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Biomedical subjects

D Fabbro

Publications and source records attributed to D Fabbro.

At least 109 records · Page 6Linked to original sources
PubMed1992

Hormone- and phorbol ester-activated protein kinase C isozymes mediate a reorganization of the actin cytoskeleton associated with prolactin secretion in GH4C1 cells.

TRH regulates PRL secretion and synthesis in GH4C1 rat pituitary cells. TRH responses are associated with activation of protein kinase C (PKC) isozymes and elevation of cytosolic calcium. To determine which PKC isozymes are involved in TRH-directed responses, we evaluated the effect of TRH on GH cell alpha-, beta-, delta-, and epsilon-PKC isozymes. Immunoblot analysis demonstrated that TRH caused rapid redistribution of all isozymes to a Triton X-100-insoluble (i.e. cytoskeletal) fraction. Corollary immunocytofluorescence studies demonstrated that redistributed PKCs accumulate in cell peripheries. Exocytosis involves reorganization of the cytoskeleton, therefore, each of the GH cell PKCs is appropriately located to phosphorylate proteins important for cytoskeleton organization. To determine the relative contributions of calcium and PKC signal transduction pathways in mediating TRH responses, the effects of potassium depolarization (which increases cytosolic calcium) and phorbol dibutyrate (which activates all PKC isozymes without increasing calcium) were compared. The data indicate that TRH-mediated reorganization of vinculin proceeds via a calcium-mediated pathway, whereas fragmentation of actin filaments proceeds via a PKC-dependent pathway. Selective down-modulation of epsilon-PKC with prolonged TRH-treatment was used to demonstrate that epsilon-PKC is not necessary for certain TRH-stimulated biological responses.

Actins↗
PubMed1992

Growth-promoting effect of a protein-free hemodialysate used in situations of hypoxia and for tissue repair as measured via stimulation of S6-kinase.

Solcoseryl is the low-molecular weight fraction of calf blood as manufactured by counterflow dialysis. This hemodialysate (HD) is in clinical use in situations involving hypoxia and for the normalization of tissue repair. The influence of the HD on ZR-75 cells was tested. These cells preferably express receptors for Epidermal Growth Factor (EGF)/Transforming Growth Factor alpha (TGF-alpha) or Somatomedin C (SMC = insulin like growth factor I resp. ILA-I) and react upon stimulation by enhancement of their S6-kinase activity, the latter being a prerequisite for growth. The functional presence of one or several of these peptide growth factors should therefore reflect in a stimulation of S6-kinase activity.

Actihaemyl↗
PubMed1992

Auditory brainstem evoked potentials (BAEPs) in lead-exposed workers.

Brainstem Evoked Potentials (BAEPs) were recorded in 49 lead exposed workers, and in a control group of 49 age- and sex-matched subjects, never exposed to neurotoxic substances. The mean duration of lead exposure was 7.4 (SD 5.6) yr. Blood lead concentration was analyzed in the morning of the experimental day (PbBc); an averaged PbB level was based on the levels of the 3 previous years (PbBm). Interpeak latency differences (IPLD) I-V, I-III and III-V were considered. The mean PbBc level was 54.6 (SD 16.1) micrograms/dl while the mean PbBm level was 53.5 (SD 15.9) micrograms/dl. Lead exposed workers showed a significant prolongation of IPLDs. IPLD I-V was longer in the subgroup with PbBm greater than 50 micrograms/dl (4.06 vs 3.98, c.l. 95% 0.00-0.16). These results are consistent with literature data and show that BAEPs may be a sensitive detector of subclinical lead effects on brainstem auditory pathways.

Adult↗
PubMed1991

Differential regulation of protein kinase C isozymes by thyrotropin-releasing hormone in GH4C1 cells.

