J Bone Miner Metab DOI 10.1007/s00774-015-0655-5
ORIGINAL ARTICLE
Osteoclasts are not activated in middle ear cholesteatoma Hiroki Koizumi · Hideaki Suzuki · Shoji Ikezaki · Toyoaki Ohbuchi · Koichi Hashida · Akinori Sakai
Received: 23 July 2014 / Accepted: 12 January 2015 © The Japanese Society for Bone and Mineral Research and Springer Japan 2015
Abstract It is unclear whether osteoclasts are present and activated in cholesteatomas. We explored the expression of messenger RNA (mRNA) for osteoclast biomarkers and regulating factors in middle ear cholesteatomas to elucidate the level of osteoclast activity in this disease. Bone powder was collected from 14 patients with cholesteatomatous and noncholesteatomatous chronic otitis media during tympanomastoidectomy, separately from cortical bone of the mastoid (clean bone powder), from bone neighboring cholesteatoma (cholesteatomatous bone powder), and from bone of the air cells and antrum of noncholesteatomatous chronic otitis media patients (noncholesteatomatous bone powder). The samples collected were soaked in TRIzol reagent, and total RNA was extracted and purified by the acid guanidinium thiocyanate–phenol–chloroform method, followed by the use of magnetic bead technology. The sample was then subjected to quantitative reverse transcription polymerase chain reaction for receptor activator of nuclear factor κB (RANK), tartrate-resistant acid phosphatase (TRAP), cathepsin K (CTSK), osteoclast-associated receptor (OSCAR), calcitonin receptor (CALCR), matrix metalloproteinase 9 (MMP9), receptor activator of nuclear factor H. Koizumi and H. Suzuki contributed equally to this work. H. Koizumi · H. Suzuki (*) · S. Ikezaki · T. Ohbuchi · K. Hashida Department of Otorhinolaryngology‑Head and Neck Surgery, School of Medicine, University of Occupational and Environmental Health, 1‑1 Iseigaoka, Yahatanishi‑ku, Kitakyushu 807‑8555, Japan e-mail: [email protected]‑u.ac.jp A. Sakai Department of Orthopaedic Surgery, School of Medicine, University of Occupational and Environmental Health, 1‑1 Iseigaoka, Yahatanishi‑ku, Kitakyushu 807‑8555, Japan
κB ligand (RANKL), and osteoprotegerin (OPG). There was no significant difference in the expression of TRAP, CTSK, OSCAR, CALCR, MMP9, or OPG among the clean, cholesteatomatous, and noncholesteatomatous bone powder. On the other hand, the expression of RANK and RANKL was significantly lower in the cholesteatomatous bone powder than in the noncholesteatomatous bone powder (P = 0.003 and P = 0.028, respectively). The RANKL mRNA/OPG mRNA ratio did not differ among the three samples. These results indicate that osteoclasts are unlikely to be activated in cholesteatomas. Bone resorption mechanisms not mediated by osteoclasts may need to be reappraised in cholesteatoma research in future studies. Keywords Middle ear cholesteatoma · Osteoclast · Bone powder · Receptor activator of nuclear factor κB · Quantitative reverse transcription polymerase chain reaction
Introduction Middle ear cholesteatoma is an epidermal cyst that typically arises from an invagination of the pars flaccida of the tympanic membrane. A deep invagination is referred to as a retraction pocket, and the pocket wall histologically consists of an inverted epidermis. The epidermis is a keratinizing stratified squamous epithelium, which exhibits “keratin debris” desquamation from its surface. As the retraction pocket extends deeply into the middle ear cavity, the pocket stores the keratin debris and develops into a cholesteatoma. The cholesteatoma then gradually erodes neighboring bone structures and further expands, leading to a vicious circle of cholesteatoma expansion. Consequently, cholesteatoma in the middle ear cavity not only causes hearing
13
loss and otorrhea, but may also induce vertigo, deafness, facial nerve paralysis, sigmoid sinus thrombosis, and even intracranial complications such as meningitis and epidural/ subdural/brain abscesses. In contrast, noncholesteatomatous otitis media rarely shows such aggressive properties. Because there is no effective conservative treatment for middle ear cholesteatomas, most patients with this disease have no choice but to undergo surgical treatment. Until the middle of the twentieth century, the “pressure necrosis” theory had been a mainstream explanation for bone resorption in cholesteatoma [1, 2], but most researchers no longer support this theory. Since the 1970s, a variety of cytokines, chemical mediators, and enzymes have been detected in cholesteatoma tissue, and these factors have been reported as possible causative factors for bone resorption. Many of these substances are thought to exert bone-resorbing action eventually through osteoclast activation. In this century, the focus of research on cholesteatoma has shifted to osteoclast activation via the receptor activator