Boutin Alisa, Eliseeva Elena, Gershengorn Marvin C, Neumann Susanne
Laboratory of Endocrinology and Receptor Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA.
Laboratory of Endocrinology and Receptor Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA
FASEB J. 2014 Aug;28(8):3446-55. doi: 10.1096/fj.14-251124. Epub 2014 Apr 10.
Thyrotropin (TSH) activation of the TSH receptor (TSHR), a 7-transmembrane-spanning receptor (7TMR), may have osteoprotective properties by direct effects on bone. TSHR activation by TSH phosphorylates protein kinases AKT1, p38α, and ERK1/2 in some cells. We found TSH-induced phosphorylation of these kinases in 2 cell lines engineered to express TSHRs, human embryonic kidney HEK-TSHR cells and human osteoblastic U2OS-TSHR cells. In U2OS-TSHR cells, TSH up-regulated pAKT1 (7.1±0.5-fold), p38α (2.9±0.4-fold), and pERK1/2 (3.1±0.2-fold), whereas small molecule TSHR agonist C2 had no or little effect on pAKT1 (1.8±0.08-fold), p38α (1.2±0.09-fold), and pERK1/2 (1.6±0.19-fold). Furthermore, TSH increased expression of osteoblast marker genes ALPL (8.2±4.6-fold), RANKL (21±5.9-fold), and osteopontin (OPN; 17±5.3-fold), whereas C2 had little effect (ALPL, 1.7±0.5-fold; RANKL, 1.3±0.6-fold; and OPN, 2.2±0.7-fold). β-Arrestin-1 and -2 can mediate activatory signals by 7TMRs. TSH stimulated translocation of β-arrestin-1 and -2 to TSHR, whereas C2 failed to translocate either β-arrestin. Down-regulation of β-arrestin-1 by siRNA inhibited TSH-stimulated phosphorylation of ERK1/2, p38α, and AKT1, whereas down-regulation of β-arrestin-2 increased phosphorylation of AKT1 in both cell types and of ERK1/2 in HEK-TSHR cells. Knockdown of β-arrestin-1 inhibited TSH-stimulated up-regulation of mRNAs for OPN by 87 ± 1.7% and RANKL by 73 ± 2.4%, and OPN secretion by 74 ± 10%. We conclude that TSH enhances osteoblast differentiation in U2OS cells that is, in part, caused by activatory signals mediated by β-arrestin-1.
促甲状腺激素（TSH）激活促甲状腺激素受体（TSHR），这是一种7次跨膜受体（7TMR），可能通过对骨骼的直接作用而具有骨保护特性。TSH对TSHR的激活在某些细胞中使蛋白激酶AKT1、p38α和ERK1/2磷酸化。我们发现在经过基因工程改造以表达TSHR的两种细胞系，即人胚肾HEK - TSHR细胞和人成骨细胞U2OS - TSHR细胞中，TSH可诱导这些激酶的磷酸化。在U2OS - TSHR细胞中，TSH上调pAKT1（7.1±0.5倍）、p38α（2.9±0.4倍）和pERK1/2（3.1±0.2倍），而小分子TSHR激动剂C2对pAKT1（1.8±0.08倍）、p38α（1.2±0.09倍）和pERK1/2（1.6±0.19倍）没有或只有很小的影响。此外，TSH增加成骨细胞标记基因ALPL（8.2±4.6倍）、RANKL（21±5.9倍）和骨桥蛋白（OPN；17±5.3倍）的表达，而C2的影响很小（ALPL，1.7±0.5倍；RANKL，1.3±0.6倍；OPN，2.2±0.7倍）。β - 抑制蛋白 - 1和 - 2可介导7TMR的激活信号。TSH刺激β - 抑制蛋白 - 1和 - 2向TSHR的转位，而C2未能使任何一种β - 抑制蛋白转位。通过小干扰RNA（siRNA）下调β - 抑制蛋白 - 1可抑制TSH刺激的ERK1/2、p38α和AKT1的磷酸化，而下调β - 抑制蛋白 - 2则增加两种细胞类型中AKT1的磷酸化以及HEK - TSHR细胞中ERK1/2的磷酸化。敲低β - 抑制蛋白 - 1可抑制TSH刺激的OPN mRNA上调87±1.7%、RANKL上调73±2.4%以及OPN分泌74±10%。我们得出结论，TSH增强U2OS细胞中的成骨细胞分化，这部分是由β - 抑制蛋白 - 1介导的激活信号所引起的。