GH4C1 cells, which express Ca(2+)-dependent alpha- and beta- as well as Ca(2+)-independent gamma-, epsilon- and zeta-protein kinase C (PKC) isozymes, provide a cell culture model for studying isozyme-specific properties and functions. Hormonal activation of PKCs regulates the differentiated functions of these cells, namely secretion and synthesis of prolactin (PRL). We previously reported that thyrotropin-releasing hormone (TRH) selectively down-modulates epsilon-PKC with no effect on alpha- or beta-PKCs (Kiley, S.C., Schaap, D., Parker, P., Hsieh, L.-L., and Jaken, S. (1990) J. Biol. Chem. 265, 15704-15712). We now extend those studies to explore the relationship between TRH-stimulated diacylglycerol (DAG) levels and epsilon-PKC down-modulation. TRH stimulates three distinct DAG phases in GH cells. Phase 1 DAG peaks at 15 s, is accompanied by a 6-fold increase in intracellular Ca2+, and causes the redistribution of alpha-, beta-, delta, and epsilon-PKC isozymes from a soluble to a detergent-insoluble particulate compartment. Phase 2 DAG peaks at 10 min, is not associated with a Ca2+ signal, and does not activate PKC by any criteria tested. Phase 3 DAG peaks at 6 h and is sustained through 12 h. This novel DAG phase is not associated with increased intracellular Ca2+. The time course of phase 3 DAG formation corresponds to the time course of TRH-stimulated epsilon-PKC down-regulation; maximal effects are observed at 6-12 h for both events. Unlike alpha-, beta-, and delta-PKCs which are preferentially distributed in the soluble fraction of resting GH cells, epsilon-PKC is also distributed in the detergent-insoluble particulate fraction. The selective compartmentalization of epsilon-PKC in the particulate fraction may render this pool uniquely susceptible to proteolytic degradation. The time course of phase 3 DAG formation and epsilon-PKC down-modulation corresponds to the time course of decreasing PRL message synthesis in GH4 cells. The data suggests that loss of epsilon-PKC may be associated with the down-regulation of prolactin synthesis and that regulation of PRL gene transcription may be an epsilon-PKC-specific function in GH cells.

Animals↗
PubMed1991

Differential recovery of protein kinase C-alpha and -epsilon isozymes after long-term phorbol ester treatment in rat renal mesangial cells.

Glomerular mesangial cells have been shown to express two protein kinase C (PKC) isozymes, PKC-alpha and PKC-epsilon. Upon long-term treatment with phorbol ester PKC-alpha is depleted faster than PKC-epsilon. Here we demonstrate that removal of phorbol ester results in a differential recovery of PKC-alpha and -epsilon isozymes. Whereas PKC-epsilon starts to recover within 1h, PKC-alpha does not begin to recover before 4 h after removal of phorbol ester. These data suggest a differential rate of protein synthesis of PKC-alpha and -epsilon. In parallel to the recovery of PKC isozymes mesangial cells also regained their functional responsiveness, i.e., stimulation of prostaglandin synthesis and feedback inhibition of angiotensin II-stimulated InsP3 formation.

Angiotensin II↗
PubMed1991

Protein kinase C down-regulation enhances cAMP-mediated induction of urokinase-type plasminogen activator mRNA in LLC-PK1 cells.

Expression of the urokinase-type plasminogen activator (uPA) gene in LLC-PK1 cells can be induced by signals mediated by both cAMP-dependent protein kinase (PKA) and Ca(2+)- and phospholipid-dependent protein kinase (PKC). We have utilized the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) to down-regulate PKC, in order to test for an effect on the PKA-mediated induction of the uPA gene expression. Incubation of cells for 24 h with 100 ng/ml TPA caused a marked decrease of PKC protein, both in cytosolic and particulate fractions, and an 85% reduction of total PKC activity. After down-regulation of PKC, uPA mRNA accumulation induced by 8-Br-cAMP was 5-10-fold higher than in control cells. Both uPA mRNA stability and uPA gene transcription rates induced by 8-Br-cAMP were increased by PKC down-regulation (6- and 1.8-fold, respectively). Although total PKA activity was reduced by 20% in extracts from PKC-depleted cells, activation of PKA by 8-Br-cAMP was 2.5-fold higher than in control cells. This enhanced activation of PKA in PKC-depleted cells also occurred in response to other cAMP derivatives and to cAMP induced endogenously by the activation of adenylate cyclase with forskolin, but was not due to down-regulation-associated changes in the rate of cAMP synthesis. Our results demonstrate that in LLC-PK1 cells, down-regulation of PKC results in an enhanced induction of uPA gene expression by cAMP-mediated signals without alterations in adenylate cyclase activity, suggesting a mechanism distal to adenylate cyclase.

1-Methyl-3-isobutylxanthine↗
PubMed1991

Possible regulatory functions of protein kinase C-alpha and -epsilon isoenzymes in rat renal mesangial cells. Stimulation of prostaglandin synthesis and feedback inhibition of angiotensin II-stimulated phosphoinositide hydrolysis.

