Potent Inducer Of Differentiation Research Articles

Autotaxin, a potent inducer of cell motility and differentiation, is differentially expressed in visceral and parietal mesothelia

The serosal mesothelium is a simple squamous epithelium lining the body cavity. It can be divided into two categories: visceral mesothelium covering abdominal organs including the omentum and parietal mesothelium lining the peritoneal wall. Earlier investigations considered visceral and parietal mesothelia the same cell type. Indeed, these tissues are continuous and share similar expression of mesothelial marker genes. However, we report a microarray analysis of murine visceral and parietal mesothelia where over 35,000 genes were differentially expressed. Specifically, Autotaxin (Atx) was expressed significantly higher in visceral versus parietal mesothelium. Atx is a secreted glycoprotein that is responsible for production of Lysophosphatidic acid (Lpa), promotes cell motility, cell survival, and angiogenesis, and has been identified as an important mediator of tumorigenesis. Here, we demonstrate visceral mesothelium migrates and differentiates into smooth muscle more readily than parietal mesothelium. Addition of Atx to parietal cells induces “visceral‐like” migration and differentiation. Finally, we identified several novel small organic molecule inhibitors of Lpa that repress Atx‐induced motility and differentiation. Importantly, as Atx plays a critical role in tumorigenesis, these inhibitors may represent new therapeutic agents for a variety of cancers including mesothelioma.Grant Funding Source: NIH

Cyp26b1Mediates Differential Neurogenicity in Axial-Specific Populations of Adult Spinal Cord Progenitor Cells

Utilization of endogenous adult spinal cord progenitor cells (SCPCs) for neuronal regeneration is a promising strategy for spinal cord repair. To mobilize endogenous SCPCs for injury repair, it is necessary to understand their intrinsic properties and to identify signaling factors that can stimulate their neurogenic potential. In this study, we demonstrate that adult mouse SCPCs express distinct combinatorial Hox genes and exhibit axial-specific stem cell properties. Lumbar-derived neurospheres displayed higher primary sphere formation and greater neurogenicity compared with cervical- and thoracic-derived neurospheres. To further understand the mechanisms governing neuronal differentiation of SCPCs from specific axial regions, we examined the neurogenic responses of adult SCPCs to retinoic acid (RA), an essential factor for adult neurogenesis. Although RA is a potent inducer of neuronal differentiation, we found that RA enhanced the generation of neurons specifically in cervical- but not lumbar-derived cells. We further demonstrate that the differential RA response was mediated by the RA-degrading enzyme cytochrome P450 oxidase b1 Cyp26b1. Lumbar cells express high levels of Cyp26b1 and low levels of the RA-synthesizing enzyme retinaldehyde dehydrogenase Raldh2, resulting in limited activation of the RA signaling pathway in these cells. In contrast, low Cyp26b1 expression in cervical spinal cord progenitor cells allows RA signaling to be readily activated upon RA treatment. The intrinsic heterogeneity and signaling factor regulation among adult SCPCs suggest that different niche factor regimens are required for site-specific mobilization of endogenous SCPCs from distinct spatial regions of the spinal cord for injury repair.

A potent small-molecule inducer of chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells

We discovered a novel small-molecule modulator 5{i,2} that can specifically induce the chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBM-MSCs). Based on our biochemical and histological studies, 5{i,2} showed more of a directed differentiation of MSCs to chondrocyte balls compared to TGF-β3.

