Down Syndrome Candidate Region 1 Isoform 1L regulated tumor growth by targeting both angiogenesis and tumor cells.

Angiogenesis is critical for solid tumor growth beyond its minimal size. Previously, we reported that Down Syndrome Candidate Region 1 isoform 1L (DSCR1-1L) was one of the most up-regulated genes in endothelial cells induced by VEGF and histamine, and regulated endothelial cell proliferation, migration and angiogenesis. However, it was not known whether DSCR1-1L played a role in tumor growth. In this study, we found that DSCR1-1L shRNAs significantly inhibited the growth of transplanted melanoma in mice and its associated tumoral angiogenesis. In the gain of function assay, overexpression of DSCR1-1L cDNA in mouse endothelium is sufficient to significantly increase the tumor initiation induced by carcinogen, the growth of xenografted tumor, and the tumor metastasis in our endothelially-expressed DSCR1-1L transgenic mice, in which angiogenesis was induced. It was the first time to find that DSCR1-1L was also expressed in various tumor cells. DSCR1-1L shRNAs inhibited, but overexpression of DSCR1-1L cDNA increased, the tumor cell proliferation and migration. Most recently, we reported that DSCR1-1L modulated angiogenesis by down-regulation of VE-cadherin expression. Here, we found that DSCR1-1L down-regulated the expression of E-cadherin. Hence, DSCR1-1L is an excellent therapeutic target for cancers by regulation of both the endothelial and tumor cells through down-regulating (V)E-cadherin. DSCR1-1L shRNAs have the potential to be developed for clinical application.

A novel transcriptional complex on the VE-cadherin promoter regulated the downregulation of VE-cadherin in the Down Syndrome Candidate Region 1 isoform 1L-mediated angiogenesis.

Angiogenesis is critical for many diseases. Previously, we reported that Down Syndrome Candidate Region 1 isoform 1L (DSCR1-1L) was one of the most up-regulated genes in endothelial cells induced by VEGF and histamine, and regulated endothelial cell proliferation and Matrigel angiogenesis in mice. However, it was not known whether DSCR1-1L regulated angiogenesis in vivo and what was the molecular mechanism underlying it. In this study, gene knockdown and overexpression models were established to study the role of DSCR1-1L in angiogenesis in vivo. Further, the downstream regulatory target of DSCR1-1L was explored with molecular biological methods in vascular endothelial cells. We found that DSCR1-1L shRNAs significantly inhibited angiogenesis induced by VEGF in mice (p < 0.0001). In the gain-of-function assay, overexpression of DSCR1-1L cDNA in mouse endothelium of EC-FH-DSCR1-1L transgenic mice was sufficient to induce angiogenesis significantly (p < 0.01). DSCR1-1L regulated angiogenesis in the early stage by down-regulation of the VE-cadherin expression through targeting its transcription, but not mRNA stability. Three DSCR1-1L-targeted DNA elements in the VE-cadherin promoter were identified by promoter reporter assays, among which, a novel specific transcriptional complex was found. The DNA sequence (CTTCTG) in the VE-cadherin promoter was identified to directly interact with proteins by Electrophoresis Mobility Shift Assays and DNase I footprint assay. Hence, DSCR1-1L is an excellent therapeutic target for angiogenic diseases through down-regulating the formation of a novel transcriptional complex on the VE-cadherin promoter. DSCR1-1L shRNAs and cDNA have the potential to be developed for clinical application. Our results also contribute significantly to the field of mechanistic studies.

Orphan nuclear receptor TR3/Nur77 biologics inhibit tumor growth by targeting angiogenesis and tumor cells.

