Tumor Necrosis Factor-α Converting Enzyme Research Articles

The evaluation of inhibitive effectiveness of the tumour necrosis factor-α converting enzyme selective inhibitors by HPLC

A novel high-performance liquid chromatography (HPLC) method based on the internal standard method was established for assaying the tumour necrosis factor-α converting enzyme (TACE) activity and matrix metalloprotease-9 (MMP-9) activity, and was used to evaluate the inhibitive effectiveness of inhibitors to TACE and MMP-9. In the assay method for TACE and MMP-9, peptides labelled with the ultraviolet group-Dpa were used as substrates. Alanine-Dpa was synthesised and was used as the internal standard for quantitative analysis. After the peptide substrates were hydrolysed by TACE (MMP-9) for 15min (25min) at 37°C, the amount of remaining substrates were determined by reversed-phased HPLC with UV detection at 353nm. The relative peak area of the substrate was linearly dependent on the substrate concentration. This method was then applied to determine the 50% inhibitory concentration (IC50) of GM6001 and inhibitor A for both TACE and MMP-9.

Inhibitory role of TACE/ADAM17 cytotail in protein ectodomain shedding

To determine if the cytotail of the principal sheddase tumor necrosis factor-α converting enzyme (TACE; ADAM17) controls protein ectodomain shedding. Site-directed mutagenesis was performed to derive TACE variants. The resulting TACE expression plasmids with amino acid substitutions in the extracellular, cysteine-rich disintegrin domain (CRD) and/or deleted cytotail, along with an expression vector for the enhanced green fluorescence protein were transfected into shedding-defective M1 mutants stably expressing transmembrane L-selectin or transforming growth factor (TGF)-α. The expression levels of the TACE substrates at the cell surface were determined by flow cytometry. Consistent with published data, a single point mutation (C600Y) in the CRD led to shedding deficiency. However, removal of the cytotail from the C600Y TACE variant partially restored ectodomain cleavage of TGF-α and L-selectin. Cytotail-deleted mutants with any other substituting amino acid residues in place of Cys600 displayed similar function compared with tail-less C600Y TACE. The cytotail plays an inhibitory role, which becomes evident when it is removed from an enzyme with another mutation that affects the enzyme function.

The role of stem cell factor and c-KIT in keloid pathogenesis: do tyrosine kinase inhibitors have a potential therapeutic role?

Keloids are fibroproliferative disorders characterized by increased deposition of extracellular matrix components. Stem cell factor (SCF) and its receptor c-KIT are expressed in a wide variety of cells and have also been demonstrated to be important modulators of the wound healing process. To examine the role of the SCF/c-KIT system in keloid pathogenesis. Immunohistochemical staining and Western blot analyses were used to examine localization and expression of SCF and c-KIT in keloid and normal skin tissue. This was followed by the detection of SCF and c-KIT expression in fibroblasts cultured in vitro and fibroblasts exposed to serum. To investigate the effect of epithelial-mesenchymal interactions, a two-chamber system was employed in which keratinocytes on membrane inserts were cocultured with the fibroblasts. SCF and c-KIT expression levels in all cell extracts and conditioned media were assayed by Western blotting. In another set of experiments, the effect of imatinib (Glivec(®), Gleevec(®); Novartis Pharma AG, Basel, Switzerland) on keloid fibroblasts was examined. SCF and c-KIT were upregulated in keloid scar tissue and in cultured fibroblasts stimulated with serum, highlighting their importance in the initial phase of wound healing. We further demonstrated that epithelial-mesenchymal interactions, mimicked by coculture of keratinocytes and fibroblasts in vitro, not only stimulated secretion of the soluble form of SCF in keloid cocultures but also brought about shedding of the extracellular domain of c-KIT perhaps by upregulation of tumour necrosis factor-α converting enzyme which was also upregulated in keloid scars in vivo and keloid cocultures in vitro. In addition keloid cocultures expressed increased levels of phosphorylated c-KIT highlighting an activation of the SCF/c-KIT system. Finally, we demonstrated that imatinib, a tyrosine kinase inhibitor, may be a possible therapeutic agent for keloids. These data indicate that the SCF/c-KIT system plays an important role in scar pathogenesis, and underscore the role of imatinib as a key therapeutic agent in keloid scars.

