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Thyrostimulin Regulates Osteoblastic Bone Formation During Early Skeletal Development.

Bassett JH, van der Spek A, Logan JG, Gogakos A, Bagchi-Chakraborty J, Murphy E, van Zeijl C, Down J, Croucher PI, Boyde A, Boelen A, Williams GR - Endocrinology (2015)

Bottom Line: However, thyrostimulin failed to induce a canonical cAMP response or activate the noncanonical Akt, ERK, or mitogen-activated protein kinase (P38) signaling pathways in primary calvarial or bone marrow stromal cell-derived osteoblasts.Furthermore, thyrostimulin did not directly inhibit osteoblast proliferation, differentiation or mineralization in vitro.These studies identify thyrostimulin as a negative but indirect regulator of osteoblastic bone formation during skeletal development.

View Article: PubMed Central - PubMed

Affiliation: Molecular Endocrinology Laboratory (J.H.D.B., J.G.L., A.G., J.B.C., E.M., G.R.W.), Department of Medicine, Imperial College London, London, W12 0NN United Kingdom; Department of Endocrinology (A.v.d.S., C.v.Z., A.Boe.), Academic Medical Centre, University of Amsterdam, 1100 DD Amsterdam, The Netherlands; Bone Biology Program (J.D., P.I.C.), Garvan Institute of Medical Research, Sydney, NSW 2010 Australia; and Centre for Oral Growth and Development (A.Boy.), Queen Mary, University of London, London, E1 4NS United Kingdom.

ABSTRACT
The ancestral glycoprotein hormone thyrostimulin is a heterodimer of unique glycoprotein hormone subunit alpha (GPA)2 and glycoprotein hormone subunit beta (GPB)5 subunits with high affinity for the TSH receptor. Transgenic overexpression of GPB5 in mice results in cranial abnormalities, but the role of thyrostimulin in bone remains unknown. We hypothesized that thyrostimulin exerts paracrine actions in bone and determined: 1) GPA2 and GPB5 expression in osteoblasts and osteoclasts, 2) the skeletal consequences of thyrostimulin deficiency in GPB5 knockout (KO) mice, and 3) osteoblast and osteoclast responses to thyrostimulin treatment. Gpa2 and Gpb5 expression was identified in the newborn skeleton but declined rapidly thereafter. GPA2 and GPB5 mRNAs were also expressed in primary osteoblasts and osteoclasts at varying concentrations. Juvenile thyrostimulin-deficient mice had increased bone volume and mineralization as a result of increased osteoblastic bone formation. However, thyrostimulin failed to induce a canonical cAMP response or activate the noncanonical Akt, ERK, or mitogen-activated protein kinase (P38) signaling pathways in primary calvarial or bone marrow stromal cell-derived osteoblasts. Furthermore, thyrostimulin did not directly inhibit osteoblast proliferation, differentiation or mineralization in vitro. These studies identify thyrostimulin as a negative but indirect regulator of osteoblastic bone formation during skeletal development.

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TSHR signaling in BMSC/osteoblast cultures. A, Primary mouse BMSC osteoblasts treated for 28 days with medium (control), medium supplemented with conditioned medium from COS-7 cells (10% vol/vol) transfected with empty vector (vector), medium supplemented with conditioned medium from COS-7 cells (10% vol/vol) transfected with GPA2/GPB5 expression vector (GPA2/GPB5), and PTH (100μM). B, Graphs showing analysis of cell proliferation (viable cells, %) after 9 and 28 days in response to treatments. C, Graphs showing analysis of osteoblast differentiation (ALP activity) after 9 and 28 days in response to treatments. D, Graphs showing analysis of osteoblast mineralization (alizarin red staining) after 9 and 28 days in response to treatments. E, Graphs showing analysis of osteoblast gene expression (Runx2, Osx, and Oc) after 9 and 28 days in response to treatments. F, cAMP responses of primary BMSC osteoblasts cultured for 9 and 28 days after no treatment (OB) or in response to treatment with forskolin, conditioned medium from COS-7 cells (10% vol/vol) transfected with empty vector (vector), or conditioned medium from COS-7 cells (10% vol/vol) transfected with GPA2/GPB5 expression vector GPA2/GPB5. G–I, Analysis of noncanonical TSHR downstream signaling pathways in response to treatment with thyrostimulin (GPA2/GPB5) for 5, 15, and 30 minutes after 9 and 28 days: (G) Akt pathway activation determined by quantitation of phospho-Akt (pAkt); −ve control, MEM medium alone; +ve control, recombinant pAkt2; (H) ERK pathway activation determined by quantitation of phospho-ERK (pERK); −ve control, MEM medium; +ve control, recombinant pERK2; (I) P38 pathway activation determined by quantitation of phospho-P38 (pP38); −ve control, MEM medium; +ve control, recombinant pP38α.
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Figure 8: TSHR signaling in BMSC/osteoblast cultures. A, Primary mouse BMSC osteoblasts treated for 28 days with medium (control), medium supplemented with conditioned medium from COS-7 cells (10% vol/vol) transfected with empty vector (vector), medium supplemented with conditioned medium from COS-7 cells (10% vol/vol) transfected with GPA2/GPB5 expression vector (GPA2/GPB5), and PTH (100μM). B, Graphs showing analysis of cell proliferation (viable cells, %) after 9 and 28 days in response to treatments. C, Graphs showing analysis of osteoblast differentiation (ALP activity) after 9 and 28 days in response to treatments. D, Graphs showing analysis of osteoblast mineralization (alizarin red staining) after 9 and 28 days in response to treatments. E, Graphs showing analysis of osteoblast gene expression (Runx2, Osx, and Oc) after 9 and 28 days in response to treatments. F, cAMP responses of primary BMSC osteoblasts cultured for 9 and 28 days after no treatment (OB) or in response to treatment with forskolin, conditioned medium from COS-7 cells (10% vol/vol) transfected with empty vector (vector), or conditioned medium from COS-7 cells (10% vol/vol) transfected with GPA2/GPB5 expression vector GPA2/GPB5. G–I, Analysis of noncanonical TSHR downstream signaling pathways in response to treatment with thyrostimulin (GPA2/GPB5) for 5, 15, and 30 minutes after 9 and 28 days: (G) Akt pathway activation determined by quantitation of phospho-Akt (pAkt); −ve control, MEM medium alone; +ve control, recombinant pAkt2; (H) ERK pathway activation determined by quantitation of phospho-ERK (pERK); −ve control, MEM medium; +ve control, recombinant pERK2; (I) P38 pathway activation determined by quantitation of phospho-P38 (pP38); −ve control, MEM medium; +ve control, recombinant pP38α.

