Effect of interleukin-6/interferon-beta 2 on glucocorticoid action in rat hepatoma cells

Reuber hepatoma cells (RHC) were treated 4 h with dexamethasone (dex), with and without simultaneous fibroblast-conditioned medium (cIL-6). A cytosol fraction, prepared in the presence of molybdate and dithiothreitol, was analyzed for [3H]dex (20 nM) binding in the presence and absence of 1 microM dex at 4 degrees C. Receptor levels declined from 76.0 fmol/mg at zero dex to 28.8 fmol/mg at 10 nM dex, and to 11.8 fmol/mg at 1 microM dex (P less than or equal to 0.05). cIL-6 plus 10 nM dex lowered binding to 18.3 fmol/mg (P less than or equal to 0.05), and treatment with cIL-6 alone diminished binding to 9.8 fmol/mg (P less than or equal to 0.05). Thus, cIL-6 diminished the number of available glucocorticoid receptors.     

Cystatin F is a cathepsin C-directed protease inhibitor regulated by proteolysis

Cystatins are a family of naturally occurring cysteine protease inhibitors, yet the target proteases and biological processes they regulate are poorly understood. Cystatin F is expressed selectively in immune cells and is the only cystatin to be synthesised as an inactive disulphide-linked dimeric precursor. Here, we show that a major target of cystatin F in different immune cell types is the aminopeptidase cathepsin C, which regulates the activation of effector serine proteases in T cells, natural killer cells, neutrophils and mast cells. Surprisingly, recombinant cystatin F was unable to inhibit cathepsin C in vitro even though overexpression of cystatin F suppressed cellular cathepsin C activity. We predicted, using structural models, that an N-terminal processing event would be necessary before cystatin F can engage cathepsin C and we show that the intracellular form of cystatin F indeed has a precise N-terminal truncation that creates a cathepsin C inhibitor. Thus, cystatin F is a latent protease inhibitor itself regulated by proteolysis in the endocytic pathway. By targeting cathepsin C, it may regulate diverse immune cell effector functions.     

Human inter-alpha-trypsin inhibitor. Synthesis and maturation in hepatoma HepG2 cells.

Inhibitor studies were performed on the two endopeptidase activities involved in proinsulin conversion in isolated insulin secretory granules [Davidson, Rhodes & Hutton (1988) Nature (London) 333, 93-96]. The active-site-directed peptides L-alanyl-L-arginyl-L-arginylmethyldimethylsulphonium and L-alanyl-L-lysyl-L-arginylmethyldimethylsulphonium inhibited these activities in accordance with the observed cleavage pattern, suggesting that the primary amino acid sequence of the dibasic site was an important determinant of the endopeptidase substrate specificities.     

Production of the cysteine proteinase inhibitor cystatin C by rat Sertoli cells.

The Sertoli cells of the rat testis produce cystatin C, a cysteine proteinase inhibitor. Primary culture of Sertoli cells secreted both unglycosylated and glycosylated forms of rat cystatin C. Despite the low concentration of cystatin C in rete testis fluid, equilibrium dissociation constants (Ki) for the interaction between cystatin C and lysosomal cathepsins indicate that this molecule could be involved in the local regulation of testicular cysteine proteinase activity which may be necessary for spermatogenesis and spermiogenesis.     

Inhibition of a cathepsin L-like cysteine protease by a chimeric propeptide-derived inhibitor

