Biomedical subjects

M Pomerance

Publications and source records attributed to M Pomerance.

17 recordsLinked to original sources

PubMed2000

Thyroid-stimulating hormone and cyclic AMP activate p38 mitogen-activated protein kinase cascade. Involvement of protein kinase A, rac1, and reactive oxygen species.

p38 mitogen-activated protein kinases (p38-MAPKs) are activated by cytokines, cellular stresses, growth factors, and hormones. We show here that p38-MAPKs are activated upon stimulation by thyroid-stimulating hormone (TSH) or cAMP. TSH caused the phosphorylation of p38-MAPK in Chinese hamster ovary cells stably transfected with the human TSH receptor but not in wild-type Chinese hamster ovary cells. The effect of TSH was fully mimicked by the adenylyl cyclase activator, forskolin, and by a permeant analog of cAMP. The effect of forskolin was reproduced in FRTL5 rat thyroid cells. TSH also stimulated the phosphorylation of MAPK kinase 3 or 6, over the same time scale as that of p38-MAPKs. TSH and forskolin stimulated the activity of the alpha-isoform of p38-MAPK assayed by phosphorylation of the transcription factor ATF2. The activity of MAPK-activated protein kinase-2 was stimulated by TSH and forskolin. This stimulation was abolished by SB203580, a specific inhibitor of p38-MAPKs. The protein kinase A inhibitor H89 inhibited the stimulation of phosphorylation of p38-MAPKs by forskolin, whereas inhibitors of protein kinase C, p70(S6k), and phosphatidylinositol 3-kinase were ineffective. Expression of the dominant negative form of Rac1, but not that of Ras, blocked forskolin-induced p38-MAPK activation. Diphenylene iodonium, a potent inhibitor of NADPH oxidase(s), and ascorbic acid, an effective free radical scavenger, suppressed TSH- or forskolin-stimulated p38-MAPK phosphorylation, indicating that the generation of reactive oxygen species plays a key role in signaling from cAMP to p38-MAPKs. Inhibition of the p38-MAPK pathway with SB203580 partially but significantly, attenuates cAMP- and TSH-induced expression of the sodium iodide symporter in FRTL-5 cells. These results point to a new signaling pathway for the G(s)-coupled TSH receptor, involving cAMP, protein kinase A, Rac1, and reactive oxygen species and resulting in the activation of a signaling kinase cascade that includes MAPK kinase 3 or 6, p38-MAPK, and MAPK-activated protein kinase-2.

Animals↗

PubMed2000

Thyroid oxidase (THOX2) gene expression in the rat thyroid cell line FRTL-5.

A cDNA encoding an NADPH oxidase flavoprotein was isolated from the rat thyroid gland. The predicted 1517-residue polypeptide was 82.5% identical to the human THOX2/DUOX2 and 74% similar to THOX1/DUOX1. Rat THOX2 lacks a stretch of 30 residues, corresponding to one exon in the human gene sequence. THOX2 mRNA was found to be expressed in cultured FRTL-5 cells. The level of THOX2 mRNA was increased by cAMP in these cells and it was decreased in the thyroids of rats treated with the antithyroid drug methimazole, unlike the TPO and NIS mRNAs. Since it was found in the intestine, duodenum, and colon, in addition to thyroid, we suggest that it be called LNOX, the new family of long homologs of NOX flavoproteins rather than THOX and/or DUOX.

Amino Acid Sequence↗

PubMed2000

The thyrotropin receptor is not involved in the activation of p42/p44 mitogen-activated protein kinases by thyrotropin preparations in Chinese hamster ovary cells expressing the human thyrotropin receptor.

