two forms of IF antigen in plants and the study of any possible physical relationships between them at the electron microscopic level. In this paper, we show that ME 101, which recognizes an epitope in the N-terminal portion of all classes of intermediate filaments, stains both forms of plant IF antigen simultaneously in tobacco suspension cells using immunofluorescence or immunogold labelling techniques. These cells contain in their cortex short (ca. l m) fibrillar bundles which stain with ME 101. These bundles appear to be independent of the microtubule-associated epitope which stains in a continuous linear manner with ME 101. When protoplasts are either cleaved open on grids or sequentially extracted with detergents prior to critical point drying, the short fibrillar bundles are specifically labelled by ME 101 tagged with colloidal gold. ME 101 also co-distributed with underlying linear filaments, which appeared to be microtubules. In addition to these structures, the cortex also contains a meshwork of variably-sized fine filaments but these are not labelled with ME 101 nor with an antibody raised against the plant cytoskeleton, which recognizes cytokeratin 8. These results confirm that the fibrillar bundles and the microtubule-associated form of plant IF antigens are present simultaneously rather than experimentally-interconvertible, and that they appear to be physically unconnected.Abbreviations DAPI 4,6-diamidino-2-phenylindole - FB fibrillar bundle - FITC fluorescein isothiocyanate - IF intermediate filaments - MTSB microtubule stabilizing buffer - TBS Tris-buffered-saline     

Extracellular finger domain modulates the response of the epithelial sodium channel to shear stress

The epithelial sodium channel (ENaC) is regulated by multiple extracellular stimuli, including shear stress. Previous studies suggest that the extracellular finger domains of ENaC α and γ subunits contain allosteric regulatory modules. However, the role of the finger domain in the shear stress response is unknown. We examined whether mutations of specific residues in the finger domain of the α subunit altered the response of channels to shear stress. We observed that Trp substitutions at multiple sites within the tract αLys-250-αLeu-290 altered the magnitude or kinetics of channel activation by shear stress. Consistent with these findings, deletion of two predicted peripheral β strands (αIle-251-αTyr-268) led to slower channel activation by shear stress, suggesting that these structures participate in the shear stress response. The effects of mutations on the shear stress response did not correlate with their effects on allosteric Na(+) inhibition (i.e. Na(+) self-inhibition), indicating a divergence within the finger domain regarding mechanisms by which the channel responds to these two external stimuli. This result contrasts with well correlated effects we previously observed at sites near the extracellular mouth of the pore, suggesting mechanistic convergence in proximity to the pore. Our results suggest that the finger domain has an important role in the modulation of channel activity in response to shear stress.     

Cloning of human,murine, and marsupial keratin 7 and a survey of K7 expression in the mouse

Keratins are cytoplasmic intermediate filament proteins expressed by epithelial cells. Keratin 7 (K7) is expressed in a wide range of epithelial structures in humans. We have cloned and fully sequenced the human and mouse K7 genes and mRNAs, and the K7 mRNA from the marsupial Potorous tridactylis, from which the widely used simple epithelial cell lines PtK1 and PtK2 are derived. Percentage identity plots comparing the mouse and human genomic sequences revealed a number of conserved CpG islands associated with the K7 gene. There was considerable conservation of introns between the two species, which may indicate the presence of intronic regulatory elements. Only the most proximal 500bp of the promoter was conserved, although an additional conserved sequence island was found 2-3kb upstream. Protein sequence comparisons between the three species allowed identification of conserved regions of the keratin variable domains that may be candidates for protein-protein interactions and/or regulatory modification. From the mouse sequence, we generated a polyclonal rabbit antibody specific for murine K7. This antibody was used to perform a survey of K7 expression in the mouse. The expression pattern was similar to the reported human distribution, with substantial expression observed in lung, bladder, mesothelium, hair follicle, and ductal structures. We also noted previously unreported expression of K7 in the gastrointestinal tract and filiform papillae of the tongue and specific K7 expression in a range of "hard" epithelial tissues. The distribution of K7 in mouse and availability of genomic sequence from the 129/Sv mouse strain will allow the generation and analysis of transgenic mice expressing mutant forms of K7 and to predict the phenotype of human genetic disorders caused by mutations in this keratin.     

