Bone resorption is an essential process for growing bone tissue, adaptating to exercise, and maintaining a healthy skeleton throughout life and is carried out by a highly specialized cell type, the osteoclast (53
). Defects in the resorptive capacity of osteoclasts lead to osteopetrosis, an inherited disease manifested clinically by a generalized increase in bone mass and, in severe cases, accompanied by secondary bone marrow deficiency and neurologic problems due to cranial nerve compression (13
). There is a wide range of clinical phenotypes in osteopetrosis, and it is becoming clear that these are associated with different genetic defects. It has long been recognized in studies of spontaneous animal models of osteopetrosis that the disease is heterogeneous, and it is now known that genetic mutations in any of the steps leading to osteoclast formation or activation of the resorption process can cause osteopetrosis (13
). By contrast, the molecular mechanisms identified to play a role in human osteopetroses are so far completely restricted to alterations in osteoclast function (1
). The 4 causative genes currently identified are all involved in the process of acidification of the extracellular compartment between the bone surface and the osteoclast. In these cases there is no deficiency in osteoclast formation, with normal or even increased numbers of osteoclasts present on the bone surface. In the case of recessive TCIRG1
loss-of-function mutations, osteoclasts fail to polarize properly and, although rudimentary clear zones may be found, there is generally no sign of ruffled border formation, nor of extracellular matrix degradation (3
), indicating that loss of function of these genes completely abrogates the secretion of acid and proteolytic enzymes. In this report, we demonstrate for the first time to our knowledge that loss-of-function mutations in the PLEKHM1
gene underlie mild osteopetrosis in the ia
rat as well as an intermediate form of human osteopetrosis. Moreover, we believe this to be the first indication of a function for Plekhm1 and highlights its crucial role in bone metabolism.
The expression of Plekhm1 protein is increased during RANKL-induced differentiation of osteoclasts and is much higher than in other cell types such as osteoblasts. Together with the fact that loss of function results in osteopetrosis, this indicates that the PLEKHM1
gene product is particularly important for osteoclast function, as is the case for other human osteopetrosis-causing genes. Microscopy analysis showed that osteoclasts from the osteopetrotic patient differentiated normally from PBMCs and attached to the surface of dentine by peripheral belts of podosomes. However, osteoclasts from the osteopetrotic patient — unlike those from her brother, who was heterozygous for the PLEKHM1
mutation — showed hardly any evidence of resorptive activity when cultured on dentine discs. These findings indicate that the Plekhm1 protein is not involved in the differentiation, attachment, or initial actin organization of osteoclasts, but rather in a later step of the bone resorption process. Although the formation of lysosomal vesicles was normal, the ruffled border was absent or underdeveloped in the patient-derived osteoclasts. The in vitro osteoclast phenotype we observed was very similar to that seen with other osteopetrotic mutations (54
). An interesting feature of osteoclasts from the patient with the PLEKHM1
mutation was an increased accumulation of the enzyme TRAP (which is highly expressed in osteoclasts), an effect also seen in the ia
). Moreover, transmission EM of the osteoclasts from the 2 siblings homozygous for the PLEKHM1
mutation demonstrated the presence of electron-dense vesicles that were identical to those seen in the ia
rat, which have previously been shown to contain TRAP (15
). These vesicles were not seen in actively resorbing osteoclasts from the heterozygous brother, which instead contained many multivesicular bodies near the ruffled border. Such bodies were much less abundant in osteoclasts from the 2 homozygous siblings, suggesting that the multivesicular bodies contain material taken up from the resorption lacuna, whereas the dense granules contain TRAP and could be secretory vesicles. Unfortunately, we did not have a bone biopsy or sufficient patient-derived cultured osteoclasts to confirm expression of TRAP in these vesicles by immunoelectron microscopy, a technique that requires different fixation and embedding of tissue than the one used for routine transmission EM. Optimization of currently available anti-TRAP antibodies for such studies in human osteoclasts is underway. However, our findings suggest that the absence of Plekhm1 disrupts processes involved in the transport and/or fusion of late endosomal vesicles and consequently disrupts the formation of the ruffled border, which in turn impacts the endocytosis that normally occurs from this membrane domain (56
). It is also possible that a defect in the maturation or trafficking of late endosomes prolongs their lifespan, allowing accumulation of their contents.
