Neurotransmitter action in osteoblasts: expression of a functional system for serotonin receptor activation and reuptake

Bone Vol. 29, No. 5 November 2001:477– 486

Neurotransmitter Action in Osteoblasts: Expression of a Functional System for Serotonin Receptor Activation and Reuptake M. M. BLIZIOTES,1,2 A. J. ESHLEMAN,1,2 X.-W. ZHANG,2 AND K. M. WIREN1,2 1 2

Portland VA Medical Center, Portland, Oregon, USA Oregon Health and Science University, Portland, Oregon, USA

tentiates 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. (Bone 29:477– 486; 2001) © 2001 by Elsevier Science Inc. All rights reserved.

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-HT1A, 5-HT1D, 5-HT2A, and 5-HT2B receptors by reverse transcription-polymerase chain reaction (RTPCR) analysis. Protein expression of the 5-HT1A, 5-HT2A, and 5-HT2B 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. GBR12935, a relatively selective dopamine transporter antagonist, had a much lower potency, as did desipramine, a selective norepinephrine transporter antagonist. The maximal [3H]5HT uptake rate in ROS cells was 110 pmol/10 min per well, with a Km value of 1.13 ␮mol/L. Imipramine and fluoxetine inhibited specific [3H]5-HT uptake with IC50 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 po-

Key Words: Bone; Osteoblast; Neurotransmitter; Serotonin; Receptor; Transporter. Introduction Neurotransmitter regulation of bone metabolism has been a topic of increasing clinical interest and investigation. Collectively, anatomical and in vitro studies suggest that bone metabolism may be influenced by the nervous system; for example, bone and periosteum have been shown to be innervated by both sympathetic and sensory nerves.10,11,18,28,32,36 –38,47,54 Anatomical studies of nerve terminals innervating bone have revealed the presence of several neuropeptides, including calcitonin generelated peptide (CGRP), vasoactive intestinal peptide, substance P, and neuropeptide Y27; glutamate-containing terminals have also been described in a dense and intimate network in bone tissue.56 Fann et al. demonstrated that bone morphogenetic proteins (BMP-2 and BMP-6) induce mRNAs for some neuropeptide and neurotransmitter synthetic enzymes in vitro.23 Vasoactive intestinal polypeptide (VIP) was shown to stimulate prostaglandin (PGE2) and cyclic AMP production in human osteoblast-like cells.50 These immunohistochemical and biochemical studies of nervous system components in bone may reflect not only sensory and vascular regulatory functions for neurotransmitters, but possibly also neurohormonal control of bone cell activities. In support of these findings, there have been reports of the effects of neurotransmitter transporter expression/deletion on bone function. We have recently explored the effects of deletion of the dopamine transporter (DAT) in mice.12 We have further demonstrated that DAT⫺/⫺ mice have reduced cancellous bone mass in vertebrae and proximal tibia. DAT⫺/⫺ animals also have a shorter femur length as well as reduced cortical thickness and bone area in the femoral diaphysis. The ultimate bending load (femoral strength) for DAT⫺/⫺ mice was found to be 30% lower than in wild-type mice. Thus, disruption of the DAT gene results in deficiencies in skeletal structure and integrity. DAT is a member of a highly homologous family of neurotransmitter transporters for bioactive amines, which includes the serotonin transporter. These transporters allow intracellular ac-

Address for correspondence and reprints: Dr. M. M. Bliziotes, Department of Veterans Affairs Medical Center (P3-ENDO), 3710 SW Veterans Hospital Road, Portland, OR 97201. E-mail: [email protected] Portions of this investigation were presented at the 21st and 22nd annual meetings of the American Society for Bone and Mineral Research, St. Louis, Missouri, USA, 1999 (Abstract SU191) and Toronto, Ontario, Canada, 2000 (Abstract SU213), respectively. © 2001 by Elsevier Science Inc. All rights reserved.

477

8756-3282/01/$20.00 PII S8756-3282(01)00593-2

478

M. M. Bliziotes et al. Serotonin receptors and reuptake in osteoblasts

cumulation of neurotransmitters by reuptake from the extracellular fluid through a sodium/chloride-dependent cotransport process (for review, see Nelson44). Presynaptic transporters that reduce neurotransmitter concentrations in the synapse are a major mechanism for terminating synaptic transmission.13 It is the augmentation of synaptic activity, by inhibition of sodiumdependent monoamine transport, which forms the basis for the mechanism of action of important antidepressant drugs. Neurotransmitter transporters are also expressed in nonneural tissue, including kidney, liver, muscle, and intestine, where they are thought to play a role in cellular signaling, metabolism, and organ function (e.g., see Hediger and Welbourne26). In osteoblast and osteocyte cells, expression and regulation of the excitatory amino acid glutamate/aspartate transporter by mechanical loading has also been described.36 Serotonin (5-HT) has been demonstrated to play a role as a regulator of craniofacial morphogenesis, which may in part be mediated by the 5-HT transporter (5-HTT). In particular, 5-HT has been shown to influence development of craniofacial mesenchyme.33 5-HTT has been localized in developing craniofacial mesenchyme of the mouse55 where it may influence the morphogenic effects of 5-HT by transporting the neurotransmitter toward epithelial uptake sites. Whole-embryo culture studies have demonstrated that craniofacial malformations may result from 5-HT uptake inhibitors,57 as well as 5-HT agonists and antagonists.34 In this study, we investigate expression of both transporter and receptors for the neurotransmitter 5-HT in osteoblastic cells. We report that cultured osteoblastic cell lines and normal differentiating rat osteoblasts express a variety of 5-HT receptors. Furthermore, 5-HTT is shown to be expressed in all osteoblastic cell lines examined. 5-HTT activity is downregulated by PMA treatment in osteoblastic cells. Finally, 5-HT potentiates PTH regulation of AP-1 activity in UMR 106-H5 cells. It is concluded that osteoblasts possess a functional system for both responding to and regulating 5-HT activity. Materials and Methods Cell Culture Media, buffers, supplements, and reagents for cell culture were obtained from Gibco BRL Life Technologies (Grand Island, NY) and Sigma Chemical Co. (St. Louis, MO). The rat osteoblastic osteosarcoma UMR 106-H5 cell line (passage 20) was cultured in minimum essential medium (MEM) containing 5% bovine calf serum (BCS) with antibiotics at 37°C in 5% CO2. The clonal Py1a cell line was derived from collagenase-digested primary rat osteoblast cells immortalized by viral infection with polyoma virus with a deficient middle T antigen necessary for transformation.22,48 The cultures synthesize type I collagen and alkaline phosphatase; respond to cortisol, insulin-like growth factor 1, and vitamin D; and progress through a differentiation paradigm, similar to that of the normal osteoblastic cells (data not shown). Primary cultures of rat osteoblasts were prepared using collagenase digestion of fetal rat calvariae, as we have previously described,8,9 modified from the original protocol developed by Lian and Stein.4 Twenty-one-day-old fetal rats (Harlan SpragueDawley, Indianapolis, IN) were killed using methods consistent with the panel on euthanasia of the American Veterinary Medical Association. Calvariae were removed and the overlying soft tissue was stripped away. Calvariae were then digested with 1 mg/mL collagenase solution (collagenase I and II; Worthington Biochemical Corp., Freehold, NJ) and 0.5 mg/mL of dispase/ protease (Sigma) for the first 30 min interval. The initial digestion products were discarded. The calvariae were then incubated

