Qingjie Li

Response of Apcmin and A33ΔNβ-cat mutant mice to treatment with tea, sulindac, and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)

There is growing interest in the potential health benefits of tea, and a recent report described the potent antimutagenic activity of white tea in comparison with green tea against several heterocyclic amines, including 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) [Mutat. Res. 495 (2001) 61]. We compared the inhibitory effects of white and green teas with sulindac, a nonsteroidal anti-inflammatory agent, in two different mouse models of intestinal tumorigenesis. In the Apc(min) mouse, white and green teas given at human-relevant concentrations (1.5% w/v, 2-min brew), and sulindac (80 ppm in the drinking water), each suppressed polyp formation by approximately 50%, and the combination of white tea plus sulindac was more effective than either treatment alone (P=0.05). Mice expressing an N-terminally truncated, oncogenic version of beta-catenin (A 33(delta N beta-cat) mutant mice) developed colonic aberrant crypt foci (ACF) spontaneously, but PhIP treatment increased the incidence and number of ACF per colon. In the normal-looking intestinal mucosa of Apc(min) and A 33(delta N beta-cat) mice, white tea plus sulindac treatment markedly attenuated the expression of beta-catenin protein, and this was recapitulated in vitro in cells transiently transfected with beta-catenin plus Tcf-4 and treated with tea or the major tea polyphenol epigallocatechin-3-gallate (EGCG). Expression of a beta-catenin/Tcf reporter was inhibited by EGCG in the transfected cells, and the beta-catenin/Tcf target genes cyclin D1 and c-jun were downregulated in vivo by tea plus sulindac treatment. Collectively, the data support a chemopreventive role for tea and sulindac against intermediate and late stages of colon cancer, via effects on the beta-catenin/Tcf signaling pathway.

Bcl-2 overexpression in PhIP-induced colon tumors: cloning of the rat Bcl-2 promoter and characterization of a pathway involving β -catenin, c-Myc and E2F1

β-Catenin/T-cell factor (Tcf) signaling is constitutively active in the majority of human colorectal cancers, and there are accompanying changes in Bcl-2 expression. Similarly, 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP)-induced colon tumors in the rat have increased β-catenin and elevated Bcl-2. To examine the possible direct transcriptional regulation of rat Bcl-2 by β-catenin/Tcf, we cloned and characterized the corresponding promoter region and found 70.1% similarity with its human counterpart, BCL2. Bcl-2 promoter activity was increased in response to LiCl and exogenous β-catenin, including oncogenic mutants of β-catenin found in PhIP-induced colon tumors. Protein/DNA arrays identified E2F1, but not β-catenin/Tcf, as interacting most strongly with the rat Bcl-2 promoter. Exogenous E2F1 increased the promoter activity of rat Bcl-2, except in mutants lacking the E2F1 sites. As expected, β-catenin induced its downstream target c-Myc, as well as E2F1 and Bcl-2, and this was blocked by siRNA to c-Myc or E2F1. These findings suggest an indirect pathway for Bcl-2 over-expression in PhIP-induced colon tumors involving β-catenin, c-Myc and E2F1.

Nuclear Myosin II Regulates the Assembly of Preinitiation Complex for ICAM-1 Gene Transcription

BACKGROUND & AIMS Actin-myosin II motor converts chemical energy into force/motion in muscle and nonmuscle cells. The phosphorylation of 20-kilodalton regulatory myosin light chain (MLC(20)) is critical to the cytoplasmic functions of these motors. We do not know whether myosin II and actins in the nucleus function as motors to generate relative motion, such as that between RNA polymerase II holoenzyme and DNA, for assembly of the preinitiation complex. METHODS The experiments were performed on primary cultures of human colonic circular smooth muscle cells and rat colonic circular muscle strips. RESULTS We show that myosin II and alpha- and beta-actins are present in the nuclei of colonic smooth muscle cells. The nuclear myosin II is tethered to recognition sequence AGCTCC (-39/-34) in the intercellular adhesion molecule 1 (ICAM-1) core promoter region. The actins are known to complex with RNA polymerase II, and they are tethered to the nucleoskeleton. The dephosphorylation of MLC(20) increases the transcription of ICAM-1, whereas its phosphorylation decreases it. Colonic inflammation suppresses nuclear myosin light chain kinase, which increases the unphosphorylated form of nuclear MLC(20), resulting in enhanced transcription of ICAM-1. CONCLUSIONS Myosin II is a core transcription factor. The phosphorylation/dephosphorylation of nuclear MLC(20) results in the sliding of myosin and actin molecules past each other, producing relative motion between DNA bound to the myosin II and RNA polymerase II holoenzyme bound to actins and nucleoskeleton.

