MerTK, a receptor tyrosine kinase (RTK) of the TYRO3/AXL/MerTK family, is expressed in myeloid lineage cells in which it acts to suppress proinflammatory cytokines following ingestion of apoptotic material. Using syngeneic mouse models of breast cancer, melanoma, and colon cancer, we found that tumors grew slowly and were poorly metastatic in MerTK -/-mice. Transplantation of MerTK -/-bone marrow, but not wild-type bone marrow, into lethally irradiated MMTV-PyVmT mice (a model of metastatic breast cancer) decreased tumor growth and altered cytokine production by tumor CD11b + cells. Although MerTK expression was not required for tumor infiltration by leukocytes, MerTK -/-leukocytes exhibited lower tumor cell-induced expression of wound healing cytokines, e.g., IL-10 and growth arrest-specific 6 (GAS6), and enhanced expression of acute inflammatory cytokines, e.g., IL-12 and IL-6. Intratumoral CD8 + T lymphocyte numbers were higher and lymphocyte proliferation was increased in tumor-bearing MerTK -/-mice compared with tumor-bearing wild-type mice. Antibody-mediated CD8 + T lymphocyte depletion restored tumor growth in MerTK -/-mice. These data demonstrate that MerTK signaling in tumor-associated CD11b + leukocytes promotes tumor growth by dampening acute inflammatory cytokines while inducing wound healing cytokines. These results suggest that inhibition of MerTK in the tumor microenvironment may have clinical benefit, stimulating antitumor immune responses or enhancing immunotherapeutic strategies.

To identify potential cell surface receptors for chicken cytotactin (CT), we have characterized the ability of recombinant fusion proteins spanning the proximal fibronectin (FN) type Ill repeats of the molecule to support attachment of glioma and carcinoma cell lines. The third FN type HI repeat, which contains the RGD tripeptide, supported cell attachment and cell spreading; however, mutation of RGD to RAD did not result in significant loss of either activity. In addition, the same repeat of mouse CT, which contains a natural mutant, RVD, also supported cell attachment and spreading, although at a lower level; both activities were increased by mutation of the RVD sequence to RGD. (10,17,18). The ability of CT to inhibit cell attachment, spreading, and migration has been termed counteradhesion (19), or antiadhesion (5). This property is not unique to CT and is shared with at least three other extracellular matrix proteins, SPARC (20,21), thrombospondin (22,23), and laminin (24). A cell attachment site in chicken CT has been mapped to the region between the second and sixth (II-VI) FN type III repeats using proteolytic fragments (16) and also by analysis of recombinant fusion proteins (19). The third FN type III repeat contains an RGD sequence that has been identified as The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.an adhesion sequence in a large number of extracellular matrix molecules (for review, ref. 25) and that binds mainly to members of the integrin family (for review, ref. 26). In CT the RGD tripeptide is not conserved among different species. It is present in the chicken (1) and human (3) homologs but is absent in the mouse, newt, and pig, where it is replaced by RVD (4), RGL (27), and RAD (28), respectively. The role of RGD in cell binding to CT has not been definitively established (14, 16, 29), although preliminary reports have suggested that CT and CT peptides can bind to members of the integrin family (30,31). We undertook the present study to clarify the role of the RGD tripeptide and integrins in cell attachment to CT. The results indicate that the third FN type III repeat, which contains the RGD sequence, functions as a cell attachment site and that multiple integrins can mediate the binding of different cells to CT.

Tyro-3, Axl, and Mer are three related receptor protein-tyrosine kinases (RPTKs) characterized by an extracellular domain exhibiting significant amino acid sequence similarity to neural cell adhesion molecules. The molecule Gas6 (for growth arrest-specific gene-6) has been shown to activate each of these receptors. Gas6 is expressed extensively in the central nervous system (CNS), suggesting that interactions between Gas6 and its receptors are likely to have physiologically relevant functions. To identify and localize the relevant Gas6/RPTK pairs, we have characterized the developmental expression of Tyro-3, Axl, and Mer in rat CNS using blotting and mRNA in situ hybridization analyses. Throughout development, Tyro-3 was the most widely expressed of the three receptors in the CNS, with Axl and Mer detected in only a limited number of sites in the adult. Tyro-3 expression was low in the embryo and increased markedly during early postnatal stages, with a time course paralleling that of synaptogenesis. Axl and Mer were expressed at low but relatively constant levels throughout development. In the cerebellum, all three receptors were found in Purkinje cells, and Tyro-3 was also detected in both granule neurons and Bergmann glia. Insofar as Gas6 has been previously shown to also be expressed by Purkinje cells, it may be engaged in both autocrine and paracrine signaling. The three receptors were also detected in cerebellar white matter, primarily during myelination. In the cortex, Tyro-3 was expressed at high levels during postnatal development and in the adult. Beginning at P6 in the hippocampus, Tyro-3 was expressed at high levels in CA1 pyramidal neurons and at lower levels in CA3 and was not detected in dentate granule neurons. Axl and Mer were found in the molecular layer of the dentate gyrus and were absent from the pyramidal and dentate granule neurons. In that Gas6 is expressed throughout the pyramidal cell layer, it may activate these cells in both an autocrine and a paracrine manner. These studies provide initial clues for elucidating the cellular functions of the Axl subfamily members and suggest potential complex Gas6/RPTK as well as RPTK/RPTK signaling interactions in the mature and developing CNS.

