Signal Transduction Mechanisms Involved In The Effects Of Thyroid Stimulating Autibody In Human Thyroid Cells

Objective: To investigate signal transduction mechanisms involved in the effects of thyroid stimulating autibody(TSAb) on the functions of cultured thyrocytes ,and to elucidate the preliminary molecular mechanism of Graves' disease(GD) mediated by TSAb .Methods: 1. With the method of enzyme-linked immunosorbent assay (ELISA) , the effects of TSAb on the PKA and PKC activities were observed in primary cultured human thyrocytes . 2. Inhibitors or activators of protein kinases (PKA and PKC) were used to observe the roles of PKA and PKC pathways on the TSAb-stimulated secretions of T3,T4 measured by radioimmunoassay . 3. The expressions of TTF-1 and PAX-8 genes in thyroid tissues and cells were evaluated with a semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) technique, and inhibitors or activators of PKA and PKC were used to observe the roles of PKA and PKC pathways on the TSAb-stimulated expressions of TTF-1 and PAX-8 genes .Results: 1. TSAb activated both PKA and PKC in thyrocytes in a dose-,time-dependent manner,and PKA activity was much higher than that of PKC. The activity peak of PKA was at 30min and PKC was at 60min . 2. The effects of TSAb on the secretions of T3,T4 in thyrocytes were mimicked by the PKA activator,and were inhibited by the PKA inhibitor,whereas the inhibitor and activator of PKC had no such effects as PKA . Although TPA alone,a PKC activator ,was devoid of any significant effect on T3,T4 secretions ,it partly inhibited secretions of T3,T4 stimulated by TSAb and the PKA activator, and these inhibitions were reversed by the PKC inhibition . 3. PAX-8 gene was highly expressed in thyroid tissues of GD, whereas TTF-1 gene expression was no significantly increased in GD by comparison to normal thyroid tissues. 4. TSAb time and dose dependently stimulated PAX-8 gene expression . Such effect of TSAb was mimicked by the PKA activator ,and was inhibited by the PKA inhibitor ,but not by the PKC inhibitor . TPA alone showed no effect on the expression of PAX-8 gene,but combined with TSAb,it inhibited the expression of PAX-8 gene elicited by TSAb,and this inhibition was reversed by the PKC inhibitor . TSAb had no effect on the expression of TTF-1 gene .Conclusion: 1. TSAb could increase the activities of PKA and PKC,and PKA activity was much higher than that of PKC ,but it is the cAMP/PKA pathway, not the activity of PKC, involved in TSAb-stimulated secretions of T3,T4 . However, there is a cross-talk between PKA and PKC signaling pathways,PKC pathway seemed to inhibit TSAb(cAMP/PKA)-mediated secretions of T3,T4 . 2. The expression of PAX-8 gene was significantly increased in GD by comparison to control tissues . TSAb also could increase the expression of PAX-8 gene by the cAMP/PKA pathway in human thyroid cells . TSAb upregulated the expression of pax-8 gene and stimulated the secretions of T3,T4 through the same signal transduction mechanisms,so it indicated that upregulation of PAX-8 gene expression by the cAMP/PKA pathway might be one of the mechanisms for TSAb-induced hyperfuctions of human thyrocytes .