Regulatory Pathways in Growth Plate Chondrocytes that Are Impacted by Matrix Vesicle microRNA Identified by Targeted RISC Pulldown and Sequencing of the Resulting Transcriptome.

During endochondral bone formation, growth plate chondrocytes are differentially regulated by various factors and hormones. As the cellular phenotype changes, the composition of the extracellular matrix is altered, including the production and composition of matrix vesicles (MV) and their cargo of microRNA. The regulatory functions of these MV microRNA in the growth plate are still largely unknown. To address this question, we undertook a targeted bioinformatics approach. A subset of five MV microRNA was selected for analysis based on their specific enrichment in these extracellular vesicles compared to the parent cells (miR-1-3p, miR-22-3p, miR-30c-5p, miR-122-5p, and miR-133a-3p). Synthetic biotinylated versions of the microRNA were produced using locked nucleic acid (LNA) and were transfected into rat growth plate chondrocytes. The resulting LNA to mRNA complexes were pulled down and sequenced, and the transcriptomic data were used to run pathway analysis pipelines. Bone and musculoskeletal pathways were discovered to be regulated by the specific microRNA, notably those associated with transforming growth factor beta (TGFß) and Wnt pathways, cell differentiation and proliferation, and regulation of vesicles and calcium transport. These results can help with understanding the maturation of the growth plate and the regulatory role of microRNA in MV.

1α,25-Dihydroxyvitamin D3 Regulates microRNA Packaging in Extracellular Matrix Vesicles and Their Release in the Matrix.

Growth plate chondrocytes are regulated by numerous factors and hormones as they mature during endochondral bone formation, including transforming growth factor beta-1 (TGFb1), bone morphogenetic protein 2 (BMP2), insulin-like growth factor-1 (IFG1), parathyroid hormone and parathyroid hormone related peptide (PTH, PTHrP), and Indian hedgehog (IHH). Chondrocytes in the growth plate's growth zone (GC) produce and export matrix vesicles (MVs) under the regulation of 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3]. 1α,25(OH)2D3 regulates MV enzyme composition genomically and 1α,25(OH)2D3 secreted by the cells acts on the MV membrane nongenomically, destabilizing it and releasing MV enzymes. This study examined the regulatory role 1α,25(OH)2D3 has over production and packaging of microRNA (miRNA) into MVs by GC cells and the release of miRNA by direct action on MVs. Costochondral cartilage GC cells were treated with 1α,25(OH)2D3 and the miRNA in the cells and MVs sequenced. We also treated MVs with 1α,25(OH)2D3 and determined if the miRNA was released. To assess whether MVs can act directly with chondrocytes and if this is regulated by 1α,25(OH)2D3, we stained MVs with a membrane dye and treated GC cells with them. 1α,25(OH)2D3 regulated production and packaging of a unique population of miRNA into MVs compared to the vehicle control population. 1α,25(OH)2D3 treatment of MVs did not release miRNA. Stained MVs were endocytosed by GC cells and this was increased with 1α,25(OH)2D3 treatment. This study adds new regulatory roles for 1α,25(OH)2D3 with respect to packaging and transport of MV miRNAs.

Selective BET inhibitor RVX-208 ameliorates periodontal inflammation and bone loss.

AIM: To determine the effects of RVX-208, a selective bromodomain and extra-terminal domain (BET) inhibitor targeting bromodomain 2 (BD2), on periodontal inflammation and bone loss. MATERIALS AND METHODS: Macrophage-like cells (RAW264.7) and human gingival epithelial cells were challenged by Porphyromonas gingivalis (Pg) with or without RVX-208. Inflammatory gene expression and cytokine production were measured by reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. RAW264.7 cells were induced to osteoclast differentiation. After RVX-208 treatment, osteoclast differentiation was evaluated by histology, tartrate-resistant-acid-phosphatase (TRAP) activity and the expression of osteoclast-specific genes. The effect of RVX-208 on osteoclast transcriptome was studied by RNA sequencing. Periodontitis was induced in rats by ligature and local RVX-208 treatment was administered every other day. Alveolar bone loss was measured by micro-computed tomography. RESULTS: RVX-208 inhibited inflammatory gene expression and cytokine production in Pg-infected cells. Osteoclast differentiation was inhibited by RVX-208, as evidenced by reduced osteoclast number, TRAP activity and osteoclast-specific gene expression. RVX-208 displayed a more selective and less profound suppressive impact on transcriptome compared with pan-BET inhibitor, JQ1. RVX-208 administration prevented the alveolar bone loss in vivo. CONCLUSIONS: RVX-208 regulated both upstream (inflammatory cytokine production) and downstream (osteoclast differentiation) events that lead to periodontal tissue destruction, suggesting that it may be a promising 'epi-drug' for the prevention of periodontitis.

