Synergistic Stimulation of Interleukin 6 Release and Gene Expression by Phorbol Esters and Interleukin 1β in Rat Cortical Astrocytes: Role of Protein Kinase C Activation and Blockade

Abstract: The involvement of protein kinase C and its interaction with interleukin 1β in the control of interleukin 6 release by cortical astrocytes was studied. The blockade of protein kinase C catalytic domain, by staurosporine, as well as the desensitization of protein kinase C by short-term phorbol 12-myristate 13-acetate pretreatment, increased the basal release of interleukin 6 by rat cortical astrocytes, whereas calphostin C, an antagonist of phorbol ester binding on protein kinase C regulatory domain, did not affect the basal release of the cytokine. The activation of protein kinase C by phorbol 12-myristate 13-acetate enhanced concentration- and time-dependently interleukin 6 release. This stimulatory action of phorbol 12-myristate 13-acetate was significantly reduced by staurosporine, by calphostin C, and by the desensitization of protein kinase C. Interleukin 1β increased interleukin 6 release in a concentration-related manner. Protein kinase C inhibition, by staurosporine or desensitization, potentiated severalfold, whereas calphostin C reduced interleukin 1β stimulation of interleukin 6 release. The treatment of cortical astrocytes with both interleukin 1β (3 ng/ml) and phorbol 12-myristate 13-acetate (10 nM) caused a synergistic stimulation of interleukin 6 release and its gene expression, an effect that was not relieved by either 20 nM staurosporine or by calphostin C but was slightly affected by protein kinase C desensitization. In conclusion, our data show that in rat cortical astrocytes the basal release of interleukin 6 is under a tonic inhibition exerted by a protein kinase C isoform or isoforms sensitive to blockade by staurosporine and desensitization but insensitive to calphostin C. Interleukin 1β stimulated interleukin 6 secretion via a mechanism that is also negatively modulated by a protein kinase C isoform or isoforms sensitive to staurosporine and desensitization. Finally, we showed that interleukin 1β and phorbol 12-myristate 13-acetate synergistically stimulated interleukin 6 release and its gene expression, operating in a manner insensitive to protein kinase C blockers and slightly reduced by protein kinase C desensitization.     

Activation of Protein Kinase C by the Capsaicin Analogue Resiniferatoxin in Sensory Neurones

Abstract: Resiniferatoxin and capsaicin are sensory neurone-specific excitotoxins that operate a common cation channel in nociceptors. Resiniferatoxin is structurally similar to capsaicin and to phorbol esters. Specific [³H]-resiniferatoxin binding, which was detected in the membrane ( K _D value 1.8 ± 0.2 n M ) but not cytosolic fraction of rat dorsal root ganglia, could not be displaced by phorbol 12,13-dibutyrate. Conversely, resiniferatoxin did not displace [³H]phorbol 12,13-dibutyrate binding in either the cytosolic or membrane fraction. Resiniferatoxin and capsaicin both caused translocation of protein kinase C in dorsal root ganglion neurones (EC₅₀ value 18 ± 3 n M ). This translocation was greatly reduced but not abolished, in the absence of external Ca²⁺, suggesting that it was secondary to Ca²⁺ entry. Resiniferatoxin also caused direct activation of a Ca²⁺- and lipid-dependent kinase (or kinases) in the cytosolic fraction of dorsal root ganglia, at concentrations (100 n M to 10 µ M ) higher than required for displacement of [³H]resiniferatoxin binding or translocation of protein kinase C. Capsaicin (up to 10 µ M ) was unable to mimic this effect. These data imply that although resiniferatoxin-induced translocation of protein kinase C in dorsal root ganglion neurones was mainly indirect, it also caused direct activation of a protein kinase C-like kinase in these cells.     

Epidermal growth factor increases P incorporation into phosphatidylcholine and protein kinase C activity in colon carcinoma cell line (HT29)

There are conflicting data about the effect of the epidermal growth factor (EGF) on protein kinase C (PKC) enzyme activity. The aim of our study was to find out which type of phospholipids [phosphatidylinositol 4,5-bisphosphate P14,5P₂ or the other phospholipids-phosphatidylcholine (PC) or phosphatidic acid (PA)] could be the source of 1,2-diacylglycerol (1,2-DAG) in PKC activation. In colon carcinoma cells (HT29) we observed a more than 2-fold increase in the PC pool and at the same time decreased tyrosine kinase activity (50%). With increasing incubation time EGF affects the pools of both phosphatidylinositols and other phospholipids parallel with the activation of the tyrosine kinase activity. EGF increases the activity of PKC in the HT29 cell line and PC could be the source of 1,2-DAG which may stimulate PKC activity.     

