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

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

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

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-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