Substance P inactivation by transglutaminase in vitro.

Gamma(glutamyl5)spermine derivative of substance P (Spm-SP) was synthesized in vitro in the presence of purified guinea pig liver transglutaminase and Ca2+. The spermine adduct of the neuropeptide was purified by HPLC on a reversed-phase column and characterized by fast atom bombardment mass spectrometry. The biological activities of Spm-SP were tested by assaying, in comparison with substance P, its ability to induce both the contractions of smooth muscle in vitro and the edema formation in vivo. Spm-SP was shown not to elicit contractile responses in the isolated rat stomach strip and duodenum and not to antagonize the spasmogenic effect evoked by the native neuropeptide. Furthermore, Spm-SP was unable, when administered into rats by plantar injection, either to provoke an acute inflammatory response in the hind limb or to antagonize the edema formation induced by a concurrent administration of substance P. These results indicate that the introduction of a large size hydrophilic moiety at the glutamine5 level negatively affects the ability of the neuropeptide to bind to its receptor(s), thus supporting the view that the hydrophobic middle portion of substance P plays a key role in receptor recognition.     

Identification of tyrosine 706 in the kinase insert as the major colony-stimulating factor 1 (CSF-1)-stimulated autophosphorylation site in the CSF-1 receptor in a murine macrophage cell line.

The receptor for colony-stimulating factor 1 (CSF-1) is a ligand-activated protein-tyrosine kinase. It has been shown previously that the CSF-1 receptor is phosphorylated on serine in vivo and that phosphorylation on tyrosine can be induced by stimulation with CSF-1. We studied the phosphorylation of the CSF-1 receptor by using the BAC1.2F5 murine macrophage cell line, which naturally expresses CSF-1 receptors. Two-dimensional tryptic phosphopeptide mapping showed that the CSF-1 receptor is phosphorylated on several different serine residues in vivo. Stimulation with CSF-1 at 37 degrees C resulted in rapid phosphorylation on tyrosine at one major site and one or two minor sites. We identified the major site as Tyr-706. The identity of Tyr-706 was confirmed by mutagenesis. This residue is located within the kinase insert domain. There was no evidence that Tyr-973 (equivalent to Tyr-969 in the human CSF-1 receptor) was phosphorylated following CSF-1 stimulation. When cells were stimulated with CSF-1 at 4 degrees C, additional phosphotyrosine-containing phosphopeptides were detected and the level of phosphorylation of the individual phosphotyrosine-containing phosphopeptides was substantially increased. In addition, we show that CSF-1 receptors are capable of autophosphorylation at six to eight major sites in vitro.     

Modulation of in vitro myelopoiesis by LGL: different effects on early and late progenitor cells

We describe here the modulatory activity of human peripheral blood natural killer (NK) cells on the growth and differentiation of myeloid progenitor cells at different stages of maturation. NK-enriched cell fractions containing 54 to 75% large granular lymphocytes (LGL) and displaying high levels of NK activity significantly inhibited the growth of late (7 day) granulocyte-macrophage colony-forming cells (CFU-GM) from about 50% of normal human bone marrow samples. However, the same fractions strongly enhanced the growth of early (14 day) stem cells from peripheral blood. Enhancing activity on early CFU-GM from blood was greater in highly purified NK cell preparations containing 96% LGL than in NK-depleted T cell preparations from the same donors. Analogous to the results when using the NK-enriched fractions, the NK-purified preparations inhibited late CFU-GM and stimulated the early ones. We conclude from these observations that human LGL have a modulatory effect on myelopoiesis depending on the maturation stage of the progenitor cell.     

Transduction of a signal for arachidonic acid metabolism by untriggered CSF-1 receptor induces an opposite effect to that induced by CSF-1 receptor and its ligand: separate regulation of phospholipase A2 and cyclooxygenase by CSF-1 receptor/CSF-1.

