The Rho-guanine nucleotide exchange factor Solo decelerates collective cell migration by modulating the Rho-ROCK pathway and keratin networks.

Collective cell migration plays crucial roles in tissue remodeling, wound healing, and cancer cell invasion. However, its underlying mechanism remains unknown. Previously, we showed that the RhoA-targeting guanine nucleotide exchange factor Solo (ARHGEF40) is required for tensile force-induced RhoA activation and proper organization of keratin-8/keratin-18 (K8/K18) networks. Here, we demonstrate that Solo knockdown significantly increases the rate at which Madin-Darby canine kidney cells collectively migrate on collagen gels. However, it has no apparent effect on the migratory speed of solitary cultured cells. Therefore, Solo decelerates collective cell migration. Moreover, Solo localized to the anteroposterior regions of cell-cell contact sites in collectively migrating cells and was required for the local accumulation of K8/K18 filaments in the forward areas of the cells. Partial Rho-associated protein kinase (ROCK) inhibition or K18 or plakoglobin knockdown also increased collective cell migration velocity. These results suggest that Solo acts as a brake for collective cell migration by generating pullback force at cell-cell contact sites via the RhoA-ROCK pathway. It may also promote the formation of desmosomal cell-cell junctions related to K8/K18 filaments and plakoglobin.

Mechanical and signaling roles for keratin intermediate filaments in the assembly and morphogenesis of Xenopus mesendoderm tissue at gastrulation.

The coordination of individual cell behaviors is a crucial step in the assembly and morphogenesis of tissues. Xenopus mesendoderm cells migrate collectively along a fibronectin (FN) substrate at gastrulation, but how the adhesive and mechanical forces required for these movements are generated and transmitted is unclear. Traction force microscopy (TFM) was used to establish that traction stresses are limited primarily to leading edge cells in mesendoderm explants, and that these forces are balanced by intercellular stresses in follower rows. This is further reflected in the morphology of these cells, with broad lamellipodial protrusions, mature focal adhesions and a gradient of activated Rac1 evident at the leading edge, while small protrusions, rapid turnover of immature focal adhesions and lack of a Rac1 activity gradient characterize cells in following rows. Depletion of keratin (krt8) with antisense morpholinos results in high traction stresses in follower row cells, misdirected protrusions and the formation of actin stress fibers anchored in streak-like focal adhesions. We propose that maintenance of mechanical integrity in the mesendoderm by keratin intermediate filaments is required to balance stresses within the tissue to regulate collective cell movements.

Tumor necrosis factor-α confers cardioprotection through ectopic expression of keratins K8 and K18.

Tumor necrosis factor-α (TNF-α), one of the major stress-induced proinflammatory cytokines, is upregulated in the heart after tissue injury, and its sustained expression can contribute to the development of heart failure. Whether TNF-α also exerts cytoprotective effects in heart failure is not known. Here we provide evidence for a cardioprotective function of TNF-α in a genetic heart failure model, desmin-deficient mice. The cardioprotective effects of TNF-α are a consequence of nuclear factor-κB (NF-κB)-mediated ectopic expression in cardiomyocytes of keratin 8 (K8) and keratin 18 (K18), two epithelial-specific intermediate filament proteins. In cardiomyocytes, K8 and K18 (K8/K18) formed an alternative cytoskeletal network that localized mainly at intercalated discs (IDs) and conferred cardioprotection by maintaining normal ID structure and mitochondrial integrity and function. Ectopic induction of K8/K18 expression in cardiomyocytes also occurred in other genetic and experimental models of heart failure. Loss of the K8/K18 network resulted in a maladaptive cardiac phenotype following transverse aortic constriction. In human failing myocardium, where TNF-α expression is upregulated, K8/K18 were also ectopically expressed and localized primarily at IDs, which did not contain detectable amounts of desmin. Thus, TNF-α- and NF-κB-mediated formation of an alternative, stress-induced intermediate filament cytoskeleton has cardioprotective function in mice and potentially in humans.

