Distinct expression patterns of ICK/MAK/MOK protein kinases in the intestine implicate functional diversity.

ICK/MRK (intestinal cell kinase/MAK-related kinase), MAK (male germ cell-associated kinase), and MOK (MAPK/MAK/MRK-overlapping kinase) are closely related serine/threonine protein kinases in the protein kinome. The biological functions and regulatory mechanisms of the ICK/MAK/MOK family are still largely elusive. Despite significant similarities in their catalytic domains, they diverge markedly in the sequence and structural organization of their C-terminal non-catalytic domains, raising the question as to whether they have distinct, overlapping, or redundant biological functions. In order to gain insights into their biological activities and lay a fundamental groundwork for functional studies, we investigated the spatio-temporal distribution patterns and the expression dynamics of ICK/MAK/MOK protein kinases in the intestine. We found that ICK/MAK/MOK proteins display divergent expression patterns along the duodenum-to-colon axis and during postnatal murine development. Furthermore, they are differentially partitioned between intestinal epithelium and mesenchyme. A significant increase in the protein level of ICK, but not MAK, was induced in human primary colon cancer specimens. ICK protein level was up-regulated whereas MOK protein level was down-regulated in mouse intestinal adenomas as compared with their adjacent normal intestinal mucosa. These data suggest distinct roles for ICK/MAK/MOK protein kinases in the regulation of intestinal neoplasia. Taken together, our findings demonstrate that the expressions of ICK/MAK/MOK proteins in the intestinal tract can be differentially and dynamically regulated, implicating a significant functional diversity within this group of protein kinases

Laparoscopic colorectal resection combined with simultaneous thermal ablation or surgical resection of liver metastasis: a retrospective comparative study

Objective Colorectal liver metastasis is a critical cause of mortality. However, the safety and long-term prognosis of simultaneous colorectal tumor resection along with hepatic lesion ablation are debated. The current analysis was conducted to further clarify the controversy. Methods In this retrospective study, we collected data of 68 patients who underwent ablation or resection for liver lesions combined with simultaneous laparoscopic primary colorectal tumor resection between September 2011 and October 2016 at the Third Affiliated Hospital of Sun Yat-sen University. Perioperative outcomes and long-term follow-up data were compared between patients in the resection and ablation groups. Results Both groups had similar surgical duration (286.70 ± 78.33 vs. 313.67 ± 80.90 min), conversion rate (2 vs. 0), total expenses (81.51 ± 20.20 vs. 82.21 ± 27.81 kRMB, p = .914) and morbidities (11 vs. 24, p = .667). However, the postoperative hospital stays (12.82 ± 9.25 vs. 8.40 ± 2.38 d) and transfusion rates (56.52% vs. 8.89%) were significantly lower in the ablation group. The long-term overall survival (p = .714), disease-free survival (p = .680) and intra-hepatic recurrent-free survival (p = .496) were comparable between both groups. Conclusion With respect to simultaneous treatment for both primary colorectal cancer and liver metastasis, hepatic lesion ablation was associated with lower blood loss and hospital stay duration than liver resection, without compromising the surgical safety and long-term prognosis

Expression of ICK/MAK/MOK proteins along the duodenum-to-colon axis in the intestine.

<p>The intestinal tube was rapidly dissected from four littermates of postnatal day 21 and segmented into duodenum (D), proximal jejunum (PJ), distal jejunum (PJ), ileum (I), and colon (C) respectively. Tissue extracts were prepared from frozen intestinal tissues. Equal amount of total proteins (50 µg) were loaded on Western blot against ICK, MAK, and MOK antibodies respectively. ERK signal was shown to demonstrate an equal distribution pattern along the duodenum-to-colon axis, whereas AKT signal was shown to demonstrate a colon-enriched distribution pattern under the normal steady-state condition.</p

Expression of ICK/MAK/MOK proteins in young adult and embryonic mouse tissues.

<p>(A) Equal amount of total proteins (50 µg) extracted from various frozen tissues of young adult C57BL/6J mice (8 week-old) were Western blotted against antibodies recognizing ICK, MAK, and MOK respectively. Western blot signal of ERK2, closely related to ICK/MAK/MOK, was also shown as a control to indicate a universal expression pattern in various young adult mouse tissues. (B) Equal amount of total proteins (50 µg) extracted from frozen tissues (lung, liver, kidney and intestine) of mouse embryos of E13.5 and E16.5 were Western blotted against antibodies recognizing ICK, MAK, and MOK respectively. Western blot signals of ERK1/2 were shown as controls to indicate a universal expression pattern in various mouse embryonic tissues.</p

Schematic illustration of the structural domains and features of murine ICK, MAK and MOK protein kinases.

<p>Although ICK, MAK and MOK protein kinases are very similar in their N-terminal catalytic domains, they differ markedly in the structural organization and features in their C-terminal non-catalytic domains. Note that the antigen peptides for ICK, MAK, and MOK proteins are all located at the C-terminal domain by design so as to ensure the antibody specificity.</p

Differential partition of ICK/MAK/MOK proteins between intestinal epithelial and mesenchymal cells.

<p>Intestinal epithelial cells and mesenchymal cells were isolated and enriched from small intestine (A) and colon (B) tissues of young adult C57BL/6J mice. ICK/MAK/MOK protein signals in these two distinct cell populations were analyzed on Western blot. The E-Cadherin signal was used as a marker for epithelial cells and the VIM (Vimentin) and α-SMA (α-Smooth Muscle Actin) signals were used as markers for mesenchymal cells. The ERK1/2 signals were shown to indicate an equal distribution pattern between the epithelial and mesenchymal cell populations.</p

ICK and MAK proteins exhibited distinct expression patterns in human colorectal tumors.

<p>(A) Tissue extracts from nine pairs of human specimens macro-dissected from primary colon tumor (T) and its surrounding normal mucosa (N) were prepared. Equal amount of total proteins (30 µg) from tissue extracts were loaded for Western blot. ERK2 signals indicate equal loading of total proteins. PCNA was used as a proliferation marker. (B) Western blot signals of ICK and MAK were quantified by densitometry and normalized against ERK2 signals. ICK and MAK protein levels in tumors relative to their adjacent normal tissues were shown as mean ± SE, n = 9, P<0.05, Paired <i>t</i>-Test.</p

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