BoneKEy-Osteovision | Commentary
Life is short, art long. Bcl-2 and osteoclast survival
Commentary on: McGill GG, Horstmann M, Widlund HR, Du J, Motyckova G, Nishimura EK, Lin YL, Ramaswamy S, Avery W, Ding HF, Jordan SA, Jackson IJ, Korsmeyer SJ, Golub TR, Fisher DE. Bcl2 regulation by the melanocyte master regulator Mitf modulates lineage survival and melanoma cell viability. Cell. 2002 Jun 14;109(6):707-18.
The recently published study by McGill et al. () shows for the first time that Bcl-2 -/- mice have an osteopetrotic phenotype. Increased bone density, accumulation of primary spongiosa, diminished marrow space and thickening of cortical bone were described in the femurs of 2-week old Bcl-2 -/- animals. Other phenotypes that were seen included gross craniofacial and skeletal abnormalities, as seen in Bcl-2 +/- heterozygotes. A link between the microphthalmia transcription factor (Mitf) and the regulation of Bcl-2 transcription in melanocytes and osteoclasts was also suggested.
Bcl-2 and other proteins from the same family (e.g., Bcl-xL, Mcl1) play an important role in the regulation of cell survival (). Bcl-2 functions as an integral mitochondrial membrane protein to prevent apoptosis. While Bcl-xL -/- and Mcl1 -/- animals die at early embryonic stages, Bcl-2 -/- animals are born viable. However, they display growth retardation, lymphoid apoptosis, hypopigmented hair, and progressive neuronal degeneration, and they eventually die at about 6 weeks from polycystic kidney disease leading to renal failure ().
The osteoclast is a short-lived cell with a life span of about 2-3 weeks (), a lifespan that correlates roughly with the resorption phase of a basic multicellular unit (BMU). The classic cytokine with the greatest influence on osteoclast survival is M-CSF, which acts through its receptor tyrosine kinase (RTK) c-fms. M-CSF is of paramount importance for osteoclast differentiation in vivo and survival in vitro, and a lack thereof results in induction and progression of osteoclast apoptosis. That this could relate to Bcl-2 was previously suggested in osteopetrotic (op/op) mice that do not express functional M-CSF (). When op/op mice carried a transgene expressing Bcl-2, osteopetrosis in long bones was largely reversed, perhaps compensating for the lack of signaling via this survival factor in the op/op osteoclast precursor. By analogy, the survival of melanocytes is found to depend greatly on the RTK c-kit and its ligand, stem cell factor (SCF). Whereas M-CSF signaling through c-fms was shown to complement defects in signaling through c-kit, it cannot compensate for defects in Mitf (). This is now explained by the finding that both c-fms () and c-kit () signal to Mitf and elicit transcriptional responses in osteoclasts and melanocytes, respectively. In retrospect, it was perhaps predictable that cytokines that promote the function and survival of the osteoclast signal through Mitf since dominant negative mutations in Mitf result in osteopetrosis (), reduce osteoclast activity () and are correlated with reduced osteoclast survival (). In relation to osteoclast activity, we know from more recent work that cathepsin K (CatK) is a target of the Mitf transcription factor family and that CatK expression is deficient in osteoclasts from the osteopetrotic Mitf mutant (). In relation to survival, M-CSF stimulation of endogenous Bcl-2 and Bcl-xL mRNA levels in osteoclasts was shown earlier in vitro, although the mechanism of this regulation was not studied in detail (). It is conceivable that osteoclast survival, and thereby the overall resorptive capacity of the osteoclastic population, is regulated at least in part by members of the Bcl-2 family. This can also be deduced from experiments using Bcl-xL over-expression in osteoclasts (). The study by McGill et al. () thus elegantly links several key observations, whereby M-CSF in osteoclasts and SCF in melanocytes signal through their respective RTKs via Mitf to regulate anti-apoptotic targets, among them Bcl-2. It is of interest to note that Receptor Activator of NF-kB ligand (RANKL), might also act in part through Mitf () to support osteoclast survival () and to stimulate CatK expression (). A complete functional deficiency of any of the above factors then results in an osteopetrotic phenotype.
Taken together, this work shows that Mitf participates in the control of bone resorption by regulating both osteoclast activity (e.g., CatK) and probably osteoclast survival (Bcl-2). In addition, the prominent role played by Bcl-2 in osteoclast biology cannot be fully compensated by other Bcl-2-like proteins. However, osteoclasts can be isolated from Mitf mi/mi and Bcl-2 -/- animals (129S-Bcl-2 tm1Sjk) indicating that this cell type does not completely depend on Mitf or Bcl-2 expression for either differentiation or survival. Lack of Bcl-2 might under this circumstance be partially compensated for by Bcl-xL or Mcl-1. Interestingly, a bone phenotype in Bcl-2 deficient mice (129S1/Sv-Bcl-2 tm1Mpin) had been reported earlier, although this phenotype was restricted to osteoblasts and, marginally, the growth plate (). Furthermore, Amling et al. () found that Bcl-2 knockout mice () exhibit premature chondrocyte maturation and terminal differentiation leading to accelerated endochondral bone formation and a reduced growth plate thickness. However, no osteopetrotic phenotype was reported in the latter two studies. At the present time it is unclear why different Bcl-2 knockout strains manifest such diverse bone phenotypes, but this may point to multiple roles of Bcl-2 in bone homeostasis.
Although McGill et al. () identified Bcl-2 as direct transcriptional target of Mitf, the expression of Bcl-2 can be modulated at different levels. Bcl-2 mRNA can undergo destabilization during apoptosis () and Bcl-2 protein levels are regulated by posttranscriptional mechanisms including translational regulation (), phosphorylation and proteasomic degradation (). These regulatory mechanisms might be of varying importance in different cell types. Translational regulation involving the mammalian target of rapamycin (mTOR), and ribosomal protein S6 kinase (S6K) () is of key importance to the survival of the osteoclast (). Both M-CSF and RANKL signal through this pathway in osteoclasts, and suppression of the pathway by rapamycin leads to osteoclast apoptosis. An apoptosis-inducing inhibitor of geranylgeranylation also suppresses S6K signaling, possibly by blocking small GTPases (). It is therefore tempting to implicate mTOR/S6K signaling in the apoptosis induced by geranylgeranylation-inhibiting bisphosphonates, although this hypothesis remains to be tested and multiple pathways may exist. The mechanism by which mTOR/S6K signaling promotes osteoclast survival is unclear, although it may be linked to the turnover of Bcl-2-like proteins such as Mcl-1. GM-CSF stimulated expression of Mcl-1 can indeed be attenuated by inhibition of the mTOR/S6K pathway ().
The study by McGill et al.() indicates a specific role for anti-apoptotic Bcl-2 and perhaps its related family members in the regulation of osteoclast survival. As we move forward, it is tempting to speculate that a gradual decrease in the level of Bcl-2 expression in the osteoclast might regulate survival, and thus bone resorption. This would limit the life span of the osteoclast within the BMU. This kind of regulation could be achieved at multiple levels and might also involve M-CSF and other cytokines synthesized by osteoclastogenesis supporting cells. In short, the study by McGill et al. () puts in place a previously missing link, tying the effects of M-CSF and RANKL on osteoclast survival to their effects on Mitf and, ultimately, Bcl-2. Remaining questions relate to the exact signaling pathways involved and to the identification of other downstream targets that are of critical importance to the osteoclast lifespan.
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