Pathological angiogenesis is a hallmark of many diseases. Previously, we reported that orphan nuclear receptor TR3/Nur77 was a critical mediator of angiogenesis to regulate tumor growth, sepsis and skin wound healing. However, none of the TR3/Nur77 targeting molecule has been in clinical trial so far. Here, we designed and generated novel TR3 shRNAs and two minigenes that had therapeutic potential for cancer treatment. In addition to extend our previous findings that tumor growth was inhibited in Nur77 knockout mice, we found that metastasis of colorectal tumor was completely inhibited in Nur77-/- mice. Tumor masses were increased ~70% and decreased ~40% in our transgenic EC-Nur77-S mice and EC-Nur77-DN mice, in which the full-length cDNA and the dominant negative mutant of TR3/Nur77 were inducibly and specifically expressed in mouse endothelium, respectively. TR3 was highly expressed in the vasculature and tumor cells of human melanoma and colorectal cancer tissues, but not in normal tissues. The novel TR3 shRNAs and two minigenes almost completely inhibited the proliferation and migration of HUVECs and human melanoma A375sm cells. Angiogenesis induced by adenoviruses expressing VEGF and melanoma growth in mice were greatly and significantly inhibited by systemically administration of adenoviruses expressing TR3 shRNAs and two minigenes. Tumor angiogenesis and the expressions of genes associated with angiogenesis were greatly regulated in tumor tissues treated with TR3 shRNAs and minigenes. Taken together, these studies demonstrated that TR3/Nur77 was a specific therapeutic target for several human cancers by targeting both tumor cells and tumor microenvironment. These TR3/Nur77 biologics inhibit angiogenesis and tumor growth, and have translational potential.

The novel axis of YAP1, transcription enhancer factor 3 and Down Syndrome Candidate Region 1 isoform 1L is a common signaling pathway downstream of several angiogenic factors.

Angiogenesis is a hallmark of many diseases. Previously, we found that Down Syndrome Candidate Region 1 Isoform 1L (DSCR1-1L) was expressed in human tumor vessels, but was not detectable in normal tissues, and played important roles in angiogenesis induced by vascular endothelial growth factor (VEGF-A165). The expressions of DSCR1-1L mRNA and protein induced by VEGF-A165 were regulated via the direct interaction of transcription enhancer factor 3 (TEF3) with DSCR1-1L promoter. However, the function and the regulation of DSCR1-1L in angiogenesis had not been completely understood. In this study, we found that the expressions of DSCR1-1L mRNA and proteins were upregulated by other angiogenic factors, including VEGF-A121, VEGF-E, histamine, PAF, the endothelial cell (EC) growth medium, and the conditional medium obtained from cancer cells, but not by PlGF, bFGF, PDGF, and serotonin. The EC proliferation, migration and elongation induced by histamine and EC growth medium were inhibited by knocking down the mRNA and protein expressions of DSCR1-1L and TEF3. The TEF3 activation was regulated by its interaction with YAP1, and translocation from cytosol to nuclei, but not by increase of protein expression, after the stimulation of VEGF, histamine and EC growth medium. YAP1 regulated the protein expression of DSCR1-1L, the proliferation, migration and elongation of ECs induced by VEGF, histamine and EC growth medium. Taken together, this study identified a novel axis of YAP1, TEF3 and DSCR1-1L that was a common signaling pathway downstream of several angiogenic factors to regulate angiogenesis, suggesting that this pathway is an excellent therapeutic target for angiogenic diseases and cancers. Our results contribute significantly to the field of mechanistic studies.

DLL4 and Jagged1 are angiogenic targets of orphan nuclear receptor TR3/Nur77.

Pathological angiogenesis is a hallmark of many diseases. Previously, we reported that orphan nuclear receptor TR3/Nur77 was a critical mediator of angiogenesis to regulate tumor growth and skin wound healing via regulating the expression of the junctional proteins and integrins. However, the molecular mechanism, by which TR3/Nur77 regulates angiogenesis is not completely understood. Here, we were the first to find that TR3/Nur77, via its various amino acid fragments, regulated the expression of DLL4 and Jagged 1 in cultured endothelial cells. DLL4 and Jagged1 mediated TR3/Nur77-induced angiogenic responses and signaling molecules, but not the expression of integrins. Instead, integrins regulated the expressions of DLL4 and Jagged1 induced by TR3/Nur77. Further, DLL4, Jagged1 and integrins α1, α2, ß3 and ß5 were regulated by TR3/Nur77 in animal sepsis models of lipopolysaccharide (LPS)-induced endotoxemia, and cecal ligation and puncture (CLP), in which, TR3/Nur77 expression was significantly and tranciently increased. Mouse survival rates were greatly increased in Nur77 knockout mice bearing both CLP and LPS models. The results elucidated a novel axis of VEGF/histamine ➔ TR3/Nur77 ➔ integrins ➔ DLL4/Jagged1 in angiogenesis, and demonstrated that TR3/Nur77 was an excellent target for sepsis. These studies supported our previous findings that TR3/Nur77 was an excellent therapeutic target, and further our understanding of the molecular mechanism, by which TR3/Nur77 regulated angiogenesis.

