Enhanced osteoblast development after continuous infusion of hPTH(1-84) in the rat

Bone Vol. 24, No. 2 February 1999:89 –94

Enhanced Osteoblast Development After Continuous Infusion of hPTH(1-84) in the Rat P. H. WATSON,1 L. J. FRAHER,1 M. KISIEL,1 D. DESOUSA,1 G. HENDY,2 and A. B. HODSMAN1 1

Departments of Medicine and Biochemistry, The University of Western Ontario and The Lawson Research Institute, St. Joseph’s Health Centre, London, Ontario, Canada 2 Calcium Research Laboratory, Royal Victoria Hospital, McGill University, Montreal, Quebec, Canada

Introduction Rats and humans respond to intermittent treatment with parathyroid hormone (PTH) with increased bone density and cancellous bone volume. In the rat, osteoblast expression of insulin-like growth factor-I (IGF-I) is elevated by intermittent PTH. We examined the effect of continuous infusion of rhPTH(1-84), a bone catabolic regime, on the IGF system in rat pelvis. Female Sprague-Dawley rats (12 weeks, 250 g) were randomly assigned to receive 0, 0.1, 1, or 5 mg/100 g body weight (b.w.) rhPTH(1-84) (0, 0.106, 1.06, or 5.305 nmol/kg) in vehicle (1% normal rat serum in saline) delivered by subcutaneous Alzet minipump. After 7 days, blood was taken for serum chemistry and pelvises were processed for immunocytochemistry. Sections of pelvis from rats continuously infused with 0.1 or 1 mg/100 g b.w. rhPTH(1-84) for 7 days did not differ significantly from those of the vehicletreated controls. However, continuous infusion of 5 mg/100 g b.w. rhPTH(1-84) resulted in a dramatic increase in cellular development, with trabeculae surrounded by many layers of large, plump osteoblasts. All pelvis osteoblasts expressed osteocalcin, but only those from rats that received 0, 0.1, or 1 mg/100 g b.w. rhPTH(1-84) showed positive staining for IGF-I. The extra-abundant osteoblasts from rats that received 5 mg/100 g b.w. rhPTH(1-84) did not stain for IGF-I. However, although all osteoblasts stained positively for IGF binding proteins (IGFBPs)-3, -4, and -5, staining for these IGFBPs increased as the dose of rhPTH(1-84) (and osteoblast number) increased. These results suggest that continuous infusion of PTH has a direct effect on osteoblast development (either recruitment or proliferation), decreases the expression of IGF-I, and enhances the expression of IGFBPs in pelvis, factors which may interact to bring about negative bone balance. (Bone 24:89 –94; 1999) © 1999 by Elsevier Science Inc. All rights reserved.

Parathyroid hormone (PTH) is a multifunctional molecule that, in bone, can either initiate bone turnover through the activation of bone metabolic units (BMUs), with the end result being the net resorption of bone, or directly activate the formation of new bone, not necessarily via the BMU.12 The current paradigm is that continuous exposure to PTH results in bone resorption, while intermittent exposure to PTH results in bone formation. However, early on in the modern investigation of PTH, it was noted in some patients with primary hyperparathyroidism (PHPT) that, although cortical bone mineral density was indeed reduced, cancellous bone appeared to be preserved and even augmented.20 Further studies have shown an increased apposition rate,16 enhanced activation frequency, and trabecular bone remodeling, leading to conservation of bone volume,5 increased cancellous bone volume with increased trabecular bone with conserved connectivity,13 and, in children, enhanced cancellous bone formation leading to osteosclerosis.3 Studies of PTH infusions in both dogs11,14 and rats8,17 have led to the conclusion that cancellous bone mass is either unchanged11,14 or reduced17 and that indices of both formation and resorption are increased.8,11,14,17 In a more recent study, Hock and Gera6 showed that in the intact rat, infusion of 4 mg/100 g hPTH(1-34) enhanced trabecular bone mass much the same as a dose of 8 mg/100 g human parathyroid hormone (hPTH)(1-34) given intermittently. This last dose had no effect when continuously infused. It was also noted that both routes of PTH administration were capable of activating parameters of bone formation independent of the PTH effect on bone resorption. Since we are interested in the anabolic actions of PTH in bone, we wished to contrast our work on anabolic treatment with PTH with continuous infusion in the rat. This initial study was undertaken to explore the effects of continuous infusion of low doses of hPTH(1-84) on the expression of insulin-like growth factor (IGF) system components and the PTH/Parathyroid hormone related peptide (PTHrP) receptor in trabecular bone osteoblasts.

