− mice

BBRC Biochemical and Biophysical Research Communications 341 (2006) 989–994 www.elsevier.com/locate/ybbrc

Impaired bone anabolic response to parathyroid hormone in Fgf2 / and Fgf2+/ mice M.M. Hurley a,*, Y. Okada b, L. Xiao a, Y. Tanaka b, M. Ito c, N. Okimoto b, T. Nakamura b, C.J. Rosen d, T. Doetschman e, J.D. Coffin f b

a University of CT Health Center, Farmington, CT, USA University of Occupational and Environmental Health, Kitakyushu, Japan c University of Nagasaki, Nagasaki, Japan d Jackson Laboratory, Bangor, ME, USA e University of Cincinnati, Cincinnati, OH, USA f University of Montana, Missoula, MT, USA

Received 9 January 2006 Available online 24 January 2006

Abstract Since parathyroid hormone (PTH) increased FGF2 mRNA and protein expression in osteoblasts, and serum FGF-2 was increased in osteoporotic patients treated with PTH, we assessed whether the anabolic effect of PTH was impaired in Fgf2 / mice. Eight-week-old Fgf2+/+ and Fgf2 / male mice were treated with rhPTH 1–34 (80 lg/kg) for 4 weeks. Micro-CT and histomorphometry demonstrated that PTH significantly increased parameters of bone formation in femurs from Fgf2+/+ mice but the changes were smaller and not significant in Fgf2 / mice. IGF-1 was significantly reduced in serum from PTH-treated Fgf2 / mice. DEXA analysis of femurs from Fgf2+/+, Fgf2+/ , and Fgf2 / mice treated with rhPTH (160 lg/kg) for 10 days showed that PTH significantly increased femoral BMD in Fgf2+/+ by 18%; by only 3% in Fgf2+/ mice and reduced by 3% in Fgf2 / mice. We conclude that endogenous Fgf2 is important for maximum bone anabolic effect of PTH in mice. Ó 2006 Elsevier Inc. All rights reserved. Keywords: Fgf2 null; Fgf2 haploinsufficient; Mice; PTH; Bone formation; DEXA; Micro-CT

Parathyroid hormone (PTH) functions to maintain normal serum calcium and is a major regulator of bone cell function [1]. PTH has been shown to increase bone formation in vivo [1], decrease bone formation in vitro [2], and stimulate bone resorption [3]. Although PTH is a potent bone resorber [4,5], it is currently utilized for the treatment of osteoporosis, since studies have shown that intermittent PTH treatment increases bone mass and strength in osteopenic rats and improves vertebral bone mass in humans [6,7]. Several mechanisms are postulated to mediate the anabolic effects of PTH. They include increased production of local growth factors [8–10], activation of bone lining

*

Corresponding author. Fax: +1 860 679 1258. E-mail address: [email protected] (M.M. Hurley).

0006-291X/$ - see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.01.044

cells [11], recruitment of osteoblast precursors [12], and reduced apoptosis [13–15]. Although the local factors that mediate the anabolic effects of PTH have not been fully defined, several candidates have been identified. Since PTH treatment increases insulin-like growth factor 1 (IGF-1) and transforming growth factor beta (TGFb) expression in bones cells [8–10], they are considered as potential mediators of the anabolic effect of PTH in bone. The anabolic effect of PTH appears to be mediated in part by IGF-1 [9] since studies in Igf1 null mice demonstrated impaired anabolic response to PTH [16]. However, the role of other anabolic growth factors such as fibroblast growth factor 2 (FGF-2) [17] in the anabolic response to PTH has not been characterized. FGF-2 regulates the proliferation and differentiation of osteoblasts in vitro [17] is stored in the extracellular matrix

990

M.M. Hurley et al. / Biochemical and Biophysical Research Communications 341 (2006) 989–994