Identification of cell surface-exposed proteins involved in the fimbria-mediated adherence of enteroaggregative Escherichia coli to intestinal cells.

Fimbria-mediated adherence to the intestinal epithelia is a key step in enteroaggregative Escherichia coli (EAEC) pathogenesis. To date, four fimbriae have been described for EAEC; aggregative adherence fimbria II (AAF/II) is the most important adherence factor for EAEC prototype strain 042. Previously, we described results showing that extracellular matrix (ECM) components might be involved in the recognition of AAF/II fimbriae by intestinal cells. In this study, we sought to identify novel potential receptors on intestinal epithelial cells recognized by the AAF/II fimbriae. Purified AafA-dsc protein, the major subunit of AAF/II fimbriae, was incubated with a monolayer of T84 cells, cross-linked to the surface-exposed T84 cell proteins, and immunoprecipitated by using anti-AafA antibodies. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of cellular proteins bound to AafA-dsc protein identified laminin (previously recognized as a potential receptor for AAF/II) and cytokeratin 8 (CK8). Involvement of the major subunit of AAF/II fimbriae (AafA protein) in the binding to recombinant CK8 was confirmed by adherence assays with purified AAF/II fimbriae, AafA-dsc protein, and strain 042. Moreover, HEp-2 cells transfected with CK8 small interfering RNA (siRNA) showed reduced 042 adherence compared with cells transfected with scrambled siRNA as a control. Adherence of 042 to HEp-2 cells preincubated with antibodies against ECM proteins or CK8 was substantially reduced. Altogether, our results supported the idea of a role of CK8 as a potential receptor for EAEC.

Keratin 8 modulates ß-cell stress responses and normoglycaemia.

Keratin intermediate filament (IF) proteins are epithelial cell cytoskeletal components that provide structural stability and protection from cell stress, among other cellular and tissue-specific functions. Numerous human diseases are associated with IF gene mutations, but the function of keratins in the endocrine pancreas and their potential significance for glycaemic control are unknown. The impact of keratins on ß-cell organisation and systemic glucose control was assessed using keratin 8 (K8) wild-type (K8(+/+)) and K8 knockout (K8(-/-)) mice. Islet ß-cell keratins were characterised under basal conditions, in streptozotocin (STZ)-induced diabetes and in non-obese diabetic (NOD) mice. STZ-induced diabetes incidence and islet damage was assessed in K8(+/+) and K8(-/-) mice. K8 and K18 were the predominant keratins in islet ß-cells and K8(-/-) mice expressed only remnant K18 and K7. K8 deletion resulted in lower fasting glucose levels, increased glucose tolerance and insulin sensitivity, reduced glucose-stimulated insulin secretion and decreased pancreatic insulin content. GLUT2 localisation and insulin vesicle morphology were disrupted in K8(-/-) ß-cells. The increased levels of cytoplasmic GLUT2 correlated with resistance to high-dose STZ-induced injury in K8(-/-) mice. However, K8 deletion conferred no long-term protection from STZ-induced diabetes and prolonged STZ-induced stress caused increased exocrine damage in K8(-/-) mice. ß-cell keratin upregulation occurred 2 weeks after treatments with low-dose STZ in K8(+/+) mice and in diabetic NOD mice, suggesting a role for keratins, particularly in non-acute islet stress responses. These results demonstrate previously unrecognised functions for keratins in ß-cell intracellular organisation, as well as for systemic blood glucose control under basal conditions and in diabetes-induced stress.

Keratin 8/18 regulation of glucose metabolism in normal versus cancerous hepatic cells through differential modulation of hexokinase status and insulin signaling.

