The Critical Function Of Orphan Nuclear Receptor TR3 In Gastrointestinal Tumor Angiogenesis

Background and Objective Angiogenesis is a hallmark of gastrointestinal tumorigenesis and metastasis including colon cancer, gastric cancer, hepatic carcinoma and cholangiocarcinoma. The vascular endothelial growth factor （VEGF） family which includes VEGF-A, VEGF-B, VEGF-C, VEGF-C, VEGF-D, VEGF-E and placenta growth factor （P1GF） and histamine are two of the most important known angiogenic factors.VEGF is very potential and relative selectivity for vascular endothelium （which express VEGFR-1 and VEGFR-2 as well as neuropilins）, and almost universal overexpression in pathological angiogenesis. VEGF-A affects multiple steps in the angiogenic cascade along with other VEGF family members. Although the humanized VEGF-A antibody Avastin showed effectiveness in treating several types of cancers, it also has significant side effects. Thus it is desirable to identifyspecific downstream targets of VEGF-A with less toxic effects.Histamine is an important biogenic amine, participating in acute inflammation. It is also an important neurotransmitter in the central nervous system. Histamine is widely expressed in mammalian tissues by neurons, macrophages, mast cells and basophils, many cancer cells, etc. The activities of histamine are mediated through 4 G-protein-coupled receptors （H1-H4） which exhibit different tissue distributions. H1 is widely distributed. And in addition to roles in the central nervous system, it also mediates the roles of histamine in immediate hypersensitivity reactions. In these reactions, histamine released from mast cells or basophils has potent effects on vascular smooth muscle cells, causing cell contraction and division, and on vascular endothelial cells （EC）, inducing microvascular permeability and, disputably, EC division. H2 also mediates neurotransmission but, additionally, gastric acid secretion and T-lymphocyte function. H3 serves primarily as a neurotransmitter, whereas the more recently discovered H4 is distributed in cells of hematopoietic lineage and mediates functions such as inflammatory cell chemotaxis, cytokine release, and T-lymphocyte activation.TR3/Nur77 belongs to nuclear receptor IV subfamily of transcription factors including Nur77, NOT1/Nurrl and NOR1 that contain three functional domains, the transactivation domain, the DNA-binding domain and the ligand-binding domain. They have highly homologous DNA binding domain, but much less homologous transactivation domains. Due to high homology in their sequences, they have redundant roles in T cell receptor （TCR）-mediated apoptosis and brown fat thermogenesis. However, they may play different roles in development. Recent studies demonstrated that TR3/Nur77 is a critical mediator of angiogenesis and its associated microvessel permeability. TR3/Nur77 is highly and transiently up-regulated by angiogenic factors that induce microvessel permeability, including VEGF-A, histamine and serotonin, in vitro and in vivo, but not by angiogenic factors, bFGF and PIGF, which are unable to induce microvessel permeability. TR3/Nur77 is also up-regulated in postnatal angiogenesis, such as tumor angiogenesis and wound healing. Inhibition of Nur77 expression in EC in vitro by means of antisense DNA or siRNA indicated that TR3/Nur77 is required for EC proliferation, migration, survival and tube formation induced by VEGF-A, histamine and serotonin in vitro. Tumor growth, angiogenesis and microvessel permeability induced by VEGF-A, histamine and serotonin were markedly inhibited in Nur77 knockout mice. However, Nur77 null mice are viable, fertile, and develop an apparently normal adult vasculature. Together these findings suggest that TR3/Nur77 is required for pathological angiogenesis, but is not essential for developmental