FES/FER kinase signaling in hematopoietic cells and leukemias

FES and FES-related (FER) comprise a unique subfamily of protein-tyrosine kinases (PTKs) that signal downstream of several classes of receptors involved in regulating hematopoietic cell development, survival, migration, and inflammatory mediator release. Activated alleles of FES are potent inducers of myeloid differentiation, however FES-deficient mice have only subtle differences in hematopoiesis. This may reflect overlapping function of other kinases such as FER. Studies of FES- and FER-deficient mice have revealed more prominent roles in regulating the activation of mature innate immune cells, including macrophages and mast cells. Recently, new insights into regulation of FES/FER kinases has emerged with the characterization of their N-terminal phospholipid-binding and membrane targeting FER/CIP4 homology-Bin/Amphyphysin/Rvs (F-BAR) and F-BAR extension (FX) domains. The F-BAR/FX domains regulate subcellular localization and FES/FER kinase activation. FES kinase activity is also enhanced upon ligand binding to its SH2 domain, which may lead to further phosphorylation of the same ligand, or other ligand-associated proteins. In mast cells, SH2 ligands of FES/FER include KIT receptor PTK, and the high affinity IgE receptor (FceRI) that trigger rapid activation of FES/FER and signaling to regulators of the actin cytoskeleton and membrane trafficking. Recently, FES/FER have also been implicated in growth and survival signaling in leukemias driven by oncogenic KIT and FLT3 receptors. With further definition of their roles in immune cells and their progenitors, FES/FER may emerge as relevant therapeutic targets in inflammatory diseases and leukemias.

Cardiac Differentiation of Human Wharton's Jelly Stem Cells – Experimental Comparison of Protocols

Cardiomyoplasty represents a promising approach for the repair of the injured heart, but is hampered by the availability of appropriate cells. Mesenchymal stem cells derived from the human umbilical cord tissue (UCMSC) can be obtained in large amounts without medical intervention, exhibit self renewal and immunological naivity as well as multipotency. In the present study, different published protocols of cardiac differentiation designed for different stem cell types were compared to differentiate UCMSC into cardiomyocyte-like cells (cUCMSC). Cardiac differentiation of UCMSC was driven by cell treatment with 5-azacytidine, oxytocin as well as by forming of embryoid bodies. The morphological and immunocytochemical analysis of cUCMSC with an extensive panel of cardiac markers showed that oxytocin is a more potent inducer of cardiac differentiation than 5-azacytidine and the forming of embryoid bodies. cUCMSC reveal a cardiomyocyte-like structure and the expression of cardiomyocyte associated proteins. The easy accessibility and the ability of UCMSC to differentiate into cells with characteristics of cardiomyocytes render UCMSC an attractive candidate for cell based therapies and cardiac tissue engineering.

Endogenous Oncogenic Nras Signaling Promotes Proliferative Exhaustion of Hematopoietic Stem Cells Through a Dose-Dependent Hyperactivation of ERK,

Abstract 3997Both monoallelic and biallelic oncogenic NRAS mutations are identified in human leukemias, suggesting a dose-dependent role of oncogenic NRAS in leukemogenesis. We recently characterized conditional mouse models that express one or two copies of constitutively active Nras G12D in the hematopoietic compartment (Nras G12D/+ and Nras G12D/G12D). Nras G12D/G12D results in stronger downstream signaling than Nras G12D/+ and consequently distinct hematopoietic phenotypes. In particular, we found that somatic expression of Nras G12D/G12D but not Nras G12D/+ leads to an acute myeloproliferative neoplasm (MPN). The development of acute MPN is associated with cytokine-evoked hyperactivation of ERK but not Stat5 and Akt in hematopoietic stem/progenitor cells (HSPCs). Interestingly, genetically altered HSCs appear to be required for initiation and maintenance of chronic and acute MPD phenotypes mediated by Nras G12D/+ and Nras G12D/G12D, respectively. Furthermore, Nras G12D/G12D-mediated signaling promotes excessive HSC proliferation and leading to HSC exhaustion. Exhaustion of HSCs is tightly associated with diminished MPN phenotypes in non-engineered recipient mice into which Nras G12D/G12D HSCs are transferred by bone marrow transplantation. To investigate the molecular mechanisms underlying the exhaustion of Nras G12D/G12D HSCs, we performed a microarray analysis using highly purified control, Nras G12D/+, and Nras G12D/G12D HSCs. To our surprise, neither p16INK4a and p15INK4b, critical cell senescence genes, or other genes or pathways known to involve in HSC self-renewal were differentially expressed in Nras G12D/G12D HSCs. In contrast, gene ontology analysis revealed significant differential expression of genes in the MEK/ERK pathway in Nras G12D/G12D HSCs. This result is consistent with our phospho-flow study showing that Nras G12D/G12D selectively hyperactivates ERK in HSPCs. It has been documented that prolonged ERK signaling is a potent inducer of differentiation in cultured PC12 cells and inhibition of normal ERK signaling by a chemical inhibitor increases self-renewal in mouse embryonic stem cells. Thus, we are currently testing whether hyperactivation of ERK promotes depletion of Nras G12D/G12D HSCs through promoting HSC differentiation and whether blocking hyperactivation of ERK restores the normal HSC function and prevents MPN development. Disclosures:No relevant conflicts of interest to declare.