Key Words: Parathyroid hormone; Osteoblast; Insulin-like growth factor-I (IGF-I); Parathyroid hormone/Parathyroid hormone related peptide (PTH/PTHrP) receptor immunocytochemistry; IGF binding proteins (IGFBPs).

Materials and Methods Animals All procedures were carried out with approval from the Standing Committee on Animal Care of The University of Western Ontario and following the guidelines set out by the Canada Council on Animal Care. Twelve intact female Sprague-Dawley rats (12 weeks of age; approximately 250 g body weight [b.w.]) were

Address for correspondence and reprints: Patricia H. Watson, Ph.D., Research Associate, Lawson Research Institute, St. Joseph’s Health Centre, 268 Grosvenor St., London, ON N6A 4V2, Canada. E-mail: [email protected] © 1999 by Elsevier Science Inc. All rights reserved.

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Table 1. Serum chemistry values Treatment Calcium (mmol/L) Phosphorus (mmol/L) Alkaline phosphatase (U/L) Creatinine (U/mol) Magnesium (mmol/L) Albumin (g/L)

Control

0.1 mg/100 g

1.0 mg/100 g

5.0 mg/100 g

2.62 6 0.12 2.55 6 0.23 160 6 66 51 6 4.6 1.04 6 0.07 33 6 3.1

2.64 6 0.05 2.48 6 0.24 123 6 32 49 6 2.0 1.09 6 0.06 35 6 2.0

2.62 6 0.17 2.54 6 0.22 256 6 254 48 6 4.6 1.06 6 0.12 31 6 5.6

2.84 6 0.48 2.27 6 0.70 209 6 55 46 6 1.0 1.04 6 0.06 28 6 5.0

Results shown are the means 6 standard deviations of n 5 3/group. Data were analyzed by ANOVA and no significant differences between treatment groups were detected.

randomly assigned to one of four treatment groups: control, and 0.1, 1, or 5 mg/100 g b.w./day (0, 0.106, 1.06, or 5.305 nmol/kg hPTH[1-84]) (Allelix Biopharmaceuticals, Mississauga, Ontario, Canada). hPTH(1-84) was delivered by osmotic minipump (Alzet) implanted subcutaneously under ketamine anesthesia. Rats were allowed food and water ad libitum for the 7 days of the experiment. Rats were decapitated following carbon dioxide narcolepsy; their blood was collected and pelvic bones were removed for histology and histochemistry.

Histomorphometry Histomorphometry was performed on 5 mm sections of decalcified, paraffin-embedded pelvis using the OsteoMeasure, Version 3.0, image analysis system (OsteoMetrics, Decatur, GA). Two parameters were determined for each animal, the osteoblast surface (Ob.S/BS; %, bone surface referent) and the number of osteoblasts (N.Ob./BS; #/mm2, bone surface referent). Data Analysis

