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Results for “Mitogens”

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At least 19 recordsLinked to original sources

PubMed2004

Mitogenic properties of pokeweed lectin-D isoforms on human peripheral blood lymphocytes: non-mitogen PL-D1 and mitogen PL-D2.

We investigated native structures and mitogenic properties of pokeweed lectin-D isoforms (PL-D1 and -D2) on human peripheral blood lymphocytes along with other isolectins (PL-A to -C). Both native PL-D isoforms appeared to behave as monomers. PL-D2 proliferated the lymphocytes like PL-C, whereas PL-D1 had no mitogenicity. PL-D1 acquired mitogenic activity after trimming of the C-terminal dipeptide.

Cell Proliferation↗

PubMed1977

Characterization of guinea pig mitogenic factors. I. Evidence that antigen-induced mitogenic factor and mitogenic factor from mixed leukocyte cultures are distinct molecular entities.

Mitogenic factor from BCG-sensitized cells stimulated with antigen (PPD) was found to have a m.w. between 20 and 25,000 daltons and an isoelectric point of about 7.5. The blastogenic activity of this factor was not affected by L-fucose or heating at 56 degrees C for up to 1 hr. Mitogenic factor obtained from supernatants of allogeneic cell mixtures (MLC-MF) on the other hand, had a m.w. at 15 to 18,000 daltons and an isoelectric point of 6.5. The blastogenic activity of MLC-MF was inhibited by 0.1 M L-fucose. The factor was stable at 56 degrees C for 1 hr. An antibody prepared against MLC-MF inhibited the MLC reaction as well as the activity of MLC-MF on non-committed cells. This antibody, however, did not affect the response of lymphocytes to PHA or PPD and had no suppressive effect on PPD-MF. The antibody was not cytotoxic and its suppressive activity in the MLC response could not be absorbed out by lymphoid cells indicating that it is probably directed against a lymphocyte activation product (MLC-MF) rather than membrane antigens. The chemical and immunologic differences exhibited by PPD-MF and MLC-MF indicate that these two lymphokines are distinct molecular entities.

Animals↗

PubMed1995

Increased expression and subcellular translocation of the mitogen activated protein kinase kinase and mitogen-activated protein kinase in Alzheimer's disease.

The sequential activation of the mitogen-activated protein kinase kinase and its substrate, the mitogen-activated protein kinase is involved in a cascade of protein kinases which link a number of cell surface signals to intracellular changes in enzyme activity and gene expression. In vitro, mitogen-activated protein kinase is able to phosphorylate the microtubule-associated protein tau at Ser-Pro and Thr-Pro sites, thereby generating abnormally hyperphosphorylated tau species that are similar to paired helical filament-tau found in Alzheimer's disease. In the present study, we analysed the levels of immunoreactive mitogen-activated protein kinase kinase and mitogen-activated protein kinase in the temporal cortex (area 22) of patients with Alzheimer's disease by means of enzyme-linked immuno-sorbent assays and compared these changes with the content of abnormally phosphorylated paired helical filament-tau. The levels of immunochemically detected mitogen-activated protein kinase kinase and mitogen-activated protein kinase were both increased in Alzheimer's disease by between 35 and 40% compared with age-matched controls. Elevation of mitogen-activated protein kinase kinase was most pronounced during early stages of Alzheimer's disease and was inversely related to the tissue content of abnormally phosphorylated paired helical filament-tau. Pronounced immunoreactivity of mitogen-activated protein kinase kinase and mitogen-activated protein kinase was present in both tangle bearing neurons and unaffected neurons of the temporal cortex. Immunoreactive neurons were most often localized in the direct vicinity of neuritic plaques. In Alzheimer's disease, the subcellular distribution of mitogen-activated protein kinase kinase and mitogen-activated protein kinase showed a striking translocation from the cytoplasmic to the nuclear compartment. It is suggested that the activation of the mitogen-activated protein kinase cascade which appears to be an early feature of Alzheimer's disease might be critically involved in self-stimulating processes of neurodegeneration and aberrant repair under these conditions.

Aged↗

PubMed1979

Mitogen-activated B-cell blasts reactive to more than one mitogen.

