Genetic networks responsive to sodium butyrate in colonic epithelial cells

We performed microarray and computational gene network analyses to identify the detailed mechanisms by which sodium butyrate (SB) induces cell growth arrest and the differentiation of mouse colonic epithelial MCE301 cells. Two thousand six hundred four differentially expressed probe sets were identified in the cells treated with 2mM SB and were classified into four groups. Of these, the gradually increased group and the gradually and remarkably decreased group contained the genetic networks for cellular development and cell cycles or canonical pathways for fatty acid biosynthesis and pyrimidine metabolism, respectively. The present results provide a basis for understanding the detailed molecular mechanisms of action of SB in colonic epithelial cells.     

Sodium butyrate alters erythropoietin glycosylation via multiple mechanisms

Recombinant human erythropoietin (rHuEPO) produced in a human kidney fibrosarcoma cell line, HT1080, was used as a model to study the effects of sodium butyrate (SB) on protein glycosylation. Treatment with 2 mM SB resulted in complex changes with respect to sugar nucleotide pools including an increase in UDP-Gal and a decrease in UDP-GlcNac. In addition, polylactosamine structures present on rHuEPO increased after SB treatment. To determine if these phenotypic changes correlated with changes in mRNA abundance, we profiled mRNA levels over a 24-h period in the presence or absence of SB using oligonucleotide microarrays. By filtering our data through a functional glycomics gene list associated with the processes of glycan degradation, glycan synthesis, and sugar nucleotide synthesis and transport we identified 26 genes with significantly altered mRNA levels. We were able to correlate the changes in message in six of these genes with measurable phenotypic changes within our system including: neu1, b3gnt6, siat4b, b3gnt1, slc17a5, and galt. Interestingly, for the two genes: cmas and gale, our measurable phenotypic changes did not correlate with changes in mRNA expression. These data demonstrate both the utility and pit falls of coupling biochemical analysis with high throughput oligonucleotide microarrays to predict how changes in cell culture environments will impact glycoprotein oligosaccharide content.     

Hepatocyte growth factor promotes colonic epithelial regeneration via Akt signaling

Hepatocyte growth factor (HGF) can promote the regeneration of injured organs, including HGF gene therapy by electroporation (EP) for liver injury. In this study, we investigated the effect of HGF on dextran sulfate sodium-induced colitis and tried to clarify the regenerative mechanisms of colonic epithelial cells and the signaling pathway involved. Colitis was induced by dextran sulfate sodium in mice, together with HGF gene transfer by EP. On day 10, the colitis was evaluated histologically and by Western blot analysis. The colonic epithelial cell line MCE301 was exposed to HGF protein, and its proliferation and activated signaling pathway were analyzed. In vivo, the histological score improved and the number of Ki-67-positive epithelial cells increased in the HGF-treated mice compared with the controls. Western blot analysis showed enhanced expression of phospho-Akt in the HGF-treated mice compared with the controls. In vitro, HGF stimulated the proliferation of MCE301 cells. There was enhanced phospho-Akt expression for more than 48 h after HGF stimulation, although phospho-ERK1/2 was enhanced for only 10 min. LY-294002 or Akt small interfering RNA suppressed cell proliferation induced by HGF. Thus HGF induces the proliferation of colonic epithelial cells via the phosphatidylinositol 3-kinase/Akt signaling pathway. HGF gene therapy can attenuate acute colitis via epithelial cell proliferation through the PI3K/Akt pathway. These data suggested that HGF gene therapy by EP may be effective for the regeneration and repair of injured epithelial cells in inflammatory bowel disease.     

Inhibition of p38 MAPK facilitates ex vivo expansion of skin epithelial progenitor cells