Activation of extracellular signal-regulated kinase ERK after hypo-osmotic stress in renal epithelial A6 cells

Activation of mitogen-activated protein (MAP) kinases has been reported to occur after a hypo-osmotic cell swelling in various types of cells. In renal epithelial A6 cells, the hypo-osmotic shock induced a rapid increase in the phosphorylation of an extracellular signal-regulated kinase (ERK)-like protein that was maximal 10 min after osmotic stress. Activation of ERK was significantly increased when hypo-osmotic stress was performed in the absence of extracellular Ca²⁺, a condition that inhibits regulatory volume decrease (RVD). Exposure of cells to PD98059, an inhibitor of the MAP kinase kinase MEK, at a concentration that fully cancelled ERK activation, did not inhibit RVD. On the contrary, RVD was abolished when osmotic shock was induced in the presence of SB203580, an inhibitor of stress-activated protein kinases (SAPKs). These results suggest that different MAP kinases are activated after hypo-osmotic stress in A6 cells. SAPKs would be involved in the control of RVD, while ERK would lead to later events, such as gene expression or energy metabolism.     

Quercetin inhibits IL-1 beta-induced ICAM-1 expression in pulmonary epithelial cell line A549 through the MAPK pathways

Quercetin is a herbal flavonoid derived from various foods of plant origin and widely used as a major constituent of nutritional supplements. Quercetin has been shown to have anti-inflammatory properties and can play a role in anti-inflammatory procedure. Intercellular adhesion molecule-1 (ICAM-1) is one of the important pro-inflammatory factors, especially in early phage of inflammation. However, the mechanisms regulating ICAM-1 expression by quercetin in human A549 cells were still unclear. In this study, the inhibitory effect of quercetin on ICAM-1 expression by interleukin-1 beta (IL-1 beta)-stimulated A549 cells was investigated, and the roles of mitogen-activated protein kinases (MAPK) pathways were explored. Quercetin attenuated IL-1 beta-induced expression of ICAM-1 mRNA and protein in a dose-dependent manner. The experiment suggested that quercetin actively inhibited inhibitory protein of nuclear factor-kappa B (I kappa B) degradation, and nuclear factor-kappa B (NF-kappa B) activity. The c-fos and c-jun, components of activator protein-1 (AP-1), were mediated by MAPK pathways. ERK and p38 were involved in the c-fos mRNA expression, and JNK was involved in the c-jun mRNA expression. The inhibitory effect of quercetin on ICAM-1 expression was mediated by the sequential attenuation of the c-fos and c-jun mRNA expressions. These inhibitory effects were partially inhibited by SB203580, a specific inhibitor of p38 MAPK, but not by PD98059, a specific inhibitors of extracellular signal-regulated kinase (ERK), and SP600125, a specific inhibitor of c-Jun-N-terminal kinase (JNK). Taken together, these results suggest that quercetin negatively modulating ICAM-1 partly dependent on MAPK pathways. Binwu Ying and Ting Yang have contributed equally to this work.     

Cadmium regulation of apoptotic and stress response genes in tumoral and immortalized epithelial cells of the human breast

Cadmium (Cd) is a widely-disseminated metal which can be imported and accumulated in living cells thereby drastically interfering with their biological mechanisms. Increasing interest has been recently focused on the elucidation of the cellular and molecular aspects of Cd-dependent regulation of gene expression and signal transduction pathways in different model system. Concerning breast cancer, very limited studies have been produced so far on the role played by Cd on estrogen receptor-negative human breast cancer cells, that are expected to be insensitive to the already-proven metallo-estrogenic effect exerted by Cd on the estrogen receptor-positive cell counterparts. Here, we have examined the effects of long-term (96h) exposure of estrogen receptor-negative MDA-MB231 malignant adenocarcinoma cells to CdCl(2) at 5muM concentration, corresponding to the IC(50) for this time of incubation, by evaluating the expression levels of genes coding for stress response factors (e.g. heat shock proteins and metallothioneins), and for apoptosis-related factors and enzymes. In parallel, we tested the gene expression pattern of immortalized HB2 breast epithelial cells, taken as non-tumoral counterpart, after the same exposure to the metal which instead did not exert any change in their cell number with respect to controls. Our cumulative results indicate that, whilst HB2 cells appear to activate defense mechanisms against metal stress principally via metallothionein massive up-regulation and appearance of the spliced form of XBP-1 message, MDA-MB231 cells seem to couple the onset of a protective reaction (e.g. up-regulation of hsp27 and metallothioneins) to the switching-on of new intracellular pathways directing cells to a kind of death which shares several aspects with the apoptotic program, such as down-regulation of Bcl-2 and over-expression of Dap kinase and several caspases.     