Abstract. The extracellular matrix molecule cytotactin is a multidomain protein that plays a role in cell migration, proliferation, and differentiation during development. To analyze the structure-function relationships of the different domains of this glycoprotein, we have prepared a series of fusion constructs in bacterial expression vectors. Results obtained using a number of adhesion assays suggest that at least four independent cell binding regions are distributed among the various cytotactin domains. Two of these are adhesive; two others appear to be counteradhesive in that they inhibit cell attachment to otherwise favorable substrates. The adhesive regions were mapped to the fibronectin type III repeats II-VI and the fibrinogen domain. The morphology of the cells plated onto these adhesive fragments differed; the cells spread on the fibronectin type III repeats as they do on fibronectin, but remained round on the fibrinogen domain. The counteradhesive properties of the molecule were mapped to the EGFlike repeats and the last two fibronectin type III repeats, VII-VIII. The latter region also contained a cell attachment activity that was observed only after proteolysis of the cells. Several cell types were used in these analyses, including fibroblasts, neurons, and glia, all of which are known to bind to cytotactin. The different domains exert their effects in a concentration-dependent manner and can be inhibited by an excess of the soluble molecule, consistent with the hypothesis that the observed properties are mediated by specific receptors. Moreover, it appears that some of these receptors are restricted to particular cell types. For example, glial cells bound better than neurons to the fibrinogen domain and fibroblasts bound better than glia and neurons to the EGF fragment. These results provide a basis for understanding the multiple activities of cytotactin and a framework for isolating different receptors that mediate the various cellular responses to this molecule.C YTOTACTIN is an extracellular matrix glycoprotein that has important functions during morphogenesis, histogenesis, and tumor formation. The molecule exhibits a site-restricted distribution during development (15, 59), which may be controlled by other developmentally important genes. The cytotactin promoter region reveals a rich array of regulatory motifs with homology to the DNA binding sequences for homeotic proteins and growth factors (39) and has recently been shown to be an in vitro target of homeodomain-containing proteins (40). The restricted spatiotemporal expression of cytotactin that results from its developmental regulation is tightly linked to a number of cellular primary processes, including adhesion (29), migration (13, 32, 69), proliferation (12, 14), differentiation (50), epithelialmesenchymal interactions (3, 4), and cell death (74).Cytotactin, which is also known as tenascin (11) mal extracellular matrix protein (6), and myotendinous antigen (8), is composed of polypeptides of 190, 200, and 220 kD when isolated from chi...