Traumatic Brain Injury Dysregulates MicroRNAs to Modulate Cell Signaling in Rat Hippocampus

Traumatic brain injury (TBI) is a common cause for cognitive and communication problems, but the molecular and cellular mechanisms are not well understood. Epigenetic modifications, such as microRNA (miRNA) dysregulation, may underlie altered gene expression in the brain, especially hippocampus that plays a major role in spatial learning and memory and is vulnerable to TBI. To advance our understanding of miRNA in pathophysiological processes of TBI, we carried out a time-course microarray analysis of microRNA expression profile in rat ipsilateral hippocampus and examined histological changes, apoptosis and synapse ultrastructure of hippocampus post moderate TBI. We found that 10 out of 156 reliably detected miRNAs were significantly and consistently altered from one hour to seven days after injury. Bioinformatic and gene ontology analyses revealed 107 putative target genes, as well as several biological processes that might be initiated by those dysregulated miRNAs. Among those differentially expressed microRNAs, miR-144, miR-153 and miR-340-5p were confirmed to be elevated at all five time points after TBI by quantitative RT-PCR. Western blots showed three of the predicated target proteins, calcium/calmodulin-dependent serine protein kinase (CASK), nuclear factor erythroid 2-related factor 2 (NRF2) and alpha-synuclein (SNCA), were concurrently down- regulated, suggesting that miR-144, miR-153 and miR-340-5p may play important roles collaboratively in the pathogenesis of TBI-induced cognitive and memory impairments. These microRNAs might serve as potential targets for progress assessment and intervention against TBI to mitigate secondary damage to the brain.

TGF-β converts Th1 cells into Th17 cells through stimulation of Runx1 expression

Differentiated CD4+ T cells preserve plasticity under various conditions. However, the stability of Th1 cells is unclear, as is whether Th1 cells can convert into Th17 cells and thereby contribute to the generation of IFN‐γ+IL‐17+CD4+ T cells, the number of which correlates with severity of colitis. We investigated whether IFN‐γ+Th1 cells can convert into Th17 cells under intestinal inflammation and the mechanisms involved. IFN‐γThy1.1+ Th1 cells were generated by culturing naïve CD4+ T cells from IFN‐γThy1.1 CBir1 TCR‐Tg reporter mice, whose TCR is specific for an immunodominant microbiota antigen, CBir1 flagellin, under Th1 polarizing conditions. IFN‐γThy1.1+ Th1 cells induced colitis in Rag−/− mice after adoptive transfer and converted into IL‐17+Th17, but not Foxp3+Treg cells in the inflamed intestines. TGF‐β and IL‐6, but not IL‐1β and IL‐23, regulated Th1 conversion into Th17 cells. TGF‐β induction of transcriptional factor Runx1 is crucial for the conversion, since silencing Runx1 by siRNA inhibited Th1 conversion into Th17 cells. Furthermore, TGF‐β enhanced histone H3K9 acetylation but inhibited H3K9 trimethylation of Runx1‐ and ROR‐γt‐binding sites on il‐17 or rorc gene in Th1 cells. We conclude that Th1 cells convert into Th17 cells under inflammatory conditions in intestines, which is possibly mediated by TGF‐β induction of Runx1.

Chronic prenatal stress epigenetically modifies spinal cord BDNF expression to induce sex-specific visceral hypersensitivity in offspring

Irritable bowel syndrome (IBS) is a heterogeneous disorder with abdominal pain as one of the primary symptoms. The etiology of IBS remains unknown. Epidemiological studies found that a subset of these patients have a history of adverse early‐life experiences. We tested the hypothesis that chronic prenatal stress (CPS) epigenetically enhances brain‐derived neurotrophic factor (BDNF) in spinal cord to aggravate colon sensitivity to colorectal distension (CRD) differentially in male and female offspring.

