The Presence Of Adenosine A2a Receptor In Thyrocytes And Its Involvement In Graves’ IgG-Induced VEGF Expression

Background:Graves Disease is a common endocrine disorder which is characterized by enlarged thyroid and thyrotoxicosis induced by elevated thyroid hormone levels. An increase in both intrathyroidal vascularity and blood velocity was often observed in GD goiter. Generally speaking, lager goiter produces more thyroid hormones. Patients with larger goiter and seriously vascularied thyroid tend to need longer drug treatment period. So it is important to study the mechanism of goiter and vascularization. For a long time, goitrogenesis in Graves disease (GD) was attributed to stimulation of TSH receptor auto-antibodies (TRAb) which interacted with TSH receptor and triggered cyclic adenosine monophosphate signaling pathway then induce thyrocytes proliferation and thyroid hormones over production. For this reason, early studied on GD goiter all focused on TRAb. In recent years, growth factors which was secreted by thyrocytes and in turn acted on thyrocytes or other surrounding cells in the way of autocrine, paracrine and endocrine got more attention. For example, vascular endothelial growth factor (VEGF).Adenosine is the metabolism product of Adenosine triphosphate (ATP) and cyclic adenosine monophosphate (cAMP). It can bind with the adenosine receptors and act through the second messager system in cells. Ledent constructed a fusion gene which containing the promoter region of the bovine thyroglobulin gene linked to the coding region of canine adenosine A2a receptor and setup transgenic mice carrying the fusion gene. All the mice developed enlarged vascularized thyroid with heavey VEGF expression and thyrotoxicosis. Therefore, we suspected that A2aR may play an important part in GD goiter formation. A2aR has been demonstrated to be the upstream regulator of VEGF expression in many tissues and cells. So far, no similar research was done in thyroid.Most of the former research about adenosine induced VEGF expression focused on angiogenesis induced by tumor or hypoxia and the key point was hypoxia inducible factor-1alpha (HIF-1alpha), a transcription factors which can bind with hypoxic response element (HRE) in VEGF promoter and prompt VEGF transcription. Recent study showed more transcription regulator independent of HIF-1alpha. For example, peroxisome-proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) and phosphorylated cAMP-response element binding protein (p-CREB). It has been demonstrated that PGC-1alpha, HIF-1alpha and p-CREB were all downstream molecular of cAMP, which was the signaling molecular of adenosine receptors. Therefore, we suspected that A2aR may prompt VEGF expression through cAMP/p-CREB/PGC-1alpha/HIF-1alpha pathway.In the present study, we tested the expression of A2aR in thyrocytes and detected the possible role it might play in GD IgG induced VEGF expression.Objectives:1. To determine A2aR expression on FRTL-5cells, primary human thyrocytes, rat thyroid, mouse thyroid and human thyroid2. To determine the effect of GD IgG and A2aR agonist on VEGF expression3. To determine the possible mechanism of VEGF elevation induced by GD IgG and A2aR agonist4. To determine the effect of conditioned media from thyrocytes treated by GD IgG and A2aR agonist on endothelial cell proliferationMethods: 1. Cell culture1)FRTL-5cells:cells were grown in6H media consisting of Coon’s modified Ham’s F12medium supplemented with5%fetal bovine serum and a mixture of six hormones according to guidance from ATCC and relative references2) Primary human thyrocytes:human thyroid tissues was cut into pieces and digested in a mixture of type I collagenase and trypsin for40-60min at37℃then cultured in DMEM/F12with newborn fetal serum and TSH3)Primary human umbilical vein endothelial cells (HUVEC):umbilical cord was digested in a mixture of trypsin and EDTA then cultured in M199medium