Marc Bissonnette

Intestinal epithelial vitamin D receptor signaling inhibits experimental colitis

The inhibitory effects of vitamin D on colitis have been previously documented. Global vitamin D receptor (VDR) deletion exaggerates colitis, but the relative anticolitic contribution of epithelial and nonepithelial VDR signaling is unknown. Here, we showed that colonic epithelial VDR expression was substantially reduced in patients with Crohns disease or ulcerative colitis. Moreover, targeted expression of human VDR (hVDR) in intestinal epithelial cells (IECs) protected mice from developing colitis. In experimental colitis models induced by 2,4,6-trinitrobenzenesulfonic acid, dextran sulfate sodium, or CD4(+)CD45RB(hi) T cell transfer, transgenic mice expressing hVDR in IECs were highly resistant to colitis, as manifested by marked reductions in clinical colitis scores, colonic histological damage, and colonic inflammation compared with WT mice. Reconstitution of Vdr-deficient IECs with the hVDR transgene completely rescued Vdr-null mice from severe colitis and death, even though the mice still maintained a hyperresponsive Vdr-deficient immune system. Mechanistically, VDR signaling attenuated PUMA induction in IECs by blocking NF-κB activation, leading to a reduction in IEC apoptosis. Together, these results demonstrate that gut epithelial VDR signaling inhibits colitis by protecting the mucosal epithelial barrier, and this anticolitic activity is independent of nonepithelial immune VDR actions.

1,25-Dihydroxyvitamin D3 Stimulates Activator Protein-1-dependent Caco-2 Cell Differentiation

1,25-Dihydroxyvitamin D3(1,25(OH)2D3) is a potential chemopreventive agent for human colon cancer. We have reported that 1,25(OH)2D3 specifically activated protein kinase C-α (PKC-α) and also caused a reduction in proliferation while increasing apoptosis and differentiation in CaCo-2 cells, a cell line derived from a human colon cancer. The mechanisms by which this secosteroid influences these important cellular processes, however, remain unclear. The transcription factor, activator protein-1 (AP-1), regulates many genes involved in these processes. Therefore, we asked whether 1,25(OH)2D3 activated AP-1 in CaCo-2 cells and, if so, by what mechanisms? 1,25(OH)2D3 caused a time-dependent increase in AP-1 DNA binding activity and significantly enhanced the protein and mRNA abundance of c-Jun, a component of AP-1. 1,25(OH)2D3 also induced a rapid and transient activation of ERK2 (where ERK is extracellular signal-regulated kinase) and a more persistent activation of JNK1 (where JNK Jun N-terminal kinase). Transfection experiments revealed that 1,25(OH)2D3 also increased AP-1 gene-transactivating activity. This AP-1 activation was completely blocked by PD 098059, a specific mitogen-activated protein kinase/ERK kinase inhibitor, as well as by a dominant negative JNK or a dominant negative Jun, indicating that the AP-1 activation induced by 1,25(OH)2D3 was mediated by ERK and JNK. Using a specific inhibitor of the Ca2+-dependent PKC isoforms, Gö6976, and CaCo-2 cells stably transfected with antisense PKC-α cDNA, demonstrated that PKC-α mediated the AP-1 activation induced by this secosteroid. Inhibition of JNK activation or c-Jun protein expression significantly reduced 1,25(OH)2D3-induced alkaline phosphatase activity, a marker of CaCo-2 cell differentiation, in secosteroid-treated cells. Taken together, the present study demonstrated that 1,25(OH)2D3 stimulated AP-1 activation in CaCo-2 cells by a PKC-α- and JNK-dependent mechanism leading to increases in cellular differentiation.

The Microbe-Derived Short Chain Fatty Acid Butyrate Targets miRNA-Dependent p21 Gene Expression in Human Colon Cancer

