Low kindlin-3 levels in osteoclasts of kindlin-3 hypomorphic mice result in osteopetrosis due to leaky sealing zones

Osteoclasts form special integrin-mediated adhesion structures called sealing zones that enable them to adhere to and resorb bone. Sealing zones consist mainly of densely packed podosomes tightly inter-connected by actin fibers. Their formation requires the presence of the hematopoietic integrin regulator kindlin-3. In the current study, we investigated osteoclasts and their adhesion structures in kindlin-3 hypomorphic mice expressing only 5-10% of kindlin-3. Low kindlin-3 expression reduces integrin activity, results in impaired osteoclast adhesion and signaling, and delays cell spreading. Despite these defects, in vitro generated kindlin-3-hypomorphic osteoclasts arrange their podosomes into adhesion patches and belts, which show abnormal podosome and actin organization. Remarkably, kindlin-3-hypomorphic osteoclasts form sealing zones when cultured on calcified matrix in vitro and on bone surface in vivo. However, functional assays as well as immunohistochemical staining and electron micrographs of bone sections showed that they fail to properly seal the resorption lacunae, which is required for secreted proteases to be able to digest bone matrix. This results in mild osteopetrosis. Our study reveals a new, hitherto understudied function of kindlin-3 as an essential organizer of integrin-mediated adhesion structures, such as sealing zones.

破骨细胞会形成一类由整合素（integrin）介导的特殊黏附结构，称为封闭带（sealing zones），这类结构可使其黏附于骨组织并介导骨吸收。封闭带主要由密集排布的足小体（podosomes）构成，这些足小体通过肌动蛋白纤维（actin fibers）彼此紧密连接。封闭带的形成依赖于造血系整合素调节蛋白kindlin-3的表达。本研究中，我们对仅表达5%-10%正常量kindlin-3的kindlin-3低功能突变（hypomorphic）小鼠体内的破骨细胞及其黏附结构进行了研究。Kindlin-3低表达会降低整合素活性，导致破骨细胞黏附与信号转导受损，并延缓细胞铺展过程。尽管存在上述缺陷，体外培养的kindlin-3低功能突变破骨细胞仍可将足小体组装为黏附斑与黏附带，但其足小体与肌动蛋白纤维的排布存在异常。值得注意的是，当在体外钙化基质（calcified matrix）与体内骨表面培养时，kindlin-3低功能突变破骨细胞仍可形成封闭带。然而，功能实验、骨组织切片的免疫组织化学染色（immunohistochemical staining）与电子显微照片（electron micrographs）结果显示，此类破骨细胞无法正常封闭吸收陷窝（resorption lacunae），而这一过程是分泌型蛋白酶（secreted proteases）降解骨基质的必要条件，最终会引发轻度骨硬化症（osteopetrosis）。本研究揭示了kindlin-3一项此前未被充分研究的新功能：它作为整合素介导黏附结构（如封闭带）的核心组织者发挥作用。