Elsevier

Bone

Volume 51, Issue 3, September 2012, Pages 578-585
Bone

Original Full Length Article
Black bear parathyroid hormone has greater anabolic effects on trabecular bone in dystrophin-deficient mice than in wild type mice

https://doi.org/10.1016/j.bone.2012.05.003Get rights and content

Abstract

Duchenne muscular dystrophy (DMD) is an X-linked neuromuscular disease that has deleterious consequences in muscle and bone, leading to decreased mobility, progressive osteoporosis, and premature death. Patients with DMD experience a higher-than-average fracture rate, particularly in the proximal and distal femur and proximal tibia. The dystrophin-deficient mdx mouse is a model of DMD that demonstrates muscle degeneration and fibrosis and osteoporosis. Parathyroid hormone, an effective anabolic agent for post-menopausal and glucocorticoid-induced osteoporosis, has not been explored for DMD. Black bear parathyroid hormone (bbPTH) has been implicated in the maintenance of bone properties during extended periods of disuse (hibernation). We cloned bbPTH and found 9 amino acid residue differences from human PTH. Apoptosis was mitigated and cAMP was activated by bbPTH in osteoblast cultures. We administered 28 nmol/kg of bbPTH 1–84 to 4-week old male mdx and wild type mice via daily (5×/week) subcutaneous injection for 6 weeks. Vehicle-treated mdx mice had 44% lower trabecular bone volume fraction than wild type mice. No changes were found in femoral cortical bone geometry or mechanical properties with bbPTH treatment in wild type mice, and only medio-lateral moment of inertia changed with bbPTH treatment in mdx femurs. However, μCT analyses of the trabecular regions of the distal femur and proximal tibia showed marked increases in bone volume fraction with bbPTH treatment, with a greater anabolic response (7-fold increase) in mdx mice than wild type mice (2-fold increase). Trabecular number increased in mdx long bone, but not wild type bone. Additionally, greater osteoblast area and decreased osteoclast area were observed with bbPTH treatment in mdx mice. The heightened response to PTH in mdx bone compared to wild type suggests a link between dystrophin deficiency, altered calcium signaling, and bone. These findings support further investigation of PTH as an anabolic treatment for DMD-induced osteoporosis.

Highlights

► Black bear PTH is a potent osteoanabolic agent in the dystrophin‐deficient mdx mouse. ► Black bear PTH is more potent in mdx mice than in wild type mice. ► Black bear PTH increases osteoblast surface and decreases osteoclast surface in mdx mice.

Introduction

Boys with Duchenne muscular dystrophy (DMD) progressively lose muscle strength and between the ages of 12 and 15 years are usually confined to a wheelchair [1]. The condition is typically fatal by 20–25 years due to respiratory or cardiac muscle failure [2]. DMD‐induced muscular necrosis and fibrosis are treated with glucocorticoids (e.g., prednisone or deflazacort) [3], [4]. DMD patients treated with glucocorticoids demonstrate a 30–50% decrease in bone mineral content and bone mineral density in the lumbar spine, hip, and long bones of the lower limbs as compared with healthy age-matched controls [3], [5], [6], [7], [8]. Greater losses occur in trabecular bone than cortical bone due to its greater surface area for remodeling activity [3]. Fracture risk increases as a result. One study found that 44% of 71 boys with DMD sustained at least one fracture; of boys over the age of 16 years, 67% sustained at least one fracture [6]. The majority of fractures occur in the proximal and distal femur in DMD patients [9], [10], [11].