containing fetal bovine serum and fibroblast growth factor4)Primary rat neurons:newborn rat brain was cut into pieces and digested in trypsin then cultured in DMEM medium containing fetal bovine serum, glucose, insulin and amino benzoic acid2. Animal modelTSHR adenovirus-induced Graves’disease mouse model:Female mice were injected intramuscularly with adenovirus expressing human TSHR3. Clinical informationUntreated GD patients had their thyroid volume measured and fasting blood obtained then their serum thyroid hormones and TRAb levels were measured by electrochemiluminescence4. RT-PCR:was adopted to determine the expression of A2aR mRNA and the mRNA of VEGF splicing isoforms.5. Protein extraction and immunoblotting analysis:was adopted to determine the expression of A2aR, p-CREB, CREB, PGC-1alpha, HIF-alpha and VEGF protein6. Immunohistochemistry and Immunofluorescence:were adopted to determine the expression of A2aR, p-CREB, PGC-1alpha, HIF-alpha and VEGF protein7. A2aR silence:was adopted to silent A2aR expression8. ELISA:was adopted to determine the expression of VEGF9. Direct immunoassay:was adopted to determine the level of cAMP10. EdU:was adopted to determine the proliferation of HUVEC Results:1. The presence of functional A2aR in thyrocytesAfter PCR amplification, distinct band of A2aR was observed in FRTL-5cells, primary human thyrocytes and rat thyroid. Western Blotting confirmed the protein expression of A2aR in FRTL-5cell and primary human thyrocytes. Immunohistochemistry and Immunofluorescence demonstrated that A2aR protein was expressed on cell membrane of FRTL-5cells, primary human thyrocytes, rat thyroid, mouse thyroid and human thyroid. A2aR stimulation induced cAMP elevation.2. A2aR agonists and GD IgG increase VEGF expression in thyrocytesWe successfully detected four isoforms in FRTL-5cells:VEGF188, VEGF164, VEGF144, VEGF120while in primary human thyrocytes:VEGF189, VEGF165, VEGF145, VEGF121. Of them, the productions of both VEGF120/121and VEGF164/165isoforms appeared to be predominant. Both A2aR specific agonist CGS21680and GD IgG elevated VEGF mRNA expression in thyrocytes. And both intracellular and extracellular VEGF protein showed the same tendency.3. The activation of the cAMP/p-CREB/PGC-1alpha/HIF-1alpha axis by the Adenosine A2aR agonist and GD IgGPhosphorylated CREB, PGC-1alpha, HIF-1alpha are all VEGF transcription promoters. Both CGS21680and GD IgG increased p-CREB, PGC-1alpha, HIF-1alpha in FRTL-5cells. Adenylyl cyclase specific agonist Forskolin showed the same tendency. Pretreatment with H89diminished the GD IgG-and CGS21680-mediated increases in PGC-1alpha and p-CREB.4. VEGF, p-CREB and PGC-1alpha increase in parallel in the GD mouse thyroidWe established a GD mouse model by intramuscular injection of human TSHR-expressing adinovirus. Robust VEGF staining was observed in their thyroid. The nuclei were intensely labeled with PGC-1alpha and p-CREB and were weakly stained for HIF-1alpha. 5. A2aR deficiency modulates the IgG-induced changes in VEGF expressionA2aR specific antagonist ZM241385partially counteracted the GD IgG-mediated up regulation of nuclear PGC-1alpha and p-CREB. Pretreatment with ZM241385attenuated the GD IgG-induced increase in VEGF mRNA in a dose-dependent manner. A2aR siRNA inhibited the GD IgG-mediated increase in VEGF and the nuclear accumulation of p-CREB, PGC-1alpha and HIF-1alpha.6. Conditioned supernates from FRTL-5cells prompted proliferation of HUVECsConditioned medium from CGS21680stimulated FRTL-5cells can significantly increase EdU positive (EdU-labeled replicating) HUVEC cells. Medium from FRTL-cells treated by GD IgG showed the same proliferation prompting tendency while ZM241385reduced it dose-dependently.Conclusions:1. Functional A2aR was expressed on cell membrane of FRTL-5cells, primary human thyrocytes, rat thyroid and human thyroid.2. GD IgG could activate cAMP/p-CREB/PGC-1alpha/HIF-1alpha axis via A2aR.3. GD