Osteoblast-Derived Matrix Vesicles Exhibit Exosomal Traits and a Unique Subset of microRNA: Their Caveolae-Dependent Endocytosis Results in Reduced Osteogenic Differentiation.

Matrix vesicles (MVs) are nano-sized extracellular vesicles that are anchored in the extracellular matrix (ECM). In addition to playing a role in biomineralization, osteoblast-derived MVs were recently suggested to have regulatory duties. The aims of this study were to establish the characteristics of osteoblast-derived MVs in the context of extracellular vesicles like exosomes, assess their role in modulating osteoblast differentiation, and examine their mechanism of uptake. MVs were isolated from the ECM of MG63 human osteoblast-like cell cultures and characterized via enzyme activity, transmission electron microscopy, nanoparticle tracking analysis, Western blot, and small RNA sequencing. Osteoblasts were treated with MVs from two different culture conditions (growth media [GM]; osteogenic media [OM]) to evaluate their effects on the differentiation and production of inflammatory markers and on macrophage polarization. MV endocytosis was assessed using a lipophilic, fluorescent dye and confocal microscopy with the role of caveolae determined using methyl-ß-cyclodextrin. MVs exhibited a four-fold enrichment in alkaline phosphatase specific activity compared to plasma membranes; were 50-150 nm in diameter; possessed exosomal markers CD63, CD81, and CD9 and endosomal markers ALIX, TSG101, and HSP70; and were selectively enriched in microRNA linked to an anti-osteogenic effect and to M2 macrophage polarization. Treatment with GM or OM MVs decreased osteoblast differentiation. Osteoblasts endocytosed MVs using a mechanism that involves caveolae. These results support the hypothesis that osteoblasts produce MVs that participate in the regulation of osteogenesis.

miR-122 and the WNT/ß-catenin pathway inhibit effects of both interleukin-1ß and tumor necrosis factor-α in articular chondrocytes in vitro.

Interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), and WNT/ß-catenin signaling cause dysregulation of rat primary articular chondrocytes (rArCs), resulting in cartilage extracellular matrix destruction and osteoarthritis (OA) progression. microRNA (miR) miR-122 represses these effects whereas miR-451 exacerbates IL-1ß-stimulated matrix metalloproteinase-13 (MMP-13) and prostaglandin E2 (PGE2) production. The goals of this study were to evaluate crosstalk between these signaling pathways and determine if miR-122 and miR-451 exert their protective/destructive effects through these pathways in an in vitro model of OA. Primary rArCs were treated with IL-1ß or TNF-α for 24 h and total DNA, MMP-13, and PGE2, as well as expression levels of miR-122 and miR-451 were measured. After 24-h transfection with miR-122, miR-451, miR-122-inhibitor, or miR-451-inhibitor, rArCs were treated with or without TNF-α for 24 h; total DNA, MMP-13, and PGE2 were measured. Similarly, cells were treated with WNT-agonist lithium chloride (LiCl), WNT-antagonist XAV-939 (XAV), or PKF-118-310 (PKF) with and without IL-1ß or TNF-α stimulation. Both IL-1ß and TNF-α-stimulation increased MMP-13 and PGE2 production. Transfection with miR-122 prevented TNF-α-stimulated increases in MMP-13 and PGE2 whereas transfection with miR-451 did not change these levels. No differences were found in MMP-13 or PGE2 production with miR-122 or miR-451 inhibitors. LiCl treatment decreased PGE2 production in cultures treated with TNF-α, but not MMP-13. XAV increased TNF-α-stimulated increases in PGE2 but not MMP-13. LiCl reduced IL-1ß-stimulated increases in MMP-13 and PGE2. XAV and PKF increased IL-1ß-stimulated increases in MMP-13 and PGE2. In this in vitro OA model, miR-122 protects against both IL-1ß and TNF-α stimulated increases in MMP-13 and PGE2 production. miR-451 does not act through the TNF-α pathway. The WNT/ß-catenin pathway regulates the effects of IL-1ß and TNF-α stimulation. This study suggests that miR-122 can be used as a treatment or prevention for OA.