(ECM) [17], and is synthesized by osteoblast/stromal cells [17]. FGF-2 is an important modulator of cartilage and bone growth and differentiation [17]. Fibroblast growth factor receptors (FGFR) are also important regulators of bone growth and development and are differentially expressed in bone and cartilage [17]. In contrast to the inhibitory effect of chronic treatment [17–19], in animal models, intermittent FGF-2 treatment stimulated bone formation in vitro [20], as well as in vivo [21]. In addition, several investigators reported that FGF-2 enhanced the growth and expression of the osteogenic phenotype of dexamethasone-treated human bone marrow-derived bonelike cells [22,23] resulting in the deposition of greater mineralized matrix than in control cultures [24]. In previous studies, we reported that the expression of FGF-2 mRNA and protein in murine osteoblasts was increased by PTH [25]. In vitro [26] and in vivo [27] studies have shown that FGF-2 also up-regulated the expression of IGF-1 in rodent osteoblasts that is implicated in the anabolic response to PTH [28]. We previously reported that Fgf2 null mice [29] developed decreased bone mass and bone formation rates with age suggesting a role for endogenous FGF-2 in maintaining bone mass [30]. In other studies we reported that Fgf2 null mice form fewer osteoclasts in culture and have an impaired hypercalcemic response to high dose acute PTH in vivo [31]. Since we recently observed that the anabolic response to PTH in humans was associated with increased serum levels of FGF-2 [32], we utilized the Fgf2 null and haploinsufficient mice to assess whether the anabolic response to PTH was impaired in these mutant mice. Materials and methods Animals. Development of Fgf2 null mice was previously described [29]. Heterozygote Fgf2+/ mice that are maintained on a Black/Swiss/129 Sv background were bred and housed in the transgenic facility in the Center for Laboratory Animal Care at the University of Connecticut Health Center. Genotyping of mice was performed using primers as previously described [29,30]. Animal protocols were approved by the University of Connecticut Health Center’s Animal Care Committee. In vivo bone formation assay. Eight-week-old male mice or 15-monthold female Fgf2+/+ and Fgf2 / mice were weighed and injected s.c. once daily with vehicle or PTH 1–34 (80 lg/kg body wt) for 4 weeks. Human recombinant PTH 1–34 was purchased from Bachem, Torrence, CA. Mice were injected with calcein (0.6 mg/kg) on day 18 and day 24 to assess new bone formation. In shorter studies, 8-week-old male Fgf2+/+, Fgf2+/ , and Fgf2 / mice were weighed and injected s.c. once daily with vehicle or PTH 1–34 (160 lg/kg body wt) for 10 days and femoral bones were harvested for measurement of bone mineral density. Measurement of bone mineral density. Femoral bones were harvested and stored in 70% ethanol. Bone mineral density (BMD) was measured by dual energy X-ray absorptiometry (DEXA; PIXimus Mouse 11 densitometer (GE Medical System, Madison WI). Micro-computerized tomography. Quantitative micro-computed tomography (Micro-CT) analysis of the metaphyseal cancellous bones of the distal femurs was performed as previously described [30] with MicroCT instrumentation (lCT20, Scanco Medical AG, Bassersdorf, Switzerland). Using two-dimensional data from scanned slices, three-dimensional analysis was conducted to calculate morphometric parameters defining trabecular bone mass and micro-architecture including bone volume

density (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), trabecular spacing (Tb.Sp), and the structure model index (SMI), an indicator of plate-like versus rod-like trabecular architecture. Bone histomorphometry. Trabecular bone architecture of the femoral metaphysis of mice was determined by dynamic histomorphometry. Bones were dehydrated in a progressive ethanol series for histological analysis to determine dynamic parameters of bone formation. The Bioquant program for histomorphometry (Bioquant-True Color Windows, Advanced Image Analysis Software, sVGA Frame Grabber Image Processing Board, Optronics DEI-470 Video Camera and Specialized Computer) uses a light microscope for histomorphometric measurements. Bone histomorphometry [33,34] was performed on femoral bones. For static histomorphometry, bones were isolated fixed in 4% paraformaldehyde at 4 °C, dehydrated in progressive concentrations of ethanol, cleared in xylene, and embedded in paraffin. Five micron sections were cut, deparaffinized, and stained for osteoclast using tartrate-resistant acid phosphatase kit (TRAP) according to the manufacturer’s instructions. Statistical analysis was performed using paired Student’s t test or analysis of variance (ANOVA) to determine differences between groups. IGF-1 radioimmunoassay. IGF-protein was measured using a commercially available kit (ALPCO, Windham, NH) as previously described [35]. IGFBPs were removed by an acid dissociation step, followed by the addition of a neutralization buffer containing excess recombinant human IGF-II that bound IGFBPs prior to immunoassay with a human antiIGF-I polyclonal antibody. The assay sensitivity is 0.01 ng/ml IGF-I. There is no cross-reactivity with IGF-II. Statistic analysis. The significance of difference between two groups was evaluated with an unpaired two-tailed Student’s t test. For the comparison among multiple groups analysis of variance (ANOVA) was used.