Microarray analysis of differentially regulated genes in human neuronal and epithelial cell lines upon exposure to type A botulinum neurotoxin

Among the seven serotypes (A–G), type A botulinum neurotoxin (BoNT/A) is the most prevalent etiologic agent and the most potent serotype to cause foodborne botulism, characterized by flaccid muscle paralysis. Upon ingestion, BoNT/A crosses epithelial cell barriers to reach lymphatic and circulatory systems and blocks acetylcholine release at the pre-synaptic cholinergic nerve terminals of neuromuscular junctions (NMJs) resulting in paralysis. One of the unique features of BoNT/A intoxication is its neuroparalytic longevity due to its persistent catalytic activity. The persistent presence of the toxin inside the cell can induce host cell responses. To understand the pathophysiology and host response at the cellular level, gene expression changes upon exposure of human HT-29 colon carcinoma (epithelial) and SH-SY5Y neuroblastoma cell lines to BoNT/A complex were investigated using microarray analysis. In HT-29 cells, 167 genes were up-regulated while 60 genes were down-regulated, whereas in SH-SY5Y cells about 223 genes were up-regulated and 18 genes were down-regulated. Modulation of genes and pathways involved in neuroinflammatory, ubiquitin–proteasome degradation, phosphatidylinositol, calcium signaling in SH-SY5Y cells, and genes relevant to focal adhesion, cell adhesion molecules, adherens and gap junction related pathways in HT-29 cells suggest a massive host response to BoNT/A. A clear differential response in epithelial and neuronal cells indicates that the genes affected may play a distinct role in BoNTs cellular mode of action, involving these two types of host cells.     

Interleukin-17 is a potent immuno-modulator and regulator of normal human intestinal epithelial cell growth

Upregulation of the T-cell derived cytokine interleukin (IL-17) was reported in the inflamed intestinal mucosa of patients with inflammatory bowel disorders. In this study, we analyzed the effect of IL-17 on human intestinal epithelial cell (HIEC) turnover and functions. Proliferation and apoptosis in response to IL-17 was monitored in HIEC (cell counts, [(3)H]thymidine incorporation method, and annexinV-PI-apoptosis assay). Signalling pathways were analyzed by Western blots, electromobility shift assay, and immunofluorescence studies. IL-17 proved to be a potent inhibitor of HIEC proliferation without any pro-apoptotic/necrotic effect. The growth inhibitory effect of IL-17 was mediated via the p38 stress kinase. Consequently, the p38-SAPkinase-inhibitor SB203580 abrogated this anti-mitotic effect. In parallel, IL-17 provoked the degradation of IkappaBalpha, allowing nuclear translocation of the p65 NF-kappaB subunit and induction of the NF-kappaB-controlled genes IL-6 and -8. IL-17 potently blocks epithelial cell turnover while at the same time amplifying an inflammatory response in a positive feedback manner.     

MAP kinase-signaling controls nuclear translocation of tripeptidyl-peptidase II in response to DNA damage and oxidative stress

Reactive oxygen species (ROS) are a continuous hazard in eukaroytic cells by their ability to cause damage to biomolecules, in particular to DNA. Previous data indicated that the cytosolic serine peptidase tripeptidyl-peptidase II (TPPII) translocates into the nucleus of most tumor cell lines in response to γ-irradiation and ROS production; an event that promoted p53 expression as well as caspase-activation. We here observed that nuclear translocation of TPPII was dependent on signaling by MAP kinases, including p38MAPK. Further, this was caused by several types of DNA-damaging drugs, a DNA cross-linker (cisplatinum), an inhibitor of topoisomerase II (etoposide), and to some extent also by nucleoside-analogues (5-fluorouracil, hydroxyurea). In the minority of tumor cell lines where TPPII was not translocated into the nucleus in response to DNA damage we observed reduced intracellular ROS levels, and the expression levels of redox defense systems were increased. Further, treatment with the ROS-inducer γ-hexa-chloro-cyclohexane (γ-HCH, lindane), an inhibitor of GAP junctions, restored nuclear translocation of TPPII in these cell lines upon γ-irradiation. Moreover, blocking nuclear translocation of TPPII in etoposide-treated cells, by using a peptide-derived inhibitor (Z-Gly-Leu-Ala-OH), attenuated expression of γ-H2AX in γ-irradiated melanoma cells. Our results indicated a role for TPPII in MAPK-dependent DNA damage signaling.     

Novel insights into the osmotic stress response of yeast

Response to hyperosmolarity in the baker's yeast Saccharomyces cerevisiae has attracted a great deal of attention of molecular and cellular biologists in recent years, from both the fundamental scientific and applied viewpoint. Indeed the underlying molecular mechanisms form a clear demonstration of the intricate interplay of (environmental) signalling events, regulation of gene expression and control of metabolism that is pivotal to any living cell. In this article we briefly review the cellular response to conditions of hyperosmolarity, with focus on the high-osmolarity glycerol mitogen-activated protein kinase pathway as the major signalling route governing cellular adaptations. Special attention will be paid to the recent finding that in the yeast cell also major structural changes occur in order to ensure maintenance of cell integrity. The intriguing role of glycerol in growth of yeast under (osmotic) stress conditions is highlighted.     

