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

Department of Molecular and Integrative Physiology, University of Michigan Medical School, Ann Arbor, MI 48109, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 01/2011; 108(4):1445-50. DOI: 10.1073/pnas.1010833108
Source: PubMed


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 β4-integrin. Treatment of K8(-/-) mice with anti-β4-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.

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    • "Histological analysis suggests that the colonic inflammation seen in K8 null mice might be the result of an epithelial rather than immune system defect [34]. In addition, K8 null mouse colonocytes display a resistance to apoptotic stimuli, which is considered a protective function [35]. Both inflammation and resistance to apoptosis was treatable with antibiotics, suggesting the primary defect lies in the intestinal epithelium, while inflammation is the result of a subsequent immune response to luminal bacteria. "
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    ABSTRACT: Keratin 8 and 18 (K8/K18) mutations have been implicated in the aetiology of certain pathogenic processes of the liver and pancreas. While some K8 mutations (K8 G62C, K8 K464N) are also presumed susceptibility factors for inflammatory bowel disease (IBD), the only K18 mutation (K18 S230T) discovered so far in an IBD patient is thought to be a polymorphism. The aim of our study was to demonstrate that these mutations might also directly affect intestinal cell barrier function. Cell monolayers of genetically engineered human colonocytes expressing these mutations were tested for permeability, growth rate and resistance to heat-stress. We also calculated the change in dissociation constant (Kd, measure of affinity) each of these mutations introduces into the keratin protein, and present the first model of a keratin dimer L12 region with in silico clues to how the K18 S230T mutation may affect keratin function. Physiologically, these mutations cause up to 30% increase in paracellular permeability in vitro. Heat-stress induces little keratin clumping but instead cell monolayers peel off the surface suggesting a problem with cell junctions. K18 S230T has pronounced pathological effects in vitro marked by high Kd, low growth rate and increased permeability. The latter may be due to the altered distribution of tight junction components claudin-4 and ZO-1. This is the first time intestinal cells have been suggested also functionally impaired by K8/K18 mutations. Although an in vitro colonocyte model system does not completely mimic the epithelial lining of the intestine, nevertheless the data suggest that K8/K18 mutations may be also able to produce a phenotype in vivo.
    Full-text · Article · Jun 2014 · PLoS ONE
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    • "Actually, a large part of the work has used hepatocytes as cell model, given that their IFs consist solely of the K8/K18 pair and that a loss of one keratin partner normally leads to the degradation of the other [10]. For instance, using cultured K8-knockout (K8-null) mouse hepatocytes and their wild-type (WT) counterparts, we have demonstrated that K8/K18 IFs contribute to the maintenance of the integrity of the hepatocyte surface membrane in response to mechanical stress [11], and modulate the hepatocyte response to various apoptotic stimuli [12]–[14]. Moreover, other lines of work on different K8-null versus WT mouse simple epithelial cells, such as those from intestine and pancreas, have revealed a K8/K18 IF involvement in epithelial cell polarity and protein targeting to subcellular compartments [15]–[16], in spite of the fact that such cell types contain the K8/K18 pair in combination with 1–2 other keratins. "
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    ABSTRACT: Keratins (Ks), the intermediate filament (IF) proteins of epithelia, are coordinately expressed as pairs in a cell-lineage and differentiation manner. Cortical thymic epithelial cells (cTECs) predominantly express the simple epithelium keratin 8/18 (K8/K18) pair, whereas medullary thymic epithelial cells (mTECs) express the stratified epithelium K5/K14 pair, with TECs exhibiting K5 and K8 at the cortico-medullary junction in mature thymus. In the work reported here, we used wild-type (WT) and K8-knockout (K8-null) mice to address the contribution of K8/K18 IFs in the maintenance of the thymic epithelial structure. K8-null thymus maintained the differential cell segregation at the cortex versus the medulla observed in WT thymus, and the distribution of immature thymocytes at the cortex. The K8/K18 loss did not affect thymocyte development. However, it massively perturbed the TEC morphology both at the cortex and the medulla, along with a prominent depletion of cTECs. Such tissue alterations coincided with an increase in apoptosis and a reduced expression of Albatross (Fas-binding factor-1), also known for its capacity to bind K8/18 IFs. In addition, the K8/K18 loss affected the distribution of K5/K14-positive mTECs, but not their differentiation status. Together, the results indicate that K8/K18 IFs constitute key promoters of the thymic epithelium integrity.
    Full-text · Article · Sep 2013 · PLoS ONE
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    • "Defect in CK19 expression affects the polarity of the cell (Salas, Rodriguez et al. 1997). In rat intestine, staining of CK8 and CK21 is observed at the cell periphery of absorptive cells while staining of CK19 is observed at the central region (Habtezion, Toivola et al. 2011). Cytokeratin filaments are also important in intercellular context. "

    Full-text · Chapter · Feb 2012
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