Localization of osteopontin in the otoconial organs of adult rats

HBIRIrlC, RCH ELSEVIER

Hearing Research 79 (1994) 99-104

Localization of osteopontin in the otoconial organs of adult rats Teiji Takemura

,,x, M a s a f u m i Yukihiko

Sakagami Kitamura

~'*, T a k a n o b u N a k a s e c, S h i n t a r o N o m u r a ~

b, T a k e s h i

Kubo

",

D~7~artments q[" '~ Otolaryngology, h Orthopaedic Surgeo' and c Pathology, Osaka Unil'ersity Medical School, 2-2 Yamadaoka, Suita City, Osaka 565, Japan Received 9 June 1993; revision received 6 January 1994; accepted 15 May 1994

Abstract

Although it is known that mammalian otoconia consist of calcium bicarbonate and organic materials, none of the protein components have been identified in mammals at the molecular level, and the mechanisms of morphogenesis and calcification of the otoconia is still unclear. In the present study, we demonstrated the presence of osteopontin (OPN) in rat otoconia by using immunohistochcmistry, and detected OPN mRNA in the sensory hair cells by a non-radioisotopic in situ hybridization technique. These results indicate that OPN is one of the protein components in rat otoconia and suggest that sensory hair cells are involved in the production of otoconia.

Key words: Ostcopontin; Otoconia; Sensory hair cell; Hybridization, in situ

1. Introduction

The otoconia, which are located on the otoconial m e m b r a n e of the saccular and utricular maculae of mammals and lagenar macula of birds, act as weightloading structures which increase the responsiveness of the otoconial m e m b r a n e to head-tilting and linear acceleration. Mammalian otoconia consist of both calcium bicarbonate, which is crystallized in the form of calcite, and organic materials composed of proteins and carbohydrates (Carlstrom et al., 1953; Wislocki and Ladmann, 1955; Lim, 1984; Gil-Loyzaga et al., 1985; Ross and Pote, 1984; Pote and Ross, 1986, 1991). Pote and Ross (1986) reported the molecular weights of major proteins of rat otoconia as 45-55 kDa and 90-100 kDa by SDS-PAGE. They also reported that 90-1(110 kDa protein was a major site of calcium binding (Pote and Ross, 1991). The mechanism of the morphogcncsis and calcification of the otoconia is still unclear despite recent work in birds that have identified certain molecular groups immunohistochemically in the otoconia (Fermin et al., 1990). Pote et al. (1993)

* C o r r e s p o n d i n g author. Fax: +81 (/16) 879-3959. 0378-5955/94/$07.0(I r¢~ 1994 Elsevier Science B.V. All rights reserved SSDI 113 7,~-5 95 5 ( 9 4 )0(1(19 7- A

purified the major protein, named otoconin-22, of the aragonitic otoconia of Xenopus laezJs, and they determined its amino acid sequence and carbohydrate composition. However, in mammalian calcitic otoconia, none of the protein components and the genes involved in the generation of otoconia have been isolated. The proteins involved in the ossification process have been identified as non-collagenous bone matrix proteins. The representative non-collagenous bone matrix proteins are osteopontin (OPN), osteonectin (ON) and osteocalcin (OC). These extraccllular matrix proteins arc thought to have important functions in the calcification of bones and teeth (Mark ct al., 1988a; Boskey, 1989). Among them, OPN is thought to be an important molecule for osteogencsis and chondrogenesis, because OPN m R N A and protein have been detected in the premature osteoblasts and chondrocytes in cmbryogenesis (Nomura et at., 1988; Mark et al., 1988a). In adult bones, OPN m R N A level is decreased with the age, whereas the expression of OPN m R N A is enhanced in the process of fracture healing in the osteoblasts in primitive woven bone and newly formed bone (Hirakawa et al., in press). OPN is characterized as a sialic acid-rich phosphorylated glycoprotein originally isolated from rat bone (Prince et al., 1987). It

