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. 2007 Apr;117(4):919-30.
doi: 10.1172/JCI30328.

Involvement of PLEKHM1 in osteoclastic vesicular transport and osteopetrosis in incisors absent rats and humans

Liesbeth Van Wesenbeeck  1 , Paul R Odgren, Fraser P Coxon, Annalisa Frattini, Pierre Moens, Bram Perdu, Carole A MacKay, Els Van Hul, Jean-Pierre Timmermans, Filip Vanhoenacker, Ruben Jacobs, Barbara Peruzzi, Anna Teti, Miep H Helfrich, Michael J Rogers, Anna Villa, Wim Van Hul
Affiliations

Involvement of PLEKHM1 in osteoclastic vesicular transport and osteopetrosis in incisors absent rats and humans

Liesbeth Van Wesenbeeck et al. J Clin Invest. 2007 Apr.

Abstract

This study illustrates that Plekhm1 is an essential protein for bone resorption, as loss-of-function mutations were found to underlie the osteopetrotic phenotype of the incisors absent rat as well as an intermediate type of human osteopetrosis. Electron and confocal microscopic analysis demonstrated that monocytes from a patient homozygous for the mutation differentiated into osteoclasts normally, but when cultured on dentine discs, the osteoclasts failed to form ruffled borders and showed little evidence of bone resorption. The presence of both RUN and pleckstrin homology domains suggests that Plekhm1 may be linked to small GTPase signaling. We found that Plekhm1 colocalized with Rab7 to late endosomal/lysosomal vesicles in HEK293 and osteoclast-like cells, an effect that was dependent on the prenylation of Rab7. In conclusion, we believe PLEKHM1 to be a novel gene implicated in the development of osteopetrosis, with a putative critical function in vesicular transport in the osteoclast.

