β-Arrestin 1 in Thyrotropin Receptor Signaling in Bone: Studies in Osteoblast-Like Cells

Abstract

A direct action of thyrotropin (TSH, thyroid-stimulating hormone) on bone precursors in humans is controversial. Studies in rodent models have provided conflicting findings. We used cells derived from a moderately differentiated osteosarcoma stably overexpressing human TSH receptors (TSHRs) as a model of osteoblast precursors (U2OS-TSHR cells) to investigate TSHR-mediated effects in bone differentiation in human cells. We review our findings that (1) TSHR couples to several different G proteins to induce upregulation of genes associated with osteoblast activity-interleukin 11 (IL-11), osteopontin (OPN), and alkaline phosphatase (ALPL) and that the kinetics of the induction and the G protein-mediated signaling pathways involved were different for these genes; (2) TSH can stimulate β-arrestin-mediated signal transduction and that β-arrestin 1 in part mediates TSH-induced pre-osteoblast differentiation; and (3) TSHR/insulin-like growth factor 1 (IGF1) receptor (IGF1R) synergistically increased OPN secretion by TSH and IGF1 and that this crosstalk was mediated by physical association of these receptors in a signaling complex that uses β-arrestin 1 as a scaffold. These findings were complemented using a novel β-arrestin 1-biased agonist of TSHR. We conclude that TSHR can signal via several transduction pathways leading to differentiation of this model system of human pre-osteoblast cells and, therefore, that TSH can directly regulate these bone cells.

Keywords: IGF1 receptor; TSH receptor; crosstalk; osteoblast-like cells; positive allosteric modulator; β-arrestin 1.

Figure 1

β-arrestin 1, but not β-arrestin 2, mediates OPN secretion. U2OS-TSHR cells were treated with non-targeting (scramble siRNA, Scr), β-arrestin 1 (ARRB1), or β-arrestin 2 (ARRB2) siRNAs for 24 h. After 72 h, the cells were incubated with 2 μM TSH in EMEM supplemented with 0.1% BSA for 5 days prior to the ELISA measurement of OPN secreted into the cell culture media (21).

Figure 2

D3-βArr is a functionally selective TSHR agonist. (A) D3-βArr is a positive allosteric modulator for TSH-induced OPN secretion. U2OS-TSHR cells were treated with increasing doses of TSH with or without 5 μM D3-βArr in EMEM with 0.1% BSA for 7 days prior to OPN measurement by ELISA in cell culture media (43). (B) The potentiating effect of D3-βArr is mediated by β-arrestin 1 in U2OS-TSHR cells. Cells were transfected with non-targeting (Scr) or β-arrestin 1 (ARRB1) siRNA, respectively. After 72 h of siRNA-mediated knockdown, cells were treated with 10 μM D3-βArr, 2 μM TSH or their combination in EMEM with 0.1% BSA for 5 days. OPN secretion was measured in conditioned media by ELISA (43).

Figure 3

TSHR and IGF1R exhibit crosstalk in U2OS-TSHR cells. (A) TSH and IGF1 synergistically stimulate OPN secretion. U2OS-TSHR cells were treated with increasing doses of recombinant human TSH (rhTSH) with or without 100 ng/ml IGF1. After 5 days, OPN secreted into cell culture media was measured by ELISA. This original figure is based on the data published in (52). (B) IGF1R and β-arrestin 1 are part of the same signaling pathway in TSHR/IGF1R crosstalk-mediated OPN secretion. U2OS-TSHR cells, in which IGF1R and β-arrestin1 (ARRB1) were silenced separately or simultaneously were treated with 10 mU/ml rhTSH. After 5 days, OPN was measured in culture media and compared to scramble siRNA (Scr) control. Knockdown efficiencies of IGF1R and ARRB1 were 95.5 and 82.9%, respectively (52).

Figure 4

TSHR/IGF1R crosstalk is dependent on receptor proximity and β-arrestin 1 stabilizes the protein complex. TSHR/IGF1R signaling complexes were detected using a Proximity Ligation Assay (PLA) and visualized with fluorescent microscopy. A PLA signal is measured when TSHR and IGF1R are in a protein complex within 40 nm of each other. PLA was performed in unstimulated U2OS-TSHR cells treated with non-targeting (A), β-arrestin 1 (ARRB1) (B), or β-arrestin 2 (ARRB2) (C) siRNA. Positive PLA signals are shown in red. SYTO9 nucleic acid staining, applied to determine the cell number, is shown in green at ×63 magnification. The knock-down of β-arrestin 1 reduced the number of positive PLA signals by 55% demonstrating this proteins stabilizing function in the TSHR/IGF1R complex (52).

Figure 5

Model of signaling pathways leading to OPN secretion in U2OS-TSHR cells. (A) TSHR-mediated upregulation of OPN. TSH-induced OPN secretion is regulated via Gα_i and β-arrestin 1-mediated signal transduction pathways. (B) β-arrestin 1 scaffolds TSHR and IGF1R to enable crosstalk. TSH and IGF1 synergistically upregulate OPN secretion. β-arrestin 1 stabilizes the receptor complex which enables TSHR/IGF1R crosstalk.