hPTH(1-84) Recombinant hPTH(1-84) (specific activity ;4000 IU/mg) was dissolved in saline containing 1% normal rat serum, sterile filtered, and loaded by syringe into osmotic minipumps which delivered the solution at a rate of 5 mL/h. Tissue At sacrifice, blood was collected and allowed to clot, and serum was frozen at 220°C until analyzed. Standard serum chemistry was performed in the hospital laboratory autoanalyzer. Pelvis bones were removed and fixed in 4% paraformaldehyde– 0.2% glutaraldehyde overnight at 4°C. Bones were washed thoroughly in 70% ethanol followed by phosphate-buffered saline (PBS) and decalcified in 10% ethylenediaminetetraacetic acid in PBS for 3 weeks at 4°C. Bones were embedded in paraffin; 5 mm sections were cut and either stained with 0.1% thionin or subjected to immunocytochemistry (ICC) and in situ hybridization (ISH). Immunocytochemistry Immunocytochemistry was performed as described elsewhere9 using the Vectastain ABC immunoperoxidase kit (Vector Labs, Torrance, CA) and diaminobenzidine (DAB) as the chromogen. Rabbit antirat osteocalcin (1/1000) was a generous gift of Dr. Modrowski (INSERM, Paris, France) and antirat IGF-I (1/1000) was obtained from Dr. B. Breir (New Zealand). Insulin-like growth factor binding protein (IGFBP)-3, -4, and -5 was detected using rabbit antihuman antibodies at a 1/100 dilution (UBI, Lake Placid, NY). Negative controls substituted nonimmune rabbit serum for the primary antibody. All sections were counterstained with Carazzi’s hematoxylin. ISH In situ hybridization was performed as described previously, following standard protocols using 35S-labeled antisense riboprobes for the PTH/PTHrP receptor1 and for IGF-I.18 Control sections were hybridized with the corresponding 35S-labeled sense probes.

Numerical data were analyzed by analysis of variance (ANOVA) followed by Tukey’s test for difference of means using SPSS 7.5 Statistics software (SPSS). Results Animals were sacrificed on day 7 of the infusion; serum chemistry is shown in Table 1. Analysis by ANOVA did not detect any significant differences in serum calcium, phosphorus, creatinine, magnesium, or albumin. Although not statistically significant, rats treated with 1 or 5 mg/100 g b.w./day hPTH(1-84) had elevated serum alkaline phosphatase levels. The general morphology of the pelvis of control and treated rats is shown in Figure 1. Morphology of cancellous tissue appeared normal in all groups except those animals receiving 5 mg/100 g b.w./day hPTH(1-84) (high dose). Sections of pelvis from animals in that group showed extensive cellular development, presumably osteoblasts, surrounding cancellous trabeculae. Table 2 shows the results of histomorphometric osteoblast measurements. Animals receiving the high dose of PTH for 7 days had significantly greater osteoblast number and osteoblast surface (bone surface referent) than animals from the control group (15.832 6 3.102 vs. 0.428 6 0.449 and 29.249 6 4.052 vs. 0.583 6 0.639, respectively; p , 0.0001). These data do not allow us to distinguish between increased proliferation and increased recruitment as the source of the enhanced osteoblast investment. The results of immunocytochemistry staining for osteocalcin, IGF-I, and IGFBP-3, -4, and -5 are shown in Figure 2. Since there was no difference in staining pattern and intensity in the pelvis between controls and animals receiving the two lowest doses of PTH (0.1 and 1 mg/100 g b.w./day), only the results from the control and high-dose-treated animals are shown. The multiple cell layers surrounding trabeculae in the pelvis of high-dose animals are osteocalcin positive (Figure 2D), as are the controls (Figure 2C), indicating that they are indeed osteoblasts. These cells were IGF-I positive in control animals (Figure 2E), as were those from animals receiving PTH at the two lower doses. However, the osteoblasts in the high-dose group (Figure 2F) showed no staining for IGF-I, a result that was highly reproducible from sample to sample. Osteoblasts in the pelvis of control

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Figure 1. General morphology of cancellous bone in the pelvis from control rats (A) and rats infused with 0.1 (B), 1.0 (C), or 5 (D) mg/100 g b.w./day hPTH(1-84). Five micrometer sections cut from paraffin-embedded tissues were stained with 0.1% thionin to demonstrate the morphology of the tissue. Note the extensive cellular development surrounding trabeculae in the pelvis of rats infused with the highest dose of PTH (arrows, [D]). KEY: m, marrow; t, trabeculum. Original magnification 356; scale bar 5 300 mm.

