Sealing Zone Research Articles

AVL-292, a Bruton's Tyrosine Kinase Inhibitor Impacts Bone Resorption by Abrogating Osteoclast Sealing Zone Formation in Multiple Myeloma

Abstract 1822The activation of Bruton's tyrosine kinase (Btk), a member of the Tec family of tyrosine kinases regulates B-cell development and activation, and plays a key role in antibody production. Interestingly, Btk and the other Tec kinase family, Tec, regulate OC differentiation via Receptor Activator of Nuclear Factor κ B (RANK) signaling. Moreover, patients with X-linked agammaglobulinemia (XLA) who harbor Btk null mutations have impaired OC function.Here we show that, a potent and specific inhibitor of Btk, AVL-292, inhibits OC function in MM patients. AVL-292 is a highly selective, covalent Btk inhibitor that potently silences Btk enzymatic activity (IC50 < 0.5nM) with high selectivity towards Btk with lack of significant inhibition against other kinases involved in BCR signaling (Syk, Lyn). We examined the mechanism of action of AVL-292 in the context of OCs function. OC derived from MM patient monocytes were assayed with or without AVL-292. Interestingly, OC function was inhibited in the presence AVL-292 as demonstrated by pit formation assay. However, mRNA expression of Cathepsin K and TRAP, markers of OC differentiation, were increased in the presence of AVL-292. These data suggest AVL-292 inhibits OC function without affecting the OC differentiation.It has been shown that BTK and Tec regulation of OC differentiation is related to calcium (Ca2+) signaling by increasing Ca2+ flux through direct phosphorylation of phospholipase C2 (PLCγ2). To delineate the mechanism of action of the BTK inhibitor against OC function, the RANK signaling proteins were detected by western blotting and Ca2+ concentration was measured by flourescence. AVL-292 inhibited phosphorylation of the BTK substrate, PLCγ2 in OCs. This was associated with an inhibition of intracellular Ca2+ release by AVL-292 which otherwise increased with RANKL stimulation in OCs.Given the effect of AVL-292 on RANK signaling we next evaluated its effect on Proline-rich tyrosine Kinase 2 (Pyk2) and c-Src. Pyk2 plays a role in OC activation and localizes to the sealing zone in OC. RANK signaling activates c-Src, which phosphorylates Pyk2. Moreover c-Src controls OC bone resorption by regulating actin organization via cortactin. Interestingly, AVL-292 inhibited c-Src and Pyk2 phosphorylation. Furthermore, AVL-292 inhibited cortactin protein and mRNA expression, and upregulated c-Cbl protein (E3 ubiquitin ligase for c-Src) expression in OCs derived from MM patient monocytes.These data demonstrate that the novel BTK inhibitor AVL-292 inhibits OC function through inhibition of OC sealing zone formation. These results suggest a potential novel mechanism of Btk inhibition with AVL-292 on osteoclast function and therefore bone resorption. Disclosures:Evans:Celgene: Employment. Singh:Celgene: Employment. Westlin:Celgene: Employment. Raje:Onyx: Consultancy; Celgene: Consultancy; Millenium: Consultancy; Acetylon: Research Funding; Amgen: Research Funding; Eli-Lilly: Research Funding.

