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Keratin 8 (K8) is the main intestinal epithelial intermediate filament protein with proposed roles for colonic epithelial cell integrity. Here, we used mice lacking K8 in intestinal epithelial cells (floxed K8 and Villin-Cre1000 and Villin-CreER t2 ) to investigate the cell-specific roles of intestinal epithelial K8 for colonocyte function and pathologies. Intestinal epithelial K8 deletion decreased K8 partner proteins, K18–K20, 75–95%, and the remaining keratin filaments were located at the colonocyte apical regions with type II K7, which decreased 30%. 2-Deoxy-2-[ 18 F]-fluoroglucose positron emission tomography in vivo imaging identified a metabolic phenotype in the lower gut of the conditional K8 knockouts. These mice developed intestinal barrier leakiness, mild diarrhea, and epithelial damage, especially in the proximal colon. Mice exhibited shifted differentiation from enterocytes to goblet cells, displayed longer crypts and an increased number of Ki67 + transit-amplifying cells in the colon. Significant proproliferative and regenerative signaling occurred in the IL-22, STAT3, and pRb pathways, with minor effects on inflammatory parameters, which, however, increased in aging mice. Importantly, colonocyte K8 deletion induced a dramatically increased sensitivity to azoxymethane-induced tumorigenesis. In conclusion, intestinal epithelial K8 plays a significant role in colonocyte epithelial integrity maintenance, proliferation regulation and tumor suppression. Graphical abstract

Keratins (K) are intermediate filament proteins important in stress protection and mechanical support of epithelial tissues. K8, K18 and K19 are the main colonic keratins, and K8-knockout (K8 −/− ) mice display a keratin dose-dependent hyperproliferation of colonic crypts and a colitis-phenotype. However, the impact of the loss of K8 on intestinal cell differentiation has so far been unknown. Here we show that K8 regulates Notch1 signalling activity and differentiation in the epithelium of the large intestine. Proximity ligation and immunoprecipitation assays demonstrate that K8 and Notch1 co-localize and interact in cell cultures, and in vivo in the colonic epithelial cells. K8 with its heteropolymeric partner K18 enhance Notch1 protein levels and activity in a dose dependent manner. The levels of the full-length Notch1 receptor (FLN), the Notch1 intracellular domain (NICD) and expression of Notch1 downstream target genes are reduced in the absence of K8, and the K8-dependent loss of Notch1 activity can be rescued with re-expression of K8/K18 in K8-knockout CRISPR/Cas9 Caco-2 cells protein levels. In vivo , K8 deletion with subsequent Notch1 downregulation leads to a shift in differentiation towards a goblet cell and enteroendocrine phenotype from an enterocyte cell fate. Furthermore, the K8 −/− colonic hyperproliferation results from an increased number of transit amplifying progenitor cells in these mice. K8/K18 thus interact with Notch1 and regulate Notch1 signalling activity during differentiation of the colonic epithelium.

Loss of keratin 8 (K8) has been shown to increase susceptibility towards colonocyte hyperproliferation and tumorigenesis. However, most colorectal cancer (CRC) mouse models require carcinogen, develop small intestinal tumors or have long latency period. The aim was to establish a genetic, colon-specific and more human like CRC model driven by loss of K8 and Apc. Colon epithelium specific targeting using the CDX2P-CreERT2 mice was used to generate K8flox/flox; CDX2P-CreERT2 and K8flox/flox; CDX2P-CreERT2; Apcflox/+ mice. Body weight and stool consistency were monitored, and colon was analyzed for tumor burden and histopathology. Keratin expression, inflammation, and proliferation were assessed using immunoblotting and immunofluorescence analysis. This data was compared to K8 expression analysis in patients with CRC using UALCAN database. K8 downregulation in adult K8flox/flox; CDX2P-CreERT2 mice triggers mild diarrhea and leads to loss of K8 and reduced partner keratin levels in a mosaic pattern in the colonic epithelium, while ileal K8 protein levels are unchanged. K8-negative colon areas display increased crypt loss and more MPO+ cells predominantly in the proximal colon. Increased colonocyte proliferation is observed as increased percentage of Ki67+ cells and lower IL-22BP protein levels throughout the colon. These mice with additional monoallelic Apc inactivation show increased colon tumor formation. In colon adenocarcinoma patients, K8 expression is decreased independent of disease type and stage, age or gender. New genetic and colon-specific mouse model with loss of K8 and Apc adequately resembles human CRC. This study also highlights a role of colonocyte K8 in maintaining colon epithelial integrity and protecting against colon tumorigenesis.Competing Interest StatementThe authors have declared no competing interest.

Keratin intermediate filaments (IFs) convey mechanical stability and protection against stress to epithelial cells, and may participate in nuclear structure and organization. Keratins are important for colon health as observed in keratin 8 knockout (K8−/−) mice, which exhibit colonic inflammation and epithelial hyperproliferation. Here, using a full body and two intestinal epithelial-specific K8−/− knockout mouse models, we determine if cytoplasmic keratins affect the nuclear structure and lamina in epithelial colonocytes. K8−/− colonocytes in vivo and in organoid cultures exhibit significantly decreased levels of the major lamins A/C, B1 and B2 in a colon-specific and cell-intrinsic manner independent of major changes in colonic inflammation or microbiota. Downregulation of K8 by siRNA in Caco-2 cells similarly decreases lamin A levels, which recover after re-expression of K8. K8 loss is associated with reduced plectin, LINC complex proteins and lamin-associated proteins, indicating a dysfunctional keratin-nuclear lamina coupling. Immunoprecipitation identifies complexes of colonocyte keratins with the LINC protein SUN2 and lamin A. Hyperphosphorylation of the lamin A-associated cell cycle regulator pRb in K8−/− colonocytes together with increased nuclear localization of the mechanosensor YAP provide a molecular mechanism for the hyperproliferation phenotype. These findings identify a novel, colonocyte-specific role for K8 in nuclear function.