Short-term treatment of mesangial cells with phorbol 12-myristate 13-acetate (PMA) decreases angiotensin II-induced InsP3 formation, but potentiates hormone-stimulated arachidonic acid release and prostaglandin (PG) E2 synthesis. Long-term treatment with PMA augments hormone-stimulated InsP3 generation (after 8 h treatment), but eliminates angiotensin II-induced arachidonic acid release and PGE2 formation (after 24 h treatment). By using specific antibodies it is observed that mesangial cells express two protein kinase C (PKC) isoenzymes, PKC-alpha and -epsilon. No PKC-beta and -gamma isoenzymes are detected. On exposure to PMA a complete depletion of PKC-alpha is observed within 8 h. In contrast, down-regulation of PKC-epsilon to 10-20% of that found in control cells requires a 24 h treatment with PMA. These results may imply that PKC-alpha mediates feedback inhibition of phosphoinositide hydrolysis, whereas PKC-epsilon is a candidate for regulating PG synthesis in mesangial cells.

Amino Acid Sequence↗
PubMed1991

Different translocation of three distinct PKC isoforms with tumor-promoting phorbol ester in human platelets.

Protein kinase C (PKC), a family of related but distinct enzymes whose cellular functions are poorly understood, acts in synergy with Ca2+ mobilization for the activation of platelets. Using specific antibodies for the different isoforms, immunoblot analysis revealed the presence in human platelets of three different PKC subtypes which specifically react with alpha, beta and zeta-PKC antibodies. Whereas the subcellular distribution of the alpha PKC remained unaffected, incubation of platelets with 1 microM PMA for 2 min resulted in a significant subcellular distribution from cytosol to membrane of beta-PKC (25%) and zeta (15%). The beta-PKC isoform is more sensitive than alpha and zeta-PKC to PMA, since 100 nM PMA resulted in a translocation of 85%, 64% and 66% respectively of a maximum translocation observed with 1 microM PMA.

Amino Acid Sequence↗
PubMed1991

Human acid beta-glucosidase. Use of inhibitory and activating monoclonal antibodies to investigate the enzyme's catalytic mechanism and saposin A and C binding sites.

Of 14 identified epitopes on human GCase (acid beta-glucosidase), monoclonal antibodies (MCABs) recognizing 3 produced inhibition and 1 resulted in activation of GCase. MCABs F1 and F2 completely, and MCAB 61 partially (approximately 70%), inhibited GCase activity. Substrates and active site-directed inhibitors (specific sphingolipid and 5-amino-5-deoxyglucose derivatives) protected the enzyme from inhibition by MCAB F1 and F2, but not that by MCAB 61. Conduritol B epoxide did not protect GCase from the inhibition by these MCABs when covalently bound to the active site. These results indicated highly specific binding requirements of MCABs F1 and F2 for residues in a complex active site. In comparison, kinetic analyses using GCase transition state analogues, N-alkyl-glucosylamines, and MCAB 61 demonstrated that this MCAB "freezes" the conformation of the enzyme and inhibits GCase by preventing formation of a conformer needed for maximal catalytic rates. The activating MCAB 122 mimicked the effects of saposin C and competed with this natural activator for residues on the enzyme. Interaction of saposin A and saposin C or MCAB 122 with GCase produced a synergistic effect leading to a marked sensitization of the enzyme to these activators. No such synergism or additivity was found for the maximal catalytic rate since it could be achieved by saturating amounts of any one or combinations of these activators. In the presence of MCAB 61, only 15 to 25% of the maximal activation of GCase was obtained by saposin C or MCAB 122, indicating that the major activation effects of these effectors derived from an induction of a GCase conformational change. These results demonstrate that saposins A and C mediate their activating effects by binding to distinct sites on GCase. Furthermore, major components of the mechanisms for catalysis and saposin C activation are due to conformational changes during the transition state. These findings have implications for understanding the perturbations of GCase function due to the missense mutations which cause Gaucher disease.

Antibodies, Monoclonal↗
PubMed1991

Effect of hypothyroidism and thyroid hormone replacement on the level of protein kinase C and protein kinase A in rat liver.

We investigated the influence of the thyroid hormone status on the levels of protein kinases C (PKC) and A (PKA) in the soluble fraction of rat liver. The immunodetectable PKC level in hypothyroid liver was elevated 7.7-fold, whereas the phorbol-ester binding capacity and the immunodetectable alpha-PKC level were increased 2.4- and 2.6-fold, respectively. Conversely, in hypothyroid livers the abundance of the regulatory type I and the catalytic subunits of PKA were lowered to 42% of the euthyroid level as determined by immunoblotting and by measuring the substrate specific phosphorylation rate of PKA. These changes in the PKC and PKA levels were reversible upon treatment with 0.5 microgram T4/100 g body weight for 2-21 days. The thyroid state dependent alterations in hepatic PKC and PKA levels may be responsible for the known changes in the response of hepatocytes to other hormonal stimuli in hypothyroidism.

