Expression Of Thyroid Stimulating Hormone β Splice Variant In Iodine Deficiency Balb/C Mice

ObjectiveIn2009, a novel splice variant of thyroid stimulating hormone (TSHβ-V) was found by Vincent BH in bone marrow (BM) of the Balb/c mice, which is codeed by3end27base of the fourth intron and the total fifth exon. and this TSHβ-V is7-fold in the thyroid following systemic virus infection while the expression of the native TSHβ has no change. The biological fuction is not clear at the moment. We design this iodine deficiency experiment model to find if this TSHβ-V would be regulated by the thyroid hormone levels in serum via detecting the TSHβ-V mRNA expression level in BM. PBL, thyroid and pituitary of iodine deficiency Balb/c mice, to get a further understanding of the function and mechanism of this immune system derived TSHβ-V in thyroid homeostasis.MCT8was an imprtant TH transporter which is involved in the secretion of TH from the thyroid gland into blood circulation and the low serum T4level observed in MCT8-deficient mice is due not only to the consumptive effect caused by the increase in5’deiodination. but also to a defect of TH secretion from thvroid gland. To further investigate the kinetics of TH secretion, we measured the MCT8mRNA and MCT8protein in the thyroid gland of iodine deficiency Balb/c mice and of iodine deficiency Balb/c mice following the administration of T3MethodsA total of120weaning Balb/c mice (half male and half female) were selected and randomly divided into two groups according to their body mass and gender (n=10). Mice of normal control group (NC group) were fed with common forage and demineralized water. Mice of the low iodine group (LI group) were fed with low-iodine forage (containing iodine20-40μg/kg, iodine-intake about0.25μg/d) and demineralized water. At the3th month.10mice of each group was killed and the rest were kept feeding to the6th month, when15mice in LI goup was selected randomly to divided into3groups (n=5). which are injected with T3(100μg/kg. one time per24h)24h.48h.72h respectively.At the end of the experiment, blood was collected to observe its thyroid stimulating hormone (TSH) and thyroid hormone changes by chemiluminescence immunoassay (CIA):bone marrow (BM), peripheral blood (PBL). thyroid gland and pituitary were collected to assay the TSHβ-V mRNA expression by SYBR GREEN fluorescent real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR). The thyroid MCT8mRNA expression was also assayed by SYBR GREEN RT-PCR. The thyroid MCT8protein was detected by western blot (WB).ResultsThree months later, the serum TT4and FT4levels in LI group mice [(0.47±0.70) nmol/L,(2.41±0.28)pmol/L] were significantly lower than those of NC group mice [(55.2±3.68)nmol/L.(32.72±1.02) pmol/L. P<0.001] and the serum TT3and FT3reduction in LI group mice [(0.76±0.08) nmol/L,(4.01±0.40) pmol/L] were relatively lower than those of NC group mice (1.10±0.06) nmol/L.(5.40±0.38) pmol/L, P<0.001]. Iodine dificiency strongly elevated the serum TSH in LI group mice [(35.67±17.39) mIU/L] than that of NC group mice [(0.24±0.10) mIU/L, P<0.001], The mRNA levels of TSHp-V in BM (0.0014±0.0006) and in PBL (0.0019±0.0011) of LI group mice were lower than those of NC group mice (0.0050±0.0005,0.0075±0.0021. P<0.001), while the mRNA levels of TSHβ-V in pituitary of LI group mice (0.2704±0.1044) was increased comparing with that of NC group mice (0.0219±0.0426. P<0.05). Comparing with NC group mice (0.0019±0.0006). there was no thyroid TSHβ-V mRNA level change in LI group mice (0.0018±0.0005. P>0.05). There was no detectable native TSHβ in BM. PBL and thvroid. The mRNA levels of native TSHβ in pituitary of LI group mice (108.1017±8.9426) was dramatically elevated comparing to that of NC group mice (2.8331±0.9199. P0.05).Six months later, compared to NC group mice. TT4and FT4level [(4.60±2.62) nmol/L.(4.72±0.78) pmol/L] in LI group mice were still lower than those of NC group mice [(46.74±10.22) nmol/L.