VEGF-C attenuates renal damage in salt-sensitive hypertension

Salt-sensitive hypertension is a major risk factor for renal impairment leading to chronic kidney disease. High-salt diet leads to hypertonic skin interstitial volume retention enhancing the activation of the tonicity-responsive enhancer-binding protein (TonEBP) within macrophages leading to vascular endothelial growth factor C (VEGF-C) secretion and NOS3 modulation. This promotes skin lymphangiogenesis and blood pressure regulation. Whether VEGF-C administration enhances renal and skin lymphangiogenesis and attenuates renal damage in salt-sensitive hypertension remains to be elucidated. Hypertension was induced in BALB/c mice by a high-salt diet. VEGF-C was administered subcutaneously to high-salt-treated mice as well as control animals. Analyses of kidney injury, inflammation, fibrosis, and biochemical markers were performed in vivo. VEGF-C reduced plasma inflammatory markers in salt-treated mice. In addition, VEGF-C exhibited a renal anti-inflammatory effect with the induction of macrophage M2 phenotype, followed by reductions in interstitial fibrosis. Antioxidant enzymes within the kidney as well as urinary RNA/DNA damage markers were all revelatory of abolished oxidative stress under VEGF-C. Furthermore, VEGF-C decreased the urinary albumin/creatinine ratio and blood pressure as well as glomerular and tubular damages. These improvements were associated with enhanced TonEBP, NOS3, and lymphangiogenesis within the kidney and skin. Our data show that VEGF-C administration plays a major role in preserving renal histology and reducing blood pressure. VEGF-C might constitute an interesting potential therapeutic target for improving renal remodeling in salt-sensitive hypertension.     

The Differential Role of Protein Kinase C Isozyme in the Rapid Induction of Neurofilament Phosphorylation by Nerve Growth Factor and Phorbol Esters in PC12 Cells

We examined the short-term regulation of the phosphorylation of the mid-sized neurofilament subunit (NF-M) by kinases which were activated in rat pheochromocytoma (PC12) cells by nerve growth factor (NGF) and/or 12-O-tetradecanoylphorbol 13-acetate (TPA). We found that NGF and TPA, alone or in combination, increased (a) the incorporation of [32P]Pi into NF-M and (b) the rate of conversion of NF-M from a poorly phosphorylated to a more highly phosphorylated form. This was not due to increased synthesis of NF-M, because NGF alone did not increase NF-M synthesis and TPA alone or TPA and NGF together inhibited the synthesis of NF-M. Further, an increase in calcium/phospholipid-dependent kinase (PKC) activity resulting from the treatment of PC12 cells with NGF and TPA was observed concomitant with the increased phosphorylation of NF-M. This PKC activity was determined to be derived from the PKC alpha and PKC beta isozymes. Finally, when PC12 cells were rendered PKC-deficient by treatment with 1 muM TPA for 24 h, NGF maintained the ability to induce an increase in NF-M phosphorylation, though not to the level attained in cells which were not PKC-deficient. These data suggest that NGF with or without TPA stimulates NF-M phosphorylation as a result of a complex series of events which include PKC-independent and PKC-dependent pathways.     

Different Mechanisms of β-Adrenoceptor Down-Regulation by Chronic Imipramine and Electroconvulsive Treatment: Possible Role for Protein Kinase C

The aim of this study was to find out how protein kinase C (PKC) is involved in down-regulation of the beta-adrenoceptor in cortical slices of rats subjected to antidepressant treatments. The responses of the cyclic AMP generating system to forskolin, isoproterenol, and noradrenaline were tested in the absence and presence of a PKC activator, 12-O-tetradecanoylphorbol 13-acetate (TPA). The antidepressive treatments applied were chronic administration of imipramine and electroconvulsive shock. The potentiating effect of the phorbol ester on cyclic AMP response to isoproterenol was retained in imipramine-treated animals and even accentuated in rats subjected to electroconvulsive treatment; the TPA effect on noradrenaline-induced cyclic AMP response was blunted in rats receiving imipramine, but augmented in those receiving electroconvulsive treatment. In imipramine-treated rats the beta-down-regulation was still evident in the presence of TPA; after electroconvulsive treatment the phorbol ester-induced potentiation was so high that no significant beta-down-regulation could be observed. No procedure affected the response to forskolin. The beta-down-regulation that develops during chronic imipramine treatment differs from that caused by chronic electroconvulsive treatment; in both cases it is not related to the direct effect on adenylate cyclase.     

Soil structural aspects of decomposition of organic matter by micro-organisms

Soil architecture is the dominant control over microbially mediated decomposition processes in terrestrial ecosystems. Organic matter is physically protected in soil so that large amounts of well-decomposable compounds can be found in the vicinity of largely starving microbial populations. Among the mechanisms proposed to explain the phenomena of physical protection in soil are adsorption of organics on inorganic clay surfaces and entrapment of materials in aggregates or in places inaccessible to microbes. Indirect evidence for the existence of physical protection in soil is provided by the occurrence of a burst of microbial activity and related increased decomposition rates following disruption of soil structures, either by natural processes such as the remoistening of a dried soil or by human activities such as ploughing. In contrast, soil compaction has only little effect on the transformation of ¹⁴C-glucose. Another mechanism of control by soil structure and texture on decomposition in terrestrial ecosystems is through their impact on microbial turnover processes. The microbial population is not only the main biological agent of decomposition in soil, it is also an important, albeit small, pool through which most of the organic matter in soil passes. Estimates on the relative importance of different mechanisms controlling decomposition in soil could be derived from results of combined tracer and modelling studies. However, suitable methodology to quantify the relation between soil structure and biological processes as a function of different types and conditions of soils is still lacking.     