Complete Genome Sequences of Rat and Mouse Segmented Filamentous Bacteria, a Potent Inducer of Th17 Cell Differentiation

Segmented filamentous bacteria (SFB) are noncultivable commensals inhabiting the gut of various vertebrate species and have been shown to induce Th17 cells in mice. We present the complete genome sequences of both rat and mouse SFB isolated from SFB-monocolonized hosts. The rat and mouse SFB genomes each harbor a single circular chromosome of 1.52 and 1.59 Mb encoding 1346 and 1420 protein-coding genes, respectively. The overall nucleotide identity between the two genomes is 86%, and the substitution rate was estimated to be similar to that of the free-living E.coli. SFB genomes encode typical genes for anaerobic fermentation and spore and flagella formation, but lack most of the amino acid biosynthesis enzymes, reminiscent of pathogenic Clostridia, exhibiting large dependency on the host. However, SFB lack most of the clostridial virulence-related genes. Comparative analysis with clostridial genomes suggested possible mechanisms for host responses and specific adaptations in the intestine.

BMP-9 Induced Osteogenic Differentiation of Mesenchymal Stem Cells: Molecular Mechanism and Therapeutic Potential

Promoting osteogenic differentiation and efficacious bone regeneration have the potential to revolutionize the treatment of orthopaedic and musculoskeletal disorders. Mesenchymal Stem Cells (MSCs) are bone marrow progenitor cells that have the capacity to differentiate along osteogenic, chondrogenic, myogenic, and adipogenic lineages. Differentiation along these lineages is a tightly controlled process that is in part regulated by the Bone Morphogenetic Proteins (BMPs). BMPs 2 and 7 have been approved for clinical use because their osteoinductive properties act as an adjunctive treatment to surgeries where bone healing is compromised. BMP-9 is one of the least studied BMPs, and recent in vitro and in vivo studies have identified BMP-9 as a potent inducer of osteogenic differentiation in MSCs. BMP-9 exhibits significant molecular cross-talk with the Wnt/ β-catenin and other signaling pathways, and adenoviral expression of BMP-9 in MSCs increases the expression of osteogenic markers and induces trabecular bone and osteiod matrix formation. Furthermore, BMP-9 has been shown to act synergistically in bone formation with other signaling pathways, including Wnt/ β-catenin, IGF, and retinoid signaling pathways. These results suggest that BMP-9 should be explored as an effective bone regeneration agent, especially in combination with adjuvant therapies, for clinical applications such as large segmental bony defects, non-union fractures, and/or spinal fusions.

Abstract 588: Silencing of MnSOD attenuates ATRA-induced neurofilament expression in neuroblastoma cells

