markers, nor hearing loss as measured by auditory brainstem response. (ABR) or distoruon product oto-âacousuc emissions (DPOAE), between wild type and ...
Parallel Mechanisms Suppress Cochlear Bone Remodeling to Protect Hearing Emmanuel Jáuregui, BA1; Omar Akil, PhD2; Claire Acevedo, PhD1,3; Faith Hall-‐Glenn, PhD1; Be?y S. Tsai, MD2; Hrishikesh A. Bale, PhD3; Ellen Liebenberg, MS1 Mary Beth Humphrey, MD, PhD5; Robert O. Ritchie, PhD3; Lawrence R. LusOg, MD2; Tamara Alliston, PhD1
CLINICAL & TRANSLATIONAL MEDICINE
1Department of Orthopaedic Surgery, University of California, San Francisco , 2Department of Otolaryngology—Head & Neck Surgery, University of California, San Francisco, University of California, San Francisco, CA
3Materials Science Division, Lawrence Berkeley NaOonal Laboratory, Berkeley, CA, 4Department of Otorhinolaryngology, University of Oklahoma Health Sciences Center, 5Department of Medicine, University of Oklahoma Health Sciences Center
Bone remodeling, a combination of bone resorption and formation, requires precise regulation of cellular and molecular signaling to maintain proper bone quality. Whereas osteoblasts deposit and osteoclasts resorb bone matrix, osteocytes both dynamically resorb and replace perilacunar bone matrix. Osteocytes secrete proteases like matrix metalloproteinase-13 (MMP13) to maintain the material quality of bone matrix through perilacunar remodeling (PLR). Deregulated bone remodeling impairs bone quality and can compromise hearing since the auditory transduction mechanism is within bone. Understanding the mechanisms regulating cochlear bone provide unique ways to assess bone quality independent of other aspects that contribute to bone mechanical behavior. Cochlear bone is singular in its regulation of remodeling by expressing high levels of osteoprotegerin. Since cochlear bone expresses a key PLR enzyme, MMP13, we examined whether cochlear bone relies on, or is protected from, osteocyte-mediated PLR to maintain hearing and bone quality using a mouse model lacking MMP13 (MMP13-/-). Therefore, we seek to investigate the cellular and molecular mechanisms that maintain cochlear bone in a mouse model deficient in MMP13, with defective long bone PLR, to evaluate whether the cochlea is uniquely dependent on MMP13mediated osteocytic PLR to maintain cochlear bone to maintain normal hearing. RANKL
Figure 2. Normal inner ear structures and ossicular chain in wild type and MMP13-/- mice. (A-F) Plastic embedded sections of cochleae showing cochlea in sagittal plane of WT (A) and MMP13-/- mice (D). Organ of Corti (B) and spiral neuroganglion (C) in WT and MMP13-/- (E and F) reveal no bony compression of neuroganglia. Toluidine blue stain of WT (G) and MMP13-/- (H) show no bony deformation. Micro-CT reconstruction show normal ossicular chain (I) and cochlea (J) in WT and MMP13-/- (K and L).
Figure 3. Hearing is maintained despite lack of MMP13. Auditory brainstem responses (ABR) on wild-type and MMP13-/- and distortion product otoacoustic emissions (DPOAE) on wild-type and MMP13-/mice at 6 months of age reveal increased variability, however no significant difference between the two groups.
Cochlear bone is singular in its regulaOon of remodeling by expressing high levels of osteoprotegerin. Since cochlear bone expresses a key PLR enzyme, MMP13, we examined whether cochlear bone relies on, or is protected from, osteocyte-‐mediated PLR to maintain hearing and bone quality using a mouse model lacking MMP13 (MMP13-‐/-‐). We invesOgated the canalicular network, collagen organizaOon, lacunar v o l u m e v i a m i c r o -‐ c o m p u t e d t o m o g r a p h y , a n d d y n a m i c histomorphometry. Despite ﬁnding defects in these hallmarks of PLR in MMP13-‐/-‐ long bones, cochlear bone revealed no diﬀerences in these markers, nor hearing loss as measured by auditory brainstem response (ABR) or distorOon product oto-‐acousOc emissions (DPOAE), between wild type and MMP13-‐/-‐ mice. Dynamic histomorphometry revealed abundant PLR by Obial osteocytes, but near absence in cochlear bone. Cochlear suppression of PLR corresponds to repression of several key PLR genes in the cochlea relaOve to long bones. These data suggest cochlear bone uniquely maintains bone quality and hearing independent of MMP13-‐mediated osteocyOc PLR. Furthermore, the cochlea employs parallel mechanisms to inhibit remodeling by osteoclasts and osteoblasts, and by osteocytes, to protect hearing. Understanding the cellular and molecular mechanisms that confer site-‐ speciﬁc control of bone remodeling have the potenOal to elucidate new pathways that are deregulated in skeletal disease.