Animals↗
PubMed1991

Tumor-promoting phorbol ester and activated Ha-ras synergistically reduce the interleukin 3 requirement in a mast cell line.

Infection of the bone marrow-derived mast cell line PB-3c with a retrovirus carrying oncogenic c-Ha-ras or v-Ha-ras reduced the interleukin 3 (IL-3) growth requirement and induced a state of tumorigenicity. In contrast, normal c-Ha-ras had no effect on the IL-3 requirement of this cell line nor did the cells become tumorigenic. A factor reduction similar to that caused by activated Ha-ras was transiently obtained with 12-O-tetradecanoylphorbol-13-acetate in the PB-3c cells expressing normal c-Ha-ras. The analogous stimulation of protein kinase C (PKC) in PB-3c cells producing oncogenic Ha-ras led to an additional reduction of the IL-3 requirement during the first 24 h. In the absence of IL-3, the prolonged exposure of the cells to 12-O-tetradecanoylphorbol-13-acetate for 72 h resulted in a stimulation of growth when activated but not when normal Ha-ras was expressed. PB-3c cell lines expressing activated Ha-ras neither revealed differences in the amounts nor in the subcellular distribution of PKC activity but displayed elevated levels of immunoreactive beta-PKC compared to the parental PB-3c cells. Upon 12-O-tetradecanoylphorbol-13-acetate treatment, a protracted down-regulation of the immunodetectable alpha-PKC as well as constitutively high levels of c-fos mRNA were observed when oncogenic Ha-ras was expressed. These data suggest the involvement of specific PKC subtypes and of c-fos in the reduction of the IL-3 requirement caused by activated Ha-ras in this particular hematopoietic cell line.

Animals↗
PubMed1991

Overexpression of the alpha-type protein kinase (PK) C in LLC-PK1 cells does not lead to a proportional increase in the induction of two 12-O-tetradecanoylphorbol-13-acetate-inducible genes.

Phorbol esters, by activating protein kinase C (PKC), induce the expression of the urokinase-type plasminogen activator (uPA) gene and the proto-oncogene c-fos in LLC-PK1 (PK1) porcine kidney epithelial cells. To investigate the role of PKC in the regulation of these two 12-O-tetradecanoylphorbol-13-acetate (TPA)-inducible genes, the alpha-type PKC, the predominant subtype present in the PK1 cells, was overexpressed in this cell line. Two clonal PK1 derivatives overexpressing the alpha PKC 15- and 20-fold, respectively, were established. Compared with the parental and control cells, only a modest but substantially sustained (2- to 3-fold) increase in the accumulation of uPA as well as c-fos mRNAs were observed by TPA in these cells. These results indicate that the extent of induction of these genes mediated by TPA was not proportional to the amounts of alpha-type PKC stably overexpressed in these cells, suggesting that factor(s) downstream of the activation of the alpha PKC appear to be rate limiting for the induction of both TPA-inducible genes in PK1 cells.

Animals↗
PubMed1989

Staurosporine stimulates expression of the urokinase-type (u-PA) plasminogen activator in LLC-PK1 cells.

In LLC-PK1 porcine epithelial cells, the urokinase-type plasminogen activator (u-PA) mRNA and protein can be induced either by stimulation of the protein kinase C (PKC) pathway using a tumor promoter (PMA) or by stimulation of the protein kinase A (PKA) pathway with calcitonin (SCT). By contrast, addition of 10(-7) M staurosporine, an inhibitor of PKC, to LLC-PK1 cells also stimulated urokinase production. In contrast to the in vitro situation (where staurosporine inhibited PKC activity), in the cell-culture system the microbial agent caused an early translocation of PKC and inhibited PKA. Addition of staurosporine together with PMA or with SCT further increased urokinase mRNA and protein synthesis. Maximal stimulation was obtained when all 3 agents were added together. We thus assume that in LLC-PK1 cells the PKA and PKC signal-transferring pathways can function independently.

Alkaloids↗
PubMed1989

Biosynthesis and posttranslational modifications of protein kinase C in human breast cancer cells.