Abstract Differentiation therapy with retinoids is a means to treat high-risk neuroblastoma patients when the tumor has recurred after initial attempts of removal by surgery, radiation, and/ or chemotherapy. A metabolite of Vitamin A, all-trans retinoic acid (ATRA) is the main signaling retinoid in vivo and one of the most potent differentiation inducers for human neuroblastoma in vitro. Manganese superoxide dismutase (MnSOD) is a mitochondrial enzyme that converts the superoxide anion to hydrogen peroxide. Previously we have reported that ATRA increased MnSOD expression and activity in SK-N-SH neuroblastoma cells. However, our current study suggests that this upregulation may limit the ability of ATRA to induce differentiation. Treatment with ATRA upregulated the expression of neurofilament (an early marker of neuronal differentiation) as early as 48 hrs. Although MnSOD activity was not significantly increased by ATRA until 72 hrs, silencing of the MnSOD gene with siRNA enhanced neurofilament expression over that of cells infected with a control siRNA construct at 48 hrs. This suggests that even basal levels of MnSOD activity can attenuate an early marker of differentiation. We also demonstrated that ATRA decreased catalase expression and increased dichlorofluoroscein fluorescence, suggesting an increase in hydrogen peroxide. Therefore, these data suggest that rather than being scavenged by catalase, hydrogen peroxide may remain present in the cell after ATRA treatment to also limit the differentiation potential of the cells. Taken together, it is apparent that the alteration of redox status by retinoids may contribute to the modulation of genes which are involved in differentiation. (Funding: Centers for Biomedical Research Excellence 1P20RR020180 (KKK) and 5P20RR016477; WV-INBRE 5P20RR016477; WV-NASA 91-175B). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 588. doi:10.1158/1538-7445.AM2011-588

MicroRNAs 10a and 10b are potent inducers of neuroblastoma cell differentiation through targeting of nuclear receptor corepressor 2

MicroRNAs function as negative regulators of posttranscriptional gene expression, having major roles in cellular differentiation. Several neuroblastoma cell lines can be induced to undergo differentiation by all-trans-retinoic acid (ATRA) and are used for modeling signaling pathways involved in this process. To identify miRNAs contributing to differentiation, we profiled 364 loci following ATRA treatment of neuroblastoma cell lines and found miR-10a and miR-10b to be highly overexpressed in SK-N-BE, LAN5 and SHSY-5Y. Ectopic overexpression of these miRNAs led to a major reprogramming of the transcriptome and a differentiated phenotype that was similar to that induced by ATRA in each of these cell lines. One of the predicted downregulated miR-10a/b targets was nuclear receptor corepressor 2 (NCOR2), a corepressor of gene transcription, which is known to suppress neurite outgrowth. NCOR2 was experimentally validated as a direct target of miR-10a/b, and siRNA-mediated inhibition of this mRNA alone resulted in neural cell differentiation. Moreover, induction of differentiation could be blocked by ectopic upregulation of NCOR2 using an expression construct lacking the miR-10a/b 3' untranslated region target site. We conclude that miR-10a/b has major roles in the process of neural cell differentiation through direct targeting of NCOR2, which in turn induces a cascade of primary and secondary transcriptional alterations, including the downregulation of MYCN.

Unique Mode of Lipogenic Activation in Rat Preputial Sebocytes

Lipoprotein delivery of fatty acids and cholesterol is linked with peroxisome proliferator-activated receptor (PPAR) activation in adipocytes and macrophages. We postulated that similar interactions exist in sebaceous epithelial cells (sebocytes) in which PPAR activation induces differentiation. High-density lipoprotein (HDL) and very low-density lipoprotein (VLDL) markedly enhanced sebocyte differentiation above that found with PPAR agonists and were more potent than explicable by their lipid content. The PPARγ antagonist GW5393 reduced sebocyte differentiation to all PPAR isoform agonists, HDL and VLDL, suggesting that the lipoprotein effect on differentiation occurs partially through activation of PPARγ. Furthermore, we found that sebocytes expressed a unique pattern of lipogenic genes. Our results demonstrate that HDL and VLDL are the most potent inducers of sebocyte differentiation tested to date, and these actions are partially inhibited by PPAR antagonists. This suggests that substrates provided by lipoproteins are targeted to sebocytes and affect their own disposition via PPAR activation.