Dual Role of α-Secretase Cleavage in the Regulation of γ-Secretase Activity for Amyloid Production

Processing of the amyloid precursor protein (APP) by β- and γ-secretases generates pathogenic β-amyloid (Aβ) peptides associated with Alzheimer disease (AD), whereas cleavage of APP by α-secretases precludes Aβ formation. Little is known about the role of α-secretase cleavage in γ-secretase regulation. Here, we show that α-secretase-cleaved APP C-terminal product (αCTF) functions as an inhibitor of γ-secretase. We demonstrate that the substrate inhibitory domain (ASID) within αCTF, which is bisected by the α-secretase cleavage site, contributes to this negative regulation because deleting or masking this domain turns αCTF into a better substrate for γ-secretase. Moreover, α-secretase cleavage can potentiate the inhibitory effect of ASID. Inhibition of γ-secretase activity by αCTF is observed in both in vitro and cellular systems. This work reveals an unforeseen role for α-secretase in generating an endogenous γ-secretase inhibitor that down-regulates the production of Aβ. Deregulation of this feedback mechanism may contribute to the pathogenesis of AD.

E0154 Expression of tumour necrosis factor-α converting enzyme and tumour necrosis factor-α in rats with alcoholic cardiomyopathy

PurposeChronic excessive consumption of alcohol causes ventricular remodelling and eventually leads to alcoholic cardiomyopahty (ACM). Tumour necrosis factor-α converting enzyme (TACE) has been identified to cleave membrane-bound tumour necrosis factor-α...

Tumor necrosis factor α-converting enzyme (TACE/ADAM17) mediates ectodomain shedding of the scavenger receptor CD163

CD163 is expressed specifically in the monocyte/macrophage lineage, where it mediates uptake of haptoglobin-hemoglobin complexes, leading to metabolism of the oxidative heme molecule. Shedding of the CD163 ectodomain from the cell surface produces a sCD163 plasma protein, and a positive correlation is seen between the sCD163 plasma level and the severity of various infectious and inflammatory diseases. In the present analysis of the phorbol ester-induced shedding of sCD163 in CD163 cDNA-transfected HEK293 cells, we used metalloproteinase inhibitors and siRNA-mediated inhibition of metalloproteinases to identify TACE/ADAM17 as an enzyme responsible for PMA-induced cleavage of the membrane-proximal region of CD163. As TACE/ADAM17-mediated shedding of TNF-α is up-regulated in macrophages subjected to inflammatory stimuli, the present results now provide a likely explanation for the strong empirical relationship between the sCD163 plasma level and infectious/inflammatory diseases relating to macrophage activity.

Roles of ROS/TACE in neutrophil elastase-induced mucus hypersecretion in NCI-H292 airway epithelial cells.

Complications arise in chronic obstructive pulmonary diseases (COPD) with excessive mucus production, especially during the exacerbation period, which contributes to airway blockage and bacterial infection. Neutrophil elastase (NE) is detected at high levels in airway secretions, and is the primary inducer of mucin production. Understanding the mechanism of NE-induced overproduction of mucin may lead to new therapies for COPD. It is known that activation of epidermal growth factor receptor (EGFR) and its downstream signaling cascade are involved in mucin production. However, the mechanism of NE-induced EGFR activation remains unclear. Tumor necrosis factor-α-converting enzyme (TACE) cleaves pro-transforming growth factor (TGF)-α in airway epithelial cells to release the mature, soluble TGF-α form, which subsequently binds to and activates EGFR. In this investigation, we demonstrate that NE-induced mucin production requires reactive oxygen species (ROS) production, which activates TACE, resulting in TGF-α shedding, and EGFR phosphorylation in NCI-H292 epithelial cells.