Like other papain-related cathepsins, congopain from Trypanosoma congolense is synthesized as a zymogen. We have previously identified a proregion-derived peptide (Pcp27), acting as a weak and reversible inhibitor of congopain. Pcp27 contains a 5-mer YHNGA motif, which is essential for selectivity in the inhibition of its mature form [Lalmanach, G., Lecaille, F., Chagas, J. R., Authié, E., Scharfstein, J., Juliano, M. A., and Gauthier, F. (1998) J. Biol. Chem. 273, 25112-25116]. In the work presented here, a homology model of procongopain was generated and subsequently used to model a chimeric 50-mer peptide (called H3-Pcp27) corresponding to the covalent linkage of an unrelated peptide (H3 helix from Antennapedia) to Pcp27. Molecular simulations suggested that H3-Pcp27 (pI = 9.99) maintains an N-terminal helical conformation, and establishes more complementary electrostatic interactions (E(coul) = -25.77 kcal/mol) than 16N-Pcp27, the 34-mer Pcp27 sequence plus the 16 native residues upstream from the proregion (E(coul) = 0.20 kcal/mol), with the acid catalytic domain (pI = 5.2) of the mature enzyme. In silico results correlated with the significant improvement of congopain inhibition by H3-Pcp27 (K(i) = 24 nM), compared to 16N-Pcp27 (K(i) = 1 microM). In addition, virtual alanine scanning of H3 and 16N identified the residues contributing most to binding affinity. Both peptides did not inhibit human cathepsins B and L. In conclusion, these data support the notion that the positively charged H3 helix favors binding, without modifying the selectivity of Pcp27 for congopain.     

Synthesis of serum proteins by a clonal strain of rat hepatoma cells

The production of rat serum proteins by a clonal strain of rat hepatoma cells (MH₁C₁) has been investigated by growing the cells in a serum-free medium. By immunodiffusion, immunoelectrophoresis and autoradiography we have demonstrated that the cells produce and secrete at least 12 rat serum proteins.     

Angiotensinogen production by rat hepatoma cells is stimulated by B cell stimulatory factor 2/interleukin-6

Angiotensinogen has been identified as one of the acute-phase reactants. In vitro studies were carried out using the Reuber H35 hepatoma cell line to identify the species of cytokines contributing to the increased synthesis of angiotensinogen in the liver. Angiotensinogen secretion by H35 cells was maximally increased 4-fold by the addition of 10(-7) M dexamethasone. Under this condition, angiotensinogen secretion was further stimulated by B cell stimulatory factor 2/interleukin-6 (IL-6, 50 U/ml), but not by interleukin-1 or interferon-alpha. In the absence of glucocorticoid, IL-6 did not affect angiotensinogen secretion by H35 cells, indicating that the presence of glucocorticoid is required for the stimulatory activity of IL-6. These results suggest that IL-6 is a mediator responsible for the increased synthesis of angiotensinogen in the liver during acute inflammation.     

The mitochondrial protease HtrA2 is regulated by Parkinson's disease-associated kinase PINK1

In mice, targeted deletion of the serine protease HtrA2 (also known as Omi) causes mitochondrial dysfunction leading to a neurodegenerative disorder with parkinsonian features. In humans, point mutations in HtrA2 are a susceptibility factor for Parkinson's disease (PARK13 locus). Mutations in PINK1, a putative mitochondrial protein kinase, are associated with the PARK6 autosomal recessive locus for susceptibility to early-onset Parkinson's disease. Here we determine that HtrA2 interacts with PINK1 and that both are components of the same stress-sensing pathway. HtrA2 is phosphorylated on activation of the p38 pathway, occurring in a PINK1-dependent manner at a residue adjacent to a position found mutated in patients with Parkinson's disease. HtrA2 phosphorylation is decreased in brains of patients with Parkinson's disease carrying mutations in PINK1. We suggest that PINK1-dependent phosphorylation of HtrA2 might modulate its proteolytic activity, thereby contributing to an increased resistance of cells to mitochondrial stress.     

Cystathionine beta-synthase is coordinately regulated with proliferation through a redox-sensitive mechanism in cultured human cells and Saccharomyces cerevisiae