Cyclooxygenase-2 Deficiency Attenuates Adipose Tissue Differentiation and Inflammation in Mice

Obesity is associated with a variety of disorders and is a significant health problem in developed countries. One factor controlling the level of adiposity is the differentiation of cells into adipocytes. Adipocyte differentiation requires expression of peroxisome proliferator-activated receptor γ (PPARγ), which is activated by ligands to regulate expression of genes involved in adipocyte differentiation. Although 15-deoxy-Δ(12,14)-prostaglandin (PG) J(2) (15d-PGJ(2)) has long been known to be a potent activator of PPARγ, the importance of its synthesis in adipose tissue in vivo is not clear. The current study utilized mice deficient in cyclooxygenase-2 (COX-2) to examine the role of COX-2-derived PGs as in vivo modulators of adiposity. As compared with strain- and age-matched wild-type controls, the genetic deficiency of COX-2 resulted in a significant reduction in total body weight and percent body fat. Although there were no significant differences in food consumption between groups, COX-2-deficient mice showed increased metabolic activity. Epididymal adipose tissue from wild-type mice produced a significantly greater level of 15d-PGJ(2), as compared with adipose tissue isolated from mice deficient in COX-2. Furthermore, production of the precursor required for 15d-PGJ(2) formation, PGD(2), was also significantly reduced in COX-2-deficient adipose tissue. The expression of markers for differentiated adipocytes was significantly reduced in adipose tissue from COX-2-deficient mice, whereas preadipocyte marker expression was increased. Macrophage-dependent inflammation was also significantly reduced in adipose tissue of COX-2-deficient mice. These findings suggest that reduced adiposity in COX-2-deficient mice results from attenuated PPARγ ligand production and adipocyte differentiation.

CCN2 is required for bleomycin‐induced skin fibrosis in mice

No therapy for fibrotic disease is available. The proadhesive matricellular protein connective tissue growth factor CCN2 is a marker of fibrotic cells; however, the specific role of CCN2 in connective tissue biology in general and in fibrogenesis in particular is unclear. The aim of this study was to assess whether adult mice bearing a smooth muscle cell/fibroblast-specific deletion of CCN2 are resistant to bleomycin-induced skin scleroderma. Cutaneous fibrosis was induced in mice by subcutaneous injection of bleomycin. Untreated control groups were injected with phosphate buffered saline. Mice bearing a fibroblast/smooth muscle cell-specific deletion of CCN2 were investigated for changes in dermal thickness, collagen content, and the number of α-smooth muscle actin (α-SMA)-positive cells. Dermal fibroblasts were isolated to assess whether the induction of collagen and α-SMA messenger RNA in response to transforming growth factor β (TGFβ) was impaired. The loss of CCN2 resulted in resistance to bleomycin-induced skin fibrosis. In response to bleomycin, wild-type mice possessed, but CCN2-deficient mice lacked, abundant α-SMA-expressing myofibroblasts within fibrotic lesions. Fibroblast responses to TGFβ, a potent inducer of myofibroblast differentiation, were not affected. Collectively, these results indicate that CCN2 is essential for bleomycin-induced skin fibrosis, likely due to a defect in myofibroblast recruitment. These data indicate that therapeutic strategies that involve blocking CCN2 in vivo may be of benefit in combating fibrotic skin disease.

The Implication of Aberrant GM-CSF Expression in Decidual Cells in the Pathogenesis of Preeclampsia

Preeclampsia is characterized by an exaggerated systemic inflammatory state as well as shallow placentation. In the decidual implantation site, preeclampsia is accompanied by an excessive number of both macrophages and dendritic cells as well as their recruiting chemokines, which have been implicated in the impairment of endovascular trophoblast invasion. Granulocyte-macrophage colony-stimulating factor is known to regulate the differentiation of both macrophages and dendritic cells, prompting both in vivo and in vitro evaluation of granulocyte-macrophage colony-stimulating factor expression in human decidua as well as in a mouse model of preeclampsia. This study revealed increased granulocyte-macrophage colony-stimulating factor expression levels in preeclamptic decidua. Moreover, both tumor necrosis factor-α and interleukin-1 β, cytokines that are implicated in the genesis of preeclampsia, markedly up-regulated granulocyte-macrophage colony-stimulating factor production in cultured first-trimester human decidual cells. The conditioned media of these cultures promoted the differentiation of both macrophages and dendritic cells from a monocyte precursor. Evaluation of a murine model of preeclampsia revealed that the decidua of affected animals displayed higher levels of immunoreactive granulocyte-macrophage colony-stimulating factor as well as increased numbers of both macrophages and dendritic cells when compared to control animals. Because granulocyte-macrophage colony-stimulating factor is a potent inducer of differentiation and activation of both macrophages and dendritic cells, these findings suggest that this factor plays a crucial role in the pathogenesis of preeclampsia.