Selective inhibition of ADAM12 catalytic activity through engineering of tissue inhibitor of metalloproteinase 2 (TIMP-2)

The disintegrin and metalloprotease ADAM12 has important functions in normal physiology as well as in diseases, such as cancer. Little is known about how ADAM12 confers its pro-tumorigenic effect; however, its proteolytic capacity is probably a key component. Thus selective inhibition of ADAM12 activity may be of great value therapeutically and as an investigative tool to elucidate its mechanisms of action. We have previously reported the inhibitory profile of TIMPs (tissue inhibitor of metalloproteinases) against ADAM12, demonstrating in addition to TIMP-3, a unique ADAM-inhibitory activity of TIMP-2. These findings strongly suggest that it is feasible to design a TIMP mutant selectively inhibiting ADAM12. With this purpose, we characterized the molecular determinants of the ADAM12-TIMP complex formation as compared with known molecular requirements for TIMP-mediated inhibition of ADAM17/TACE (tumour necrosis factor alpha-converting enzyme). Kinetic analysis using a fluorescent peptide substrate demonstrated that the molecular interactions of N-TIMPs (N-terminal domains of TIMPs) with ADAM12 and TACE are for the most part comparable, yet revealed strikingly unique features of TIMP-mediated ADAM12 inhibition. Intriguingly, we found that removal of the AB-loop in N-TIMP-2, which is known to impair its interaction with TACE, resulted in increased affinity to ADAM12. Importantly, using a cell-based epidermal growth factor-shedding assay, we demonstrated for the first time an inhibitory activity of TIMPs against the transmembrane ADAM12-L (full-length ADAM12), verifying the distinctive inhibitory abilities of N-TIMP-2 and engineered N-TIMP-2 mutants in a cellular environment. Taken together, our findings support the idea that a distinctive ADAM12 inhibitor with future therapeutic potential can be designed.

Strategy for generation of new TACE inhibitors: pharmacophore and counter pharmacophore modeling to remove non-selective hits

Present study describes the ligand-based molecular modeling along with virtual screening (VS) approach for generation of new tumor necrosis factor-α converting enzyme (TACE) inhibitors. In ligand-based molecular modeling, two statistically reliable HipHop pharmacophore models Hip1 and counter pharmacophore (CP1) were generated using training set of 3 and 2 molecules, respectively. CP1 was generated using inhibitors of MMP-1 (matrix metalloproteinase-1), an important enzyme involved in musculoskeletal degradation. VS was performed with model Hip1 in in-house database of 1.2 million molecules. In addition, the retrieved molecules were screened with CP1. The combination of both models helped for generating new improved TACE inhibitor molecules.

A Presenilin-1 Mutation Identified in Familial Alzheimer Disease with Cotton Wool Plaques Causes a Nearly Complete Loss of γ-Secretase Activity

Mutations in presenilin-1 and presenilin-2 (PS1 and PS2) are the most common cause of familial Alzheimer disease. PS1 and PS2 are the presumptive catalytic components of the multisubunit gamma-secretase complex, which proteolyzes a number of type I transmembrane proteins, including the amyloid precursor protein (APP) and Notch. APP processing by gamma-secretase produces beta-amyloid peptides (Abeta40 and Abeta42) that accumulate in the Alzheimer disease brain. Here we identify a pathogenic L435F mutation in PS1 in two affected siblings with early-onset familial Alzheimer disease characterized by deposition of cerebral cotton wool plaques. The L435F mutation resides in a conserved C-terminal PAL sequence implicated in active site conformation and catalytic activity. The impact of PS1 mutations in and around the PAL motif on gamma-secretase activity was assessed by expression of mutant PS1 in mouse embryo fibroblasts lacking endogenous PS1 and PS2. Surprisingly, the L435F mutation caused a nearly complete loss of gamma-secretase activity, including >90% reductions in the generation of Abeta40, Abeta42, and the APP and Notch intracellular domains. Two nonpathogenic PS1 mutations, P433L and L435R, caused essentially complete loss of gamma-secretase activity, whereas two previously identified pathogenic PS1 mutations, P436Q and P436S, caused partial loss of function with substantial reductions in production of Abeta40, Abeta42, and the APP and Notch intracellular domains. These results argue against overproduction of Abeta42 as an essential property of presenilin proteins bearing pathogenic mutations. Rather, our findings provide support for the hypothesis that pathogenic mutations cause a general loss of presenilin function.

