L. Shannon Holliday

The Glycolytic Enzyme Aldolase Mediates Assembly, Expression, and Activity of Vacuolar H-ATPase*

Vacuolar H+-ATPases (V-ATPases) are a family of highly conserved proton pumps that couple hydrolysis of cytosolic ATP to proton transport out of the cytosol. How ATP is supplied for V-ATPase-mediated hydrolysis and for coupling of proton transport is poorly understood. We have reported that the glycolytic enzyme aldolase physically associates with V-ATPase (Lu, M., Holliday, L. S., Zhang, L., Dunn, W. A., and Gluck, S. L. (2001) J. Biol. Chem. 276, 30407–30413). Here we show that aldolase interacts with three different subunits of V-ATPase (subunits a, B, and E). The binding sites for the V-ATPase subunits on aldolase appear to be on distinct interfaces of the glycolytic enzyme. Aldolase deletion mutant cells were able to grow in medium buffered at pH 5.5 but not at pH 7.5, displaying a growth phenotype similar to that observed in V-ATPase subunit deletion mutants. Abnormalities in V-ATPase assembly and protein expression observed in aldolase deletion mutant cells could be fully rescued by aldolase complementation. The interaction between aldolase and V-ATPase increased dramatically in the presence of glucose, suggesting that aldolase may act as a glucose sensor for V-ATPase regulation. Taken together, these findings provide functional evidence that the ATP-generating glycolytic pathway is directly coupled to the ATP-hydrolyzing proton pump through physical interaction between aldolase and V-ATPase.

Vacuolar H+-ATPase Binding to Microfilaments REGULATION IN RESPONSE TO PHOSPHATIDYLINOSITOL 3-KINASE ACTIVITY AND DETAILED CHARACTERIZATION OF THE ACTIN-BINDING SITE IN SUBUNIT B

Vacuolar H+-ATPase (V-ATPase) binds microfilaments, and that interaction may be mediated by an actin binding domain in subunit B of the enzyme. To test for possible physiologic functions of the actin binding activity of V-ATPase, early responses of resorbing osteoclasts to inhibition of phosphatidylinositol 3-kinase activity by wortmannin and LY294002 were examined. Rapid co-localization between V-ATPase and F-actin was demonstrated by immunocytochemistry, and corresponding association between V-ATPase and F-actin in immunoprecipitations and pelleting assays was detected. This response was reversed as osteoclasts recovered resorptive activity after inhibitors were removed. By expressing and characterizing fusion proteins containing segments of the actin-binding amino-terminal regions of the B subunits of V-ATPase, we mapped the actin-binding site to a 44-amino acid domain. An 11-amino acid segment with a sequence similar to the actin-binding site of human profilin I was detected within this region. 13-Mers containing these profilin-like segments bound actin in fluorescent anisotropy studies and competed with profilin for binding to actin. Using site-directed mutagenesis, the 11-amino acid profilin-like actin-binding motifs (amino acids 49–59 of B1 and 55–65 of B2) were replaced with an 11-amino acid spacer with a sequence based on the homologous sequence from subunit B of Pyrococcus horikoshii, an organism that lacks an actin cytoskeleton. These substitutions eliminated the actin-binding activity of the B subunit fusion proteins. In summary, binding between V-ATPase and F-actin in osteoclasts occurs in response to blocking phosphatidylinositol 3-kinase activity. This response was fully reversible. The actin binding activities of the B subunits of V-ATPase required 11-amino acid actin-binding motifs that are similar in sequence to the actin-binding site of mammalian profilin I.

Actin-related protein 2/3 complex is required for actin ring formation.

Actin rings are vital for osteoclastic bone resorption, and actin‐related protein 2/3 complex is a pivotal regulator of actin polymerization. Actin‐related protein 2/3 complex was found in the podosomes of actin rings. A short interfering RNA knocked down expression of actin‐related protein 2 in osteoclasts and disrupted actin rings, suggesting that the complex is crucial to actin ring formation.

Multifunctional role of osteopontin in directing intrafibrillar mineralization of collagen and activation of osteoclasts.

