Resorption of auditory ossicles and hearing loss in mice lacking osteoprotegerin

doi:10.1016/j.bone.2006.01.155

Bone

Volume 39, Issue 2, August 2006, Pages 414-419

https://doi.org/10.1016/j.bone.2006.01.155 Get rights and content

Abstract

Bones conduct sound in the middle ear. The three ossicles—the malleus, incus, and stapes—form a chain that transmits vibrations from the tympanic membrane to the oval window of the inner ear. Little is known about bone remodeling events in these ossicles and about potential effects of osteoporosis on hearing loss. Osteoclastic bone resorption is enhanced in Opg^−/− mice lacking osteoprotegerin, which is a soluble decoy receptor for the osteoclastogenic cytokine RANKL. We asked whether auditory ossicles are resorbed in Opg^−/− mice, and whether these mice suffer from impaired auditory function. All three ossicles in Opg^−/− mice showed thinning, especially at the malleal manubrium and incus body. Most notably, unlike in the case in wild-type mice, the junction between the stapes and the otic capsule was fixed in Opg^−/− mice, and the stapedial footplate was thinner and broader. Radiological analyses revealed that malleal cortical thickness was positively correlated with tibial bone mineral density in Opg^−/− and control littermate mice. Furthermore, progressive hearing loss was detected in Opg^−/− mice starting at 6 to 15 weeks of age. These data suggest that osteoprotegerin plays a crucial role in hearing by protecting the auditory ossicles and otic capsule from osteoclastic bone resorption.

Introduction

The three ossicles in the middle ear, the malleus, incus, and stapes, are formed mainly by endochondral ossification of the mesenchyme from the first and second branchial arches [1], [2]. The manubrium (handle) of the malleus attaches to the tympanic membrane, while the footplate of the stapes attaches to the oval window of the cochlea. The stapedial foot is mobile and transmits vibrations to the perilymph, the fluid in the inner ear. The inner ear is contained in the otic capsule of the temporal bone, which is the hardest bone in the body.

Bone mineral density (BMD) is determined by the balance between bone resorption by osteoclasts and formation by osteoblasts. Genetic studies of osteopetrotic mice reveal a number of molecules essential for osteoclastogenesis. Osteoclasts differentiate from precursors of the monocyte–macrophage lineage in the presence of the two membrane bound cytokines, macrophage-colony stimulating factor (M-CSF) and RANKL (receptor activator of nuclear factor-κB ligand, also called osteoclast differentiation factor or TRANCE) [3]. The RANKL receptor is a tumor necrosis factor receptor superfamily member known as RANK encoded by the Tnfrsf11A gene. RANK signaling in osteoclast precursors activates a series of osteoclastogenic transcription factors including NF-κB, c-Fos/AP-1, and NFATc1 [4], [5], [6], [7], [8], [9]. The osteoclastogenic activity of RANKL is masked by the soluble decoy receptor osteoprotegerin (OPG, also called osteoclast inhibitory factor), encoded by Tnfrsf11B [10], [11]. In bone remodeling, BMD is maintained by a coupling of osteoclastic bone resorption with subsequent osteoblastic formation [12].

In human populations, the incidence of osteoporotic hip fracture increases exponentially with age [13]. Age-related hearing loss, or presbycusis, affects more than one third of individuals above the age of 75 [14]. Although a link between osteoporosis and hearing loss has been suggested [15], [16], [17], recent epidemiological studies reveal no correlation of hearing loss and osteoporosis in elderly women [18], a finding that seems counterintuitive given that hearing largely depends on bone.

Opg^−/− mice develop osteopenia due to enhanced differentiation of osteoclasts [19], [20], [21], [22]. To gain deeper insight into the role of bone remodeling in hearing, we asked if auditory ossicles are susceptible to osteoclastic bone resorption in Opg^−/− mice and whether auditory function is impaired.

Section snippets

Mice

Female Opg^−/− and heterozygous control mice on a C57BL6 background were purchased from Clea Japan. All experiments were conducted in accordance with institutional review board-approved protocols.