Specific MicroRNAs Found in Extracellular Matrix Vesicles Regulate Proliferation and Differentiation in Growth Plate Chondrocytes.

Matrix vesicles (MVs) are extracellular organelles produced by growth plate cartilage cells in a zone-specific manner. MVs are similar in size to exosomes, but they are tethered to the extracellular matrix (ECM) via integrins. Originally associated with matrix calcification, studies now show that they contain matrix processing enzymes and microRNA that are specific to their zone of maturation. MVs produced by costochondral cartilage resting zone (RC) chondrocytes are enriched in microRNA 503 whereas those produced by growth zone (GC) chondrocytes are enriched in microRNA 122. MVs are packaged by chondrocytes under hormonal and factor regulation and release of their contents into the ECM is also under hormonal control, suggesting that their microRNA might have a regulatory role in growth plate proliferation and maturation. To test this, we selected a subset of these enriched microRNAs and transfected synthetic mimics back into RC and GC cells. Transfecting growth plate chondrocytes with select microRNA produced a broad range of phenotypic responses indicating that MV-based microRNAs are involved in the regulation of these cells. Specifically, microRNA 122 drives both RC and GC cells toward a proliferative phenotype, stabilizes the matrix and inhibits differentiation whereas microRNA 22 exerts control over regulatory factor production. This study demonstrates the strong regulatory capability possessed by unique MV enriched microRNAs on growth plate chondrocytes and their potential for use as therapeutic agents.

Advanced Glycation End Products Are Retained in Decellularized Muscle Matrix Derived from Aged Skeletal Muscle.

Decellularized tissues are biocompatible materials that engraft well, but the age of their source has not been explored for clinical translation. Advanced glycation end products (AGEs) are chemical cross-links that accrue on skeletal muscle collagen in old age, stiffening the matrix and increasing inflammation. Whether decellularized biomaterials derived from aged muscle would suffer from increased AGE collagen cross-links is unknown. We characterized gastrocnemii of 1-, 2-, and 20-month-old C57BL/6J mice before and after decellularization to determine age-dependent changes to collagen stiffness and AGE cross-linking. Total and soluble collagen was measured to assess if age-dependent increases in collagen and cross-linking persisted in decellularized muscle matrix (DMM). Stiffness of aged DMM was determined using atomic force microscopy. AGE levels and the effect of an AGE cross-link breaker, ALT-711, were tested in DMM samples. Our results show that age-dependent increases in collagen amount, cross-linking, and general stiffness were observed in DMM. Notably, we measured increased AGE-specific cross-links within old muscle, and observed that old DMM retained AGE cross-links using ALT-711 to reduce AGE levels. In conclusion, deleterious age-dependent modifications to collagen are present in DMM from old muscle, implying that age matters when sourcing skeletal muscle extracellular matrix as a biomaterial.

Titanium implant surface properties enhance osseointegration in ovariectomy induced osteoporotic rats without pharmacologic intervention.