Bone Vol. 29, No. 5 November 2001:477– 486

with digestion solution for three 30 min digestions, and the pooled cells obtained were plated at 8000 cells/cm2. All cultures were maintained in a humidified 5% CO2 environment at 37°C. Cells were grown in MEM with 10% BCS for the first week. Beginning at day 7, when the cultures reached confluence, 50 ␮g/mL ascorbic acid was added in BGJb media for appropriate matrix deposition. From day 14 on, the cells were maintained with 3 mmol/L ␤-glycerol phosphate (for appropriate mineralization) and 50 ␮g/mL ascorbic acid in 10% fetal bovine serum (FBS)/BGJb media. In a previous study we characterized both the heterogeneity and the differentiation of primary rat osteoblast cultures.9 Histochemical staining of the cultures on day 22 indicated that more than 80%–90% of the cells were positive for the osteoblast marker alkaline phosphatase. Differentiation of the cultures is reflected both by differential gene expression and changes in alkaline phosphatase activity, and can be characterized in three distinct stages: proliferation (to days 10 –12); matrix maturation (to days 20 –23); and mineralization (to day 30). Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Total RNA was isolated by the single-step acid guanidinium isothiocyanate-phenol-choloroform extraction method.17 Fulllength cDNA was synthesized using RNaseH-reverse transcriptase (Superscript II RT, Life Technologies, Rockville, MD) and an oligo(dT)12-18 primer (Life Technologies) with 4 ␮g total RNA. PCR primers for 5-HTT (5-HTT), 5-HT1A, 5-HT1D, 5-HT2A, and 5-HT2B receptors and for the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were designed using OLIGO software (National Biosciences, Plymouth, MN). GAPDH expression was used as an internal control to correct for differences in RNA loading and efficiency of amplification. The fragments generated by RT-PCR using these primers were subcloned into pGEM-T Easy Vector (Promega), grown in DH10B competent cells (Life Technologies), and subsequently sequenced using the ABI-377 dye terminator cyclesequencing system. The initial primers used for the 5-HT1-class receptors were selected from a region of high homology between six different members of this class (A, B, C, D, E, and F). The primers used were: forward, 5⬘-GCCAACTATCTVATCGGCTCCTT-3⬘; reverse, 5⬘-TAGAARGCBCCVAAVGTGGAGTA-3⬘, with R ⫽ A/G, V ⫽ A/C/G, and B ⫽ C/G/T. RT-PCR using these primers gave an expected fragment of 404 bp. This fragment was subcloned and clones were sequenced. Comparisons of the sequences obtained using a BLAST search indicated similarity with sequences from 5-HT1A and 5-HT1D receptors, respectively. RT-PCR was then carried out with specific 5-HT1A and 5-HT1D sequences, as well as sequences for 5-HT2A and 5-HT2B receptors (Table 1). Primers were selected based on their melting temperatures, and did not show significant homology to nontarget sequences in the BLAST database. Amplification was performed with 2.5 U of Taq-DNA polymerase (Life Technologies) and 1/20th volume of the reverse transcriptase reaction in a final volume of 50 ␮L. PCR was carried out for 30 cycles in a Perkin-Elmer 9600 thermocycler. Each cycle consisted of denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec, and extension at 72°C for 30 sec. A final extension step was carried out at 72°C for 10 min. Parallel reactions were performed with GAPDH primers using 25 cycles while still in the linear range for amplification.31 Negative controls with no RT product were routinely performed (data not shown). The RT-PCR products were analyzed by electrophoresis on a 1% agarose gel and visualized by staining with ethidium bromide. The identity of all RT-PCR products was confirmed by sequence analysis.

Bone Vol. 29, No. 5 November 2001:477– 486

M. M. Bliziotes et al. Serotonin receptors and reuptake in osteoblasts

479

Table 1. Primers used for reverse transcription-polymerase chain reaction (RT-PCR)

Primer

Sequence

Position

Basepairs

Accession no. (GeneBank)

r5-HTT r5-HTT

FW RV

5⬘-TGGCGGTTTCCTTACATATGC-3⬘ 5⬘-GATTACGGTGAAGATGAGCAC-3⬘

422–442 893–913

492

X63253

r5-HT1A r5-HT1A

FW RV

5⬘-CAGGTGCTCAACAAGTGGACCCT-3⬘ 5⬘-GGCTGTCCGTTCAGGCTCTTCTT-3⬘

411–433 873–895

485

J05276

r5-HT1D r5-HT1D

FW RV

5⬘-ACCACCACCCGTACCTGGAACTT-3⬘ 5⬘-AGAGGGAGGGTGGGTTCAGGATT-3⬘

800–822 1186–1208

409

M89953

r5-HT2A r5-HT2A

FW RV

5⬘-TATGCTGCTGGGTTTCCTTGTC-3⬘ 5⬘-CACAAAAGAGCCTATGAGAACA-3⬘

419–440 761–782

364

M30705

r5-HT2B r5-HT2B

FW RV

5⬘-ATGTTTGAGGCTACATGGCCC-3⬘ 5⬘-ACTGCCAAAGCGGTCCTTTGT-3⬘

569–589 851–871

303

X66842

rGAPDH FW rGAPDH RV

5⬘-CGGCAAGTTCAATGGCACAGT-3⬘ 5⬘-TCATACTTGGCAGGTTTCTCC-3⬘

183–203 771–791

609

M17701

Immunoblot Protocol For assessment of 5-HT receptor protein expression, whole cell extracts in lysis buffer (137 mmol/L NaCl, 20 mmol/L Tris-HCl [pH 8.0], 2 mmol/L ethylene-diamine tetraacetic acid [EDTA], 10 mmol/L NaF, 1% NP-40, 10% glycerol, 1 mmol/L PMSF, 1 ␮g/mL leupeptin, 1 mmol/L Na3VO4) were pelleted by centrifugation (400g), and 25 ␮g of supernatant protein was subjected to sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 10% gels. Proteins were transferred to nitrocellulose membranes (5-HT1A and 5-HT2B) or PVDF membranes (5-HT2A), and immunoblots were performed using goat polyclonal antibodies against the 5-HT1A receptor (Santa Cruz Biotechnology, Santa Cruz, CA) and mouse monoclonal antibodies against the 5-HT2A or 5-HT2B receptors (BD PharMingen, San Diego, CA). Secondary antibodies were either donkey anti-goat (Santa Cruz Biotechnology) for the 5-HT1A receptor or donkey anti-mouse (Amersham) for the 5-HT2A and 5-HT2B receptors. Blots were developed with the ECL (5-HT1A and 5-HT2B) or ECF (5-HT2A) western blotting detection reagent from Amersham (Buckinghamshire, UK). 5-HTT Binding Assays ROS 17/2.8 cells were grown to confluence in 150-mm-diameter tissue culture dishes in a humidified 5% CO2 environment at 37°C. The medium was then removed from the plates, cells were washed with 10 mL of phosphate-buffered saline, 10 mL lysis buffer (2 mmol/L HEPES, 1 mmol/L EDTA [pH 7.4]) was added, and plates were placed on ice for 10 min. Cells were scraped from plates and centrifuged for 20 min at 30,000g. The pellet was resuspended in 5 mL of 0.32 mol/L sucrose with a Polytron homogenizer at setting 7 for 5 sec. Assays contained an aliquot of membrane preparation (approximately 30 ␮g protein, which resulted in binding ⬍10% of the total radioactivity), drug, and [125I]RTI-55 (60 –70 pmol/L final concentration) in a final volume of 250 ␮L. Krebs-HEPES assay buffer (25 mmol/L HEPES, 122 mmol/L NaCl, 5 mmol/L KCl, 1.2 mmol/L MgSO4, 2.5 mmol/L CaCl2, 1 ␮mol/L pargyline, 100 ␮mol/L tropolone, 2 mg glucose/mL, 0.2 mg ascorbic acid/mL [pH 7.4]) was used for all assays. Specific binding was defined as the difference in binding observed in the absence or presence of 5 ␮mol/L imipramine. Membranes were preincubated with drugs at room tem-