Abstract. Cytotactin, an extracellular glycoprotein found in neural and nonneural tissues, influences a variety of cellular phenomena, particularly cell adhesion and cell migration. Northern and Western blot analysis and in situ hybridization were used to determine localization of alternatively spliced forms of cytotactin in neural and nonneural tissues using a probe (CT) that detected all forms of cytotactin mRNA, and one (VbVc) that detected two of the differentially spliced repeats homologous to the type III repeats of fibronectin.In the brain, the levels of mRNA and protein increased from E8 through El5 and then gradually decreased until they were barely detectable by P3. Among the three cytotactin mRNAs (7.2, 6.6, and 6.4 kb) detected in the brain, the VbVc probe hybridized only to the 7.2-kb message. In isolated cerebella, the 220-kD polypeptide and 7.2-kb mRNA were the only cytotactin species present at hatching, indicating that the 220-kD polypeptide is encoded by the 7.2-kb message that contains the VbVc alternatively spliced insert. In situ hybridization showed cytotactin mRNA in glia and glial precursors in the ventricular zone throughout the central nervous system. In all regions of the nervous system, cytotactin mRNAs were more transient and more localized than the polypeptides. For example, in the radial glia, cytotactin mRNA was observed in the soma whereas the protein was present externally along the glial fibers. In the telencephalon, cytotactin mRNAs were found in a narrow band at the edge of a larger region in which the protein was widespread. Hybridization with the VbVc probe generally overlapped that of the CT probe in the spinal cord and cerebellum, consistent with the results of Northern blot analysis. In contrast, in the outermost tectal layers, differential hybridization was observed with the two probes.In nonneural tissues, hybridization with the CT probe, but not the VbVc probe, was detected in chondroblasts, tendinous tissues, and certain mesenchymal cells in the lung. In contrast, hybridization with both probes was observed in smooth muscle and lung epithelium. Both epithelium and mesenchyme expressed cytotactin mRNA in varying combinations: in the choroid plexus, only epithelial cells expressed cytotactin mRNA; in kidney, only mesenchymal cells; and in the lung, both of these cell types contained cytotactin mRNA.These spatiotemporal changes during development suggest that the synthesis of the various alternatively spliced cytotactin mRNAs is responsive to tissuespecific local signals and prompt a search for functional differences in the various molecular forms of the protein.

The TAM family of receptor protein tyrosine kinases comprises three known members, namely Tyro3, Axl, and Mer. These receptors are widely expressed in the nervous system, including by oligodendrocytes, the cell type responsible for myelinating the CNS. We examined the potential role of the TAM family and of their principle cognate ligand, Gas6 (growth arrest gene 6), in modulating the phenotype of the cuprizone model of demyelination. We found that the expression profiles of Axl, Mer, and Gas6 mRNA were increased in the corpus callosum in a temporal profile correlating with the increased migration and proliferation of microglia/macrophages in this model. In contrast, expression of Tyro3 decreased, correlating with the loss of oligodendrocytes. Gas6 both promoted in vitro survival of oligodendrocytes (39.3 Ϯ 3.1 vs 11.8 Ϯ 2.4%) and modulated markers of activation in purified cultures of microglia (tumor necrosis factor ␣ mRNA expression was reduced ϳ48%). In Gas6 Ϫ/Ϫ mice subjected to cuprizone-challenge, demyelination was greater than in control mice, within the rostral region of the corpus callosum, as assessed by luxol fast blue staining (myelination reduced by 36%) and by ultrastructural analysis. An increased loss of Gst-(glutathione S-transferase-)-positive oligodendrocytes was also identified throughout the corpus callosum of Gas6 Ϫ/Ϫ mice. Microglial marker expression (ionized calcium-binding adapter molecule 1) was increased in Gas6 Ϫ/Ϫ mice but was restricted to the rostral corpus callosum. Therefore, TAM receptor activation and regulation can independently influence both oligodendrocyte survival and the microglial response after CNS damage.

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N-arachidonoyl glycine is an endogenous arachidonoyl amide that activates the orphan G protein-coupled receptor (GPCR) GPR18 in a pertussis toxin (PTX)-sensitive manner and produces antinociceptive and antiinflammatory effects. It is produced by direct conjugation of arachidonic acid to glycine and by oxidative metabolism of the endocannabinoid anandamide. Based on the presence of enzymes that conjugate fatty acids with glycine and the high abundance of palmitic acid in the brain, we hypothesized the endogenous formation of the saturated Nacyl amide N-palmitoyl glycine (PalGly). PalGly was partially purified from rat lipid extracts and identified using nanohigh-performance liquid chromatography/hybrid quadrupole time-of-flight mass spectrometry. Here, we show that PalGly is produced after cellular stimulation and that it occurs in high levels in rat skin and spinal cord. PalGly was up-regulated in fatty acid amide hydrolase knockout mice, suggesting a pathway for enzymatic regulation. PalGly potently inhibited heat-evoked firing of nociceptive neurons in rat dorsal horn. In addition, PalGly induced transient calcium influx in native adult dorsal root ganglion (DRG) cells and a DRG-like cell line (F-11). The effect of PalGly on the latter cells was characterized by strict structural requirements, PTX sensitivity, and dependence on the presence of extracellular calcium. PalGlyinduced calcium influx was blocked by the nonselective calcium channel blockers ruthenium red, 1-(␤-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl)-1H-