Morphological analyses

Mouse skulls were fixed in 4% paraformaldehyde. For macroscopic analysis, auditory ossicles were isolated by removing the temporal bone, stained for tartrate-resistant acid phosphatase (TRAP) activity using the Leukocyte Acid Phosphatase Kit (Sigma), and observed using a SMZ1500, stereoscopic zoom

Results

To examine the morphology of the auditory ossicles in Opg^−/− mice, we isolated mallei, incudes, and stapes from the middle ear cavities of 10-week-old Opg^−/− mice and from wild-type and heterozygous controls. We observed that the junction between the stapes and the oval window of the cochlea was tighter in Opg^−/− mice compared to control mice. Ossicles from Opg^−/− and control mice were stained for TRAP activity, which is a marker for osteoclasts and resorption lacunae. Compared to wild-type and

Discussion

In adult Opg^−/− mice, we observed erosion of the malleus, incus, and stapes. Furthermore, TRAP activity was detected in all the three ossicles and the otic capsule of Opg^−/− mice, indicating that osteoclastic bone resorption of auditory ossicles is elevated. It is unclear whether certain specific areas within each ossicle are preferentially resorbed or not. We also observed that Opg^−/− mice show progressive hearing loss. The precise mechanisms of hearing loss in these mice are currently

Acknowledgments

We thank Shumpei Niida, Kyoji Ikeda, and Minako Sato for helpful discussions and Neelanjan Ray and Elise Lamar for critical reading of the manuscript. This work is supported by Grant-in-Aid for Young Scientists B (17791198 to SK) and Grant-in-Aid for Scientific Research B (17390420 to KM) from JSPS, and a Keio University Special Grant-in-Aid for Innovative Collaborative Research Projects.

References (37)

M. Mallo
Formation of the middle ear: recent progress on the developmental and molecular mechanisms
Dev. Biol.
(2001)
G. Karsenty et al.
Reaching a genetic and molecular understanding of skeletal development
Dev. Cell
(2002)
H. Takayanagi et al.
Induction and activation of the transcription factor NFATc1 (NFAT2) integrate RANKL signaling in terminal differentiation of osteoclasts
Dev. Cell
(2002)
K. Matsuo et al.
Nuclear factor of activated T-cells (NFAT) rescues osteoclastogenesis in precursors lacking c-Fos
J. Biol. Chem.
(2004)
W.S. Simonet et al.
Osteoprotegerin: a novel secreted protein involved in the regulation of bone density
Cell
(1997)
T.J. Martin et al.
Osteoclast-derived activity in the coupling of bone formation to resorption
Trends Mol. Med.
(2005)
K. Clark et al.
Age-related hearing loss and bone mass in a population of rural women aged 60 to 85 years
Ann. Epidemiol.
(1995)
A. Mizuno et al.
Severe osteoporosis in mice lacking osteoclastogenesis inhibitory factor/osteoprotegerin
Biochem. Biophys. Res. Commun.
(1998)
K. Janssens et al.
An intermediate form of juvenile Paget's disease caused by a truncating TNFRSF11B mutation
Bone
(2005)
Z. Zhang et al.
Malleal processus brevis is dispensable for normal hearing in mice
Dev. Dyn.
(2003)

S.L. Teitelbaum et al.

Genetic regulation of osteoclast development and function

Nat. Rev. Genet.

(2003)

G. Franzoso et al.

Requirement for NF-κB in osteoclast and B-cell development

Genes Dev.

(1997)

A.E. Grigoriadis et al.

c-Fos: a key regulator of osteoclast-macrophage lineage determination and bone remodeling

Science

(1994)

K. Matsuo et al.

Fosl1 is a transcriptional target of c-Fos during osteoclast differentiation

Nat. Genet.

(2000)

H. Yasuda et al.

Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL

Proc. Natl. Acad. Sci. U. S. A.

(1998)

C. Cooper

Epidemiology of osteoporosis

P.F. Adams et al.

Current estimates from the National Health Interview Survey, 1991

Vital Health Stat.

(1992)

R.I. Henkin et al.

Hearing loss in patients with osteoporosis and Paget's disease of bone

Am. J. Med. Sci.

(1972)

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