perature for 10 min prior to addition of [125I]RTI-55, unless indicated otherwise. The reaction was incubated for 90 min at room temperature in the dark and was terminated by filtration through Wallac Filtermat A filters using a 96-well Tomtech cell harvester. Scintillation fluid (50 ␮L) was added to each filtered spot and radioactivity remaining on the filter was determined using a Wallac 1205 Betaplate or 1405 MicroBeta scintillation counter. Competition experiments were conducted with duplicate determinations for each point. Saturation binding experiments were conducted in duplicate by diluting the specific activity of [125I]RTI-55 with unlabeled ligand having a concentration of 0.01–20 nmol/L. [3H]5-HT Uptake Assays ROS 17/2.8 cells exhibited robust [3H]5-HT uptake when attached to cell culture plates; however, following removal from the plates, no specific uptake was measurable using filtration methodology. We therefore performed uptake experiments with attached cells. ROS 17/2.8 and UMR 106-H5 cells were grown on 24 well tissue culture plates until confluent. Medium was removed, and Krebs HEPES buffer (400 ␮L) and drugs or vehicle were added to each well. The plates were placed in a 30°C water bath for 10 min. Uptake was initiated by the addition of [3H]5-HT (20 nmol/L final concentration), and incubations were carried out for 10 min with the ROS 17/2.8 cells, and for 15 min with the UMR 106-H5 cells. The assay was terminated by pouring off the reaction mixture and washing twice with ice-cold phosphate-buffered saline. Trichloroacetic acid (3%, 0.5 mL) was added to each well, and transferred to scintillation vials after 15 min. Scintillation fluid was added to each vial, and radioactivity was counted on a Beckman LS 3801 scintillation counter. Each assay was conducted in triplicate, at least three times. For determination of Vmax and Km values for [3H]5-HT uptake, the specific activity of [3H]5-HT was diluted by addition of 5-HT at concentrations ranging from 0.03 to 10 ␮mol/L. Transient Transfections Twenty-four hours prior to transfection, confluent UMR 106-H5 cells were split in MEM medium containing 5% BCS. The cells were then harvested by trypsinization, counted, and diluted to 9700 cells/␮L. Approximately 5.4 ⫻ 106 cells were mixed with

480

M. M. Bliziotes et al. Serotonin receptors and reuptake in osteoblasts

Bone Vol. 29, No. 5 November 2001:477– 486

20 ␮g of pGCAT B ⫻ 4 DNA (containing four tandem repeats of an Ap-1 consensus sequence linked to the CAT coding sequence [a gift from Dr. B. Waslyk, Strasbourg, France]) and 20 ␮g of the ␤-galactosidase expression plasmid pSV-␤-galactosidase (Promega, Madison, WI). The AP-1 sequence is derived from the murine polyomavirus PEA1 site.58 Cells were exposed to a controlled electrical field of 960 microfarads at 230 V in a Bio-Rad Gene Pulser with capacitance extender (Bio-Rad Laboratories, Richmond, CA). Each electroporation was diluted in 24 mL of serum-containing media and divided into twelve 35 mm dishes, used in different experimental conditions. Cells were incubated in normal media for 20 h, washed for 3 min on three occasions with supplemented serum-free media (SSFM; containing 0.6 mmol/L ascorbic acid), and treated for 6 h with either 1 ␮mol/L serotonin or vehicle (10 ␮mol/L pargyline) in SSFM. Then, 100 ng/mL of parathyroid hormone (PTH) or vehicle was added 1 h before the extracts were harvested for chloramphenicol acetyltransferase (CAT), ␤-galactosidase, and protein determinations. CAT Assays CAT activity was determined by the fluor-diffusion method. [3H]acetyl coenzyme A (200 mCi/mmol, CAT assay grade) was purchased from DuPont NEN (Boston, MA). Cultures were lysed in 300 ␮L of reporter lysis buffer (Promega, Madison, WI). CAT activity was measured in 50 ␮L of cell extract after inactivation of endogenous acetylases by incubation at 65°C for 15 min. The extract was mixed with 200 ␮L of chloramphenicol at 0.4 mg/mL and [3H]acetyl-coenzyme A at 2.5 ␮Ci/mL in Tris-HCl (pH 7.8). The reaction mixture was overlaid with 1 mL of organic scintillation fluid (Econofluor, NEN) and counted repeatedly over a 2–3 h period in a liquid scintillation counter. CAT activity was determined from the linear portion of the slope. The ␤-galactosidase (␤-gal) activity was determined colorimetrically by using 150 ␮L of cell extract. The assay buffer contained 60 mmol/L sodium phosphate (pH 7.5), 1 mmol/L MgCl2, 0.67 mg/mL o-nitrophenyl-␤-D-galactopyranoside, and 40 mmol/L ␤-mercaptoethanol. A standard curve containing 2–100 ␮U of ␤-galactosidase was run with each assay. Assay buffer was incubated with cell extract for 60 min at 37°C and the reaction was stopped by the addition of Na2CO3 to a final concentration of 625 mmol/L. Absorbance readings were taken at 420 nm. There was no endogenous ␤-gal activity in any cell line analyzed. Protein concentrations were determined in the samples by the BCA protein assay (Pierce, Rockford, IL). All CAT activity determinations were normalized to ␤-gal activity to correct for differences in transfection efficiency. The data shown represent the mean ⫾ SEM from triplicate samples generally, and were performed in independent transfections three times. Values obtained for CAT activity were corrected to values for ␤-gal activity expressed as counts per million (cpm) per minute per milliunit ␤-gal, and then normalized to control values. Data Analysis PRISM software, version 3.0 (GraphPad Software, San Diego, CA), was used to analyze all saturation and competition binding data as well as for statistical analyses. IC50 values were converted to Ki values using the Cheng–Prusoff equation.15 PMA treatment data were analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s post hoc analysis. Significance was set at p ⬍ 0.05.

Figure 1. 5-HTT and 5-HT receptor mRNA expression in osteoblastic cells by RT-PCR analyses. (A) 5-HTT mRNA expression in confluent normal rat osteoblastic (rOB) cultures, and clonal osteoblastic cells Py1a, ROS 17/2.8, and UMR 106-H5. Mouse brain (B), rat liver (Li), and rat lung (Lu) were included as positive (brain and lung) and negative (liver) controls. All samples were analyzed in duplicate. The predicted size for the 5-HTT is 492 bp. (B) Characterization of 5-HT receptor mRNA expression in osteoblastic cells. RT-PCR was performed as described in Materials and Methods. The PCR primers employed in these studies are shown in Table 1. Products were electrophoresed on 1% agarose gels and visualized by staining with ethidium bromide. The sizes (in basepairs) of the specific amplification products are shown to the right of the gels.