Invited commentary

The debate over how best to treat the internal iliac arteries (IIAs) during endovascular repair of aorto-iliac aneurysms is a long-standing one. In this article, the authors advocate an interesting strategy of simple coverage of the IIA with an endograft without embolization of the IIA beforehand. Their early and midterm (mean follow-up, 33 months) results with this practice are acceptable when compared to a control group of patients who underwent IIA coverage with coil embolization. No difference was reported between the two groups with regard to incidence of buttock claudication, endoleak rate, and rate of secondary interventions. This technique seems to simplify the aneurysm repair by shortening the procedure time and requiring less contrast administration and radiation, all of which is of potential great benefit to the patient. In fact, the authors make such a convincing case that this technique works so well, that after reading this article, one might find themselves asking, “Why would anyone ever embolize the IIA again?” I would offer a word of caution when interpreting these very good results. This is not a practice that should be widely accepted and applied to all patients. In fact, the authors themselves state that indications for simple IIA coverage without embolization should only be performed in patients with an adequate sealing zone of at least 5 mm in the distal common iliac artery (proximal to the IIA and iliac bifurcation), a sealing “ring” in the distal common iliac artery, or an IIA <5 mm in diameter, and that the endograft should be oversized to the seal zone or “ring” by 15% to 20%. In other words, there must be apposition of the endograft to the distal common iliac artery and iliac bifurcation when the endograft is crossing and covering the IIA for this technique to be effective. Furthermore, they go on to concede that they do not recommend simple IIA coverage when there are large common iliac arteries (>30 mm) and no seal zone. These are important points to keep in mind. Many patients with aorto-iliac aneurysms will not have iliac anatomy conducive to using this technique. It should also be emphasized that even in patients with anatomy that meet the authors' criteria, this technique is not without risks and complications. Although, as the authors propose, in theory, there is the potential for a lower incidence of buttock claudication for simple IIA coverage compared to IIA embolization where branches of the IIA may be affected, this was not borne out in their results with an equal rate of buttock claudication in both groups (NE: 13% vs CE: 12%). However, the major concern over IIA coverage without embolization is not the possible sequelae of IIA occlusion (buttock claudication, gluteal necrosis) because that would happen with IIA embolization as well. The major concern is a type II endoleak in the setting of a common iliac artery aneurysm. In this series, the overall endoleak rate was 9% with 10 of the 112 patients in the NE group experiencing a type II endoleak. In 7 of these 10 patients, the type II endoleak originated from the covered IIA. This has the potential for disastrous consequences. In addition, all three of the aneurysm-related deaths in this series directly resulted from endoleak formation and subsequent aneurysm rupture. I applaud the authors for thinking “outside of the box” in an attempt to find ways to simplify endovascular repair of aorto-iliac aneurysms while maintaining its safety and efficacy. Like all surgical treatment strategies, their results must be interpreted with caution before widespread acceptance and application. When confronted with various treatment options, it is often more important to select the appropriate patient for the treatment rather than the appropriate treatment for the patient. Outcomes of endovascular aneurysm repair with selective internal iliac artery coverage without coil embolizationJournal of Vascular SurgeryVol. 56Issue 2PreviewEndovascular aneurysm repair (EVAR) of abdominal aortic aneurysms (AAAs) that also involve the common iliac artery (CIA) typically is accomplished by endograft limb extension into the external iliac artery (EIA). In order to prevent endoleak, the internal iliac artery (IIA) is usually embolized, or alternatively a branched limb is deployed. However, IIA embolization is associated with longer operative time and increased use of contrast and radiation. It has been our practice not to routinely coil embolize the IIA. Full-Text PDF Open Archive

Substrate adhesion regulates sealing zone architecture and dynamics in cultured osteoclasts.

The bone-degrading activity of osteoclasts depends on the formation of a cytoskeletal-adhesive super-structure known as the sealing zone (SZ). The SZ is a dynamic structure, consisting of a condensed array of podosomes, the elementary adhesion-mediating structures of osteoclasts, interconnected by F-actin filaments. The molecular composition and structure of the SZ were extensively investigated, yet despite its major importance for bone formation and remodelling, the mechanisms underlying its assembly and dynamics are still poorly understood. Here we determine the relations between matrix adhesiveness and the formation, stability and expansion of the SZ. By growing differentiated osteoclasts on micro-patterned glass substrates, where adhesive areas are separated by non-adhesive PLL-g-PEG barriers, we show that SZ growth and fusion strictly depend on the continuity of substrate adhesiveness, at the micrometer scale. We present a possible model for the role of mechanical forces in SZ formation and reorganization, inspired by the current data.

Myosin X Regulates Sealing Zone Patterning in Osteoclasts through Linkage of Podosomes and Microtubules

Osteoclasts use actin-rich attachment structures in place of focal adhesions for adherence to bone and non-bone substrates. On glass, osteoclasts generate podosomes, foot-like processes containing a core of F-actin and regulatory proteins that undergo high turnover. To facilitate bone resorption, osteoclasts generate an actin-rich sealing zone composed of densely packed podosome-like units. Patterning of both podosomes and sealing zones is dependent upon an intact microtubule system. A role for unconventional myosin X (Myo10), which can bind actin, microtubules, and integrins, was examined in osteoclasts. Immunolocalization showed Myo10 to be associated with the outer edges of immature podosome rings and sealing zones, suggesting a possible role in podosome and sealing zone positioning. Further, complexes containing both Myo10 and beta-tubulin were readily precipitated from osteoclasts lysates. RNAi-mediated suppression of Myo10 led to decreased cell and sealing zone perimeter, along with decreased motility and resorptive capacity. Further, siRNA-treated cells could not properly position podosomes following microtubule disruption. Osteoclasts overexpressing dominant negative Myo10 microtubule binding domains (MyTH4) showed a similar phenotype. Conversely, overexpression of full-length Myo10 led to increased formation of podosome belts along with larger sealing zones and enhanced bone resorptive capacity. These studies suggest that Myo10 plays a role in osteoclast attachment and podosome positioning by direct linkage of actin to the microtubule network.