As differentiated cells, hepatocytes primarily metabolize glucose for ATP production through oxidative phosphorylation of glycolytic pyruvate, whereas proliferative hepatocellular carcinoma (HCC) cells undergo a metabolic shift to aerobic glycolysis despite oxygen availability. Keratins, the intermediate filament (IF) proteins of epithelial cells, are expressed as pairs in a lineage/differentiation manner. Hepatocyte and HCC (hepatoma) cell IFs are made solely of keratins 8/18 (K8/K18), thus providing models of choice to address K8/K18 IF functions in normal and cancerous epithelial cells. Here, we demonstrate distinctive increases in glucose uptake, glucose-6-phosphate formation, lactate release, and glycogen formation in K8/K18 IF-lacking hepatocytes and/or hepatoma cells versus their respective IF-containing counterparts. We also show that the K8/K18-dependent glucose uptake/G6P formation is linked to alterations in hexokinase I/II/IV content and localization at mitochondria, with little effect on GLUT1 status. In addition, we find that the insulin-stimulated glycogen formation in normal hepatocytes involves the main PI-3 kinase-dependent signaling pathway and that the K8/K18 IF loss makes them more efficient glycogen producers. In comparison, the higher insulin-dependent glycogen formation in K8/K18 IF-lacking hepatoma cells is associated with a signaling occurring through a mTOR-dependent pathway, along with an augmentation in cell proliferative activity. Together, the results uncover a key K8/K18 regulation of glucose metabolism in normal and cancerous hepatic cells through differential modulations of mitochondrial HK status and insulin-mediated signaling.

Absence of keratin 8 confers a paradoxical microflora-dependent resistance to apoptosis in the colon.

Keratin 8 (K8) is a major intermediate filament protein present in enterocytes and serves an antiapoptotic function in hepatocytes. K8-null mice develop colonic hyperplasia and colitis that are reversed after antibiotic treatment. To investigate the pathways that underlie the mechanism of colonocyte hyperplasia and the normalization of the colonic phenotype in response to antibiotics, we performed genome-wide microarray analysis. Functional annotation of genes that are differentially regulated in K8(-/-) and K8(+/+) isolated colon crypts (colonocytes) identified apoptosis as a major altered pathway. Exposure of K8(-/-) colonocytes or colon organ ("organoid") cultures, but not K8(-/-) small intestine organoid cultures, to apoptotic stimuli showed, surprisingly, that they are resistant to apoptosis compared with their wild-type counterparts. This resistance is not related to inflammation per se because T-cell receptor α-null (TCR-α(-/-)) and wild-type colon cultures respond similarly upon induction of apoptosis. Following antibiotic treatment, K8(-/-) colonocytes and organ cultures become less resistant to apoptosis and respond similarly to the wild-type colonocytes. Antibiotics also normalize most differentially up-regulated genes, including survivin and ß4-integrin. Treatment of K8(-/-) mice with anti-ß4-integrin antibody up-regulated survivin, and induced phosphorylation of focal adhesion kinase with decreased activation of caspases. Therefore, unlike the proapoptotic effect of K8 mutation or absence in hepatocytes, lack of K8 confers resistance to colonocyte apoptosis in a microflora-dependent manner.

Identification of proteins responsible for the multiple drug resistance in 5-fluorouracil-induced breast cancer cell using proteomics analysis.

PURPOSE: This study aimed to explore the mechanism of multi-drug resistance (MDR) in 5-fluorouracil (5-FU)-induced breast cancer cell MCF-7. METHODS: MCF-7 cells were exposed in stepwise escalating concentration of 5-FU to develop the resistant cell line, MCF-7/5-FU. Biological and molecular characteristics of the cells were studied through MTT, flow cytometry, real-time PCR, western-blot, and the global protein profiles between MCF-7/5-FU and parental MCF-7 were compared using proteomic approach. Then some of the differentially expressed proteins were validated by western-blot. In addition, the role of 14-3-3sigma was validated using gene transfection. RESULTS: Drug resistance of MCF-7/5-FU cells to 5-FU, MX, cDDP, ADM, TAXOL all increased significantly compared with MCF-7 cells and that maybe related to BCRP, but not MDR1 and MRP1. Differentially expressed proteins between MCF-7/5-FU and MCF-7 cells were identified; 12 proteins were up-regulated and 18 proteins were down-regulated in MCF-7/5-FU cells. Expressive levels of some proteins in western-blot validation were consistent with the results in proteomic analysis. Enforced 14-3-3sigma expression can increase the sensitivity of MCF-7/5-FU cells to 5-FU and cDDP. CONCLUSION: MDR of MCF-7/5-FU likely associated with differentially expressed proteins and 14-3-3sigma may play a positive role in chemotherapy. These findings may provide theoretical support for the prediction of chemotherapeutic response and reverse of MDR.