One of three splenic B cells of 2- to 3-mo-old C57BL/6J or C3H/Tif mice are activated either by lipopolysaccharide (LPS) or by lipoprotein (LP) to grow and to mature to IgM-secreting cells. When mitogen-activated blast cells, after a 2-d activation period, are separated from nonactivated, small cells by velocity sedimentation, and no mitogen is readded, the blasts do not continue to grow but do continue to secrete IgM for several days. When the homologous mitogen is added for restimulation, the blast cells continue for several days to divide every 18 h and to develop IgM-secreting, plaque-forming cells. Frequency analyses at limiting dilutions of reactive B-blasts show that one cell in approximately equal to 1.2--1.5 blasts continue to grow and mature in the presence of the homologous mitogen, either LPS or LP. When the B-cell blasts obtained in a first activation period with either LPS or LP are restimulated with a heterologous mitogen, LPS, LP, Nocardia mitogen or mitogens contained in fetal calf serum a high proportion of the blasts continue to grow and mature to IgM-secreting cells. Frequency analyses show for LPS- or LP-blasts, restimulated in a heterologous fashion with either LP or LPS, that one cell in 1.35--1.5 blasts continue to divide and to mature to IgM-secreting cells. C3H/HeJ-splenic B-cells, which are LPS nonresponders, can only be activated to blast cells by LP. These LP-activated blasts can be restimulated by the homologous LP and by Nocardia mitogen and mitogens of fetal calf serum, but not by LPS. The results indicate that the majority of splenic B cells of 2- to 3-mo-old C57BL/6J or C3H/Tif mice are reactive to more than one B-cell mitogen. B cells, therefore, can possess in their surface membrane multireactive mitogen-receptor complexes which regulate growth and maturation.

Animals↗

PubMed1988

Characteristics of the mitogenic activity found in the supernatant of pokeweed mitogen stimulated human mononuclear cells.

Human peripheral blood mononuclear cells were stimulated with pokeweed mitogen (PWM), washed, and cultured in fresh mitogen-free culture medium; a second set of peripheral blood mononuclear cells was used to assess the mitogenic activity of supernatants of these cultures. The effects of this stimulation resembled those of a normal PWM stimulation, including the formation of blast cells, proliferation of lymphoid cells, and immunoglobulin synthesis. Our findings led us to perform experiments to investigate the nature of this mitogenic factor. The molecular weight of the mitogenic activity was found in the region of 700 kD. Additional experiments indicated that human alpha 2-macroglobulin was not responsible for the high molecular weight of the mitogenic factor. Protein digestion or precipitation with trichloroacetic acid only partially abolished the mitogenicity of the mitogenic supernatant. Comparison of the molecular weight of the mitogenic factor with that of the PWM in culture medium showed that the mitogenic activity in the supernatants could be caused by PWM associated with a high molecular weight component.

B-Lymphocytes↗

PubMed1981

Hyporesponsiveness of canine bronchoalveolar lymphocytes to mitogens: II. Analysis of macromolecular synthesis, T-cell antigen, and mitogen-binding.

Canine bronchoalveolar lymphocytes (BAL) and peripheral blood lymphocytes (PBL) were prepared by filtration over nylon wool columns and were studied for their ability to: (a) synthesize protein, RNA, and DNA after mitogen stimulation; (b) express T-cell antigen; and (c) bind radiolabeled mitogen. Bronchoalveolar lymphocytes were shown to be markedly hyporesponsive to mitogen stimulation as assessed by DNA synthesis after 72 hr of culture or by protein and RNA synthesis after 24 hr of culture. The hyporesponsiveness of BAL was not due to a decrease in the number of T-cells; results of an indirect immunofluorescent assay employing a rabbit antiserum indicated similar percentages of T-cells in BAL (78%) and PBL (79%). The hyporesponsiveness was not due to abnormalities in mitogen binding by BAL; results of a radiolabeled mitogen-binding assay indicated essentially no difference in mitogen receptor number or affinity between BAL and PBL. Thus, the initial interaction bronchoalveolar T lymphocytes and mitogen appears to be normal. This normal interaction with mitogen, however, fails to trigger an appropriate level of macromolecular synthesis by BAL. This suggests that the hyporesponsiveness of BAL is due to an abnormality in one of the early metabolic processes induced by the interaction of a mitogen with the cell membrane.