Orphan nuclear receptor TR3/Nur77 differentially regulates the expression of integrins in angiogenesis.

Pathological angiogenesis is a hallmark of many diseases. Previously, we reported that orphan nuclear receptor TR3/Nur77 (human homolog, Nur77, mouse homolog) is a critical mediator of angiogenesis to regulate tumor growth and skin wound healing via down-regulating the expression of the junctional proteins and integrin ß4. However, the molecular mechanism, by which TR3/Nur77 regulated angiogenesis, was still not completely understood. In this report by analyzing the integrin expression profile in endothelial cells, we found that the TR3/Nur77 expression highly increased the expression of integrins α1 and ß5, decreased the expression of integrins α2 and ß3, but had some or no effect on the expression of integrins αv, α3, α4, α5, α6, ß1 and ß7. In the angiogenic responses mediated by TR3/Nur77, integrin α1 regulated endothelial cell proliferation and adhesion, but not migration. Integrin ß5 shRNA inhibited cell migration, but increased proliferation and adhesion. Integrin α2 regulated all of the endothelial cell proliferation, migration and adhesion. However, integrin ß3 did not play any role in endothelial cell proliferation, migration and adhesion. TR3/Nur77 regulated the transcription of integrins α1, α2, ß3 and ß5, via various amino acid fragments within its transactivation domain and DNA binding domain. Furthermore, TR3/Nur77 regulated the integrin α1 promoter activity by directly interacting with a novel DNA element within the integrin α1 promoter. These studies furthered our understanding of the molecular mechanism by which TR3/Nur77 regulated angiogenesis, and supported our previous finding that TR3/Nur77 was an excellent therapeutic target for pathological angiogenesis. Therefore, targeting TR3/Nur77 inhibits several signaling pathways that are activated by various angiogenic factors.

Expressions of Orphan Nuclear Receptor TR3/Nur77 in Chronic Hepatopathy and Its Clinical Significance.

OBJECTIVE: Although great success has been achieved in cancer treatment, current cancer therapies, including anti-tumorigenesis and anti-angiogenesis, still face the problems of insufficient efficacy, resistance and intrinsic refractoriness, in addition to their toxic side effects. There is a demand to identify additional targets that can be blocked to turn off the downstream effects of most, if not all, pathways. Our studies suggest that orphan nuclear receptor TR3 (human)/Nur77 (mouse) is such a target. Most recently, we reported that TR3/Nur77 expression in human hepatic cancer tissues correlates well with tumor progress, suggesting that TR3 is a specific therapeutic target for hepatic cancers. However, the correlation of TR3/Nur77 expression in hepatocellular carcinoma (HCC) with chronic hepatitis has not been studied. METHODS: The expression of TR3/Nur77 was analyzed in human primary hepatic cancer specimens from patients that have complete medical records with Immunohistochemically staining. The statistical analysis was used to access the significance of TR3 expression in tumor tissues, cirrhosis tissues and chronic hepatitis tissues with and without hepatitis B virus infection (HBV(+) and HBV(-)), which were obtained from para-tumor tissues. RESULTS: The positive rates of TR3/Nur77 expression in hepatocellular carcinoma, cancerous liver cirrhosis and chronic hepatitis are 66.67%, 30%, and 20%, respectively, which are statistic significant (p<0.05). The positive rates of TR3/Nur77 expression in hepatocellular carcinoma are statistic significant (p<0.05) with 81.25% and 20% in HBV (+) or HBV (-), respectively. CONCLUSION: The positive expression rate of TR3/Nur77 in hepatocellular carcinoma is higher than that in chronic hepatitis and cirrhosis. The positive rate of TR3/Nur77 expression in hepatocellular carcinoma is higher with HBV infection than that without infection. Our results suggest that TR3/Nur77 plays an important role in the progression of chronic hepatitis, and the occurrence and development of HCC.

Orphan Nuclear Receptor TR3/Nur77 is a Specific Therapeutic Target for Hepatic Cancers.