The mouse hematopoietic cell line, 32D, was transfected with c-fms, which encodes for the CSF-1 receptor, a tyrosine kinase (TK). In the absence of CSF-1, transfected cells show moderate levels of arachidonic acid (AA) release and produce a substantial amount of prostaglandin E2 (PGE2) in comparison with the original cell line. Exposure of transfected cells to CSF-1, while inducing a substantial increase in arachidonate release, nevertheless resulted in inhibition of PGE2 production. Addition of ST638, a tyrosine kinase inhibitor, to cells transfected with c-fms in the absence of CSF-1 inhibited PGE2 production within 10-60 min. Its addition to the same cells in the presence of CSF-1 induced an opposite effect, but required longer treatment (24 h). In either cell type, AA release was not affected by this agent. These data indicate that CSF-1 may regulate cyclooxygenase activity. The different effect of CSF-1 receptor on PGE2 production in the presence or absence of CSF-1 and the opposite effect of a tyrosine kinase inhibitor on PGE2 suggest that both the receptor alone or the receptor-ligand complex may transduce an active, but different, signal through tyrosine phosphorylation. CSF-1 receptor and CSF-1 may exert separate, but related, effects on phospholipase A2 and cyclooxygenase activity which, in concert, or along with other tyrosine kinases, regulate prostaglandin production.     

Substance P, neurokinin A, and neurokinin B induce generation of IL-1-like activity in P388D1 cells. Possible relevance to arthritic disease

Near nanomolar concentrations of substance P induce production of IL-1 or an IL-1-like activity in the mouse macrophage cell line P388D1. Moreover, this could be accomplished with the carboxyl-terminal octapeptide substance P4-11, and could be inhibited with the substance P antagonist [D-Pro2, D-Trp7,9]-substance P. Two other mammalian neurokinins, neurokinin A and neurokinin B, were also found to induce secretion of IL-1-like activity in P388D1 cells. These findings suggest that activation of immune cells by neuromodulators can contribute to the maintenance of the chronic inflammatory state and the immunopathology observed in arthritic disease mediated by IL-1. The results also suggest that one approach to the treatment of rheumatoid arthritis might be to attempt to inhibit the local effects of immuno-modulatory neuropeptides, specifically the neurokinins, in affected joints.     

Synthesis of membrane-bound colony-stimulating factor 1 (CSF-1) and downmodulation of CSF-1 receptors in NIH 3T3 cells transformed by cotransfection of the human CSF-1 and c-fms (CSF-1 receptor) genes.

NIH 3T3 cells cotransfected with the human c-fms proto-oncogene together with a 1.6-kilobase cDNA clone encoding a 256-amino-acid precursor of the human mononuclear phagocyte colony-stimulating factor CSF-1 (M-CSF) undergo transformation by an autocrine mechanism. The number of CSF-1 receptors on the surface of transformed cells was regulated by ligand-induced receptor degradation and was inversely proportional to the quantity of CSF-1 produced. A tyrosine-to-phenylalanine mutation at position 969 near the receptor carboxyl terminus potentiated its transforming efficiency in cells cotransfected by the CSF-1 gene but did not affect receptor downmodulation. CSF-1 was synthesized as an integral transmembrane glycoprotein that was rapidly dimerized through disulfide bonds. The homodimer was externalized at the cell surface, where it underwent proteolysis to yield the soluble growth factor. Trypsin treatment of viable cells cleaved the plasma membrane form of CSF-1 to molecules of a size indistinguishable from that of the extracellular growth factor, suggesting that trypsinlike proteases regulate the rate of CSF-1 release from transformed cells. The data raise the possibility that this form of membrane-bound CSF-1 might stimulate receptors on adjacent cells through direct cell-cell interactions.     

Role of dimerization and modification of the CSF-1 receptor in its activation and internalization during the CSF-1 response.

We have used kinetic and cross-linking approaches to study CSF-1-induced changes in the structure and function of the CSF-1R. Addition of CSF-1 to cells stimulates or stabilizes non-covalent CSF-1R dimerization resulting in activation of the CSF-1R kinase and the tyrosine phosphorylation of the receptor and certain cytoplasmic proteins. The non-covalent dimers become covalently linked via disulfide bonds and/or are subsequently further modified. These modified forms are selectively internalized. Pre-treatment of cells with the alkylating agent, iodoacetic acid (IAA), selectively inhibits covalent dimerization, modification and internalization but enhances protein tyrosine phosphorylation. It is proposed that ligand-induced non-covalent dimerization activates the CSF-1R kinase, whereas the covalent dimerization and subsequent modification lead to kinase inactivation, phosphotyrosine dephosphorylation and internalization of the receptor--ligand complex.     