Colonic microbiota ferment non-absorbed dietary fiber to produce prodigious amounts of short chain fatty acids (SCFAs) that benefit the host through a myriad of metabolic, trophic, and chemopreventative effects. The chemopreventative effects of the SCFA butyrate are, in part, mediated through induction of p21 gene expression. In this study, we assessed the role of microRNA(miRNA) in butyrates induction of p21 expression. The expression profiles of miRNAs in HCT-116 cells and in human sporadic colon cancers were assessed by microarray and quantitative PCR. Regulation of p21 gene expression by miR-106b was assessed by 3′ UTR luciferase reporter assays and transfection of specific miRNA mimics. Butyrate changed the expression of 44 miRNAs in HCT-116 cells, many of which were aberrantly expressed in colon cancer tissues. Members of the miR-106b family were decreased in the former and increased in the latter. Butyrate-induced p21 protein expression was dampened by treatment with a miR-106b mimic. Mutated p21 3′UTR-reporter constructs expressed in HCT-116 cells confirmed direct miR-106b targeting. Butyrate decreased HCT-116 proliferation, an effect reversed with the addition of the miR-106b mimic. We conclude that microbe-derived SCFAs regulate host gene expression involved in intestinal homeostasis as well as carcinogenesis through modulation of miRNAs.

Titanium Dioxide in the Service of the Biomedical Revolution

Tijana Rajh,*,† Nada M. Dimitrijevic,† Marc Bissonnette,‡ Tamara Koritarov,†,§ and Vani Konda‡ †Center for Nanoscale Materials, Argonne National Laboratory, 9700 South Cass Avenue, Argonne, Illinois 60540, United States ‡Department of Medicine, The University of Chicago Medicine, 5841 South Maryland Avenue, MC 4076, Chicago, Illinois 60637, United States School of Medicine, Boston University, 72 East Concord Street, Boston, Massachusetts 02118, United States

miR‐143 and miR‐145 are downregulated in ulcerative colitis: Putative regulators of inflammation and protooncogenes

Background: miR‐143 and miR‐145 are believed to function as colon cancer tumor suppressors, as they inhibit colon cancer cell growth and are downregulated in sporadic colonic tumors. We speculated that miR‐143 and miR‐145 might also be downregulated and contribute to malignant transformation of colonic epithelium in longstanding ulcerative colitis (UC). Methods: Biopsies were obtained 20 cm proximal to the anus from individuals with quiescent UC and from normal controls. RNA and proteins were extracted and measured. miR‐143 and miR‐145 were quantified by real‐time polymerase chain reaction (PCR) and miR‐145 was also assessed by in situ hybridization. Putative targets of these miRNAs, K‐RAS, API5, MEK‐2 (miR‐143), and IRS‐1 (miR‐145) were determined by western blotting. To assess the effects of miR‐143 and miR‐145 on these predicted targets, HCT116 and HCA‐7 colorectal cancer cells were transfected with miR‐143 and miR‐145 and expression levels of these proteins were measured. Results: In UC, miR‐143 and miR‐145 were significantly downregulated 8.3‐fold (3.4–20.1) (P < 0.0001) and 4.3‐fold (2.3–7.8) (P < 0.0001), respectively, compared to normal colon. In contrast, IRS‐1, K‐RAS, API5, and MEK‐2 were upregulated in UC, consistent with their assignments as targets of these miRNAs. Furthermore, transfected miR‐143 and miR‐145 significantly downregulated these proteins in HCT116 or HCA‐7 cells. Conclusions: Compared to normal colonic mucosa, in chronic UC miR‐143 and miR‐145 were significantly downregulated and their predicted targets, IRS‐1, K‐RAS, API5, and MEK‐2 were upregulated. We postulate that loss of these tumor suppressor miRNAs predispose to chronic inflammation and neoplastic progression in IBD. (Inflamm Bowel Dis 2011;)

Vitamin D receptor is not required for the rapid actions of 1,25-dihydroxyvitamin D3 to increase intracellular calcium and activate protein kinase C in mouse osteoblasts

The rapid, non‐genomic actions of 1,25‐dihydroxyvitamin D3 [1,25(OH)2D3] have been well described, however, the role of the nuclear vitamin D receptor (VDR) in this pathway remains unclear. To address this question, we used VDR(+/+) and VDR(−/−) osteoblasts isolated from wild‐type and VDR null mice to study the increase in intracellular calcium ([Ca2+]i) and activation of protein kinase C (PKC) induced by 1,25(OH)2D3. Within 1 min of 1,25(OH)2D3 (100 nM) treatment, an increase of 58 and 53 nM in [Ca2+]i (n = 3) was detected in VDR(+/+) and VDR(−/−) cells, respectively. By 5 min, 1,25(OH)2D3 caused a 2.1‐ and 1.9‐fold increase (n = 6) in the phosphorylation of PKC substrate peptide acetylated‐MBP4–14 in VDR(+/+) and VDR(−/−) osteoblasts. The 1,25(OH)2D3‐induced phosphorylation was abolished by GF109203X, a general PKC inhibitor, in both cell types, confirming that the secosteroid induced PKC activity. Moreover, 1,25(OH)2D3 treatment resulted in the same degree of translocation of PKC‐α and PKC‐δ, but not of PKC‐ζ, from cytosol to plasma membrane in both VDR(+/+) and VDR(−/−) cells. These experiments demonstrate that the 1,25(OH)2D3‐induced rapid increases in [Ca2+]i and PKC activity are neither mediated by, nor dependent upon, a functional nuclear VDR in mouse osteoblasts. Thus, VDR is not essential for these rapid actions of 1,25(OH)2D3 in osteoblasts.