OBJECTIVE: Although great success has been achieved in cancer treatment, current cancer therapies, including anti-tumorigenesis and anti-angiogenesis, still face the problems of insufficient efficacy, resistance and intrinsic refractoriness, in addition to their toxic side effects. There is a demand to identify additional targets that can be blocked to turn off the downstream effects of most, if not all, pathways. Our previous studies suggest that orphan nuclear receptor TR3 (human) / Nur77 (mouse) is such a target. However, the correlation of TR3 expression and clinical tumor progression has not been studied. METHODS: The expression of TR3 was analysed in human primary hepatic cancer specimens from patients that have complete medical records with Immunohistochemical staining. The statistical analysis was used to assess the significance of TR3 expression in tumor tissues, paratumor tissues and normal tissues, and to investigate the correlation of TR3 expression and clincopathologic characteristics. RESULTS: TR3 is highly expressed in human hepatic cancer tissues, but not in normal liver tissues. The positive expression yields of TR3 are 67.67% (14/21), 19.05% (4/21) and 0% (0/10) in cancer tissues, para cancer tissues, and normal liver tissue, respectively, which are statistic significant (χ2=17.07, p<0.005). The expression of TR3 is significantly higher in cancer tissues than in para cancer tissues χ2=9.722, p<0.005) and in normal tissues (p<0.0005). The levels of TR3 expression in human hepatic cancer tissues correlates well with tumors that are at low/middle degree of tumor differentiation and have portal vein thrombosis, metastasis and recurrence, but not with age, gender, tumor number and Alpha-fetal protein (AFP) volume. CONCLUSION: The results indicate that TR3 is a specific therapeutic target for hepatic cancers.

Magnetic Resonance Imaging Identifies Differential Response to Pro-Oxidant Chemotherapy in a Xenograft Model.

Induction of oxidative stress is a key component of cancer therapy. Pro-oxidant drugs have been demonstrated to enhance the efficacy of radiotherapy and chemotherapy. An emerging concept is that therapeutic outcomes are dictated by the differential redox buffering reserve in subpopulations of malignant cells, indicating the need for noninvasive biomarkers of tumor redox that can be used for dose identification and response assessment in a longitudinal setting. Magnetic resonance imaging (MRI) enhanced with the thiol-binding contrast agent Gd-LC6-SH, and hemodynamic response imaging (HRI) in combination with hypercapnia and hyperoxia were investigated as biomarkers of the pharmacodynamics of the small molecule pro-oxidant imexon (IMX). Human multiple myeloma cell lines 8226/S and an IMX-resistant variant, 8226/IM10, were established as contralateral tumors in SCID mice. T1slope, an MRI measure of the washout rate of Gd-LC6-SH, was significantly lower post-IMX therapy in 8226/S tumors compared with vehicle controls, indicating treatment-related oxidization of the tumor microenvironment, which was confirmed by analysis of tumor tissue for thiols. T1slope and ex vivo assays for thiols both indicated a more reduced microenvironment in 8226/IM10 tumors following IMX therapy. HRI with hypercapnia challenge revealed IMX inhibition of vascular dilation in 8226/S tumors but not 8226/IM10 tumors, consistent with decreased immunohistochemical staining for smooth muscle actin in treated 8226/S tumors. MRI enhanced with Gd-LC6-SH, and HRI coupled with a hypercapnic challenge provide noninvasive biomarkers of tumor response to the redox modulator imexon.

AP-1 transcription factor mediates VEGF-induced endothelial cell migration and proliferation.

VEGF, upon binding to its endothelial cell specific receptors VEGF-R1 and VEGF-R2, can induce endothelial cell migration, proliferation and angiogenesis. However, the molecular mechanism of these effects still remains unclear. In this study, we investigated whether VEGF promotes human umbilical vascular endothelial cell (HUVEC) migration and proliferation through activator protein-1 transcription factor (AP-1) family. We first showed that VEGF induces immediate-early genes AP-1 family gene expression differentially with the profound induction of JunB (both mRNA and protein) under various conditions (PBS, DMSO or control adenoviruses). The increase in AP-1 mRNA expression occurs primarily at the transcriptional level. Inhibition of AP-1 DNA binding activity by adenovirus expressing a potent dominant negative form of c-Fos (Afos) significantly attenuated VEGF-induced HUVEC migration and proliferation and cyclin D1 expression. Knockdown of JunB with adenovirus expressing JunB shRNA reduces VEGF-induced JunB expression and attenuated HUVEC migration. However the shJunB-expressing virus has no effect on VEGF-induced cyclin D1 protein expression and proliferation. These results suggest that VEGF-induced endothelial migration is mediated primarily by induction of JunB whereas the promotion of endothelial proliferation by VEGF is mediated by JunB-independent AP-1 family members.