OBJECTIVES: This study determined whether implant surfaces that promote osseointegration in normal rats can promote osseointegration in osteoporotic rats without pharmacologic intervention. MATERIALS AND METHODS: Virgin female 8-month-old CD Sprague Dawley rats (N = 25) were ovariectomized. At 6 weeks, microstructured/non-nanostructured/hydrophobic, microstructured/nanostructured/hydrophobic, or microstructured/nanostructured/hydrophilic Ti implants (Ø2.5 × 3.5 mm; Institut Straumann AG, Basel, Switzerland) were placed in the distal metaphysis of each femur. At 28 days, bone quality and implant osseointegration were assessed using microCT, histomorphometrics, and removal torque values (RTVs). Calvarial osteoblasts were isolated and cultured for 7 days on Ø15 mm Ti disks processed to exhibit similar surface characteristics as the implants used for the in vivo studies. The phenotype was assessed by measuring the production of osteocalcin, osteoprotegerin, osteopontin, BMP2, VEGF, and RANKL. RESULTS: Microstructured/nanostructured/hydrophilic implants promoted increased bone-to-implant contact and RTVs in vivo and increased osteoblastic marker production in vitro compared to microstructured/non-nanostructured/hydrophobic and microstructured/nanostructured/hydrophobic implants, suggesting that osseointegration occurs in osteoporotic animals, and implant surface properties improve its rate. CONCLUSIONS: Although all modified implants were able to osseointegrate in rats with OVX-induced osteoporosis without pharmacologic intervention, the degree of osseointegration was greater around microstructured/nanostructured/hydrophilic implant surfaces. These results suggest that when appropriate microstructure is present, hydrophilicity has a greater influence on Ti implant osseointegration compared to nanostructures. Moreover, modified implant surfaces can exert their control over the altered bone turnover observed in osteoporotic patients to stimulate functional osseointegration. These results provide critical insight for developing implants with improved osseointegration in patients with metabolic disorders of bone remodeling.

Acellular mineralized allogenic block bone graft does not remodel during the 10 weeks following concurrent implant placement in a rabbit femoral model.

OBJECTIVES: Due to bone loss, endosseous implants often require addition of a bone graft to support adequate primary fixation, bone regeneration, and osseointegration. The aim of this study was to compare effectiveness of autogenic and allogenic bone grafts when used during simultaneous insertion of the implant. MATERIALS AND METHODS: 4-mm-diameter rabbit diaphyseal bone autografts or allografts (n = 16/group) with a 3.2-mm pre-drilled hole in the center were placed into a 4 mm defect in the proximal femur of 3.5 kg male New Zealand White rabbits. Machined 3.2 × 10 mm grit-blasted, acid-etched titanium-aluminum-vanadium (Ti6Al4V) implants were placed. Control implants were placed into progressively drilled 3.2-mm holes in the contralateral limbs. Post-insertion day 70, samples were analyzed by micro-CT and calcified histology, or by mechanical torque and push-out testing followed by decalcified histology. RESULTS: Both grafts were integrated with the native bone. Micro-CT showed less bone volume (BV) and bone volume/total volume (BV/TV) in the allograft group, but histology showed no differences in BV or BV/TV between groups. Allograft lacked living cells, whereas autograft was cellularized. No difference was found in maximum removal torque between groups. Compressive loading at the graft-to-bone interface was significantly lower in allograft compared with autograft groups. CONCLUSIONS: There was less bone in contact with the implant and significantly less maximum compressive load in the allograft group compared with autograft. The allograft remained acellular as demonstrated by empty lacunae. Taken together, block allograft implanted simultaneously with an implant produces a poorer quality bone compared with autograft.

Integrin-α7 signaling regulates connexin 43, M-cadherin, and myoblast fusion.

Regenerative medicine treatments for severe skeletal muscle injuries are limited, resulting in persistent functional deficits. Clinical options include neglecting the wound with the expectation that fibrosis will develop or using an autologous muscle graft with minimal functional improvement. A regenerative matrix can be used, but muscle fiber development on these matrices remains a challenge in vivo. Here, we explored the fundamental mechanisms that mediate cell-substrate signaling and its effect on cell-cell communication during myoblast fusion and tube formation to improve outcomes following implantation of matrices used to stimulate muscle regeneration. We previously reported that integrin-α7 was increased on anisotropic biomaterials, suggesting a role for α7ß1 signaling in myoblast communication via connexin 43 and M-cadherin. Our results demonstrated that α7 silencing blocked expression of myogenic differentiation factor 1 (Myod), myogenin (Myog), myogenic factor 6 (Myf6), myosin heavy chain type 1 (Myh1), and transmembrane protein 8c (Tmem8c), indicating that myoblast fusion was inhibited. Expression of α5 and M-cadherin decreased but ß1 and connexin 43 increased. We examined protein production and observed reduced extracellular-signal regulated kinase 1/2 (ERK) in α7-silenced cells that correlated with upregulation of connexin 43 and M-cadherin, suggesting a compensatory pathway. These results indicate that α7 signaling plays a critical role in ex vivo fusion and implicates a relationship with connexin 43 and M-cadherin.

Rapid steroid hormone actions via membrane receptors.