Ex vivo expansion of skin epithelial stem cells has long attracted great interest because of the potential utilization in transplantation and gene therapy. The use of cultured stem or progenitor cells was limited by the lack of applicable culturing system with both satisfactory expansion efficacy and well suppressed differentiation ex vivo. The p38 mitogen-activated protein kinase (MAPK) pathways are responsible for cell growth and differentiation process. We investigated the function of p38 inhibitor SB203580 in the ex vivo expansion of skin epithelial progenitor cells by comparing media with or without addition of this inhibitor. Our results showed that the culturing medium with murine 3T3 feeder layers added with 10 μM SB203580 was more effective in promoting clonal growth of human skin epithelial progenitors or stem cells than the conventional medium without SB203580. The clone initial day in cells treated with 10 μM SB203580 came 2 d earlier with higher colony formation efficiency. The skin epithelial progenitor cells treated with 10 μM SB203580 formed clones that were uniformly smaller in size, longer in sustained proliferation, shorter in clone doubling time, higher in S-phase cells percentage, and lower in levels of differentiation markers such as K10 along with higher levels of stem-cell-associated markers such as p63, K15, and ABCG2 than those cultured in the conventional medium. Collectively, these results indicate that the p38 MAPK pathways inhibitor SB203580 can be used as a culture medium additive that helps to achieve more effective ex vivo expansion of skin epithelial progenitor cells.     

Isolation and in vitro expansion of human colonic stem cells

Here we describe the isolation of stem cells of the human colonic epithelium. Differential cell surface abundance of ephrin type-B receptor 2 (EPHB2) allows the purification of different cell types from human colon mucosa biopsies. The highest EPHB2 surface levels correspond to epithelial colonic cells with the longest telomeres and elevated expression of intestinal stem cell (ISC) marker genes. Moreover, using culturing conditions that recreate the ISC niche, a substantial proportion of EPHB2-high cells can be expanded in vitro as an undifferentiated and multipotent population.     

Differential susceptibility to epithelial-mesenchymal transition (EMT) of alveolar, bronchial and intestinal epithelial cells in vitro and the effect of angiotensin II receptor inhibition

The generation of myofibroblasts via epithelial-mesenchymal transition (EMT), a process through which epithelial cells lose their polarity and become motile mesenchymal cells, is a proposed contributory factor in fibrosis of a number of organs. Currently, it remains unclear to what extent epithelia of the upper airways and large intestine are susceptible to this process. Herein, we investigated the ability of model cell lines of alveolar (A549), bronchial (Calu-3) and colonic (Caco-2) epithelial cells to undergo EMT when challenged with transforming growth factor-β1 (TGF-β1) and other pro-inflammatory cytokines. Western blot and immunofluorescence microscopy demonstrated that A549 cells readily underwent EMT, as evidenced by a spindle-like morphology, increase in the mesenchymal marker, vimentin, and down-regulation of E-cadherin, an epithelial marker. In contrast, neither Calu-3 nor Caco-2 cells exhibited morphological changes nor alterations in marker expression associated with EMT. Moreover, whilst stimulation of A549 cells enhanced migration and reduced their proliferative capacity, no such effect was observed in epithelial cell lines of the bronchus or colon. In addition, concomitant treatment of A549 cells with telmisartan, an angiotensin II receptor antagonist with antifibrotic properties, was found to reduce cytokine-induced collagen I production and cell migration, although expression levels of vimentin and E-cadherin remained unaltered. Mechanistically, telmisartan failed to inhibit phosphorylation of Smad2/3. Together, these results, using representative in vitro models of the alveolus, bronchus and colon, tentatively suggest that epithelial cell plasticity and susceptibility to EMT may differ depending on its tissue origin. Furthermore, our investigations point to the beneficial effect of telmisartan in partial abrogation of alveolar EMT.     

A method for the rapid establishment of normal adult mammalian colonic epithelial cell cultures

Summary Normal colonic epithelial cell cultures of mammalian origin are required to facilitate the study of both normal cellular functions as well as pathogenesis of certain (human) colonic diseases. To date, little information is available regarding the growth requirements of colonic epithelial cells in culture of eitehr animal or human origin. Such data would enable the development of a long-term culture system for these cells. In this study, we present methodology that results in the establishment of homogeneous cultures of adult rabbit colonic epithelial reproducibly, quickly, and in quantity. The epithelial nature of the cultures is unambiguously established by intermediate filament typing using antikeratin antibodies. Such culutres can now be used for a variety of functional studies as well as to investigate the growth requirements of colonic epithelial in culture. This work was supported by the Blinder Foundation for Crohn’s Disease Research, Harbor UCLA IBD Center (AM 36200) and grant AM 27806 from the National Institutes of Health, Bethesda, MD.     