Results We assessed whether PTH induced trabecular bone formation was impaired in Fgf2 / mice. We first examined the effect of PTH in growing Fgf2 knockout and WT male mice. Knockout mice were healthy with normal fertility. The mice (8-week-old male mice) were given daily subcutaneous injections of bPTH (1–34) (80 lg/kg) or vehicle for 4 weeks after which bones were harvested for Micro-CT and histological analyses. Although Micro-CT (Fig. 1 and Table 1), analysis of femoral bones from vehicle-treated Fgf2 / mice revealed a small reduction in trabecular

Fig. 1. Micro-CT of the effect of administration of rhPTH (80 lg/kg body wt) on bone formation in Fgf2+/+ and Fgf2 / mice. Eight-week-old male Fgf2+/+ and Fgf2 / mice were injected subcutaneously once daily for 4 weeks with either vehicle or RhPTH as described under experimental procedures. Representative Micro-CT 3-dimensional image of femurs harvested from each treatment group and genotype are shown.

M.M. Hurley et al. / Biochemical and Biophysical Research Communications 341 (2006) 989–994 Table 1 Micro-CT analysis of parameters of bone formation in femoral metaphysis of 8-week-old Fgf2+/+ and Fgf2 / rhPTH (80 lg/kg body wt) Fgf2+/+

BV/TV Tb.N Tb.Sp SMI

Fgf2 /

Vehicle

PTH

27.0 5.7 130.0 1.9

37.1 6.6 95.0 1.3

(2.4) (0.3) (9.3) (0.1)

(2.1) (0.1) (4.9) (0.2)

male mice treated for 4 weeks with ANOVA (Effect of PTH)

Vehicle

PTH

19.9 4.7 185.0 2.4

25.4 5.2 150.0 1.9

(2.9) (0.4) (31.8) (0.2)

991

(3.4) (0.4) (21.3) (0.6)

+/+

/

<0.05 <0.05 <0.05 <0.05

NS NS NS NS

N = 6 bones/group. Values are means with (SE) in parentheses. One-way ANOVA and post hoc test (Tukey–Kramer).

bone volume (BV/TV) and trabecular number (Tb.N), these changes were not significant and are consistent with our earlier reports that Fgf2 / mice develop low bone mass with age [30]. PTH increased femoral BV/TV and trabecular number (Tb.N) by 27% and 14%, respectively, in Fgf2+/+ mice with no significant increases in Fgf2 / mice. PTH treatment also significantly reduced trabecular separation (Tb.Sp) and structure model index (SMI) in Fgf2+/+ but not in Fgf2 / mice. Static histomorphometric analysis confirmed the Micro-CT results in that femoral BV/TV was significantly increased by 41% in femoral bones from Fgf2+/+ but insignificantly increased in bones from Fgf2 / mice (Table 2A). Trabecular thickness was also significantly increased by PTH in Fgf2+/+ but was unchanged in Fgf2 / mice. As shown in Table 2B, dynamic histomorphometry demonstrated that PTH caused a significant increase in double-labeled surface,

mineralized surface, bone formation rate, and osteoblast surface in Fgf2+/+ but not Fgf2 / mice. Similar to our observation in young male mice, we observed that in vivo administration of PTH to 15month-old female mice induced an anabolic response in femoral bones of Fgf2+/+ but not Fgf2 / mice. As shown in Table 3A, BV/TV and TbN were significantly increased in PTH-treated femurs from female Fgf2+/+ mice when compared with vehicle-treated femurs from Fgf2+/+ mice. Tb.Sp was also significantly reduced by PTH in femoral bones of Fgf2+/+ mice. In contrast, there was no significant effect of PTH on these parameters in the Fgf2 / mice. Dynamic histomorphometric analysis of femurs of these 15-month-old female mice showed that double-labeled surface, mineralized surface, and bone formation rate were significantly increased in PTH-treated Fgf2+/+ but not Fgf2 / mice (Table 3B).

