Evidence that the Cells Responsible for Marrow Fibrosis in a Rat Model for Hyperparathyroidism Are Preosteoblasts
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《内分泌学杂志》
Department of Nutrition and Exercise Science (S.L., R.T.T.), Oregon State University, Corvallis, Oregon 97331
Life Sciences Division (J.D.S.), Universities Space Research Association, Houston, Texas 77058
Abstract
We examined proliferation of cells associated with PTH-induced peritrabecular bone marrow fibrosis in rats as well as the fate of those cells after withdrawal of PTH. Time-course studies established that severe fibrosis was present 7 d after initiation of a continuous sc PTH infusion (40 μg/kg·d). To ascertain cell proliferation, rats were coinfused for 1 wk with PTH (treated) or vehicle (control) and [3H]thymidine (1.5 mCi/rat). Groups of control and treated rats were killed immediately (d 0) and 1 wk (d 7) later. Few osteoblasts (Obs) and osteocytes in treated and control groups were radiolabeled on d 0. Peritrabecular cells expressing a fibroblastic (Fb) phenotype and surrounded by an extracellular matrix were not present in controls on either d 0 or d 7. Multiple cell layers of Fbs lined most (70%) of the bone surface on d 0 in treated rats and nearly all (85%) of the Fbs were radiolabeled. Fbs had entirely disappeared from bone surfaces on d 7. Eighty-five percent of the Obs on and 73% of the osteocytes within the active remodeling sites were radiolabeled. Immunohistochemistry revealed that Fbs induced by PTH treatment produced osteocalcin, osteonectin, and core binding factor-1. These data provide compelling evidence that Fbs recruited to bone surfaces in response to a continuous PTH infusion undergo extensive proliferation, express osteoblast-specific proteins, and produce an extracellular matrix that is similar to osteoid. After restoration of normal PTH levels, Fbs differentiated to Obs, providing further evidence that Fbs are preosteoblasts.
Introduction
INTERMITTENT (TRANSIENT) TREATMENT with PTH increases osteoblast (Ob) number, bone formation rate, and bone mass in humans and laboratory animals (1, 2). In contrast, hyperparathyroidism (HPT) is associated with continuously increased PTH levels, and continuous PTH results in parathyroid bone disease. Depending on PTH levels and additional factors, including availability of calcium, phosphorus, and 1,25-dihydroxyvitamin D, this disease is characterized by osteomalacia, focal bone resorption, increased bone formation, and peritrabecular marrow fibrosis (osteitis fibrosa) (3, 4, 5, 6).
The cellular and molecular mechanisms that mediate the remarkably different effects of intermittent and continuous PTH are important but incompletely understood. Intermittent PTH can lead to increases in bone mass and strength with a corresponding decrease in fracture risk (6, 7), whereas continuous PTH impairs bone quality and can result in bone pain and pathological fractures (6, 8, 9). Parathyroid bone disease is commonly associated with secondary HPT caused by renal failure. Treatment options in these patients are limited and their prognosis is often poor (8, 9, 10). The recent introduction of intermittent PTH as a treatment for osteoporosis provides an additional incentive to understand the mechanisms for the differential actions of intermittent and continuous PTH on target cells in bone. The efficacy of intermittent PTH requires rapid metabolism; even mildly impaired metabolic clearance of the active hormone has the potential to lead to adverse skeletal effects (10, 11).
Intermittent PTH increases bone formation in rats by increasing Ob number and Ob activity (12). The initial rapid increase in Ob number does not require cell proliferation, indicating that the Obs originate from a postmitotic population of cells (12, 13). Bone-lining cells are postmitotic cells derived from Obs and are ideally situated to be rapidly mobilized to express the Ob phenotype in response to PTH (14). Alternatively, there may be a population of postmitotic-committed preosteoblasts that respond to PTH by progressing to Obs.
Continuous infusion of PTH increases bone formation in rats (15, 16, 17, 18), presumably by mechanisms similar to intermittent PTH. As is the case in patients with parathyroid bone disease, continuous PTH treatment of rats results in the accumulation of poorly mineralized extracellular matrix onto bone surfaces. This abnormal matrix is produced by cells having a fibroblastic (Fb) phenotype and contributes to the impaired bone quality associated with HPT (11, 12, 15, 18). The goals of the present studies were to investigate whether cell proliferation contributes to bone marrow fibrosis in the rat model for HPT and determine the fate of the Fb and fibrotic extracellular matrix after normalization of PTH levels.
Materials and Methods
Animals
All animal experiments were approved by the Institutional Animal Care and Use Committee. Six-month-old female Sprague Dawley rats of approximately 260 g were obtained from Harlan Sprague Dawley, Inc. (Indianapolis, IN). They were fed standard rat chow containing 0.95% calcium, 0.67% phosphorus, and 4.5 IU/g vitamin D3 (Laboratory Rodent Diet 5001, LabDiet, St. Louis, MO) ad libitum and maintained under a 12-h light, 12-h dark cycle.
Experiment 1: time-course effects of continuous PTH
Three experimental studies of 1-, 2-, and 4-wk duration were performed to establish a time course for the effects of continuous PTH and its withdrawal on cancellous bone histomorphometry. In the 1-wk duration study, rats were divided into five groups of seven to eight animals per group. They were implanted sc using 1-wk duration osmotic pumps (Alza Corp., Mountainview, CA) loaded to deliver human PTH 1–34 (Bachem Inc., Torrance, CA) at a continuous rate of 40 μg/kg·d (treated) or vehicle (control) containing 150 mM NaCl, 1 mM HCl, and 2% heat-inactivated rat serum. Control rats were killed on d 7, whereas the continuous PTH animals were killed on d 1, 3, 5, and 7. Tetracycline HCl (20 mg/kg; Sigma, St. Louis, MO) and calcein (20 mg/kg; Sigma) were given as an aqueous solution by perivascular tail vein injection (0.1 ml) to control rats and 1-wk duration continuous PTH animals on d 0 and 6.
Rats in 2- and 4-wk duration studies were divided into three groups, vehicle (control), continuous PTH, and continuous PTH (1 wk duration) followed by PTH withdrawal (1 or 3 wk duration). The 2- and 4-wk duration continuous PTH infused rats were implanted sc with 2-wk duration osmotic pumps containing either vehicle or PTH. We had shown that PTH was stable in sc implanted osmotic pumps for 2 wk but had not tested longer intervals (our unpublished data). Therefore, a new pump was replaced after 2 wk in the 4-wk duration study. The rats received fluorochrome labels, tetracycline, and calcein 8 and 1 d, respectively, before being killed.