100

/~ I'akemura et aL /ttearing Research 79 (1994) 99-104

contains the Arg-Gly-Asp ( R G D ) amino acid motif sequence and is considered as an extracellular protein associated with cell binding or adhesion of cells to the mineralized matrix (Pytela et al., 1986; Tamkum et al., 1986). From the analysis of amino acid sequence, OPN has putative binding sites to calcium (Prince, 1989, Gorski, 1992), and to hydroxyapatite which is a component of calcified bone matrix (Oldberg et al., 1986; Prince et al., 1987). From these observations, OPN is considered to be biologically involved in the attachment of osteoblasts to the extracellular matrix. Recent studies have revealed that OPN is involved not only in the physiological calcifying process such as osteogenesis but also in some pathological calcifying processes. Kohri et al. (1992) reported that OPN was a component of a urinary stone in the kidney. They also indicated that the level of OPN m R N A was enhanced by a treatment of sodium glyoxylate which induced the intrarenal crystallization of calcium oxalate (Kohri et al. 1993). We recently found (Hirota et al., 1993) that OPN m R N A was detected in the macrophages around the atheromatous plaques of human adult aortae and its expression was in parallel with the severity of atherosclerosis. These results strongly suggest that OPN plays some role in calcification process of urinary stones and atherosclerosis. In this study, the presence of OPN in the otoconial organ of the rat was investigated immunohistochemically, in order to clarify whether O P N was involved in the generation of otoconia in adult mammals. Furthermore, OPN mRNA-expressing cells were identified in the decalcified inner ear of adult rats by the in situ hybridization technique.

2. Materials and methods Materials and tissue preparation Sprague-Dawley (SD) rats, each weighing 200 to 250 g, were purchased from the Shizuoka Laboratory Animal Center (Hamamatsu, Japan). The rats were sacrificed under ether anesthesia, and the bone of the labyrinth together with the surrounding tissue was fixed

with 4% paraformaldehyde in 0.1 M phosphate buffer (PB) overnight at 4°C. The samples were then dehydrated with 70, 80, 90 and 100% ethanol and defatted with chloroform : ethanol = 1 : 1 solution. They were then hydrated with 100, 90, 80 and 70% ethanol, and decalcified with 20% E D T A (pH 7.4) for 14 days. The decalcified tissues were dehydrated with 70, 80, 90 and 100% ethanol again and embedded in paraffin. All solutions were prepared with 0.02% D E P C (diethylpyrocarbonate, SIGMA, St. Louis, USA) treated and autoclaved water. Sections 4 ~ m in thickness were cut and mounted on slides coated with 3-(triethoxylosilyl)propylamin (Merck, Schuchardt, Munich, FRG), which they were stored at 4°C until use. Probe preparation for in situ hybridization D i g o x i g e n i n ( D I G ) - l l - U T P - l a b e l e d single-strand R N A probes were prepared using a D I G R N A labeling kit (Boehringer Mannheim G m b H Biochemica, Mannheim, F R G ) according to the manufacturer's instructions. A 1.2 kb fragment of mouse O P N c D N A subcloned into a pGEM-1 plasmid was linearized with E c o R I endonuclease and transcribed with SP6 R N A polymerase to generate a 1.2 kb long antisense probe. To generate a sense probe, it was linearized with HindlII endonuclease and transcribed with T7 R N A polymerase. Similarly, a 1.0 kb fragment of mouse osteonectin (ON) cDNA, subcloned as described above, was linearized with E c o R I endonuclease and transcribed with SP6 R N A polymerase to generate a 1.0 kb antisense probe. For generation of the mouse osteocalcin (OC) probe, a 0.47 kb fragment of mouse osteocalcin c D N A obtained by reverse transcription followed by polymerase chain reaction (RT-PCR) was subcloned into a Bluescript I pKS-plasmid. This plasmid was either linearized with HindlII endonuclease and transcribed with T7 R N A polymerase to generate an approximately 0.5 kb antisense probe, or linearized with E c o R I endonuclease and transcribed with T3 R N A polymerase to generate the sense probe. In situ hybridization Hybridization and detection were carried out by a previously described method (Hirota et al., 1992) using