Substance P enhances the proliferation of rat anterior pituitary cells in vitro

The undecapeptide substanceP(SP) was shown to be intimately involved in both the structural and functional aspects of the anterior pituitary.Yet,in addition to its influences on hormonal secretion,SP may well possess more actions in this master gland.The present study was ftherefore aimed to investigate the effect of SP on the proliferation of rat anterior pituitary cells in primary culture,It was found that SP could dose-dependently increase the incorporation of tritiated thymidine(3H-TdR) into cultured anterior pituitary cells.Other mammalian tachykinins such as neurokinin A and neurokinin B had similar effect but to varying degrees.The equipotent analogue of SP,Norleucine^11-SP(Nle^11-SP),also acted likewise.with its action antagonizable by spantide,a SP receptor blocker.To further characterize the nature of cells responsive to the challenge of SP,immunocytochemical staining against S-100 protein and some adenohypophyseal hormones was performed alone or plus autoradiography.The results showed that the percentage of S-100 proteinimmunorective cells was apparently elevated by the addtion of Nle^11-Sp for 48h,which indicates a preferential proliferation of folliculo-stellate cells under the regime .This was confirmed by increases in immunocytochemical or autoradiographical labelling indices of anterior pituitary Substance P and anterior pituitary cell proliferation.Cells treated similarly.Taken together,These results reveal that the trophic action of SP observed previously in other tissues is also present at least in cultured rat anterior pituitary cells.with responding cells being predominantly folliculo-stellate cells as typified by S-100 proteinimmunoreactivity.Therefore,an intra-pituitary trophicaction of SP in vivo could be anticipated.     

miR-1254 inhibits progression of glioma in vivo and in vitro by targeting CSF-1

The role of miRNAs (microRNAs) has been implicated in glioma initiation and progression, although the inherent biochemical mechanisms still remain to be unravelled. This study strived to evaluate the association between CSF-1 and miR-1254 and their effect on advancement of glioma cells. The levels of miR-1254 in glioma cells and tissues were determined by real-time RT-PCR. Proliferation, apoptosis and cell cycle arrest, invasion and migration, were assessed by CCK-8 assay, colony formation assay, flow cytometry, transwell assay and wound-healing assay, respectively. The targeted relationship between miR-1254 and CSF-1 was confirmed by dual-luciferase reporter assay. The effects of CSF-1 on cellular functions were also assessed. The in vivo effect of miR-1254 on the formation of a tumour was explored by using the mouse xenograft model. We found in both glioma tissues and glioma cells, the down-regulated expressions of miR-1254 while that of CSF-1 was abnormally higher than normal level. The target relationship between CSF-1 and miR-1254 was validated by dual-luciferase reporter assay. The CSF-1 down-regulation or miR-1254 overexpression impeded the invasion, proliferation and migratory ability of U251 and U87 glioma cells, concurrently occluded the cell cycle and induced cell apoptosis. Moreover, in vivo tumour development was repressed due to miR-1254 overexpression. Thus, CSF-1 is targeted directly by miR-1254, and the miR-1254/CSF-1 axis may be a potential diagnostic target for malignant glioma.     

Transforming growth factor-beta causes partial inhibition of interleukin 1-stimulated cartilage degradation in vitro

We show that purified human transforming growth factor-beta (1-10ng/ml) inhibits interleukin 1-stimulated loss of proteoglycan from cartilage in vitro. Inhibition is incomplete, as interleukin 1 retains the ability to cause a dose dependent stimulation of proteoglycan release in the presence of high levels of transforming growth factor-beta (100ng/ml) although both basal and interleukin 1-stimulated levels can be reduced by up to 50 per cent. This observation, together with its ability to stimulate proteoglycan synthesis and to stimulate proteinase inhibitor production, suggests a possible role for transforming growth factor-beta in limiting cartilage proteoglycan loss in inflammatory conditions such as rheumatoid arthritis.     

Characterization of the Substance P (NK-1) Receptor in Tunicamycin-Treated Transfected Cells Using a Photoaffinity Analogue of Substance P

Abstract: Chinese hamster ovary cells expressing the N -glycosylated substance P (NK-1) receptor were treated with the glycosylation inhibitor tunicamycin and photolabeled with ¹²⁵I-Bolton-Hunter- p -benzoyl- l -phenylalanine⁸-substance P. Two radioactive proteins of M_r 80,000 and 46,000, representing the glycosylated and nonglycosylated substance P (NK-1) receptor, respectively, were observed. The IC₅₀ for the inhibition of photolabeling of both receptor forms was 0.3 ± 0.1 n M for substance P and 30 ± 5 n M for neurokinin A (substance K). Thus, glycosylation of the substance P (NK-1) receptor has no detectable effect on the affinity of the substance P (NK-1) receptor for substance P or neurokinin A (substance K).     