Cystathionine beta-synthase (CBS) catalyzes the condensation of serine with homocysteine to form cystathionine and occupies a crucial regulatory position between the methionine cycle and the biosynthesis of cysteine by transsulfuration. Analysis of CBS activity under a variety of growth conditions indicated that CBS is coordinately regulated with proliferation in both yeast and human cells. In batch cultures of Saccharomyces cerevisiae, maximal CBS activities were observed in the exponential phase of cells grown on glucose, while growth-arrested cultures or those growing non-fermentatively on ethanol or glycerol had approximately 3-fold less activity. CBS activity assays and Western blotting indicated that growth-specific regulation of CBS is evolutionarily conserved in a range of human cell lines. CBS activity was found to be maximal during proliferation and was reduced two- to five-fold when cells became quiescent at confluence. In cultured HepG2 cells, the human CBS gene is induced by serum and basic fibroblast growth factor and is downregulated, but not abolished, by contact inhibition, serum-starvation, nutrient depletion, or the induction of differentiation. Consequently, for certain cell types, CBS may represent a novel marker of both differentiation and proliferation. The intracellular level of the CBS regulator compound, S-adenosylmethionine, was found to reflect the proliferation status of both yeast and human cells, and as such, constitutes an additional mechanism for proliferation-specific regulation of human CBS. Our data indicates that screening compounds for the ability to affect transsulfuration in cultured cell models must take proliferation status into account to avoid masking regulatory interactions that may be of significance in vivo.     

Expression of α-amylases, carbohydrate metabolism, and autophagy in cultured rice cells is coordinately regulated by sugar nutrient

A rice suspension cell culture system has been established to study how sugar depletion regulates α-amylase expression, carbohydrate metabolism, and other physiological and cellular changes. It is shown here that a group of 44 kDa α-amylases are constitutively expressed whether or not the cells are starved of sucrose. However, expression of a new group of α-amylases of 46 kDa is dramatically induced when cells are starved of sucrose. Cellular sugar and starch were rapidly consumed and metabolic activity was decreased in the starved cells. Extensive autophagy also occurred in the starved cells, which caused an increase in vacuolar volume and degradation of cytoplasmic constituents including amyloplasts. Immunocytochemical studies revealed that α-amylases are localized in starch granules within amyloplasts, in cell walls, and in some of the vacuoles. The presence of putative signal sequences in the N-termini of nine rice α-amylases suggests hitherto unidentified pathways for import of α-amylases into amyloplasts. The studies show that differential α-amylase expression, carbohydrate metabolism, metabolic activity, and vacuolar autophagy are coordinately regulated by the sugar level in the medium. As the starved suspension cells exhibit some sugar-regulated characteristics of α-amylase expression in germinating rice embryos as well as physiological changes similar to those in senescing cells, this system represents an ideal tool for studying cellular, biochemical, and molecular biological aspects of α-amylase gene regulation, carbohydrate metabolism, senescence, and protein targeting in plants.     

A cluster of disease resistance genes in Arabidopsis is coordinately regulated by transcriptional activation and RNA silencing

The RPP5 (for recognition of Peronospora parasitica 5) locus in the Arabidopsis thaliana Columbia strain contains a cluster of paralogous disease Resistance (R) genes that play important roles in innate immunity. Among the R genes in this locus, RPP4 confers resistance to two races of the fungal pathogen Hyaloperonospora parasitica, while activation of SNC1 (for suppressor of npr1-1, constitutive 1) results in the resistance to another race of H. parasitica and to pathovars of the bacterial pathogen Pseudomonas syringae through the accumulation of salicylic acid (SA). Here, we demonstrate that other Columbia RPP5 locus R genes can be induced by transgenic overexpression of SNC1, which itself is regulated by a positive amplification loop involving SA accumulation. We also show that small RNA species that can target RPP5 locus R genes are produced in wild-type plants and that these R genes can be cosuppressed in transgenic plants overexpressing SNC1. Steady state expression levels of SNC1 increase in some mutants (dcl4-4, ago1-36, and upf1-5) defective in RNA silencing as well as in transgenic plants expressing the P1/Helper Component-Protease viral suppressor of RNA silencing. However, steady state levels of small RNA species do not change in mutants that upregulate SNC1. These data indicate many Columbia RPP5 locus R genes can be coordinately regulated both positively and negatively and suggest that the RPP5 locus is poised to respond to pathogens that disturb RNA silencing.     