Mentions: To investigate further whether the effects of thyrostimulin on bone formation are likely to be mediated by primary actions on osteoblast precursor cells, studies were also performed in BMSC/osteoblasts cultured for 9 and 28 days (Figure 8). Thyrostimulin had no effect on the number of viable BMSC osteoblasts, ALP activity, nodule formation, mineralization or marker gene expression at both time points (Figure 8, A–E). Thyrostimulin also had no effect on noncanonical TSHR downstream signaling pathways (Figure 8, F–I), although there was a trend for inhibition of AKT activation after 30 minutes treatment (P = .051) in day-28 BMSC osteoblasts (Figure 8G, right panel).


Thyrostimulin Regulates Osteoblastic Bone Formation During Early Skeletal Development.

Bassett JH, van der Spek A, Logan JG, Gogakos A, Bagchi-Chakraborty J, Murphy E, van Zeijl C, Down J, Croucher PI, Boyde A, Boelen A, Williams GR - Endocrinology (2015)

TSHR signaling in BMSC/osteoblast cultures. A, Primary mouse BMSC osteoblasts treated for 28 days with medium (control), medium supplemented with conditioned medium from COS-7 cells (10% vol/vol) transfected with empty vector (vector), medium supplemented with conditioned medium from COS-7 cells (10% vol/vol) transfected with GPA2/GPB5 expression vector (GPA2/GPB5), and PTH (100μM). B, Graphs showing analysis of cell proliferation (viable cells, %) after 9 and 28 days in response to treatments. C, Graphs showing analysis of osteoblast differentiation (ALP activity) after 9 and 28 days in response to treatments. D, Graphs showing analysis of osteoblast mineralization (alizarin red staining) after 9 and 28 days in response to treatments. E, Graphs showing analysis of osteoblast gene expression (Runx2, Osx, and Oc) after 9 and 28 days in response to treatments. F, cAMP responses of primary BMSC osteoblasts cultured for 9 and 28 days after no treatment (OB) or in response to treatment with forskolin, conditioned medium from COS-7 cells (10% vol/vol) transfected with empty vector (vector), or conditioned medium from COS-7 cells (10% vol/vol) transfected with GPA2/GPB5 expression vector GPA2/GPB5. G–I, Analysis of noncanonical TSHR downstream signaling pathways in response to treatment with thyrostimulin (GPA2/GPB5) for 5, 15, and 30 minutes after 9 and 28 days: (G) Akt pathway activation determined by quantitation of phospho-Akt (pAkt); −ve control, MEM medium alone; +ve control, recombinant pAkt2; (H) ERK pathway activation determined by quantitation of phospho-ERK (pERK); −ve control, MEM medium; +ve control, recombinant pERK2; (I) P38 pathway activation determined by quantitation of phospho-P38 (pP38); −ve control, MEM medium; +ve control, recombinant pP38α.
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Related In: Results  -  Collection