Differential function and regulation of orphan nuclear receptor TR3 isoforms in endothelial cells.

TR3 has been reported to be an excellent target for angiogenesis therapies. We reported three TR3 transcript variant messenger RNAs (mRNAs) are expressed in human umbilical vein endothelial cell (HUVEC) and are differentially regulated by vascular endothelial growth factor (VEGF). TR3 transcript variant 1 (TR3-TV1) and variant 2 (TR3-TV2) encoding the same TR3 isoform 1 protein (TR3-iso1) that was named TR3 has been extensively studied. However, the function of TR3 isoform 2 protein (TR3-iso2) encoded by TR3 transcript variant 3 (TR3-TV3) is still not known. Here, we clone and express the novel TR3-iso2 protein and find that expression of TR3-iso2, in contrast to TR3-iso1, inhibits endothelial cell proliferation induced by VEGF-A, histamine, and phorbol-12-myristate-13-acetate (PMA). The differential function of TR3-iso2 correlates with the down-regulation of cyclin D1. However, TR3-iso2 plays similar roles in endothelial cell migration and monolayer permeability as TR3-iso1. We further demonstrate that several intracellular signaling pathways are involved in histamine-induced TR3 transcript variants, including histamine receptor H1-mediated phospholipase C (PLC)/calcium /calcineurin/protein kinase C (PKC)/protein kinase D (PKD) pathway and ERK pathway, as well as histamine receptor H3-mediated PKC-ERK pathway. Further, expressions of TR3-TV1, TR3-TV2, and TR3-TV3 by VEGF and histamine are regulated by different promoters, but not by their mRNA stability.

Requirement of novel amino acid fragments of orphan nuclear receptor TR3/Nur77 for its functions in angiogenesis.

Pathological angiogenesis is a hallmark of many diseases. We demonstrated that TR3/Nur77 is an excellent target for pro-angiogenesis and anti-angiogenesis therapies. Here, we report that TR3 transcriptionally regulates endothelial cell migration, permeability and the formation of actin stress fibers that is independent of RhoA GTPase. 1) Amino acid residues 344-GRR-346 and de-phosphorylation of amino acid residue serine 351 in the DNA binding domain, and 2) phosphorylation of amino acid residues in the 41-61 amino acid fragment of the transactivation domain, of TR3 are required for its induction of the formation of actin stress fibers, cell proliferation, migration and permeability. The 41-61 amino acid fragment contains one of the three potential protein interaction motifs in the transactivation domain of TR3, predicted by computational modeling and analysis. These studies further our understanding of the molecular mechanism, by which TR3 regulates angiogenesis, identify novel therapeutic targeted sites of TR3, and set the foundation for the development of high-throughput screening assays to identify compounds targeting TR3/Nur77 for pro-angiogenesis and anti-angiogenesis therapies.

Orphan nuclear receptor TR3/Nur77 improves wound healing by upregulating the expression of integrin ß4.