RANKL Up-regulates Brain-type Creatine Kinase via Poly(ADP-ribose) Polymerase-1 during Osteoclastogenesis

Receptor activator of nuclear factor κB ligand (RANKL) is the key regulator for osteoclast formation and function. During osteoclastogenesis, RANKL-stimulated signals differentially modulate expression of a large number of proteins. Using proteomics approaches, we identified that brain-type cytoplasmic creatine kinase (Ckb) was greatly induced in mature osteoclasts. Ckb has been shown to contribute to osteoclast function. However, the mechanisms of Ckb regulation and the contribution of other isoforms of creatine kinase during RANKL-induced osteoclastogenesis are unknown. We found that Ckb was the predominant isoform of creatine kinase during osteoclastogenesis. Real-time PCR confirmed that RANKL induced ckb mRNA expression by over 40-fold in primary mouse bone marrow macrophages and Raw 264.7 cells. The RANKL-responsive region was identified within the -0.4- to -0.2-kb 5'-flanking region of the ckb gene. Affinity binding purification followed by mass spectrometry analysis revealed that poly(ADP-ribose) polymerase-1 (PARP-1) bound to the -0.4/-0.2-kb fragment that negatively regulated expression of ckb in response to RANKL stimulation. Electrophoretic mobility shift assays with PARP-1-specific antibody located the binding site of PARP-1 to the TTCCCA consensus sequence. The expression of PARP-1 was reduced during RANKL-induced osteoclastogenesis, concurrently with increased expression of Ckb. Consistently, knockdown of PARP-1 by lentivirus-delivered shRNA enhanced ckb mRNA expression. The activity of PARP-1 was determined to be required for its inhibitory effect on the ckb expression. In summary, we have demonstrated that PARP-1 is a negative regulator of the ckb expression. Down-regulation of PARP-1 is responsible for the up-regulation of ckb during RANKL-induced osteoclastogenesis.

Liver-specific cytochrome P450 CYP2C22 is a direct target of retinoic acid and a retinoic acid-metabolizing enzyme in rat liver

Several cytochrome P450 (CYP) enzymes catalyze the C4-hydroxylation of retinoic acid (RA), a potent inducer of cell differentiation and an agent in the treatment of several diseases. Here, we have characterized CYP2C22, a member of the rat CYP2C family with homology to human CYP2C8 and CYP2C9. CYP2C22 was expressed nearly exclusively in hepatocytes, where it was one of the more abundant mRNAs transcripts. In H-4-II-E rat hepatoma cells, CYP2C22 mRNA was upregulated by all-trans (at)-RA, and Am580, a nonmetabolizable analog of at-RA. In comparison, in primary human hepatocytes, at-RA increased CYP2C9 but not CYP2C8 mRNA. Analysis of the CYP2C22 promoter region revealed a RA response element (5'-GGTTCA-(n)5-AGGTCA-3') in the distal flanking region, which bound the nuclear hormone receptors RAR and RXR and which was required for transcriptional activation response of this promoter to RA in CYP2C22-luciferase-transfected RA-treated HepG2 cells. The cDNA-expressed CYP2C22 protein metabolized [3H]at-RA to more polar metabolites. While long-chain polyunsaturated fatty acids competed, 9-cis-RA was a stronger competitor. Our studies demonstrate that CYP2C22 is a high-abundance, retinoid-inducible, hepatic P450 with the potential to metabolize at-RA, providing additional insight into the role of the CYP2C gene family in retinoid homeostasis.