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Figure 8: TSHR signaling in BMSC/osteoblast cultures. A, Primary mouse BMSC osteoblasts treated for 28 days with medium (control), medium supplemented with conditioned medium from COS-7 cells (10% vol/vol) transfected with empty vector (vector), medium supplemented with conditioned medium from COS-7 cells (10% vol/vol) transfected with GPA2/GPB5 expression vector (GPA2/GPB5), and PTH (100μM). B, Graphs showing analysis of cell proliferation (viable cells, %) after 9 and 28 days in response to treatments. C, Graphs showing analysis of osteoblast differentiation (ALP activity) after 9 and 28 days in response to treatments. D, Graphs showing analysis of osteoblast mineralization (alizarin red staining) after 9 and 28 days in response to treatments. E, Graphs showing analysis of osteoblast gene expression (Runx2, Osx, and Oc) after 9 and 28 days in response to treatments. F, cAMP responses of primary BMSC osteoblasts cultured for 9 and 28 days after no treatment (OB) or in response to treatment with forskolin, conditioned medium from COS-7 cells (10% vol/vol) transfected with empty vector (vector), or conditioned medium from COS-7 cells (10% vol/vol) transfected with GPA2/GPB5 expression vector GPA2/GPB5. G–I, Analysis of noncanonical TSHR downstream signaling pathways in response to treatment with thyrostimulin (GPA2/GPB5) for 5, 15, and 30 minutes after 9 and 28 days: (G) Akt pathway activation determined by quantitation of phospho-Akt (pAkt); −ve control, MEM medium alone; +ve control, recombinant pAkt2; (H) ERK pathway activation determined by quantitation of phospho-ERK (pERK); −ve control, MEM medium; +ve control, recombinant pERK2; (I) P38 pathway activation determined by quantitation of phospho-P38 (pP38); −ve control, MEM medium; +ve control, recombinant pP38α.
Mentions: To investigate further whether the effects of thyrostimulin on bone formation are likely to be mediated by primary actions on osteoblast precursor cells, studies were also performed in BMSC/osteoblasts cultured for 9 and 28 days (Figure 8). Thyrostimulin had no effect on the number of viable BMSC osteoblasts, ALP activity, nodule formation, mineralization or marker gene expression at both time points (Figure 8, A–E). Thyrostimulin also had no effect on noncanonical TSHR downstream signaling pathways (Figure 8, F–I), although there was a trend for inhibition of AKT activation after 30 minutes treatment (P = .051) in day-28 BMSC osteoblasts (Figure 8G, right panel).

Bottom Line: However, thyrostimulin failed to induce a canonical cAMP response or activate the noncanonical Akt, ERK, or mitogen-activated protein kinase (P38) signaling pathways in primary calvarial or bone marrow stromal cell-derived osteoblasts.Furthermore, thyrostimulin did not directly inhibit osteoblast proliferation, differentiation or mineralization in vitro.These studies identify thyrostimulin as a negative but indirect regulator of osteoblastic bone formation during skeletal development.

View Article: PubMed Central - PubMed

Affiliation: Molecular Endocrinology Laboratory (J.H.D.B., J.G.L., A.G., J.B.C., E.M., G.R.W.), Department of Medicine, Imperial College London, London, W12 0NN United Kingdom; Department of Endocrinology (A.v.d.S., C.v.Z., A.Boe.), Academic Medical Centre, University of Amsterdam, 1100 DD Amsterdam, The Netherlands; Bone Biology Program (J.D., P.I.C.), Garvan Institute of Medical Research, Sydney, NSW 2010 Australia; and Centre for Oral Growth and Development (A.Boy.), Queen Mary, University of London, London, E1 4NS United Kingdom.

ABSTRACT
The ancestral glycoprotein hormone thyrostimulin is a heterodimer of unique glycoprotein hormone subunit alpha (GPA)2 and glycoprotein hormone subunit beta (GPB)5 subunits with high affinity for the TSH receptor. Transgenic overexpression of GPB5 in mice results in cranial abnormalities, but the role of thyrostimulin in bone remains unknown. We hypothesized that thyrostimulin exerts paracrine actions in bone and determined: 1) GPA2 and GPB5 expression in osteoblasts and osteoclasts, 2) the skeletal consequences of thyrostimulin deficiency in GPB5 knockout (KO) mice, and 3) osteoblast and osteoclast responses to thyrostimulin treatment. Gpa2 and Gpb5 expression was identified in the newborn skeleton but declined rapidly thereafter. GPA2 and GPB5 mRNAs were also expressed in primary osteoblasts and osteoclasts at varying concentrations. Juvenile thyrostimulin-deficient mice had increased bone volume and mineralization as a result of increased osteoblastic bone formation. However, thyrostimulin failed to induce a canonical cAMP response or activate the noncanonical Akt, ERK, or mitogen-activated protein kinase (P38) signaling pathways in primary calvarial or bone marrow stromal cell-derived osteoblasts. Furthermore, thyrostimulin did not directly inhibit osteoblast proliferation, differentiation or mineralization in vitro. These studies identify thyrostimulin as a negative but indirect regulator of osteoblastic bone formation during skeletal development.

Show MeSH
Related in: MedlinePlus