Table 2A Static histomorphometric parameters of femur metaphysis of 8-week-old Fgf2+/+ and Fgf2 / body wt) Fgf2+/+

BV/TV Tb.N Tb.Th Tb.Sp

male mice treated for 4 weeks with rhPTH (80 lg/kg

Fgf2 /

ANOVA (Effect of PTH)

Vehicle

PTH

Vehicle

PTH

+/+

/

6.30 (0.45) 1.63 (0.06) 38.9 (2.43) 583 (24)

10.6 (1.04) 1.95 (0.12) 53.7 (2.84) 475 (35)

4.23 (0.31) 1.26 (0.04) 34.2 (3.06) 768 (26)

5.43 (0.47) 1.38 (0.06) 39.8 (3.68) 692 (29)

<0.05 NS <0.05 NS

NS NS NS NS

N = 6 bones/group. Values are means with (SE) in parentheses. One-way ANOVA and post hoc test (Tukey–Kramer).

Table 2B Dynamic histomorphometric parameters of femur metaphysis of 8-week-old Fgf2+/+ and Fgf2 / body wt) Fgf2+/+

D-LS/BS MS/BS BFR/BS Ob.S/BS Oc.N/BS Oc.S/BS

male mice treated for 4 weeks with rhPTH (80 lg/kg

Fgf2 /

ANOVA (Effect of PTH)

Vehicle

PTH

Vehicle

PTH

+/+

/

7.04 (0.59) 15.2 (1.03) 24.8 (1.64) 10.4 (0.73) 2.42 (0.23) 6.0 (0.4)

14.0 (0.80) 27.1 (1.50) 47.6 (2.61) 21.4 (1.57) 3.34 (0.34) 8.0 (0.9)

5.27 (0.37) 12.4 (0.41) 17.8 (1.34) 8.41 (1.14) 1.91 (0.20) 5.1 (0.6)

5.5 (0.47) 14.0 (1.04) 21.0 (1.92) 10.80 (1.12) 2.26 (0.15) 5.6 (0.4)

<0.05 <0.05 <0.05 <0.05 NS NS

NS NS NS NS NS NS

N = 6 bones/group. Values are means with (SE) in parentheses. One-way ANOVA and post hoc test (Tukey–Kramer).

992

M.M. Hurley et al. / Biochemical and Biophysical Research Communications 341 (2006) 989–994

Table 3A Static histomorphometric parameters of femur of 15-month-old female Fgf2+/+ and Fgf2 / Fgf2+/+

BV/TV TbN Tb.Th Tb.Sp

mice treated with rhPTH for 4weeks

Fgf2 /

ANOVA (Effect of PTH)

Vehicle

PTH

Vehicle

PTH

+/+

/

3.00 (0.32) 0.85 (0.09) 35.8 (1.74) 1262 (142)

10.27 (1.20) 2.18 (0.19) 46.4 (2.25) 438 (37)

3.17 (0.36) 0.91 (0.09) 34.5 (0.96) 1152 (113)

4.24 (0.35) 1.11 (0.09) 38.7 (2.34) 914 (84)

<0.05 <0.05 <0.05 <0.05

NS NS NS NS

N = 6 bones/group. Values are means with (SE) in parentheses. One-way ANOVA and post hoc test (Tukey–Kramer).

Table 3B Dynamic histomorphometric parameters of femur of 15-month-old female Fgf2+/+ and Fgf2 / Fgf2+/+

D-LS/BS MS/BS BFR/BS

mice treated with rhPTH for 4 weeks

Fgf2 /

ANOVA (Effect of PTH)

Vehicle

PTH

Vehicle

PTH

+/+

/

10.2 ± (1.57) 22.1 ± (2.26) 33.3 ± (4.78)

17.1 ± (1.35) 34.5 ± (2.83) 56.2 ± (4.55)

6.38 ± (0.61) 15.7 ± (0.86) 22.7 ± (1.48)

7.71 ± (1.22) 18.4 ± (2.57) 27.5 ± (3.56)

<0.05 <0.05 <0.05

NS NS NS

N = 6 bones/group. Values are means with (SE) in parentheses. One-way ANOVA and post hoc test (Tukey–Kramer).