Results A variety of osteoblastic cells were analyzed for mRNA expression of 5-HTT and 5-HT receptors using RT-PCR. The primers used for the 5-HTT and 5-HT receptors 5-HT1A, 5-HT1D, 5-HT2A, and 5-HT2B are shown in Table 1. All primers were chosen to amplify regions specific for the various proteins. 5-HTT mRNA was expressed in all osteoblastic cell lines examined, including ROS 17/2.8, UMR 106-01 osteoblastic osteosarcoma lines, immortalized clonal Py1a osteoblastic cells, and normal rOB cells, at day 22 (Figure 1A, 5-HTT band). RT-PCR generated the expected 492 bp fragment; the PCR-amplified fragment was sequenced and confirmed to be authentic rat 5-HTT. Expression in nonossified tissues was seen in controls (brain, lung, and liver as described). We also investigated the expression of 5-HT receptor mRNAs by RT-PCR (Figure 1B). The initial primers used for the 5-HT1class receptors were selected from a region of high homology between six different members of this class (A, B, C, D, E, and

Bone Vol. 29, No. 5 November 2001:477– 486

M. M. Bliziotes et al. Serotonin receptors and reuptake in osteoblasts

481

Figure 2. Immunoblot analysis of 5-HT receptor expression in osteoblastic cells. Cell lysates (25 ␮g protein) from ROS 17/2.8, UMR 106-H5, and primary cultures of osteoblasts from neonatal rat calvariae (day 25 or 32 in culture) were subjected to SDS-PAGE on 10% gels. Immunoblots were carried out as described in Materials and Methods, using mouse monoclonal antibodies against the 5-HT2A receptor (A) and the 5-HT2B receptor (B), or goat polyclonal antibodies against the 5-HT1A receptor (C). The relative molecular mass of significant bands identified on the blots are indicated to the left of the gels. Lane 1: rat hippocampus; lane 2: ROS 17/2.8 cells; lane 3: UMR 106-H5 cells; lane 4: rat calvarial osteoblasts.

F). RT-PCR using these primers gave an expected fragment of 404 bp; subcloning of this fragment and sequencing of several clones revealed sequences consistent with either 5-HT1A or 5-HT1D receptors. Specific primers for 5-HT1A and 5-HT1D were then employed. As shown, 5-HT1A, 5-HT1D, 5-HT2A, and 5-HT2B receptor mRNAs were expressed in ROS cells. All amplified fragments were the expected size (indicated on the right of Figure 1B). The same receptors were also expressed in the UMR cells. We confirmed expression of all of the aforementioned 5-HT receptors in Py1a and confluent rOB cultures at day 22. All PCR fragments were confirmed to be identical to the published sequences for the respective proteins by sequence analysis. Western blot analysis confirmed protein expression of the 5-HT1A, 5-HT2A, and 5-HT2B receptors in the osteoblastic cells (Figure 2). For the 5-HT2A receptor, a monoclonal antibody raised against a fusion protein containing amino acids 1–72 of the human 5-HT2A receptor recognized a band of ⵒ61 kDa in rat hippocampus, ⵒ72 kDa in ROS and UMR cells, and ⵒ65 kDa in day 32 rOB cells (Figure 2A). This antibody has been reported to recognize bands of 53 and 58 kDa in rat brain.24 A monoclonal antibody against human 5-HT2B receptor recognized a doublet of 80 – 85 kDa in rat hippocampus, ROS, and UMR; a single band

of ⵒ85 kDa was seen in day 25 rOB cells (Figure 2B). A smaller band of 53 kDa was also seen in rat hippocampus, and additional bands ranging from 47 to 74 kDa were seen in ROS and UMR cells. An antibody against the aminoterminus of the rat receptor recognized a band of 52 kDa in rat brain.19 An antipeptide antibody against a carboxyterminal sequence of the human 5-HT1A receptor recognized a band of ⵒ65 kDa in extracts of rat hippocampus, ROS 17/2.8, and UMR 106-H5 cells (Figure 2C). No bands were detected in day 25 rOB cells. Varied results have been reported for the molecular weight of the 5-HT1A receptor. A polyclonal antibody directed against residues 170 –186 of the rat receptor recognized bands at 49.5, 42, and 39 kDa on immunoblots of crude hippocampal proteins,5 whereas another antibody against residues 258 –274 recognized bands of 40 and 70 kDa in hippocampal membranes.3 Binding and uptake studies to confirm functional expression of 5-HTT were first performed in the ROS 17/2.8 osteoblastic cell line. Antagonist and substrate affinities for the 5-HTT, as well as the density of 5-HTT, were evaluated using [125I]RTI-55 binding to membrane preparations. [125I]RTI-55 is a stable cocaine analog that has high affinity for 5-HT, dopamine, and norepinephrine transporters. ROS cells expressed a relatively high density of nanomolar affinity [125I]RTI-55 binding sites,

482

M. M. Bliziotes et al. Serotonin receptors and reuptake in osteoblasts

Bone Vol. 29, No. 5 November 2001:477– 486

Table 2. Binding and uptake of neurotransmitters to ROS 17/2.8, UMR 106-H5, and rOB (primary rat osteoblast) cellsa

Compound Cocaine Desmethyl imipramine (DMI) Dopamine Fluoxetine GBR-12935 Imipramine Mazindol Methylphenidate Norepinephrine Serotonin RTI-55

[125I]RTI-55 binding in ROS membranes Ki (nmol/L) ⫾ SEM 279 ⫾ 71 115 ⫾ 23 730 ⫾ 110 ␮mol/L 1.61 ⫾ 0.35 321 ⫾ 83 6.6 ⫾ 1.9 39 ⫾ 11 25,200 ⫾ 5800 3.4 ⫾ 1.0 mmol/L 128 ⫾ 18 0.97 ⫾ 0.36

[3H]5-HT uptake in ROS cells IC50 (nmol/L) ⫾ SEM 348 ⫾ 53 1120 ⫾ 250 442 ⫾ 47 ␮mol/L 58 ⫾ 16 4470 ⫾ 910 124 ⫾ 11 287 ⫾ 64 58,400 ⫾ 9100 1.14 ⫾ 0.09 mmol/L 1760 ⫾ 240 1.38 ⫾ 0.44

[125I]RTI-55 binding in UMR membranes Ki (nmol/L) ⫾ SEM 115 ⫾ 21 120 ⫾ 17 330 ⫾ 25 ␮mol/L 6.6 ⫾ 1.5 800 ⫾ 200 13.1 ⫾ 3.4 63.7 ⫾ 7.1 11,100 ⫾ 3800 591 ⫾ 54 ␮mol/L 221 ⫾ 31 0.25 ⫾ 0.05

[3H]5-HT uptake in UMR cells IC50 (nmol/L) ⫾ SEM 273 ⫾ 64 700 ⫾ 180 194 ⫾ 34 ␮mol/L 35 ⫾ 11 4300 ⫾ 1100 45 ⫾ 15 74 ⫾ 11 42,800 ⫾ 9300 570 ⫾ 130 ␮mol/L 610 ⫾ 110 0.56 ⫾ 0.13

[3H]5-HT uptake in rOB cells IC50 (nmol/L) ⫾ SEM 340 ⫾ 270

19.8 ⫾ 4.1 85 ⫾ 12

2210 ⫾ 960

a

With ROS and UMR cells, typical binding experiments had 1800 and 1100 specific and 80 and 50 nonspecific, cpm per assay, respectively, and typical uptake experiments had 20,000 and 13,000 specific and 800 and 1000 nonspecific, cpm per well, respectively. Kd values for [125I]RTI-55 of 3.16 nmol/L for ROS and 0.513 nmol/L for UMR were used in the Cheng–Prusoff correction for Ki values. All binding experiments were conducted in duplicate, all uptake experiments were conducted in triplicate (N ⫽ 3– 6). 5-HTT selective compounds have the following relative affinities for the DAT, NET, and 5-HTT: fluoxetine 5-HTT ⬎⬎ NET ⬎ DAT; imipramine 5-HTT ⬎⬎ NET ⬎⬎ DAT. NET selective compounds: DMI NET ⬎ 5-HTT ⬎⬎ DAT; mazindol NET ⬎ DAT ⬎ 5-HTT. DAT selective compounds: GBR 12935 DAT ⬎ 5-HTT ⫽ NET; methylphenidate DAT ⬎ NET ⬎ 5-HTT.