All rats were anesthetized with ketamine HCl (50 mg/kg)/xylazine HCl (5 mg/kg). Blood samples were collected and allowed to clot at room temperature for 60 min before centrifugation. Serum was aliquoted and stored at –70 C before analysis. The animals were then killed by cardiectomy and tibiae were removed. Tibiae were fixed in 70% ethanol for bone histomorphometry.
Experiment 2: radioautography
This experiment was performed to investigate the role of cell proliferation in the origin of continuous PTH-induced Ob lineage cells and Fb using [3H]thymidine incorporation to detect cells entering the S phase of the cell cycle. Rats were divided into four groups of three rats per group, two controls, and two PTH-treated groups. The rats were implanted sc with osmotic pumps containing 1.5 mCi [methyl-3H]thymidine (specific activity; 86 Ci/mmol, Amersham Pharmacia Biotech, Piscataway, NJ) in aqueous solution with 2% ethanol for 1 wk. The [3H]thymidine labels DNA of cells that progress through the cell cycle. The rats were coinfused with vehicle or PTH at 40 μg/kg·d for 1 wk. Vehicle (control) and PTH groups were either killed immediately (d 0) or 1 wk (d 7) after treatment (Fig. 1). Femora were removed and fixed in 10% neutral buffered formalin overnight for radioautography.
Experiment 3: immunohistochemistry
This experiment was performed to investigate whether PTH-induced Fbs express the osteoblast transcription factor core binding factor 1 (cbfa1) and whether the extracellular matrix surrounding Fb contains the bone matrix proteins osteocalcin and osteonectin. Rats were divided into three groups of four rats per group: controls, 1-wk duration PTH, and 1-wk duration PTH followed by 1-wk duration withdrawal. They were implanted sc with osmotic pumps containing either vehicle or PTH as described in experiment 1. The rats were then killed. Tibiae were removed, fixed in 5 solution for 2 h, and immersed overnight in 10% neutral buffered formalin for immunohistochemistry.
Serum PTH and calcium (Ca) measurement
Serum PTH concentration was determined using a rat PTH immunoradiometric assay kit from Immutopics International (San Clemente, CA), which detects intact (1–84) and N-terminal (1–34) forms of rat PTH and has approximately 100% cross-reactivity to human PTH 1–34. Total serum Ca was measured by the Central Clinical Laboratory at the Mayo Clinic (Rochester, MN) using a 717 automated system (Hitachi, Hialeah, FL).
Bone histomorphometry
The proximal tibial metaphyses were dehydrated, infiltrated, and embedded in glycol methacrylate. Undecalcified 5-μm-thick sections were cut using a microtome (model 2050 Supercut; Reichert-Jung, Heidelberg, Germany) and mounted unstained for dynamic measurements. Consecutive sections were toluidine blue stained to quantitate bone cell and Fb measurements. Histomorphometric parameters were measured using an Osteomeasure image analysis system (OsteoMetrics, Atlanta, GA) coupled to a photomicroscope and personal computer and expressed using standard nomenclature (19). A sampling site of 2.8 mm2 was established in the secondary spongiosa at 0.5 mm below the middle of the growth plate. Cancellous bone volume was assessed as a percentage of total tissue volume (BV/TV, percent). Trabecular thickness (Tb.Th, μm), trabecular number (Tb.N, mm–1), and trabecular separation (Tb.Sp, μm) were calculated. Double-labeled surface, expressed as percentage of bone surface (dL.S/BS), was defined as the trabecular surface covered with tetracycline and calcein labels. Mineral apposition rate (MAR, micromoles per day) was derived from the mean distance between fluorescent labels divided by the labeling interval. Bone formation rate was defined as the product of dL.S/BS and MAR and expressed per bone surface (BFR/BS, cubic micrometers per square micrometers per day), bone volume (BFR/BV, percent per day), and tissue volume (BFR/TV, percent per day). Ob surface (Ob.S/BS, percent) was reported as a percentage of total bone surface lined by a palisade of plump cuboidal cells located immediately adjacent to the thin layer of osteoid in direct physical contact with the bone surface. Osteoclasts (Ocs) surface (Oc.S/BS, percent) was determined as the percentage of cancellous bone surface covered by multinucleated (two or more nuclei) cells. Fb surface (Fb.S/BS, percent) was defined as the cancellous bone surface lined by multiple layers of elongated or fusiform cells surrounded by extracellular matrix. The fibroblast phenotype of the cells responsible for osteitis fibrosa in rats after continuous PTH was confirmed by transmission electron microscopy.
Radioautography
Distal femoral metaphyses were decalcified in 5% formic acid in 10% formalin, infiltrated, and embedded in JB-4 (Polysciences, Inc., Warrington, PA). Five-micron-thick sections were cut, attached to slides, dipped into melted (40 C) 1:1 diluted Ilford K5D emulsion in water (Polysciences), air dried, and kept in a light-sealed box at 4 C for 4 months. Sections were developed in Kodak D-19 (Sigma) for 4 min, fixed in Kodak fixer (Sigma) for 4 min, rinsed in water for 10 min, and stained with toluidine blue. Radiolabeled Ob, osteocytes (Ocys), and Fbs were counted as being labeled if they had at least five silver grains over the nucleus. The data were expressed as percent cells of each type labeled for Obs and Fbs and percent labeled within actively forming bone remodeling sites for Ocys. Actively forming remodeling sites were identified as the area between a bone surface lined by osteoblasts and a cement line.
Immunohistochemistry
Immunohistochemical staining for osteonectin (20) was performed with rabbit antisera (LF-23); staining for osteocalcin was performed with a primary goat polyclonal antibody (Biomedical Technologies, Stoughton, MA) and a secondary antibody from Vector Laboratories (Burlingame, CA); and staining for cbfa1 was performed with a specific rabbit antisera (21).
Deparaffinized sections (5 μm) were pretreated by trypsin digestion [0.06% in PBS (pH 7.4) at room temperature followed by 0.2% glycine in PBS], peroxidase activity block (3% H2O2 in 100% methanol at room temperature), and hyaluronidase digestion [250U/ml in 0.1 M sodium acetate buffer with 0.85% NaCl (pH 5.5) at 37 C]. Nonspecific binding was blocked with normal serum before sections were incubated overnight with specific antisera or antibody diluted in the same blocking buffer. After a thorough washing with Tris-HCl buffer, sections were incubated at room temperature for 60 min with secondary antibody. For the detection of osteonectin, the secondary antibody (goat antirabbit Vectastain Elite ABC kit, Vector Laboratories) was diluted 0.5% (vol/vol) in blocking buffer, amplified with the avidin-biotin-complex (Vectastain Elite ABC kit), and visualized by incubation in diaminobenzidine HCl (Sigma; 0.025% in Tris-buffered saline, 0% BSA, and 0.1% hydrogen peroxide). Cbfa1 and osteocalcin detection was performed with a 60-min incubation with peroxidase-labeled antibody (Envision Plus system, DakoCytomation, Carpinteria, CA) at room temperature and a 5-min development in diaminobenzidine-substrate solution (DakoCytomation). Tissue sections were counterstained with methyl green (osteonectin and osteocalcin) or hematoxylin (cbfa1).