Animals↗

PubMed1984

Mitogenic activities of synthetic lipid A analogs and suppression of mitogenicity of lipid A.

The effect of synthetic lipid A analogs on murine spleen cells was studied. The preparations represented D-glucosamine and D-glucosaminyl-beta 1,6-D-glucosamine disaccharide derivatives substituted in different combinations by ester- and amide-bound fatty acids and by phosphate groups. Significant mitogenic activity was demonstrated with a number of synthetic disaccharide preparations; however, their potency was lower than that of lipid A. The synthetic preparations were not mitogenic for spleen cells from C3H/HeJ mice. Furthermore, the mitogenicity of the synthetic preparations was abolished after binding with polymyxin B. The results indicate that for expression of mitogenicity, a phosphate group at position 1 of the reducing glucosamine and amide-bound acyloxyacyl residues are important factors. Some of the synthetic preparations containing the diglucosamine backbone and expressing relatively low mitogenicity suppressed B-cell mitogenicity of lipid A. Although these preparations were lytic for erythrocytes, they did not affect the viability of the splenic lymphocytes. Suppression was seen when the synthetic preparations were added simultaneously with or after the lipid A mitogen, but optimal suppression was expressed when the preparations were added to the system 3 h before lipid A. Washing of the cells before the addition of lipid A did not affect the results. The suppression was not due to the induction of suppressor cells by the synthetic preparations. The disaccharide preparations did not inhibit T-cell mitogenicity of concanavalin A. In contrast to the disaccharide preparations, the monosaccharide preparations suppressed mitogenicity of both lipid A and concanavalin A, probably because of their direct toxicity for lymphocytes.

Animals↗

PubMed1981

Induction of human immunoglobulin secretion. I. Synergistic effect of B cell mitogen Cowan I plus T cell mitogens or factors.

Synergistic effects of B cell mitogen Staphylococcus bacteria strain Cowan I (Cowan I) plus T cell mitogens in generating immunoglobulin-secreting cells (ISC) from human peripheral blood mononuclear cells (MNC) were investigated. ISC were assayed by reverse plaque-forming cells with protein A-coated red blood cells. Low concentrations of pokeweed mitogen (PWM) plus Cowan I gave superadditive effects on ISC induction, generating 3 to 10 times as many ISC as optimal amounts of either mitogen alone. The mitogens together and separately showed similar kinetics of ISC; synergy was observed at every day tested. Plaque-forming cells of IgM, IgG, and IgA classes all showed strong synergy, together routinely representing 20% of initial MNC. At day 7 of culture, over 80% of non-E-rosetting cells were ISC. Cell donors tested gave these strong responses even if they were low responders to either mitogen alone. Cowan I plus other T cell mitogens, PHA, Con A, and protein A, also provided good signals for B cell activation. Cowan I induced marked proliferation of purified B cells, but T cell-helper signals were required for differentiation to ISC. T cell-helper factor, induced by PWM or PHA, also showed synergistic effects with Cowan I in induction of ISC. Purified B cells did not respond to T cell-helper factor(s) alone to proliferate or differentiate to ISC. These results indicate that optimal ISC induction occurs with a B cell mitogen plus T cell signals acting synergistically.

Antibody-Producing Cells↗

PubMed1983

Further characterization of mitogen-induced autorosette-forming cells: correlation with T-cell subsets and with lymphocyte proliferative responses to mitogens.

