A New Heterozygous Mutation (R714C) of the Osteopetrosis Gene, Pleckstrin Homolog Domain Containing Family M (With Run Domain) Member 1 (PLEKHM1), Impairs Vesicular Acidification and Increases TRACP Secretion in Osteoclasts

Abstract

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. Introduction: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. Materials and Methods: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. Results: 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. TRACPcontaining conditioned medium was able to increase osteoblast alkaline phosphatase, suggesting the focal osteosclerosis is a result of increased osteoclast-osteoblast coupling. Conclusions: 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.

PLEKHM1/DEF8/RAB7 complex regulates lysosome positioning and bone homeostasis

“…Full-length Plekhm1 and the R 714 C mutant of Plekhm1 rescued the morphology and bone resorbing activity of Plekhm1 -/-osteoclasts (Supplemental Figure 7, A-C). However, in contrast to the findings in human osteoclasts (34), R 714 C mutation in murine osteoclasts failed to exhibit gain of function in vitro. These findings show that PLEKHM1 plays an intrinsic role in regulation of osteoclast function and each structural domain is functionally essential.…”

“…An R 714 C mutation in a human patient increases TRAP secretion (34). We generated recombinant retroviruses expressing HA-tagged full-length murine PLEKHM1 as well as a series of mutants with either mutations in human patients and rats or deletions of individual structural domains (Supplemental Figure 5A).…”

EMBnet.journal 18 Suppl. A

“…Nevertheless, the study of complex mechanisms needs a deeper level of data integration, in particular the analysis of the bone and chondral pathologies plays a critical role for understanding molecular mechanisms and regulative interactions. Among the pathologies considered in our database we can list: osteoporosis [20], osteogenesis imperfecta [21], osteopetrosis [22], osteoarthritis [23] and juvenile Paget's disease [24]. This represents a crucial aspect of the OsteoChondroDB, since no other resource tries to structure bone related biomolecular data relying on healthy and pathological conditions.…”

Regulation of lysosome biogenesis and functions in osteoclasts

“…In this case, however, the defect appeared to be at the level of endosomal acidification. 65 Finally, proteins implicated in autophagy such as Atg5 and LC3 have been shown to localize at the ruffled border in resorbing osteoclasts. Osteoclasts lacking Atg5 fail to localize Rab7-positive lysosomes to the ruffled border and have reduced resorptive capacity.…”