Intestinal Cell Kinase Is a Novel Participant in Intestinal Cell Signaling Responses to Protein Malnutrition

<div><p>Nutritional deficiency and stress can severely impair intestinal architecture, integrity and host immune defense, leading to increased susceptibility to infection and cancer. Although the intestine has an inherent capability to adapt to environmental stress, the molecular mechanisms by which the intestine senses and responds to malnutrition are not completely understood. We hereby report that intestinal cell kinase (ICK), a highly conserved serine/threonine protein kinase, is a novel component of the adaptive cell signaling responses to protein malnutrition in murine small intestine. Using an experimental mouse model, we demonstrated that intestinal ICK protein level was markedly and transiently elevated upon protein deprivation, concomitant with activation of prominent pro-proliferation and pro-survival pathways of Wnt/β-catenin, mammalian target of rapamycin (mTOR), mitogen-activated protein kinase (MAPK), and protein kinase B (PKB/Akt) as well as increased expression of intestinal stem cell markers. Using the human ileocecal epithelial cell line HCT-8 as an <i>in</i><i>vitro</i> model, we further demonstrated that serum starvation was able to induce up-regulation of ICK protein in intestinal epithelial cells in a reversible manner, and that serum albumin partially contributed to this effect. Knockdown of ICK expression in HCT-8 cells significantly impaired cell proliferation and down-regulated active β-catenin signal. Furthermore, reduced ICK expression in HCT-8 cells induced apoptosis through a caspase-dependent mechanism. Taken together, our findings suggest that increased ICK expression/activity in response to protein deprivation likely provides a novel protective mechanism to limit apoptosis and support compensatory mucosal growth under nutritional stress.</p></div

ICK deficiency in HCT-8 cells induced apoptosis via the caspase-dependent mechanism.

<p>(A) HCT-8 cells expressing either the ICK-specific shRNA (shICK) or the control shRNA (shCTL) were assessed for the number of apoptotic cells using Annexin V staining. The relative fold change of Annexin V stain-positive cells was shown as mean ± SE, n = 4, **P<0.01. Similar results were obtained from two independent experiments. (B) Equal amount of total proteins from cell extracts was used on Western blot against antibodies recognizing key components in the caspase pathway as indicated. Western blot signals were quantified using densitometry and shown as the fold change after normalization.</p

Serum albumin as a supplement to the starvation medium significantly lowered starvation-induced ICK protein level.

<p>(A) The albumin protein content from 1 µl of the complete medium (10% FBS), the starvation medium (0.2% FBS), or the starvation medium supplemented with either 0.25% or 0.5% BSA was analyzed and shown in a Coomassie blue-stained SDS-Gel. (B) HCT-8 cells were grown either in the complete medium containing 10% serum, or in the starvation medium containing 0.2% serum for 40 min, or in the starvation medium for 20 min followed by in the starvation medium supplemented with either 0.25% or 0.5% purified bovine serum albumin (BSA) for 20 min. Equal amount of total proteins from cell extracts were Western blotted against ICK and β-actin antibodies respectively. After densitometry quantification and normalization against β-actin, the fold change of the ICK protein level relative to the complete medium condition was shown as mean ± SE, n = 3, *P<0.05, ^#P<0.01. Similar results were obtained from two independent experiments.</p

Serum-starvation of HCT-8 ileocecal epithelial cells <i>in</i><i>vitro</i> induces acute up-regulation of ICK protein in a reversible manner.

<p> (A) HCT-8 cells were starved in growth medium containing 1% serum for various time points. Equal amount of total proteins from cell extracts were Western blotted against ICK and β-actin antibodies as indicated. After densitometry quantification and normalization against β-actin, the fold change of the ICK protein abundance relative to time zero during serum starvation was shown as mean ± SE, n = 2, *P<0.05. Similar results were obtained from three independent experiments. (B) HCT-8 cells were grown either in the complete medium containing 10% serum, or in the starvation medium containing 0.2% serum for 40 min, or in the starvation medium for 20 min followed by in the recovery medium containing either 2% or 10% serum for 20 min. Equal amount of total proteins from cell extracts were Western blotted against ICK and β-actin antibodies respectively. After densitometry quantification and normalization against β-actin, the fold change of the ICK protein level relative to the complete medium condition was shown as mean ± SE, n = 3, *P<0.05, <i>N.S.</i>, not significant. Similar results were obtained from three independent experiments.</p

Protein malnutrition induces up-regulation of key signaling pathways that are related to intestinal cell growth and survival.