EGFR Signals Downregulate Tumor Suppressors miR-143 and miR-145 in Western Diet–Promoted Murine Colon Cancer: Role of G1 Regulators

Epidermal growth factor receptors (EGFR) contribute to colonic tumorigenesis in experimental models of colon cancer. We previously showed that EGFR was also required for colonic tumor promotion by Western diet. The goal of this study was to identify EGFR-regulated microRNAs that contribute to diet-promoted colonic tumorigenesis. Murine colonic tumors from Egfrwt and hypomorphic Egfrwa2 mice were screened using micro RNA (miRNA) arrays and miR-143 and miR-145 changes confirmed by Northern, real-time PCR, and in situ analysis. Rodent and human sporadic and ulcerative colitis (UC)-associated colon cancers were examined for miR-143 and miR-145. Effects of EGFR on miR-143 and miR-145 expression were assessed in murine and human colonic cells and their putative targets examined in vitro and in vivo. miR-143 and miR-145 were readily detected in normal colonocytes and comparable in Egfrwt and Egfrwa2 mice. These miRNAs were downregulated in azoxymethane and inflammation-associated colonic tumors from Egfrwt mice but upregulated in Egfrwa2 tumors. They were also reduced in human sporadic and UC colon cancers. EGFR signals suppressed miR-143 and miR-145 in human and murine colonic cells. Transfected miR-143 and miR-145 inhibited HCT116 cell growth in vitro and in vivo and downregulated G1 regulators, K-Ras, MYC, CCND2, cdk6, and E2F3, putative or established targets of these miRNAs. miRNA targets Ras and MYC were increased in colonic tumors from Egfrwt but not Egfrwa2 mice fed a Western diet. EGFR suppresses miR-143 and miR-145 in murine models of colon cancer. Furthermore, Western diet unmasks the tumor suppressor roles of these EGFR-regulated miRNAs. Mol Cancer Res; 9(7); 960–75. ©2011 AACR.

Elevated protein expression of cyclin D1 and Fra-1 but decreased expression of c-Myc in human colorectal adenocarcinomas overexpressing β-catenin

Mutations of the adenomatous polyposis coli tumor suppressor gene, or its downstream target β‐catenin, have been implicated in the initiation of most sporadic human colorectal epithelial neoplasms. These mutations, in turn, lead to aberrant nuclear accumulation of β‐catenin and subsequent activation of the β‐catenin/Tcf transcription factor complex. In vitro studies utilizing cultured human colon cancer cell lines have identified c‐myc, cyclin D1 and fra‐1 as target genes of β‐catenin/Tcf signaling. In our study, 12 cases of human colorectal adenocarcinomas were examined by Western immunoblotting analysis and immunohistochemical staining to specifically investigate whether the protein expression of these target genes was indeed altered in vivo by β‐catenin dysregulation. The results show that the protein level of β‐catenin was significantly increased in all 12 tumors (3.4 ± 1.0‐fold increase compared to the control normal mucosa by Western immunoblotting, p < 0.05), and this increase was associated with positive nuclear staining by immunohistochemistry in 10 cases. Increased levels of expression of cyclin D1 and Fra‐1 proteins were also demonstrated in every tumor (9.0 ± 2.7 and 3.3 ± 0.9‐fold increases compared to normal mucosa, respectively). Surprisingly, the protein level of c‐Myc was significantly decreased in all tumors examined by 49 ± 19% (p < 0.05), but the c‐myc mRNA level was increased in 8 of 12 tumors when compared to that in normal mucosa by RT‐PCR. Immunohistochemical staining performed on these carcinomas and additional 27 colorectal carcinomas further demonstrated that the protein expression level of c‐Myc and β‐catenin nuclear localization were not correlated. Moreover, 15 of 20 colorectal adenomas exhibited positive nuclear β‐catenin immunostaining, among which 11 also exhibited increased c‐Myc protein expression. These data thus support the notion that upregulation of cyclin D1 and Fra‐1 in human colorectal adenocarcinomas is driven by abnormally expressed β‐catenin. However, the regulation of c‐myc expression in colorectal tumors appears to be more complex. While dysregulated β‐catenin may cause a transcriptional upregulation of the c‐myc gene, the c‐Myc protein expression appears to be further regulated by a posttranscriptional mechanism(s) during the process of neoplastic progression.

