Neurotransmitter action in osteoblasts: expression of a functional system for serotonin receptor activation and reuptake.
Neurotransmitter regulation of bone metabolism has been the subject of increasing interest and investigation. Because serotonin (5-HT) plays a role as a regulator of craniofacial morphogenesis, we investigated the expression and function of 5-HT receptors and the 5-HT transporter (5-HTT) in bone. Primary cultures of rat osteoblasts (rOB) and a variety of clonal osteoblastic cell lines, including ROS 17/2.8, UMR 106-H5, and Py1a, showed mRNA expression for 5-HTT as well as the 5-HT(1A), 5-HT(1D), 5-HT(2A), and 5-HT(2B) receptors by reverse transcription-polymerase chain reaction (RT-PCR) analysis. Protein expression of the 5-HT(1A), 5-HT(2A), and 5-HT(2B) receptors was confirmed by immunoblot. 5-HTT binding sites were assessed in ROS 17/2.8 and UMR 106-H5 cells by binding of the stable cocaine analog [125I]RTI-55, which showed a relatively high density of nanomolar affinity binding sites. Imipramine and fluoxetine, antagonists with specificity for 5-HTT, showed the highest potency to antagonize [125I]RTI-55 binding in ROS and UMR cells. GBR-12935, a relatively selective dopamine transporter antagonist, had a much lower potency, as did desipramine, a selective norepinephrine transporter antagonist. The maximal [3H]5-HT uptake rate in ROS cells was 110 pmol/10 min per well, with a K(m) value of 1.13 micromol/L. Imipramine and fluoxetine inhibited specific [3H]5-HT uptake with IC(50) values in the nanomolar range. In normal differentiating rOB cultures, 5-HTT functional activity was observed initially at day 25, and activity increased almost eightfold by day 31. In mature rOB cultures, the estimated density of [125I]RTI-55 binding sites was 600 fmol/mg protein. Functional downregulation of transporter activity was assessed after PMA treatment, which caused a significant 40% reduction in the maximal uptake rate of [3H]5-HT, an effect that was prevented by pretreatment with staurosporine. The affinity of 5-HT for the transporter was significantly increased following PMA treatment. We assessed the functional significance of expression of the 5-HT receptors by investigating the interaction between 5-HT and parathyroid hormone (PTH) signaling. 5-HT potentiates the PTH-induced increase in AP-1 activity in UMR cells. These results demonstrate that osteoblastic cells express a functional serotonin system, with mechanisms for responding to and regulating uptake of 5-HT.
Bliziotes
M M
MM
Portland VA Medical Center, Portland, Oregon 97201, USA. bliziote@ohsu.edu
Eshleman
A J
AJ
Zhang
X W
XW
Wiren
K M
KM
D000818:Animals; D002273:Carcinogens / Q000494:pharmacology; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects; D015870:Gene Expression / Q000502:physiology; D007457:Iodine Radioisotopes; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000378:metabolism; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism*; D012516:Osteosarcoma; D010281:Parathyroid Hormone / Q000502:physiology; D012333:RNA, Messenger / Q000032:analysis; D011869:Radioligand Assay; D051381:Rats; D044346:Receptor, Serotonin, 5-HT1D; D044402:Receptor, Serotonin, 5-HT2A; D044403:Receptor, Serotonin, 5-HT2B; D011985:Receptors, Serotonin / Q000235:genetics* / Q000378:metabolism; D044263:Receptors, Serotonin, 5-HT1; D012701:Serotonin / Q000493:pharmacokinetics*; D050486:Serotonin Plasma Membrane Transport Proteins; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014316:Tritium; D014407:Tumor Cells, Cultured
D002273:Carcinogens; D002352:Carrier Proteins; D007457:Iodine Radioisotopes; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D010281:Parathyroid Hormone; D012333:RNA, Messenger; D044346:Receptor, Serotonin, 5-HT1D; D044402:Receptor, Serotonin, 5-HT2A; D044403:Receptor, Serotonin, 5-HT2B; D011985:Receptors, Serotonin; D044263:Receptors, Serotonin, 5-HT1; D050486:Serotonin Plasma Membrane Transport Proteins; C493232:Slc6a4 protein, rat; D014316:Tritium; D012701:Serotonin; D013755:Tetradecanoylphorbol Acetate