A mutation of keratin 18 within the coil 1A consensus motif causes widespread keratin aggregation but cell type-restricted lethality in mice

Mutations in genes encoding epidermal keratins cause skin disorders, while those in internal epithelial keratins, such as K8 and K18, are risk factors for liver diseases. The effect of dominant mutations in K8 or K18 during embryonic development and tissue homeostasis has not been examined so far. Here we demonstrate that the dominant mutation hK18 R89C, that is highly similar to hK14 R125C, causing EBS in humans, leads to cell type-specific lethality in mice, depending on the ratio of mutant to endogenous keratins. Mice expressing hK18 R89C in the absence of endogenous K19 and K18 died at mid-gestation from defects in trophoblast giant cells, accompanied by haematomas. A single, endogenous K18 allele rescued embryonic lethality but caused aggregation of keratins in all adult internal epithelia, surprisingly without spontaneous cell fragility. Closer analysis revealed that both filaments and aggregates coexisted in the same cell, depending on the ratio of mutant to endogenous keratins. Our results demonstrate that balanced overexpression of a wild-type keratin rescued the lethal consequences of a dominant-negative mutation. This has important implications for therapy approaches of keratinopathies, suggesting that suppressing the mutant allele is not necessary in vivo.     

Up-regulation of neural stem cell markers suggests the occurrence of dedifferentiation in regenerating spinal cord

Following tail amputation in urodele amphibians, an ependymal tube, that resembles a developing neural tube, forms from ependymal cells that migrate from the cord stump and elongates by cell proliferation. Expression of the keratin pair 8 and 18 has been observed in the developing urodele nervous system and is maintained in the ependymal cells of the mature cord. We show here that expression of these keratins is not unique to urodeles, but is also observed in the radial glia of the human spinal cord, suggesting that these proteins might play a role both in neural development and regeneration. Analysis of their expression in the regenerating spinal cord following tail amputation shows that their expression, as well as that of glial fibrillary acidic protein (GFAP), is maintained in the ependymal tube during regeneration, though differences in their levels of expression are observed along the anteroposterior axis and appear to be related to the progression of morphogenesis. In addition, we show that following tail amputation the ependymal tube expresses the neural stem cell markers nestin and vimentin, which are undetectable in normal urodele spinal cord. This up-regulation of neural stem cell markers shows that the ependymal cells undergo a phenotypic change. Whereas maintenance of keratin and GFAP expression in the adult ependyma may reflect a higher plasticity of these cells in adult urodeles than in other vertebrates, re-expression of markers of early neural development suggests the occurrence of a dedifferentiation process in the spinal cord in response to injury.Edited by J. Campos-Ortega     

Dynamics of Phosphorylation and Assembly of the High Molecular Weight Neurofilament Subunit in NB2a/d1 Neuroblastoma

In neuronal systems thus far studied, newly synthesized neurofilament subunits rapidly associate with the Triton-insoluble cytoskeleton and subsequently undergo extensive phosphorylation. However, in the present study we demonstrate by biochemical and immunological criteria that NB2a/d1 neuroblastoma cells also contain Triton-soluble, extensively phosphorylated 200-kDa high molecular weight neurofilament subunits (NF-H). High-speed centrifugation (100,000 g) of the Triton-soluble fraction for 1 h sedimented some, but not all, soluble NF-H subunits; immunoelectron microscopic analyses of the resulting pellet indicated that a portion of the NF-H subunits in this fraction are assembled into (Triton-soluble) neurofilaments. When cells were pulse labeled for 15 min with [35S]methionine, radiolabel was first associated with the Triton-soluble 200-kDa NF-H variants. Because only extensively phosphorylated NF-H subunits migrate at 200 kDa, whereas hypophosphorylated subunits migrate instead at 160 kDa, these findings suggest that some newly synthesized subunits were phosphorylated before they polymerized. In pulse-chase analyses, radiolabeled 200-kDa NF-H migrated into the 100,000 g particulate fraction of Triton-soluble extracts before its arrival in the Triton-insoluble cytoskeleton. Undifferentiated cells, which do not possess axonal neurites and lack a significant amount of Triton-insoluble, extensively phosphorylated NF-H, contain a sizeable pool of Triton-soluble extensively phosphorylated NF-H subunits and polymers. We interpret these data to indicate that the integration of newly synthesized NF-H into the cytoskeleton occurs in a progression of distinct stages, and that assembly of NF-H into neurofilaments and integration into the Triton-insoluble cytoskeleton are not prerequisites for the incorporation of certain phosphate groups on these polypeptides.(ABSTRACT TRUNCATED AT 250 WORDS)