Several forms of protein kinase C with molecular masses of 74-, 77-, and 80-kDa were detected in subcellular fractions of human breast cancer MDA-MB-231 cells which express the alpha-type protein kinase C. Several lines of evidence indicated that the 74-kDa is the precursor of the 77- and 80-kDa protein kinase C forms. (i) Pulse-labeling experiments revealed that protein kinase C is synthesized on membranes as a 74-kDa protein that can be chased into the 77- and the 80-kDa protein kinase C forms. (ii) The primary translation product of protein kinase C displayed an apparent molecular size of 74-kDa as determined by in vitro translation of poly(A)+ RNA from MDA-MB-231 cells. (iii) Incubation with serine/threonine-specific protein phosphatases (potato acid phosphatase and phosphatase 1 or 2A) resulted in the complete dephosphorylation of the 77-kDa to the 74-kDa protein kinase C form. Protein kinase C appears to be synthesized in membranes as an unphosphorylated and presumably inactive 74-kDa form that is converted into the active 77- and 80-kDa protein kinase C by post-translational modification involving at least two phosphorylation steps. The first phosphorylation is probably achieved by a specific, yet unidentified, "protein kinase C kinase" since the 74-kDa protein kinase C species did not undergo autophosphorylation and was neither a substrate for the purified protein kinase C, S6 kinase, phosphorylase kinase, casein kinase II, nor for the catalytic subunit of cAMP-dependent protein kinase. Except for phosphorylase kinase and the catalytic subunit of the cAMP-dependent protein kinase, phosphorylation of the 77-kDa protein kinase C form with purified protein kinase C (autophosphorylation), S6 kinase or casein kinase II shifted the molecular mass of the 77-kDa protein kinase C to 80-kDa. Prolonged exposure of MDA-MB-231 cells to phorbol 12-myristate 13-acetate not only leads to a complete down-regulation of protein kinase C activity but also to an accumulation of 74-kDa protein kinase C due to a retarded conversion of the 74-kDa into the 77- and 80-kDa protein kinase C forms in these cells. Our data indicate that tumor promoters additionally interfere with the posttranslational processing that converts the 74-kDa protein kinase C precursor into the 77- and 80-kDa forms of the enzyme.

Breast Neoplasms↗
PubMed1989

EGF induces biphasic S6 kinase activation: late phase is protein kinase C-dependent and contributes to mitogenicity.

Detailed kinetics reveal that EGF-induced S6 kinase activation is biphasic: an early phase appears at 10-15 min, followed by a late phase between 30 and 60 min. Both activities exhibit the same chromatographic behavior and sensitivity to phosphatase 2A. Direct activation of protein kinase C by TPA induces only late phase activity. Down-regulation of protein kinase C leads to loss of both TPA- and EGF-induced late phase activity, while the early phase is unaffected. The loss of late phase kinase activity results in decreased EGF-induced S6 phosphorylation, protein synthesis, and cell growth. The results indicate that EGF differentially regulates S6 kinase activation by two distinct signaling pathways and that loss of the late or protein kinase C-dependent phase leads to a diminished mitogenic response.

Animals↗
PubMed1989

A derivative of staurosporine (CGP 41 251) shows selectivity for protein kinase C inhibition and in vitro anti-proliferative as well as in vivo anti-tumor activity.

Analogues of staurosporine were synthesized and their ability to inhibit protein kinases was examined. Staurosporine is a potent but non-selective inhibitor of in vitro protein kinase C(PKC) activity (IC50 6.0 nM). The derivative CGP 41 251 had reduced PKC activity with an IC50 of 50 nM but showed a high degree of selectivity when assayed for inhibition of cyclic AMP-dependent protein kinase (IC50 2.4 microM), S6 kinase (IC50 5.0 microM) and tyrosine-kinase-specific activity of epidermal growth factor receptor (IC50 3.0 microM). Staurosporine and CGP 41 251 exerted growth inhibition in the human bladder carcinoma line T-24, human promyelocytic leukemia line HL-60 and bovine corneal endothelial cells at concentrations which correlated well with in vitro PKC inhibition. In addition, both compounds inhibited the release of H2O2 from human monocytes pre-treated with 12-O-tetradecanoyl-phorbol-13-acetate at non-toxic concentrations. In vivo anti-tumor activity was examined in T-24 human bladder carcinoma xenografts in athymic nude mice. Tumor growth inhibition tests revealed significant anti-tumor activity (2p less than 0.001) at 1/10 of the maximum tolerated doses for both compounds. By contrast, a closely related derivative of staurosporine (CGP 42 700) was inactive at concentrations of over 100 microM in all in vitro enzyme and anti-proliferative assays as well as in animal tumor models. Our data suggest an association between PKC inhibition and anti-proliferative and anti-tumor activity.

Alkaloids↗