Fig. 1. Decalcified sections of the otoconial organs, treated with (A, B, C) or without (D) anti-rat OPN antibody. (A): Lower magnificationof the utricle (U) and saccule (S). (B): Higher magnification of the utricle. Stronglypositive reaction is observed in the otoconia (O), while the gelatin layer (GL) is faintly stained. (C): Higher magnification of the saccule. The same pattern of reaction as shown in B is seen. (D): Without primary antibodies. No reaction is observed. U: utricle, S: saccule, ELS: endolymphatic space, O: otoconia, GL: gelatin layer, SE: sensory epithelium. Scale bars, (A): 500 ~m; (B, C and D): 50 p.m. Fig. 2. Decalcified sections through the utricle (U) and saccule (S) were hybridized with antisense (A, B, C) or sense (D) strands of OPN probes. (A): Lower magnificationof the utricle (U) and saccule (S). (B): Higher magnification of the utricle. A positive signal is detected in the cytoplasm of the sensory hair cells (SHC), but not in that of the supporting cells (SC). (C): Higher magnification of the saccule. The same pattern as in B is seen. The box is higher magnification of arrowhead. A positive signal is observed in the cytoplasmof the sensory cell. (D): Control (sense probe). No signal is detected in the sensory epithelium. U:utricle, S:saccule, SHC: sensory hair cells, SC: supporting cells, ELS: endolymphaticspace, SE: sensory epithelium. Scale bars, (A): 500/xm; (B, C and D): 50 ~m.

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a Nucleic Acid Detection Kit (Boehringer Mannheim GmbH Biochemica, Mannheim, FRG) according to the manufacturer's instruction with minor modifications. All solutions used for hybridization and pretreatment were treated with 0.02% DEPC and autoclaved. All glassware was baked at 180°C for over 3 h to inactivate RNase. Sections were deparaffinized, hydrated at room temperature, washed twice with PB, and incubated with proteinase K (Boehringer Mannheim) at 2 / z g / m l in 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA for 30 min at 37°C. After the proteinase K treatment, the sections were re-fixed with freshly prepared 4% paraformaldehyde in 0.1 M PB for 15 min, washed twice with 0.1 M PB for 2 min, and treated with 0.2 N HCI to inactivate internal alkaline phosphatase. They were then acetylated with 0.25% acetic anhydrate in 0.1 M triethanolamine (pH 8.0) for 10 min. Subsequently, they were rinsed twice in 0.1 M PB for 2 min, dehydrated with 70, 80, 90, 95, and 100% ethanol, and air-dried. The hybridization solution contained 50% deionized formamide, 10% dextran sulfate, lx Denhardt's solution, 600 mM NaC1, 0.25% SDS, 250 /zg/ml of Escherichia coli tRNA (proteinase treated), 10 mM DTT (dithiothreitol), and 0.1 to 0.2 /xg/ml of digoxigeninUTP-labeled RNA probe. Fifty /xl of hybridization solution was placed on each section, covered with parafilm and incubated at 50°C for 16 h in a moisture chamber. After hybridization, the parafilm was removed and the slides were washed briefly in 5 × SSC at 50°C. They were then washed in 50% formamide, and 2 × SSC at 50°C for 30 min. After rinsing slides with 1 × TES [10 mM Tris HC1 (pH 7.6), 1 mM EDTA and 0.5 M NaC1] for 15 min at 37°C, they were treated with 2 X SSC for 20 min at 50°C and with 0.2 x SSC for 20 min twice at 50°C. The slides were rinsed briefly with 1 × TS (100 mM Tris HCI pH 7.5, 150 mM NaC1) and treated with blocking solution [1 x TS containing 1.5% (w/v) non-fat, non-sugar purified dry milk powder] for 60 min at room temperature. They were rinsed again with l × TS briefly and treated with alkaline phosphatase-conjugated anti-digoxigenin antibody (1.5 U/ml) for 30 min at room temperature, then washed twice with 1 x TS for 15 min. They were then rinsed briefly with 1 × TSM [100 mM Tris HCI (pH 9.5), 100 mM NaC1 and 50 mM MgC12] and reacted with color solution [340 n g / m l nitro blue tetrazolium (NBT), 175 ng/ml 5-bromo-4-chloro3-indolyl phosphate (BCIP) in 1 × TSM] overnight at room temperature. The reaction was stopped by the addition of 10 mM Tris HC1 (pH 8.0) and 1 mM EDTA, and the slides counterstained with hematoxylin.

Immunohistochemistry Sections were deparaffinized, dehydrated with methanol, and treated with 0.3% H 2 0 : in methanol to

inactivate internal peroxidase. After blocking with horse serum, the sections were exposed to mouse monoclonal antibody against rat OPN (Development Studies Hybridoma Bank, Iowa City, USA) at 4°C overnight. After washing with PBS, they were reacted with biotinylated anti-mouse lgG horse antibody from a Vectastain ABC kit (Vector Labs, Burlingame, CA, USA), followed by an avidin-biotin-peroxidase complex solution (ABC reagent from the kit). Color reaction was carried out with DAB (p-dimethyl-aminoazobenzene) for 4 min. The sections were counterstained with hematoxylin after washing with PBS.