Statistical analysis
Results are expressed as mean ± SEM. All measurements were compared across the different time points of treatments by one-way ANOVA to determine differences between groups. Pair-wise comparisons between groups were then conducted using the Fisher’s protected least significant difference post hoc test. Statistical significance was defined as P < 0.05.
Results
Experiment 1
The time-course effects of continuous PTH on body weight and serum chemistry are shown in Table 1. Animals exhibited modest weight loss during the first week of continuous PTH infusion, but body weight returned toward normal during longer duration PTH treatment and after withdrawal of PTH. Immunoreactive serum PTH was unchanged in the control groups during the 4-wk duration time-course study. Serum PTH was increased within 1 d of beginning continuous PTH treatment and remained elevated throughout treatment. PTH levels returned to near normal values within 1 wk of discontinuation of PTH treatment. Continuous infusion with PTH resulted in increases in serum Ca, which became significant within 3 d. Serum Ca returned to normal within 1 wk after discontinuation of PTH treatment.
The time-course effects of continuous PTH on static and dynamic bone histomorphometry are shown in Tables 2 and 3. BV/TV and Tb.Th were increased and Tb.Sp was decreased in rats treated with PTH for 2 and 4 wk as well as in rats in the 1-wk duration PTH/3-wk duration withdrawal group. Tb.N was increased in rats after the 4 wk infusion of PTH. Photomicrographs showing the morphology of cancellous bone in control, 1-wk duration PTH, 2-wk duration PTH, and 4-wk duration PTH, and representative Ob, Oc, and Fb are shown in Fig. 2.
The time-course effects of PTH infusion on Ob.S/BS, Oc.S/BS, and Fb.S/BS are shown in Table 2. Continuous PTH resulted in a time-dependent increase in Ob.S/BS, which reached a peak of approximately 6-fold on d 5 and gradually returned to the control value by wk 4. Ob.S/BS was increased in rats 1 wk after discontinuation of continuous PTH (1 wk treatment) but returned to normal by 3 wk. In contrast, Oc.S/BS was significantly increased in continuous PTH-treated rats after 5 d and remained elevated for 4 wk of treatment. Oc.S/BS 1 and 3 wk after PTH infusion did not differ from control groups. Peritrabecular Fbs were not observed in control rats. Fbs were first detected on d 3 of the PTH infusion and were present thereafter in treated rats. Peritrabecular Fbs had completely disappeared within 1 wk of discontinuing PTH treatment.
Bone formation in rats continuously infused with PTH (BFR/BS, BFR/BV, and BFR/TV) was increased after 2 and 4 wk. Bone formation was increased 1 wk after discontinuation of PTH treatment but had returned to normal by 3 wk. The increased bone formation in PTH-treated rats was due to a approximately 2-fold increase in MAR and approximately 10-fold increase in dL.S/BS.
Experiment 2
The effects of continuous PTH on [3H]thymidine-labeled Ob, Fb, and Ocy are shown in Figs. 3 and 4. In Fig. 3, representative radioautographs from control and continuous PTH-treated rats are shown; the data are shown in Fig. 4. Groups of control and treated rats were killed 0 and 7 d after cotreatment for 7 d with continuous PTH and radiolabeled thymidine. Negligible numbers of Obs and Ocys were labeled in either treatment group on d 0, but most bone marrow cells were labeled. Peritrabecular Fbs were not present in the control, but 85% of the numerous Fbs induced by continuous PTH were labeled. Seven days after withdrawal of PTH and [3H]thymidine cotreatment, 85% of the Obs on and 73% of the Ocys within active remodeling sites were radiolabeled, numbers dramatically greater than in the controls (15 and 4%, respectively). Very few radiolabeled bone marrow cells were detected in either treatment group, presumably due to continued cell proliferation with accompanying dilution of the radioactivity.
Experiment 3
Immunohistochemical staining was performed to qualitatively assess the spatial localization of osteonectin, osteocalcin, and cbfa1 (Fig. 5). Osteonectin was localized to the Obs in the controls and throughout the peritrabecular unmineralized extracellular matrix in PTH-treated rats. The cbfa1 was localized to the Obs in controls, whereas the Fbs were uniformly positive for cbfa1 in the PTH-treated rats. One week after withdrawal of PTH treatment, cbfa1 was highly expressed in the Obs. Lower levels of staining were present in the newly formed Ocys. The osteocalcin was detected in the Obs and mineralized bone matrix in controls. The fibrous extracellular matrix and Fbs immediately adjacent to trabecular surfaces but not Fbs close to marrow in PTH-treated rats were strongly positive for osteocalcin.
Discussion
Continuous infusion of PTH resulted in profound changes in gene expression and cellular populations within rat bone (11, 15, 18). Within 5 d, there were large increases in Ob, Oc, and Fb surfaces, and the Fbs were surrounded by an extensive volume of extracellular unmineralized fibrous matrix. During HPT in humans, the parathyroid glands secrete PTH (1–84) and N terminally truncated PTH. However, continuous administration of PTH (1–34) was sufficient to induce parathyroid bone disease in the rat. Cessation of PTH treatment resulted in similarly remarkable changes; within 1 wk Oc surface had returned to normal and no peritrabecular fibrosis was detected. These findings suggest that restoration of normal PTH levels is correlated with rapid correction of the skeletal manifestations of parathyroid bone disease.
The effects of HPT on bone turnover depend, in part, on available calcium. In agreement with earlier work, continuous PTH resulted in hypercalcemia as well as increases in bone formation and bone resorption (11, 12, 15, 16, 17, 18). In the present study, continuous PTH treatment for 4 wk resulted in a large increase in BV/TV. The anabolic effects of continuous PTH on bone matrix production in hypercalcemic rats outweighed the catabolic effects on bone resorption. These findings are in agreement with earlier work (17, 18) and contrast to the bone loss observed in calcium-deficient HPT rats, in which there was a net increase in bone resorption (22, 23).
BV/TV increased after discontinuation of PTH treatment. This was, in part, because the increases in Ob surface and bone formation rate induced by continuous PTH persisted longer than the increase in Oc surfaces.