Spontaneous autologous rosette-forming cells (ARFC), which form rosettes with autologous erythrocytes, have been of interest as a subset of thymus-derived lymphocytes (T cells). An association of these cells with concanavalin A (Con A)-induced ARFC has been suggested. Furthermore, the Con A-induced ARFC have been shown to be a suppressor T-cell subset in the Con A-generated suppressor system. We have previously reported the induction of ARFC from T cells by several T-cell mitogens such as phytohemagglutinin-P (PHA) and allogeneic non-T cells other than Con A. In the present report, we further characterized the mitogen-induced ARFC and have extended the study to patients with systemic lupus erythematosus (SLE). We have found that ARFC are also inducible from peripheral blood T cells by pokeweed mitogen (PWM). Studies of T-cell surface markers on the ARFC using OKT monoclonal antibodies confirmed the induction of ARFC from both OKT4- and OKT8-reactive T cells by either Con A, PHA, or PWM stimulation. However, OKT4-reactive T cells were the major cellular source of the ARFC induced by all of the mitogens. In studies of SLE patients, proportions of both Con A- and PWM-induced ARFC were found to be significantly low in PBL of SLE patients treated with moderate or large doses of prednisone, with or without concomitant immunosuppressants, but not in SLE patients without such treatment. Proportional analysis of the T cells and their subsets suggested association of these alterations in the mitogen-induced ARFC with the OKT4-reactive T cells, since a significant decrease in the OKT4-reactive T-cell subset was demonstrated in the PBL of these patients. Proportions of PHA-induced ARFC, however, were not significantly different between SLE patients and healthy adults. Moreover, positive correlations of the mitogen-induced ARFC with lymphocyte proliferative responses to each mitogen were established in both SLE patients and healthy adults. These results further support our previous observation that suggest the receptors for autologous erythrocytes are enhanced or reexpressed on those T cells which are highly activated by mitogens.

Antibodies, Monoclonal↗

PubMed1976

Activation of purified human T cells by mitogens: diminished mitogen-induced deoxyribonucleic acid synthesis in human T cells compared with autologous peripheral blood lymphocytes.

Human thymus-derived (T) cells were isolated from peripheral blood after rosette formation with neuraminidase-treated sheep erythrocytes (SRBC). After separation on Ficoll-hypaque, SRBC were removed from T cells by treatment with tris(hydroxymethyl)aminomethane-NH4Cl. Human T cells and autologous peripheral blood lymphocytes (PBL) were then incubated with phytohemagglutinin, concanavalin A, or pokeweed mitogen. Human T cells, in the absence of other cell types, responded with less deoxyribonucleic acid (DNA) synthesis (measured by uptake of [3H]thymidine) than equal numbers of autologous PBL. Further experimentation established that, compared with autologous PBL, the diminished capacity of human T cells to be activated by mitogens was due neither to differences in the mitogen dose-response relationship nor to the time of peak DNA synthesis of T cells or autologous PBL. Fragments or components of SRBC were not detected on human T cells, and treatment of the T cell-SRBC mixture with tris(hydroxymethyl)aminomethane-NH4Cl did not contribute to the results. Increased cell density or a period of preculture before addition of mitogen also did not influence the degree of decreased DNA synthesis in human T cells compared with the response of autologous PBL incubated with the same mitogens. When mixed with the cells remaining at the Ficoll-media interface, purified human T cells did not suppress the mitogenic response of this cell mixture to PHA or concanavalin A. The data indicated that human T cells, in the absence of other cell types, were activated by mitogens to a lesser degree than autologous PBL. Furthermore, T cells responded with DNA synthesis after direct T cell-mitogen interaction.

Cell Separation↗

PubMed1981

Mitogenic and co-mitogenic properties of hemin.

We report here that hemin, an agent known to induce cellular differentiation in Friend erythroleukemia cells, induces mitogenesis in human T cells and enhances mitogenic responses to supraoptimal concentrations of Con A. Mitogenesis induced by suboptimal or optimal concentrations of Con A is not affected by concentrations of hemin that markedly potentiate responses to supraoptimal amounts of Con A. Maximum mitogenic responses are reached 4 to 5 days after initiation of the cultures. The mitogenic response is macrophage dependent, whereas enhancement of responses to supraoptimal concentrations of Con A is not. Indeed, a greater degree of enhancement is observed after removal of adherent cells. Responses to other mitogens (phytohemagglutinin, wheat germ agglutinin, and sodium periodate) are also enhanced by hemin. Compounds that are metabolically or structurally related to hemin are neither mitogenic nor co-mitogenic. Protoporphyrin IX, which is nonmitogenic, is rendered mitogenic by the chemical insertion of iron to form hemin. Hemin does not affect Con A binding to lymphocytes. These findings indicate that hemin is a macrophage-dependent T cell mitogen with the unusual and remarkable property of overcoming the inhibition of mitogenesis induced by high concentrations of Con A. Since hemin has a limited number of metabolic effects, it provides a useful probe for determination of molecular events associated with lymphocyte activation.