Myosin X Regulates Sealing Zone Patterning in Osteoclasts through Linkage of Podosomes and Microtubules

Myosin X Regulates Sealing Zone Patterning in Osteoclasts through Linkage of Podosomes and Microtubules

Natural History of the Iliac Arteries after Endovascular Abdominal Aortic Aneurysm Repair and Suitability of Ectatic Iliac Arteries as a Distal Sealing Zone

To investigate the natural history of dilated common iliac arteries (CIA) exposed to pulsatile blood flow after endovascular abdominal aortic aneurysm repair (EVAR) and the suitability of ectatic iliac arteries as sealing zones using flared iliac limbs. Follow-up computed tomograms of 102 CIAs in 60 EVAR patients were investigated. Diameter changes in CIAs < or =16 mm (group 1) were compared with changes in vessels where a dilated segment >16 mm in diameter continued to be exposed to pulsatile blood flow (group 2). Within group 2, cases in which the stent terminated proximal to the dilated artery segment (2a) were compared with those that had been treated with a flared limb (2b). The mean CIA diameter increased by 1.0+/-1.0 mm in group 1 (p<0.001 versus immediately after EVAR) and by 1.5+/-1.7 mm in group 2 (p<0.001 versus immediately after EVAR) within an average follow-up of 43.6+/-18.0 months. Diameter increase was more pronounced in dilated CIAs (p=0.048), and it was not significantly different between groups 2a and 2b (p=0.188). No late distal type I endoleak or stent-graft migration associated with CIA ectasia was observed. Dilatation of the CIA is significant after EVAR, and it is more pronounced in ectatic iliac arteries. Although ectatic iliac arteries appear to be suitable sealing zones in the short term, continued follow-up is mandatory.

Natural History of the Iliac Arteries After Endovascular Abdominal Aortic Aneurysm Repair and Suitability of Ectatic Iliac Arteries as a Distal Sealing Zone

Natural History of the Iliac Arteries After Endovascular Abdominal Aortic Aneurysm Repair and Suitability of Ectatic Iliac Arteries as a Distal Sealing Zone

CD44 and β3 Integrin Organize Two Functionally Distinct Actin-based Domains in Osteoclasts

The actin cytoskeleton of mature osteoclasts (OCs) adhering to nonmineralized substrates is organized in a belt of podosomes reminiscent of the sealing zone (SZ) found in bone resorbing OCs. In this study, we demonstrate that the belt is composed of two functionally different actin-based domains: podosome cores linked with CD44, which are involved in cell adhesion, and a diffuse cloud associated with beta3 integrin, which is involved in cell adhesion and contraction. Wiskott Aldrich Syndrome Protein (WASp) Interacting Protein (WIP)-/- OCs were devoid of podosomes, but they still exhibited actin clouds. Indeed, WIP-/- OCs show diminished expression of WASp, which is required for podosome formation. CD44 is a novel marker of OC podosome cores and the first nonintegrin receptor detected in these structures. The importance of CD44 is revealed by showing that its clustering restores podosome cores and WASp expression in WIP-/- OCs. However, although CD44 signals are sufficient to form a SZ, the presence of WIP is indispensable for the formation of a fully functional SZ.

The Architecture of the Adhesive Apparatus of Cultured Osteoclasts: From Podosome Formation to Sealing Zone Assembly

BackgroundOsteoclasts are bone-degrading cells, which play a central role in physiological bone remodeling. Unbalanced osteoclast activity is largely responsible for pathological conditions such as osteoporosis. Osteoclasts develop specialized adhesion structures, the so-called podosomes, which subsequently undergo dramatic reorganization into sealing zones. These ring-like adhesion structures, which delimit the resorption site, effectively seal the cell to the substrate forming a diffusion barrier. The structural integrity of the sealing zone is essential for the cell ability to degrade bone, yet its structural organization is poorly understood.Principal FindingsCombining high-resolution scanning electron microscopy with fluorescence microscopy performed on the same sample, we mapped the molecular architecture of the osteoclast resorptive apparatus from individual podosomes to the sealing zone, at an unprecedented resolution. Podosomes are composed of an actin-bundle core, flanked by a ring containing adhesion proteins connected to the core via dome-like radial actin fibers. The sealing zone, hallmark of bone-resorbing osteoclasts, consists of a dense array of podosomes communicating through a network of actin filaments, parallel to the substrate and anchored to the adhesive plaque domain via radial actin fibers.SignificanceThe sealing zone of osteoclasts cultured on bone is made of structural units clearly related to individual podosomes. It differs from individual or clustered podosomes in the higher density and degree of inter-connectivity of its building blocks, thus forming a unique continuous functional structure connecting the cell to its extracellular milieu. Through this continuous structure, signals reporting on the substrate condition may be transmitted to the whole cell, modulating the cell response under physiological and pathological conditions.

