Zhang Lei, Bowen Timothy, Grennan-Jones Fiona, Paddon Carol, Giles Peter, Webber Jason, Steadman Robert, Ludgate Marian
Centre for Endocrine & Diabetes Sciences, Cardiff University, Cardiff CF14 4XN, United Kingdom.
J Biol Chem. 2009 Sep 25;284(39):26447-55. doi: 10.1074/jbc.M109.003616. Epub 2009 Jul 24.
The thyrotropin receptor (TSHR) is expressed during lineage-specific differentiation (e.g. adipogenesis) and is activated by TSH, thyroid-stimulating antibodies, and gain-of-function mutations (TSHR*). Comparison of gene expression profiles of nonmodified human preadipocytes (n = 4) with the parallel TSHR* population revealed significant up-regulation of 27 genes including hyaluronan (HA) synthases (HAS) 1 and 2. The array data were confirmed by quantitative PCR of HAS1 and HAS2 and enzyme-linked immunosorbent assay measurement of HA; all values were significantly increased (p < 0.03) in TSHR*-expressing preadipocytes (n = 10). Preadipocytes (n = 8) treated with dibutyryl (db)-cAMP display significantly increased HAS1 and HAS2 transcripts, HAS2 protein, and HA production (p < 0.02). HAS1 or HAS2 small interfering RNA treatment of db-cAMP-stimulated preadipocytes (n = 4) produced 80% knockdown in HAS1 or 61% knockdown in HAS2 transcripts (compared with scrambled), respectively; the corresponding HA production was reduced by 49 or 38%. Reporter assays using A293 cells transfected with HAS1 promoter-driven plasmids containing or not containing the proximal CRE and treated with db-cAMP revealed that it is functional. Chromatin immunoprecipitation, using a cAMP-responsive element-binding protein antibody, of db-cAMP-treated preadipocytes (n = 4) yielded products for HAS1 and HAS2 with relative fold increases of 3.3 +/- 0.8 and 2.6 +/- 0.9, respectively. HA accumulates in adipose/connective tissues of patients with thyroid dysfunction. We investigated the contributions of TSH and thyroid-stimulating antibodies and obtained small (9-24%) but significant (p < 0.02) increases in preadipocyte HA production with both ligands. Similar results were obtained with a TSHR monoclonal antibody lacking biological activity (p < 0.05). We conclude that TSHR activation is implicated in HA production in preadipocytes, which, along with thyroid hormone level variation, explains the HA overproduction in thyroid dysfunction.
促甲状腺激素受体（TSHR）在谱系特异性分化过程（如脂肪生成）中表达，并可被促甲状腺激素、促甲状腺刺激抗体及功能获得性突变（TSHR*）激活。将未修饰的人前脂肪细胞（n = 4）与平行的TSHR细胞群体的基因表达谱进行比较，发现包括透明质酸（HA）合成酶（HAS）1和2在内的27个基因显著上调。通过对HAS1和HAS2进行定量PCR以及对HA进行酶联免疫吸附测定，证实了芯片数据；在表达TSHR的前脂肪细胞（n = 10）中，所有值均显著升高（p < 0.03）。用二丁酰（db）-cAMP处理的前脂肪细胞（n = 8）显示HAS1和HAS2转录本、HAS2蛋白及HA产生均显著增加（p < 0.02）。对经db-cAMP刺激的前脂肪细胞（n = 4）进行HAS1或HAS2小干扰RNA处理，HAS1转录本分别降低80%，HAS2转录本降低61%（与乱序序列相比）；相应的HA产生分别减少49%或38%。使用转染了含或不含近端CRE的HAS1启动子驱动质粒的A293细胞进行报告基因检测，并经db-cAMP处理，结果显示其具有功能。使用cAMP反应元件结合蛋白抗体对经db-cAMP处理的前脂肪细胞（n = 4）进行染色质免疫沉淀，得到HAS1和HAS2的产物，相对倍数分别增加3.3±0.8和2.6±0.9。HA在甲状腺功能障碍患者的脂肪/结缔组织中蓄积。我们研究了促甲状腺激素和促甲状腺刺激抗体的作用，发现两种配体均可使前脂肪细胞HA产生小幅（9 - 24%）但显著（p < 0.02）增加。使用缺乏生物活性的TSHR单克隆抗体也得到了类似结果（p < 0.05）。我们得出结论，TSHR激活与前脂肪细胞中HA产生有关，这与甲状腺激素水平变化一起，解释了甲状腺功能障碍中HA的过度产生。
