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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 primary calvarial osteoblasts. A, Primary mouse 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 osteoblast 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 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 7: TSHR signaling in primary calvarial osteoblasts. A, Primary mouse 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 osteoblast 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 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 whether thyrostimulin directly inhibits calvarial osteoblast function, cell proliferation, differentiation, and mineralization assays were performed (Figure 7). Primary calvarial osteoblasts were cultured for 9 and 28 days in the presence of conditioned medium from COS-7 cells transfected with vector alone or with medium from cells coexpressing GPA2 and GPB5. Continuous treatment with PTH was used as a positive control for inhibition of osteoblast activity. Thyrostimulin had no effect on the number of viable osteoblasts, ALP activity or nodule formation and mineralization at both time points (Figure 7, A–D) and did not affect levels of expression of Runx2, Osx, or Oc osteoblast marker gene mRNAs (Figure 7E). Persisting thyrostimulin activity was confirmed in the conditioned medium at completion of the 28-day culture period by determining the cAMP response in TSHR-expressing CHO cells (data not shown). To investigate whether thyrostimulin activates noncanonical TSHR downstream signaling pathways in calvarial osteoblasts, analysis of the Akt, ERK, and P38 (P38 MAPK) pathways was performed (Figure 7, F–I). Thyrostimulin had no effect on these downstream pathways in cells cultured for 9 or 28 days.


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 primary calvarial osteoblasts. A, Primary mouse 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 osteoblast 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 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 7: TSHR signaling in primary calvarial osteoblasts. A, Primary mouse 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 osteoblast 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 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 whether thyrostimulin directly inhibits calvarial osteoblast function, cell proliferation, differentiation, and mineralization assays were performed (Figure 7). Primary calvarial osteoblasts were cultured for 9 and 28 days in the presence of conditioned medium from COS-7 cells transfected with vector alone or with medium from cells coexpressing GPA2 and GPB5. Continuous treatment with PTH was used as a positive control for inhibition of osteoblast activity. Thyrostimulin had no effect on the number of viable osteoblasts, ALP activity or nodule formation and mineralization at both time points (Figure 7, A–D) and did not affect levels of expression of Runx2, Osx, or Oc osteoblast marker gene mRNAs (Figure 7E). Persisting thyrostimulin activity was confirmed in the conditioned medium at completion of the 28-day culture period by determining the cAMP response in TSHR-expressing CHO cells (data not shown). To investigate whether thyrostimulin activates noncanonical TSHR downstream signaling pathways in calvarial osteoblasts, analysis of the Akt, ERK, and P38 (P38 MAPK) pathways was performed (Figure 7, F–I). Thyrostimulin had no effect on these downstream pathways in cells cultured for 9 or 28 days.

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