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Title: PTHrP participates in the bone destruction of middle ear cholesteatoma via promoting macrophage differentiation into osteoclasts induced by RANKL.
Author: Xie S, Jin L, Fu J, Yuan Q, Yin T, Ren J, Liu W.
Journal: Zhong Nan Da Xue Xue Bao Yi Xue Ban; 2024 May 28; 49(5):655-666. PubMed ID: 39174879.
Abstract:
OBJECTIVES: Progressive bone resorption and destruction is one of the most critical clinical features of middle ear cholesteatoma, potentially leading to various intracranial and extracranial complications. However, the mechanisms underlying bone destruction in middle ear cholesteatoma remain unclear. This study aims to explore the role of parathyroid hormone-related protein (PTHrP) in bone destruction associated with middle ear cholesteatoma. METHODS: A total of 25 cholesteatoma specimens and 13 normal external auditory canal skin specimens were collected from patients with acquired middle ear cholesteatoma. Immunohistochemical staining was used to detect the expressions of PTHrP, receptor activator for nuclear factor-kappa B ligand (RANKL), and osteoprotegerin (OPG) in cholesteatoma and normal tissues. Tartrate-resistant acid phosphatase (TRAP) staining was used to detect the presence of TRAP positive multi-nucleated macrophages in cholesteatoma and normal tissues. Mono-nuclear macrophage RAW264.7 cells were subjected to interventions, divided into a RANKL intervention group and a PTHrP+ RANKL co-intervention group. TRAP staining was used to detect osteoclast formation in the 2 groups. The mRNA expression levels of osteoclast-related genes, including TRAP, cathepsin K (CTSK), and nuclear factor of activated T cell cytoplasmic 1 (NFATc1), were measured using real-time polymerase chain reaction (real-time PCR) after the interventions. Bone resorption function of osteoclasts was assessed using a bone resorption pit analysis. RESULTS: Immunohistochemical staining showed significantly increased expression of PTHrP and RANKL and decreased expression of OPG in cholesteatoma tissues (all P<0.05). PTHrP expression was significantly positively correlated with RANKL, the RANKL/OPG ratio, and negatively correlated with OPG expression (r=0.385, r=0.417, r=-0.316, all P<0.05). Additionally, the expression levels of PTHrP and RANKL were significantly positively correlated with the degree of bone destruction in cholesteatoma (r=0.413, r=0.505, both P<0.05). TRAP staining revealed a large number of TRAP-positive cells, including multi-nucleated osteoclasts with three or more nuclei, in the stroma surrounding the cholesteatoma epithelium. After 5 days of RANKL or PTHrP+RANKL co-intervention, the number of osteoclasts was significantly greater in the PTHrP+RANKL co-intervention group than that in the RANKL group (P<0.05), with increased mRNA expression levels of TRAP, CTSK, and NFATc1 (all P<0.05). Scanning electron microscopy of bone resorption pits showed that the number (P<0.05) and size of bone resorption pits on bone slices were significantly greater in the PTHrP+RANKL co-intervention group compared with the RANKL group. CONCLUSIONS: PTHrP may promote the differentiation of macrophages in the surrounding stroma of cholesteatoma into osteoclasts through RANKL induction, contributing to bone destruction in middle ear cholesteatoma. 目的: 骨质进行性吸收破坏是中耳胆脂瘤最重要的临床特征之一，可导致一系列颅内外并发症，而目前中耳胆脂瘤骨破坏的机制尚未明确。本研究旨在探究甲状旁腺激素相关蛋白(parathyroid hormone-related protein，PTHrP)参与中耳胆脂瘤骨破坏的机制。方法: 收集后天性中耳胆脂瘤患者的25例胆脂瘤标本和13例外耳道正常皮肤组织标本。采用免疫组织化学染色方法检测PTHrP、核因子κB受体活化因子配体(receptor activator for nuclear factor-kappa B ligand，RANKL)和骨保护素(osteoprotegerin，OPG)在中耳胆脂瘤和外耳道正常皮肤组织中的表达，抗酒石酸酸性磷酸酶(tartrate-resistant acid phosphatase，TRAP)染色法检测中耳胆脂瘤和外耳道正常皮肤组织中是否存在TRAP阳性多核巨噬细胞。