CSF-1 is broadly expressed and regulates macrophage and osteoclast development. The action and expression of IL-34, a novel CSF-1R ligand, were investigated in the mouse. As expected, huIL-34 stimulated macrophage proliferation via the huCSF-1R, equivalently to huCSF-1, but was much less active at stimulating mouse macrophage proliferation than huCSF-1. Like muCSF-1, muIL-34 and a muIL-34 isoform lacking Q81 stimulated mouse macrophage proliferation, CSF-1R tyrosine phosphorylation, and signaling and synergized with other cytokines to generate macrophages and osteoclasts from cultured progenitors. However, they respectively possessed twofold and fivefold lower affinities for the CSF-1R and correspondingly, lower activities than muCSF-1. Furthermore, muIL-34, when transgenically expressed in a CSF-1-dependent manner in vivo, rescued the bone, osteoclast, tissue macrophage, and fertility defects of Csf1(op)/(op) mice, suggesting similar regulation of CSF-1R-expressing cells by IL-34 and CSF-1. Whole-mount IL34 in situ hybridization and CSF-1 reporter expression revealed that IL34 mRNA was strongly expressed in the embryonic brain at E11.5, prior to the expression of Csf1 mRNA. QRT-PCR revealed that compared with Csf1 mRNA, IL34 mRNA levels were lower in pregnant uterus and in cultured osteoblasts, higher in most regions of the brain and heart, and not compensatorily increased in Csf1(op/op) mouse tissues. Thus, the different spatiotemporal expression of IL-34 and CSF-1 allows for complementary activation of the CSF-1R in developing and adult tissues.

CSF-1 is the major regulator of tissue macrophage development and function. A GM-CSF-dependent, CSF-1 receptor (CSF-1R)-deficient F4/80(hi)Mac-1(+)Gr1(-)CD11c(+) bone marrow macrophage (BMM) line (MacCsf1r-/-) was developed to study the roles of the eight intracellular CSF-1R tyrosines phosphorylated upon receptor activation. Retroviral expression of the wild-type CSF-1R rescued the CSF-1-induced survival, proliferation, differentiation, and morphological characteristics of primary BMM. Mutation of all eight tyrosines failed to rescue, whereas the individual Y --> F mutants (544, 559, 697, 706, 721, 807, 921, 974) rescued these CSF-1-inducible phenotypes to varying degrees. The juxtamembrane domain Y559F and activation loop Y807F mutations severely compromised proliferation and differentiation, whereas Y706, Y721F, and Y974F mutations altered morphological responses, and Y706F increased differentiation. Despite their retention of significant in vitro tyrosine kinase activity, Y559F and Y807F mutants exhibited severely impaired in vivo receptor tyrosine phosphorylation, consistent with the existence of cellular mechanisms inhibiting CSF-1R tyrosine phosphorylation that are relieved by phosphorylation of these two sites. The MacCsf1r-/- macrophage line will facilitate genetic and proteomic approaches to CSF-1R structure/function studies in the major disease-related CSF-1R-expressing cell type.

SummaryColony stimulating factor-1 (CSF-1) regulates macrophage morphology and motility, as well as mononuclear phagocytic cell proliferation and differentiation. The CSF-1 receptor (CSF-1R) transduces these pleiotropic signals through autophosphorylation of eight intracellular tyrosine residues. We have used a novel bone-marrow-derived macrophage cell line system to examine specific signaling pathways activated by tyrosine-phosphorylated CSF-1R in macrophages. Screening of macrophages expressing a single species of CSF-1R with individual tyrosine-to-phenylalanine residue mutations revealed striking morphological alterations upon mutation of Y721. M-/-.Y721F cells were apolar and ruffled poorly in response to CSF-1. Y721-P-mediated CSF-1R signaling regulated adhesion and actin polymerization to control macrophage spreading and motility. Moreover, the reduced motility of M-/-.Y721F macrophages was associated with their reduced capacity to enhance carcinoma cell invasion. Y721 phosphorylation mediated the direct association of the p85 subunit of phosphoinositide 3-kinase (PI3K) with the CSF-1R, but not that of phospholipase C (PLC) 2, and induced polarized PtdIns(3,4,5)P 3 production at the putative leading edge, implicating PI3K as a major regulator of CSF-1-induced macrophage motility. The Y721-P-motif-based motility signaling was at least partially independent of both Akt and increased Rac and Cdc42 activation but mediated the rapid and transient association of an unidentified ~170 kDa phosphorylated protein with either Rac-GTP or Cdc42-GTP. These studies identify CSF-1R-Y721-P-PI3K signaling as a major pathway in CSF-1-regulated macrophage motility and provide a starting point for the discovery of the immediate downstream signaling events.