Mineralized collagen composites are of interest because they have the potential to provide a bone-like scaffold that stimulates the natural processes of resorption and remodeling. Working towards this goal, our group has previously shown that the nanostructure of bone can be reproduced using a polymer-induced liquid-precursor (PILP) process, which enables intrafibrillar mineralization of collagen with hydroxyapatite to be achieved. This prior work used polyaspartic acid (pASP), a simple mimic for acidic non-collagenous proteins, to generate nanodroplets/nanoparticles of an amorphous mineral precursor which can infiltrate the interstices of type-I collagen fibrils. In this study we show that osteopontin (OPN) can similarly serve as a process-directing agent for the intrafibrillar mineralization of collagen, even though OPN is generally considered a mineralization inhibitor. We also found that inclusion of OPN in the mineralization process promotes the interaction of mouse marrow-derived osteoclasts with PILP-remineralized bone that was previously demineralized, as measured by actin ring formation. While osteoclast activation occurred when pASP was used as the process-directing agent, using OPN resulted in a dramatic effect on osteoclast activation, presumably because of the inherent arginine-glycine-aspartate acid ligands of OPN. By capitalizing on the multifunctionality of OPN, these studies may lead the way to producing biomimetic bone substitutes with the capability of tailorable bioresorption rates.

Actin Binding Activity of Subunit B of Vacuolar H + -ATPase Is Involved in Its Targeting to Ruffled Membranes of Osteoclasts

Adeno‐associated virus was used to transduce primary mouse osteoclasts with the B1 isoform of vacuolar H+‐ATPase. B1, which is not normally expressed in osteoclasts, was correctly targeted to ruffled membranes of resorbing osteoclasts. Mutant subunit B1 that lacked a functional actin‐binding site did not accumulate in ruffled membranes.

Identification of Enoxacin as an Inhibitor of Osteoclast Formation and Bone Resorption by Structure-Based Virtual Screening

An interaction between the B2 subunit of vacuolar H(+)-ATPase (V-ATPase) and microfilaments is required for osteoclast bone resorption. An atomic homology model of the actin binding site on B2 was generated and molecular docking simulations were performed. Enoxacin, a fluoroquinolone antibiotic, was identified and in vitro testing demonstrated that enoxacin blocked binding between purified B2 and microfilaments. Enoxacin dose dependently reduced the number of osteoclasts differentiating in mouse marrow cultures stimulated with 1,25-dihydroxyvitamin D(3), as well as markers of osteoclast activity, and the number of resorption lacunae formed on bone slices. Enoxacin inhibited osteoclast formation at concentrations where osteoblast formation was not altered. In summary, enoxacin is a novel small molecule inhibitor of osteoclast bone resorption that acts by an unique mechanism and is therefore an attractive lead molecule for the development of a new class of antiosteoclastic agents.

Formation of GW/P bodies as marker for microRNA-mediated regulation of innate immune signaling in THP-1 cells

GW bodies (GWB or P bodies) are cytoplasmic foci thought to result from microRNA (miRNA) regulation of messenger RNA (mRNA) targets and subsequent mRNA degradation. The purpose of this study is to examine the effects of lipopolysaccharide (LPS) stimulation of human monocytes on GWB formation, miRNA induction, miRNA target regulation and downstream cytokine and chemokine expression. In response to LPS stimulation, the number of GWB consistently increased by twofold at 8 h after stimulation and this increase was abolished when the miRNA‐effector proteins Rck/p54 or argonaute 2 were depleted. As the level of miR‐146a increased from 19‐fold up to 100‐fold during LPS stimulation, the transfection of a miR‐146a mimic into THP‐1 cells was examined to determine whether miR‐146a alone can induce similar changes in GWB. The results showed transfected miR‐146a could produce a comparable increase in the number of GWB and this was accompanied by a reduction in major cytokines/chemokines induced by LPS. These data show that the increase in size and number of GWB may serve as a biomarker for miRNA‐mediated gene regulation, and miR‐146a has a significant role in the regulation of LPS‐induced cytokine production in THP‐1 cells.

Vacuolar H+-ATPase activity and expression in mouse bone marrow cultures.

