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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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Mouse and human GPA2, GPB5, CGA, TSHB, and TSHR mRNA expression. A, Gpa2 and Gpb5 mRNA, and 18s rRNA, expression in skeletal and nonskeletal mouse tissues during development at P1, P14, and P28. −ve ctrl, no reverse transcriptase control. B, Cga, Tshb, Tshr, Gpa2, and Gpb5 mRNA expression in mouse primary osteoblasts and osteoclasts cultured in the presence or absence of 100nM T3. C, CGA, TSHB, TSHR, GPA2, and GPB5 mRNA expression in human primary osteoblasts and osteoclasts in the presence or absence of 100nM T3. Noncontiguous lanes are separated by a white line. Cga, glycoprotein hormones, α; Tshb, TSHβ; Tshb-sv, TSHβ splice variant; Tshr, TSHR; Gpa2, glycoprotein hormone α2; Gpb5, glycoprotein hormone β5.
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Figure 1: Mouse and human GPA2, GPB5, CGA, TSHB, and TSHR mRNA expression. A, Gpa2 and Gpb5 mRNA, and 18s rRNA, expression in skeletal and nonskeletal mouse tissues during development at P1, P14, and P28. −ve ctrl, no reverse transcriptase control. B, Cga, Tshb, Tshr, Gpa2, and Gpb5 mRNA expression in mouse primary osteoblasts and osteoclasts cultured in the presence or absence of 100nM T3. C, CGA, TSHB, TSHR, GPA2, and GPB5 mRNA expression in human primary osteoblasts and osteoclasts in the presence or absence of 100nM T3. Noncontiguous lanes are separated by a white line. Cga, glycoprotein hormones, α; Tshb, TSHβ; Tshb-sv, TSHβ splice variant; Tshr, TSHR; Gpa2, glycoprotein hormone α2; Gpb5, glycoprotein hormone β5.

Mentions: Expression of both Gpa2 and Gpb5 mRNAs was readily identified in skulls and long bones from newborn WT mice. Expression of Gpa2 was reduced by P14 and barely detectable at P28, whereas expression of Gpb5 was only present in skulls at P14 but was absent from both skulls and long bones at P28 (Figure 1A). Neither CGA nor TSHB mRNAs (encoding the α- and β-subunits of TSH, the established hormone that binds and activates TSHR) were expressed in mouse or human primary osteoblast or osteoclast cultures (Figure 1, B and C). TSHR mRNA was expressed in mouse and human osteoblasts and mouse osteoclasts but was at the limit of detection in human osteoclasts. GPA2 was expressed in both mouse and human osteoblasts and osteoclasts. Four distinct human GPA2 RT-PCR products were amplified from brain mRNA but only 2 were expressed in osteoblasts and osteoclasts. Sequencing revealed the size differences resulted from retention of intron 2 (182 bp), intron 3 (116 bp), or both introns 2 and 3 (Supplemental Figure 1A). Inclusion of intron 2 results in an open reading frame terminated by a stop codon after 93 bp, whereas inclusion of intron 3 results in an open reading frame terminated by a stop codon after 69 bp. The GPA2 mRNAs expressed in human osteoblasts and osteoclasts consisted of the full-length correctly spliced GPA2 mRNA (283 bp) and an unspliced transcript (581 bp), in which introns 2 and 3 are retained (Supplemental Figure 1A). By contrast, Gpb5 was expressed only at low levels early in differentiation in mouse osteoblasts but was not detected in mouse osteoclasts (Figure 1B), whereas GPB5 mRNA expression was clearly detectable in human osteoblasts and osteoclasts (Figure 1C).


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)

Mouse and human GPA2, GPB5, CGA, TSHB, and TSHR mRNA expression. A, Gpa2 and Gpb5 mRNA, and 18s rRNA, expression in skeletal and nonskeletal mouse tissues during development at P1, P14, and P28. −ve ctrl, no reverse transcriptase control. B, Cga, Tshb, Tshr, Gpa2, and Gpb5 mRNA expression in mouse primary osteoblasts and osteoclasts cultured in the presence or absence of 100nM T3. C, CGA, TSHB, TSHR, GPA2, and GPB5 mRNA expression in human primary osteoblasts and osteoclasts in the presence or absence of 100nM T3. Noncontiguous lanes are separated by a white line. Cga, glycoprotein hormones, α; Tshb, TSHβ; Tshb-sv, TSHβ splice variant; Tshr, TSHR; Gpa2, glycoprotein hormone α2; Gpb5, glycoprotein hormone β5.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4541616&req=5

Figure 1: Mouse and human GPA2, GPB5, CGA, TSHB, and TSHR mRNA expression. A, Gpa2 and Gpb5 mRNA, and 18s rRNA, expression in skeletal and nonskeletal mouse tissues during development at P1, P14, and P28. −ve ctrl, no reverse transcriptase control. B, Cga, Tshb, Tshr, Gpa2, and Gpb5 mRNA expression in mouse primary osteoblasts and osteoclasts cultured in the presence or absence of 100nM T3. C, CGA, TSHB, TSHR, GPA2, and GPB5 mRNA expression in human primary osteoblasts and osteoclasts in the presence or absence of 100nM T3. Noncontiguous lanes are separated by a white line. Cga, glycoprotein hormones, α; Tshb, TSHβ; Tshb-sv, TSHβ splice variant; Tshr, TSHR; Gpa2, glycoprotein hormone α2; Gpb5, glycoprotein hormone β5.
Mentions: Expression of both Gpa2 and Gpb5 mRNAs was readily identified in skulls and long bones from newborn WT mice. Expression of Gpa2 was reduced by P14 and barely detectable at P28, whereas expression of Gpb5 was only present in skulls at P14 but was absent from both skulls and long bones at P28 (Figure 1A). Neither CGA nor TSHB mRNAs (encoding the α- and β-subunits of TSH, the established hormone that binds and activates TSHR) were expressed in mouse or human primary osteoblast or osteoclast cultures (Figure 1, B and C). TSHR mRNA was expressed in mouse and human osteoblasts and mouse osteoclasts but was at the limit of detection in human osteoclasts. GPA2 was expressed in both mouse and human osteoblasts and osteoclasts. Four distinct human GPA2 RT-PCR products were amplified from brain mRNA but only 2 were expressed in osteoblasts and osteoclasts. Sequencing revealed the size differences resulted from retention of intron 2 (182 bp), intron 3 (116 bp), or both introns 2 and 3 (Supplemental Figure 1A). Inclusion of intron 2 results in an open reading frame terminated by a stop codon after 93 bp, whereas inclusion of intron 3 results in an open reading frame terminated by a stop codon after 69 bp. The GPA2 mRNAs expressed in human osteoblasts and osteoclasts consisted of the full-length correctly spliced GPA2 mRNA (283 bp) and an unspliced transcript (581 bp), in which introns 2 and 3 are retained (Supplemental Figure 1A). By contrast, Gpb5 was expressed only at low levels early in differentiation in mouse osteoblasts but was not detected in mouse osteoclasts (Figure 1B), whereas GPB5 mRNA expression was clearly detectable in human osteoblasts and osteoclasts (Figure 1C).

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