Study On Biological Characteristic Of Human Novel TSHβ Splice Variant

ObjectiveThyroid-stimulating hormone (TSH) is a key protein in the control of thyroid function. TSH consists of a common α-subunit and a unique β-subunit, the latter being responsible for hormone specificity. In2009, the first functional splice variant of TSHβ was identified in mouse and human. TSHβ splice variant (TSHβv) may have the different effects on immune and neuroendocrine homeostatic regulation from the native TSHβ,which have not been investigated to date. In the present study, the basic biological characteristics, expression regulation of TSHβv and the relationship between autoimmune thyroid disease and TSHβv will be explored systematically.Methods1. Study of TSHβv in blood:After the serum samples were predisposed, TSHβv protein was detected by immunoprecipitation. TSHβv gene and protein expressions in human peripheral blood leukocyte (PBL) were identified by RT-PCR, Western Blot and immunofluorescene staining, respectively.2. TSHβv dimerized with TSHa or not: hTSHβv and hTSHa stable expression CHO cell lines were constructed. After cocultured, co-immunoprecipitation method was used to detect whether TSHβv can dimerize with TSHa.3. Expression alterations of TSHβv in human PBL under different pathophysiological condition:Total RNA was isolated from PBL of HT-and GD-patients. Real Time PCR was used to detect TSHβv mRNA expression differentiations. Human PBL were isolated and induced by single100nMTRH,100nMT3, lOmg/L LPS and dexamethasone at various concentrations (10-6,10-7and10-8mol/L) seperately. TSHβv gene expression differentiations were detected at different time points.4. Prokaryotic expression and purification of recombinated TSHβv protein:PET-28a-TSHβv was constructed and TSHβv protein was purified by Immuno-affinity chromatography.Results 1. TSHβv not native TSHβ was expressed in human PBL. TSHβv was identified in serum for the first time.2. Recombinant plasmids pcDNA3.1D/V5-His-TOPO-TSHα and pSELECT-GFPzeo-TSHpv were successfully constructed. hTSHα and hTSH(3v were expressed in CHO cells by transfection. TSHβv dimerize with TSHa was identified by Co-IP.3. Compared with the control group, mRNA expressions of TSHβv were higher in PBL of HT patients without prednisone therapy (n=10, P<0.0001) and HT patients undergoing prednisone therapy with longer duration of illness (≥18months)(n=5, P=0.023).That pattern was reversed in PBL of HT patients undergoing prednisone therapy with shorter duration of illness (≤9months)(n=8, P<0.0001). TSHPv mRNA expression was down-regulated in PBL of GD patients (P<0.0001). TSHβv expression level in hPBL was correlated negatively with FT3level (r=-0.635, P<0.0001), FT4level (r=-0.760, P<0.0001) and positively with TSH (r=0.975, P<0.0001) in serum.4. TSHβv mRNA expression increased rapidly at6h induced by LPS (P=0.003). The inhibition of dexamethason on TSHβv mRNA expression was in dose-and time-dependent manner.5. TSHβv mRNA expression was up-regulated by TRH and down-regulated by T3.6. About7.4mg hTSHβv protein was purified.Conclusions1. TSHβv can exert adjustive function by telecrine and paracrine.2. TSHβ splice variant can exert biological function in target organ by dimerized with TSHa.3. TSHβv may take part in the pathological processes of HT and GD. TSHβv expression may be regulated by hypothalamus-pituitary-thyroid axis under particular circumstances. PBL production of TSHβv could be involved in immune-neuroendocrine regulation network in the inflammatory responses induced by LPS.4. Purified TSHβ splice variant will provide the important material foundation for furtherly study the biological function and expression regulation of TSHβ splice variant. Our findings characterized the first functional TSHβv, which may offer a new perspective for understanding autoimmune thyroiditis and immune-endocrine interactions network.