Steroid hormones regulate a wide variety of physiological and developmental functions. Traditional steroid hormone signaling acts through nuclear and cytosolic receptors, altering gene transcription and subsequently regulating cellular activity. This is particularly important in hormonally-responsive cancers, where therapies that target classical steroid hormone receptors have become clinical staples in the treatment and management of disease. Much progress has been made in the last decade in detecting novel receptors and elucidating their mechanisms, particularly their rapid signaling effects and subsequent impact on tumorigenesis. Many of these receptors are membrane-bound and lack DNA-binding sites, functionally separating them from their classical cytosolic receptor counterparts. Membrane-bound receptors have been implicated in a number of pathways that disrupt the cell cycle and impact tumorigenesis. Among these are pathways that involve phospholipase D, phospholipase C, and phosphoinositide-3 kinase. The crosstalk between these pathways has been shown to affect apoptosis and proliferation in cardiac cells, osteoblasts, and chondrocytes as well as cancer cells. This review focuses on rapid signaling by 17ß-estradiol and 1α,25-dihydroxy vitamin D3 to examine the integrated actions of classical and rapid steroid signaling pathways both in contrast to each other and in concert with other rapid signaling pathways. This new approach lends insight into rapid signaling by steroid hormones and its potential for use in targeted drug therapies that maximize the benefits of traditional steroid hormone-directed therapies while mitigating their less desirable effects.

Comparable responses of osteoblast lineage cells to microstructured hydrophilic titanium-zirconium and microstructured hydrophilic titanium.

OBJECTIVES: Although titanium (Ti) is commonly used for dental implants, Ti alloy materials are being developed to improve their physical material properties. Studies indicate that osteoblast differentiation and maturation of human mesenchymal stem cells (MSCs) and normal human osteoblasts (NHOsts) respond to microstructured Ti and titanium-aluminum-vanadium (Ti6Al4V) surfaces in a similar manner. The goal of this study was to determine whether this is the case for osteoblast lineage cells grown on microstructured TiZr surfaces and whether their response is affected by surface nanotexture and hydrophilicity. MATERIALS AND METHODS: Grade 4 Ti and TiZr (13-17% Zr) disks were modified by large grit sand-blasting and acid-etching with storage in saline solution, resulting in a complex microstructured and hydrophilic surface corresponding to the commercially available implants SLActive® and Roxolid® SLActive® (Institut Straumann AG, Basel, Switzerland). The subsequent Ti modSLA and TiZr modSLA surfaces were characterized and osteogenic markers were measured. RESULTS: Evaluation of physical parameters revealed that the fabrication method was capable of inducing a microstructured and hydrophilic surface on both the Ti and TiZr disks. Overall, the surfaces were similar, but differences in nanostructure morphology/density and surface chemistry were detected. On Ti modSLA and TiZr modSLA, osteoblastic differentiation and maturation markers were enhanced in both MSCs and NHOsts, while inflammatory markers decreased compared with TCPS. CONCLUSIONS: These results indicate a similar positive cell response of MSCs and NHOsts when cultured on Ti modSLA and TiZr modSLA. Both surfaces were hydrophilic, indicating the importance of this property to osteoblast lineage cells.

Dental implant surface chemistry and energy alter macrophage activation in vitro.

OBJECTIVES: To determine the effects of dental implant surface chemistry and energy on macrophage activation in vitro. MATERIALS AND METHODS: Disks made from two clinically used implant materials (titanium [Ti], titanium zirconium alloy [TiZr]) were produced with two different surface treatments (sandblast/acid-etch [SLA], hydrophilic-SLA [modSLA]). Surface roughness, energy, and chemistry were characterized. Primary murine macrophages were isolated from 6- to 8-week-old male C57Bl/6 mice and cultured on test surfaces (Ti SLA, TiZr SLA, Ti modSLA, TiZr modSLA) or control tissue culture polystyrene. mRNA was quantified by quantitative polymerase chain reaction after 24 h of culture. Pro- (IL-1ß, IL-6, and TNF-α) and anti-inflammatory (IL-4, IL-10) protein levels were measured by ELISA after 1 or 3 days of culture. RESULTS: Quantitatively, microroughness was similar on all surfaces. Qualitatively, nanostructures were present on modSLA surfaces that were denser on Ti than on TiZr. modSLA surfaces were determined hydrophilic (high-energy surface) while SLA surfaces were hydrophobic (low-energy surface). Cells on high-energy surfaces had higher levels of mRNA from anti-inflammatory markers characteristic of M2 activation compared to cells on low-energy surfaces. This effect was enhanced on the TiZr surfaces when compared to cells on Ti SLA and Ti modSLA. Macrophages cultured on TiZr SLA and modSLA surfaces released more anti-inflammatory cytokines. CONCLUSIONS: The combination of high-energy and altered surface chemistry present on TiZr modSLA was able to influence macrophages to produce the greatest anti-inflammatory microenvironment and reduce extended pro-inflammatory factor release.