We examined whether PTH differentially modulated serum calcium and serum levels of IGF-1 in Fgf2 / mice. Serum calcium and IGF-1 were measured at time of sacrifice in mice treated with bPTH (1–34) (80 lg/kg) or vehicle for 4 weeks. As shown in Table 4, although baseline serum calcium was slightly increased in Fgf2 / mice, it was not significant relative to Fgf2+/+ mice. Serum calcium was not significantly altered in response to 4 weeks of PTH treatment in either genotype. Although serum IGF-1 levels were similar in vehicle-treated mice of both genotypes, the levels were significantly reduced in PTH-treated Fgf2 / mice (Table 4). Limited studies were performed to assess whether the anabolic response to PTH was also impaired in Fgf2 haplo-insufficient mice and to determine whether a higher concentration of PTH would increase bone mass as measured by DEXA analysis of BMD. For these studies, 8week-old Fgf2+/+, Fgf2+/ , and Fgf2 / mice were weighed and injected once daily with vehicle or PTH 1– 34 (160 lg/kg) body wt for 10 days. Mice were weighed, sacrificed, and femurs were excised. As shown in Fig. 2, PTH significantly increased BMD by 18% in femurs from Fgf2+/+ mice but there was no significant effect in the Fgf2+/ or Fgf2 / mice.

Discussion We postulated that the impaired anabolic response to PTH is due in part to reduced availability of local anabolic factors such as FGF-2. We previously showed that PTH transcriptionally increased the production of FGF-2 in murine osteoblasts [25] and our observation that increased levels of serum FGF-2 paralleled increases in serum markers of bone formation such as osteocalcin and alkaline phosphatase in osteoporotic patients treated with PTH [32] suggested a possible role for FGF-2 in the anabolic response to PTH. We therefore examined whether in the absence of endogenous FGF-2 (produced and released by stromal cells) [17]), bone formation was impaired in Fgf2 / mice in response to PTH. Our results clearly demonstrate that in contrast to Fgf2+/+ mice, 4 weeks of treatment with PTH (80 lg/kg body wt) did not significantly increase trabecular bone volume as determined by MicroCT or histomorphometric parameters of bone formation in male Fgf2 / mice. Reduced bone formation in Fgf2 / mice was not due to increased osteoclast activity since osteoclast number/bone surface was not increased by PTH in femoral bones of either genotype. The reduced BMD observed in Fgf2 haplo-insufficient mice further

Table 4 Effect of 4 weeks of rhPTH (80 lg/kg/body wt) on biochemical markers in sera from Fgf2+/+ and Fgf2 / Fgf2+/+

Ca+/+ IGF-1

male mice

Fgf2 /

ANOVA (Effect of PTH)

Vehicle

PTH

Vehicle

PTH

+/+

/

9.01 (0.80) 351.3 (5.01)

9.53 (0.08) 329.8 (14.9)

10.66 (0.74) 345.5 (7.2)

9.80 (0.45) 303.1 (8.7)

NS NS

NS <0.02

N = 9 samples/group. Values are means with (SE) in parentheses.

M.M. Hurley et al. / Biochemical and Biophysical Research Communications 341 (2006) 989–994

Fig. 2. DEXA analysis of the effect of subcutaneous administration of PTH (160 lg/kg body wt) on femoral bone mineral density in Fgf2+/+, Fgf2+/ , and Fgf2 / mice. Eight-week-old male Fgf2+/+, Fgf2+/ , and Fgf2 / mice were injected subcutaneously once daily for 10 days with either vehicle or rhPTH as described under Materials and methods. Femurs were harvested and bone densitometry was determined by Piximus-DEXA as described under Materials and methods. Values are means ± SEM for 6 bones/group *Significantly different from Fgf2+/+ mice. p < 0.05.