HPT patients have an increased fracture risk, largely due to a reduction in bone quality (24). PTH-induced osteomalacia, peritrabecular fibrosis, focal bone resorption, and high bone turnover may all contribute to diminished bone quality. In patients, HPT is associated with increases, decreases, or no change in cancellous bone mass, with the last being the most common outcome (25, 26). Additional factors that influence bone volume in HPT patients are not well described. Several studies have reported that parathyroidectomy in these patients leads to increased bone formation and bone mass as well as resolution of osteitis fibrosa (27, 28). In this regard, HPT patients respond similarly to rats after normalization of PTH levels. However, long-duration suppression of PTH in patients with chronic renal failure through the use of calcium-based phosphate binders and vitamin D therapy can result in adynamic bone disease (29).
The 430% increase in Ob.S/BS measured during the first 5 d of continuous PTH treatment was not associated with Ob proliferation. As was observed for intermittent PTH treatment (12), very few of the continuous PTH-induced Obs had passed through S phase of the cell cycle. Ob apoptosis has been reported in some studies to be inhibited by intermittent PTH, whereas in others apoptosis was increased (30, 31). A change in Ob life span is one pathway by which Ob number could be altered without a corresponding change in [3H]thymidine-labeled Ob. However, Ob turnover in adults is very low. Consistent with this view, only a small percentage of the Obs in vehicle-treated rats became labeled after continuous exposure to radioactive thymidine. Because Obs have a long life span (weeks to months), compared with the time interval required for continuous PTH treatment to dramatically increase their number (days), a change in their death rate would have negligible impact on this early increase in Ob number. A more plausible explanation for the rapid increase in osteoblast surface is modulation of postmitotic osteoblast lineage cells such as bone-lining cells and committed preosteoblasts to express the osteoblast phenotype.
Most of the peritrabecular Fbs induced by continuous PTH were labeled with 3H-thymidine, indicating that they had passed through S phase of the cell cycle during the treatment protocol. Thus, continuous PTH is a potent stimulator of Fb proliferation. PTH has been reported to have effects on a number of fibroblast populations, and PTH receptors have been described on dermal fibroblasts (32). Platelet-derived growth factor (PDGF) A is associated with pathological tissue fibrosis in several organs (33, 34) and PDGF-A expression is increased in rat bone by continuous PTH (15). It is possible that the recruitment of Fbs to bone surfaces in continuous PTH-treated rats is in response to paracrine factors that are overexpressed in target cells after continuous infusion of PTH.
The gene expression profile of rat long bones by DNA microarray indicated that continuous PTH up-regulated the mRNA levels for osteocalcin and osteonectin (35). In addition, PTH significantly increased the gene expression for decorin, lysyl oxidase, collagen I, collagen III, glypican I, and FGF receptor, which are associated with tissue remodeling and fibrosis in other tissues (36, 37, 38, 39, 40, 41, 42). Trapidil, a PDGF signaling antagonist, blunts peritrabecular fibrosis in the rat model for chronic HPT (15). Trapidil antagonized the increase in expression of several genes in cancellous bone, including lysyl oxidase (our unpublished results). PDGF-A signaling, therefore, might play a key role in PTH-induced tissue remodeling.
Obs are specialized fibroblasts that differ from all other fibroblasts in that they produce bone matrix (43). Fully differentiated Obs are morphologically distinct from other Fbs, whereas preosteoblasts are not. The latter were recognized in this study by their location on bone surfaces, expression of osteoblast marker proteins, and ability to differentiate into Obs. The detection of the latter process was greatly facilitated in this study by prelabeling the PTH-induced preosteoblast population with radioactive thymidine.
The labeled Obs and Ocys observed at the end of the study had to have been derived from cells that went through S phase 1–2 wk earlier. Furthermore, these cells were arrested in G0 phase of the cell cycle after labeling; otherwise the label would have been diluted by subsequent cell divisions and, as was the case of most hematopoietic cells, would no longer be detectable.
Discontinuation of PTH treatment resulted in the rapid disappearance of radiolabeled Fbs as well as the matrix that surrounded these cells and a corresponding increase in [3H]thymidine-labeled Obs and Ocys located on and within mineralized bone matrix, respectively. These findings provide nearly unequivocal evidence that the labeled Fbs had differentiated to Obs and Ocys and are in agreement with an earlier study in mice with surgery-induced renal failure (44). Osteocalcin, osteonectin, and cbfa 1, osteoblast marker peptides (45, 46), were localized to Fb cells or the surrounding extracellular matrix by immunohistochemistry, further supporting the conclusion that Fb cells are preosteoblasts.
The origin of the Fbs targeted by PTH is under investigation but as of yet is unknown. PTH regulates many genes related to Ob differentiation, including bone morphogenic proteins and members of the Wnt signaling system (35, 47). Mesenchymal stem cells are a candidate because they are capable of differentiating to Obs (48) and PTH-responsive spindle-shaped bone marrow Obs might participate in regulation of hematopoietic stem cell niche (49). However, it is equally possible that PTH targets a cell population that is already committed to the Ob lineage.
In summary, we have shown that PTH treatment can increase Ob surface by at least two distinct cellular mechanisms: 1) rapid modulation of postmitotic cells to express the Ob phenotype; and 2) attraction of marrow Fbs to bone surfaces, expansion of the Fb population by proliferation, and differentiation of these cells to Obs. The former mechanism occurs after both intermittent and continuous PTH treatment. The latter largely unrecognized mechanism requires longer exposure to elevated PTH levels and may represent a target for the development of a new class of drugs. Targeted recruitment and differentiation of Fbs to Obs may provide a novel approach to reverse osteoporosis.
Acknowledgments
The authors thank Ms. Lori Rolbiecki for typing this manuscript and Ms. Peggy Backup for editorial assistance. We also thank Dr. Larry Fisher (National Institute of Dental and Craniofacial Research, Bethesda, MD) and Dr. Gerard Karsenty (Baylor College of Medicine, Houston, TX) for providing antisera used for the immunohistochemical detection of osteoblast protein markers, LF-23 osteonectin, and cbfa1, respectively.
Footnotes
This work was supported by National Institutes of Health Grant AR48833 and National Aeronautics and Space Administration Grant NAG9-1458.
Abbreviations: BFR/BS, Bone formation rate expressed per bone surface; BFR/BV, bone formation rate expressed per bone volume; BFR/TV, bone formation rate expressed per tissue volume; BV/TV, bone volume expressed per tissue volume; cbfa1, core binding factor 1; dL.S/BS, double-labeled surface expressed per bone surface; Fb, fibroblast; Fb.S/BS, Fb surface expressed per bone surface; HPT, hyperparathyroidism; MAR, mineral apposition rate; Ob, osteoblast; Ob.S/BS, Ob surface expressed per bone surface; Oc, osteoclast; Oc.S/BS, Oc surface expressed per bone surface; Ocy, osteocyte; Tb.N, trabecular number; Tb.Sp, trabecular separation; Tb.Th, trabecular thickness.