with a Bmax value of 715 ⫾ 96 fmol/mg protein and a Kd value of 3.16 ⫾ 0.54 nmol/L. To pharmacologically characterize the binding site labeled by [125I]RTI-55, competition assays were conducted using 11 different compounds (Table 2). Two antagonists with specificity for 5-HTT, imipramine, and fluoxetine, showed the highest potency in ROS cells with affinities of 6.6 and 1.6 nmol/L, respectively (column 1 of Table 2). In contrast, GBR-12935, a relatively selective dopamine transporter antagonist, had a much lower potency (321 nmol/L), as did desipramine, a selective norepinephrine transporter antagonist (115 nmol/L). As shown in Figure 3A, the potencies for all drugs tested compared favorably with values measured in HEK 293 cells heterologously expressing the human 5-HTT (also known as hSERT, see Eshleman et al.20). In contrast, the correlations with inhibitory potencies observed between ROS 17/2.8 cells and HEK 293 cells heterologously expressing either the human dopamine transporter or the human norepinephrine transporter were quite low (Spearman’s r ⫽ 0.19 and 0.65, respectively; data not shown). Thus, the pharmacological characterization of the 5-HT binding site is highly consistent with 5-HTT. To measure functional capacity of 5-HTT in osteoblastic cells, we performed uptake studies with [3H]5-HT in ROS 17/2.8 and UMR 106-H5 cultures. The specific uptake rate was linear with respect to time from 2 min through 30 min for the ROS cells, and through 20 min for UMR cells. At 10 min, nonspecific uptake was ⬍10% of specific uptake. The maximal [3H]5-HT uptake rate for ROS 17/2.8 cells was 110 ⫾ 19 pmol/10 min/ well, with a Km value of 1.13 ⫾ 0.16 ␮mol/L; for UMR 106-H5 cells, the maximal uptake was 10.0 ⫾ 2.2 pmol/15 min per well, and the Km was 480 ⫾ 110 nmol/L. Inhibition of specific [3H]5-HT uptake by imipramine and fluoxetine also demonstrated high affinity of these antagonists for 5-HTT in ROS and UMR cells, with IC50 values in the nanomolar range, again comparable to 5-HTT (hSERT)-expressing HEK cells (Figure 3B). The rank order of potency of neurotransmitters for inhibition of [3H]5-HT uptake was 5-HT ⬎ dopamine ⬎ norepinephrine. Combined, the pharmacological profile of these binding and uptake studies indicates that both ROS 17/2.8 and UMR 106-H5 cells express functional 5-HTT, confirming mRNA expression studies. We confirmed 5-HTT expression in normal differentiating rat calvarial osteoblasts (rOB) using [3H]5-HT uptake. 5-HTT ex-

pression was observed initially at day 25, and uptake increased by almost eightfold at day 31 (data not shown). IC50 values for inhibition of [3H]5-HT uptake at day 31 in rOB culture determined by imipramine and fluoxetine competition were quite comparable to values obtained for the ROS 17/2.8 cells (85 ⫾ 12 nmol/L and 19.8 ⫾ 4.1 nmol/L, respectively; see column 5 of Table 2). The uptake rate of 20 nmol/L [3H]5-HT was about 3% of that seen in the ROS cells (52 ⫾ 3 fmol/10 min per well with rOB cells compared with 1540 ⫾ 110 fmol/10 min per well with ROS cells). Binding sites were also determined in the rOB cultures at day 36 in highly differentiated osteocyte-like cells. The estimated density of [125I]RTI-55 binding sites in rOB cells at day 36 was 600 fmol/mg protein, slightly below that for confluent ROS cells. With either endogenously or heterologously expressed 5-HTT, 5-HT uptake can be downregulated in a time- and concentration-dependent manner by acute exposure to phorbol esters, which activate protein kinase C (PKC). Thus, PKC activators rapidly elevate the basal level of 5-HTT phosphorylation and downregulate 5-HTT activity.53 This decrease can be reversed by pretreatment with staurosporine, an inhibitor of protein kinases.2,43,52,53 To determine whether the 5-HTT in ROS 17/2.8 cells can be similarly regulated by the PKC pathway, cells were preincubated for 10 min with vehicle or staurosporine, followed by a 2 h incubation with the phorbol ester PMA (100 nmol/L) or vehicle (0.1% dimethysulfoxide [DMSO]). The incubation medium and drugs were removed and [3H]5-HT uptake was measured for 10 min at 30°C. As shown in Figure 4, incubation with PMA caused a significant 40% reduction in the maximal uptake rate (from 48 to 29 pmol/10 min per well; p ⬍ 0.001, using one-way ANOVA followed by Tukey’s post hoc analysis). The decrease was prevented by pretreatment with 1 ␮mol/L staurosporine (42 pmol/10 min per well, p ⬍ 0.05). The affinity of 5-HT for transporter increased following PMA treatment (Km values: control 980 ⫾ 150 nmol/L, PMA treatment 493 ⫾ 31 nmol/L; p ⬍ 0.05, one-way ANOVA followed by Tukey’s post hoc analysis). Pretreatment with staurosporine also partially prevented this decrease in Km value (Km ⫽ 900 ⫾ 160 nmol/L; p ⬍ 0.05 for staurosporine vs. PMA treatment). We next explored the functional correlates of 5-HT receptor expression in osteoblastic cells. 5-HT regulates immediate early gene responses in a number of cell types and tissues. In uterine

Bone Vol. 29, No. 5 November 2001:477– 486

M. M. Bliziotes et al. Serotonin receptors and reuptake in osteoblasts

483

Figure 4. Regulation of 5-HTT activity in osteoblastic cells by the protein kinase C (PKC) pathway. ROS 17/2.8 cultures were preincubated for 10 min with vehicle or staurosporine (ST), a protein kinase C inhibitor, followed by a 2 h incubation with control (0.1% DMSO), or the phorbol ester PMA (100 nmol/L), an activator of PKC (filled squares: controls; open squares: PMA; filled circles: ST ⫹ PMA). The incubation medium and drugs were removed, and [3H]5-HT uptake was measured for 10 min at 30°C as described in Materials and Methods. Data are the mean ⫾ SEM for triplicate samples, and each assay was conducted at least three times. The maximal uptake rate was 48 pmol/10 min per well; PMA treatment caused a 40% reduction to 29 pmol/10 min per well (p ⬍ 0.001). The decrease was prevented by pretreatment with 1 ␮mol/L staurosporine at 42 pmol/10 min per well (p ⬍ 0.05).

Discussion

Figure 3. Correlation of inhibitory potency of antagonists and substrates in ROS 17/2.8 cells and HEK-hSERT cells consistent with 5-HTT expression in bone cells. (A) Ki values for inhibition of [125I]RTI-55 binding. (B) IC50 values for inhibition of [3H]5-HT uptake. ROS cell data were taken from Table 2; HEK-hSERT cell data was reported in Eshleman et al.20 Spearman’s nonparametric correlational analysis was used. Conditions and analyses of the binding assays for the two cell lines were identical except for a slightly larger amount of membrane protein used in the ROS cell assays. The conditions for uptake were slightly different; that is, uptake assays with ROS cells were conducted with attached cells at 30°C, whereas uptake assays with HEK-hSERT cells were conducted with detached cells using a filtration assay at room temperature. Preincubation and incubation times were identical.

smooth muscle cells, 5-HT regulates interleukin-1␣ gene expression through AP-1 activation29; in rat cerebellar granule cells, a 5-HT2A receptor agonist (DOI) increases AP-1 and CRE transcription factor binding activity.14 We investigated the effects of 5-HT on AP-1 activity using a transient transfection assay with a CAT reporter gene fused to tandem AP-1 binding sequences in UMR 106-H5 osteoblastic cells. We also evaluated the effect of serotonin on AP-1 activity induced by PTH in the same model. As shown in Figure 5, 6 h of incubation with 5-HT at a concentration of 1 ␮mol/L produced a nonsignificant 1.7 ⫾ 0.1-fold increase (mean ⫾ SEM) in AP-1 activity in UMR 106-H5 cells. One hour of exposure to 100 ng/mL PTH produced a similar effect. However, preincubation for 5 h with 5-HT (1 ␮mol/L) followed by the addition for 1 h of 100 ng/mL PTH produced a significant 2.3-fold increase of AP-1 activity over control (p ⬍ 0.05, Kruskal–Wallis test with post hoc analysis by Dunn’s multiple comparison test). Thus, 5-HT potentiates the PTH-induced increase in AP-1 activity in osteoblastic cells.

