Del Fattore Andrea, Fornari Rachele, Van Wesenbeeck Liesbeth, de Freitas Fenna, Timmermans Jean-Pierre, Peruzzi Barbara, Cappariello Alfredo, Rucci Nadia, Spera Giovanni, Helfrich Miep H, Van Hul Wim, Migliaccio Silvia, Teti Anna
Department of Experimental Medicine, University of L'Aquila, Italy.
J Bone Miner Res. 2008 Mar;23(3):380-91. doi: 10.1359/jbmr.071107.
We studied phenotypic and cellular aspects in a patient with a heterozygous mutation of the PLEKHM1 gene and obtained some indications regarding the role of the protein in bone cell function. Plekhm1 is involved in osteoclast endosomal vesicle acidification and TRACP exocytosis, contributing to events involved in osteoclast-osteoblast cross-talk.
The gene PLEKHM1 encodes a nonsecretory adaptor protein that localizes to endosomal vesicles. A highly truncated Plekhm1 protein was previously found in a patient with intermediate autosomal recessive osteopetrosis.
We describe a new heterozygous mutation in the PLEKHM1 gene in a patient presenting with low vertebral and femoral T-scores and areas of focal sclerosis. Clinical evaluation, mutational analysis, assessment of in vitro osteoclast morphology and activity, transfection studies, and evaluation of osteoclast-osteoblast cross-talk were carried out.
Direct DNA sequencing showed a heterozygous C to T substitution on cDNA position 2140 of the PLEKHM1 gene, predicted to lead to an R714C mutant protein. The mutation was not found in 104 control chromosomes. In vitro, patient's osteoclasts showed normal formation rate, morphology, number of nuclei, and actin rings but lower TRACP activity and higher endosomal pH than control osteoclasts. The patient had high serum PTH and TRACP, despite low TRACP activity in osteoclasts in vitro. HEK293 cells overexpressing either wildtype or Plekhm1-R714C showed no difference in localization of the variants, and co-transfection with a TRACP vector confirmed low TRACP activity in cells carrying the R714C mutation. RAW 264.7 osteoclast-like cells expressing the Plekhm1-R714C variant also showed low TRACP activity and reduced ability to acidify endosomal compartments compared with cells expressing the wildtype protein. Reduced intracellular TRACP was caused by increased protein secretion rather than reduced expression. TRACP-containing conditioned medium was able to increase osteoblast alkaline phosphatase, suggesting the focal osteosclerosis is a result of increased osteoclast-osteoblast coupling.
We provide further evidence for a role of Plekhm-1 in osteoclasts by showing that a novel mutation in PLEKHM1 is associated with a complex bone phenotype of generalized osteopenia combined with "focal osteosclerosis." Our data suggest that the mutation affects endosomal acidification/maturation and TRACP exocytosis, with implications for osteoclast-osteoblast cross-talk.
我们研究了一名携带PLEKHM1基因杂合突变患者的表型和细胞方面，并获得了有关该蛋白在骨细胞功能中作用的一些线索。Plekhm1参与破骨细胞内体囊泡酸化和抗酒石酸酸性磷酸酶（TRACP）的胞吐作用，促进破骨细胞与成骨细胞相互作用相关的事件。
PLEKHM1基因编码一种定位于内体囊泡的非分泌衔接蛋白。先前在一名患有中间型常染色体隐性骨硬化症的患者中发现了一种高度截短的Plekhm1蛋白。
我们描述了一名患有低椎体和股骨T值以及局灶性硬化区域的患者中PLEKHM1基因的一种新的杂合突变。进行了临床评估、突变分析、体外破骨细胞形态和活性评估、转染研究以及破骨细胞与成骨细胞相互作用的评估。
直接DNA测序显示PLEKHM1基因cDNA位置2140处存在杂合的C到T替换，预计会导致R714C突变蛋白。在104条对照染色体中未发现该突变。在体外，患者的破骨细胞显示出正常的形成速率、形态、核数量和肌动蛋白环，但与对照破骨细胞相比，TRACP活性较低且内体pH值较高。尽管体外破骨细胞中TRACP活性较低，但患者的血清甲状旁腺激素（PTH）和TRACP水平较高。过表达野生型或Plekhm1 - R714C的人胚肾293（HEK293）细胞在变体定位上没有差异，并且与TRACP载体共转染证实携带R714C突变的细胞中TRACP活性较低。与表达野生型蛋白的细胞相比，表达Plekhm1 - R714C变体的RAW 264.7破骨细胞样细胞也显示出较低的TRACP活性和酸化内体区室的能力降低。细胞内TRACP减少是由于蛋白质分泌增加而非表达减少所致。含有TRACP的条件培养基能够增加成骨细胞碱性磷酸酶，表明局灶性骨硬化是破骨细胞与成骨细胞偶联增加的结果。
我们通过表明PLEKHM1中的一种新突变与全身性骨质减少合并“局灶性骨硬化”的复杂骨表型相关，为Plekhm - 1在破骨细胞中的作用提供了进一步证据。我们的数据表明该突变影响内体酸化/成熟和TRACP胞吐作用，对破骨细胞与成骨细胞相互作用有影响。