Effects of low-frequency ultrasound treatment of titanium surface roughness on osteoblast phenotype and maturation.

OBJECTIVE: Low-frequency ultrasound is widely used in the treatment of chronically infected wounds. To investigate its feasibility as a method for in situ restoration of metal implant surfaces in cases of peri-implantitis, we evaluated how low-frequency ultrasound affected surface properties of and response of human osteoblast-like MG63 cells to titanium (Ti). MATERIAL AND METHODS: Three Ti surfaces [hydrophobic/smooth (pretreatment, PT); hydrophobic/rough (sandblasted/acid-etched, SLA); and hydrophilic/rough (SLA processed and stored hydrophilicity, mSLA)] were subjected to 25 kHz ultrasound for 10 min/cm2 . Substrate roughness, chemical composition, and wettability were analyzed before and after ultrasound application. Osteoblastic maturation of cells on sonicated disks was compared to cells on untreated disks. RESULTS: Ultrasound treatment altered the topography of all surfaces. Contact angles were reduced, and chemical compositions were altered by ultrasound on PT and SLA surfaces. Cell response to sonicated PT was comparable to untreated PT. Alkaline phosphatase was increased on sonicated SLA compared to untreated SLA, whereas DNA, osteocalcin, BMP2, osteoprotegerin, and VEGF-A were unchanged. Cells produced less osteocalcin and BMP2 on sonicated mSLA than on untreated mSLA, but no other parameters were affected. CONCLUSIONS: These results show that low-frequency ultrasound altered Ti surface properties. Osteoblasts were sensitive to the changes induced by ultrasound treatment. The data suggest that the effect is to delay differentiation, but it is unclear whether this delay will prevent osseointegration. These results suggest that low-frequency ultrasound may be useful for treating implant surfaces in situ leading to successful re-osseointegration of implants affected by peri-implantitis.

Laser-Sintered Constructs with Bio-inspired Porosity and Surface Micro/Nano-Roughness Enhance Mesenchymal Stem Cell Differentiation and Matrix Mineralization In Vitro.

Direct metal laser sintering can produce porous Ti-6Al-4V orthopedic and dental implants. The process requires reduced resources and time and can provide greater structural control than machine manufacturing. Implants in bone are colonized by mesenchymal stem cells (MSCs), which can differentiate into osteoblasts and contribute to osseointegration. This study examined osteoblast differentiation and matrix mineralization of human MSCs cultured on laser-sintered Ti-6Al-4V constructs with varying porosity and at different time scales. 2D solid disks and low, medium and high porosity (LP, MP, and HP) 3D constructs based on a human trabecular bone template were laser sintered from Ti-6Al-4V powder and further processed to have micro- and nanoscale roughness. hMSCs exhibited greater osteoblastic differentiation and local factor production on all 3D porous constructs compared to 2D surfaces, which was sustained for 9 days without use of exogenous factors. hMSCs cultured for 8 weeks on MP constructs in osteogenic medium (OM), OM supplemented with BMP2 or collagen-coated MP constructs in OM exhibited bone-like extracellular matrix mineralization. Use of bio-inspired porosity for the 3D architecture of additively manufactured Ti-6Al-4V enhanced osteogenic differentiation of hMSCs beyond surface roughness alone. This study suggests that a 3D architecture may enhance the osseointegration of orthopedic and dental implants in vivo.

Estrogen receptor-alpha 36 mediates the anti-apoptotic effect of estradiol in triple negative breast cancer cells via a membrane-associated mechanism.