Hepatocyte growth factor enhances IL-1β stimulated IL-8 secretion by Caco-2 epithelial cells

Hepatocyte growth factor (HGF) can induce proliferation and migration of intestinal epithelial cells and has also been shown to be important in wound healing of inflamed mucosal tissues. HGF is known to be expressed along with interleukin-1 (IL-1) by inflamed mucosal tissues, yet the effect of HGF on IL-1-induced proinflammatory cytokine responses by colonic epithelial cells is unknown. In this report, we have examined the effect of HGF on IL-1-induced secretion of IL-8 by the Caco-2 colonic epithelial cell line. HGF stimulation alone had no effect on the secretion of IL-8 by the Caco-2 cells. However, culture of the cells with HGF and suboptimal levels of IL-1 resulted in a significant enhancement of IL-8 secretion compared to cells cultured with IL-1 alone. A similar effect was seen with HGF and IL-1 simulation of monocyte chemoattractant protein-1 secretion by the rat IEC-6 intestinal epithelial cell line. The enhancing effect of HGF was seen regardless of whether the culture medium contained serum or not. Simultaneous stimulation with HGF and IL-1 was required for the enhancing effect as cells pretreated with HGF for 24 h and then stimulated with IL-1 alone secreted IL-8 levels similar to that of cells stimulated with IL-1 alone. These results suggest that in addition to wound healing, HGF may play a role in the IL-1-induced chemokine response of epithelial cells in inflamed mucosal tissues.     

Endothelin-3 stimulates survival of goblet cells in organotypic cultures of fetal human colonic epithelium

Cells within the normal human colonic epithelium undergo a dynamic cycle of growth, differentiation, and death. The organotypic culture system of human fetal colonic epithelial cells seeded on top of collagen gels with embedded colonic fibroblasts allowed prolonged culture of the colonic epithelial cells (Kalabis J, Patterson MJ, Enders GM, Marian B, Iozzo RV, Rogler G, Gimotty PA, Herlyn M. FASEB J 17: 1115-1117, 2003). Herein, we have evaluated the role of endothelin-3 (ET3) and both cognate endothelin receptors (ETRA, ETRB) for human colonic epithelial cell growth and survival. ET3 was produced continuously by the fibroblasts as a result of adenovirus-mediated gene transfer. The presence and function of the endothelin receptors (ETRs) in epithelial cells was evaluated by [(3)H]thymidine incorporation using primary epithelial cells in monoculture and by immunohistochemistry on human fetal and adult paraffin-embedded tissues. In organotypic culture, ET3 increased the number of goblet cells but not of enteroendocrine cells. The increase in goblet cells was caused by prolonged cell survival and differentiation. The inhibition of both ETRA and ETRB significantly decreased the number of goblet cells and proliferation in epithelial cells, whereas the number of enteroendocrine cells remained unchanged. ET3 induced activation of IkappaB and MAPK in the epithelial cells, suggesting that these signaling pathways mediate its proproliferation and prosurvival activities. Our results demonstrate that ET3 is involved in regulating human colonic epithelial cell proliferation and survival, particularly for goblet cells, and may be an important component of colonic homeostasis.     

ADAM-15 inhibits wound healing in human intestinal epithelial cell monolayers

The disintegrin metalloproteases (or ADAMs) are membrane-anchored glycoproteins that have been implicated in cell-cell or cell-matrix interactions and in proteolysis of molecules on the cell surface. The expression and/or the pathophysiological implications of ADAMs are not known in intestinal epithelial cells. Therefore, our aim was to investigate the expression and the role of ADAMs in intestinal epithelial cells. Expression of ADAMs was assessed by RT-PCR, Western blot analysis, and immunufluorescence experiments. Wound-healing experiments were performed by using the electric cell substrate impedence sensing technology. Our results showed that ADAMs-10, -12, and -15 mRNA are expressed in the colonic human cell lines Caco2-BBE and HT29-Cl.19A. An ADAM-15 complementary DNA cloned from Caco2-BBE poly(A)+ RNA, and encompassing the entire coding region, was found to be shorter and to present a different region encoding the cytoplasmic tail compared with ADAM-15 sequence deposited in the database. In Caco2-BBE cells and colonic epithelial cells, ADAM-15 protein was found in the apical, basolateral, and intracellular compartments. We also showed that the overexpression of ADAM-15 reduced cell migration in a wound-healing assay in Caco2-BBE monolayers. Our data show that 1) ADAM-15 is expressed in human intestinal epithelia, 2) a new variant of ADAM-15 is expressed in a human intestinal epithelial cell line, and 3) ADAM-15 is involved in intestinal epithelial cells wound-healing processes. Together, these results suggest that ADAM-15 may have important pathophysiological roles in intestinal cells.     