Abstract 191: Azacitidine induces differentiation of acute myeloid leukemia cell lines along the granulocytic/monocytic lineage

Abstract Acute myeloid leukemia (AML) is characterized by blast cells that are unable to mature into functional, terminally-differentiated hematopoietic cells. Inducing leukemic cells to differentiate restores a natural cell death program and inhibits proliferation. Azacitidine (AZA; 5-azacytidine; Vidaza) is a cytidine nucleoside analog used clinically for the treatment of myelodysplastic syndromes (MDS) and AML. AZA therapy was recently shown to significantly increase median overall survival in higher-risk MDS and World Health Organization AML (20-30% bone marrow blasts) patients compared with conventional care regimens, and a phase III clinical trial of AZA in patients with more advanced AML has been activated. We have shown previously that AZA induces dose-dependent cytotoxicity to AML cell lines; however, at sub-micromolar AZA concentrations, complete cell kill is not achieved. To explore an additional anti-leukemic mechanism of AZA in AML, we assessed the effect of AZA on induction of AML cell differentiation in vitro. AML cell lines, encompassing several FAB classifications, were evaluated, using all-trans retinoic acid (ATRA) and 1,25-dihydroxyvitamin D3 (VD3), two potent inducers of AML cell differentiation, as control compounds. Differentiation along the granulocytic/monocytic lineage was assayed by CD11b expression (antigen detection by flow cytometry and mRNA by Luminex) and nitroblue tetrazolium (NBT) reduction. ATRA, VD3, and AZA induced CD11b RNA and protein expression and NBT reduction in HL-60 and AML-193 cell lines. Gene expression profiles (GEPs) in HL-60 and AML-193 cells revealed significant overlap in the genes regulated by AZA-treatment vs. VD3- or ATRA-treatment. GEPs of HL-60 and AML-193 cells treated with AZA strongly correlated with publicly-available gene sets representing differentiated eosinophils, neutrophils, and monocytes, but negatively correlated with those of differentiated erythrocytes and megakaryocytes. Similar studies in primary AML cells are ongoing. Our results demonstrate that AZA can induce cellular differentiation of AML cell lines along the granulocytic/monocytic lineage in vitro, and suggest that cellular differentiation may contribute as one of multiple mechanisms of AZA's anti-leukemic activity in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 191.

Abstract 287: Proteolytic regulation of PDGF-D activity by matriptase in human prostate cancer cells