Cytokeratin 8 silencing in human nasopharyngeal carcinoma cells leads to cisplatin sensitization.

By comparing protein profiles of nasopharyngeal carcinoma HONE1 cells to transformed nasopharyngeal epithelial NP 69 cells, several clusters of differentially expressed proteins were identified. The increased expression of cytokeratin 8 (CK8) and pyruvate kinase M2 was a common feature in four NPC cell lines compared to the two transformed epithelial cell lines. Suppression of CK8 was associated with the sensitivity to cisplatin in HONE1 cells; while overexpression of CK8 provided resistance to cisplatin-mediated apoptosis; and this protection occurred through an enhanced phosphorylation of c-Jun NH(2)-terminal kinase (JNK). Our findings implicate an underlying molecular mechanism in which CK8 is required for cisplatin resistance.

Keratins let liver live: Mutations predispose to liver disease and crosslinking generates Mallory-Denk bodies.

Keratin polypeptides 8 and 18 (K8/K18) are the cytoskeletal intermediate filament proteins of hepatocytes while K8/K18/K19 are the keratins of hepatobiliary ductal cells. Hepatocyte K8/K18 are highly abundant and behave as stress proteins with injury-inducible expression. Human association studies show that K8/K18 germline heterozygous mutations predispose to end-stage liver disease of multiple etiologies ( approximately 3 fold increased risk), and to liver disease progression in patients with chronic hepatitis C infection. These findings are supported by extensive transgenic mouse and ex vivo primary hepatocyte culture studies showing that K8 or K18 mutations predispose the liver to acute or subacute injury and promote apoptosis and fibrosis. Mutation-associated predisposition to liver injury is likely related to mechanical and nonmechanical keratin functions including maintenance of cell integrity, protection from apoptosis and oxidative injury, serving as a phosphate sponge, regulation of mitochondrial organization/function and protein targeting. These functions are altered by mutation-induced changes in keratin phosphorylation, solubility and filament organization/reorganization. Keratins are also the major constituents of Mallory-Denk bodies (MDBs). A toxin-induced K8>K18 ratio, and keratin crosslinking by transglutaminase-2 play essential roles in MDB formation. Furthermore, intracellular or cell-released K18 fragments, generated by caspase-mediated proteolysis during apoptosis serve as markers of liver injury. Therefore, K8 and K18 are cytoprotective stress proteins that play a central role in guarding hepatocytes from apoptosis. Keratin involvement in liver disease is multi-faceted and includes modulating disease progression upon mutation, formation of MDBs in response to unique forms of injury, and serving as markers of epithelial cell death.

The possible role of cytokeratin 8 in cadmium-induced adaptation and carcinogenesis.

Chronic exposures to cadmium compounds are carcinogenic. It was hypothesized that the development of resistance to cadmium may drive carcinogenesis. This is achieved by selection of resistant cells in which the apoptotic response is significantly attenuated. The induction of cadmium resistance in rat lung epithelial cells (LEC) was used to explore the mechanisms of cadmium-induced adaptation and carcinogenesis. Our previous results showed that LECs developed resistance to apoptosis during cadmium adaptation possibly due to perturbation of the c-Jun NH(2)-terminal kinase pathway. Here, we further study these cells by comparative proteomics. Interestingly, we showed that two intermediate filament proteins, cytokeratin 8 (CK8) and cytokeratin 14 (CK14), were increased significantly and stably maintained only in the adapted cells but not in cadmium-treated parental cells. It has been documented that CK8/cytokeratin 18 provided resistance to tumor necrosis factor (TNF)-induced apoptosis and CK14 may function as an inhibitor of TNF-TNF receptor 1 (TNFR1) signaling through an association with TNFR1-associated death domain protein, suggesting that up-regulation of CK8 and CK14 may be responsible for apoptotic resistance. Finally, we showed that small interfering RNA-specific knockdown of CK8 in cadmium-adapted cells attenuated the cadmium resistance, indicating the potential role of CK8 in cadmium resistance. This acquired self-resistance to apoptosis could account for cadmium-induced carcinogenesis, as this promotes neoplastic cell survival as well as subsequent clonal expansion and then progression of tumor development. Thus, increased expression of these cytokeratins represents an adaptive survival mechanism that resists cadmium-induced apoptosis and it is unprecedented that cells respond to long-term cadmium exposure by modulating keratin dynamics.