In this study we have demonstrated that the 5-HTT and multiple 5-HT receptors are expressed in a variety of osteoblastic cells. This is the first report of expression of 5-HT receptors and the 5-HTT in bone. Serotonin thus joins other neurotransmitters that have been described as having receptors and/or transporters expressed in osseous tissue.21,36 We also demonstrated regulation of 5-HTT activity by PKC activation in osteoblastic cells. Neurotransmitter expression in nerve terminals penetrating densely into bone has been described.27,56 Therefore, locally

Figure 5. 5-HT potentiates PTH-induced AP-1 activity in UMR 106-H5 osteoblastic cells. UMR 106-H5 cells were transfected with pGCAT B ⫻ 4 DNA and the ␤-galactosidase expression plasmid pSV-␤-galactosidase. Twenty hours later, the cells were preincubated with 5-HT (1 ␮mol/L) or vehicle for 5 h, and then 100 ng/mL bPTH(1-34) or vehicle added for 1 h in serum-free medium. Extracts were then harvested for CAT, ␤-galactosidase, and protein determinations. All CAT activity determinations were normalized to ␤-gal activity to correct for differences in transfection efficiency. Data are mean ⫾ SEM from triplicate samples, and represent three independent transfections. The mean value for the control (5-HT vehicle/PTH vehicle) samples was 211 ⫾ 94 cpm/min per milliunit (n ⫽ 3). *p ⬍ 0.05 by Kruskal–Wallis test with Dunn’s multiple comparison test for post hoc analysis.

484

M. M. Bliziotes et al. Serotonin receptors and reuptake in osteoblasts

produced neurotransmitters may act as signaling molecules and have direct effects on bone cells. Combined, these results demonstrate that osteoblastic cells express both a mechanism for responding to and regulating uptake of 5-HT, and thus represents a functional serotonin system in bone. Previously, 5-HT has been demonstrated to play a role as a regulator of craniofacial morphogenesis. Early craniofacial morphogenesis involves intercellular interactions that regulate neural crest cell migration,45 mesenchymal growth,6 and differentiation.25 It has been suggested that 5-HT may regulate migration of cranial neural crest cells and their mesenchymal derivatives in the mouse embryo.41 Furthermore, 5-HTT has been localized in developing craniofacial mesenchyme of the mouse55 where it may mediate the morphogenic effects of 5-HT by transporting the neurotransmitter toward epithelial uptake sites. Shuey et al. found that inhibition of 5-HT uptake into craniofacial epithelia interferes with serotonergic regulation of epithelial-mesenchymal interactions, which are important for normal craniofacial morphogenesis.57 5-HT has also been shown to directly influence craniofacial mesenchyme.33 In mandibular mesenchyme cells, 5-HT has been found to promote expression of cartilage core protein by activation of 5-HT3 or 5-HT1A receptors, to inhibit production of tenascin (an extracellular matrix molecule produced by perichondrial and periosteal cells35), and to either promote or inhibit synthesis of the calcium binding protein S-100.40 These studies suggest that 5-HT may have profound effects on craniofacial development at a variety of levels. Our investigation also characterized expression of 5-HT system components in osteoblastic cells. Previously, 5-HTT had been localized to bone marrow.42 Herein, we have documented the expression of several 5-HT receptor types in a variety of clonal osteoblastic cell lines, and in normal primary osteoblast cultures. 5-HT receptors are linked to a variety of signal transduction pathways, including multiple G-protein-associated effector systems, phospholipase C activation, and Ca2⫹ release (for review, see Peroutka49). We did not, however, characterize the potential expression in osteoclastic cells. We noted some heterogeneity in the size of proteins recognized by the antibody raised against the 5-HT2B receptor (Figure 2B). Specifically, the osteoblastic osteosarcoma cell lines ROS 17/2.8 and UMR 106-H5 were shown to express an immunoreactive protein of ⵒ80 kDa, which has not been seen in rat hippocampus or primary rat calvarial osteoblasts. Isoforms of other 5-HT receptors have been described, which arise due to either alternative splicing or differences in glycosylation. For example, 5-HT3 receptor splice variants have been reported in the rat, and the expression of these isoforms is regulated in the central nervous system during development.39 A splice variant of the 5-HT4 receptor has been described that displays some differential responsivity to antagonists in comparison to other 5-HT4 isoforms.7 Both the 5-HT1A3 and the 5-HT2C receptors1 have isoforms that arise from different glycosylation patterns. We are not aware of any evidence for alternative splicing or differential glycosylation of 5-HT2B receptors, but an explanation of such findings remains an intriguing possibility. Future work in our lab will explore this issue. We have also described the expression of 5-HTT in all osteoblastic cells examined. In the differentiating primary cultures of fetal rat calvarial osteoblasts (rOB), the expression of 5-HTT mRNA was observed in confluent cultures. However, functional 5-HTT activity was not detected until relatively late in culture (day 25), during which cells are secreting osteocalcin and undergoing mineralization.46 This result suggests that transporter uptake of 5-HT may influence events late in the differentiation paradigm of the osteoblast. We further showed that 5-HTT activity is downregulated by treatment with PMA, an activator of

Bone Vol. 29, No. 5 November 2001:477– 486

the protein kinase C system. This posttranslational modulation of 5-HTT function via activation of second messenger pathways may be a mechanism for short-term functional regulation or plasticity of the serotonin uptake system in osteoblasts. We have begun to explore the functional correlates of 5-HT receptor and 5-HTT expression in osteoblastic cells. 5-HT regulates immediate early gene responses in a number of cell types and tissues. We have demonstrated that a 6 h incubation with 5-HT potentiates the PTH-induced increase in AP-1 activity in osteoblastic cells. This has physiological relevance in that PTH induces collagenase production in osteoblastic cells through a mechanism that involves an AP-1 consensus-binding sequence.51 Interestingly, the stimulation correlates with CREB binding activity and implicates phosphorylation of CREB as a possible mechanism for transcriptional activation of interstitial collagenase. This suggests a testable hypothesis for the mechanism whereby serotonin potentiates the PTH effect on AP-1 activity, and potentially on regulation of collagenase expression. Expression in bone cells of another neurotransmitter transporter was recently described.36 Messenger RNA and protein for the excitatory amino acid glutamate/aspartate transporter was found in osteoblasts and osteocytes. Regulation of the glutamate transporter protein was demonstrated by mechanical loading; that is, mechanical loading of rat ulna produced a downregulation of glutamate transporter in cortical bone, whereas upregulation of the protein occurred on the periosteal surface. These data suggest that the glutamate transporter may be involved in coupling mechanical loading to skeletal modeling; thus, the investigators proposed that regulation of glutamate transport may be an early response of osteocytes to mechanical loading of bone.36 Furthermore, mRNA expression for a range of glutamate receptor subtypes in various osteoblast cell lines and primary cultures and in osteoclasts16,47 has been described. In addition, functional glutamate receptors have been demonstrated in mammalian osteoclasts,21 and specific N-methyl-D-aspartate (NMDA) antagonists have been shown to prevent formation of the osteoclast sealing zone required for bone resorption.30 It has also been shown that glutamate-containing fibers, among others, are present as a dense and intimate network in bone tissue.56 Together, these studies suggest a functional system for glutamate in bone. We have previously demonstrated that disruption of the dopamine transporter (DAT) gene in mice results in deficiencies in skeletal structure and integrity by mechanisms that have yet to be defined.12 These earlier studies, combined with the results presented here, suggest that neurotransmitters, through their respective transporters and receptors, may play a significant but underappreciated role as signaling molecules that modulate skeletal health. Furthermore, the data suggest that one level of neurotransmitter action may be through direct or indirect effects on differentiating osteoblasts/osteocytes. Our findings regarding expression of 5-HT receptors and 5-HTT in immortalized and transformed osteoblastic cells have been confirmed in normal differentiating rat osteoblasts. We do not know the functional correlates of our findings in vivo, however. Future work in our laboratory will be directed toward investigating the biological role of serotonin in the regulation of osteoblast differentiation.