Specific Antagonists of NMDA Receptors Prevent Osteoclast Sealing Zone Formation Required for Bone Resorption

N-Methyl-d-aspartate (NMDA) glutamate receptors, widely distributed in the nervous system, have recently been identified in bone. They are expressed and are functional in osteoclasts. In the present work, we have studied the effects of specific antagonists of NMDA receptors on osteoclast activation and bone resorption. Using an in vitro assay of bone resorption, we showed that several antagonists of NMDA receptors binding to different sites of the receptor inhibit bone resorption. Osteoclast activation requires adhesion to the bone surface, cytoskeletal reorganization and survival. We demonstrated by autoradiography that the specific NMDA receptor channel blocker, MK 801, binds to osteoclasts. This antagonist had no effect on osteoclast attachment to bone and did not induce osteoclast apoptosis. In contrast, MK 801 rapidly decreased the percentage of osteoclasts with actin ring structures that are associated with actively resorbing osteoclasts. These results suggest that NMDA receptors expressed by osteoclasts may be involved in adhesion-induced formation of the sealing zone required for bone resorption.

Stable Association of PYK2 and p130Cas in Osteoclasts and Their Co-localization in the Sealing Zone

Bone resorption is initiated by osteoclast attachment to the mineralized matrix, cytoskeletal reorganization, cellular polarization, and the formation of the sealing zone. The present study examines the interaction between PYK2 and p130(Cas) (Crk-associated substrate), suggested to be part of the signaling pathway initiated by osteoclast adhesion. Using murine osteoclast-like cells (OCLs) and their mononuclear precursors (pOCs), generated in a co-culture of bone marrow and osteoblastic MB1.8 cells, we show that: 1) p130(Cas) is tyrosine-phosphorylated upon adhesion of pOCs to vitronectin or ligation of beta3 integrins; 2) p130(Cas) colocalizes with PYK2 and the cytoskeletal proteins F-actin, vinculin, and paxillin in the podosomal-rich ring-like structures of OCLs plated on glass and in the sealing zone in actively resorbing OCLs on bone; 3) p130(Cas) and PYK2 form a stable complex in pOCs, independent of tyrosine phosphorylation of either molecule, and this complex is present in Src (-/-) OCLs, in which neither protein is phosphorylated or associated with the osteoclast adhesion structure; 4) the association of p130(Cas) and PYK2 is mediated by the SH3 domain of p130(Cas) and the C-terminal domain of PYK2. These findings suggest that p130(Cas) and its association with PYK2 may play an important role in the adhesion-dependent signaling that leads to cytoskeletal reorganization and formation of the sealing zone during osteoclast activation.

Inhibition of Bone Resorption in Vitroby a Peptide Containing the Cadherin Cell Adhesion Recognition Sequence HAV Is Due to Prevention of Sealing Zone Formation

Cadherins are transmembrane glycoproteins usually mediating homophilic calcium-dependent cell–cell adhesion in a variety of cells and species. Allclassical cadherins share common structural and functional properties, one of which is the cell adhesion recognition (CAR) sequence HAV (His-Ala-Val). In the present work we have studied the effects of the cadherin CAR sequence-containing hexapeptide AHAVSE on osteoclasts, the main bone resorbing cells in well-characterized pit formation assay. The primary attachment of osteoclasts to bone surface is not affected by the peptide, suggesting that it is not mediated by cadherins. However, treatment of osteoclast cultures with AHAVSE peptide decreased the number of resorption pits and the total resorbed area without affecting the mean size of resorption pits. Furthermore, we show rapid inactivation of osteoclasts with AHAVSE, which is seen as a decrease in the percentage of osteoclasts with actin rings. Double staining of pan-cadherin antibody with actin and vinculin localized cadherin-like molecule in the sealing zone area of osteoclasts. These results suggest that the tight attachment of osteoclasts to the bone surface in the sealing zone area may be mediated by cadherin-like molecules and that the decrease of resorption in AHAVSE-treated osteoclast cultures is due to prevention of sealing zone formation.