17ß-Estradiol can promote the growth and development of several estrogen receptor (ER)-negative breast cancers. The effects are rapid and non-genomic, suggesting that a membrane-associated ER is involved. ERα36 has been shown to mediate rapid, non-genomic, membrane-associated effects of 17ß-estradiol in several cancer cell lines, including triple negative HCC38 breast cancer cells. Moreover, the effect is anti-apoptotic. The aim of this study was to determine if ERα36 mediates this anti-apoptotic effect, and to elucidate the mechanism involved. Taxol was used to induce apoptosis in HCC38 cells, and the effect of 17ß-estradiol pre-treatment was determined. Antibodies to ERα36, signal pathway inhibitors, ERα36 deletion mutants, and ERα36-silencing were used prior to these treatments to determine the role of ERα36 in these effects and to determine which signaling molecules were involved. We found that the anti-apoptotic effect of 17ß-estradiol in HCC38 breast cancer cells is in fact mediated by membrane-associated ERα36. We also showed that this signaling occurs through a pathway that requires PLD, LPA, and PI3K; Gαs and calcium signaling may also be involved. In addition, dynamic palmitoylation is required for the membrane-associated effect of 17ß-estradiol. Exon 9 of ERα36, a unique exon to ERα36 not found in other identified splice variants of ERα with previously unknown function, is necessary for these effects. This study provides a working model for a mechanism by which estradiol promotes anti-apoptosis through membrane-associated ERα36, suggesting that ERα36 may be a potential membrane target for drug design against breast cancer, particularly triple negative breast cancer.

Signaling components of the 1α,25(OH)2D3-dependent Pdia3 receptor complex are required for Wnt5a calcium-dependent signaling.

Wnt5a and 1α,25(OH)2D3 are important regulators of endochondral ossification. In osteoblasts and growth plate chondrocytes, 1α,25(OH)2D3 initiates rapid effects via its membrane-associated receptor protein disulfide isomerase A3 (Pdia3) in caveolae, activating phospholipase A2 (PLA2)-activating protein (PLAA), calcium/calmodulin-dependent protein kinase II (CaMKII), and PLA2, resulting in protein kinase C (PKC) activation. Wnt5a initiates its calcium-dependent effects via intracellular calcium release, activating PKC and CaMKII. We investigated the requirement for components of the Pdia3 receptor complex in Wnt5a calcium-dependent signaling. We determined that Wnt5a signals through a CaMKII/PLA2/PGE2/PKC cascade. Silencing or blocking Pdia3, PLAA, or vitamin D receptor (VDR), and inhibition of calmodulin (CaM), CaMKII, or PLA2 inhibited Wnt5a-induced PKC activity. Wnt5a activated PKC in caveolin-1-silenced cells, but methyl-beta-cyclodextrin reduced its stimulatory effect. 1α,25(OH)2D3 reduced stimulatory effects of Wnt5a on PKC in a dose-dependent manner. In contrast, Wnt5a had a biphasic effect on 1α,25(OH)2D3-stimulated PKC activation; 50ng/ml Wnt5a caused a 2-fold increase in 1α,25(OH)2D3-stimulated PKC but higher Wnt5a concentrations reduced 1α,25(OH)2D3-stimulated PKC activation. Western blots showed that Wnt receptors Frizzled2 (FZD2) and Frizzled5 (FZD5), and receptor tyrosine kinase-like orphan receptor 2 (ROR2) were localized to caveolae. Blocking ROR2, but not FZD2 or FZD5, abolished the stimulatory effects of 1α,25(OH)2D3 on PKC and CaMKII. 1α,25(OH)2D3 membrane receptor complex components (Pdia3, PLAA, caveolin-1, CaM) interacted with Wnt5a receptors/co-receptors (ROR2, FZD2, FZD5) in immunoprecipitation studies, interactions that changed with either 1α,25(OH)2D3 or Wnt5a treatment. This study demonstrates that 1α,25(OH)2D3 and Wnt5a mediate their effects via similar receptor components and suggests that these pathways may interact.

Accuracy of computer-guided implantation in a human cadaver model.