Adult↗

PubMed1980

Characterization of human lymphocyte surface receptors for mitogenic and non-mitogenic substances.

To compare the receptor patterns for mitogenic and non-mitogenic substances, surface glycoproteins of human lymphocytes were labelled with the lactoperioxidase-catalysed iodination technique and with a galactose oxidase-tritiated sodium borohydride technique. Labelled cells were detergent-solubilized, and the lysates were allowed to react with insolubilized purified mitogenic lectins, phytohaemagglutinin, leucoagglutinin and an insolubilized non-mitogenic lectin, oxidized leucoagglutinin. Lectin-reactive proteins were eluted with sodium dodecyl sulphate (SDS) buffer. Cell membrane components reactive with anti-lymphocyte globulin (ALG) were retrieved by indirect immunoprecipitation with protein-A-bearing staphylococcus Cowan I strain (SaCI). Lectin- and ALG-reactive proteins were analysed by SDS polyacrylamide gel electrophoresis. Iodinated glycoproteins regularly showed four major components with molecular weights of 120,000, 70,000, 60,000 and 43,000 daltons, respectively, on 7% gels. An additional broad peak in the molecular weight range 20,000--35,000 daltons was found on 10% gels. Tritiated glycoproteins also showed four major components with MW 120,000, 70,000, 60,000 AND 42,000, RESPECTIVely, which reacted with lectin and ALG. In addition, ALG reacted with some glycoproteins with MW between 150,000 and 230,000 daltons. On 10% gels additional lectin- and ALG-binding glycoproteins with MW around 30,000 daltons were found. The similarity in structures bound by mitogenic and non-mitogenic substances indicates that lymphocyte activation may depend on some property conferred by the mitogen.

Antilymphocyte Serum↗

PubMed1982

Stimulation of mouse lymphocytes by a mitogen derived from Mycoplasma arthritidis. III. Ir gene control of lymphocyte transformation correlates with binding of the mitogen to specific Ia-bearing cells.

The supernatant from Mycoplasma arthritidis broth cultures (MAS) contains a T cell mitogen that is under Ir gene control. Responsiveness to this mitogen is dictated by the I-E/I-C subregion of the major histocompatibility complex and is dependent upon adherent radioresistant Ia+ accessory cells from responding haplotype animals. In this study, we established that MAS could be removed from culture supernatants by absorption with spleen cells from mice that themselves are responsive to the mitogen (k and d haplotypes), but activity is not removed by spleen cells from mouse strains that are nonresponsive to the mitogen (b, q, and s haplotypes). Absorption studies with lymphoid cells from congenic and recombinant strain mice established that absorption of the mitogen was itself linked to the I-E/I-C subregion of the major histocompatibility complex. Thymocytes from responding haplotype strains were incapable of removing MAS activity, and spleen cells devoid of Thy-1-positive cells retained their full absorbing capacity. The ability to effectively absorb MAS activity was abrogated by the pretreatment of spleen cells with anti-Ia antiserum and complement. Furthermore, the ability of spleen cells from responding haplotype strains to respond to MAS was blocked by the addition of anti-Ia serum to the cell cultures. Whereas the latter treatment resulted in an almost complete elimination of MAS responsiveness, the ability of similarly treated spleen cells to respond to the mitogens PHA and Con A was only minimally depressed. These results are consistent with our hypothesis that the mitogenic moiety of MAS actually binds to I-E/I-C-coded Ia antigens.

Animals↗

PubMed1999

Association of the Ras to mitogen-activated protein kinase signal transduction pathway with microfilaments. Evidence for a p185(neu)-containing cell surface signal transduction particle linking the mitogenic pathway to a membrane-microfilament association site.