Expression of a Y559F Mutant CSF-1 Receptor in M1 Myeloid Cells: A Role for Src Kinases in CSF-1 Receptor-Mediated Differentiation

We have established two M1 myeloid cell lines, M1/WT cells overexpressing the wild-type CSF-1 receptor and M1/Y559F cells expressing a specific tyrosine mutant. M1/WT cells differentiated in response to CSF-1, with a reduction in their proliferative capacity. CSF-1-mediated differentiation was partially abrogated in the M1/Y559F cells, with a less marked reduction in proliferative capacity. The Src tyrosine kinases c-Src, c-Yes, c-Fyn, and c-Hck were tyrosine phosphorylated in the M1/WT cells in response to CSF-1 and bound to the WT CSF-1R through their SH2 domains. Binding of the Src kinases to the CSF-1 receptor was greatly reduced in the M1/Y559F cells. CSF-1-mediated activation of STAT3 was also abrogated in the M1/Y559F cell line. Treatment of M1/WT cells with the Src family inhibitor PP2 resulted in an inhibition of CSF-1-mediated differentiation, equivalent to that observed in the M1/Y559F cells. These data suggest that the reduced Src binding observed in the M1/Y559F cells may contribute to their reduced ability to differentiate.     

Human acidic ribosomal phosphoproteins P0, P1, and P2: analysis of cDNA clones, in vitro synthesis, and assembly.

cDNA clones encoding three antigenically related human ribosomal phosphoproteins (P-proteins) P0, P1, and P2 were isolated and sequenced. P1 and P2 are analogous to Escherichia coli ribosomal protein L7/L12, and P0 is likely to be an analog of L10. The three proteins have a nearly identical carboxy-terminal 17-amino-acid sequence (KEESEESD(D/E)DMGFGLFD-COOH) that is the basis of their immunological cross-reactivity. The identities of the P1 and P2 cDNAs were confirmed by the strong similarities of their encoded amino acid sequences to published primary structures of the homologous rat, brine shrimp, and Saccharomyces cerevisiae proteins. The P0 cDNA was initially identified by translation of hybrid-selected mRNA and immunoprecipitation of the products. To demonstrate that the coding sequences are full length, the P0, P1, and P2 cDNAs were transcribed in vitro by bacteriophage T7 RNA polymerase and the resulting mRNAs were translated in vitro. The synthetic P0, P1, and P2 proteins were serologically and electrophoretically identical to P-proteins extracted from HeLa cells. These synthetic P-proteins were incorporated into 60S but not 40S ribosomes and also assembled into a complex similar to that described for E. coli L7/L12 and L10.     

Priming of normal human neutrophils by tachykinins: tuftsin-like inhibition of in vitro chemotaxis stimulated by formylpeptide or interleukin-8.

Tachykinins have priming effects on polymorphonuclear neutrophils, since they may activate the neutrophils to exhibit an exaggerated inflammatory response to phlogistic mediators. In order to investigate mechanisms involved in this action, we determined the influence of substance P and neurokinin A on chemotaxis of human neutrophils towards gradients of formymethionyl-leucyl-phenylalanine or recombinant human interleukin-8. As seen with other neutrophil-priming agents such as tumor necrosis factor-alpha, exposure of neutrophils to substance P or neurokinin A had an inhibitory effect upon a stimulated migration, with effective concentrations being in the nanomolar range. Tuftsin, a known neutrophil activating peptide, similarly inhibited stimulated migration. Analysis of structure-activity relationships revealed that activity of tachykinins is located in amino-terminal, tuftsin-like sequences. The inhibition of stimulated migration was partly reversed by (Pro1)-tuftsin, a partial tuftsin receptor antagonist, which suggests that the effects of amino-terminal tachykinins involves activation of tuftsin receptors of neutrophils.     

Substance P, neurokinin A and calcitonin gene-related peptide during development of the rat gastrointestinal tract.