of nuclear factor κB ligand (RANKL)/receptor activator of nuclear factor κB (RANK)/osteoprotegerin (OPG) system in connection with inflammatory products [3–5]. Although the presence of osteoclasts in cholesteatoma has been documented by several authors [6–8], many other researchers have found no or few osteoclasts in this lesion [9–16]. Thus, whether osteoclasts are present and play a role in bone resorption in cholesteatomas remains controversial. In the present study, we explored the expression of messenger RNA (mRNA) for osteoclast markers in middle ear cholesteatomas in comparison with noncholesteatomatous otitis media and normal cortical bone in an attempt to elucidate the level of osteoclast activity in these tissues.
J Bone Miner Metab
bone neighboring cholesteatoma (cholesteatomatous bone powder), and from the bone of the air cells and antrum of noncholesteatomatous chronic otitis media patients (noncholesteatomatous bone powder). The clean bone powder was collected from eight patients: four patients with cholesteatomatous otitis media, three patients with noncholesteatomatous chronic otitis media, and one patient who underwent transmastoid facial nerve decompression. Written informed consent was obtained from all patients, and the study was approved by the Institutional Review Board of the University of Occupational and Environmental Health. Isolation of total RNA For quantitative reverse transcription (qRT) polymerase chain reaction (PCR), the sample collected was soaked in 1 ml TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and sonicated using an ultrasonic homogenizer (Taitec, Saitama, Japan). Chloroform (200 μl) was added to the homogenate, and after it had been thoroughly shaken, the mixture was centrifuged at 21,880g for 15 min at 4 °C. The aqueous layer was transferred to another tube, and total RNA was extracted by the acid guanidinium-thiocyanate– phenol–chloroform method and cleaned with a BioRobot EZ1 system (QIAGEN, Hilden, Germany) [18, 19], which allows fully automated extraction and purification of nucleic acids by magnetic bead technology. The purity of RNA was assessed by determining the ratio of light absorption at 260 nm to that at 280 nm. The RNA concentration was determined from A260. Quantitative reverse transcription polymerase chain reaction
Materials and methods Patients and sample collection We enrolled 14 patients (14 ears) with chronic otitis media with or without cholesteatoma who underwent tympanomastoidectomy in our department. There were nine men and five women, ranging in age from 14 to 82 years, with an average age of 55.4 years, and they consisted of eight patients with cholesteatomatous otitis media, five patients with noncholesteatomatous chronic otitis media, and one patient who underwent transmastoid facial nerve decompression. While drilling the mastoid bone during surgery, we collected ground bone, so-called bone powder, by irrigation–suction using the mesh filter of a transfusion set (Terumo, Tokyo, Japan) as described previously [17]. The bone powder sample was collected separately from the cortical bone of the mastoid without contamination from cholesteatoma/mucosa (clean bone powder), from the
13
The total RNA was reverse transcribed to complementary DNA (cDNA) with a high-capacity RNA-to-cDNA kit (Applied Biosystems, Foster City, CA), which uses random primers. The qRT-PCR analysis was performed with an Applied Biosystems StepOnePlus real-time PCR system using TaqMan fast universal PCR master mix (Applied Biosystems) according to the manufacturer’s specifications. The TaqMan gene expression assays for RANK (also known as TNFRSF11A; assay identification number Hs00187192_m1), tartrate-resistant acid phosphatase (TRAP) (also known as ACP5; assay identification number Hs00243522_m1), cathepsin K (CTSK; assay identification number Hs00166156_ m1), osteoclast-associated receptor (OSCAR; assay identification number Hs01100185_m1), calcitonin receptor (CALCR; assay identification number Hs00907738_m1), matrix metalloproteinase 9 (MMP9; assay identification number Hs00234579_m1), RANKL (also known as TNFSF11; assay identification number Hs00243522_m1),
J Bone Miner Metab P=0.464 P=0.529
0.16
0.12 0.1 0.08 0.06 0.04 0.02 0
clean (n=8)
non-cholesteatoma (n=5)
P=0.306 P=0. 462
Ra o of TRAP mRNA/GAPDH mRNA
1.2
Results
cholesteatoma (n=8)
Fig. 1 Expression of RANK messenger RNA (mRNA) in the clean, cholesteatomatous, and noncholesteatomatous bone powder. The asterisk indicates statistical significance
Statistics Data are shown as a dot plot with a median. The statistical significance of differences was analyzed using the Mann– Whitney U test. The statistical significance of correlation coefficients between osteoclast biomarkers was examined by a t test. P values less than 0.05 were considered significant.