选取小鼠单核巨噬细胞RAW264.7细胞进行干预，分为RANKL干预组和PTHrP+RANKL共同干预组，采用TRAP染色法检测2组破骨细胞的生成情况，实时聚合酶链反应(real-time polymerase chain reaction，real-time PCR)检测干预后2组破骨细胞相关基因TRAP、组织蛋白酶K(cathepsin K，CTSK)和活化T细胞核因子1(nuclear factor of activated T cell cytoplasmic 1，NFATc1)的mRNA表达水平，骨吸收陷窝实验检测2组破骨细胞的骨吸收功能。结果: 免疫组织化学染色结果显示，PTHrP和RANKL在中耳胆脂瘤组织中的表达均显著增高，OPG表达降低(均P<0.05)，且PTHrP的表达与RANKL、RANKL/OPG比值均呈显著正相关，与OPG表达呈显著负相关(分别r=0.385、r=0.417、r=-0.316，均P<0.05)。同时，PTHrP、RANKL的表达水平与中耳胆脂瘤的骨破坏程度均呈显著正相关(分别r=0.413、r=0.505，均P<0.05)。TRAP染色结果显示中耳胆脂瘤上皮周围基质中有大量TRAP阳性细胞，并存在细胞核数量为3个或3个以上的TRAP阳性破骨细胞。RANKL或PTHrP+RANKL联合干预5 d后，与RANKL干预组相比，PTHrP+RANKL联合干预组的破骨细胞数量显著增加(P<0.05)，且破骨细胞相关基因TRAP、CTSK和NFATc1的mRNA表达水平均升高(均P<0.05)。骨吸收陷窝扫描电镜结果显示RANKL干预组、PTHrP+RANKL联合干预组的骨片表面均形成骨吸收陷窝；与RANKL干预组相比，PTHrP+RANKL联合干预组的骨片表面骨吸收陷窝数量显著增加(P<0.05)，面积也更大。结论: PTHrP可能通过促进RANKL诱导胆脂瘤组织周围基质中的巨噬细胞分化为破骨细胞，参与中耳胆脂瘤骨破坏。. OBJECTIVE: Progressive bone resorption and destruction is one of the most critical clinical features of middle ear cholesteatoma, potentially leading to various intracranial and extracranial complications. However, the mechanisms underlying bone destruction in middle ear cholesteatoma remain unclear. This study aims to explore the role of parathyroid hormone-related protein (PTHrP) in bone destruction associated with middle ear cholesteatoma. METHODS: A total of 25 cholesteatoma specimens and 13 normal external auditory canal skin specimens were collected from patients with acquired middle ear cholesteatoma. Immunohistochemical staining was used to detect the expressions of PTHrP, receptor activator for nuclear factor-kappa B ligand (RANKL), and osteoprotegerin (OPG) in cholesteatoma and normal tissues. Tartrate-resistant acid phosphatase (TRAP) staining was used to detect the presence of TRAP positive multi-nucleated macrophages in cholesteatoma and normal tissues. Mono-nuclear macrophage RAW264.7 cells were subjected to interventions, divided into a RANKL intervention group and a PTHrP+ RANKL co-intervention group. TRAP staining was used to detect osteoclast formation in the 2 groups. The mRNA expression levels of osteoclast-related genes, including TRAP, cathepsin K (CTSK), and nuclear factor of activated T cell cytoplasmic 1 (NFATc1), were measured using real-time polymerase chain reaction (real-time PCR) after the interventions. Bone resorption function of osteoclasts was assessed using a bone resorption pit analysis. RESULTS: Immunohistochemical staining showed significantly increased expression of PTHrP and RANKL and decreased expression of OPG in cholesteatoma tissues (all P<0.05). PTHrP expression was significantly positively correlated with RANKL, the RANKL/OPG ratio, and negatively correlated with OPG expression (r=0.385, r=0.417, r=-0.316, all P<0.05). Additionally, the expression levels of PTHrP and RANKL were significantly positively correlated with the degree of bone destruction in cholesteatoma (r=0.413, r=0.505, both P<0.05). TRAP staining revealed a large number of TRAP-positive cells, including multi-nucleated osteoclasts with three or more nuclei, in the stroma surrounding the cholesteatoma epithelium. After 5 days of RANKL or PTHrP+RANKL co-intervention, the number of osteoclasts was significantly greater in the PTHrP+RANKL co-intervention group than that in the RANKL group (P<0.05), with increased mRNA expression levels of TRAP, CTSK, and NFATc1 (all P<0.05). Scanning electron microscopy of bone resorption pits showed that the number (P<0.05) and size of bone resorption pits on bone slices were significantly greater in the PTHrP+RANKL co-intervention group compared with the RANKL group. CONCLUSION: PTHrP may promote the differentiation of macrophages in the surrounding stroma of cholesteatoma into osteoclasts through RANKL induction, contributing to bone destruction in middle ear cholesteatoma.

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