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Life Sciences Division (J.D.S.), Universities Space Research Association, Houston, Texas 77058
Abstract
We examined proliferation of cells associated with PTH-induced peritrabecular bone marrow fibrosis in rats as well as the fate of those cells after withdrawal of PTH. Time-course studies established that severe fibrosis was present 7 d after initiation of a continuous sc PTH infusion (40 μg/kg·d). To ascertain cell proliferation, rats were coinfused for 1 wk with PTH (treated) or vehicle (control) and [3H]thymidine (1.5 mCi/rat). Groups of control and treated rats were killed immediately (d 0) and 1 wk (d 7) later. Few osteoblasts (Obs) and osteocytes in treated and control groups were radiolabeled on d 0. Peritrabecular cells expressing a fibroblastic (Fb) phenotype and surrounded by an extracellular matrix were not present in controls on either d 0 or d 7. Multiple cell layers of Fbs lined most (70%) of the bone surface on d 0 in treated rats and nearly all (85%) of the Fbs were radiolabeled. Fbs had entirely disappeared from bone surfaces on d 7. Eighty-five percent of the Obs on and 73% of the osteocytes within the active remodeling sites were radiolabeled. Immunohistochemistry revealed that Fbs induced by PTH treatment produced osteocalcin, osteonectin, and core binding factor-1. These data provide compelling evidence that Fbs recruited to bone surfaces in response to a continuous PTH infusion undergo extensive proliferation, express osteoblast-specific proteins, and produce an extracellular matrix that is similar to osteoid. After restoration of normal PTH levels, Fbs differentiated to Obs, providing further evidence that Fbs are preosteoblasts.
Introduction
INTERMITTENT (TRANSIENT) TREATMENT with PTH increases osteoblast (Ob) number, bone formation rate, and bone mass in humans and laboratory animals (1, 2). In contrast, hyperparathyroidism (HPT) is associated with continuously increased PTH levels, and continuous PTH results in parathyroid bone disease. Depending on PTH levels and additional factors, including availability of calcium, phosphorus, and 1,25-dihydroxyvitamin D, this disease is characterized by osteomalacia, focal bone resorption, increased bone formation, and peritrabecular marrow fibrosis (osteitis fibrosa) (3, 4, 5, 6).
The cellular and molecular mechanisms that mediate the remarkably different effects of intermittent and continuous PTH are important but incompletely understood. Intermittent PTH can lead to increases in bone mass and strength with a corresponding decrease in fracture risk (6, 7), whereas continuous PTH impairs bone quality and can result in bone pain and pathological fractures (6, 8, 9). Parathyroid bone disease is commonly associated with secondary HPT caused by renal failure. Treatment options in these patients are limited and their prognosis is often poor (8, 9, 10). The recent introduction of intermittent PTH as a treatment for osteoporosis provides an additional incentive to understand the mechanisms for the differential actions of intermittent and continuous PTH on target cells in bone. The efficacy of intermittent PTH requires rapid metabolism; even mildly impaired metabolic clearance of the active hormone has the potential to lead to adverse skeletal effects (10, 11).
Intermittent PTH increases bone formation in rats by increasing Ob number and Ob activity (12). The initial rapid increase in Ob number does not require cell proliferation, indicating that the Obs originate from a postmitotic population of cells (12, 13). Bone-lining cells are postmitotic cells derived from Obs and are ideally situated to be rapidly mobilized to express the Ob phenotype in response to PTH (14). Alternatively, there may be a population of postmitotic-committed preosteoblasts that respond to PTH by progressing to Obs.
Continuous infusion of PTH increases bone formation in rats (15, 16, 17, 18), presumably by mechanisms similar to intermittent PTH. As is the case in patients with parathyroid bone disease, continuous PTH treatment of rats results in the accumulation of poorly mineralized extracellular matrix onto bone surfaces. This abnormal matrix is produced by cells having a fibroblastic (Fb) phenotype and contributes to the impaired bone quality associated with HPT (11, 12, 15, 18). The goals of the present studies were to investigate whether cell proliferation contributes to bone marrow fibrosis in the rat model for HPT and determine the fate of the Fb and fibrotic extracellular matrix after normalization of PTH levels.
Materials and Methods
Animals
All animal experiments were approved by the Institutional Animal Care and Use Committee. Six-month-old female Sprague Dawley rats of approximately 260 g were obtained from Harlan Sprague Dawley, Inc. (Indianapolis, IN). They were fed standard rat chow containing 0.95% calcium, 0.67% phosphorus, and 4.5 IU/g vitamin D3 (Laboratory Rodent Diet 5001, LabDiet, St. Louis, MO) ad libitum and maintained under a 12-h light, 12-h dark cycle.
Experiment 1: time-course effects of continuous PTH
Three experimental studies of 1-, 2-, and 4-wk duration were performed to establish a time course for the effects of continuous PTH and its withdrawal on cancellous bone histomorphometry. In the 1-wk duration study, rats were divided into five groups of seven to eight animals per group. They were implanted sc using 1-wk duration osmotic pumps (Alza Corp., Mountainview, CA) loaded to deliver human PTH 1–34 (Bachem Inc., Torrance, CA) at a continuous rate of 40 μg/kg·d (treated) or vehicle (control) containing 150 mM NaCl, 1 mM HCl, and 2% heat-inactivated rat serum. Control rats were killed on d 7, whereas the continuous PTH animals were killed on d 1, 3, 5, and 7. Tetracycline HCl (20 mg/kg; Sigma, St. Louis, MO) and calcein (20 mg/kg; Sigma) were given as an aqueous solution by perivascular tail vein injection (0.1 ml) to control rats and 1-wk duration continuous PTH animals on d 0 and 6.
Rats in 2- and 4-wk duration studies were divided into three groups, vehicle (control), continuous PTH, and continuous PTH (1 wk duration) followed by PTH withdrawal (1 or 3 wk duration). The 2- and 4-wk duration continuous PTH infused rats were implanted sc with 2-wk duration osmotic pumps containing either vehicle or PTH. We had shown that PTH was stable in sc implanted osmotic pumps for 2 wk but had not tested longer intervals (our unpublished data). Therefore, a new pump was replaced after 2 wk in the 4-wk duration study. The rats received fluorochrome labels, tetracycline, and calcein 8 and 1 d, respectively, before being killed.
All rats were anesthetized with ketamine HCl (50 mg/kg)/xylazine HCl (5 mg/kg). Blood samples were collected and allowed to clot at room temperature for 60 min before centrifugation. Serum was aliquoted and stored at –70 C before analysis. The animals were then killed by cardiectomy and tibiae were removed. Tibiae were fixed in 70% ethanol for bone histomorphometry.