P=0.003 *
0.14
Ra o of RANK mRNA/GAPDH mRNA
and OPG (also known as TNFRSF11B; assay identification number Hs00900358_m1), and for glyceraldehyde 3-phosphate dehydrogenase (GAPDH; assay identification number Hs99999905_m1) as a housekeeping gene were purchased from Applied Biosystems. Complementary DNA (cDNA; 10 ng/µl) was mixed with TaqMan universal PCR master mix with AmpErase (uracil N-glycosylase) and the primer/probe set of the TaqMan gene expression assays, and the mixture was subjected to PCR amplification with real-time detection. The thermal cycler conditions were as follows: holding at 95 °C for 2 min, followed by two-step PCR of 40 cycles at 95 °C for 1 s followed by 60 °C for 20 s. Each sample was assayed in duplicate. The measured threshold cycle (CT) was normalized by subtracting CT for GAPDH of each sample from that for RANK, TRAP, CTSK, OSCAR, CALCR, MMP9, RANKL, and OPG. From the ΔCT value obtained, the ratio of target mRNA to GAPDH mRNA was calculated by the following formula: Target mRNA/GAPDH mRNA ratio = 2−ΔCT.
P=0.661
1
0.8
0.6
0.4
0.2
The wet weight of the collected bone powder samples ranged from 150 to 790 mg, with a median of 420 mg. The total amount of extracted RNA ranged from 0.80 to 10.32 μg, with a median of 3.89 μg. The A260/A280 ratio of the final RNA eluates ranged from 1.77 to 1.81, and GAPDH mRNA was detected in all samples, indicating that the purity and quality of the isolated RNA were acceptable. The expression of RANK, TRAP, CTSK, OSCAR, CALCR, MMP9, RANKL, and OPG mRNA is shown in Figs. 1, 2, 3, 4, 5, 6, 7, and 8. There was no significant difference in the expression of TRAP, CTSK, OSCAR, CALCR, MMP9, or OPG mRNA among the clean, cholesteatomatous, and noncholesteatomatous bone powder. On the other hand, the expression of RANK and RANKL mRNA was significantly lower in the cholesteatomatous bone powder than in the noncholesteatomatous bone powder (Fig. 1, P = 0.003, and Fig. 7, P = 0.028, respectively). The RANKL mRNA/OPG mRNA ratio did not differ among the three samples (Fig. 9). These results indicate that the presence and activity of osteoclasts in the cholesteatoma are no more than those in the noncholesteatomatous lesion and normal cortical bone.