3. Results

Localization of OPN by immunohistochemical study When sections of decalcified adult rat inner ear were stained with anti-rat OPN monoclonal antibody, positive reaction was detected in the saccular and utricular maculae (Fig. IA). At higher magnification, strongly positive reaction was localized in the polygonal and circular structures which were characteristic of the otoconia, while gelatin layer was faintly stained (Figs. 1B, C). No reaction was detected in the sensory epithelium when the adjacent section was incubated without primary antibody (Fig. 1D).

In situ hybridization analysis In situ hybridization was carried out to identify OPN mRNA expressing cells in the otoconial organs. Deparaffinized sections of the inner ear were hybridized with antisense probe encoding OPN cDNA. Positive signal was detected in the sensory epithelium of the saccule and the utricle (Fig. 2A). At higher magnification, positive signal was detected in the cytoplasm of the cells that were located on the apical part of the sensory epithelium facing the endolymphatic space (Figs. 2B, C), whereas the signal was not detected in the basal part of the sensory epithelium, in which the supporting cells were located. These observations suggest that OPN mRNA-positive cells are sensory hair cells of the saccule and utricle. No signal was detected in the sensory epithelium when the adjacent slide was hybridized with the OPN sense probe (Fig. 2D) or the antisense probes encoding ON and OC cRNA (data not shown).

4. Discussion

In the present study, we investigated the localization of OPN protein and mRNA in the otoconial organ using immunohistochemistry and in situ hybridization

T. Takemura et al. / Hearing Research 79 (1994) 99-104

histochemistry. Mark et al. (1988b) found that OPN was present in the otoconial membrane of the saccule and utricle using immunohistochemistry. However, they did not describe whether it was located in the gelatin layer or in the otoconia. In the present study, the otoconia themselves were demonstrated to contain OPN. Swanson et al. (1989) examined the gene expression of OPN in the embryonic mouse inner ear using in situ hybridization. Although signals were identified in the sensory epithelium of the vestibular apparatus, the resolution of their results was poor, and it could not be determined which cells expressed OPN mRNA. In our study, signals were detected in the apical part of the sensory epithelium, and the sensory hair cells expressed OPN mRNA. By in situ hybridization, strong signal was detected in the sensory hair cells, but by immunohistochemistry, positive signal was located in otoconia. This result is consistent to the previous finding that OPN is a secreted protein (Prince et al., 1987), and strongly suggests that OPN is produced in sensory hair cells and is secreted from those cells into the endolymph. The existence of metabolism in adult otoconia has not been demonstrated. However, previous investigations using calcium 45 strongly suggested that adult otoconia were in a dynamic state (Preston et al., 1975; Ross, 1979; Ross and Williams, 1979). In morphogenesis of embryonic otoconia, there has been a dominant hypothesis that otoconia are produced by the sensory epithelium, particularly supporting cells of otoconial organ (see review of Lim, 1985). In adult stage, there have been some reports that adult otoconia are also related with the supporting cells of the sensory epithelium. Harada et a1.(1979) found the small granular substance containing calcium within and beneath the surface of supporting cells in the sensory epithelium of matured cat by scanning electron microscopy. They speculated that the substance was the precursor of otoconia. Tachibana et al. (1992) detected glucuronic acid-containing glycosaminoglycan, which was one of the components of otoconia, in the supporting cells of adult Mongolian gerbil using a hyaluronidase-gold labeling technique. However, our results indicate that OPN, one of the proteinous components of otoconia, is produced by the sensory hair ceils of the saccule and utricle, and suggest that sensory hair cells are involved in the generation of otoconia together with supporting cells. Since OPN was immunohistochemically detected mainly in the outer surface of otoconia (Figs. 1B,C), it may be located richly on the peripheral area of otoconia and it may throw light on the producing process of the otoconia. Further investigation of OPN in the inner ear from the aspect of OPN as adhesion molecule may contribute to clarifying the process of otoconia formation.

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Acknowledgement The authors would like to express their hearty thanks to Emeritus Professor Toru Matsunaga, M.D., Osaka University Medical School, for his continuous encouragement and support.

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