Acknowledgments: This work was supported in part by the Medical Research Service of the Department of Veterans Affairs, and NIH Grant DK54415 to M.B. The authors gratefully acknowledge the excellent technical support and encouragement provided by Anne Chapman Evans and Les Alberque throughout the course of these studies. We also thank Dr. Laurie Vessely for assistance and critical reading of the manuscript.

Bone Vol. 29, No. 5 November 2001:477– 486

M. M. Bliziotes et al. Serotonin receptors and reuptake in osteoblasts

References 1. Abramowski, D. and Staufenbiel, M. Identification of the 5-hydroxytryptamine2C receptor as a 60-kDa N-glycosylated protein in choroid plexus and hippocampus. J Neurochem 65:782–790; 1995. 2. Anderson, G. and Horne, W. Activators of protein kinase C decrease serotonin transport in human platelets. Biochim Biophys Acta 1137:331–337; 1992. 3. Anthony, T. and Azmitia, E. Molecular characterization of antipeptide antibodies against the 5-HT1A receptor: Evidence for state-dependent antibody binding. Mol Brain Res 50:277–284; 1997. 4. Aronow, M., Gerstenfeld, L., Owen, T., Tassinari, M., Stein, G., and Lian, J. Factors that promote progressive development of the osteoblast phenotype in cultured fetal rat calvarial cells. J Cell Physiol 143:213–221; 1990. 5. Azmitia, E., Yu, I., Akbari, H., Kheck, N., Whitaker-Azmitia, P., and Marshak, D. Antipeptide antibodies against the 5-HT1A receptor. J Chem Neuroanat 5:289 –298; 1992. 6. Bailey, L., Minkoff, R., and Koch, W. Relative growth rates of maxillary mesenchyme in the chick embryo. J Craniofac Genet Dev Biol 8:167–177; 1988. 7. Bender, E., Pindon, A., van Oers, I., Zhang, Y.-B., Gommeren, W., Verhasselt, P., Jurzak, M., Leysen, J., and Luyten, W. Structure of the human serotonin 5-HT4 receptor gene and cloning of a novel 5-HT4 splice variant. J Neurochem 74:478 – 489; 2000. 8. Birnbaum, R., Bowsher, R., and Wiren, K. Changes in insulin-like growth factor-I expression and secretion during the proliferation and differentiation of normal rat osteoblasts. J Endocrinol 144:251–259; 1995. 9. Birnbaum, R. and Wiren, K. Changes in insulin-like growth factor-binding protein expression and secretion during the proliferation, differentiation, and mineralization of primary cultures of rat osteoblasts. Endocrinology 135:223– 230; 1994. 10. Bjurholm, A., Kreicbergs, A. E. B., and Schultzberg, M. Substance P- and CGRP-immunoreactive nerves in bone. Peptides 9:165–171; 1988. 11. Bjurholm, A., Kreicbergs, A., Terenius, L., Goldstein, M., and Schultzberg, M. Neuropeptide Y-, tyrosine hydroxylase- and vasoactive intestinal polypeptideimmunoreactive nerves in bone and surrounding tissues. J Auton Nerv Syst 25:119 –125; 1988. 12. Bliziotes, M., McLoughlin, S., Gunness, M., Fumagalli, F., Jones, S., and Caron, M. Bone histomorphometric and biomechanical abnormalities in mice homozygous for deletion of the dopamine transporter. Bone 26:15–19; 2000. 13. Bohm, S., Grady, E., and Bunnett, N. Regulatory mechanisms that modulate signalling by G-protein-coupled receptors. Biochem J 322:1–18; 1997. 14. Chalecka-Franaszek, E., Chen, H., and Chuang, D. 5-Hydroxytryptamine2A receptor stimulation induces activator protein-1 and cyclic AMP-responsive element-binding with cyclic AMP-responsive element-binding protein and JunD as common components in cerebellar neurons. Neuroscience 88:885– 898; 1999. 15. Cheng, Y. and Prusoff, W. Relationship between the inhibition constant (Ki) and the concentration of inhibitor which causes 50 percent inhibition (I50) of an enzymatic reaction. Biochem Pharmacol 22:3099 –3108; 1973. 16. Chenu, C., Serre, C., Raynal, C., Burt-Pichat, B., and Delmas, P. Glutamate receptors are expressed by bone cells and are involved in bone resorption. Bone 22:295–299; 1998. 17. Chomczynski, P. and Sacchi, N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156 –159; 1987. 18. Duncan, C. and Shim, S.-S. The autonomic nerve supply of bone. J Bone Jt Surg 58-B:323–330; 1977. 19. Duxon, M., Flanigan, T., Reavley, A., Baxter, G., Blackburn, T., and Fone, K. Evidence for expression of the 5-hydroxytryptamine-2B receptor protein in the rat central nervous system. Neuroscience 76:323–329; 1997. 20. Eshleman, A., Carmolli, M., Cumbay, M., Martens, C., Neve, K., and Janowsky, A. Characteristics of drug interactions with recombinant biogenic amine transporters expressed in the same cell type. J Pharmacol Exp Ther 289:877– 885; 1999. 21. Espinosa, L., Itzstein, C., Cheynel, H., Delmas, P., and Chenu, C. Active NMDA glutamate receptors are expressed by mammalian osteoclasts. J Physiol (Lond) 518:47–53; 1999. 22. Fall, P., Breault, D., and Raisz, L. Inhibition of collagen synthesis by prostaglandins in the immortalized rat osteoblastic cell line Py1a: Structure-activity relations and signal transduction mechanisms. J Bone Miner Res 9:1935–1943; 1994. 23. Fann, M. and Patterson, P. Depolarization differentially regulates the effects of

24.

25.

26. 27.

28.

29.

30.

31.

32.

33. 34.

35.

36.

37.

38. 39.

40.

41. 42.

43.

44. 45. 46.