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Figures

Figure 1
Figure 1. The Plekhm1 gene and protein.
(A) Genomic structure of the rat plekhm1 gene and the ia mutation. The plekhm1 gene consists of 12 exons (boxes) with the start codon located in exon 2. In ia rats, 1 cytosine (located in a stretch of 6 cytosines, underlined) is deleted in exon 5 of the plekhm1 gene. This results in a frameshift mutation followed by 5 divergent amino acids and a stop codon (asterisk). (B) Domain structure of the Plekhm1 protein. The Plekhm1 protein consists of a RUN domain, 2 PH domains, and a cysteine-rich (CYS) domain. The positions of the ia and human mutations are indicated.
Figure 2
Figure 2. Expression pattern of Plekhm1.
(A) The plekhm1 gene was expressed in all tissues examined, as demonstrated by a rat cDNA multiple tissue panel. (B) Western blot results of the Plekhm1 protein (confirmed by subsequent incubation of the same blot with the anti-FLAG antibody; data not shown) demonstrated that the Plekhm1 protein was expressed in human monocytes (hOCL) and osteoblasts (hOB). Moreover, expression of Plekhm1 increased during differentiation of human monocytes into osteoclasts by treatment with RANKL.
Figure 3
Figure 3. Mutation analysis of a family with autosomal-recessive osteopetrosis.
(A) Sequencing analysis showed a homozygous G→A transition at position +1 of intron 3 of the PLEKHM1 gene in the patient and the youngest brother. (B) Full-leg radiograph from the affected patient: cortical sclerosis of the pelvic bones, particularly at the iliac wings. Note the band-like sclerosis of the vertebral endplates (rugger jersey spine) and the inhomogeneous sclerosis at the metadiaphyses of the distal femora, tibiae and fibulae, and proximal fibulae and tibiae. Also note the broadening of the involved segments of the long bones (“Erlenmeyer flask” deformity). (C) Radiograph of the right femur of the youngest brother at 2 years of age showing the presence of a dense metaphyseal band at the distal metaphysis. (D) RT-PCR amplification across exons 3–4 of the PLEKHM1 gene. The sequence of the corresponding PCR products is presented. Lane 1, size marker; lane 2, father; lane 3, mother; lane 4, affected patient; lane 5, unrelated control; lane 6, blank. (E) Western blot analysis demonstrated that the Plekhm1 protein was not present in osteoclast lysates from the affected patient, in contrast to lysates from a healthy individual.
Figure 4
Figure 4. Characterization of the osteoclasts from the family with autosomal-recessive osteopetrosis.
Cells were cultured on dentine discs (A–D) or plastic (E and F) in the presence of RANKL for 10 days and then fixed, stained, and analyzed by confocal microscopy (A–D) or conventional microscopy (E and F). (A) Staining of F-actin with FITC-phalloidin (green stain), acidic vesicles with lysotracker (red stain), and osteoclast membrane with anti-VNR antibodies (blue stain). (B) Staining of the dentine surface with FL-ALN (green stain) and F-actin with TRITC-phalloidin (red stain). Dark areas correspond to resorption pits (asterisks). (C) Staining of the dentine surface with FL-ALN and nuclei with Sytox Green (green); F-actin with TRITC-phalloidin (red); osteoclast membrane with anti-VNR antibodies (blue). A–C represent 1-μm xy optical sections; D represents zx reconstructions of osteoclasts in C. Scale bar: 10 μm. (E and F) Staining for TRAP in osteoclasts. Original magnification, ×10 (E) and ×40 (F).
Figure 5
Figure 5. Electron microscopy of osteoclasts generated from the family with autosomal-recessive osteopetrosis.
(A and B) Osteoclasts were formed from all 3 siblings, then fixed and analyzed by scanning EM or transmission EM. (A) Scanning EM images show that the oldest brother produced normal, actively resorbing osteoclasts excavating deep resorption pits (denoted by asterisks), whereas both the patient and the youngest brother formed large, very flat osteoclasts with little evidence of resorption. (B) Transmission EM demonstrated that both the patient and the youngest brother produced osteoclasts containing large numbers of electron-dense granules (black arrows), whereas the resorbing osteoclasts formed from the oldest brother contained many multivesicular bodies (white arrows). (C) The electron-dense vesicles were identical in ultrastructure to those seen in the ia rat, which are known to contain TRAP. Scale bars: 100 μm (A), 1 μm (B), 0.2 μm (C).
Figure 6
Figure 6. Subcellular localization of Plekhm1 in HEK 293 cells.
HEK293 cells were transfected with plasmids, then fixed 24 hours later and analyzed by confocal microscopy. (A) Both Plekhm1-EGFP and Plekhm1-dsRedM displayed a diffuse cytoplasmic distribution, but were also clearly associated with intracellular vesicles, which frequently became abnormally enlarged. (B–E) HEK293 cells were cotransfected with Plekhm1-dsRedM and EGFP-Rab5 (B), EGFP-Rab6 (C), EGFP-Rab7 (D), or EGFP-Rab9 (E). Plekhm1 did not colocalize with either Rab5 or Rab6, but partially colocalized with Rab9 and completely colocalized to intracellular vesicles with Rab7. Interestingly, Plekhm1 showed more pronounced localization to these Rab7-positive vesicles when cotransfected with EGFP-Rab7. (F and G) HEK293 cells were transfected with EGFP-Rab7 and Plekhm1-dsRedM mutants corresponding to the mutations found in the osteopetrotic patient (hplekhm1; F) and the ia rat (rplekhm1; G). Neither mutant form of Plekhm1 colocalized with Rab7. Panels represent 1-μm xy optical sections. Scale bars: 10 μm.
Figure 7
Figure 7. Localization of Plekhm1 to endosomes is dependent on prenylated, active Rab7.
HEK293 cells were incubated for 24 hours in the presence of the Rab GGTase inhibitor 3-PEHPC (1 mM) immediately following transfection with EGFP-Rab7 and Plekhm1-dsRedM. (A and B) The vesicular localization of both Rab7 and Plekhm1 (A) was disrupted by 3-PEHPC (B). (C) Acidic vesicles were stained with lysotracker (LT) prior to fixation. No effect of 3-PEHPC on the integrity of the endosomal/lysosomal compartment was observed. (D and E) HEK293 cells were transfected with Plekhm1-dsRedM and constitutively active EGFP–Rab7-Q67L (D) or dominant-negative EGFP–Rab7-T22N (E). Plekhm1 and Rab7-Q67L colocalized on intracellular vesicles, whereas Rab7-T22N and plekhm1 were both diffusely distributed throughout the cytoplasm. Panels represent 1-μm xy optical sections. Scale bars: 10 μm.
Figure 8
Figure 8. Plekhm1 colocalizes to late endosomes/lysosomes with Rab7 in prefusion osteoclasts.
(A–D) Prefusion human osteoclasts were transfected with Plekhm1-EGFP or EGFP-Rab7 and then analyzed 24 hours later by confocal microscopy. We also labeled acidic vesicles with lysotracker red (A), late endosomes/lysosomes with TRITC-dextran (B), and recycling endosomes with Alexa Fluor 633–transferrin (C) prior to fixation. Plekhm1 was associated mainly with intracellular vesicles that accumulated lysotracker and endocytosed dextran (A and B, arrows) but not transferrin, indicating that plekhm1 is localized to late endosomes/lysosomes. (D) Osteoclast-like cells were counterstained with an antibody to the VNR (red stain) and the nuclei stained with TO-PRO-3 iodide (blue stain). Both EGFP-Rab7 and Plekhm1-EGFP were localized to intracellular vesicles in VNR-positive osteoclasts. (E) Prefusion osteoclast cells were cotransfected with Plekhm1-dsRedM and EGFP-Rab7, then stained with an anti-VNR antibody. Plekhm1 and Rab7 almost completely colocalize on intracellular vesicles in VNR-positive prefusion osteoclast cells. Panels represent 1-μm xy optical sections. Scale bars: 5 μm.
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