Tissue repair/wound healing, in which angiogenesis plays an important role, is a critical step in many diseases including chronic wound, myocardial infarction, stroke, cancer, and inflammation. Recently, we were the first to report that orphan nuclear receptor TR3/Nur77 is a critical mediator of angiogenesis and its associated microvessel permeability. Tumor growth and angiogenesis induced by VEGF-A, histamine, and serotonin are almost completely inhibited in Nur77 knockout mice. However, it is not known whether TR3/Nur77 plays any roles in wound healing. In these studies, skin wound-healing assay was performed in 3 types of genetically modified mice having various Nur77 activities. We found that ectopic induction of Nur77 in endothelial cells of mice is sufficient to improve skin wound healing. Although skin wound healing in Nur77 knockout mice is comparable to the wild-type control mice, the process is significantly delayed in the EC-Nur77-DN mice, in which a dominant negative Nur77 mutant is inducibly and specifically expressed in mouse endothelial cells. By a loss-of-function assay, we elucidate a novel feed-forward signaling pathway, integrin ß4 → PI3K → Akt → FAK, by which TR3 mediates HUVEC migration. Furthermore, TR3/Nur77 regulates the expression of integrin ß4 by targeting its promoter activity. In conclusion, expression of TR3/Nur77 improves wound healing by targeting integrin ß4. TR3/Nur77 is a potential candidate for proangiogenic therapy. The results further suggest that TR3/Nur77 is required for pathologic angiogenesis but not for developmental/physiologic angiogenesis and that Nur77 and its family members play a redundant role in normal skin wound healing.

Differential regulation of orphan nuclear receptor TR3 transcript variants by novel vascular growth factor signaling pathways.

Angiogenesis is a hallmark of many diseases, including cancer, ischemic heart disease, inflammation, and others. It is well known that vascular endothelial growth factor (VEGF) is the most important angiogenic factor. Recently, we demonstrated that orphan nuclear receptor TR3 (mouse Nur77 and rat NGFI-B) plays critical roles in tumor growth and angiogenesis induced by VEGF-A in vitro and in vivo. However, the signaling pathways that mediate the expression of TR3 induced by VEGF are still not completely understood. Here we reported that 3 TR3 transcript variants (TR3-TVs) are expressed at differential levels, and regulated differentially in endothelial cells. While the expression of TR3-TV1 is relatively low, the expression of TR3-TV2 is up-regulated markedly, and the expression of TR3-TV3 is up-regulated moderately in endothelial cells induced by VEGF-A. The kinetics of the induction of these TR3-TVs is different. We also found that several signaling pathways, including calcium-PLC-PKC-PKD1 pathway, NF-κB pathway, and MAP kinase (ERK, p38, and JNK) pathways are important for VEGF-A-induced TR3-TV2 and TR3-TV3 mRNA induction. More important, we found that VEGF-A or VEGF-E, but not VEGF-B, nor placenta growth factor (PlGF), induces the phosphorylation of insulin-like growth factor-1 receptor (IGF-1R) and the interaction of VEGF receptor 2/kinase insert domain receptor (VEGFR2/KDR) with IGF-1R, which mediates the expression of TR3-TV2, but not TR3-TV3. Taking together, we demonstrate that TR3-TVs are differentially regulated by VEGF-A and identify a novel signaling pathway by which VEGF-A and VEGF-E, but neither VEGF-B, nor PlGF, induce the interaction of VEGFR2/KDR with IGF-1R, resulting in IGF-1R transactivation to induce the high level expression of TR3-TV2. Our data not only elucidate the signaling pathways by which TR3-TVs are regulated, but extend the molecular mechanism, by which VEGF-A-induced angiogenesis. These studies should permit the development of screening assays for compounds that inhibit VEGF signaling.

The vascular permeabilizing factors histamine and serotonin induce angiogenesis through TR3/Nur77 and subsequently truncate it through thrombospondin-1.

Angiogenesis plays an important role in cancer and in many other human diseases. Vascular endothelial growth factor-A (VEGF-A), the best known angiogenic factor, was originally discovered as a potent vascular permeability factor (VPF), suggesting that other vascular permeabilizing agents, such as histamine and serotonin, might also have angiogenic activity. We recently demonstrated that, like VEGF-A, histamine and serotonin up-regulate the orphan nuclear receptor and transcription factor TR3 (mouse homolog Nur77) and that TR3/Nur77 is essential for their vascular permeabilizing activities. We now report that histamine and serotonin are also angiogenic factors that, at low micromolar concentrations, induce endothelial cell proliferation, migration and tube formation in vitro, and angiogenesis in vivo. All of these responses are mediated through specific histamine and serotonin receptors, are independent of VEGF-A, and are directly dependent on TR3/Nur77. Initially, the angiogenic response closely resembled that induced by VEGF-A, with generation of "mother" vessels. However, after ~10 days, mother vessels began to regress as histamine and serotonin, unlike VEGF-A, up-regulated the potent angiogenesis inhibitor thrombospondin-1, thereby triggering a negative feedback loop. Thus, histamine and serotonin induce an angiogenic response that fits the time scale of acute inflammation.