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Receptor tyrosine kinase (RTK) activation involves ligandinduced receptor dimerization and transphosphorylation on tyrosine residues. Colony-stimulating factor-1 (CSF-1)-induced CSF-1 receptor (CSF-1R) tyrosine phosphorylation and ubiquitination were studied in mouse macrophages. Phosphorylation of CSF-1R Tyr-559, required for the binding of Src family kinases (SFKs), was both necessary and sufficient for these responses and for c-Cbl tyrosine phosphorylation and all three responses were inhibited by SFK inhibitors. In c-Cbldeficient macrophages, CSF-1R ubiquitination and tyrosine phosphorylation were substantially inhibited. Reconstitution with wild-type, but not ubiquitin ligase-defective C381A c-Cbl rescued these responses, while expression of C381A c-Cbl in wild-type macrophages suppressed them. Analysis of site-directed mutations in the CSF-1R further suggests that activated c-Cbl-mediated CSF-1R ubiquitination is required for a conformational change in the major kinase domain that allows amplification of receptor tyrosine phosphorylation and full receptor activation. Thus the results indicate that CSF-1-mediated receptor dimerization leads to a Tyr-559/SFK/c-Cbl pathway resulting in receptor ubiquitination that permits full receptor tyrosine phosphorylation of this class III RTK in macrophages.

This unit provides protocols for measuring the abundance and growth of macrophage precursors in agar cultures and the proliferation of isolated mature macrophages in vitro, by either direct cell counting or by DNA measurement. Methods for the immunohistochemical identification of macrophages and the determination of their proliferative status in vivo by immunofluorescence are also included. It also describes methods for characterization of macrophage differentiation through the immunofluorescence analysis of cell surface expression of CSF-1 receptor.

Background: The therapeutic efficacy of cisplatin-based chemotherapy for non-small cell lung cancer (NSCLC) is limited by drug resistance. In NSCLC, hyperactivation of nuclear factor erythroid 2-related factor 2 (Nrf2) counteracts oxidative stress to promote chemoresistance. Metformin-mediated downregulation of Nrf2 plays a pivotal role in overcoming drug resistance in NSCLC cells. Therefore, a deeper understanding of the molecular mechanisms of combination therapy and the role of Nrf2 in chemotherapeutic response is critical to clinical translation. Methods:The effects of combination therapy with metformin and cisplatin on cell proliferation and apoptosis, intracellular reactive oxygen species (ROS) levels, and xenograft tumor formation were analyzed in NSCLC cells. Co-immunoprecipitation (co-IP) and Phos-tag assays were used to explore the mechanism of metformin-mediated Nrf2 suppression. Immunohistochemical (IHC) staining was performed to detect Nrf2 expression in matched tumor samples before and after neoadjuvant chemotherapy.Results: Metformin was observed to synergistically augment cisplatin-induced cytotoxicity by strongly inhibiting the level of Nrf2, thereby weakening the antioxidant system and detoxification ability of Nrf2 and enhancing ROS-mediated apoptosis in NSCLC. The synergistic antitumor effect of combination therapy is blocked by treatment with the ROS scavenger N-acetyl cysteine (NAC) as well as overexpression of Nrf2 and its downstream antioxidant protein. Mechanistically, metformin extensively dephosphorylates Nrf2 by attenuating the interaction between Nrf2 and extracellular signal-regulated kinases 1/2 (ERK1/2), which then restores its polyubiquitination and accelerates its proteasomal degradation. Moreover, for the first time, an association of non-decreased Nrf2 expression in patients after neoadjuvant chemotherapy with poor survival and chemoresistance in NSCLC was revealed.Conclusions: Our findings illustrate the mechanism of metformin-mediated Nrf2 degradation through posttranslational modifications (PTMs), which weakens the ROS defense system in NSCLC. Fluctuations in Nrf2 expression have a strong predictive ability for chemotherapeutic response in neoadjuvant NSCLC patients. Targeting of the Nrf2 pathway could be a therapeutic strategy for overcoming chemoresistance, with metformin as the first choice for this strategy.

Background Junctional adhesion molecule-like protein (JAML) is a member of the junctional adhesion molecule family and mediates migration of immune cells, but its function in cancers remains unclear. This study aimed to evaluate the role of JAML in the prognosis and immune infiltrates of lung adenocarcinoma (LUAD). Material/Methods JAML expressions in LUAD tissues and normal tissues were compared using The Cancer Genome Atlas (TCGA) database and datasets from the Gene Expression Omnibus (GEO) database. The influence of JAML expression on prognosis was analyzed by Kaplan-Meier curve and Cox regression model. Interactive and functional analyses of JAML were performed by LinkedOmics and GeneMANIA databases. TIMER2.0, TISIDB, and GEPIA2 databases were used to investigate the correlation between JAML expression and immune infiltrates. Results JAML expression was decreased in LUAD ( P <0.001), and lower JAML expression was associated with worse outcomes of LUAD patients. High JAML expression was the protective factor for overall survival (OS) (HR 0.706, 95% CI 0.500–0.997, P =0.048). Interactive and functional analyses suggested that co-expressed genes with JAML have an obvious link to immune-related pathways. In addition, JAML expression was positively associated with infiltrating levels of CD8+ T cells, CD4+ T cells, B cells, dendritic cells, macrophages, and neutrophils, and had significant correlations with diverse immune marker sets in LUAD. Conclusions JAML expression was significantly correlated with prognosis and immune infiltrates. These preliminary findings suggested JAML could be considered as a potential prognostic biomarker and therapeutic target for LUAD.