Effects Of Immunoglobulin G And Insulin-Like Growth Factor-1 (IGF-1) On Thyroid Goiter Formation In Graves' Disease As Well As Concerned Signaling Pathway

Background:Graves' disease (GD) is a kind of common autoimmune thyroid disease. Diffusethyroid goiter is one of the characteristic features of GD. The bigger the thyroid volumes are, the longer the period of medical treatment for GD is. So it is very important to study the mechanism of thyroid goiter formation to find effective therapeutic targets for this disease.In GD, there exist autoantibodies (immunoglobulinG, IgG) against thyrotropin (TSH) receptor (TSHR), named thyrotropin receptor antibody (TRAb). These antibodies are believed to act via the TSHR and mimic the role of TSH to bring biological effect through signaling cascades in thyroid and thus alter thyroid growth and function. Furthermore, previous studies also showed that growth factors, especially insulin-like growth factor-1 (IGF-1), play important roles in thyroid cell growth and human thyroid diseases. IGF-1 is an important hypertrophic factor for thyroid cells. It regulates cell proliferation and a vast variety of differentiated cell functions. Our previous research showed that in patients with untreated GD, there was a significant increase in serum IGF-1 levels, and localized IGF-1 expression in thyroid tissues was higher in those patients compared with healthy subjects. Meanwhile, thyroid volumes were positively correlated with IGF-1 levels in untreated patients. All indicate that IGF-1 is actively involved in thyroid goiter formation. Since TRAb and IGF-1 are all correlated with the formation of thyroid goiter and those autoantibodies are the initiative reason for GD, we speculate that those IgGs could influence localized IGF-1 levels.An imbalance between the proliferation and the apoptosis of thyroid cells may be crucial for goiter formation. It has been known that cellular Fas associated death domain-like interleukin-1β-converting enzyme (FLICE)-inhibitory protein (FLIP) could block Fas-induced apoptosis. In GD, upregulation of FLIP leads to thyroid goiter because of an absence of thyrocyte apoptosis; on the other hand, the role of Cyclin D1 in proliferative thyroid diseases also get great attention. Cyclin D1 is a G₁ phase cell cycle protein. Accumulation of this kind of protein by stimulation of extracellular signals promotes cell proliferation. It has been reported that in thyroid papillary carcinomas, the expression of Cyclin D1 was elevated, which was thought to be a contributory factor to thyroid tumorigenesis. But in normal thyroid cells, information regarding the relationship between IGF-1 and FLIP / Cyclin D1 as well as concerned signaling pathway are uncertain.Phosphatidylinositol 3-kinase (PI3K) is a kind of important second messenger that is related with cellular signal transduction. Evidence from several cell lines indicates that PI3K mediates the actions of IGF-1. So it is thought to be a key regulator in the actions of IGF-1.In addition, a growing body of evidence demonstrates that nuclear factor-κB (NF-κB) is a key regulator of the transcription of genes that control cellular proliferation and antiapoptosis. Some research found that IGF-1 induced upregulation of NF-κB activity, corresponding with a proliferative response in different cell types. But whether NF-κB is involved in the process of IGF-1 induced proliferation of thyroid cells needs further investigation. Objectives:1.The effect of serum IgG from Graves'patients on IGF-1 mRNA and protein expression and cell cycle progression in thyroid cells;2.The effect of IGF-1 on expression levels of cyclinD1, FLIP, cell cycle progression and NF-κB-DNA binding activity;3.The possible PI3K/ NF-κB signaling pathway: the effect of PI3K and /or NF-κB inhibitors on expression levels of cyclinD1 and FLIP.Methods:1. Cell culture: FRTL-5 thyroid cells (ATCC catalog number CRTL-1486) were grown in 6H medium consisting of Coon's modified Ham's F12 medium supplemented with 5% fetal bovine serum and a mixture of six hormones: bovine thyrotropin (1 milliunit/ml), insulin (10μg/ml), hydrocortisol (0.4 ng/ml), transferrin (5μg/ml), glycyl-L-histidyl-L-lysine acetate (10 ng/ml), and somatostatin (10 ng/ml). Fresh medium was added to all cells every 2 or 3 days, and cell passaging was done every 7-10 days. As appropriate for the experiments to be performed, the cells were shifted to 4H medium containing no thyrotropin, no insulin, and 0.2% calf serum 2 days before they were used. Then different treatments were added for 24 h.2. Measure of thyroid volumes: Survey the length, width, thickness of the right side, left side and gorge of thyroid of Graves'patients. The total thyroid volumes were the sum of the volume of the right side, the left side and the gorge part.3. Preparation of crude IgG fractions: 500μl serum samples were mixed with 20% (wt/vol; final concentration, 15%) polyethylene glycol (mol weight, 4000), and followed by centrifugation at 2800 g for 20 min. The pellet was dissolved in F12k medium. IgGs from GD patients and normal control were pooled separately (GD-IgG). Patients with GD were divided into two groups according to their goiter size (using a volume of 40 cm³ as the cut-off) and IgG was precipitated.4. Effects of IgG on IGF-1 expression: mRNA level of IGF-1 was measured using Real-Time PCR and Reverse-Transcription (RT)-PCR; Protein level was further measured using the method of Immunoprecipitation and Western blot.5. Effect of IGF-1 on cell proliferation, apoptosis and concerned PI3K/NF-κB signaling pathway: For some experiments, the PI3K inhibitor LY294002 (10 MM) or the NF-κB inhibitor BAY11-7082 (50 MM) was added 1 h prior to IGF-1 treatment. Then, cells were treated with IGF-1 for 24 h. mRNA