We studied whether bovine pituitary thyrotropin (bTSH) or human recombinant thyrotropin (rhTSH) stimulated p42/p44 mitogen-activated protein kinases (MAPKs) in Chinese hamster ovary cells expressing human thyrotropin receptor (CHO-hTSHR cells). We show that p42/p44 MAPK phosphorylation was induced by both TSH preparations at similar levels in CHO-hTSHR cells and in wild-type CHO cells. In contrast, cyclic adenosine monophosphate (cAMP) production was stimulated by TSH only in CHO-hTSHR cells, demonstrating that p42/p44 MAPK stimulation was independent of the TSH receptor. Moreover, similar results were obtained with two other cell lines: the FRTL-5 thyroid cell line and the CCL39 fibroblast cell line. Maximal stimulation of p42/p44 MAPK phosphorylation was observed after a 5- to 10-minute incubation with bTSH and rhTSH preparations. At this time, the phosphorylation of GST-Elk1 was also increased in a time- and concentration-dependent manner by bTSH preparations. The phosphorylation of p42/p44 MAPKs was abolished by PD 98059 and GF 109203X, indicating the involvement of MAPK kinases (MEK 1/2) and protein kinase C. In contrast, the activation of p42/p44 MAPKs was insensitive to H89, to cholera toxin and to pertussis toxin. These data suggest that the protein kinase A pathway was not implicated in p42/p44 MAPK activation by TSH preparations. Moreover, Gs or Gi/Go proteins do not appear to participate in p42/p44 MAPK activation. We also showed that these TSH preparations failed to induce activation of c-Jun NH2 terminal kinase. We therefore conclude that the commercial TSH preparations used in this study contained factor(s) responsible for the specific activation of p42/p44 MAPKs by a TSH receptor-independent mechanism.

Animals↗

PubMed1996

The Ras-GTPase-activating protein SH3 domain is required for Cdc2 activation and mos induction by oncogenic Ras in Xenopus oocytes independently of mitogen-activated protein kinase activation.

The Ras-GTPase-activating protein (RasGAP) is an important modulator of p21ras - dependent signal transduction in Xenopus oocytes and in mammalian cells. We investigated the role of the RasGAP SH3 domain in signal transduction with a monoclonal antibody against the SH3 domain of RasGaP. This antibody prevented the activation of the maturation-promoting factor complex (cyclin B-p34cdc2) by oncogenic Ras. The antibody appears to be specific because as little as 5 ng injected per oocyte reduced the level of Cdc2 activation by 50% whereas 100 ng of nonspecific immunoglobulin G did not affect Cdc2 activation. The antibody blocked the Cdc2 activation induced by oncogenic Ras but not that induced by progesterone, which acts independently of Ras. A peptide corresponding to positions 317 to 326 of a sequence in the SH3 domain of human RasGAP blocked Cdc2 activation, whereas a peptide corresponding to positions 273 to 305 of a sequence in the N-terminal moiety of the SH3 domain of RasGAP had no effect. The antibody did not block the mitogen-activated protein (MAP) kinase cascade (activation of MAPK/ERK kinase [MEK], MAP kinase, and S6 kinase p90rsk). Surprisingly, injection of the negative MAP kinase mutant protein ERK2 K52R (containing a K-to-R mutation at position 52) blocked the Cdc2 activation induced by oncogenic Ras as well as blocking the activation of MAP kinase. Thus, MAP kinase is also implicated in the regulation of Cdc2 activity. In this study, we further investigated the regulation of the synthesis of the c-mos oncogene product, which is necessary for the activation of Cdc2. We report that the synthesis of the c-mos oncogene product, which is necessary for the activation antibody to the SH3 domain of RasGAP and by injecting the negative MAP kinase mutant protein ERK2 K52R. These results suggest that oncogenic Ras activates two signaling mechanisms: the MAP kinase cascade and a signaling pathway implicating the SH3 domain of RasGAP. These mechanisms might control Mos protein expression implicated in Cdc2 activation.

Animals↗

PubMed1995

Effects of growth factors on phosphatidylinositol-3 kinase in astroglial cells.