Three Dimensional Molecular Field Analysis of Thiomorpholine Analogs of TACE Inhibitors Using Receptor Based Alignment

Thiomorpholine analogs (TML) have been identified as novel class of potent tumor necrosis factor-α converting enzyme (TACE) inhibitors. A computational strategy based on molecular docking studies, followed by MFA analysis has been performed to elucidate the atomic details of the TACE/TML interactions and to identify the most important features impacting TACE inhibitory activity of TMLs. The generated MFA model resulted to be well predictive, and gave r2 test 0.723, conventional r2 0.982 and r2 cv 0.811. The 3D-QSAR field contributions and the structural features of the TACE binding site showed a good correlation. These studies will be useful to design new TACE inhibitors with improved potency. Keywords: TACE inhibitors, Rheumatoid arthritis, 3D-QSAR, Molecular Field Analysis (MFA), Docking

Anthranilate derivatives as TACE inhibitors: Docking based CoMFA and CoMSIA analyses

Anthranilic acid based derivatives (ANTs) have been identified as a novel class of potent tumor necrosis factor-α converting enzyme (TACE) inhibitors. A computational strategy based on molecular docking studies, followed by CoMFA and CoMSIA analyses has been performed to elucidate the atomic details of the TACE/ANT interactions and also to identify the most important features impacting TACE inhibitory activity of ANTs. The CoMSIA model resulted to be slightly more predictive than CoMFA model, and gave conventional r² 0.991, r²(cv) 0.793, q² 0.777, SEE 0.050, F-value 655.610, and r²(test) 0.871. The 3D-QSAR field contributions and the structural features of the TACE binding site showed a good correlation. These studies will be useful to design new TACE inhibitors with improved potency.

ADAM-17 over-expression in gallbladder carcinoma correlates with poor prognosis of patients

The ADAMs is a multi-functional gene family of membrane proteins possessing a disintegrin and metalloprotease domain. They have potential implications for the metastasis of human tumor cells via cell adhesion and protease activities. However, no studies have yet comprehensively examined the expression of ADAMs in gallbladder carcinoma. The aim of this study was to test the hypothesis that ADAM-17 (otherwise known as tumor necrosis factor-α converting enzyme) is involved in the progression of gallbladder carcinoma. Two hundreds samples of gallbladder carcinoma and sixty non-cancerous gallbladder samples were used to measure the expression of total ADAM-17 by enzyme-linked immunosorbent assay, and the precursor and active forms by western blotting analysis. Expression of ADAM-17 was significantly increased in tumors with high histological grade and pT stage compared with low histological grade and pT stage tumors and was not associated with patients' gender, age, histological type, and resection margin involvement. Patients with high expression of ADAM-17 had a significantly shorter overall survival compared with those with low expression. Significantly, the prognostic impact of ADAM-17 was independent of conventional prognostic factors for gallbladder carcinoma. The current study demonstrated that the over-expression of ADAM-17 in patients with gallbladder carcinoma was linked closely with histological grade, pT stage and prognosis, and thus provides further impetus for exploiting ADAM-17 as new target for the treatment of gallbladder carcinoma.

P38 mitogen-activated protein kinase activation during platelet storage: consequences for platelet recovery and hemostatic function in vivo

AbstractPlatelets undergo several modifications during storage that reduce their posttransfusion survival and functionality. One important feature of these changes, which are known as platelet storage lesion, is the shedding of the surface glycoproteins GPIb-α and GPV. We recently demonstrated that tumor necrosis factor-α converting enzyme (TACE/ADAM17) mediates mitochondrial injury-induced shedding of adhesion receptors and that TACE activity correlates with reduced posttransfusion survival of these cells. We now confirm that TACE mediates receptor shedding and clearance of platelets stored for 16 hours at 37°C or 22°C. We further demonstrate that both storage and mitochondrial injury lead to the phosphorylation of p38 mitogen-activated kinase (MAPK) in platelets and that TACE-mediated receptor shedding from mouse and human platelets requires p38 MAP kinase signaling. Protein kinase C, extracellular regulated-signal kinase MAPK, and caspases were not involved in TACE activation. Both inhibition of p38 MAPK and inactivation of TACE during platelet storage led to a markedly improved posttransfusion recovery and hemostatic function of platelets in mice. p38 MAPK inhibitors had only minor effects on the aggregation of fresh platelets under static or flow conditions in vitro. In summary, our data suggest that inhibition of p38 MAPK or TACE during storage may significantly improve the quality of stored platelets.