Interleukin-6 induces keratin expression in intestinal epithelial cells: potential role of keratin-8 in interleukin-6-induced barrier function alterations.

Keratin 8 (K8) and keratin-18 (K18) are the major intermediate filament proteins in the intestinal epithelia. The regulation and function of keratin in the intestinal epithelia is largely unknown. In this study we addressed the role and regulation of K8 and K18 expression by interleukin 6 (IL-6). Caco2-BBE cell line and IL-6 null mice were used to study the effect of IL-6 on keratin expression. Keratin expression was studied by Northern blot, Western blot, and confocal microscopy. Paracellular permeability was assessed by apical-to-basal transport of a fluorescein isothiocyanate dextran probe (FD-4). K8 was silenced using the small interfering RNA approach. IL-6 significantly up-regulated mRNA and protein levels of K8 and K18. Confocal microscopy showed a reticular pattern of intracellular keratin localized to the subapical region after IL-6 treatment. IL-6 also induced serine phosphorylation of K8. IL-6 decreased paracellular flux of FD-4 compared with vehicle-treated monolayers. K8 silencing abolished the decrease in paracellular permeability induced by IL-6. Administration of dextran sodium sulfate (DSS) significantly increased intestinal permeability in IL-6-/- mice compared with wild type mice given DSS. Collectively, our data demonstrate that IL-6 regulates the colonic expression of K8 and K18, and K8/K18 mediates barrier protection by IL-6 under conditions where intestinal barrier is compromised. Thus, our data uncover a novel function of these abundant cytoskeletal proteins, which may have implications in intestinal disorders such as inflammatory bowel disease wherein barrier dysfunction underlies the inflammatory response.

Keratin 8 modulation of desmoplakin deposition at desmosomes in hepatocytes.

Keratins, the intermediate filament proteins of epithelial cells, connect to desmosomes, the cell-cell adhesion structures at the surface membrane. The building elements of desmosomes include desmoglein and desmocollin, which provide the actual cell adhesive properties, and desmoplakins, which anchor the keratin intermediate filaments to desmosomes. In the work reported here, we address the role of keratin 8 in modulating desmoplakin deposition at surface membrane in mouse hepatocytes. The experimental approach is based on the use of keratin 8- and keratin 18-null mouse hepatocytes as cell models. In wild-type mouse hepatocytes, desmoplakin is aligned with desmoglein and keratin 8 at the surface membrane. In keratin 8-null hepatocytes, the intermediate filament loss leads to alterations in desmoplakin distribution at the surface membrane, but not of desmoglein. Intriguingly, a significant proportion of keratin 18-null hepatocytes express keratin 8 at the surface membrane, associated with a proper desmoplakin alignment with desmoglein at desmosomes. A Triton treatment of the monolayer reveals that most of the desmoplakin present in either wild-type, keratin 8- or keratin 18-null hepatocytes is insoluble. Deletion analysis of keratin 8 further suggests that the recovery of desmoplakin alignment requires the keratin 8 rod domain. In addition, similarly to other works revealing a key role of desmoplakin phosphorylation on its interaction with intermediate filaments, we find that the phosphorylation status of the keratin 8 head domain affects desmoplakin distribution at desmosomes. Together, the data indicate that a proper alignment/deposition of desmoplakin with keratins and desmoglein in hepatocytes requires keratin 8, through a reciprocal phosphoserine-dependent process.