Growth factors differently regulate astroglial cell differentiation and proliferation. In an effort to understand the early intracellular events promoted by growth factors in astroglial cells, we have determined the effects of insulin-like growth factor I (IGF1), insulin, platelet-derived growth factor (PDGF), epidermal growth factor (EGF) and fibroblast growth factors (FGFs) on phosphatidylinositol-3 kinase (PI(3)-kinase). In astroglial cells cultured in serum-free medium, IGF1, PDGF, and EGF, which stimulate cell proliferation, increased PI(3)-kinase activity immunoprecipitated with anti-phosphotyrosine antibodies as shown by thin layer chromatography and high performance liquid chromatography. FGFa and FGFb, which strongly stimulate proliferation, glutamine synthetase, and deiodinase activities and modify cell morphology, have no effect on PI(3)-kinase activity. Addition of 1 nM PDGF, 10 nM IGF1, or 100 nM EGF to the culture medium rapidly stimulated PI(3)-kinase activity which declined slowly after 2 min. The stimulation of PI(3)-kinase increased with growth factor concentration. The maximum increase in PI(3)-kinase activity occurred with 50 nM IGF1, 1 nM PDGF, or 100 nM EGF. Since insulin was active only at high concentration (1 microM), its effect was probably mediated through IGF1 receptors and not through insulin receptors. IGF1 and PDGF, to a lesser degree, also increased the PI(3)-kinase activity associated with pp60c-src protein. Immunoblots performed with an antibody directed against the p85-subunit of the PI(3)-kinase confirmed that IGF1 increased the number of PI(3)-kinase molecules associated with phosphotyrosine-containing proteins or with c-src protein. Each growth factor affects in a different manner the association of PI(3)-kinase with phosphotyrosine-containing proteins and with pp60c-src and thus probably modulates intracellular signals downstream of PI(3)-kinase in astroglial cells.

Animals↗

PubMed1994

MAP kinase cascade in astrocytes.

We have studied in cultured rat astroglial cells MAP kinases, known for their role in intracellular signal transduction. The MAP kinase activity was stimulated by growth factors (FGFb, FGFa, EGF, PDGF, and IGF1), by a phorbol ester (TPA) activating-protein kinase C (PKC), by a neuropeptide (endothelin-1), and by a neuromediator (carbachol). Astrocytes pretreated for 18 h with TPA were still stimulated by growth factors and endothelin, suggesting that down-regulated isoforms of PKC are not involved in MAP kinase activation. In contrast, the small effect of carbachol was suppressed by TPA pretreatment. Astrocytes contained two proteins (p41 and p44) recognized by MAP kinase antibody. These proteins were phosphorylated on tyrosine residues in the cytosols of stimulated astrocytes. The kinetics of MAP kinase activation by FGFb and IGF1 were very different. FGFb promoted a rapid activation of MAP kinase (about 10 min) plus a prolonged phase that lasted at least 12 h. IGF1 produced only a rapid transient peak of activation at about 20 min. Hence, extracellular signals might generate different effects in astrocytes by differentially modulating the MAP kinase cascade. On a Mono Q column the growth factor-stimulated MAP kinase activity was separated into two peaks containing p41 and p44. Stimulation of astrocytes altered the elution pattern of p44 as a result of its phosphorylation. An ATP-dependent MAP kinase activator (MW = 40-45 kDa) was found in fractions of FGFb-stimulated cells which were not retained on Mono Q column, indicating the existence of a MAP kinase kinase (MEK) in astrocytes. C-Raf, identified in other cells as a MAP kinase kinase kinase, was also present in astrocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Growth factor-regulated phosphatidylinositol-3-kinase in astrocytes. Involvement of pp60c-src.

