Multiple Transduction Pathways Mediate Thyrotropin Receptor Signaling in Preosteoblast-Like Cells

Abstract

It has been shown that the TSH receptor (TSHR) couples to a number of different signaling pathways, although the Gs-cAMP pathway has been considered primary. Here, we measured the effects of TSH on bone marker mRNA and protein expression in preosteoblast-like U2OS cells stably expressing TSHRs. We determined which signaling cascades are involved in the regulation of IL-11, osteopontin (OPN), and alkaline phosphatase (ALPL). We demonstrated that TSH-induced up-regulation of IL-11 is primarily mediated via the Gs pathway as IL-11 was up-regulated by forskolin (FSK), an adenylyl cyclase activator, and inhibited by protein kinase A inhibitor H-89 and by silencing of Gαs by small interfering RNA. OPN levels were not affected by FSK, but its up-regulation was inhibited by TSHR/Gi-uncoupling by pertussis toxin. Pertussis toxin decreased p38 MAPK kinase phosphorylation, and a p38 inhibitor and small interfering RNA knockdown of p38α inhibited OPN induction by TSH. Up-regulation of ALPL expression required high doses of TSH (EC50 = 395nM), whereas low doses (EC50 = 19nM) were inhibitory. FSK-stimulated cAMP production decreased basal ALPL expression, whereas protein kinase A inhibition by H-89 and silencing of Gαs increased basal levels of ALPL. Knockdown of Gαq/11 and a protein kinase C inhibitor decreased TSH-stimulated up-regulation of ALPL, whereas a protein kinase C activator increased ALPL levels. A MAPK inhibitor and silencing of ERK1/2 inhibited TSH-stimulated ALPL expression. We conclude that TSH regulates expression of different bone markers via distinct signaling pathways.

Discovery of a Positive Allosteric Modulator of the Thyrotropin Receptor: Potentiation of Thyrotropin-Mediated Preosteoblast Differentiation In Vitro

“…The knockdown of b-Arr 1 confirmed our previous finding that b-Arr 1-mediated signaling plays a pivotal role in TSH-induced OPN secretion (Boutin et al, 2014). Recently, we showed that multiple TSHR-activated signaling pathways lead to OPN secretion (Boutin et al, 2016). More importantly, we demonstrated that the potentiating effect of D3-bArr is solely mediated by b-Arr 1 and not by b-Arr 2 (Fig.…”

“…It has been known that the TSHR can interact with members of all G protein families (Laugwitz et al, 1996), and we showed previously (Boutin et al, 2014) that TSHR activation by TSH will lead to b-Arr 1-mediated signaling. Specifically, we showed that TSHR-induced upregulation of osteoblast gene markers in U2OS-TSHR cells, like PTH effects in bone precursors (Gesty-Palmer et al, 2009), is mediated by G protein-and b-arrestinmediated pathways (Boutin et al, 2014(Boutin et al, , 2016, with TSHR acting via b-Arr 1 and PTH1R via b-Arr 2. We demonstrated that TSH-induced upregulation of interleukin 11 is primarily mediated via G s -cAMP-protein kinase A, ALPL upregulation is predominantly regulated by activation of G q/11 -phosphokinase C-ERK1/2 and OPN expression, and secretion is mediated by b-Arr 1 and G i -p38a mitogen-activated protein kinase cascades (Boutin et al, 2016).…”

“…Therefore, we next determined if the PAM effect of D3-bArr on b-Arr 1 translocation to the TSHR will translate into potentiation of TSH-induced upregulation of osteoblast-specific genes osteopontin (OPN) and alkaline phosphatase (ALPL). For these experiments, we used U2OS-TSHR cells that are derived from a human osteosarcoma and are a model of osteoblast precursors that we used previously (Boutin et al, 2014(Boutin et al, , 2016. U2OS cells are frequently used to study the regulation of osteoblastic genes.…”

“…However, the role of TSH in human bone homeostasis is still unclear (Mazziotti et al, 2005;Giusti et al, 2007;Karga et al, 2010;Leader et al, 2014). To gain insight into the effects of TSH in human cells, we used a preosteoblast-like cell line derived from a human osteosarcoma made to stably express wild-type TSHR, U2OS-TSHR cells (Boutin et al, 2014(Boutin et al, , 2016. U2OS cells have been shown to differentiate into osteoblasts (Salvatori et al, 2009).…”

Thyroid Status, Quality of Life, and Mental Health in Patients on Hemodialysis

“…Although multiple coexisting mechanisms may lead to impaired physical function in patients on dialysis (e.g., older age, comorbidities, and inflammation) (43), it is plausible that myalgia, reduced muscle strength, higher oxygen requirements during physical activity, and refractory anemia ensuing from hypothyroidism may be potent yet under-recognized contributors (8,(44)(45)(46)(47)(48). Indeed, it is established that thyroid hormones have direct action in nearly every tissue, including the skeletal muscle, bone and cartilage, and the heart, and emerging data suggest TSH may have extrathyroidal effects on these end organs given the presence of TSH receptors in skeletal muscle, bone, and brain (49)(50)(51).…”