Phosphorylation and ubiquitination of oncogenic mutants of β-catenin containing substitutions at Asp32

β-Catenin, a member of the Wnt signaling pathway, is downregulated by glycogen synthase kinase-3β (GSK-3β)-dependent phosphorylation of Ser/Thr residues in the N-terminus of the protein, followed by ubiquitination and proteosomal degradation. In human and rodent cancers, mutations that substitute one of the critical Ser/Thr residues in the GSK-3β region of β-catenin stabilize the protein and activate β-catenin/TCF/LEF target genes. This study examined three oncogenic β-catenin mutants from rat colon tumors containing substitutions adjacent to amino-acid residue Ser33, a key target for phosphorylation by GSK-3β. Compared with wild-type β-catenin (WT), the β-catenin mutants D32G, D32N, and D32Y strongly activated TCF-4-dependent transcription in HEK293 cells, and there was accumulation of β-catenin in the cell lysates. Immunoblotting with phosphospecific antibodies indicated that there was little if any effect on the phosphorylation of Ser37, Thr41 or Ser45; however, the phosphorylation of Ser33 appeared to be affected in the β-catenin mutants. Specifically, antiphospho-β-catenin 33/37/41 antibody identified high, intermediate and low expression levels of phosphorylated β-catenin in cells transfected with D32G, D32N and D32Y, respectively. Experiments with the proteosome inhibitor N-acetyl-Leu-Leu-norleucinal (ALLN) revealed ubiquitinated bands on all three mutant β-catenins, as well as on WT β-catenin. The relative order of ubiquitination was WT>D32G>D32N>D32Y, in parallel with findings from the phosphorylation studies. These results are discussed in the context of previous studies, which indicated that amino-acid residue D32 lies within the ubiquitination recognition motif of β-catenin.

Mutational Analysis of Ctnnb1 and Apc in Tumors from Rats Given 1,2-Dimethylhydrazine or 2-Amino-3-methylimidazo[4,5-f]quinoline: Mutational ‘Hotspots’ and the Relative Expression of β-Catenin and c-jun

There is growing interest in β‐catenin and its role in various human cancers. We recently reported that 2‐amino‐3‐methylimidazo[4,5‐f]quinoline (IQ)‐ and 1,2‐dimethylhydrazine (DMH)‐induced colon tumors in the rat contain mutations in Ctnnb1, the gene for β‐catenin, but the mutation spectrum was influenced by postinitiation exposure to chlorophyllin (CHL) and indole‐3‐carbinol (I3C) [Blum et al., Carcinogenesis 2001;22:315–320]. The present paper describes a follow‐up study in which all of the target organs for IQ‐ and DMH‐induced tumorigenesis were screened; Ctnnb1 mutations were found in 44 of 119 DMH‐induced colon tumors, six of 13 IQ‐induced colon tumors, 28 of 81 DMH‐induced small intestine tumors, none of five IQ‐induced small intestine tumors, four of 106 IQ‐induced liver tumors, none of 14 DMH‐induced Zymbals gland tumors, none of 24 IQ‐induced Zymbals gland tumors, and none of 29 IQ‐induced skin tumors. In tumors from rats given carcinogen alone, or carcinogen plus CHL or I3C, Ctnnb1 mutations frequently substituted amino acids adjacent to Ser33, a critical Ser/Thr residue in the glycogen synthase kinase‐3β regulatory domain of β‐catenin. However, substitution of critical Ser/Thr residues themselves was detected in only three of 24 (12.5%) of the tumors from rats given carcinogen alone, compared with 23 of 58 (40%) of the tumors from rats given carcinogen and treated postinitiation with I3C or CHL (P < 0.02). More than 50 of the colon tumors with wild‐type β‐catenin were examined further for their Apc status; the overall frequency of Apc mutations was <10%, and these genetic changes occurred exclusively in the ‘Mutation Cluster Region’ of Apc. A subset of colon tumors also was examined for expression of β‐catenin and c‐jun; these proteins were overexpressed in all tumors containing Ctnnb1 mutations, but the expression was highest in tumors with Ctnnb1 mutations affecting Thr41 and Ser45 residues in the glycogen synthase kinase‐3β region of β‐catenin. Thus, Ctnnb1 mutations occurred more frequently than Apc mutations in colon and small intestine tumors of the rat, and certain mutations upregulated β‐catenin/T‐cell factor target genes more effectively than others, perhaps influencing the response to phytochemicals administered postinitiation.