animals were positive for IGFBP-3, -4, and -5 (Figure 2G,I,K, respectively). Sections of pelvis from the high-dose group stained very intensely for all three IGFBPs tested (Figure 2H,J,L), most likely owing to the presence of more cells capable of producing these proteins. It was also apparent that megakaryocytes and a subpopulation of marrow stromal cells also stain positively for the IGF system peptides studied. Representative ISH results are shown in Figure 3. Very low levels of hybridization for the PTH/PTHrP receptor were present in pelvis sections from control (Figure 3C) and the two low-dose (results not shown) groups, while sections from the high-dose group had an intense signal around bony trabeculae (Figure 3D). When sections were probed for the presence of IGF-I mRNA, all showed positive signals despite not detecting the presence of IGF-I protein in the high-dose group (control and high-dose groups are shown in Figure 3E,F, respectively).

Discussion We have shown that a short (7 day) infusion of 5 mg/100 g b.w./day (;5.3 nmol/kg/day) hPTH(1-84) (but not 0.1 or 1.0 mg/100 g b.w./day) in the intact female rat results in extensive osteoblast development around trabeculae in the pelvis, similar to our previous results with intermittent injections of 8 mg/100 g b.w./day (;10 nmol/kg/day) hPTH(1-34).18 Whole trabeculae, and not just scalloped surfaces, are surrounded by these multiple layers of large, plump osteoblasts (Figure 1). These results suggest that there is recruitment and/or proliferation on quiescent surfaces in addition to the usual recruitment to freshly made resorption cavities. The observation that these cells are also osteocalcin positive indicates that they are mature, differentiated osteoblasts.15 Since PTH increases bone turnover, the observation of more bone-forming surfaces with active osteoblasts is not

Table 2. Selected histomorphometric measures

Treatment Ob.S/BS (%) N.Ob/BS (/mm2)

Control

0.1 mg/100 g b.w. hPTH(1-34)

1.0 mg/100 g b.w. hPTH(1-34)

5.0 mg/100 g b.w. hPTH(1-34)

0.583 6 0.639 0.428 6 0.449

1.015 6 0.471 0.776 6 0.321

1.457 6 0.566 1.0373 6 0.261

29.249 6 4.052a 15.832 6 3.102a

Results are the means 6 standard deviations of n 5 3/group. ANOVA showed that treatment with hPTH(1-34) significantly affected both histomorphometric parameters (p , 0.0001). a p , 0.0001 vs. control.

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Figure 3. In situ hybridization in the pelvis of rats treated with 0 or 5 mg/100 g b.w./day hPTH(1-84). Five micrometer sections cut from paraffin-embedded tissues were examined for the presence of PTH/PTHrP receptor (C,D) and IGF-I (E,F) transcripts using the appropriate 35S-labeled antisense riboprobe. Control sections were hybridized with the corresponding 35S-labeled sense riboprobes, and a representative result—in this case, using the IGF-I sense probe—is shown in (A,B). Note the presence of IGF-I transcripts after PTH infusion (F). Arrows point to areas of specific hybridization in osteoblasts. KEY: m, marrow; t, trabeculum. Original magnification 3140 except (C,D), where original magnification 3224; scale bar 5 100 mm.