Cochlear Bone ñOPG -‐-‐| Osteoclast
??? -‐-‐| Osteocytes
Mineralized Bone Matrix
Figure 4. MMP13 is necessary for collagen organizaHon in long bones, not in cochlear bone. Polarized light birefringence of cochleae and femora reveal collagen ﬁbril organizaOon. Both WT and MMP13-‐/-‐ cochleae reveal well-‐organized collagen ﬁbrils (A-‐C). Plot of the distribuOon of orientaOon of collagen ﬁbrils in cochlear bone (D) reveal no diﬀerences between MMP13-‐/-‐ (36.6±10.4o) and WT mice (33.5±7.2o), p>0.05. In femora (E-‐G), collagen organizaOon is not well organized throughout MMP13-‐/-‐ mice (G) compared to WT (F). For MMP13-‐/-‐ femora, the distribuOon of collagen orientaOon shows a wider, less well-‐deﬁned peak (H), suggesOng poorer organizaOon by width at half-‐maximum analysis, (71.8±30.6 vs. 34.3±4.8o, MMP13-‐/-‐ and WT, respecOvely; p=0.0002).
Tibia Femur Cochlea
Figure 1: MMP13 is expressed at multiple stages of development in wild-type mouse cochlea. Immunohistochemical stain for MMP13 protein in wild-type mice cochlea from embryonic day (E)15 to postnatal day (P)60. Black arrows show areas of most intense staining of MMP13 in cochlear bone. Plus IgG negative control stain of cochlea at P60.
SOST ATPg1 ATPd2 MMP13 MMP14 CatK
Figure 6. CriHcal PLR factors are expressed to a lesser degree in the cochlea compared to long bones. qPCR expression levels from p60 WT male mice. The relaOve expression levels of MMP13 and other important PLR genes (CatK, ATPd2, MMP14) in the cochlea are lower than in other bones, suggesOng suppressed PLR acOvity in the cochlea relaOve to long bones.
Figure 5. Cochlear canalicular network is maintained without MMP13, however is required in femur. Lacuno-‐canalicular networks in cochleae (A-‐B) and femora (D-‐E) stained with silver nitrate (in black) in male p60 mice. QuanOﬁcaOon of the lacuno-‐canalicular area, using ImageJ, reveals no signiﬁcant diﬀerence between the two groups (0.260±0.04µm2 vs. 0.235±0.08µm2, MMP13-‐/-‐ and WT, respecOvely, p>0.05) (C). Silver nitrate staining of axial femur secOon reveals a robust, radiaOng canalicular network from lacunae in WT femora (D). MMP13-‐/-‐ mice reveal a less extensive, blunted canalicular network (E). MMP13-‐/-‐ mice have a signiﬁcantly smaller lacuno-‐canalicular area (0.143±0.04 µm2) normalized to bone area, compared to WT mice (0.256±0.10 µm2; p=0.0002) (F). 0.002
ñSOST -‐-‐| Osteoblast
Osteocyte Perilacunar Remodeling Markers
MMP13 Collagen org. Mineralization Canalicular Network org.
+/+ ✓ ✓
+/+ ✓ ✓
Methods Detailed methods are described with these references: Tang4, Chang5, and Busse6. Brieﬂy, the procedures include: Histological analysis: Cochleae were collected, decalciﬁed, embedded in paraﬃn, secOoned, and stained with picro-‐sirius red for polarized-‐ birefringence analysis of collagen orientaOon and analysed with OrientaOonJ an ImageJ plug-‐in. Silver nitrate stain was used to visualize the lacuno-‐ canalicular network and quanOﬁed with ImageJ. MMP13 protein was examined via immunohistochemistry. High-‐resoluHon micro computed tomography was used to scan cochleae and to determined lacunar volume. Auditory brainstem responses & distorHon product oto-‐acusHc emissions were measured to assess hearing in 6-‐month-‐old mice.
100 200 300 Lacunar Volume (mm3)
Figure 7. Lacunar volume in cochlear bone is unaﬀected by MMP13 deﬁciency. RepresentaOve 3D volume rendered image of micro-‐CT scan of the cochlea. No signiﬁcant diﬀerences in the average lacunar volumes in the cochlea despite the lack of MMP13 was found.
Funding sources: Hearing Research, Inc. compeOOve award. NIH R01 DE019284 (to Alliston). References 1. Zehnder A, et al. The Laryngoscope, 2005. 2. van Bezooijen R, et al. Journal of Experimental Medicine, 2004. 3. Wysolmerski J. Bone, 2013. 4. Tang S, et al. Journal of Bone and Mineral Research, 2012. 5. Chang J, et al. EMBO Rep, 2010. 6. Busse B, et al. Science Transla?onal Medicine, 2013.