Keratins Are Altered in Intestinal Disease-Related Stress Responses

Keratin (K) intermediate filaments can be divided into type I/type II proteins, which form obligate heteropolymers. Epithelial cells express type I-type II keratin pairs, and K7, K8 (type II) and K18, K19 and K20 (type I) are the primary keratins found in the single-layered intestinal epithelium. Keratins are upregulated during stress in liver, pancreas, lung, kidney and skin, however, little is known about their dynamics in the intestinal stress response. Here, keratin mRNA, protein and phosphorylation levels were studied in response to murine colonic stresses modeling human conditions, and in colorectal cancer HT29 cells. Dextran sulphate sodium (DSS)-colitis was used as a model for intestinal inflammatory stress, which elicited a strong upregulation and widened crypt distribution of K7 and K20. K8 levels were slightly downregulated in acute DSS, while stress-responsive K8 serine-74 phosphorylation (K8 pS74) was increased. By eliminating colonic microflora using antibiotics, K8 pS74 in proliferating cells was significantly increased, together with an upregulation of K8 and K19. In the aging mouse colon, most colonic keratins were upregulated. In vitro, K8, K19 and K8 pS74 levels were increased in response to lipopolysaccharide (LPS)-induced inflammation in HT29 cells. In conclusion, intestinal keratins are differentially and dynamically upregulated and post-translationally modified during stress and recovery.</p

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 Are Altered in Intestinal Disease-Related Stress Responses

Keratin (K) intermediate filaments can be divided into type I/type II proteins, which form obligate heteropolymers. Epithelial cells express type I-type II keratin pairs, and K7, K8 (type II) and K18, K19 and K20 (type I) are the primary keratins found in the single-layered intestinal epithelium. Keratins are upregulated during stress in liver, pancreas, lung, kidney and skin, however, little is known about their dynamics in the intestinal stress response. Here, keratin mRNA, protein and phosphorylation levels were studied in response to murine colonic stresses modeling human conditions, and in colorectal cancer HT29 cells. Dextran sulphate sodium (DSS)-colitis was used as a model for intestinal inflammatory stress, which elicited a strong upregulation and widened crypt distribution of K7 and K20. K8 levels were slightly downregulated in acute DSS, while stress-responsive K8 serine-74 phosphorylation (K8 pS74) was increased. By eliminating colonic microflora using antibiotics, K8 pS74 in proliferating cells was significantly increased, together with an upregulation of K8 and K19. In the aging mouse colon, most colonic keratins were upregulated. In vitro, K8, K19 and K8 pS74 levels were increased in response to lipopolysaccharide (LPS)-induced inflammation in HT29 cells. In conclusion, intestinal keratins are differentially and dynamically upregulated and post-translationally modified during stress and recovery

In vivo imaging of reactive oxygen and nitrogen species in murine colitis

Traditional techniques analyzing mouse colitis are invasive, laborious, or indirect. Development of in vivo imaging techniques for specific colitis processes would be useful for monitoring disease progression and/or treatment effectiveness. The aim was to evaluate the applicability of the chemiluminescent probe L-012, which detects reactive oxygen and nitrogen species, for in vivo colitis imaging.Two genetic colitis mouse models were used; K8 knockout (K8(-/-)) mice, which develop early colitis and the nonobese diabetic mice, which develop a transient subclinical colitis. Dextran sulphate sodium was used as a chemical colitis model. Mice were anesthetized, injected intraperitoneally with L-012, imaged, and quantified for chemiluminescent signal in the abdominal region using an IVIS camera system.K8(-/-) and nonobese diabetic mice showed increased L-012-mediated chemiluminescence from the abdominal region compared with control mice. L-012 signals correlated with the colitis phenotype assessed by histology and myeloperoxidase staining. Although L-012 chemiluminescence enabled detection of dextran sulphate sodium-induced colitis at an earlier time point compared with traditional methods, large mouse-to-mouse variations were noted. In situ and ex vivo L-012 imaging as well as [18F]FDG-PET imaging of K8(-/-) mice confirmed that the in vivo signals originated from the distal colon. L-012 in vivo imaging showed a wide variation in reactive oxygen and nitrogen species in young mice, irrespective of K8 genotype. In aging mice L-012 signals were consistently higher in K8(-/-) as compared to K8(+/+) mice.In vivo imaging using L-012 is a useful, simple, and cost-effective tool to study the level and longitudinal progression of genetic and possibly chemical murine colitis

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

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