1,25 dihydroxyvitamin D3 stimulates phospholipase C-gamma in rat colonocytes: role of c-Src in PLC-gamma activation.

Our laboratory has previously demonstrated that 1,25-dihydroxyvitamin D3 (1,25[OH]2D3) rapidly stimulated polyphosphoinositide (PI) hydrolysis, raised intracellular Ca2+, and activated two Ca2+-dependent protein kinase C (PKC) isoforms, PKC-alpha and -betaII in the rat large intestine. We also showed that the direct addition of 1,25(OH)2D3 to isolated colonic membranes failed to stimulate PI hydrolysis, but required secosteroid treatment of intact colonocytes, suggesting the involvement of a soluble factor. Furthermore, this PI hydrolysis was restricted to the basal lateral plasma membrane of these cells. In the present studies, therefore, we examined whether polyphosphoinositide-phospholipase C-gamma (PI-PLC-gamma), a predominantly cytosolic isoform of PI-PLC, was involved in the hydrolysis of colonic membrane PI by 1,25(OH)2D3. This isoform has been shown to be activated and membrane-associated by tyrosine phosphorylation. We found that 1,25(OH)2D3 caused a significant increase in the biochemical activity, particulate association, and the tyrosine phosphorylation of PLC-gamma, specifically in the basal lateral membranes. This secosteroid also induced a twofold increase in the activity of Src, a proximate activator of PLC-gamma in other cells, with peaks at 1 and 9 min in association with Src tyrosine dephosphorylation. 1,25(OH)2D3 also increased the physical association of activated c-Src with PLC-gamma. In addition, Src isolated from colonocytes treated with 1,25(OH)2D3, demonstrated an increased ability to phosphorylate exogenous PLC-gamma in vitro. Inhibition of 1,25(OH)2D3-induced Src activation by PP1, a specific Src family protein tyrosine kinase inhibitor, blocked the ability of this secosteroid to stimulate the translocation and tyrosine phosphorylation of PLC-gamma in the basolateral membrane (BLM). Src activation was lost in D deficiency, and was reversibly restored with the in vivo repletion of 1,25(OH)2D3. These studies demonstrate for the first time that 1,25(OH)2D3 stimulates PLC-gamma as well as c-Src in rat colonocytes, and indicate that PLC-gamma is a direct substrate of secosteroid-activated c-Src in these cells.

Protein kinase C α modulates growth and differentiation in Caco-2 cells

Abstract Background & Aims: Caco-2 cells have been used extensively to elucidate events involved in intestinal cell proliferation and differentiation. Because individual isoforms of protein kinase C (PKC) and p21 waf1 , a cyclin-dependent kinase inhibitor, may regulate these processes, their role(s) on the growth and differentiation of Caco-2 cells were assessed. Methods: Protein abundance and subcellular distribution of several PKC isoforms, as well as the expression of p21 waf1 , were examined in preconfluent and postconfluent cells. Results: In cells at confluence (~7 days postplating) and during their postconfluent phase (up to 20 days postplating), both total protein expression of PKC-α and its particulate distribution increased compared with their 3-day postplated counterparts. These findings were in agreement with those obtained by immunocytochemistry of PKC-α. In contrast, neither the total expression nor the subcellular distribution of PKC-βI, -βII, -δ, or -ζ changed significantly during these time periods. In addition, the expression of p21 waf1 , which can be induced by PKC-α, increased in postconfluent cells. Conclusions: PKC-α, but not other isoforms of PKC, may modulate the proliferation and differentiation of Caco-2 cells. This regulation appears to be mediated, at least in part, via a mechanism involving p21 waf1 . GASTROENTEROLOGY 1998;114:503-509