or physiological angiogenesis.However, the signaling pathways that mediate the expression of TR3/Nur77 induced by VEGF-A and Histamine remain unclear. In this study, using Realtime PCR, shRNA, and western blotting techniques, we detected the expression of TR3/Nur77 induced by VEGF-A and Histamine, and explored the potential molecular mechnism.Part I Effect of VEGF-A and Histamine on the expression of TR3/Nur77 IntroductionObjective1. To identify the three TR3 transcript variants and their difference in structure.2. To detect the effect of VEGF-A and Histamine on the expression of TR3/Nur77.3. To detect the effect of the other inflammation factors and growth factors on the expression of TR3/Nur77.Methods1. The Genebank database was searched to find out the transcription variants TR3 gene encodes and picked up specific Realtime PCR primers for them.2. The three TR3 transcript variants were identified by Realtime PCR and western blot.3. The effect of the other inflammation factors and growth factors on the expression of TR3/Nur77 in different cancer cells was detected by Realtime PCR.4. Unpaired t test, and the Mann-Whitney test were used, as appropriate, to determine statistical significance.Results1. The three TR3 transcript variants in the Genebank database and their Realtime PCR primersTR3 gene is located on chromosome 12（NC₀00012.12）, and in the Genebank database, there are three transcription variants. TR3 transcript variant 1 （TR3-TV1） consists of exons 3-10, lacking of exons 1 and 2, whereas TR3 transcript variant 2 （TR3-TV2） lacks of exons 1,2 and 4, and is composed exons 3,5-10. TR3 transcript variant 3 （TR3-TV3） contains exons 1,2 and 5-10, without exons 3 and 4. TR3-TV1 and TR3-TV2 encode the same 59.8 KDa TR3-isoforml （TR3-isol） protein with translation starting site ATG locates in exon 5, whereas TR3-TV3 uses a translation starting site in exon 2, resulting in a 61.2 KDa TR3-isoform2 （TR3-iso2） protein with 13 amino acids longer than TR3-isol protein. We designed Realtime PCR primers that would specifically amplify these three TR3 transcript variants and studied the expression by Realtime PCR. The PCR products were analyzed with DNA gel electrophoresis and were confirmed to be correct by DNA sequencing.2. Expression and up-regulation of TR3 transcript variants in various cell linesThe three TR3 transcript variants are expressed at different levels. The TR3-TV1 is expressed at the lowest levels and TR3-TV3 is expressed in the highest levels in both HUVEC and HDMVEC. We further tested whether all three TR3 transcript variants are regulated by VEGF-A. TR3-TV1, TV2 and TV3 are induced about 2 fold,100 fold and 5 fold, respectively in HUVEC. In HDMVEC, the induction is much higher than that in HUVEC, with 100 fold,400 fold and 20 fold for TR3-TV1, TR3-TV2 and TR3-TV3, respectively. VEGF-A121 and histamme have similar effect, while the effect of histamine is much stronger. Further the induction of TR3-TV1 and TR3-TV2 are peaked at 1 h, but that of TR3-TV3 is peaked at 2 hour, after VEGF-A stimuling. To confirm whether TR3-iso2 protein is expressed, we developed an antibody against the 13 amino acids in the N-terminus of TR3-iso2. The level of TR3-iso2 protein is increased in both HUVEC and HDMVEC by VEGF-A. Similar results are obtained with the antibody against both TR3 isoforms. However, multiple TR3 bands were detected in HDMVEC, imply that TR3 is highly phosphorylated. Our data demonstrate that TR3 transcript variants are expressed and induced by VEGF-A at different levels in endothelial cells.3. The effect of the other inflammation factors and growth factors on the expression of TR3/Nur77We also stimulated HUVECs, HCT116, PC3 and LnCap by growth factor—EGF and other inflammatory factors including TNF-a, PMA, LPS and CHX. The results showed that EGF could