Experiment 2: radioautography
This experiment was performed to investigate the role of cell proliferation in the origin of continuous PTH-induced Ob lineage cells and Fb using [3H]thymidine incorporation to detect cells entering the S phase of the cell cycle. Rats were divided into four groups of three rats per group, two controls, and two PTH-treated groups. The rats were implanted sc with osmotic pumps containing 1.5 mCi [methyl-3H]thymidine (specific activity; 86 Ci/mmol, Amersham Pharmacia Biotech, Piscataway, NJ) in aqueous solution with 2% ethanol for 1 wk. The [3H]thymidine labels DNA of cells that progress through the cell cycle. The rats were coinfused with vehicle or PTH at 40 μg/kg·d for 1 wk. Vehicle (control) and PTH groups were either killed immediately (d 0) or 1 wk (d 7) after treatment (Fig. 1). Femora were removed and fixed in 10% neutral buffered formalin overnight for radioautography.
Experiment 3: immunohistochemistry
This experiment was performed to investigate whether PTH-induced Fbs express the osteoblast transcription factor core binding factor 1 (cbfa1) and whether the extracellular matrix surrounding Fb contains the bone matrix proteins osteocalcin and osteonectin. Rats were divided into three groups of four rats per group: controls, 1-wk duration PTH, and 1-wk duration PTH followed by 1-wk duration withdrawal. They were implanted sc with osmotic pumps containing either vehicle or PTH as described in experiment 1. The rats were then killed. Tibiae were removed, fixed in 5 solution for 2 h, and immersed overnight in 10% neutral buffered formalin for immunohistochemistry.
Serum PTH and calcium (Ca) measurement
Serum PTH concentration was determined using a rat PTH immunoradiometric assay kit from Immutopics International (San Clemente, CA), which detects intact (1–84) and N-terminal (1–34) forms of rat PTH and has approximately 100% cross-reactivity to human PTH 1–34. Total serum Ca was measured by the Central Clinical Laboratory at the Mayo Clinic (Rochester, MN) using a 717 automated system (Hitachi, Hialeah, FL).
Bone histomorphometry
The proximal tibial metaphyses were dehydrated, infiltrated, and embedded in glycol methacrylate. Undecalcified 5-μm-thick sections were cut using a microtome (model 2050 Supercut; Reichert-Jung, Heidelberg, Germany) and mounted unstained for dynamic measurements. Consecutive sections were toluidine blue stained to quantitate bone cell and Fb measurements. Histomorphometric parameters were measured using an Osteomeasure image analysis system (OsteoMetrics, Atlanta, GA) coupled to a photomicroscope and personal computer and expressed using standard nomenclature (19). A sampling site of 2.8 mm2 was established in the secondary spongiosa at 0.5 mm below the middle of the growth plate. Cancellous bone volume was assessed as a percentage of total tissue volume (BV/TV, percent). Trabecular thickness (Tb.Th, μm), trabecular number (Tb.N, mm–1), and trabecular separation (Tb.Sp, μm) were calculated. Double-labeled surface, expressed as percentage of bone surface (dL.S/BS), was defined as the trabecular surface covered with tetracycline and calcein labels. Mineral apposition rate (MAR, micromoles per day) was derived from the mean distance between fluorescent labels divided by the labeling interval. Bone formation rate was defined as the product of dL.S/BS and MAR and expressed per bone surface (BFR/BS, cubic micrometers per square micrometers per day), bone volume (BFR/BV, percent per day), and tissue volume (BFR/TV, percent per day). Ob surface (Ob.S/BS, percent) was reported as a percentage of total bone surface lined by a palisade of plump cuboidal cells located immediately adjacent to the thin layer of osteoid in direct physical contact with the bone surface. Osteoclasts (Ocs) surface (Oc.S/BS, percent) was determined as the percentage of cancellous bone surface covered by multinucleated (two or more nuclei) cells. Fb surface (Fb.S/BS, percent) was defined as the cancellous bone surface lined by multiple layers of elongated or fusiform cells surrounded by extracellular matrix. The fibroblast phenotype of the cells responsible for osteitis fibrosa in rats after continuous PTH was confirmed by transmission electron microscopy.
Radioautography
Distal femoral metaphyses were decalcified in 5% formic acid in 10% formalin, infiltrated, and embedded in JB-4 (Polysciences, Inc., Warrington, PA). Five-micron-thick sections were cut, attached to slides, dipped into melted (40 C) 1:1 diluted Ilford K5D emulsion in water (Polysciences), air dried, and kept in a light-sealed box at 4 C for 4 months. Sections were developed in Kodak D-19 (Sigma) for 4 min, fixed in Kodak fixer (Sigma) for 4 min, rinsed in water for 10 min, and stained with toluidine blue. Radiolabeled Ob, osteocytes (Ocys), and Fbs were counted as being labeled if they had at least five silver grains over the nucleus. The data were expressed as percent cells of each type labeled for Obs and Fbs and percent labeled within actively forming bone remodeling sites for Ocys. Actively forming remodeling sites were identified as the area between a bone surface lined by osteoblasts and a cement line.
Immunohistochemistry
Immunohistochemical staining for osteonectin (20) was performed with rabbit antisera (LF-23); staining for osteocalcin was performed with a primary goat polyclonal antibody (Biomedical Technologies, Stoughton, MA) and a secondary antibody from Vector Laboratories (Burlingame, CA); and staining for cbfa1 was performed with a specific rabbit antisera (21).
Deparaffinized sections (5 μm) were pretreated by trypsin digestion [0.06% in PBS (pH 7.4) at room temperature followed by 0.2% glycine in PBS], peroxidase activity block (3% H2O2 in 100% methanol at room temperature), and hyaluronidase digestion [250U/ml in 0.1 M sodium acetate buffer with 0.85% NaCl (pH 5.5) at 37 C]. Nonspecific binding was blocked with normal serum before sections were incubated overnight with specific antisera or antibody diluted in the same blocking buffer. After a thorough washing with Tris-HCl buffer, sections were incubated at room temperature for 60 min with secondary antibody. For the detection of osteonectin, the secondary antibody (goat antirabbit Vectastain Elite ABC kit, Vector Laboratories) was diluted 0.5% (vol/vol) in blocking buffer, amplified with the avidin-biotin-complex (Vectastain Elite ABC kit), and visualized by incubation in diaminobenzidine HCl (Sigma; 0.025% in Tris-buffered saline, 0% BSA, and 0.1% hydrogen peroxide). Cbfa1 and osteocalcin detection was performed with a 60-min incubation with peroxidase-labeled antibody (Envision Plus system, DakoCytomation, Carpinteria, CA) at room temperature and a 5-min development in diaminobenzidine-substrate solution (DakoCytomation). Tissue sections were counterstained with methyl green (osteonectin and osteocalcin) or hematoxylin (cbfa1).