Anti-Inflammatory Bioactivities of Honokiol through Inhibition of Protein Kinase C, Mitogen-Activated Protein Kinase, and the NF-κB Pathway To Reduce LPS-Induced TNFα and NO Expression

Much recent research has demonstrated that honokiol, a phenolic compound originally isolated from Magnolia officinalis, has potent anticancer activities; however, the detailed molecular mechanism of its anti-inflammatory activity has not yet been fully addressed. In this study we demonstrated that honokiol inhibited lipopolysaccharide (LPS)-induced tumor necrosis factor-alpha secretion in macrophages, without affecting the activity of the tumor necrosis factor-alpha converting enzyme. At the same time, honokiol not only inhibited nitric oxide expression in LPS-stimulated murine macrophages but also inhibited the LPS-induced phosphorylation of ERK1/2, JNK1/2, and p38. By means of confocal microscope analysis we demonstrated that phosphorylation and membrane translocation of protein kinase C-alpha, as well as NF-kappaB activation, were inhibited by honokiol in LPS-stimulated macrophages. Furthermore, it was found that honokiol neither antagonizes the binding of LPS to cells nor alters the cell surface expression of toll-like receptor 4 and CD14. Our current results have exhaustively described the anti-inflammatory properties of honokiol, which could lead to the possibility of its future pharmaceutical application in the realm of immunomodulation.

Essential Roles of IL-6 Trans-Signaling in Colonic Epithelial Cells, Induced by the IL-6/Soluble–IL-6 Receptor Derived from Lamina Propria Macrophages, on the Development of Colitis-Associated Premalignant Cancer in a Murine Model

Activation of the IL-6/Stat3 via IL-6 trans-signaling plays an important role in the pathogenesis of inflammatory bowel disease. Colitis-associated cancer (CAC) is a large bowel cancer and occurs with long-standing inflammatory bowel disease. The role of the IL-6/Stat3 in the development of CAC has not been fully understood. We investigate whether IL-6 trans-signaling contributes to the development of CAC using a mouse colitis-associated premalignant cancer (CApC) model. Chronic colitis (CC) was induced in BALB/c mice using dextran sodium sulfate. CApC was induced by dextran sodium sulfate treatment to CC-affected mice. IL-6 expression was determined by quantitative RT-PCR and immunofluorescence staining in colon. Phospho-Stat3 expression was examined by Western blotting and immunofluorescence analysis. The expression of IL-6 receptors (i.e., the IL-6R alpha-chain and gp130) and tumor necrosis factor-alpha converting enzyme in the colon was examined by laser-capture microdissection and immunofluorescence staining. Soluble IL-6R alpha (sIL-6R alpha) was examined by Western blotting of epithelial cell-depleted colonic tissues. We also investigated whether a soluble gp130-Fc fusion protein could prevent CApC. IL-6 expression was increased in the colon of CC- and CApC-affected mice and was restricted to lamina propria-macrophages. The expression of IL-6R alpha and tumor necrosis factor-alpha converting enzyme was increased in the lamina propria CD11b-macrophages of CC-affected mice. sIL-6R alpha expression was also increased in these tissues. Reduced levels of IL-6R alpha generation were observed in the colonic epithelial cells of CC- and CApC-affected mice and were associated with the increased expression of gp130 and phospho-Stat3. Treatment with soluble gp130Fc significantly reduced the CApC. IL-6 trans-signaling in epithelial cells induced by macrophage-derived IL-6/sIL-6R alpha plays a crucial role in the development of CAC.

Analysis of Gene Expression Profiles in Fatal Hepatic Failure After Hepatectomy in Mice

We developed 90%-hepatectomized mice that were the fatal model, and analyzed the gene expression profiles using a complementary DNA (cDNA) microarray to clarify the mechanisms of hepatic failure after excessive hepatectomy. Ribonucleic acid (RNA)s from the remnant hepatic tissue of 70%- and 90%-hepatectomized mice were labeled with fluorescent dyes, and hybridized to the Riken set of 39,168 full-length enriched mouse cDNA arrays. The gene expression profiles in 90%- and 70%-hepatectomized mice were analyzed by scanning date for fluorescent dye signals. The down-regulated genes in 90%-hepatectomized mice were genes activating extracellular matrix (ECM) remodeling (matrix metalloproteinases, laminins, and integrins), genes related to cytokines (tumor necrosis factor α converting enzyme, and Janus kinase 3) that were related to the priming, genes related to growth factor (heparin-binding epidermal growth factor-like growth factor and others), and genes promoting cell cycle progression (cyclin D1, D2, and E2) that were related to the progression of hepatocytes. The up-regulated genes were genes inhibiting ECM remodeling [plasminogen activator inhibitors (PAIs)]. Hepatic failure after hepatectomy was characterized by the inhibition of hepatic cell cycle priming and progression both induced by ECM remodeling in liver regeneration. Particularly, the overexpression of PAIs was thought to play the major role in the first step of inhibition of ECM remodeling.