0
clean (n=8)
cholesteatoma (n=8)
non-cholesteatoma (n=5)
Fig. 2 Expression of TRAP messenger RNA (mRNA) in the clean, cholesteatomatous, and noncholesteatomatous bone powder
The expression of mRNA for osteoclast biomarkers in individual samples is listed in Table 1. Of the six osteoclast biomarkers examined (RANK, TRAP, CTSK, OSCAR, CALCR, and MMP9), significant correlation was seen between four pairs; CTSK/MMP9 (r = 0.808, P
ORIGINAL ARTICLE
Osteoclasts are not activated in middle ear cholesteatoma Hiroki Koizumi · Hideaki Suzuki · Shoji Ikezaki · Toyoaki Ohbuchi · Koichi Hashida · Akinori Sakai
Received: 23 July 2014 / Accepted: 12 January 2015 © The Japanese Society for Bone and Mineral Research and Springer Japan 2015
Abstract It is unclear whether osteoclasts are present and activated in cholesteatomas. We explored the expression of messenger RNA (mRNA) for osteoclast biomarkers and regulating factors in middle ear cholesteatomas to elucidate the level of osteoclast activity in this disease. Bone powder was collected from 14 patients with cholesteatomatous and noncholesteatomatous chronic otitis media during tympanomastoidectomy, separately from cortical bone of the mastoid (clean bone powder), from bone neighboring cholesteatoma (cholesteatomatous bone powder), and from bone of the air cells and antrum of noncholesteatomatous chronic otitis media patients (noncholesteatomatous bone powder). The samples collected were soaked in TRIzol reagent, and total RNA was extracted and purified by the acid guanidinium thiocyanate–phenol–chloroform method, followed by the use of magnetic bead technology. The sample was then subjected to quantitative reverse transcription polymerase chain reaction for receptor activator of nuclear factor κB (RANK), tartrate-resistant acid phosphatase (TRAP), cathepsin K (CTSK), osteoclast-associated receptor (OSCAR), calcitonin receptor (CALCR), matrix metalloproteinase 9 (MMP9), receptor activator of nuclear factor H. Koizumi and H. Suzuki contributed equally to this work. H. Koizumi · H. Suzuki (*) · S. Ikezaki · T. Ohbuchi · K. Hashida Department of Otorhinolaryngology‑Head and Neck Surgery, School of Medicine, University of Occupational and Environmental Health, 1‑1 Iseigaoka, Yahatanishi‑ku, Kitakyushu 807‑8555, Japan e-mail: [email protected]‑u.ac.jp A. Sakai Department of Orthopaedic Surgery, School of Medicine, University of Occupational and Environmental Health, 1‑1 Iseigaoka, Yahatanishi‑ku, Kitakyushu 807‑8555, Japan
κB ligand (RANKL), and osteoprotegerin (OPG). There was no significant difference in the expression of TRAP, CTSK, OSCAR, CALCR, MMP9, or OPG among the clean, cholesteatomatous, and noncholesteatomatous bone powder. On the other hand, the expression of RANK and RANKL was significantly lower in the cholesteatomatous bone powder than in the noncholesteatomatous bone powder (P = 0.003 and P = 0.028, respectively). The RANKL mRNA/OPG mRNA ratio did not differ among the three samples. These results indicate that osteoclasts are unlikely to be activated in cholesteatomas. Bone resorption mechanisms not mediated by osteoclasts may need to be reappraised in cholesteatoma research in future studies. Keywords Middle ear cholesteatoma · Osteoclast · Bone powder · Receptor activator of nuclear factor κB · Quantitative reverse transcription polymerase chain reaction
Introduction Middle ear cholesteatoma is an epidermal cyst that typically arises from an invagination of the pars flaccida of the tympanic membrane. A deep invagination is referred to as a retraction pocket, and the pocket wall histologically consists of an inverted epidermis. The epidermis is a keratinizing stratified squamous epithelium, which exhibits “keratin debris” desquamation from its surface. As the retraction pocket extends deeply into the middle ear cavity, the pocket stores the keratin debris and develops into a cholesteatoma. The cholesteatoma then gradually erodes neighboring bone structures and further expands, leading to a vicious circle of cholesteatoma expansion. Consequently, cholesteatoma in the middle ear cavity not only causes hearing
13
loss and otorrhea, but may also induce vertigo, deafness, facial nerve paralysis, sigmoid sinus thrombosis, and even intracranial complications such as meningitis and epidural/ subdural/brain abscesses. In contrast, noncholesteatomatous otitis media rarely shows such aggressive properties. Because there is no effective conservative treatment for middle ear cholesteatomas, most patients with this disease have no choice but to undergo surgical treatment. Until the middle of the twentieth century, the “pressure necrosis” theory had been a mainstream explanation for bone resorption in cholesteatoma [1, 2], but most researchers no longer support this theory. Since the 1970s, a variety of cytokines, chemical mediators, and enzymes have been detected in cholesteatoma tissue, and these factors have been reported as possible causative factors for bone resorption. Many of these substances are thought