β-Arrestin-1 mediates thyrotropin-enhanced osteoblast differentiation.
Thyrotropin (TSH) activation of the TSH receptor (TSHR), a 7-transmembrane-spanning receptor (7TMR), may have osteoprotective properties by direct effects on bone. TSHR activation by TSH phosphorylates protein kinases AKT1, p38α, and ERK1/2 in some cells. We found TSH-induced phosphorylation of these kinases in 2 cell lines engineered to express TSHRs, human embryonic kidney HEK-TSHR cells and human osteoblastic U2OS-TSHR cells. In U2OS-TSHR cells, TSH up-regulated pAKT1 (7.1±0.5-fold), p38α (2.9±0.4-fold), and pERK1/2 (3.1±0.2-fold), whereas small molecule TSHR agonist C2 had no or little effect on pAKT1 (1.8±0.08-fold), p38α (1.2±0.09-fold), and pERK1/2 (1.6±0.19-fold). Furthermore, TSH increased expression of osteoblast marker genes ALPL (8.2±4.6-fold), RANKL (21±5.9-fold), and osteopontin (OPN; 17±5.3-fold), whereas C2 had little effect (ALPL, 1.7±0.5-fold; RANKL, 1.3±0.6-fold; and OPN, 2.2±0.7-fold). β-Arrestin-1 and -2 can mediate activatory signals by 7TMRs. TSH stimulated translocation of β-arrestin-1 and -2 to TSHR, whereas C2 failed to translocate either β-arrestin. Down-regulation of β-arrestin-1 by siRNA inhibited TSH-stimulated phosphorylation of ERK1/2, p38α, and AKT1, whereas down-regulation of β-arrestin-2 increased phosphorylation of AKT1 in both cell types and of ERK1/2 in HEK-TSHR cells. Knockdown of β-arrestin-1 inhibited TSH-stimulated up-regulation of mRNAs for OPN by 87 ± 1.7% and RANKL by 73 ± 2.4%, and OPN secretion by 74 ± 10%. We conclude that TSH enhances osteoblast differentiation in U2OS cells that is, in part, caused by activatory signals mediated by β-arrestin-1.
Laboratory of Endocrinology and Receptor Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA.
Alisa
A
Boutin
Laboratory of Endocrinology and Receptor Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA.
Elena
E
Eliseeva
Laboratory of Endocrinology and Receptor Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA.
Marvin C
MC
Gershengorn
Laboratory of Endocrinology and Receptor Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA susannen@intra.niddk.nih.gov.
Susanne
S
Neumann
D019390:Arrestins / Q000502:physiology*; D001859:Bone Neoplasms / Q000473:pathology; D045744:Cell Line, Tumor; D015972:Gene Expression Regulation, Neoplastic; D057809:HEK293 Cells; D006801:Humans; D009363:Neoplasm Proteins / Q000502:physiology; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects*; D053495:Osteopontin / Q000378:metabolism; D012516:Osteosarcoma / Q000473:pathology; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000494:pharmacology; D011989:Receptors, Thyrotropin / Q000502:physiology; D011993:Recombinant Fusion Proteins; D015398:Signal Transduction / Q000502:physiology; D013972:Thyrotropin / Q000494:pharmacology / Q000502:physiology*; D000071559:beta-Arrestin 1; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins
C000605253:ARRB1 protein, human; C000605254:ARRB2 protein, human; D019390:Arrestins; D009363:Neoplasm Proteins; D034741:RNA, Small Interfering; D011989:Receptors, Thyrotropin; D011993:Recombinant Fusion Proteins; D000071559:beta-Arrestin 1; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins; D053495:Osteopontin; D013972:Thyrotropin; D011494:Protein Kinases
TSH receptor; bone; osteopontin; phosphokinases; signaling