Molecular modeling and biological effects of peptidomimetic inhibitors of TACE activity

We investigated the molecular basis of specificity for the interaction between tumor necrosis factor-α converting enzyme (TACE) and peptidomimetic inhibitors. Four novel peptidomimetic TACE inhibitors (8a–d) were designed and synthesized by introducing a substituted sulfur group and a hydrophobic group to a novel matrix metalloprotease (MMP) inhibitor. Inhibition was determined by in vitro lipopolysaccharide (LPS) cytotoxicity tests in HL-60 cell lines and by measuring the expression of mTNF-α using FCM techniques and immunohistochemistry in vivo. We simulated the interaction of the inhibitors with the 3D structure of the TACE active site in the Brookhaven Protein Database (PDB). The four inhibitors (8a–d) inhibited activity by 9.1%, 54.5%, 27.3%, and 54.5%, respectively. 8b and 8d showed significant in vitro inhibition in cytotoxicity tests, which corresponded to the molecular docking results. 8d also showed inhibitory activity in vivo. We explored the interface between enzyme and substrate by combining bioinformatics with experimental observations to further the development of specific TACE inhibitors to reduce inflammatory responses.

Generation of Selective TACE Inhibitors: Ligand and Structure Based Molecular Modeling, Virtual Screening, Counter Pharmacophore Screening to Get Selective Molecules

AbstractThis study describes the ligand based as well as structure based molecular modeling and virtual screening of selective tumor necrosis factor‐α converting enzyme (TACE) inhibitors. In ligand based molecular modeling, two statistically reliable pharmacophore models HypoA1 and HypoB1 were generated using a same training set of 22 molecules. HypoA1 consists of two hydrogen bond acceptor and three hydrophobic groups whereas HypoB1 consists of one hydrogen bond donor, one ring aromatic and three hydrophobic groups. Virtual screening was performed with both models in in‐house database of 1.2 million molecules. To remove non selective hits from screened molecules, a counter pharmacophore was generated using inhibitors of MMP‐1, an important enzyme involved in musculoskeletal degradation. In structure based molecular modeling, docking analysis was performed to explore the important interactions between ligands and protein. On comparison, HypoA1 and HypoB1 were found to be complementing with results of docking analysis suggesting high reliability of both models for their use in virtual screening/designing of new molecules.

GM-CSF and IL-4 induce dendritic cell differentiation and disrupt osteoclastogenesis through M-CSF receptor shedding by up-regulation of TNF-α converting enzyme (TACE)

AbstractMonocytes give rise to macrophages, osteoclasts (OCs), and dendritic cells (DCs). Macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-kappaB (RANK) ligand induce OC differentiation from monocytes, whereas granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) trigger monocytic differentiation into DCs. However, regulatory mechanisms for the polarization of monocytic differentiation are still unclear. The present study was undertaken to clarify the mechanism of triggering the deflection of OC and DC differentiation from monocytes. GM-CSF and IL-4 abolished monocytic differentiation into OCs while inducing DC differentiation even in the presence of M-CSF and RANK ligand. GM-CSF and IL-4 in combination potently up-regulate tumor necrosis factor-α (TNF-α) converting enzyme (TACE) and activity in monocytes, causing ectodomain shedding of M-CSF receptor, resulting in the disruption of its phosphorylation by M-CSF as well as the induction of osteoclastogenesis from monocytes by M-CSF and RANK ligand. Interestingly, TACE inhibition robustly causes the resumption of the surface expression of M-CSF receptor on monocytes, facilitating M-CSF–mediated phosphorylation of M-CSF receptor and macrophage/OC differentiation while impairing GM-CSF– and IL-4–mediated DC differentiation from monocytes. These results reveal a novel proteolytic regulation of M-CSF receptor expression in monocytes to control M-CSF signaling and monocytic differentiation into macrophage/OC-lineage cells or DCs.