to exert bone-resorbing action eventually through osteoclast activation. In this century, the focus of research on cholesteatoma has shifted to osteoclast activation via the receptor activator of nuclear factor κB ligand (RANKL)/receptor activator of nuclear factor κB (RANK)/osteoprotegerin (OPG) system in connection with inflammatory products [3–5]. Although the presence of osteoclasts in cholesteatoma has been documented by several authors [6–8], many other researchers have found no or few osteoclasts in this lesion [9–16]. Thus, whether osteoclasts are present and play a role in bone resorption in cholesteatomas remains controversial. In the present study, we explored the expression of messenger RNA (mRNA) for osteoclast markers in middle ear cholesteatomas in comparison with noncholesteatomatous otitis media and normal cortical bone in an attempt to elucidate the level of osteoclast activity in these tissues.
J Bone Miner Metab
bone neighboring cholesteatoma (cholesteatomatous bone powder), and from the bone of the air cells and antrum of noncholesteatomatous chronic otitis media patients (noncholesteatomatous bone powder). The clean bone powder was collected from eight patients: four patients with cholesteatomatous otitis media, three patients with noncholesteatomatous chronic otitis media, and one patient who underwent transmastoid facial nerve decompression. Written informed consent was obtained from all patients, and the study was approved by the Institutional Review Board of the University of Occupational and Environmental Health. Isolation of total RNA For quantitative reverse transcription (qRT) polymerase chain reaction (PCR), the sample collected was soaked in 1 ml TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and sonicated using an ultrasonic homogenizer (Taitec, Saitama, Japan). Chloroform (200 μl) was added to the homogenate, and after it had been thoroughly shaken, the mixture was centrifuged at 21,880g for 15 min at 4 °C. The aqueous layer was transferred to another tube, and total RNA was extracted by the acid guanidinium-thiocyanate– phenol–chloroform method and cleaned with a BioRobot EZ1 system (QIAGEN, Hilden, Germany) [18, 19], which allows fully automated extraction and purification of nucleic acids by magnetic bead technology. The purity of RNA was assessed by determining the ratio of light absorption at 260 nm to that at 280 nm. The RNA concentration was determined from A260. Quantitative reverse transcription polymerase chain reaction
Materials and methods Patients and sample collection We enrolled 14 patients (14 ears) with chronic otitis media with or without cholesteatoma who underwent tympanomastoidectomy in our department. There were nine men and five women, ranging in age from 14 to 82 years, with an average age of 55.4 years, and they consisted of eight patients with cholesteatomatous otitis media, five patients with noncholesteatomatous chronic otitis media, and one patient who underwent transmastoid facial nerve decompression. While drilling the mastoid bone during surgery, we collected ground bone, so-called bone powder, by irrigation–suction using the mesh filter of a transfusion set (Terumo, Tokyo, Japan) as described previously [17]. The bone powder sample was collected separately from the cortical bone of the mastoid without contamination from cholesteatoma/mucosa (clean bone powder), from the
13
The total RNA was reverse transcribed to complementary DNA (cDNA) with a high-capacity RNA-to-cDNA kit (Applied Biosystems, Foster City, CA), which uses random primers. The qRT-PCR analysis was performed with an Applied Biosystems StepOnePlus real-time PCR system using TaqMan fast universal PCR master mix (Applied Biosystems) according to the manufacturer’s specifications. The TaqMan gene expression assays for RANK (also known as TNFRSF11A; assay identification number Hs00187192_m1), tartrate-resistant acid phosphatase (TRAP) (also known as ACP5; assay identification number Hs00243522_m1), cathepsin K (CTSK; assay identification number Hs00166156_ m1), osteoclast-associated receptor (OSCAR; assay identification number Hs01100185_m1), calcitonin receptor (CALCR; assay identification number Hs00907738_m1), matrix metalloproteinase 9 (MMP9; assay identification number Hs00234579_m1), RANKL (also known as TNFSF11; assay identification number Hs00243522_m1),
J Bone Miner Metab P=0.464 P=0.529
0.16
0.12 0.1 0.08 0.06 0.04 0.02 0
clean (n=8)
non-cholesteatoma (n=5)
P=0.306 P=0. 462
Ra o of TRAP mRNA/GAPDH mRNA
1.2
Results
cholesteatoma (n=8)
Fig. 1 Expression of RANK messenger RNA (mRNA) in the clean, cholesteatomatous, and noncholesteatomatous bone powder. The asterisk indicates statistical significance
Statistics Data are shown as a dot plot with a median. The statistical significance of differences was analyzed using the Mann– Whitney U test. The statistical significance of correlation coefficients between osteoclast biomarkers was examined by a t test. P values less than 0.05 were considered significant.