increase the expression of all the 3 TR3 transcript variants. It is the most obvious for TR3-TV2 and peaked at the time point of lhour. The other inflammatory factors can also enhance their expression. PMA can increase the expression TR3-TV2 in HUVEC by approximately 400 times, while CHX has an effect mainly on TR3-TV3, increasing by about 30 times. These data showed that these TR3 transcript variants also exit in these cells and can be induced by different factors.Conclusion1、There are three TR3 transcript variants, which are expressed at differential levels.2、The expression of the three TR3 transcript variants can be up-regulated markedly by VEGF-A and histamine, especially for TR3 transcript variant 2 （TR3-TV2）.3、TR3 transcript variants also exit in other cancer cells and can be induced by different factors. inflammatory factors can also enhance their expression. PMA can increase the expression TR3-TV2 in HUVEC by approximately 400 times, while CHX has an effect mainly on TR3-TV3, increasing by about 30 times. These data showed that these TR3 transcript variants also exit in these cells and can be induced by different factors.Conclusion1、There are three TR3 transcript variants, which are expressed at differential levels.2、The expression of the three TR3 transcript variants can be up-regulated markedly by VEGF-A and histamine, especially for TR3 transcript variant 2 （TR3-TV2）.3、TR3 transcript variants also exit in other cancer cells and can be induced by different factors.Part II The molecular mechanism of TR3/Nur77 expression induced by VEGF-A and HistamineObjective1. To detect the potential signaling pathways that regulate the expression of TR3 transcript variants.2. To investigate expression of TR3-TV2 and TR3-TV3 induced by various VEGF family members and identify the VEGF receptors getting involved.3. To detect role of IGF-1R for the expression of TR3-TV2, but not TR3-TV3.4. To investigate the histamine receptor types mediating histamine-induced TR3 expression and explore the effect of IGF-1R in this process.Methods1. Realtime PCR was used to detect the effect of inhibitors of several common signaling pathways and shRNAs on the expression of TR3 induced by VEGF-A and histamine.2. Realtime PCR was used to detect the effect of VEGF family members on the expression of TR3 and the downstream target MAPK was detected by western blot.3. Realtime PCR and western blot were used to detect the effect of VEGF on the expression of TR3 in VEGF receptor chimeric system.4. Realtime PCR and western blot were used to detect the role of IGF-1R in the VEGF-A-induced TR3 expression.5. Western blot was used to detect IGF-1R phosphorylation by VEGF and VEGFR2/KDR--IGF-1R interaction.6. Realtime PCR and western blot were used to examine the histamine receptor types mediating histamine-induced TR3 expression and explore the effect of IGF-1R in this process.Results1. Signaling pathways that regulate VEGF-A-induced TR3 transcript variants expressionwe studied the specific regulation of each TR3 transcription variant by Realtime PCR with their specific primers and found that BAPTA/AM and cyclosporine-A completely inhibits the expression of TR3-TV2 and TR3-TV3 expression induced by VEGF-A. However, the calmodulin antagonist, W-7 and the CaM kinase â…¡ inhibitor KN62 have no effect on VEGF-A-induced TR3-TV2 and TR3-TV3 expression. Further, the PLC inhibitor U-73122, PLC dominant negative control （PLC-DN）, the PKC inhibitor GF109203X and down7regulation of PKC expression by prolonged PMA treatment, PKCδ dominant negative mutant （PKCδ-DN）, PKD inhibitor （CID2011756）, PKD isoform 1 shRNA （shPKD）, and MEK inhibitor （PD98059）, IκBα shRNA （shIκBα）, the p38 inhibitor SB203580 or the JNK inhibitor JNK Inhibitor â…¡ almost completely inhibit the expression of TR3-TV2 and TR3-TV3 induced by VEGF-A. However, none