We examined vacuolar H+‐ATPase (V‐ATPase) structure, enzymatic properties, and protein and mRNA expression from mouse marrow cultured in the presence or absence of 1,25‐dihydroxyvitamin D3 (1,25(OH)2D3), which stimulates formation of bone‐resorptive osteoclasts. V‐ATPases from osteoclast‐containing cultures were similar in ion and inhibitor sensitivities to the enzyme from kidney‐derived sources. Immunopurified V‐ATPase from 1,25(OH)2D3‐stimulated cultures exhibited 20‐fold greater ATPase activity than the enzyme from unstimulated cultures, which do not contain osteoclasts. In contrast, 1,25(OH)2D3‐treated cultures contained only 2‐fold more assembled V‐ATPase, as determined by immunoprecipitation. Quantitative reverse transcription‐polymerase chain reaction (RT‐PCR) and immunoblot analysis similarly showed ∼2‐fold increases of V‐ATPase mRNA and protein levels in 1,25(OH)2D3‐treated cultures. The bulk of the relative difference in V‐ATPase activity between the two cultures was due to a 10‐fold difference in enzyme specific activity. Quantitative RT‐PCR also revealed that expression levels of V‐ATPase mRNAs reflected the stoichiometry of enzyme subunits in the assembled complex. These data indicate that in mouse bone marrow cultures, V‐ATPase expression is controlled at the level of mRNA, and that increases in subunit expression and assembly cannot account for the 20‐fold difference in enzyme activity in osteoclast‐containing cultures. Therefore, osteoclast V‐ATPase activity may be regulated by subtle alterations in enzyme structure or associated factors.

Propolis inhibits osteoclast maturation

Propolis, a natural product produced by the honey bee, has been successfully used in medicine as an anti-inflammatory and antimicrobial agent. Traumatic injuries to the teeth, especially avulsion injuries, present a challenging situation for the clinician because of post-treatment complications, such as inflammatory and/or replacement resorption. Agents that reduce osteoclast numbers and activity may be useful in the treatment of traumatic injuries to the teeth. In this study, we evaluated propolis as an anti-resorptive agent. Calcitriol-stimulated mouse marrow cultures, which contain both osteoclasts and osteoblasts, were exposed to the ethanol extracts of propolis or vehicle control and stained for tartrate-resistant acid phosphatase (TRAP)-activity to identify osteoclasts. A significant, dose-dependent reduction in multinuclear TRAP+ cells was demonstrated, although the propolis treatment accommodated cell growth and survival (P < 0.05). Propolis also reduced the formation of actin rings in pure cultures of RAW 264.7 osteoclast-like cells, suggesting that it exerts direct actions on osteoclast maturation. In summary, our data suggest that propolis inhibits late stages of osteoclast maturation including fusion of osteoclasts precursors to form giant cells and formation of actin rings. This supports the hypothesis that it may prove useful as a medicament to reduce resorption associated with traumatic injuries to the teeth.

Enoxacin directly inhibits osteoclastogenesis without inducing apoptosis

Background: Enoxacin inhibits vacuolar H+-ATPase binding to microfilaments and bone resorption. Results: Enoxacin inhibits osteoclastogenesis without triggering apoptosis, induces changes in the proteolytic regulation of proteins, and alters the localization of key proteins involved in osteoclast function. Conclusion: Enoxacin inhibits osteoclastogenesis by a mechanism that involves changes in post-translational processing and targeting of proteins. Significance: A new type of direct inhibitor of osteoclasts has been identified. Enoxacin has been identified as a small molecule inhibitor of binding between the B2-subunit of vacuolar H+-ATPase (V-ATPase) and microfilaments. It inhibits bone resorption by calcitriol-stimulated mouse marrow cultures. We hypothesized that enoxacin acts directly and specifically on osteoclasts by disrupting the interaction between plasma membrane-directed V-ATPases, which contain the osteoclast-selective a3-subunit of V-ATPase, and microfilaments. Consistent with this hypothesis, enoxacin dose-dependently reduced the number of multinuclear cells expressing tartrate-resistant acid phosphatase (TRAP) activity produced by RANK-L-stimulated osteoclast precursors. Enoxacin (50 μm) did not induce apoptosis as measured by TUNEL and caspase-3 assays. V-ATPases containing the a3-subunit, but not the “housekeeping” a1-subunit, were isolated bound to actin. Treatment with enoxacin reduced the association of V-ATPase subunits with the detergent-insoluble cytoskeleton. Quantitative PCR revealed that enoxacin triggered significant reductions in several osteoclast-selective mRNAs, but levels of various osteoclast proteins were not reduced, as determined by quantitative immunoblots, even when their mRNA levels were reduced. Immunoblots demonstrated that proteolytic processing of TRAP5b and the cytoskeletal protein l-plastin was altered in cells treated with 50 μm enoxacin. Flow cytometry revealed that enoxacin treatment favored the expression of high levels of DC-STAMP on the surface of osteoclasts. Our data show that enoxacin directly inhibits osteoclast formation without affecting cell viability by a novel mechanism that involves changes in posttranslational processing and trafficking of several proteins with known roles in osteoclast function. We propose that these effects are downstream to blocking the binding interaction between a3-containing V-ATPases and microfilaments.