Growth factors differently regulate astroglial cell differentiation and proliferation. In an effort to understand the early intracellular events promoted by growth factors in astroglial cells, we have determined the effects of IGF1, insulin, PDGF, EGF and FGFs on phosphatidylinositol-3 kinase. IGF1, PDGF and EGF which stimulate cell proliferation of astroglial cells, increased phosphatidylinositol-3 kinase activity immunoprecipitated with anti-phosphotyrosine antibodies as shown by thin layer chromatography and high performance liquid chromatography. FGFa and FGFb, which strongly stimulate proliferation of astroglial cells, have no effect on phosphatidylinositol-3 kinase activity. Addition of 1 nM PDGF, 10 nM IGF1 or 100 nM EGF to the culture medium rapidly stimulated phosphatidylinositol-3 kinase activity which declined slowly after 2 min. The stimulation of phosphatidylinositol-3 kinase increased with growth factor concentration. The maximum increase in phosphatidylinositol-3 kinase activity occurred with 50 nM IGF1, 1 nM PDGF or 100 nM EGF. Since insulin was active only at high concentration (1 microM), its effect was probably mediated through IGF1 receptors and not through insulin receptors. Treatment with IGF1-plus-EGF, had an additive effect on PI(3)-kinase activity, PDGF-plus-IGF1 did not. IGF1 and PDGF, to a lesser degree, also increased the phosphatidylinositol-3 kinase activity associated with pp60c-src protein. Immunoblots performed with an antibody directed against the p85-subunit of the phosphatidylinositol-3 kinase confirmed that IGF1 increased the number of phosphatidylinositol-3 kinase molecules associated with phosphotyrosine-containing proteins or with c-src protein. Each growth factor affects in a different manner the association of phosphatidylinositol-3 kinase with phosphotyrosine-containing proteins and with c-src protein and thus probably modulates intracellular signals downstream of phosphatidylinositol-3 kinase in astroglial cells.

Animals↗

PubMed1992

Stimulation of mitogen-activated protein kinase by oncogenic Ras p21 in Xenopus oocytes. Requirement for Ras p21-GTPase-activating protein interaction.

p21ras plays an important role in the control of cell proliferation. The molecular mechanisms implicated are unknown. We report that the injection of oncogenic Lys12 Ras into Xenopus laevis oocytes promoted the activation of mitogen-activated protein kinase (MAP kinase) after a lag of about 90 min. MAP kinase activity was 10-fold higher 4 h after injection of oncogenic Lys12 Ras than after injection of nononcogenic Gly12 Ras. The stimulated MAP kinase activity remained at a plateau for at least 18 h. Maximal stimulation was obtained with 5 ng of Lys12 Ras, which is similar to the amount that elicits germinal vesicle breakdown. DEAE-Sephacel chromatography of extracts from Lys12 Ras-injected oocytes showed one peak of MAP kinase. MAP kinase activation by Lys12 Ras was associated with tyrosine phosphorylation of MAP kinase (p42). As previously shown, the S6-kinase II (likely pp90rsk), which is activated in vitro by MAP kinase, was also activated by oncogenic Lys12 Ras. Lys12 Ras with an additional mutation (Glu38) in the effector region that binds GTPase-activating protein (GAP) did not promote MAP kinase or S6 kinase activations. Thus, GAP may be involved downstream to Ras in these activation processes. Our results indicate that the Ras-GAP complex promotes MAP kinase activation in oocytes. This supports the idea that Ras-GAP controls MAP kinase, a kinase implicated in the action of various stimuli.

Animals↗

PubMed1991

Early effect of BCNU on rat astrocytes. Inhibition of S6 kinase activation by growth factors.

In primary cultures of astrocytes, methylmethane, 2-N-methyl 9-hydroxy-ellepticinium acetate, ditercalinium, 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea and 1,3 bis (2-chloroethyl)-1-nitrosourea (BCNU) blocked to various extents the activation of S6 kinase by acidic fibroblast growth factor and insulin [or insulin-like growth factor 1 (IGF1)]. The effects of the most active agent, BCNU, were time and concentration dependent. Pretreatment of cells with 50 microM BCNU for 1 hr completely prevented S6 kinase activation by growth factors for at least 2 days. The S6 kinase activity of unstimulated cells was slightly affected. S6 kinase activation by 12-O-tetradecanoylphorbol 13 acetate was also strongly impaired by treating cells with BCNU whereas activation by 8-bromo-cyclic AMP was slightly reduced. Cyclic AMP-dependent protein kinase and phospholipid and Ca(2+)-dependent protein kinase were unaffected. BCNU had no direct effect on IGF1 binding to cell surface receptors or on the S6 kinase activity of cell cytosols.