of the PI-3 kinase inhibitors, dominant negative mutants of PI3K and Akt have any effect on TR3-TV2 and TR3-TV3 induction induced by VEGF-A. Our data indicate that the Ca2+-PLC-PKC-PKD1 pathway, NF-κB pathway and MAP kinases （ERK, p38 and JNK） pathway, but not PI3K-Akt pathway, play a critical role in the expression of TR3-TV2 and TR3-TV3 induced by VEGF-A.2. Signaling pathways that regulate histamine-induced TR3 transcript variants expressionIn the study, we found that BAPTA/AM, and cyclosporine-A completely inhibits the expression of TR3-TV2 and TR3-TV3 expression induced by histamine. However, the calmodulin antagonist, W-7 and the CaM kinase â…¡ inhibitor KN62 have no effect on histamine-induced TR3-TV2 and TR3-TV3 expression. Further, the PLC inhibitor U-73122, PLC dominant negative control （PLC-DN）, the PKC inhibitor GF109203X and down-regulation of PKC expression by prolonged PMA treatment, PKCδ dominant negative mutant （PKCδ-DN）, PKD inhibitor （CID2011756）, PKD isoform 1 shRNA （shPKD）, and MEK inhibitor （PD98059） almost completely inhibit the expression of TR3-TV2 and TR3-TV3 induced by VEGF-A. However, none of the PI-3 kinase inhibitors, dominant negative mutants of PI3K and Akt, IκBα shRNA （shIκBα）, the p38 inhibitor SB203580 or the JNK inhibitor JNK Inhibitor II have any effect on TR3-TV2 and TR3-TV3 induction induced by histamine. Our data indicate that the Ca2+-PLC-PKC-PKD1 pathway and MAP kinases （ERK, p38 and JNK） pathway, but neither PI3K-Akt pathway nor NF-κB pathway play a critical role in the expression of TR3-TV2 and TR3-TV3 induced by histamine.3. Expression of TR3-TV2 and TR3-TV3 induced by various VEGF family membersWe further studied the four VEGF receptors that are required for VEGF-induced expression of TR3 transcript variants. VEGF-A interacts with VEGFRl/Flt-1, VEGFR2/KDR, neuropilin 1（NP-1） and neuropilin 2 （NP-2）, while VEGF-B and P1GF interact with VEGFR1/Flt-1, NP-1 and NP-2, but VEGF-E interacts with VEGFR2/KDR. We found that VEGF-A and VEGF-E up-regulates both TR3-TV2 and TR3-TV3, while VEGF-B and P1GF are unable to induce the expression of either TR3-TV2 or TR3-TV3. Our data indicate that it is VEGFR2/KDR, but not VEGFR1/Flt-1, nor NP-1, nor NP-2, that mediates the expression of TR3-TV2 and TR3-TV3 induced by VEGF-A. The Realtime RT-PCR results indicate that VEGF-B and P1GF have no effect on the expression of TR3-TV2 and TR3-TV3 induced by VEGF-A or VEGF-E even though VEGF-B or P1GF is able to induce MAPK phosphorylation in the same conditions. These results suggest that VEGFR2/KDR, but not VEGFR1/Flt-1, nor neuropilins, mediates TR3-TV2 and TR3-TV3 expression induced by VEGF.4. Expression of TR3-TV2, but not TR3-TV3, cannot be induced to higher level in VEGF receptor chimeric systemHUVEC were transduced with these fusion receptors individually or in combination and treated with EGF, instead of VEGF-A, so that the signaling pathways that are mediated by each receptor can be distinguished. The data showed that, in HUVEC transduced with EGDR, EGF induces TR3-TV2 mRNA about 15 fold compare to the control,75% less than that in HUVEC induced by VEGF-A or VEGF-E, while the expression of TR3-TV3 induced by EGF is 5 fold compared to the control, similar to that induced by VEGF-A and VEGF-E. Further, combination of EGLT and EGNP-1 with EGDR has little effect on the expression of TR3-TV2 and TR3-TV3 induced by EGF. We then tested whether there is any defect of EGF-EGDR system in our current studies. The data showed that MAPK phosphorylation is highly induced in HUVEC stimulated with VEGF-A and in HUVEC that were transduced with EGDR and stimulated with EGF, but not in HUVEC transduced with Lac Z and stimulated with EGF. Our data suggest that, in addition to VEGFR2/KDR, other receptor（s） that is not associated with EGDR may be involved