OBJECTIVES: To examine the accuracy of computer-guided implantation using a human cadaver model with reduced experimental variability. MATERIALS AND METHODS: Twenty-eight (28) dental implants representing 12 clinical cases were placed in four cadaver heads using a static guided implantation template. All planning and surgeries were performed by one clinician. All radiographs and measurements were performed by two examiners. The distance of the implants from buccal and lingual bone and mesial implant or tooth was analyzed at the apical and coronal levels, and measurements were compared to the planned values. RESULTS: No significant differences were seen between planned and implanted measurements. Average deviation of an implant from its planning radiograph was 0.8 mm, which is within the range of variability expected from CT analysis. CONCLUSIONS: Guided implantation can be used safely with a margin of error of 1 mm.

Sex-specific response of rat costochondral cartilage growth plate chondrocytes to 17ß-estradiol involves differential regulation of plasma membrane associated estrogen receptors.

Both male and female rat growth plate chondrocytes express estrogen receptors (ERs); however 17ß-estradiol (E2) induces membrane responses leading to activation of phospholipase A2 (PLA2), phospholipase C (PLC), prostaglandin E2 (PGE2) production, protein kinase C (PKC), and ultimately mitogen protein kinase (MAPK) only in female cells. This study investigated if these sex-specific responses are due to differences in the actual ERs or in downstream signaling. Western blots and flow cytometry of costochondral cartilage resting zone chondrocytes (RCs) showed 2-3 times more ERα in plasma membranes (PMs) from female cells than male cells. Tunicamycin blocked E2-dependent ER-translocation to the PM, indicating palmitoylation was required. Co-immunoprecipitation showed E2 induced complex formation between ER isoforms only in female RCs. To examine if the lack of response in PKC and PGE2 in males is due to differences in signaling, we examined involvement of ERs and the role of PLC and PLA2. Selective ERα (propylpyrazole triol, PPT) and ERß (diarylproprionitrile, DPN) agonists activated PKC in female RCs only. The PLC inhibitor, U73122 blocked E2's effect on PKC and the cytosolic PLA2 inhibitor, AACOCF3 inhibited the effect on PGE2 in female RCs, confirming involvement of PLC and PLA2 in the mechanism. The PLC activator, m-3M3FßS activated PKC and PLAA peptide increased PGE2 levels in male and female RCs, showing that the signaling pathways are present. These data indicate that differences in membrane ER amount, localization, translocation and interaction are responsible for the sexual dimorphic response to E2.

Rapid 1α,25(OH)2D 3 membrane-mediated activation of Ca²âº/calmodulin-dependent protein kinase II in growth plate chondrocytes requires Pdia3, PLAA and caveolae.

1α,25-Dihydroxy vitamin D3 [1α,25(OH)2D3] regulates growth zone chondrocytes (GC) via classical steroid hormone receptor-mediated gene transcription and by initiating rapid membrane-mediated signaling pathways. 1α,25(OH)2D3 initiates its membrane effects via its specific membrane-associated receptor (Pdia3) in caveolae. 1α,25(OH)2D3 binding to Pdia3 leads to phospholipase-A2 (PLA2)-activating protein (PLAA) activation, stimulating PLA2, resulting in prostaglandin E2 (PGE2) release and protein kinase C activation. Recently, we reported that 1α,25(OH)2D3 rapidly activates Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) in GC cells. However, the roles of Pdia3, PLAA and caveolae in 1α,25(OH)2D3-dependent rapid activation of CaMKII are not clear. The aim of the present study was to evaluate the roles of Pdia3, PLAA and caveolae in 1α,25(OH)2D3 membrane-stimulated CaMKII activation. Pre-treating chondrocytes from the growth zone of the rat costochondral cartilage with antibodies against PLAA or Pdia3 blocked activation of CaMKII by 1α,25(OH)2D3. PLAA peptide rapidly activated CaMKII in GC cells. Caveolae disruption abolished CaMKII activation in response to 1α,25(OH)2D3 or PLAA peptide treatment. Immunoprecipitation studies showed increased CaM binding to PLAA in response to 1α,25(OH)2D3. The results indicated that Pdia3, PLAA and caveolae are required for rapid 1α,25(OH)2D3 membrane-mediated activation of CaMKII. 1α,25(OH)2D3 signaling via Pdia3 receptor triggered the interaction between PLAA and CaM suggesting that CaM may play a major role linking PLAA to CaMKII in membrane-mediated actions of 1α,25(OH)2D3.