Paradoxical regulation of ChAT and nNOS expression in animal models of Crohn's colitis and ulcerative colitis

Morphological and functional changes in the enteric nervous system (ENS) have been reported in inflammatory bowel disease. We examined the effects of inflammation on the expression of choline acetyltransferase (ChAT) and nNOS in the muscularis externae of two models of colonic inflammation, trinitrobenzene sulfonic acid (TNBS)-induced colitis, which models Crohns disease-like inflammation, and DSS-induced colitis, which models ulcerative Colitis-like inflammation. In TNBS colitis, we observed significant decline in ChAT, nNOS, and protein gene product (PGP) 9.5 protein and mRNA levels. In DSS colitis, ChAT and PGP9.5 were significantly upregulated while nNOS levels did not change. The nNOS dimer-to-monomer ratio decreased significantly in DSS- but not in TNBS-induced colitis. No differences were observed in the percentage of either ChAT (31 vs. 33%)- or nNOS (37 vs. 41%)-immunopositive neurons per ganglia or the mean number of neurons per ganglia (55 ± 5 vs. 59 ± 5, P > 0.05). Incubation of the distal colon muscularis externae in vitro with different types of inflammatory mediators showed that cytokines decreased ChAT and nNOS expression, whereas H₂O₂, a component of oxidative stress, increased their expression. NF-κB inhibitor MG-132 did not prevent the IL-1β-induced decline in either ChAT or nNOS expression. These findings showed that TNBS- and DSS-induced inflammation differentially regulates the expression of two critical proteins expressed in the colonic myenteric neurons. These differences are likely due to the exposure of the myenteric plexus neurons to different combinations of Th1-type inflammatory mediators and H₂O₂ in each model.

Promotion versus suppression of rat colon carcinogenesis by chlorophyllin and chlorophyll: Modulation of apoptosis, cell proliferation, and β-catenin/Tcf signaling

The carcinogens 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 1,2-dimethylhydrazine (DMH) induce colon tumors in the rat that contain mutations in beta-catenin, but the mutation pattern can be influenced by exposure to dietary phytochemicals, such as the water-soluble derivative of chlorophyll called chlorophyllin. Whereas chlorophyllin is an effective blocking agent during the initiation phase, post-initiation responses depend upon the exposure protocol, and can be influenced by the initiating agent and the concentration of chlorophyllin. Post-initiation treatment with 0.001% chlorophyllin (w/v) in the drinking water promoted colon carcinogenesis in the rat, but much higher concentrations (1.0% chlorophyllin) led to suppression. Bromodeoxyuridine and terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) indices revealed that the promotional concentration of 0.001% chlorophyllin increased the ratio of cell proliferation to apoptosis in the colonic crypts, whereas concentrations in the range 0.0l-1.0% chlorophyllin modestly reduced this ratio. Molecular studies showed that the spectrum of beta-catenin mutations was markedly different in chlorophyllin-promoted colon tumors--many of the mutations led to direct substitutions of critical Ser/Thr residues within the glycogen synthase kinase-3beta (GSK-3beta) region, whereas in all other groups, including DMH and IQ controls, the mutations typically affected amino acids adjacent to Ser(33). Substitution of critical Ser/Thr residues caused beta-catenin and c-Jun proteins to be markedly over-expressed compared with tumors in which the mutations substituted amino acid residues flanking these critical Ser/Thr sites. In a separate study, rats were exposed to IQ or azoxymethane (AOM), a metabolite of DMH, and they were treated post-initiation with chlorophyllin, chlorophyll, copper, or phytol in the diet. Natural chlorophyll (0.08%) suppressed AOM- and IQ-induced aberrant crypt foci (ACF), whereas chlorophyllin had no effect and copper promoted the number of small ACF induced by IQ. The results suggest that further investigation of the dose-response for suppression versus promotion by chlorophyll and chlorophyllin is warranted, including studies of the beta-catenin/Tcf signaling pathway and its influence on cell proliferation and apoptosis in the colonic crypt.