supports an important role for FGF-2 in the anabolic response to PTH in mice. Furthermore, the higher concentration of PTH (160 lg/kg body wt) used in these studies did not rescue the impaired anabolic response to PTH in either Fgf2+/ or Fgf2 / mice although this concentration of PTH administered for 10 days was previously shown to increase BMD in growing mice [16]. The results also show that reduced bone formation in response to PTH was not gender specific since similar results were observed in old female Fgf2 / mice. Metabolic studies showed that there were no significant differences in serum calcium, suggesting that reduced bone formation in Fgf2 / was not due to changes in this parameter. We as well as others previously reported that FGF-2 stimulated the production of other local anabolic factors such as IGF-1 [26,27]. Therefore, we reasoned that in the absence of FGF-2, reduced bone formation in response to PTH may not be due directly to a lack of FGF-2 but due to a reduction in the production or availability of growth factors such as IGF-1 that several investigators have clearly demonstrated plays a role in the anabolic response to PTH [7]. Interestingly, treatment with PTH did not increase serum IGF-1 in either genotype. There was actually a small but insignificant reduction in serum IGF-1 in response to PTH in Fgf2+/+ mice. Previous studies showed that in vivo PTH treatment for 15 days increased BMD but reduced serum IGF-1 in growing female rats [36]. Other investigators observed no change in serum IGF-1 in male rats treated with PTH for 4 or 8 weeks [9]. Interestingly, there was a significant reduction in serum IGF-1 in response to PTH in Fgf2 / mice.

993

Whether this contributed to reduced bone formation in response to PTH in the Fgf2 / mice is unclear since local production of IGF-1 by osteoblasts is probably more important in bone formation [9]. The mechanism(s) by which knockout of FGF-2 impairs the anabolic response to PTH is not clear. PTH enhances the differentiation of osteoblast progenitors, activates lining cells, and inhibits apoptosis [11–15]. The studies of Nishida et al. [37] proposed that increased bone formation by intermittent PTH administration is due to the stimulation of proliferation and differentiation of osteoprogenitor cells in bone marrow. Studies by Jilka et al. [13], demonstrated that PTH increased osteoblast longevity by decreasing their rate of apoptosis. FGF-2 is a potent mitogen for osteoblast progenitors and is anti-apoptotic for osteoblast precursors [17]. It is possible that in the absence of endogenous FGF-2 the overall number of mature osteoblasts capable of new bone formation in response to PTH is reduced. Studies to assess whether reduced proliferation and/or increased osteoblasts apoptosis contributes to reduced anabolic response to PTH in Fgf2 mutant mice are in progress. Acknowledgment This work is supported in part by NIH Grant AG021189 to M.M. Hurley. References [1] J.M. Hock, J.R. Hummert, R. Boyce, J. Fonseca, L.G. Raisz, Resorption is not essential for the stimulation of bone growth by hPTH-(1–34) in rats in vivo, J. Bone Miner. Res. 4 (3) (1989) 449–458. [2] B.E. Kream, D. LaFrancis, D.N. Petersen, C. Woody, S. Clark, D.W. Rowe, A. Lichtler, Parathyroid hormone represses a1(l) collagen promoter activity in cultured calvariae from neonatal transgenic mice, Mol. Endocrinol. 7 (1993) 399–408. [3] E. Canalis, J.M. Hock, L.G. Raisz, Anabolic and catabolic effects of parathyroid hormone on bone and interactions with growth factors, The Parathyroids, Chapter 4 (1994) 65–82. [4] U.S. Masiukiewicz, K.L. Insogna, The role of parathyroid hormone in the pathogenesis and prevention of osteoporosis, Aging. Clin. Res. 10 (1998) 232–239. [5] J.M. Hock, I. Gera, Effects of continous and intermittent administration and inhibition of resorption on the anabolic response of bone to parathyroid hormone, J. Bone Miner. Res. 7 (1992) 65–72. [6] R.M. Neer, C.D. Arnaud, J.R. Zanchetta, R. Prince, G.A. Gaich, J.Y. Reginster, A.B. Hodsman, E.F. Ericksen, S. Ish-Shalom, H.K. Genant, O. Wang, B.H. Mitlak, Effects of parathyroid hormone (1– 34) on fractures and bone mineral density in postmenopausal women with osteoporosis, N. Eng. J. Med. 10 (2001) 1434–1441. [7] C.J. Rosen, J.P. Bilezikian, Anabolic therapy for osteoporosis, J. Clin. Endocrinol. Metab. 86 (2001) 957–964. [8] E. Canalis, M. Centrella, W. Burch, T.L. McCarthy, Insulin-like growth factor I mediates selective anabolic effects of parathyroid hormone in bone cultures, J. Clin. Invest. 83 (1989) 60–65. [9] J. Pfeilschifter, F. Laukhuf, B. Muller-Beckmann, W.F. Blum, T. Pfister, R. Zeigler, Parathyroid hormone increases the concentration of insulin-like growth factor-1 and transforming growth factor beta 1 in rat bone, J. Clin. Invest. 96 (1995) 767–774. [10] B.E. Kream, D.N. Petersen, L.G. Raisz, Parathyroid hormone blocks the stimulatory effect of insulin- like growth factor-I on collagen

