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

Andrea Del Fattore, Rachele Fornari, Liesbeth Van Wesenbeeck, Fenna De Freitas, Jean Pierre Timmermans, Barbara Peruzzi, Alfredo Cappariello, Nadia Rucci, Giovanni Spera, Miep H. Helfrich, Wim Van Hul, Silvia Migliaccio, Anna Teti

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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. TRACP-containing 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 exocvtosis, with implications for osteoclast-osteoblast cross-talk.

Original languageEnglish
Pages (from-to)380-391
Number of pages12
JournalJournal of Bone and Mineral Research
Volume23
Issue number3
DOIs
Publication statusPublished - Mar 2008

Fingerprint

Osteopetrosis
Osteoclasts
Mutation
Genes
Osteoblasts
Osteosclerosis
Proteins
Transfection
platelet protein P47
Bone and Bones
Metabolic Bone Diseases
HEK293 Cells
Exocytosis
Sclerosis
Mutant Proteins
Conditioned Culture Medium
Thigh
DNA Sequence Analysis
Alkaline Phosphatase
Actins

Keywords

  • Bone resorption
  • Osteoclast
  • Osteopetrosis
  • Osteoporosis
  • PLEKHM1 gene
  • TRACP

ASJC Scopus subject areas

  • Surgery

Cite this

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. / 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.

In: Journal of Bone and Mineral Research, Vol. 23, No. 3, 03.2008, p. 380-391.

Research output: Contribution to journalArticle

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. / 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. In: Journal of Bone and Mineral Research. 2008 ; Vol. 23, No. 3. pp. 380-391.
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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. TRACP-containing 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 exocvtosis, with implications for osteoclast-osteoblast cross-talk.",
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T1 - 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

AU - Del Fattore, Andrea

AU - Fornari, Rachele

AU - Van Wesenbeeck, Liesbeth

AU - De Freitas, Fenna

AU - Timmermans, Jean Pierre

AU - Peruzzi, Barbara

AU - Cappariello, Alfredo

AU - Rucci, Nadia

AU - Spera, Giovanni

AU - Helfrich, Miep H.

AU - Van Hul, Wim

AU - Migliaccio, Silvia

AU - Teti, Anna

PY - 2008/3

Y1 - 2008/3

N2 - 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. TRACP-containing 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 exocvtosis, with implications for osteoclast-osteoblast cross-talk.

AB - 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. TRACP-containing 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 exocvtosis, with implications for osteoclast-osteoblast cross-talk.

KW - Bone resorption

KW - Osteoclast

KW - Osteopetrosis

KW - Osteoporosis

KW - PLEKHM1 gene

KW - TRACP

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