Abstract Platelet-derived growth factor (PDGF) proteins are potent inducers of cell proliferation, transformation, and differentiation. PDGF-D, a newly identified PDGF ligand, has a unique two-domain structure with an NH2-terminal CUB domain and a COOH-terminal PDGF/VEGF growth factor domain (GD). Proteolytic removal of the CUB domain is required for PDGF-D to activate its cognate β-PDGF receptor (β-PDGFR). We previously showed that the human prostate cancer cells, LNCaP and PC3, are able to process the latent PDGF-D into an active growth factor. We found that urokinase plasminogen activator (uPA), a secreted serine protease, is the activator of latent PDGF-D in PC3 cells. However, in LNCaP cells, which do not express uPA, latent PDGF-D was shown to be processed pericellularly instead of by a secreted protease. By microarray analysis, matriptase, a type II transmembrane serine protease, was found to be the putative serine protease in LNCaP cells to process latent PDGF-D. In this study, to test whether latent PDGF-D is a substrate of matriptase, time- and dose-dependent experiments of incubating purified recombinant PDGF-D with purified catalytic domain of matriptase were performed. Full-length PDGF-D dimer (FL-D) was processed into growth factor domain dimer (GD-D) in a two-step manner involving generation of an intermediate hemidimer (HD) composed of one full-length monomer (FL-M) and one growth factor domain monomer (GD-M). Through mutagenesis analyses, we identified YR247GR249↓S in the hinge region as a matriptase cleavage site for the proteolytic activation of PDGF-D. Interestingly, FL PDGF-D was first processed into 18kDa GD and then was further processed into 15kDa GD, indicating there is a matriptase cleavage site within PDGF-D growth factor domain apart from the one in the hinge region. Mutagenesis analyses identified R340RGR343 ↘A within growth factor domain to be a matriptase cleavage site. Through sequence alignment of PDGF-D GD with PDGF-B, the matriptase cleavage site R340RGR343 A is implicated in the interaction of PDGF-D GD with β-PDGFR. The subsequent β-PDGFR activation study confirmed that matriptase-mediated cleavage at R340RGR343A results in deactivation of PDGF-D. Interestingly, although PDGF-D HD is a biological inactive form of PDGF-D, HD is able to be activated by pericellular serine protease. More interestingly, PDGF-D HD can function as a dominant-negative ligand of PDGF-B/β-PDGFR signaling, presumably through its binding to one subunit of β-PDGFR, thereby inhibiting PDGF-B-mediated receptor dimerization. Lastly, we report that while PDGF-D FL-D is easily detected in the conditioned medium, a preferred deposition of PDGF-D HD and GD was detected in the extracellular matrix of LNCaP-PDGF-D cells. Collectively, this study provides molecular insights into matriptase-mediated complex regulation of PDGF-D activity and extracellular localization in human prostate carcinoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 287.

Structural, cellular, and molecular evaluation of bone erosion in experimental models of rheumatoid arthritis: Assessment by μCT, histology, and serum biomarkers

Bone erosion is a clinical endpoint for various diseases including rheumatoid arthritis. In this paper, we used rodent arthritis models with severe bone erosion to examine the structural, cellular, and molecular aspects of the inflammation-driven bone resorption process. Our data show that bone loss is observed only in chronically, severely inflamed joints. The most severely affected anatomic sites were the metatarsal phalangeal joint and tarsal bones of the paw. The magnitude of the inflammation-driven bone erosion was dependent on both the duration of inflammatory response and the severity of the joint swelling response. The application of micro-computed tomography well demonstrated the therapeutic benefit of anti-IL-17A in protection of bones from erosion. Alterations in the cellular profile of the joint occurred prior to any major structural deterioration of the bone. Receptor activator for nuclear factor κB ligand, a potent inducer of osteoclast differentiation and bone resorption, was elevated in animals coincident with severe arthritis initiation. The experimental approaches and concepts outlined in this paper provide a valuable process to evaluate and quantify therapies that modulate rodent arthritis-associated bone-erosion models.

Novel role of galectin-4: A potent inducer of monocytes differentiation and activation. (47.12)

Abstract Galectin-4 (Gal-4) is one of β-galactoside-binding proteins (Galectin family) and is specifically expressed in the gastrointestinal tracts. Although intensive studies have been done on other isoforms of Galectin (Gal-1, -3, -9), the role of Gal-4 in immune system remains unknown. Here, we found that Gal-4 specifically affects the differentiation and activation of monocytes. In addition to prolonging monocytes cell survival, Gal-4 induced morphological changes of monocytes into macrophage-like cells. Notably, Gal-4 entered into the cytoplasm of monocytes after binding and increased the production of pro-inflammatory cytokine such as IL-6 and TNF-a. Gal-4 also increased the expression of co-stimulatory molecule such as CD40, 80 and CD86 in CD14+ monocytes. Furthermore, we provide the evidence that Gal-4 mediated its modulatory function through MAPK signaling cascade (p38 and c-Jun) via MyD88 by Western blot analyses. As such, Gal-4 did not increase macrophage differentiation markers in MyD88-deficient monocytes from MyD88 knockout mice. In conclusion, our study is the first to describe the specific effect of galectin-4 on monocytes and further suggests that Gal-4 expression might modulate mucosal immunity via monocytes activation and differentiation.