IgG-induced VEGF expression and could be partially attributed to A2aR activation. Background:Graves Disease is a common endocrine disorder which is characterized by enlarged thyroid and thyrotoxicosis induced by elevated thyroid hormone levels. An increase in both intrathyroidal vascularity and blood velocity was often observed in GD goiter. Generally speaking, lager goiter produces more thyroid hormones. Patients with larger goiter and seriously vascularied thyroid tend to need longer drug treatment period. So it is important to study the mechanism of goiter and vascularization. For a long time, goitrogenesis in Graves disease (GD) was attributed to stimulation of TSH receptor auto-antibodies (TRAb) which interacted with TSH receptor and triggered cyclic adenosine monophosphate signaling pathway then induce thyrocytes proliferation and thyroid hormones over production. For this reason, early studied on GD goiter all focused on TRAb. In recent years, growth factors which was secreted by thyrocytes and in turn acted on thyrocytes or other surrounding cells in the way of autocrine, paracrine and endocrine got more attention. For example, vascular endothelial growth factor (VEGF).Adenosine is the metabolism product of Adenosine triphosphate (ATP) and cyclic adenosine monophosphate (cAMP). It can bind with the adenosine receptors and act through the second messager system in cells. Ledent constructed a fusion gene which containing the promoter region of the bovine thyroglobulin gene linked to the coding region of canine adenosine A2a receptor and setup transgenic mice carrying the fusion gene. All the mice developed enlarged vascularized thyroid with heavey VEGF expression and thyrotoxicosis. Therefore, we suspected that A2aR may play an important part in GD goiter formation. A2aR has been demonstrated to be the upstream regulator of VEGF expression in many tissues and cells. So far, no similar research was done in thyroid.Most of the former research about adenosine induced VEGF expression focused on angiogenesis induced by tumor or hypoxia and the key point was hypoxia inducible factor-1alpha (HIF-1alpha), a transcription factors which can bind with hypoxic response element (HRE) in VEGF promoter and prompt VEGF transcription. Recent study showed more transcription regulator independent of HIF-1alpha. For example, peroxisome-proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) and phosphorylated cAMP-response element binding protein (p-CREB). It has been demonstrated that PGC-1alpha, HIF-1alpha and p-CREB were all downstream molecular of cAMP, which was the signaling molecular of adenosine receptors. Therefore, we suspected that A2aR may prompt VEGF expression through cAMP/p-CREB/PGC-1alpha/HIF-1alpha pathway.In the present study, we tested the expression of A2aR in thyrocytes and detected the possible role it might play in GD IgG induced VEGF expression.Objectives:1. To determine A2aR expression on FRTL-5cells, primary human thyrocytes, rat thyroid and human thyroid2. To determine the effect of GD IgG and A2aR agonist on VEGF expression3. To determine the possible mechanism of VEGF elevation induced by GD IgG and A2aR agonist4. To determine the effect of conditioned media from thyrocytes treated by GD IgG and A2aR agonist on endothelial cell proliferationMethods:1. Cell culture1)FRTL-5cells:cells were grown in6H media consisting of Coon’s modified Ham’s F12medium supplemented with5%fetal bovine serum and a mixture of six hormones according to guidance from ATCC and relative references2) Primary human thyrocytes:human thyroid tissues was cut into pieces and digested in a mixture of type I collagenase and trypsin for40-60min at37℃then cultured in DMEM/F12with newborn fetal serum and TSH3)Primary human umbilical vein endothelial cells (HUVEC):umbilical cord was digested in a mixture of trypsin and EDTA then cultured in M199medium containing fetal bovine serum and fibroblast growth