Relationships of growth,stable carbon isotope composition and anatomical properties of leaf and xylem in seven mulberry cultivars: a hint towards drought tolerance

• The fast growth of mulberry depends on high water consumption, but considerable variations in drought tolerance exist across different cultivars. Physiological and anatomical mechanisms are important to plant survival under drought. However, few research efforts have been made to reveal the relationships of these two aspects in relation to drought tolerance.
• In this study, growth rates, leaf functional physiology and anatomical characteristics of leaf and xylem of 1‐year‐old saplings of seven mulberry cultivars at a common garden were compared. Their relationships were also explored.
• Growth, leaf physiology and anatomy were significantly different among the tested cultivars. Foliar stable carbon isotope composition (δ¹³C) was negatively correlated with growth rates, and closely related to several leaf and xylem anatomical traits. Particularly, leaf thickness, predicted hydraulic conductivity and vessel element length jointly contributed 77% of the variability in δ¹³C. Cultivar Wupu had small stomata, intermediate leaf thickness, the smallest hydraulically weighted vessel diameter and highest vessel number, and higher δ¹³C; Yunguo1 had high abaxial stomatal density, low specific leaf area, moderate hydraulic conductivity and δ¹³C; these are beneficial features to reduce leaf water loss and drought‐induced xylem embolism in arid areas. Cultivar Liaolu11 had contrasting physiological and anatomical traits compared with the previous two cultivars, suggesting that it might be sensitive to drought.
• Our findings indicate that growth and δ¹³C are closely associated with both leaf and xylem anatomical characteristics in mulberry, which provides fundamental information to assist evaluation of drought tolerance in mulberry cultivars and in other woody trees.

     

Heterochromatin and cytogenetic polymorphisms in Cebus apella (Cebidae,Platyrrhini)

Cytogenetic studies have been carried out in 39 specimens of C. apella of different origins. Three different morphologies, one affecting the long arm of chromosome 4 and two affecting pair 17, have been detected. In each case, they can be related by paracentric inversions. Heterochromatin polymorphisms affecting terminal or interstitial C+ regions have also been observed. The length of the terminal heterochromatic region in the long arms of chromosome 11 is variable in C. apella sp., in C. a. paraguayanus and absent in the C. a. nigritus specimens studied. Interstitial C + bands can be observed in the long arms of the biarmed chromosomes 4 and 6, and in the long arms of the acrocentric pairs 12, 13, 17, 18, 19, 20, and 21. Interstitial C + bands in the long arms of chromosomes 4, 12, 17, and 19 are present in all animals studied, although their size is variable, especially in the case of chromosomes 17 and 19. © 1995 Wiley-Liss, Inc.     

The most prevalent genetic cause of ALS-FTD,C9orf72 synergizes the toxicity of ATXN2 intermediate polyglutamine repeats through the autophagy pathway

The most common genetic cause for amyotrophic lateral sclerosis and frontotemporal dementia (ALS-FTD) is repeat expansion of a hexanucleotide sequence (GGGGCC) within the C9orf72 genomic sequence. To elucidate the functional role of C9orf72 in disease pathogenesis, we identified certain molecular interactors of this factor. We determined that C9orf72 exists in a complex with SMCR8 and WDR41 and that this complex acts as a GDP/GTP exchange factor for RAB8 and RAB39, 2 RAB GTPases involved in macroautophagy/autophagy. Consequently, C9orf72 depletion in neuronal cultures leads to accumulation of unresolved aggregates of SQSTM1/p62 and phosphorylated TARDBP/TDP-43. However, C9orf72 reduction does not lead to major neuronal toxicity, suggesting that a second stress may be required to induce neuronal cell death. An intermediate size of polyglutamine repeats within ATXN2 is an important genetic modifier of ALS-FTD. We found that coexpression of intermediate polyglutamine repeats (30Q) of ATXN2 combined with C9orf72 depletion increases the aggregation of ATXN2 and neuronal toxicity. These results were confirmed in zebrafish embryos where partial C9orf72 knockdown along with intermediate (but not normal) repeat expansions in ATXN2 causes locomotion deficits and abnormal axonal projections from spinal motor neurons. These results demonstrate that C9orf72 plays an important role in the autophagy pathway while genetically interacting with another major genetic risk factor, ATXN2, to contribute to ALS-FTD pathogenesis.