Microvilli of the aggressive 13762 ascites mammary adenocarcinoma contain a large, microfilament-associated signal transduction particle whose scaffolding is a stable glycoprotein complex (Li, Y., Hua, F., Carraway, K. L., and Carraway, C. A. C. (1999) J. Biol. Chem. 274, 25651-25658) associated with the growth factor receptor p185(neu). The receptor is constitutively tyrosine-phosphorylated in the cells and microvilli, predicting that it should recruit mitogenic pathway components to this membrane-microfilament interaction site. Immunoprecipitation of cell lysates with anti-phosphotyrosine and immunoblotting showed phosphorylated forms of the mitogenic pathway proteins Shc and MAPK in addition to p185(neu), suggesting that the Ras to MAPK mitogenic pathway is activated. Immunoblotting of p185(neu)-containing microvillar fractions revealed the presence in each of stably associated Shc, Grb-2, Sos, Ras, Raf, mitogen-activated protein kinase kinase, and mitogen-activated protein kinase/extracellular signal-regulated kinase, as well as the transcription factor-phosphorylating kinase Rsk. All of these pathway components co-immunoprecipitated with p185(neu) from cleared lysates of microvilli solubilized under microfilament-depolymerizing conditions. The recruitment of constitutively phosphorylated p185(neu) and the activated mitogenic pathway proteins to this membrane-microfilament interaction site provides a physical model for integrating the assembly of the mitogenic pathway with the transmission of growth factor signal to the cytoskeleton. This linkage is probably a requisite step in the global cytoskeleton remodeling accompanying mitogenesis.

Actin Cytoskeleton↗

PubMed1978

Mitogen induction of murine C-type viruses. IV. Effects of lipoprotein E. coli, pokeweed mitogen and dextran sulphate.

Lipoprotein E. coli, a B-cell mitogen, is identified as a new agent inducing the release of endogenous C-type virus from mouse spleen cells. Like lipopolysaccharide, a previously identified inducer, this compound has a synergistic effect with 5-bromo-2'-deoxyuridine. Induced virus has the characteristic density as well as morphology of C-type viruses. Budding viruses are detected on cultured BALB/c cells by electron microscopy 2 to 4 days following culturing in the presence of lipoprotein. Pokeweed mitogen, a compound mitogenic for T- and B-cells was negative when tested for virus induction, both alone and in combination with 5-bromo-2'-deoxyuridine. Dextran sulphate, another B-cell mitogen, was negative for induction as well. However, when, combined with lipopolysaccharide, it enhanced the virus release induced by this mitogen. In contrast, no additive effects were observed either by combining dextran sulphate with other virus amplifying mitogens or by combinations of mitogens which both have virus-inducing ability. This finding is discussed with respect to B-cell differentiation.

Animals↗

PubMed1981

Procoagulant activity of human mononuclear leukocytes: dissociation of the effect of mitogens on procoagulant activity and mitogen-stimulated lymphocyte proliferation.

The role of mononuclear cells in generating procoagulant activity was examined by incubating Ficoll-Hypaque-separated mononuclear leukocytes with or without mitogens (phytohemagglutinin, pokeweed mitogen, and concanavalin A). The procoagulant activity was assayed by a modification of a one-stage plasma recalcification time. Significant procoagulant activity developed after 24 hr incubation and was dose dependent; mitogens alone had no effect on the clotting time. The increase in activity was paralleled by the increase in tritiated thymidine incorporation into replication DNA. However, mitomycin C had little inhibitory effect on the development of procoagulant activity, whereas thymidine incorporation was inhibited. The major procoagulant activity was associated with intact cells and not the conditioned supernatant. The removal of adherent mononuclear cells (mostly monocytes) by polystyrene bead columns abolished the procoagulant activity, whereas purification of mononuclear leukocyte populations for monocytes markedly increased the activity as compared to purified lymphocytes. The procoagulant activity was shown to act by the extrinsic limb of the coagulation sequence because of substitution factor VII-deficient plasma for normal plasma resulted in marked depression of procoagulant activity, whereas factor VIII-deficient plasma resulted in a clotting time only minimally longer then normal plasma. Thus, although procoagulant activity in cultures of mononuclear cells is stimulated by the mitogen reagent, these studies suggest that the activity may not be the result of the mitogenic effect on lymphocytes per se. Whether it is a direct effect of the mitogen on the adherent cell or is an effect of a contaminant of the mitogen reagent, such as endotoxin, remains to be determined.