994

[11]

[12]

[13]

[14]

[15]

[16]

[17]

[18]

[19]

[20]

[21]

[22]

[23]

[24]

M.M. Hurley et al. / Biochemical and Biophysical Research Communications 341 (2006) 989–994 synthesis in cultured 21-day fetal rat calvariae, Bone 11 (1990) 411– 415. H. Dobnig, R.T. Turner, Evidence that intermittent treatment with parathyroid hormone increases bone formation in adult rats by activation of bone lining cells, Endocrinology 136 (1995) 3632– 3638. T. Shizuya, S. Yokose, M. Hori, T. Noda, T. Suda, S. Yoshiki, A. Yamaguchi, Parathyroid hormone exerts disparate effects on osteoblast differentiation depending on exposure time in rat osteoblastic cells, J. Clin. Invest. 99 (1997) 2961–2970. R.L. Jilka, R.S. Weinstein, T. Bellido, P. Roberson, A.M. Parfitt, S.C. Manolagos, Increased bone formation by prevention of osteoblast apoptosis with parathyroid hormone, J. Clin. Invest. 104 (1999) 439– 446. D. Stanislaus, X. Yang, J.D. Liang, J. Wolfe, R.L. Cain, J.E. Onyia, N. Falla, P. Marder, J.P. Bidwell, W. Queener, J.M. Hock, In vivo Regulation of Apoptosis in metaphyseal trabecular bone of young rats by synthetic human parathyroid hormone (1.34) fragment, Bone 27 (2000) 209–218. H. Chen, B. Demilrap, A. Schneider, A. Koh, C. Silve, C. Wang, L.K. McCauley, Parathyroid hormone and parathyroid hormone related peptide exert both pro and anti-apoptotic effects in mesenchymal cells, J. Biol. Chem. 277 (2002) 19374–19380. N. Miyakoshi, Y. Kasukawa, T. Linkart, D.J. Baylink, S. Mohan, Evidence that the anabolic effect of PTH on bone require IGF-1 in growing mice, Endocrinology 142 (2001) 4349–4356. M.M. Hurley, P. Marie, R. Florkiewicz, Fibroblast growth factor and fibroblast growth factor Receptor families, in: J.P. Bilezikian, L.G. Raisz, G. Rodan (Eds.), Principles of Bone Biology, Academic Press, San Diego, 2002, pp. 825–852. T.L. McCarthy, M. Centrella, E. Canalis, Effects of fibroblast growth factors on deoxyribonucleic acid and collagen synthesis in rat parietal bone cells, Endocrinology 125 (1989) 2118–2126. M.M. Hurley, C. Abreu, J.R. Harrison, A. Lichtler, L.G. Raisz, B.E. Kream, Basic fibroblast growth factor inhibits type I collagen gene expression in osteoblastic MC3T3-E1 cells, J. Biol. Chem. 268 (1993) 5588–5593. E. Canalis, M. Centrella, T. McCarthy, Effects of basic fibroblast growth factor on bone formation in vitro, J. Clin. Invest. 81 (1988) 1572–1577. H. Mayahara, T. Ito, H. Nagai, H. Miyajima, R. Tsukuda, S. Takatomi, J. Mizoguchi, K. Kato, In vivo stimulation of endosteal bone formation by basic fibroblast growth factor in rats, Growth Factors 9 (1993) 73–80. S. Pri-Chen, S. Pitaru, F. Lokiec, N. Savion, Basic fibroblast growth factor enhances the growth and expression of the osteogenic phenotype of dexamethasone-treated human bone marrow-derived bone-like cells in culture, Bone 23 (1998) 111–117. S. Berrada, F. Lefebvre, M.F. Harmand, The effect of recombinant human basic fibroblast growth factor RHFGF-2 on human osteoblasts in growth and phenotype expression, In Vitro Cell. Dev. Biol. Animal 31 (1995) 698–702. I. Martin, A. Muraglia, G. Campanile, R. Cancedda, R. Quarto, Fibroblast growth factor-2 supports ex vivo expansion and mainte-