The exact function of Plekhm1 is unknown. Part of Plekhm1 has previously been described as the AP162 protein, which was suggested to function as an intracellular adaptor modulating apoptotic signals in colon tissue (23
). However, the recombinant AP162 protein in the previously reported experiments lacks 130 amino acids at the N-terminal end of the Plekhm1 protein, indicating that full-length Plekhm1 has not been studied before. The putative role of the Plekhm1 protein in intracellular vesicular transport is supported by the presence of a RUN domain and 2 PH domains, which are found in proteins involved in intracellular signaling pathways of Ras-like small GTPases (29
). In particular, the RUN domain has been identified in proteins that interact with small GTPases of the Rab family, which are master regulators of vesicular transport. These proteins include Iporin, a ubiquitously expressed protein that interacts with Rab1b (30
), and Rabip4, a Rab4 effector that is associated with early endosomes (34
). Rab GTPases are known to play a critical role in the regulation of osteoclast activity (57
); in particular, Rab3D and Rab7 have been shown to be essential for osteoclastic resorption (58
We found further evidence for a role of the Plekhm1 protein in vesicular transport using overexpression studies in HEK293 cells, which demonstrated that Plekhm1 was partially associated with intracellular vesicles. These vesicles were identified as late endosomes or lysosomes, since Plekhm1 colocalizes with the late endosomal/lysosomal Rab7 and Rab9, but not with the early endosome–associated Rab5 or the Golgi-localized Rab6. Moreover, coexpression of Plekhm1 and Rab7 resulted in a striking redistribution of Plekhm1 to the Rab7-positive vesicles and a concomitant loss of the diffuse, cytoplasmic Plekhm1. This recruitment of Plekhm1 to late endosomes/lysosomes in cells expressing or overexpressing Rab7 is likely the result of an interaction between these proteins rather than changes in the nature of the compartment, since overexpression of another late endosomal/lysosomal Rab GTPase, Rab9, did not alter the distribution of Plekhm1, despite the fact that these proteins showed some colocalization. Importantly, we found that Plekhm1 also localized to late endosomes/lysosomes in prefusion osteoclasts, since it localized to acidic vesicles to which endocytosed dextran, but not transferrin (a marker of recycling endosomes), was transported. Moreover, Plekhm1 colocalized with Rab7 in VNR-positive prefusion osteoclasts, again suggesting an interaction between these 2 proteins. Interestingly, when transfected alone Plekhm1 showed a much more striking vesicular (and therefore less cytoplasmic) localization in these cells than in HEK293 cells, possibly because higher endogenous levels of Rab7 in prefusion osteoclasts enables greater association with late endosomes/lysosomes.
The correct localization of Rab GTPases to specific subcellular membranes is dependent on posttranslational prenylation, which involves the attachment of geranylgeranyl isoprenoid groups to 1 or, more commonly, 2 C-terminal cysteine residues by the enzyme Rab GGTase (50
). Rab prenylation can be blocked selectively using the specific Rab GGTase inhibitor 3-PEHPC, thereby preventing the membrane localization of Rab proteins (62
). Inhibition of Rab prenylation with 3-PEHPC disrupted the vesicular localization of both Rab7 and Plekhm1 without affecting the distribution of acidic vesicles, indicating that Plekhm1 requires Rab prenylation for its late endosomal/lysosomal localization and therefore most likely interacts with Rab7 on these vesicles. Finally, we studied the distribution of Plekhm1 when coexpressed with GTPase-deficient (active) or dominant-negative mutants of Rab7, which are predominantly associated with vesicular structures (similar to wild-type Rab7) or localized in the cytosol, respectively (41
). Whereas Plekhm1 colocalized with GTPase-deficient Rab7 on intracellular vesicles, coexpression of the dominant-negative Rab7 mutant and Plekhm1 resulted in loss of the endosomal localization of both Rab7 and Plekhm1, offering further evidence that Plekhm1 interacts with Rab7 and is recruited to late endosomes/lysosomes by prenylated, GTP-bound Rab7.
Recruitment to late endosomes/lysosomes is a characteristic that Plekhm1 shares with other proteins that have been identified as Rab7 effectors, such as Rab-interacting lysosomal protein (RILP) and Rabring7 (63
). However, these Rab7-interacting proteins also cause perinuclear clustering of the Rab7-positive vesicles (implicating them in transport of these vesicles), an effect that we did not see in Plekhm1-overexpressing cells. Since Plekhm1 overexpression caused enlargement of these endosomes, it is possible that it is involved in Rab7-mediated fusion of these vesicles rather than their transport.
It has previously been shown that Rab7 is necessary for osteoclast function, since blocking Rab7 expression in osteoclasts using antisense oligodeoxynucleotides disrupted actin ring formation and targeting of vesicles to the ruffled border and furthermore inhibited bone resorption in vitro (59
). These effects may be mediated by altered activity of Rac1, which has recently been suggested to be a direct effector of Rab7 in osteoclasts (65
). Interestingly, the defective ruffled border and extended clear zone that is seen in osteoclasts lacking Rab7 (59
) are also characteristic features of osteoclasts in the ia
), offering further evidence that Rab7 and Plekhm1 are involved in the same vesicular transport processes in osteoclasts. However, although the osteoclasts generated from the patient with the PLEKHM1
mutation clearly had a defective ruffled border, we could not discern any extension of the clear zone.
Although the exact role of Plekhm1 in bone resorption will require further investigation, our data strongly implicate Plekhm1 as a component of Rab7-regulated late endosomal trafficking in osteoclasts. Determining the exact function of the Plekhm1 protein may reveal new therapeutic approaches to osteopetrosis, osteoporosis, and other osteoclast diseases. Although there was no evidence of clinical symptoms other than osteopetrosis in the patient lacking Plekhm1 protein, it remains to be determined whether Plekhm1 plays a role in vesicular transport in other cell types.