(28.80±4.81) pmol/L. P<0.001]. and there was no statistical differences in TT3. FT3level between the two groups. The serum TSH in LI group mice [(3.22±1.68) mIU/L] was still much higher than that in NC group mice [(0.08±0.03) mIU/L. P<0.001]. There were no statistical differences in the TSHβ-V mRNA level in BM and PBL between the two groups (P>0.05). The TSHβ-V mRNA level in thyroid of LI group mice (0.0003±0.0003) was down-regulated compared with that of NC group mice (0.0014±0.0007. P<0.05). There was no detectable native TSHβ in BM. PBL and thyroid. Comparing to the NC group mice (0.5709±0.0430), the TSHβ-V mRNA level in pituitary of LI group mice (1.5851.±0.3979. P<0.05) was still elevated. The native TSHβ mRNA level in pituitary of LI group mice (29.7621±4.9983) was still higher than that of NC group mice (1.1996±0.3849. P<0.05).After T3administration. TT4. TT3. FT4. FT3level were steadly increased and had been higher than LI group mice since24hours after injection, equal to NC group mice at48hours and higher than NC group mice at72hours after the injection. TSH had been suppressed to NC group level since48h after T3injection. However, compared with LI group mice, the TSHβ-V mRNA expression in thyroid and pituitary of the three T3administration time-points was still down-regulated, the native TSHβ mRNA expression was steadly decreased, and had return to NC group level since48h.Three months later, compared to NC group (0.6223±0.1570), iodine deficiency up-regulated the thyroid MCT8mRNA expression (1.5201±0.2969. P<0.05).Six months later, the thyroid MCT8mRNA expression was also increased in LI group mice (0.6254±0.2872) compared to NC group mice (0.2282±0.0582. P<0.05) which is consistent to the WB result. Following the administration of T3. compared to LI group mice (0.6254±0.2872), the thyroid MCT8mRNA expression of the three time-points [24h (0.2900=0.0506).48h (0.2438=0.0914).72h (0.1402=0.0526)] were significantly suppressed (P<0.05). but had no statistic difference when they were compared to NC group mice (0.2282±0.0582. P>0.05).Conclusions1Three months later, the serum TT4and FT4levels in LI group mice were significantly suppressed while the reduction of serum TT3and FT3in LI group mice were relatively slighter. Iodine deficiency strongly elevated the serum TSH in LI group mice. Six months later, the serum TT3. FT3level returned to normal, but TT4and FT4level in LI group mice were still lower with still higher TSH level. After T3administration, the concentration of serum TT4, TT3. FT4and FT3were steadly increased with TSH steadly decreased. The performance of the classic HPT axis regulation was contributed to these phenomena.2Low iodine diet induced hypothyroidism supresed the proliferation, differentiation and migration of lymphocyte in the BM. PBL and thyroid, which caused the reduction of TSHfJ-V mRNA expression in these tissues. Six months later. TT3and FT3level was back to normal (TT4and FT4level was still very low). TSHβ-V mRNA expression in BM and PBL was increased. The difference of TSHβ-V mRNA expression in BM and PBL between3-months and6-months may relate to the BM and PBL lymphocyte proliferation and differentiation ability which was affected by lack of thvroid hormone.3Six months later, TT3and FT3level was back to normal (TT4and FT4was still very low). TSHβ-V mRNA expression in the thyroid was still repressed. The fact that TSHβ-V mRNA expression down-regulated in thyroid combind with TSHβ mRNA expression elevated in pituitary implies that TSHβ-V may play a negative role on thyroid function. The fact that reversion of thyroid hormone level through T3administration didn’t recover the TSHβ-V mRNA expression in the thyroid implies that the serum thyroid homone has no direct effect on the TSHβ-V mRNA expression in the thyroid4After long-term low iodine diet, there was a compensatory increase in thyroid MCT8mRNA and protein expression to enhance its capability to transport TH from thvroid to blood circulation.