Substance P, neurokinin A and calcitonin gene-related peptide (CGRP) were determined in the stomach and small intestine of rats during late foetal development and up to 35 days postnatal life. Concentrations of substance P in stomach and intestine increased from 14 gestational days to 3 days postpartum, and declined thereafter. Concentrations of neurokinin A in stomach declined from 14 days gestation over the period 3-35 postnatal days. In the intestine, concentrations of neurokinin A increased steadily from 14 days gestation to 21-35 postnatal days. Concentrations of CGRP in stomach and intestine declined from 14 days gestation to 7 postnatal days. Thereafter, concentrations of CGRP increased in both stomach and intestine. Total contents of each of the three peptides increased progressively with gestational and postnatal age in parallel with increasing stomach and intestinal weights. The results demonstrate different patterns of change in the concentrations of substance P, neurokinin A and CGRP during the dynamic phases of growth and maturation of the gastrointestinal tract in the foetal and postnatal rat.     

Prognostic Significance of Substance P and Neurokinin-1 Receptor in Bladder Cancer

Background:Bladder cancer is one of the most common genitourinary cancers with significant mortality. Finding reliable tumor markers and potential drug targets can improve early diagnosis, prognosis, and more effective therapeutic protocols. Previous studies have reported the involvement of the substance P (SP)/neurokinin-1 receptor (NK-1R) system in cancers. The potential prognostic role and the interaction of SP and NK-1R in bladder tumor are yet to be elucidated.Methods:Serum samples from 22 primarily diagnosed patients with bladder cancer as well as 22 healthy controls were examined for SP level using ELISA method. Tissue distribution of NK-1R in tumor samples and their adjacent normal tissues was evaluated through immunohistochemistry.Results:Serum SP levels in patients with bladder cancer were higher than the healthy group (p< 0.001) and had a significant correlation with NK-1R staining intensity (p< 0.001), percentage of stained cells (p< 0.001), and NK-1R tissue distribution. Also, the immunoreactivity of NK-1R in cancer samples increased significantly without correlation with tumor characteristics. However, no significant association was found between SP and NK-1R levels with clinical characteristics including tumor size (p= 0.33), tumor stage (p= 0.29), grade (p= 0.93), NK-1R staining intensity (p= 0.53), and percentage of stained cells (p= 0.32).DiscussionAccording to our findings, despite the lack of association between SP and NK-1R with clinical characteristics of bladder cancer, their serum levels were higher in patients with bladder cancer. Further studies are needed to confirm the potential prognostic role of SP and NK-1R in bladder cancer.Key Words: Biomarker, Bladder cancer, Neurokinin-1 receptor, Substance P, Prognosis     

Transgenic expression of stromal cell-derived factor-1/CXC chemokine ligand 12 enhances myeloid progenitor cell survival/antiapoptosis in vitro in response to growth factor withdrawal and enhances myelopoiesis in vivo

Hemopoiesis is regulated in part by survival/apoptosis of hemopoietic stem/progenitor cells. Exogenously added stromal cell-derived factor-1 ((SDF-1)/CXC chemokine ligand (CXCL)12) enhances survival/antiapoptosis of myeloid progenitor cells in vitro. To further evaluate SDF-1/CXCL12 effects on progenitor cell survival, transgenic mice endogenously expressing SDF-1/CXCL12 under a Rous sarcoma virus promoter were produced. Myeloid progenitors (CFU-granulocyte-macrophage, burst-forming unit-erythroid, CFU-granulocyte-erythrocyte-megakaryocyte-monocyte) from transgenic mice were studied for in vitro survival in the context of delayed addition of growth factors. SDF-1-expressing transgenic myeloid progenitors were enhanced in survival and antiapoptosis compared with their wild-type littermate counterparts. Survival-enhancing effects were due to release of low levels of SDF-1/CXCL12 and mediated through CXCR4 and G(alpha)i proteins as determined by ELISA, an antagonist to CXCR4, Abs to CXCR4 and SDF-1, and pertussis toxin. Transgenic effects of low SDF-1/CXCR4 may be due to synergy of SDF-1/CXCL12 with other cytokines; low SDF-1/CXCL12 synergizes with low concentrations of other cytokines to enhance survival of normal mouse myeloid progenitors. Consistent with in vitro results, progenitors from SDF-1/CXCL12 transgenic mice displayed enhanced marrow and splenic myelopoiesis: greatly increased progenitor cell cycling and significant increases in progenitor cell numbers. These results substantiate survival effects of SDF-1/CXCL12, now extended to progenitors engineered to endogenously produce low levels of this cytokine, and demonstrate activity in vivo for SDF-1/CXCL12 in addition to cell trafficking.