in the expression of TR3-TV2, but not TR3-TV3, induced by VEGF-A.5. Requirement of IGF-1R for the expression of TR3-TV2, but not TR3-TV3, nor phosphorylation of MAPK stimulated by VEGF-AHUVEC were treated with or without VEGFR2/KDR kinase inhibitor SU1498, IGF-1R kinase inhibitor AG1024, or transduced with shKDR or shIGF-1R2 and then stimulated with VEGF-A. Realtime PCR data show that AG1024 and shIGF-1R2 significantly inhibit the up-regulation of TR3-TV2, but not TR3-TV3, while shKDR almost completely inhibits the up-regulation of both TR3-TV2 and TR3-TV3, induced by VEGF-A. Surprisingly, SU1498 increases the expression of TR3-TV2, but has no effect on the expression of TR3-TV3. In combination of the data obtained with the chimeric fusion system, our data demonstrate that VEGFR2/KDR mediates VEGF-A-induced TR3-TV3 expression. However, VEGFR2/KDR or EGDR is not sufficient to induce the expression of TR3-TV2 to higher level, whereas IGF-1R, which is missed in the EGDR system, is required for fully up-regulation of TR3-TV2 by VEGF-A. Our studies also demonstrate that IGF-1R is not required for MAPK phosphorylation induced by VEGF-A.6. IGF-1R phosphorylation and interaction of VEGFR2/KDR and IGF-1R induced VEGF-AWe further studied the mechanism by which IGF-1R regulates VEGF-A signaling. Serum-starved HUVEC were stimulated with VEGF-A, VEGF-B, VEGF-E and P1GF. Cellular extracts were analyzed by immunoblot with an antibody against phosphorylated IGF-1Rβ. VEGF-A and VEGF-E, but neither VEGF-B, nor PlGF, induce IGF-1R phosphorylation. The IGF-1R phosphorylation induced by VEGF-A is completely inhibited by shKDR, but not by SU1498 or AG1024. Our data demonstrate that VEGF-A and VEGF-E induce IGF-1R phosphorylation, but EGF is unable to induce IGF-1R phosphorylation in HUVEC transduced with EGDR. Cellular extracts were immunoprecipitated with antibodies against VEGFR2/KDR or IGF-1Rα and immunoblotted with antibodies against phosphorylated IGF-1Rβ, IGF-1Rα or VEGFR2/KDR, respectively. The data showed that phosphorylated IGF-1Rβ and IGF-1Rα are detected in the VEGFR2/KDR immunoprecipitated complex after VEGF-A stimulation. Also VEGFR2/KDR was detected in the IGF-1Rα immunoprecipitated complex in VEGF-A-treated cells. Our data show that VEGF-A induces physical interaction of VEGFR2/KDR and IGF-1Rα/IGF-1Rβ.7. The histamine receptor types mediating histamine-induced TR3 expression and explore the effect of IGF-1R in this process.Serum-starved HUVEC were pretreated by H1, H2 and H4 receptor inhibitor, and then stimulated with histamine. The data showed that histamine-induced TR3-TV2 expression is mediated mainly by H1 receptor, while TR3-TV3 expression is mediated mainly by H4 receptor. Moreover, we found that IGF-1R blocker AG1024 and shIGF01R can inhibit the induction of histamine to TR3 expression, demonstrating that IGF-1R played partly a role in histamine-induced TR3 expression. However, the immunoblotting data showed that histamine can not phosphorylate IGF-1R, while AG1024 can inhibit ERK phosphorylation. Our data show that the induction of histamine to TR3 expression is mediated mainly by H1 and H4, and IGF-1R seemed to have played a role during this process, while the mechanism is still unknown.Conclusion1、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. While calcium-PLC-PKC-PKD1 pathway got involed in the histamine-induced TR3-TV2 and TR3-TV3 mRNA induction.2、VEGFR2/KDR, but not VEGFR1/Flt-1, nor NP-1, nor NP-2 mediates the expression of TR3-TV2 and TR3-TV3 induced by VEGF-A.3、VEGF-A and VEGF-E induce IGF-1R phosphorylation, and result in the high expression of TR3-TV2 by inducing physical interaction of VEGFR2/KDR and IGF-1Ra/IGF-1Rp.4、The histamine-induced TR3 expression is mediated mainly by H1 and H4, and IGF-1R seemed to have played a role during this process.