Animals↗

PubMed1991

Thyroid hormone action: induction of morphological changes and protein secretion in astroglial cell cultures.

The effects of triiodothyronine (T3) on cell morphology and protein secretion were examined in astrocytes cultured in a chemically defined medium devoid of other hormones and growth factors. The flat polygonal astrocytic cells treated with T3 (1-50 nM) and maintained in non-renewed medium cultures were progressively transformed into process-bearing cells. These changes were initially observed 3 days after the end of T3 treatment and accounted for more than 50% of the cells 7-8 days thereafter. The proteins secreted by the T3-stimulated cells were analyzed on SDS-PAGE after cell labeling for 4.5 h with [35S]methionine. The effect of T3 on protein secretion was dose-dependent. Half-maximal stimulation was reached with 0.2-0.5 nM hormone and the proteins of 46, 59, 67, 78, 85 and 140 kDa were over-secreted (greater than 300% of control). These results were only obtained when the cell medium was not renewed after T3 treatment.

Animals↗

PubMed1990

Effects of transforming growth factor beta 1 on astroglial cells in culture.

The effects of transforming growth factor beta 1 (TGF beta 1) on DNA synthesis and functional differentiation of astroglial cells cultured in serum-free medium were investigated. TGF beta 1 diminished and delayed the peak of DNA synthesis induced by serum. TGF beta 1-treated cells were larger than control cells. This factor delayed the appearance of process-bearing cells induced by acidic fibroblast growth factor treatment and also affected the astrocyte-specific enzyme glutamine synthetase (GS), whose accumulation is under hydrocortisone (HC) control. TGF beta 1 inhibited the induction of GS activity by HC in a dose- and time-dependent manner. Moreover, pretreatment with TGF beta 1 for 4 h maintained the inhibition of GS activity for approximately 16 h after removal of this factor from culture medium. These results suggest that TGF beta 1 may be an important regulator of astrocyte growth and differentiation.

Animals↗

PubMed1989

Activation of S6 kinase in astroglial cells by FGFa and FGFb.

Basic (b) and acidic (a) forms of the fibroblast growth factor (FGF) promoted a rapid increase of the cytosolic S6 kinase activity in astroglial cells. S6 kinase activity was maximal 10 min after addition of the factors to cell cultures and remained at this level for at least 30 min. Half-activation of the enzyme was obtained with 3 ng/ml FGFa. Heparin (100 micrograms/ml) potentiated the response to suboptimal concentrations of FGFa. This growth factor appeared to stimulate an astroglial S6 kinase resembling that stimulated by insulin, IGF1, TPA and cAMP. Although FGFb is more potent than FGFa in stimulating proliferation of Chinese hamster lung fibroblasts (CCL39), it was not more efficient than FGFa in stimulating the S6 kinase activity of astroglial cells.

Animals↗

PubMed1989

[A model for studying the transmission of information produced by certain growth factors: activation mechanisms of S6 kinase in cultured astrocytes].

Treatment of cultured astrocytes from 2-day-old rat cerebral hemispheres with insulin, somatomedin C (IGF1), thrombin and acidic or basic fibroblast growth factors promoted a rapid activation of a cytosolic protein kinase (S6 kinase) which phosphorylates ribosomal protein S6. The phorbol ester (TPA) also triggered a rapid increase in S6 kinase activity. Two agonists of adenylate cyclase activity (forskolin and isoproterenol) and the cyclic AMP analog (dibutyryl cAMP) also stimulated the same S6 kinase. These observations support the idea that several pathways might promote the activation of the same entity that is regarded as one of the primary targets of signals elicited by growth factors.

Animals↗

PubMed1988

Insulin and insulin-like growth factor 1 receptors during postnatal development of rat brain.