entirely unexpected. However, the unexpectedly large increase in osteoblast number may explain, in part, previous observations of increased cancellous bone volume, enhanced activation frequency, and increased cancellous bone formation in patients with PHPT.3,5,13,16,20 Bone formation rates in PHPT patients were 1.5 to 2 times normal,5,16,20 yet our study indicates disproportionately large osteoblast numbers which may account for the preservation of bone in PHPT patients. This study is the first to examine the effects of short-term PTH infusion on the IGF system in trabecular osteoblasts. The IGFBPs IGFBP-3, -4, and -5, which are present in the osteoblasts of control animals, were abundantly detected in the osteoblasts from the PTH-infused rats suggesting that these are normally functioning mature osteoblasts capable of secreting IGFBPs.2 All three binding proteins examined in this study are capable of binding IGF-I with high affinity when not bound to either membrane or matrix themselves.7 IGF-I bound to any of the binding proteins may not be immunologically recognized by the antibody used in this study. This fact may explain the absence of immunologically detectable IGF-I in the osteoblasts of rats infused with 5 mg/100 g b.w./day hPTH(1-84). IGFBP-4 is an inhibitor of IGF-I biological activity in osteoblast-like cells4,7 and its enhanced expression in this study may also partly explain

why continuous infusion of PTH is not anabolic.17 Since infusion of PTH has a net negative effect on bone,17 the absence of available IGF-I protein and, therefore, IGF-I-mediated bone formation10 offers one possible explanation for this phenomenon. There was no reduction in the ability of osteoblasts from PTH-infused rats to produce the mRNA for IGF-I. Hock and Gera6 suggested that although PTH infusion increases IGF-I transcripts in osteoblasts, perhaps secretion of the peptide occurs only in the absence of PTH, thus explaining the anabolic nature of intermittent treatments. Since IGF-I peptide was not immunologically detectable in this study, in addition to the consequences of the altered IGFBP expression noted above, there may also be an inability to translate IGF-I message into peptide in the continuous presence of PTH. Very low levels of detectable mRNA for the PTH/PTHrP receptor were found in pelvic osteoblasts of control animals. Previous studies have also shown that flat (inactive) osteoblasts do not express the PTH/PTHrP receptor transcript to any great degree.10 We previously reported that cancellous osteoblasts in both ovariectomized (ovx) rats and ovx rats treated intermittently with PTH express very large amounts of PTH/PTHrP receptor mRNA.19 It appears that PTH induces its own receptor expression in the rat regardless of mode of administration. This study

Figure 2 (See previous page). Immunocytochemistry in the pelvis of rats treated with 0 or 5 mg/100 g b.w./day hPTH(1-84). Five micrometer sections cut from paraffin-embedded tissues were examined for the presence of osteocalcin (OC; [C,D]), IGF-I (E,F), IGFBP-3 (G,H), IGFBP-4 (I,J), and IGFBP-5 (K,L). Control sections were treated identically except for the omission of primary antibody (A,B). Note the absence of staining for IGF-I after PTH infusion (F). Arrows point to osteoblasts. KEY: m, marrow; t, trabeculum. Original magnification 3140; scale bar 5 100 mm.

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did not address the issue of PTH/PTHrP receptor trafficking or binding, so no conclusions as to what the downstream mechanism is for the net difference PTH effect can be made. The most significant observation of this study is that continuous infusion of 5 mg/100 g b.w./day hPTH(1-84) (;5.3 nmol/ kg/day) in the intact rat gives rise to increased proliferation and recruitment and differentiation of osteoblasts around pelvis trabeculae, similar to our previous findings in ovx rats treated intermittently with 8 mg/100 g b.w./day (;10 nmol/kg/day) hPTH(1-34).18 Hock and Gera6 obtained comparable anabolic effects in rats infused with 4 mg/100 g b.w./day (;5 nmol/kg/ day) but not 8 mg/100 g b.w./day (;10 nmol/kg/day) hPTH(134). However, our results demonstrate that a significant bone growth factor, IGF-I, appears to be absent from the system. The lack of detectable IGF-I peptide after PTH infusion may explain why both intermittent and continuous treatment with PTH activate osteoblast recruitment and development, but only intermittent PTH consistently leads to increased bone formation.

Acknowledgment: This study was supported by Grant MT-13199 from The Medical Research Council of Canada.

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12. 13.

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Date Received: August 4, 1998 Date Revised: October 9, 1998 Date Accepted: October 9, 1998