Redox-active magnetic resonance imaging contrast agents: studies with thiol-bearing 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracetic acid derivatives.

The synthesis and structure-activity relationships of a homologous series of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid gadolinium(III) complexes bearing thiol-terminated alkyl side chains from three to nine carbons in length are reported. The observed binding with human serum albumin (HSA) of the compounds having C-3 through C-7 side chain lengths was inhibited by homocysteine in a manner consistent with single-site binding. The observed binding with HSA of the compounds having C-8 and C-9 side chain lengths was only partly inhibited by homocysteine, consistent with multisite binding. The binding affinity of the C-7 compound could be related to the HSA oxidation state. 2D 1H-1H NMR TOCSY provided evidence of covalent binding of the europium analog of the C-6 compound to HSA-Cys34. The longitudinal water-proton MRI relaxivities of the gadolinium complexes at 7 T increased upon binding to HSA. On the basis of these results, the C-6 and C-7 compounds were identified as promising redox-sensitive MRI contrast agents.

Substance P induces CCN1 expression via histone deacetylase activity in human colonic epithelial cells.

We have shown that substance P (SP) and its neurokinin-1 receptor (NK-1R) regulate intestinal angiogenesis by increasing expression of protein CYR61 (the cysteine-rich angiogenic inducer 61, or CCN1) in colonic epithelial cells. However, the mechanism involved in SP-induced CCN1 expression has not been studied, and the outcome of increased CCN1 expression in the development of colitis is not fully understood. Because histone deacetylase (HDAC) modulates transcription of several genes involved in inflammation, we investigated participation of HDAC in SP-induced CCN1 expression in human colonic epithelial NCM460 cells overexpressing NK-1R (NCM460-NK-1R) and in primary colonocytes. SP increased HDAC activity with deacetylation and dephosphorylation of nucleosome protein histone H3 in NCM460-NK-1R and/or primary colonocytes. Histone deacetylation and dephosphorylation was observed in colonic mucosa from irritable bowel disease patients. Similarly, colonic mucosal tissues from mice exposed to dextran sulfate sodium showed histone H3 deacetylation and dephosphorylation and increased HDAC activity that was reversed by the NK-1R antagonist CJ-12255. SP-induced increased CCN1 expression in NCM460-NK-1R cells was abolished by pharmacological HDAC inhibition. HDAC overexpression activated basal and SP-induced CCN1 promoter activity. Intracolonic CCN1 overexpression significantly ameliorated dextran sulfate sodium-induced colitis, with reduction of proinflammatory cytokine expression in mice. Thus, SP-mediated CCN1 expression in the inflamed human and mouse colon involves increased HDAC activity. Our results strongly suggest that increased CCN1 expression may be involved in mucosal healing during colitis.

Orphan nuclear transcription factor TR3/Nur77 regulates microvessel permeability by targeting endothelial nitric oxide synthase and destabilizing endothelial junctions.

Low-level basal vascular permeability (BVP) provides nutrients to normal tissues, and increased vascular permeability is characteristic of inflammation and cancer. We recently reported that VEGF-A, a potent vascular permeabilizing and angiogenic factor, exerts much of its angiogenic activity by up-regulating expression of TR3/Nur77, an orphan nuclear transcription factor, in vascular endothelial cells (EC). To determine whether TR3/Nur77 had a more general role in regulating vascular permeability, we found that histamine, serotonin, and platelet-activating factor, small molecule vascular permeabilizing agents, also increased TR3/Nur77 expression acutely in EC. BVP and the acute vascular hyperpermeability (AVH) induced by these vascular permeabilizing factors were greatly decreased in Nur77(-/-) mice, and both BVP and AVH correlated with Nur77 expression levels in several different mouse strains. BVP and AVH were enhanced in transgenic mice in which Nur77 was selectively overexpressed in vascular EC, whereas both were suppressed in mice overexpressing dominant-negative Nur77. Chronic vascular hyperpermeability (CVH) was induced long before the onset of angiogenesis in a modified, in vivo Matrigel assay that included PT67 cells packaging retroviruses expressing Nur77-sense, whereas inclusion of cells packaging viruses expressing Nur77-antisense prevented VEGF-A-induced CVH. TR3/Nur77 modulated vascular permeability by increasing endothelial nitric-oxide synthase expression and by downregulating several EC junction proteins that maintain vascular homeostasis. Both functions required TR3/Nur77 transcriptional activity. Taking these data together, TR3/Nur77 is up-regulated by several vascular permeabilizing agents and has critical roles in mediating BVP, AVH, and CVH.

