Osteoblast regulation via ligand-activated nuclear trafficking of the oxytocin receptor.
We report that oxytocin (Oxt) receptors (Oxtrs), on stimulation by the ligand Oxt, translocate into the nucleus of osteoblasts, implicating this process in the action of Oxt on osteoblast maturation. Sequential immunocytochemistry of intact cells or isolated nucleoplasts stripped of the outer nuclear membrane showed progressive nuclear localization of the Oxtr; this nuclear translocation was confirmed by monitoring the movement of Oxtr-EGFP as well as by immunogold labeling. Nuclear Oxtr localization was conclusively shown by Western immunoblotting and MS of nuclear lysate proteins. We found that the passage of Oxtrs into the nucleus was facilitated by successive interactions with β-arrestins (Arrbs), the small GTPase Rab5, importin-β (Kpnb1), and transportin-1 (Tnpo1). siRNA-mediated knockdown of Arrb1, Arrb2, or Tnpo1 abrogated Oxt-induced expression of the osteoblast differentiation genes osterix (Sp7), Atf4, bone sialoprotein (Ibsp), and osteocalcin (Bglap) without affecting Erk phosphorylation. Likewise and again, without affecting pErk, inhibiting Arrb recruitment by mutating Ser rich clusters of the nuclear localization signal to Ala abolished nuclear import and Oxtr-induced gene expression. These studies define a previously unidentified mechanism for Oxtr action on bone and open possibilities for direct transcriptional modulation by nuclear G protein-coupled receptors.
Department of Basic Medical Science, Neurosciences and Sensory Organs, University of Bari Aldo Moro Medical School, Bari 70126, Italy; Department of Clinical and Experimental Medicine, University of Foggia, Foggia 71122, Italy;
Adriana
A
Di Benedetto
Mount Sinai Bone Program and Department of Medicine, Mount Sinai School of Medicine, New York, NY 10029;
Li
L
Sun
Department of Chemistry, University of Bari Aldo Moro, Bari 70126, Italy; and.
Carlo G
CG
Zambonin
Department of Basic Medical Science, Neurosciences and Sensory Organs, University of Bari Aldo Moro Medical School, Bari 70126, Italy;
Roberto
R
Tamma
Department of Basic Medical Science, Neurosciences and Sensory Organs, University of Bari Aldo Moro Medical School, Bari 70126, Italy;
Beatrice
B
Nico
Department of Chemistry, University of Bari Aldo Moro, Bari 70126, Italy; and.
Cosima D
CD
Calvano
Department of Basic Medical Science, Neurosciences and Sensory Organs, University of Bari Aldo Moro Medical School, Bari 70126, Italy;
Graziana
G
Colaianni
Mount Sinai Bone Program and Department of Medicine, Mount Sinai School of Medicine, New York, NY 10029;
Yaoting
Y
Ji
Department of Clinical and Experimental Medicine, University of Foggia, Foggia 71122, Italy;
Giorgio
G
Mori
Department of Basic Medical Science, Neurosciences and Sensory Organs, University of Bari Aldo Moro Medical School, Bari 70126, Italy;
Maria
M
Grano
Mount Sinai Bone Program and Department of Medicine, Mount Sinai School of Medicine, New York, NY 10029;
Ping
P
Lu
Department of Basic Medical Science, Neurosciences and Sensory Organs, University of Bari Aldo Moro Medical School, Bari 70126, Italy;
Silvia
S
Colucci
Mount Sinai Bone Program and Department of Medicine, Mount Sinai School of Medicine, New York, NY 10029;
Tony
T
Yuen
Departments of Pediatrics and maria.new@mssm.edu mone.zaidi@mountsinai.org.
Maria I
MI
New
Department of Basic Medical Science, Neurosciences and Sensory Organs, University of Bari Aldo Moro Medical School, Bari 70126, Italy;
Alberta
A
Zallone
Mount Sinai Bone Program and Department of Medicine, Mount Sinai School of Medicine, New York, NY 10029; Structural and Chemical Biology, Mount Sinai School of Medicine, New York, NY 10029 maria.new@mssm.edu mone.zaidi@mountsinai.org.
Mone
M
Zaidi
D021581:Active Transport, Cell Nucleus / Q000502:physiology*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D019390:Arrestins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005786:Gene Expression Regulation / Q000502:physiology; D008024:Ligands; D020935:MAP Kinase Signaling System; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D009685:Nuclear Envelope / Q000378:metabolism*; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D010006:Osteoblasts / Q000378:metabolism*; D010012:Osteogenesis / Q000235:genetics / Q000502:physiology*; D010121:Oxytocin / Q000502:physiology*; D010766:Phosphorylation; D017354:Point Mutation; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D034741:RNA, Small Interfering / Q000494:pharmacology; D018045:Receptors, Oxytocin / Q000737:chemistry / Q000172:deficiency / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012694:Serine / Q000737:chemistry; D028961:beta Karyopherins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D000071559:beta-Arrestin 1; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins; D020696:rab5 GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism
C000605255:Arrb1 protein, mouse; C000605258:Arrb2 protein, mouse; D019390:Arrestins; C094999:Kpnb1 protein, mouse; D008024:Ligands; D008565:Membrane Proteins; D009687:Nuclear Proteins; C576074:OXTR protein, mouse; D034741:RNA, Small Interfering; D018045:Receptors, Oxytocin; D011993:Recombinant Fusion Proteins; C000597150:Tnpo1 protein, mouse; D028961:beta Karyopherins; D000071559:beta-Arrestin 1; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins; D012694:Serine; D010121:Oxytocin; D048049:Extracellular Signal-Regulated MAP Kinases; D020696:rab5 GTP-Binding Proteins
G protein-coupled receptor; nuclear translocation; transcriptional regulation