P=0.003 *
0.14
Ra o of RANK mRNA/GAPDH mRNA
and OPG (also known as TNFRSF11B; assay identification number Hs00900358_m1), and for glyceraldehyde 3-phosphate dehydrogenase (GAPDH; assay identification number Hs99999905_m1) as a housekeeping gene were purchased from Applied Biosystems. Complementary DNA (cDNA; 10 ng/µl) was mixed with TaqMan universal PCR master mix with AmpErase (uracil N-glycosylase) and the primer/probe set of the TaqMan gene expression assays, and the mixture was subjected to PCR amplification with real-time detection. The thermal cycler conditions were as follows: holding at 95 °C for 2 min, followed by two-step PCR of 40 cycles at 95 °C for 1 s followed by 60 °C for 20 s. Each sample was assayed in duplicate. The measured threshold cycle (CT) was normalized by subtracting CT for GAPDH of each sample from that for RANK, TRAP, CTSK, OSCAR, CALCR, MMP9, RANKL, and OPG. From the ΔCT value obtained, the ratio of target mRNA to GAPDH mRNA was calculated by the following formula: Target mRNA/GAPDH mRNA ratio = 2−ΔCT.
P=0.661
1
0.8
0.6
0.4
0.2
The wet weight of the collected bone powder samples ranged from 150 to 790 mg, with a median of 420 mg. The total amount of extracted RNA ranged from 0.80 to 10.32 μg, with a median of 3.89 μg. The A260/A280 ratio of the final RNA eluates ranged from 1.77 to 1.81, and GAPDH mRNA was detected in all samples, indicating that the purity and quality of the isolated RNA were acceptable. The expression of RANK, TRAP, CTSK, OSCAR, CALCR, MMP9, RANKL, and OPG mRNA is shown in Figs. 1, 2, 3, 4, 5, 6, 7, and 8. There was no significant difference in the expression of TRAP, CTSK, OSCAR, CALCR, MMP9, or OPG mRNA among the clean, cholesteatomatous, and noncholesteatomatous bone powder. On the other hand, the expression of RANK and RANKL mRNA was significantly lower in the cholesteatomatous bone powder than in the noncholesteatomatous bone powder (Fig. 1, P = 0.003, and Fig. 7, P = 0.028, respectively). The RANKL mRNA/OPG mRNA ratio did not differ among the three samples (Fig. 9). These results indicate that the presence and activity of osteoclasts in the cholesteatoma are no more than those in the noncholesteatomatous lesion and normal cortical bone.
0
clean (n=8)
cholesteatoma (n=8)
non-cholesteatoma (n=5)
Fig. 2 Expression of TRAP messenger RNA (mRNA) in the clean, cholesteatomatous, and noncholesteatomatous bone powder
The expression of mRNA for osteoclast biomarkers in individual samples is listed in Table 1. Of the six osteoclast biomarkers examined (RANK, TRAP, CTSK, OSCAR, CALCR, and MMP9), significant correlation was seen between four pairs; CTSK/MMP9 (r = 0.808, P