Cathelicidin signaling via the Toll-like receptor protects against colitis in mice.

BACKGROUND & AIMS: Cathelicidin (encoded by Camp) is an antimicrobial peptide in the innate immune system. We examined whether macrophages express cathelicidin in colons of mice with experimental colitis and patients with inflammatory bowel disease, and we investigated its signaling mechanisms. METHODS: Quantitative, real-time, reverse-transcription polymerase chain reaction (PCR), bacterial 16S PCR, immunofluorescence, and small interfering RNA (siRNA) analyses were performed. Colitis was induced in mice using dextran sulfate sodium (DSS); levels of cathelicidin were measured in human primary monocytes. RESULTS: Expression of cathelicidin increased in the inflamed colonic mucosa of mice with DSS-induced colitis compared with controls. Cathelicidin expression localized to mucosal macrophages in inflamed colon tissues of patients and mice. Exposure of human primary monocytes to Escherichia coli DNA induced expression of Camp messenger RNA, which required signaling by extracellular signal-regulated kinase (ERK); expression was reduced by siRNAs against Toll-like receptor (TLR)9 and MyD88. Intracolonic administration of bacterial DNA to wild-type mice induced expression of cathelicidin in colons of control mice and mice with DSS-induced colitis. Colon expression of cathelicidin was significantly reduced in TLR9(-/-) mice with DSS-induced colitis. Compared with wild-type mice, Camp(-/-) mice developed a more severe form of DSS-induced colitis, particularly after intracolonic administration of E coli DNA. Expression of cathelicidin from bone marrow-derived immune cells regulated DSS induction of colitis in transplantation studies in mice. CONCLUSIONS: Cathelicidin protects against induction of colitis in mice. Increased expression of cathelicidin in monocytes and experimental models of colitis involves activation of TLR9-ERK signaling by bacterial DNA. This pathway might be involved in the pathogenesis of ulcerative colitis.

Insulin-like growth factor-1 receptor transactivation modulates the inflammatory and proliferative responses of neurotensin in human colonic epithelial cells.

Neurotensin (NT) is a gastrointestinal neuropeptide that modulates intestinal inflammation and healing by binding to its high-affinity receptor NTR1. The dual role of NT in inflammation and healing is demonstrated in models of colitis induced by Clostridium difficile toxin A and dextran sulfate sodium, respectively, and involves NF-κB-dependent IL-8 expression and EGF receptor-mediated MAPK activation in human colonocytes. However, the detailed signaling pathways involved in these responses remain to be elucidated. We report here that NT/NTR1 coupling in human colonic epithelial NCM460 cells activates tyrosine phosphorylation of the insulin-like growth factor-1 receptor (IGF-1R) in a time- and dose-dependent manner. NT also rapidly induces Src tyrosine phosphorylation, whereas pretreatment of cells with the Src inhibitor PP2 before NT exposure decreases NT-induced IGF-1R phosphorylation. In addition, inhibition of IGF-1R activation by either its specific antagonist AG1024 or siRNA against IGF-1 significantly reduces NT-induced IL-8 expression and NF-κB-dependent reporter gene expression. Pretreatment with AG1024 also inhibits Akt activation and apoptosis induced by NT. Silencing of Akt expression by siRNA also substantially attenuates NT-induced IL-8 promoter activity and NF-κB-dependent reporter gene expression. This is the first report to indicate that NT transactivates IGF-1R and that this response is linked to Akt phosphorylation and NF-κB activation, contributing to both pro-inflammatory and tissue repair signaling pathways in response to NT in colonic epithelial cells. We propose that IGF-1R activation represents a previously unrecognized key pathway involved in the mechanisms by which NT and NTR1 modulate colonic inflammation and inflammatory bowel disease.