The amount of keratins matters for stress protection of the colonic epithelium

Keratins (K) are important for epithelial stress protection as evidenced by keratin mutations predisposing to human liver diseases and possibly inflammatory bowel diseases. A role for K8 in the colon is supported by the ulcerative colitis-phenotype with epithelial hyperproliferation and abnormal ion transport in K8-knockout (K8-/-) mice. The heterozygote knockout (K8+/-) colon appears normal but displays a partial ion transport-defect. Characterizing the colonic phenotype we show that K8+/- colon expresses ~50% less keratins compared to K8 wild type (K8+/+) but de novo K7 expression is observed in the top-most cells of the K8+/- and K8-/- crypts. The K8+/- colonic crypts are significantly longer due to increased epithelial hyperproliferation, but display no defects in apoptosis or inflammation in contrast to K8-/-. When exposed to colitis using the dextran sulphate sodium-model, K8+/- mice showed higher disease sensitivity and delayed recovery compared to K8+/+ littermates. Therefore, the K8+/- mild colonic phenotype correlates with decreased keratin levels and increased sensitivity to experimental colitis, suggesting that a sufficient amount of keratin is needed for efficient stress protection in the colonic epithelia

Keratins regulate β‐cell mitochondrial morphology, motility, and homeostasis

Loss of the epithelial intermediate filament protein keratin 8 (K8) in murine β cells leads to irregular insulin vesicles and decreased insulin levels. Because mitochondria are central in glucose-stimulated insulin secretion, the relationship between keratins and β-cell mitochondrial function and morphology was investigated. β cells in murine K8-knockout (K8−/−) islets of Langerhans have increased numbers of mitochondria, which are rounder and have diffuse cristae, as seen by electron microscopy. The mitochondrial network in primary cultured K8−/− β cells is more fragmented compared with K8+/+ mitochondria, correlating with decreased levels of mitofusin 2 and the mitofusin 2- and keratin-binding protein trichoplein. K8−/− β-cell mitochondria have decreased levels of total and mitochondrial cytochrome c, which correlates with a reduction in electron transport complexes I and IV. This provokes loss of mitochondrial membrane potential and reduction of ATP and insulin amount, as seen in K8−/− β cells. Mitochondria in K8 wild-type β cells and MIN6 insulinoma cells overexpressing K8 and 18 are more stationary compared with mitochondria in keratin-deficient cells. In conclusion, keratins, likely through trichoplein-mitofusin interactions, regulate both structural and dynamic functions of β-cell mitochondria, which could have implications for downstream insulin secretion

K8^+/- mice have increased crypt length.

<p>A. The K8^<b>+/-</b> baseline morphology was studied by H&E staining of the distal colon (DC) and proximal colon (PC) and the crypt lengths were measured (black arrows). B. Crypt lengths in PC and DC were measured from digital photographs of H&E stained colon sections (n = 3) taken with Zeiss Axiovert 200M microscope and processed using ImageJ software. The crypt lengths are expressed as mean length ± SD, calculated based on crypts from 3 animals / genotype.</p

Summary of the K8^+/- colonic phenotype.

<p>ND, not determined.</p><p>Summary of the K8^+/- colonic phenotype.</p

Increased number of proliferating cells in K8^+/- but unaltered anoikis of colonic crypts.

<p>A. and B. Mice were injected with BrdU (Bromodeoxyuridine, 5-bromo-2-deoxyuridine) i.p. 4 hours before sacrifice and proliferative cells were stained with anti-BrdU and quantified. Proliferating cells were significantly increased in both K8^<b>+/-</b> and K8^<b>-/-</b> mice DC (distal colon; A. b, c) and PC (proximal colon; A. e, f) compared to K8^<b>+/+</b> mice (A. a, d). Scale (a, d) = 50 μm. Brackets indicate the proliferative cell zone at the bottom of the crypt. B. The fraction of proliferative cells was quantified by counting the number of proliferative cells in relation to the total number of cells in the colonic crypts. Increased number of BrdU positive cells directly indicates the hyperproliferation in K8^<b>+/-</b> and K8^<b>-/-</b> colon. *** p<0.001. C. The level of apoptosis was assessed from K8^<b>+/-</b> and K8^<b>-/-</b> colon lysates by immunoblotting for cleaved caspase 7 (cCasp7) directly after excising from the mouse or after 1 hour of incubation at 37°C in cell culture medium. Hsc70 and caspase 7 (Casp7) were used as loading controls.</p

K8^+/- mice have decreased levels of colonic keratins and K8 mRNA.

<p>A. Total lysates of colon from K8^<b>+/+</b>, K8^<b>+/-</b> and K8^<b>-/-</b> mice (n = 3) were analyzed for the amount of K8, K18, K19 and K20 using SDS-PAGE and western blotting. Equal amount of protein was loaded to each well and Hsc70 was used as loading control. B. Keratin protein expression was quantified using ImageJ software. C. RT-PCR was performed to measure keratin mRNA levels in total RNA isolated from colon scrapings. Target genes were amplified using specific primers and KAPA probe Fast ABI Prism qPCR mix. Gene expression levels were normalized to the housekeeping gene β-actin. * = P < 0.05. Results are given as means ± SEM.</p