factor4)Primary rat neurons:newborn rat brain was cut into pieces and digested in trypsin then cultured in DMEM medium containing fetal bovine serum, glucose, insulin and amino benzoic acid2. Animal modelTSHR adenovirus-induced Graves’ disease mouse model:Female mice were injected intramuscularly with adenovirus expressing human TSHR3. Clinical informationUntreated GD patients had their thyroid volume measured and fasting blood glucose obtained then their serum thyroid hormones and TRAb levels were measured by electrochemiluminescence4. A2aR silenceA small interfering RNA oligonucleotide was transfected to FRTL-5cells with electroporation to silent A2aR expression.5. Protein extraction and immunoblotting analysis:was adopted to determine the expression of A2aR, p-CREB, CREB, PGC-1alpha, HIF-alpha and VEGF protein6. Immunohistochemistry and Immunofluorescence:were adopted to determine the expression of A2aR, p-CREB, CREB, PGC-1alpha, HIF-alpha and VEGF protein7. ELISA:was adopted to determine the expression of VEGF8. Direct immunoassay:was adopted to determine the level of cAMP9. EdU:was adopted to determine the proliferation of HUVECResults: 1. The presence of functional A2aR in thyrocytesAfter PCR amplification, distinct band of A2aR was observed in FRTL-5cells, primary human thyrocytes and rat thyroid. Western Blotting confirmed the protein expression of A2aR in FRTL-5cell and primary human thyrocytes. Immunohistochemistry and Immunofluorescence demonstrated that A2aR protein was expressed on cell membrane of FRTL-5cells, primary human thyrocytes, rat thyroid, mouse thyroid and human thyroid. A2aR stimulation induced cAMP elevation.2. A2aR agonists and GD IgG increase VEGF expression in thyrocytesWe successfully detected four isoforms in FRTL-5cells:VEGF188, VEGF164, VEGF144, VEGF120while in primary human thyrocytes:VEGF189, VEGF165, VEGF145, VEGF121. Of them, the productions of both VEGF120/121and VEGF164/165isoforms appeared to be predominant. Both A2aR specific agonist CGS21680and GD IgG elevated VEGF mRNA in thyrocytes. And both intracellular and extracellular VEGF protein showed the same tendency.3. The activation of the cAMP/p-CREB/PGC-1alpha axis by the Adenosine A2aR agonist and GD IgGPhosphorylated CREB, PGC-1alpha, HIF-1alpha are all VEGF transcription promoters. Both CGS21680and GD IgG increased p-CREB, PGC-1alpha, HIF-1alpha in FRTL-5cells. Adenylyl cyclase specific agonist Forskolin showed the same tendency. Pretreatment with H89diminished the GD IgG-and CGS21680-mediated increases in PGC-1alpha and p-CREB.4. VEGF, p-CREB and PGC-1alpha increase in parallel in the GD mouse thyroidWe established a GD mouse model by intramuscular injection of human TSHR-expressing adinovirus. Robust VEGF staining was observed in their thyroid. The nuclei were intensely labeled with PGC-1alpha and p-CREB and were weakly stained for HIF-1alpha. 5. A2aR deficiency modulates the IgG-induced changes in VEGF expressionA2aR specific antagonist ZM241385partially counteracted the GD IgG-mediated up regulation of nuclear PGC-1alpha and p-CREB. Pretreatment with ZM241385attenuated the GD IgG-induced increase in VEGF mRNA in a dose-dependent manner. A2aR SiRNA inhibited the GD IgG-mediated increase in VEGF and the nuclear accumulation of p-CREB, PGC-1alpha and HIF-1alpha.6. Conditioned supernates from FRTL-5cells prompted proliferation of HUVECsConditioned medium from CGS21680stimulated FRTL-5cells can significantly increase EdU positive (EdU-labeled replicating) HUVEC cells. Medium from FRTL-cells treated by GD IgG showed the same proliferation prompting tendency while ZM241385reduced it dose-dependently.Conclusions:1. Functional A2aR was expressed on cell membrane of FRTL-5cells, primary human thyrocytes, rat thyroid and human thyroid.2. GD IgG could activate cAMP/p-CREB/PGC-1alpha/HIF-1alpha axis via A2aR.3. GD IgG-induced VEGF expression and could be partially attributed to A2aR activation.