The binding of insulin and insulin-like growth factor 1 (IGF1) to high-affinity sites in the brain of rats aged 2-37 days was studied. Specific binding of insulin and IGF1 was assessed using tracer concentrations of 125I-insulin or 125I-IGF1. Sites for insulin and IGF1 were distinguished in these conditions as shown by competition experiments. The Kd were 3.6 nM (insulin) and 2.0 nM (IGF1). These values did not change significantly over the age range studied. The numbers of high-affinity binding sites for insulin and IGF1 were similar in adult animals. IGF1 binding was higher than the insulin binding in 2-day-old animals. The binding capacity for both insulin and IGF1 decreased from birth to age 15 and days remained stable thereafter. Tyrosine kinase activity, which is associated with these receptors, was measured using the artificial substrate poly (Glu, Tyr). It decreased over the first 15 days of life and remained stable thereafter. Autophosphorylation of the receptors confirmed this result. This decrease appears to be due to changes in the numbers of the two types of receptors, and is probably a reflection mainly of the variation in the number of IGF1 receptors. Similar results for insulin and IGF1 binding as well as tyrosine kinase activity were obtained with hypothyroid rats.

Aging↗

PubMed1988

Activation of an S6 kinase from rat astroglial cells by cAMP.

Forskolin and isoproterenol, agonists of adenylate cyclase activity, and dibutyryl cyclic AMP, stimulated an S6 kinase activity in astroglial cells. This activity was insensitive to the thermostable inhibitor of cyclic AMP-dependent protein kinase and had the same behaviour on a DEAE-Sephacel column as the mitogen stimulated S6 kinase. These observations support the idea that the cyclic AMP cascade, as well as various growth factors, can activate S6 kinase.

Animals↗

PubMed1988

Properties of the 12-O-tetradecanoylphorbol-13-acetate-stimulated S6 kinase from rat astroglial cells.

The S6 kinase activity of astroglial cells in primary culture stimulated by 12-O-tetradecanoylphorbol-13-acetate (TPA) has been studied. This activity was eluted as a single peak at 0.15 M NaCl from a DEAE-Sephacel column. The chromatography of this peak on phosphocellulose revealed an activity eluted at 0.15 M NaCl. This partially purified enzyme had a sedimentation coefficient of 3.7S; Km values were 2 X 10(-5) M for ATP and 10(-6) M for 40S ribosomal subunits. The optimal Mg2+ concentration requirement was 2-3 mM. Mn2+ and Co2+ could substitute for Mg2+ (optimum concentrations 1.5 and 0.8 mM, respectively), but these cations were strong inhibitors in the presence of Mg2+. The enzyme was inhibited by N-ethylmaleimide, indicating that it contained thiol groups. This S6 kinase used ATP, but not GTP, as a phosphate donor, and exhibited great specificity for S6 as phosphate acceptor. Whole histones and protamine were slightly phosphorylated whereas phosvitin, histone H1, and surprisingly the peptide Arg-Arg-Leu-Ser-Ser-Leu-Arg-Ala were not phosphorylated. The TPA-stimulated S6 kinase resembles the insulin-, fibroblast growth factor- and cyclic AMP-stimulated enzymes, suggesting that several pathways might activate the same entity.

Adenosine Triphosphate↗

PubMed1986

Activation of S6 kinase activity in astrocytes by insulin, somatomedin C and TPA.

Treatment of cultured astrocytes from 2-day-old rat cerebral hemispheres with insulin or somatomedin C (IGF1) promoted a rapid activation of a cytosolic protein kinase which phosphorylates ribosomal protein S6. Phosphorylation of substrates currently used for protein kinase assays (histone H2B and phosvitin) was not stimulated. Neither the cyclic AMP-dependent protein kinase activity nor that of protein kinase C was modified. Treatment of these astrocytes with TPA also promoted a rapid increase in S6 kinase activity in the cytosolic fraction. Simultaneously, protein kinase C disappeared from the cytosol. Neither cyclic AMP-dependent protein kinase activity nor phosvitin kinase activity was modified. The effects of insulin, IGF1 and TPA were also observed in the presence of cycloheximide. Cycloheximide also potentiated their effects. These data indicate that S6 kinase activity in astrocytes is promoted from a pre-existing molecule via the tyrosine kinase-insulin receptor and suggest that protein kinase C is implicated in the process.

Animals↗