Adult↗

PubMed1997

Signal transduction pathways involved in the mitogenic activity of pleiotrophin. Implication of mitogen-activated protein kinase and phosphoinositide 3-kinase pathways.

Pleiotrophin (PTN) is a developmentally regulated protein which exhibits neurite-outgrowth, mitogenic, and angiogenic properties. It has also been shown to be involved in tumor growth and metastasis. Here we used primary BEL (bovine epithelial lens) cells to investigate the signal transduction pathways involved in the mitogenic activity of recombinant PTN. PTN was purified from conditioned media of SW-13 cells transfected with the human PTN cDNA. We show that inhibitors of tyrosine kinase, mitogen-activated protein kinase, or phosphoinositide (PI) 3-kinase inhibit DNA synthesis stimulated by PTN. Analysis of tyrosine-phosphorylated proteins following PTN stimulation showed phosphorylation of two novel 190- and 215-kDa proteins in addition to SHC, ERK1, and ERK2. A mobility shift of phosphorylated ERK1 and ERK2 was detected with a panERK antibody confirming the phosphorylation of the two ERKs. Furthermore, in vitro immunocomplex kinase assay with Akt1, a natural substrate of PI 3-kinase, showed an activation of the kinase following PTN stimulation and a reversal by the PI 3-kinase inhibitor wortmannin. We conclude that the mitogenic activity of PTN is dependent on tyrosine kinase activation and utilizes the mitogen-activated protein kinase and the PI 3-kinase pathways to transduce a mitogenic signal.

Animals↗

PubMed1996

Serotonin activates the mitogen-activated protein kinase pathway in vascular smooth muscle: use of the mitogen-activated protein kinase kinase inhibitor PD098059.

Serotonin (5-hydroxytryptamine, 5-HT) promotes changes in vascular smooth muscle contractility and is a vascular smooth muscle mitogen. The hypothesis that 5-HT-induced arterial contraction is partially dependent on tyrosine kinase activation was tested in this study. Specifically, we examined the role of the tyrosine kinase mitogen-activated protein kinase kinase in 5-HT-induced vascular contraction by using a novel inhibitor of mitogen-activated protein kinase kinase PD098059. Helical strips of rat aorta, mesenteric and tail artery denuded of endothelium were mounted in tissue baths for measurement of isometric contractile force. 5-HT-induced contraction in all arteries was mediated by 5-HT2A receptors as ketanserin (3-30 nM) was a competitive antagonist in all arteries (pKB = 8.58-8.96). Genistein (5 x 10(-6) M) and tyrphostinB42 (3 x 10(-5) M), two unrelated tyrosine kinase inhibitors, shifted 5-HT-induced contraction toward the right in all arteries (approximately eight-fold). By contrast, daidzein (5 x 10(-6) M), the inactive isomer of genistein, did not reduce contraction to 5-HT in any artery. These findings suggest that 5-HT-induced activation of tyrosine kinase(s) may be a signal transduction pathway used by vascular 5-HT2A receptors. PD098059 (1 x 10(-5) M) reduced contraction to 5-HT in all arteries, indicating that 1) the tyrosine kinase(s) inhibited by genistein and tyrphostin42 probably include mitogen-activated protein kinase kinase and 2) activation of MEK is important for 5-HT-induced contraction. Western analyses of aortic strips contracted to 5-HT (10(-5) M) indicates that tyrosyl-phosphorylation of the mitogen activated protein kinases Erk-1 and Erk-2 was increased (approximately 200% and 15% of basal levels, respectively) and PD098059 (1 x 10(-5) M) significantly reduced 5-HT-stimulated tyrosyl-phosphorylation of Erk-1. Thus, these data support the use of the mitogen-activated protein kinase pathway by 5-HT in causing arterial contraction and demonstrates the usefulness of specific inhibition of the mitogen-activated protein kinase pathway.

Animals↗