485

bone morphogenetic proteins (BMP)-2, BMP-6 and activin A on sympathetic neuronal phenotype. J Neurochem 63:2074 –2079; 1994. Guillet-Deniau, I., Burnol, A.-F., and Girard, J. Identification and localization of a skeletal muscle serotonin 5-HT2A receptor coupled to the Jak/STAT pathway. J Biol Chem 272:14825–14829; 1997. Hall, B. Tissue interactions and the initiation of osteogenesis and chondrogenesis in the neural crest-derived mandibular skeleton of the embryonic mouse as seen in isolated murine tissues and in recombinations of murine and avian tissues. J Embryol Exp Morphol 58:251–264; 1980. Hediger, M. and Welbourne, T. Introduction: Glutamate transport and metabolism. Am J Physiol 277:F477–F480; 1999. Hill, E. L. and Elde, R. Distribution of CGRP-, VIP-, D␤H-, SP-, and NPY-immunoreactive nerves in the periosteum of the rat. Cell Tissue Res 264:469 – 480; 1991. Hohmann, E., Elde, R., Rysavy, J., Einzig, S., and Gebhard, R. Innervation of periosteum and bone by sympathetic vasoactive intestinal peptide-containing nerve fibers. Science 232:868 – 871; 1986. Huang, T., Vinci, J., Lan, L., Jeffrey, J., and Wilcox, B. Serotonin-inducible transcription of interleukin-1alpha in uterine smooth muscle cells requires an AP-1 site: Cloning and partial characterization of the rat IL-alpha promoter. Mol Cell Endocrinol 152:21–35; 1999. Itzstein, C., Espinosa, L., Delmas, P., and Chenu, C. Specific antagonists of NMDA receptors prevent osteoclast sealing zone formation required for bone resorption. Biochem Biophys Res Commun 268:201–209; 2000. Kassem, M., Okasaki, R., Harris, S., Spelsberg, T., Conover, C., and Riggs, B. Estrogen effects on insulin-like growth factor gene expression in a human osteoblastic cell line with high levels of estrogen receptor. Calcif Tissue Int 62:60 – 66; 1998. Kruger, L., Silverman, J., Mantyh, P., Sternini, C., and Brecha, N. Peripheral patterns of calcitonin gene-related peptide general somatic sensory innervation: Cutaneous and deep terminations. J Comp Neurol 280:291; 1989. Lauder, J. Neurotransmitters as growth regulatory signals: Role of receptors and second messengers. Trends Neurosci 16:233–240; 1993. Lauder, J., Moiseiwitsch, J., Liu, J., and Ji, W. Serotonin in development and pathophysiology. In: Lou, H., Greisen, G., and Larsen, J., Eds. Brain Lesions in the Newborn: Alfred Benzon Symposium. Vol. 37. Copenhagen: Monksgaard; 1994; 60 –75. Mackie, E., Thesleff, I., and Chiquet-Ehrismann, R. Tenascin is associated with chondrogenic and osteogenic differentiation in vivo and promotes chondrogenesis in vitro. J Cell Biol 105:2569 –2579; 1987. Mason, D. J., Suva, L. J., Genever, P. G., Patton, A. J., Steuckle, S., Hillam, R. A., and Skerry, T. M. Mechanically regulated expression of a neural glutamate transporter in bone: A role for excitatory amino acids as osteotropic agents? Bone 20:199 –205; 1997. Milgram, J. and Robinson, R. An electron microscopic demonstration of unmyelinated nerves in the haversian canals of the adult dog. Bull Johns Hopkins Hosp 117:163–173; 1965. Miller, M. and Kashara, M. Observations on the innervation of human long bones. Anat Rec 145:13–23; 1963. Miquel, M.-C., Emerit, M., Gingrich, J., Nosjean, A., Hamon, M., and El Mestikawy, S. Developmental changes in the differential expression of two serotonin 5-HT3 receptor splice variants in the rat. J Neurochem 65:475– 483; 1995. Moiseiwitsch, J. and Lauder, J. Regulation of gene expression in cultured embryonic mouse mandibular mesenchyme by serotonin antagonists. Anat Embryol 195:71–78; 1997. Moiseiwitsch, J. and Lauder, J. Serotonin regulates mouse cranial neural crest migration. Proc Natl Acad Sci USA 92:7182–7186; 1995. Mortensen, O., Kristensen, A., Rudnick, G., and Wiborg, O. Molecular cloning, expression and characterization of a bovine serotonin transporter. Brain Res Mol Brain Res 71:120 –126; 1999. Myers, C., Lazo, J., and Pitt, B. Translocation of protein kinase C is associated with inhibition of 5-HT uptake by cultured endothelial cells. Am J Physiol 257:L253–L258; 1989. Nelson, N. The family of Na⫹/Cl⫺ neurotransmitter transporters. J Neurochem 71:1785–1803; 1998. Noden, D. Interactions and fates of avian craniofacial mesenchyme. Development 103:121–140; 1988. Owen, T., Aronow, M., Shalhoub, V., Barone, L., Wilming, L., Tassinari, M., Kennedy, M., Pockwinse, S., Lian, J., and Stein, G. Progressive development of the rat osteoblast phenotype in vitro: Reciprocal relationships in expression of genes associated with osteoblast proliferation and differentiation during formation of the bone extracellular matrix. J Cell Physiol 143:213–221; 1990.

486

M. M. Bliziotes et al. Serotonin receptors and reuptake in osteoblasts

47. Patton, A., Genever, P., Birch, M., Suva, L., and Skerry, T. Expression of an N-methyl-D-aspartate-type receptor by human and rat osteoblasts and osteoclasts suggests a novel glutamate signaling pathway in bone. Bone 22:645– 649; 1998. 48. Pavlin, D., Lichtler, A., Bedalov, A., Kream, B., Harrison, J., Thomas, H., Gronowicz, G., Clark, S., Woody, C., and Rowe, D. Differential utilization of regulatory domains within the alpha 1(I) collagen promoter in osseous and fibroblastic cells. J Cell Biol 116:227–236; 1992. 49. Peroutka, S. Molecular biology of serotonin (5-HT) receptors. Synapse 18: 241–260; 1994. 50. Rahman, S., Dobson, P., Bunning, R., Russell, R., and Brown, B. The regulation of connective tissue metabolism by vasoactive intestinal polypeptide. Reg Peptides 37:111–121; 1992. 51. Rajakumar, R. and Quinn, C. Parathyroid hormone induction of rat interstitial collagenase mRNA in osteosarcoma cells is mediated through an AP-1-binding site. Mol Endocrinol 10:867– 878; 1996. 52. Ramamoorthy, S. and Blakely, R. Phosphorylation and sequestration of serotonin transporters are differentially modulated by psychostimulants. Science 285:763–766; 1999. 53. Ramamoorthy, S., Giovanetti, E., Qian, Y., and Blakely, R. Phosphorylation and regulation of antidepressant-sensitive serotonin transporters. J Biol Chem 273:2458 –2466; 1998.

Bone Vol. 29, No. 5 November 2001:477– 486 54. Reimann, I. and Christensen, S. A histological demonstration of nerves in subchondral bone. Acta Orthop Scand 48:345–352; 1977. 55. Schroeter, S., Lauder, J., and Blakely, R. Ontogeny of the mouse serotonin transporter. Soc Neurosci Abstr 20:1317; 1994. 56. Serre, C., Farlay, D., Delmas, P., and Chenu, C. Evidence for a dense and intimate innervation of the bone tissue, including glutamate-containing fibers. Bone 25:623– 629; 1999. 57. Shuey, D., Sadler, T., and Lauder, J. Serotonin as a regulator of craniofacial morphogenesis: Site specific malformations following exposure to serotonin uptake inhibitors. Teratology 46:367–378; 1992. 58. Wasylyk, B., Imler, J., Chatton, B., Schatz, C., and Wasylyk, C. Negative and positive factors determine the activity of the polyoma virus enhancer ␣ domain in undifferentiated and differentiated cell types. Proc Natl Acad Sci USA 85:7952–7956; 1988.

Date Received: August 11, 2000 Date Accepted: May 1, 2001 Date Revised: May 4, 2001