level of FLIP was measured using Real-Time PCR and RT-PCR; FLIP, cyclinD1 and IκB protein levels were further measured using Western blot. NF-κB -DNA binding activity was performed by the method of EMSA.6. Effect of IgG/IGF-1 on cell cycle progression: Cell cycle analysis was performed by using Fluorescence Activated Cell Sorter (FACS).Results:Relationship between TRAb levels and thyroid volumes: Among all the 34 patients with GD, 30 showed positive TRAb levels; while in 30 normal controls, TRAb levels were all negative. Furthermore, with the increase of thyroid volumes of GD patients, their serum TRAb values increased accordingly. Statistical analysis showed that there was a positive correlation between TRAb levels and thyroid volumes.Effect of IgG on IGF-1 expression in thyroid cells: Real-time results demonstrated that GD-IgG increased the mRNA level of IGF-1, as compared with IgG obtained from normal individuals (p<0.05). Immunoprecipitation followed by immunodetection with IGF-1 antibody showed that the expression level of this growth factor became increased after GD-IgG treatment. Meanwhile, GD-IgG from those who have larger thyroid volumes had more obvious effect on upregulating IGF-1 expression than IgG from those patients with a goiter volume smaller than 40 cm³ (p<0.05).Effect of IGF-1 on cyclinD1, FLIP and concerned signaling pathway:â‘ Results of the RT-PCR showed that IGF-1 increased FLIP mRNA levels compared with those of control cells that were not treated with IGF-1. When we validated this change with real-time PCR, a quantitative method with high-sensitivity, we found FLIP mRNA levels increased by 30% with 10 ng/ml IGF-1 and 50% with 50 ng/ml IGF-1 compared with control group. Western blot results showed that IGF-1 stimulated FLIP protein expression in a dose-dependant manner. It also demonstrated a significant increase in Cyclin D1 protein level in IGF-1-treated group (p<0.05)â‘¡RT-PCR and real-time PCR showed that LY294002 pretreatment decreased the IGF-1 -induced FLIP mRNA content to 70% of that observed in cells treated with IGF-1 alone (P < 0.05). This reduction was paralleled by a significant decrease in FLIP protein level. Results also suggested that LY294002 (10μM) inhibited by 40% the IGF-1-induced increase in Cyclin D1 protein.(3) To study the effect of IGF-1 on NF-κB, Western blot was performed on the cytoplasmic protein of thyroid cells with an antibody to IκBα. Treatment of FRTL cells with 50 ng/ml IGF-1 resulted in a decrease of IκBα. expression as compared with untreated group (p<0.05). Meanwhile, LY294002 pretreatment diminished the degradation of IκBα, induced by IGF-1 (p<0.05).(4) EMSA results showed that treatment with IGF-1 resulted in strong NF-κB-specific gel-retarded bands in nuclear samples extracted from FRTL cells relative to untreated cells. To investigate whether PI3K is also involved in increased NF-κB-DNA binding activity stimulated by IGF-1, PI3K inhibitor LY294002 was added, and faint gel-retarded bands were found in the nuclear extracts, indicating involvement of PI3K in IGF-1 's effect on NF-κB.(5) Western blot results showed that inhibition of NF-κB by BAY11-7082 prior to IGF-1 treatment decreased IGF-1-induced FLIP and cyclinD1 protein expression.Effect of IgG/IGF-1 on cell cycle progression: When FRTL cells were rendered quiescent by 48h incubation in starvation medium, 79.5±4.3% of cells were in G₀/G₁ phase, 17.7±3.0% were in G₂/M phase, and almost no cells were in S phase. In quiescent FRTL cells incubated with 100μg/ml IgG from Graves'patients for a 24 h period, the proportion of cells in G₀/G₁ phase was decreased to 73.9±2.6%, the proportion in S phase was increased to 9.3±1.9%, and the proportion in G₂/M phase was increased to 17±1.7%. When cells were stimulated with normal IgG for 24 h, no significant changes were seen in cell cycle progression as compared with starvation group. In quiescent FRTL cells incubated with 50 ng/ml IGF-1 for a 24 h period, the proportion of cells in G₀/G₁ phase was decreased to 73.9±2.3%, the proportion in S phase was increased to a small extent (6.2±1.2%), and the proportion in G₂/M phase was increased to 19.9±1.9%. However, in the presence of inhibitors, the IGF-1-mediated cell cycle progression was blocked. Inhibition of the PI3K pathway with LY294002 or the blockage of NF-κB activity with BAY11-7082 decreased progression from G₀/G₁ to S phase: the proportion of cells in S phase was decreased to 3.4±1.1% and 2.9±1.7% respectively, and the proportion of cells in G₀/G₁ was increased to 79.4±3.2 % and 78.3±3.0%.Cell morphology: After starvation, most cells begin to show vacuolated (because of lack of nutrient). Dead cell debris floated in the medium. When treated with IgG/IGF-1 for 24h, dead cell debris decreased and most cells recovered and started to grow again.Conclusions:â‘ GD-IgG upregulates IGF-1 mRNA and protein expression in thyroid cells. This regulation was tightly associated with thyroid volumes of Graves' patients. GD-IgG also promotes cell cycle progression. All indicate that IgG from Graves'patients promotes the proliferation of thyroid cells, at least in part, via upregulation of IGF-1 expression.(2) When FRTL thyroid cells are stimulated with IGF-1, the amount of IκBαdecreased , with a parallel enhancement of NF-κB-DNA binding activity in the nuclei. This suggests that IGF-1 influences transcription and translation of target genes through affecting NF-κB activity.(3) IGF-1 upregulates antiapoptotic protein, FLIP ,and cell cycle relavant protein, cyclinD1, through PI3K/NF-κB signaling cascade.This signaling pathway is also tightly related with cell cycle changes. This may be one possible signaling mechanism for the proliferative and antiapoptotic role of IGF-1 on thyroid cells.