Synthesis of amino acid cofactor in cysteine dioxygenase is regulated by substrate and represents a novel post-translational regulation of activity

Cysteine dioxygenase (CDO) catalyzes the conversion of cysteine to cysteinesulfinic acid and is important in the regulation of intracellular cysteine levels in mammals and in the provision of oxidized cysteine metabolites such as sulfate and taurine. Several crystal structure studies of mammalian CDO have shown that there is a cross-linked cofactor present in the active site of the enzyme. The cofactor consists of a thioether bond between the gamma-sulfur of residue cysteine 93 and the aromatic side chain of residue tyrosine 157. The exact requirements for cofactor synthesis and the contribution of the cofactor to the catalytic activity of the enzyme have yet to be fully described. In this study, therefore, we explored the factors necessary for cofactor biogenesis in vitro and in vivo and examined what effect cofactor formation had on activity in vitro. Like other cross-linked cofactor-containing enzymes, formation of the Cys-Tyr cofactor in CDO required a transition metal cofactor (Fe(2+)) and O(2). Unlike other enzymes, however, biogenesis was also strictly dependent upon the presence of substrate. Cofactor formation was also appreciably slower than the rates reported for other enzymes and, indeed, took hundreds of catalytic turnover cycles to occur. In the absence of the Cys-Tyr cofactor, CDO possessed appreciable catalytic activity, suggesting that the cofactor was not essential for catalysis. Nevertheless, at physiologically relevant cysteine concentrations, cofactor formation increased CDO catalytic efficiency by approximately 10-fold. Overall, the regulation of Cys-Tyr cofactor formation in CDO by ambient cysteine levels represents an unusual form of substrate-mediated feed-forward activation of enzyme activity with important physiological consequences.     

Linoleate and linolenate desaturation by rat hepatoma cells

Morris 7777 rat hepatoma cells in culture possess high delta 6 and delta 5 desaturase activities over linolenic acid added to the medium as albumin or alpha-fetoprotein complexes. After 2 hours incubation with [1-14C] linolenic acid (7 microM), around 40% of the radioactivity was recovered in other polyene fatty acids, mainly pentaenes. After 24 hours incubation with this substrate the polyene derivatives raised to more than 60%. However, [1-14C] linoleic acid was poorly converted to other polyene fatty acids. Linoleic acid up to 58 microM concentration in the medium do not inhibited linolenic acid desaturation. Long-term supplementation with 50 microM linoleic or linolenic acid, which modified the fatty acid profile of hepatoma lipids, enhanced the desaturase activities against linoleic acid. Desaturase activities were not affected by the fatty acid protein carrier, alpha-fetoprotein or albumin.     

ERK is regulated by sodium-proton exchanger in rat aortic vascular smooth muscle cells

The purposes of this study were to test 1) the relationship between two widely studied mitogenic effector pathways, and 2) the hypothesis that sodium-proton exchanger type 1 (NHE-1) is a regulator of extracellular signal-regulated protein kinase (ERK) activation in rat aortic smooth muscle (RASM) cells. Angiotensin II (Ang II) and 5-hydroxytryptamine (5-HT) stimulated both ERK and NHE-1 activities, with activation of NHE-1 preceding that of ERK. The concentration-response curves for 5-HT and Ang II were superimposable for both processes. Inhibition of NHE-1 with pharmacological agents or by isotonic replacement of sodium in the perfusate with choline or tetramethylammonium greatly attenuated ERK activation by 5-HT or Ang II. Similar maneuvers significantly attenuated 5-HT- or Ang II-mediated activation of MEK and Ras but not transphosphorylation of the epidermal growth factor (EGF) receptor. EGF receptor blockade attenuated ERK activation, but not NHE-1 activation by 5-HT and Ang II, suggesting that the EGF receptor and NHE-1 work in parallel to stimulate ERK activity in RASM cells, converging distal to the EGF receptor but at or above the level of Ras in the Ras-MEK-ERK pathway. Receptor-independent activation of NHE-1 by acute acid loading of RASM cells resulted in the rapid phosphorylation of ERK, which could be blocked by pharmacological inhibitors of NHE-1 or by isotonic replacement of sodium, closely linking the proton transport function of NHE-1 to ERK activation. These studies identify NHE as a new regulator of ERK activity in RASM cells.