Activity of β2-adrenergic receptor in oral squamous cell carcinoma is mediated by overexpression of the ADRBK2 gene: a pilot study

The β2-adrenergic receptor is most frequently involved in carcinogenic processes. Earlier studies have established a relation between the β2-adrenergic receptor and various characteristics of cancer including cell proliferation, apoptosis, chemotaxis, metastasis, tumor growth and angiogenesis. Our goal was to determine differential expression of the genes involved in adrenergic receptors using DNA microarrays and to confirm their under- or overexpression using real-time quantitative PCR. Five of the nine genes investigated showed significantly altered expression levels in tumor cells (p < 0.05). The gene product with the highest Z-score (restrictive statistical technique for selection of appropriate genes to study) was ADRBK2. Significantly, most of the overexpressed genes were related to β-adrenergic receptors. Real-time PCR analysis confirmed the up regulation observed in the microarrays, which indicated overexpression in 100% of the tumors. In oral squamous cell carcinomas, malignant cells and surrounding tissue overexpress the ADRBK2 gene.     

The major colonic cell mitogen extractable from colonic mucosa is an N terminally extended form of basic fibroblast growth factor.

Colonic growth factors (CGFs) were extracted from porcine intestinal epithelium and mucosa. Under acidic conditions, very little mitogenic activity (as assayed using murine 3T3 fibroblasts and a human colonic cell line) was extractable. However, by extracting at neutral or slightly alkaline pH, significant mitogenic activity for both the murine fibroblasts and human colonic carcinoma cell line could be detected. CGFs are present throughout the intestine and cecum. The epithelial mucosa of the distal colorectal region appeared to contain mitogens which were more potent for the colonic cells than the 3T3 fibroblasts. Purification of CGFs from the colonic mucosa required removal of associated mucin by pH precipitation prior to chromatographic fractionation. It was then possible to develop a complete purification (390,000-fold) scheme for the major CGF, an 18-kDa protein which bound to heparin-Sepharose. N-terminal sequence analysis yielded a single sequence (Q)SPGGAMAAGSITTLPALP, i.e. an N-terminally extended form of basic fibroblast growth factor. Apart from the substitution of Gly in bovine basic fibroblast growth factor by a Ser in porcine CGF, the proteins are identical. A similar extraction procedure using purified human colonic crypt epithelial cells yielded a mitogen for the human colonic cell line with similar chromatographic properties.     

Ablation of Doublecortin-Like Kinase 1 in the Colonic Epithelium Exacerbates Dextran Sulfate Sodium-Induced Colitis

Doublecortin-like kinase 1 (Dclk1), a microtubule-associated kinase, marks the fifth lineage of intestinal epithelial cells called tuft cells that function as tumor stem cells in Apc mutant models of colon cancer. In order to determine the role of Dclk1 in dextran sulfate sodium (DSS) induced colonic inflammation both intestinal epithelial specific Dclk1 deficient (Villin^Cre;Dclk1^f/f) and control (Dclk1^f/f) mice were fed 3% DSS in drinking water for 9 days, allowed to recover for 2 days, and killed. The clinical and histological features of DSS-induced colitis were scored and immunohistochemical, gene expression, pro-inflammatory cytokines/chemokines, and immunoblotting analyses were used to examine epithelial barrier integrity, inflammation, and stem and tuft cell features. In DSS-induced colitis, Villin^Cre;Dclk1^f/f mice demonstrated exacerbated injury including higher clinical colitis scores, increased epithelial barrier permeability, higher levels of pro-inflammatory cytokines and chemokines, decreased levels of Lgr5, and dysregulated Wnt/b-Catenin pathway genes. These results suggest that Dclk1 plays an important role in regulating colonic inflammatory response and colonic epithelial integrity.