Taksum Cheng

Myocyte Enhancer Factor 2 and Microphthalmia-associated Transcription Factor Cooperate with NFATc1 to Transactivate the V-ATPase d2 Promoter during RANKL-induced Osteoclastogenesis

The V-ATPase d2 protein constitutes an important subunit of the V-ATPase proton pump, which regulates bone homeostasis; however, currently little is known about its transcriptional regulation. Here, in an attempt to understand regulation of the V-ATPase d2 promoter, we identified the presence of NFATc1, microphthalmia-associated transcription factor (MITF)- and myocyte enhancer factor 2 (MEF2)-binding sites within the V-ATPase d2 promoter using complementary bioinformatic analyses, chromatin immunoprecipitation, and electromobility shift assay. Intriguingly, activation of the V-ATPase d2 promoter by NFATc1 was enhanced by either MEF2 or MITF overexpression. By comparison, coexpression of MITF and MEF2 did not further enhance V-ATPase d2 promoter activity above that of expression of MITF alone. Consistent with a role in transcriptional regulation, both NFATc1 and MITF proteins translocated from the cytosol to the nucleus during RANKL-induced osteoclastogenesis, whereas MEF2 persisted in the nucleus of both osteoclasts and their mononuclear precursors. Targeted mutation of the putative NFATc1-, MITF-, or MEF2-binding sites in the V-ATPase d2 promoter impaired its transcriptional activation. Additionally retroviral overexpression of MITF or MEF2 in RAW264.7 cells potentiated RANKL-induced osteoclastogenesis and V-ATPase d2 gene expression. Based on these data, we propose that MEF2 and MITF function cooperatively with NFATc1 to transactivate the V-ATPase d2 promoter during RANKL-induced osteoclastogenesis.

Cytoplasmic terminus of vacuolar type proton pump accessory subunit Ac45 is required for proper interaction with V(0) domain subunits and efficient osteoclastic bone resorption.

Solubilization of mineralized bone by osteoclasts is largely dependent on the acidification of the extracellular resorption lacuna driven by the vacuolar (H+)-ATPases (V-ATPases) polarized within the ruffled border membranes. V-ATPases consist of two functionally and structurally distinct domains, V1 and V0. The peripheral cytoplasmically oriented V1 domain drives ATP hydrolysis, which necessitates the translocation of protons across the integral membrane bound V0 domain. Here, we demonstrate that an accessory subunit, Ac45, interacts with the V0 domain and contributes to the vacuolar type proton pump-mediated function in osteoclasts. Consistent with its role in intracellular acidification, Ac45 was found to be localized to the ruffled border region of polarized resorbing osteoclasts and enriched in pH-dependent endosomal compartments that polarized to the ruffled border region of actively resorbing osteoclasts. Interestingly, truncation of the 26-amino acid residue cytoplasmic tail of Ac45, which encodes an autonomous internalization signal, was found to impair bone resorption in vitro. Furthermore, biochemical analysis revealed that although both wild type Ac45 and mutant were capable of associating with subunits a3, c, c″, and d, deletion of the cytoplasmic tail altered its binding proximity with a3, c″, and d. In all, our data suggest that the cytoplasmic terminus of Ac45 contains elements necessary for its proper interaction with V0 domain and efficient osteoclastic bone resorption.

Loss of Protein Kinase C-δ Protects against LPS-Induced Osteolysis Owing to an Intrinsic Defect in Osteoclastic Bone Resorption