Like boys with DMD, mdx mice lack dystrophin [12], [13], [14]. These animals exhibit myopathic lesions characteristic of those seen in DMD patients [15], [16]. Mdx mice display larger body sizes and limb muscles, though these muscles are heavily fibrosed and less capable of high muscular forces [17]. At 3 weeks of age, mdx femurs display ~ 40% reductions in the trabecular area, trabecular area fraction, and trabecular thickness as compared to age-matched wild type mice. They show decreases in osteoblast surface and increases in osteoclast surface area, leading to increased resorption and decreased formation [18]. In 4 week old male mdx mice, the metaphysis of the tibia showed decreases in bone volume fraction of 35%, with a decrease in trabecular number and increase in trabecular spacing. One study found no difference between mdx and wild type cortical thickness in the femur at 4 months of age [17]. However, at 6 months, mdx mice have decreased cortical bone properties relative to wild type mice and increased osteoclast number [18]. Whole bone breaking force and deformation at fracture are reduced in mdx mice regardless of age [19]. At 24 months of age, mdx mice show greater declines (relative to wild type mice) in trabecular and cortical properties in the tibia than at 7 weeks of age [20].

Treatments for DMD-related osteoporosis have not been widely explored. Treating DMD patients with the bisphosphonate alendronate prevented further decreases in bone mineral density over a two‐year period [21]. The impaired osteoblast function seen in both DMD patients and mdx mice suggests that an anabolic treatment for osteoporosis (i.e., PTH therapy) would be an effective therapeutic for increasing bone mass in these patients. Indeed, PTH treatment leads to greater reductions in fracture risk than alendronate in patients with glucocorticoid-induced osteoporosis [22]. Our laboratory has been investigating a novel PTH analog linked to the preservation of bone during disuse. Black bears (Ursus americanus) hibernate for 6 months of the year, and do not experience disuse-related osteoporosis [23], [24], [25]. Black bear parathyroid hormone (bbPTH) has been positively correlated to bone formation markers during hibernation and is implicated in the mechanism of bone preservation during disuse [23], [25], [26]. Furthermore, bbPTH 1–34 causes greater reductions in serum‐starved induced caspase activity in osteoblasts than hPTH 1–34 [27]. Thus, bbPTH may be well suited to improve bone mass in cases of dystrophin deficiency that impair mobility. We hypothesized that bbPTH treatment in mdx mice would demonstrate improved bone properties compared to vehicle‐treated mice, and that bbPTH treatment would restore mdx bone properties to wild type levels.

Section snippets

PCR cloning and sequencing of bbPTH

Genomic DNA was extracted from black bear whole blood samples using the GenomicPrep Blood DNA Isolation Kit (Amersham Biosciences, Piscataway, NJ). The genomic DNA was used for PCR amplification of PTH using consensus primers designed based on alignment of eight full-length mammalian PTH sequences available in GenBank including bovine (Bos taurus, AAA30749), cat (Felis catus, Q9GL67), dog (Canis familiaris, P52212), human (Homo sapiens, NP_000306), macaque (Macaca fascicularis, Q9XT35), mouse (

Cell studies

All three concentrations of bbPTH increased cAMP production in MC3T3 cells compared to vehicle controls (p < 0.0001). No difference was observed between 10 nM bbPTH and 30 nM bbPTH, but 100 nM bbPTH increased cAMP production more than the lower doses (p < 0.0001) (Fig. 2). Caspase 3/7 activity was reduced (p < 0.0001) in serum‐starved MC3T3 cells treated with both concentrations of bbPTH, with 100 nM bbPTH decreasing caspase 3/7 to unstarved control values (Fig. 3).

Bone marrow stromal cells

Alizarin red staining showed greater (p =

Discussion

We cloned the gene for black bear PTH and found that nine amino acid residues were different from human PTH 1–84. We recombinantly produced bbPTH 1–84 and found that it activates cAMP production as has been previously reported for human PTH [35]. The cAMP/protein kinase A pathway is believed to be responsible for the majority of PTH induced increases in histological and serum indices of bone formation [36], [37], [38]. PTH also produces osteoanabolic activity via anti-apoptosis mechanisms in

Acknowledgments

The authors gratefully acknowledge Dr. Mike Vaughan for the black bear blood samples, Matt Nelsen and Yinan Yuan for assistance with bbPTH cloning, and funding from Aursos Inc. and NIH (DK078407).

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