[25]

[26]

[27]

[28]

[29]

[30]

[31]

[32]

[33]

[34]

[35]

[36]

[37]

nance of osteogenic precursors from human bone marrow, Endocrinology 138 (1997) 4456–4462. M.M. Hurley, S. Tetradis, Y.F. Huang, J. Hock, B.E. Kream, L.G. Raisz, M.G. Sabbieti, Parathyroid hormone regulates the expression of fibroblast growth factor-2 mRNA and fibroblast growth factor receptor mRNA in osteoblastic cells, J. Bone Miner. Res. 14 (1999) 776–783. X. Zhang, T. Sobue, M.M. Hurley, FGF-2 increases colony formation, PTH receptor, and IGF-1 mRNA in mouse marrow stromal cell cultures, Biochem. Biophys. Res. Commun. 290 (2002) 526–531. R.A. Power, U.T. Iwanec, T.J. Wronski, Changes in gene expression associated with the bone anabolic effects of basic fibroblast growth factor, Bone 31 (2002) 143–148. M. Noda, R. Vogel, Fibroblast growth factor enhances type1 b1 transforming growth factor gene expression in osteoblastic cells, J. Cell Biol. 109 (1989) 2529–2535. M. Zhou, R.L. Sutliff, R.J. Paul, J.N. Lorenz, J.B. Hoying, C.C. Haundenschild, Y. Moying, J.D. Coffin, L. Kong, E.G. Kranias, W. Luo, G.P. Boivin, J.J. Duffy, S.A. Pawlowski, T. Doetschman, Fibroblast growth factor 2 control of vascular tone, Nat. Med. 4 (1998) 201–207. A. Montero, Y. Okada, M. Tomita, M. Ito, H. Tsurukami, T. Nakamura, T. Doetschman, J.D. Coffin, M.M. Hurley, Disruption of the fibroblast growth factor-2 gene results in decreased bone mass and bone formation, J. Clin. Invest. 105 (2000) 1085–1093. Y. Okada, A. Montero, X. Zhang, T. Sobue, J. Lorenzo, T. Doetschman, J.D. Coffin, M.M. Hurley, Impaired osteoclast formation in bone marrow cultures of Fgf2 null mice in response to parathyroid hormone, J. Biol. Chem. 278 (2003) 21258–21266. M. Hurley, W. Yao, N.E. Lane, Changes in serum fibroblast growth factor 2 in patients with glucocorticoid-induced osteoporosis treated with human parathyroid hormone (1–34), Osteoporos Int. 16 (12) (2005) 2080–2084. A.M. Parfitt, M.K. Drezner, F.H. Glorieux, J.K. Kanis, H. Malluche, P.J. Meunier, S.M. Ott, R.R. Recker, Bone histomorphometry: standardization of nomenclature, symbols and units, J. Bone Miner. Res. 6 (1987) 595–610. H.Z. Ke, H.A. Simmons, C.M. Pirie, D.T. Crawford, D.D. Thompson, Droloxifene, a new estrogen antagonist/agonist, prevents bone loss in ovariectomized rats, Endocrinology 136 (1995) 2435–2441. C.G. Rosen, C.L. Ackert-Bicknell, M.L. Adamo, K.L. Schultz, J. Rubin, L.R. Donahue, L.G. Horton, K.M. Delahunty, W.J. Beamer, J. Sipos, D. Clemons, T. Nelson, M.L. Bouxsein, M. Horowitz, Congenic mice with low serum IGF-1 have increased body fat, reduced bone mineral density and an altered osteoblast differentiation program, Bone 35 (2004) 1046–1058. A. Toromanoff, P. Ammann, J.L. Riond, Early effects of short-term parathyroid hormone administration on bone mass, mineral content and strength in female rats, Bone 22 (1998) 217–223. S. Nishida, A. Yamaguchi, N. Tanizawa, N. Endo, T. Mashiba, Y. Uchiyama, T. Suda, S. Yoshiki, H.E. Takahashi, Increased bone formation by intermittent parathyroid hormone administration is due to the stimulation of proliferation and differentiation of osteoprogenitor cells in bone marrow, Bone 15 (1994) 717–723.