Statistical analysis
Results are expressed as mean ± SEM. All measurements were compared across the different time points of treatments by one-way ANOVA to determine differences between groups. Pair-wise comparisons between groups were then conducted using the Fisher’s protected least significant difference post hoc test. Statistical significance was defined as P < 0.05.
Results
Experiment 1
The time-course effects of continuous PTH on body weight and serum chemistry are shown in Table 1. Animals exhibited modest weight loss during the first week of continuous PTH infusion, but body weight returned toward normal during longer duration PTH treatment and after withdrawal of PTH. Immunoreactive serum PTH was unchanged in the control groups during the 4-wk duration time-course study. Serum PTH was increased within 1 d of beginning continuous PTH treatment and remained elevated throughout treatment. PTH levels returned to near normal values within 1 wk of discontinuation of PTH treatment. Continuous infusion with PTH resulted in increases in serum Ca, which became significant within 3 d. Serum Ca returned to normal within 1 wk after discontinuation of PTH treatment.
The time-course effects of continuous PTH on static and dynamic bone histomorphometry are shown in Tables 2 and 3. BV/TV and Tb.Th were increased and Tb.Sp was decreased in rats treated with PTH for 2 and 4 wk as well as in rats in the 1-wk duration PTH/3-wk duration withdrawal group. Tb.N was increased in rats after the 4 wk infusion of PTH. Photomicrographs showing the morphology of cancellous bone in control, 1-wk duration PTH, 2-wk duration PTH, and 4-wk duration PTH, and representative Ob, Oc, and Fb are shown in Fig. 2.
The time-course effects of PTH infusion on Ob.S/BS, Oc.S/BS, and Fb.S/BS are shown in Table 2. Continuous PTH resulted in a time-dependent increase in Ob.S/BS, which reached a peak of approximately 6-fold on d 5 and gradually returned to the control value by wk 4. Ob.S/BS was increased in rats 1 wk after discontinuation of continuous PTH (1 wk treatment) but returned to normal by 3 wk. In contrast, Oc.S/BS was significantly increased in continuous PTH-treated rats after 5 d and remained elevated for 4 wk of treatment. Oc.S/BS 1 and 3 wk after PTH infusion did not differ from control groups. Peritrabecular Fbs were not observed in control rats. Fbs were first detected on d 3 of the PTH infusion and were present thereafter in treated rats. Peritrabecular Fbs had completely disappeared within 1 wk of discontinuing PTH treatment.
Bone formation in rats continuously infused with PTH (BFR/BS, BFR/BV, and BFR/TV) was increased after 2 and 4 wk. Bone formation was increased 1 wk after discontinuation of PTH treatment but had returned to normal by 3 wk. The increased bone formation in PTH-treated rats was due to a approximately 2-fold increase in MAR and approximately 10-fold increase in dL.S/BS.
Experiment 2
The effects of continuous PTH on [3H]thymidine-labeled Ob, Fb, and Ocy are shown in Figs. 3 and 4. In Fig. 3, representative radioautographs from control and continuous PTH-treated rats are shown; the data are shown in Fig. 4. Groups of control and treated rats were killed 0 and 7 d after cotreatment for 7 d with continuous PTH and radiolabeled thymidine. Negligible numbers of Obs and Ocys were labeled in either treatment group on d 0, but most bone marrow cells were labeled. Peritrabecular Fbs were not present in the control, but 85% of the numerous Fbs induced by continuous PTH were labeled. Seven days after withdrawal of PTH and [3H]thymidine cotreatment, 85% of the Obs on and 73% of the Ocys within active remodeling sites were radiolabeled, numbers dramatically greater than in the controls (15 and 4%, respectively). Very few radiolabeled bone marrow cells were detected in either treatment group, presumably due to continued cell proliferation with accompanying dilution of the radioactivity.
Experiment 3
Immunohistochemical staining was performed to qualitatively assess the spatial localization of osteonectin, osteocalcin, and cbfa1 (Fig. 5). Osteonectin was localized to the Obs in the controls and throughout the peritrabecular unmineralized extracellular matrix in PTH-treated rats. The cbfa1 was localized to the Obs in controls, whereas the Fbs were uniformly positive for cbfa1 in the PTH-treated rats. One week after withdrawal of PTH treatment, cbfa1 was highly expressed in the Obs. Lower levels of staining were present in the newly formed Ocys. The osteocalcin was detected in the Obs and mineralized bone matrix in controls. The fibrous extracellular matrix and Fbs immediately adjacent to trabecular surfaces but not Fbs close to marrow in PTH-treated rats were strongly positive for osteocalcin.
Discussion
Continuous infusion of PTH resulted in profound changes in gene expression and cellular populations within rat bone (11, 15, 18). Within 5 d, there were large increases in Ob, Oc, and Fb surfaces, and the Fbs were surrounded by an extensive volume of extracellular unmineralized fibrous matrix. During HPT in humans, the parathyroid glands secrete PTH (1–84) and N terminally truncated PTH. However, continuous administration of PTH (1–34) was sufficient to induce parathyroid bone disease in the rat. Cessation of PTH treatment resulted in similarly remarkable changes; within 1 wk Oc surface had returned to normal and no peritrabecular fibrosis was detected. These findings suggest that restoration of normal PTH levels is correlated with rapid correction of the skeletal manifestations of parathyroid bone disease.
The effects of HPT on bone turnover depend, in part, on available calcium. In agreement with earlier work, continuous PTH resulted in hypercalcemia as well as increases in bone formation and bone resorption (11, 12, 15, 16, 17, 18). In the present study, continuous PTH treatment for 4 wk resulted in a large increase in BV/TV. The anabolic effects of continuous PTH on bone matrix production in hypercalcemic rats outweighed the catabolic effects on bone resorption. These findings are in agreement with earlier work (17, 18) and contrast to the bone loss observed in calcium-deficient HPT rats, in which there was a net increase in bone resorption (22, 23).
BV/TV increased after discontinuation of PTH treatment. This was, in part, because the increases in Ob surface and bone formation rate induced by continuous PTH persisted longer than the increase in Oc surfaces.