<p>C57BL/6 mice at postnatal day 28 were fed with an isocaloric low-protein (2% protein) diet as compared with the regular diet containing 20% protein for a period of 5 days. (A) Equal amount of total proteins from ileum were Western blotted against antibodies recognizing key components in various signaling pathways as indicated as well as intestinal stem cell markers Lgr5 and Bmi1. The β-actin signal indicates equal loading of total proteins from intestinal tissue extracts. The doublet bands recognized by the S6K1 antibody may represent two alternatively spliced isoforms. (B) After densitometry quantification and normalization against β-actin, the fold change of the protein level relative to the control day zero was shown as mean ± SE, n = 3, *P<0.05, ^#P<0.01. Similar results were obtained from three independent experiments.</p

Correlations between urinary metabolites and inflammatory biomarkers.

<p> Spearman’s correlation heatmap between the urinary metabolites identified in the OPLS-DA models and the levels of inflammatory biomarkers LCN-2 and MPO on day 14 post infection. Only significant correlations following <i>P</i> value adjustment are shown (Benjamini-Hochberg for 5% false discovery rate).</p

Prolonged weight loss following <i>Campylobacter</i> infection.

<p><b>Panel A.</b> Mice fed either HC, or dPD had transient weight loss following infection, but dZD-fed infected mice had significant weight loss (* dZD infected vs uninfected days 2–14 post infection; P<0.001). While mice on dPD showed weight loss with no diarrhea, mice on HC had non-bloody soft stools on days 1–3 post infection, and mice on dZD had persistent bloody diarrhea on days 2–11 post infection. <b>Panel B.</b> Increasing <i>Campylobacter</i> detected in stool for the duration of the experiment (* dZD infected vs HC or dPD infected days 7&11 post infection; P<0.0001). <b>Panel C.</b> Images of stool following <i>Campylobacter</i> infection. Stool collected from HC (images from day 1 and 3 post infection) and dZD fed mice progressed from soft to bloody diarrhea by day 3 post infection (images from day 3 and 7 post infection). (N = 8/group).</p

Inflammatory biomarkers following <i>Campylobacter</i> infection.

<p>The inflammatory biomarkers myeloperoxidase (MPO) and lipocalin-2 (LCN-2) were measured in cecal contents of antibiotic pretreated mice. LCN-2 was significantly elevated in dZD <i>C</i>. <i>jejuni</i> infected mice on both day 2 and 14 post infection, * dZD infected vs dZD uninfected; P<0.05. MPO was also significantly increased in dZD-fed infected mice on both day 2 and 14 post infection, ** dZD infected vs dZD uninfected; P<0.01.</p

Figure 1

<p>A. Body weight gain (% initial weight) from experimental uninfected and undernourished groups under a low protein diet. APOE 4/4 targeted replacement (APOE 4/4 TR) mice (n = 17) showed 643 a better growth response in comparison with APOE 3/3 targeted replacement (APOE 3/3 TR) mice (n = 8). <b>B.</b> Body weight gain (% initial infection weight) from experimental mice challenged by a compounded malnutrition and <i>Cryptosporidium parvum</i> insult. Undernourished mice were orally inoculated with 10⁷- unexcysted oocysts diluted in 100 µl of PBS. APOE deficient mice show impaired growth following <i>Cryptosporidium parvum</i> infection as compared to the other groups. Results are shown as mean ±SEM.</p

Quantitative real-time PCR assays from experimental mice for the following ileal mRNA transcripts: (A) cationic amino acid transporter (CAT-1); (B) arginase 1; (C) Toll-like receptor 9 (TLR9); and (D) Inducible nitric oxide synthase (iNOS).

<p>Experimental mice were challenged by a compounded malnutrition and <i>Cryptosporidium parvum</i> insult and samples were harvested on day 7 post-<i>C. parvum inoculum</i>. Wild-type, APOE knockout, and APOE targeted replacement mice (APOE 3/3 TR and APOE 4/4 TR) were orally inoculated with 10⁷- unexcysted oocysts diluted in 100 µl of PBS. Groups have at least 4 per groups and the results are shown as mean ±SEM and expressed after β-actin normalization.</p