Bone remodeling is intrinsically regulated by cell signaling molecules. The Protein Kinase C (PKC) family of serine/threonine kinases is involved in multiple signaling pathways including cell proliferation, differentiation, apoptosis and osteoclast biology. However, the precise involvement of individual PKC isoforms in the regulation of osteoclast formation and bone homeostasis remains unclear. Here, we identify PKC-δ as the major PKC isoform expressed among all PKCs in osteoclasts; including classical PKCs (−α, −β and −γ), novel PKCs (−δ, −ε, −η and −θ) and atypical PKCs (−ι/λ and −ζ). Interestingly, pharmacological inhibition and genetic ablation of PKC-δ impairs osteoclastic bone resorption in vitro. Moreover, disruption of PKC-δ activity protects against LPS-induced osteolysis in mice, with osteoclasts accumulating on the bone surface failing to resorb bone. Treatment with the PKC-δ inhibitor Rottlerin, blocks LPS-induced bone resorption in mice. Consistently, PKC-δ deficient mice exhibit increased trabeculae bone containing residual cartilage matrix, indicative of an osteoclast-rich osteopetrosis phenotype. Cultured ex vivo osteoclasts derived from PKC-δ null mice exhibit decreased CTX-1 levels and MARKS phosphorylation, with enhanced formation rates. This is accompanied by elevated gene expression levels of cathepsin K and PKC −α, −γ and −ε, as well as altered signaling of pERK and pcSrc416/527 upon RANKL-induction, possibly to compensate for the defects in bone resorption. Collectively, our data indicate that PKC-δ is an intrinsic regulator of osteoclast formation and bone resorption and thus is a potential therapeutic target for pathological osteolysis.

Calmodulin interacts with Rab3D and modulates osteoclastic bone resorption

Calmodulin is a highly versatile protein that regulates intracellular calcium homeostasis and is involved in a variety of cellular functions including cardiac excitability, synaptic plasticity and signaling transduction. During osteoclastic bone resorption, calmodulin has been reported to concentrate at the ruffled border membrane of osteoclasts where it is thought to modulate bone resorption activity in response to calcium. Here we report an interaction between calmodulin and Rab3D, a small exocytic GTPase and established regulator osteoclastic bone resorption. Using yeast two-hybrid screening together with a series of protein-protein interaction studies, we show that calmodulin interacts with Rab3D in a calcium dependent manner. Consistently, expression of a calcium insensitive form of calmodulin (i.e. CaM1234) perturbs calmodulin-Rab3D interaction as monitored by bioluminescence resonance energy transfer (BRET) assays. In osteoclasts, calmodulin and Rab3D are constitutively co-expressed during RANKL-induced osteoclast differentiation, co-occupy plasma membrane fractions by differential gradient sedimentation assay and colocalise in the ruffled border as revealed by confocal microscopy. Further, functional blockade of calmodulin-Rab3D interaction by calmidazolium chloride coincides with an attenuation of osteoclastic bone resorption. Our data imply that calmodulin- Rab3D interaction is required for efficient bone resorption by osteoclasts in vitro.

Bafilomycin A1 Attenuates Osteoclast Acidification and Formation, Accompanied by Increased Levels of SQSTM1/p62 Protein

Vacuolar proton pump H+‐adenosine triphosphatases (V‐ATPases) play an important role in osteoclast function. Further understanding of the cellular and molecular mechanisms of V‐ATPase inhibition is vital for the development of anti‐resorptive drugs specifically targeting osteoclast V‐ATPases. In this study, we observed that bafilomycin A1, a naturally‐occurring inhibitor of V‐ATPases, increased the protein level of SQSTM1/p62, a known negative regulator of osteoclast formation. Consistently, we found that bafilomycin A1 diminishes the intracellular accumulation of the acidotropic probe lysotracker in osteoclast‐like cells; indicative of reduced acidification. Further, bafilomycin A1 inhibits osteoclast formation with attenuation of cell fusion and multi‐nucleation of osteoclast‐like cells during osteoclast differentiation. Taken together, these data indicate that bafilomycin A1 attenuates osteoclast differentiation in part via increased levels of SQSTM1/p62 protein, providing further mechanistic insight into the effect of V‐ATPase inhibition in osteoclasts. J. Cell. Biochem. 117: 1464–1470, 2016.

Evaluation of Intraoperative Retention of Autologous Chondrocytes on Type I/III Collagen Scaffold (Ortho-ACI™) for Cartilage Repair

Methods and Materials We tested cell retention on the scaffold by confocal microscopy at 7, 15, 20, 40, 60, 90 and 120 minutes after seeding, and the molecular profile (collagen II, aggrecan, Sox9, HAPLN1) of chondrocytes seeded at 20 minutes and 4 days (preoperative seeding method). Fifteen OrthoACITM patients with 25 cartilage defects were assessed by arthroscopic or magnetic resonance imaging (MRI). Graft repair was graded as excellent, good, poor or no infill. Associations between repair outcome and case variables were also investigated.