HPT patients have an increased fracture risk, largely due to a reduction in bone quality (24). PTH-induced osteomalacia, peritrabecular fibrosis, focal bone resorption, and high bone turnover may all contribute to diminished bone quality. In patients, HPT is associated with increases, decreases, or no change in cancellous bone mass, with the last being the most common outcome (25, 26). Additional factors that influence bone volume in HPT patients are not well described. Several studies have reported that parathyroidectomy in these patients leads to increased bone formation and bone mass as well as resolution of osteitis fibrosa (27, 28). In this regard, HPT patients respond similarly to rats after normalization of PTH levels. However, long-duration suppression of PTH in patients with chronic renal failure through the use of calcium-based phosphate binders and vitamin D therapy can result in adynamic bone disease (29).
The 430% increase in Ob.S/BS measured during the first 5 d of continuous PTH treatment was not associated with Ob proliferation. As was observed for intermittent PTH treatment (12), very few of the continuous PTH-induced Obs had passed through S phase of the cell cycle. Ob apoptosis has been reported in some studies to be inhibited by intermittent PTH, whereas in others apoptosis was increased (30, 31). A change in Ob life span is one pathway by which Ob number could be altered without a corresponding change in [3H]thymidine-labeled Ob. However, Ob turnover in adults is very low. Consistent with this view, only a small percentage of the Obs in vehicle-treated rats became labeled after continuous exposure to radioactive thymidine. Because Obs have a long life span (weeks to months), compared with the time interval required for continuous PTH treatment to dramatically increase their number (days), a change in their death rate would have negligible impact on this early increase in Ob number. A more plausible explanation for the rapid increase in osteoblast surface is modulation of postmitotic osteoblast lineage cells such as bone-lining cells and committed preosteoblasts to express the osteoblast phenotype.
Most of the peritrabecular Fbs induced by continuous PTH were labeled with 3H-thymidine, indicating that they had passed through S phase of the cell cycle during the treatment protocol. Thus, continuous PTH is a potent stimulator of Fb proliferation. PTH has been reported to have effects on a number of fibroblast populations, and PTH receptors have been described on dermal fibroblasts (32). Platelet-derived growth factor (PDGF) A is associated with pathological tissue fibrosis in several organs (33, 34) and PDGF-A expression is increased in rat bone by continuous PTH (15). It is possible that the recruitment of Fbs to bone surfaces in continuous PTH-treated rats is in response to paracrine factors that are overexpressed in target cells after continuous infusion of PTH.
The gene expression profile of rat long bones by DNA microarray indicated that continuous PTH up-regulated the mRNA levels for osteocalcin and osteonectin (35). In addition, PTH significantly increased the gene expression for decorin, lysyl oxidase, collagen I, collagen III, glypican I, and FGF receptor, which are associated with tissue remodeling and fibrosis in other tissues (36, 37, 38, 39, 40, 41, 42). Trapidil, a PDGF signaling antagonist, blunts peritrabecular fibrosis in the rat model for chronic HPT (15). Trapidil antagonized the increase in expression of several genes in cancellous bone, including lysyl oxidase (our unpublished results). PDGF-A signaling, therefore, might play a key role in PTH-induced tissue remodeling.
Obs are specialized fibroblasts that differ from all other fibroblasts in that they produce bone matrix (43). Fully differentiated Obs are morphologically distinct from other Fbs, whereas preosteoblasts are not. The latter were recognized in this study by their location on bone surfaces, expression of osteoblast marker proteins, and ability to differentiate into Obs. The detection of the latter process was greatly facilitated in this study by prelabeling the PTH-induced preosteoblast population with radioactive thymidine.
The labeled Obs and Ocys observed at the end of the study had to have been derived from cells that went through S phase 1–2 wk earlier. Furthermore, these cells were arrested in G0 phase of the cell cycle after labeling; otherwise the label would have been diluted by subsequent cell divisions and, as was the case of most hematopoietic cells, would no longer be detectable.
Discontinuation of PTH treatment resulted in the rapid disappearance of radiolabeled Fbs as well as the matrix that surrounded these cells and a corresponding increase in [3H]thymidine-labeled Obs and Ocys located on and within mineralized bone matrix, respectively. These findings provide nearly unequivocal evidence that the labeled Fbs had differentiated to Obs and Ocys and are in agreement with an earlier study in mice with surgery-induced renal failure (44). Osteocalcin, osteonectin, and cbfa 1, osteoblast marker peptides (45, 46), were localized to Fb cells or the surrounding extracellular matrix by immunohistochemistry, further supporting the conclusion that Fb cells are preosteoblasts.
The origin of the Fbs targeted by PTH is under investigation but as of yet is unknown. PTH regulates many genes related to Ob differentiation, including bone morphogenic proteins and members of the Wnt signaling system (35, 47). Mesenchymal stem cells are a candidate because they are capable of differentiating to Obs (48) and PTH-responsive spindle-shaped bone marrow Obs might participate in regulation of hematopoietic stem cell niche (49). However, it is equally possible that PTH targets a cell population that is already committed to the Ob lineage.
In summary, we have shown that PTH treatment can increase Ob surface by at least two distinct cellular mechanisms: 1) rapid modulation of postmitotic cells to express the Ob phenotype; and 2) attraction of marrow Fbs to bone surfaces, expansion of the Fb population by proliferation, and differentiation of these cells to Obs. The former mechanism occurs after both intermittent and continuous PTH treatment. The latter largely unrecognized mechanism requires longer exposure to elevated PTH levels and may represent a target for the development of a new class of drugs. Targeted recruitment and differentiation of Fbs to Obs may provide a novel approach to reverse osteoporosis.
Acknowledgments
The authors thank Ms. Lori Rolbiecki for typing this manuscript and Ms. Peggy Backup for editorial assistance. We also thank Dr. Larry Fisher (National Institute of Dental and Craniofacial Research, Bethesda, MD) and Dr. Gerard Karsenty (Baylor College of Medicine, Houston, TX) for providing antisera used for the immunohistochemical detection of osteoblast protein markers, LF-23 osteonectin, and cbfa1, respectively.
Footnotes
This work was supported by National Institutes of Health Grant AR48833 and National Aeronautics and Space Administration Grant NAG9-1458.
Abbreviations: BFR/BS, Bone formation rate expressed per bone surface; BFR/BV, bone formation rate expressed per bone volume; BFR/TV, bone formation rate expressed per tissue volume; BV/TV, bone volume expressed per tissue volume; cbfa1, core binding factor 1; dL.S/BS, double-labeled surface expressed per bone surface; Fb, fibroblast; Fb.S/BS, Fb surface expressed per bone surface; HPT, hyperparathyroidism; MAR, mineral apposition rate; Ob, osteoblast; Ob.S/BS, Ob surface expressed per bone surface; Oc, osteoclast; Oc.S/BS, Oc surface expressed per bone surface; Ocy, osteocyte; Tb.N, trabecular number; Tb.Sp, trabecular separation; Tb.Th, trabecular thickness.
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