Insulin-like growth factor-binding protein-5 is cleaved by physiological concentrations of thrombin

Insulin-like growth factor (IGF)-binding protein-5 (IGFBP-5) is cleaved by a serine protease that is secreted by fibroblasts and porcine smooth muscle cells (pSMC) in culture. To investigate whether other serine proteases could cleave this substrate at physiologically relevant concentrations, we determined the proteolytic effects of thrombin on IGFBP-5. Human alpha-thrombin (0.0008 NIH U/ml) cleaved IGFBP-5 into 24-, 23-, and 20-kDa non-IGF-I-binding fragments. Cleavage occurred at a physiologically relevant thrombin concentration. The effect was specific for IGFBP-5, as other forms of IGFBPs, e.g. IGFBP-1, IGFBP-2, and IGFBP-4 were not cleaved by thrombin. Although IGFBP-3 was cleaved by thrombin, this effect required a 50-fold greater thrombin concentration. [35S]Methionine labeling followed by immunoprecipitation confirmed that IGFBP-5 that was constitutively synthesized by pSMC cultures was also degraded by thrombin into 24-, 23-, and 20-kDa fragments. The binding of IGF-I to IGFBP-5 partially inhibited IGFBP-5 degradation by thrombin, and an IGF analog that does not bind to IGFBP-5 had no effect. Thrombin did not account for the serine protease activity that had been shown previously to be present in pSMC-conditioned medium. This was proven by showing that 1) no immunoreactive thrombin could be detected in the pSMC-conditioned medium; 2) the IGFBP-5 fragments that were generated by thrombin showed three cleavage sites (Arg192-Ala193, Arg156-Ile157, and Lys120-His121), whereas the serine protease in conditioned medium cleaves IGFBP-5 at a different site; and 3) hirudin had no effect on IGFBP-5 cleavage by the protease in pSMC medium; however, it inhibited IGFBP-5 degradation by thrombin. To determine the physiological significance of IGFBP-5 cleavage, the effect of an IGFBP-5 mutant that is resistant to cleavage by the pSMC protease and has been shown to inhibit IGF-I actions in pSMC was determined. This mutant inhibited IGF-I-stimulated DNA synthesis, but if thrombin was added simultaneously, IGF-I was fully active. In summary, physiological concentrations of thrombin degrade IGFBP-5. Degradation can be blocked by hirudin and is partially inhibited by IGF-I binding. Generation of active thrombin in vessel walls may be a physiologically relevant mechanism for controlling IGF-I bioactivity.     

Phorbol ester tumor promoters regulate insulin-like growth factor-binding protein-4 proteolysis

Cultured human fibroblasts secrete a specific protease that alters extracellular insulin-like growth factor-binding protein-4 (IGFBP-4) structure and function. This enzyme appears to be secreted in a latent form and requires IGFs for activation. To study regulation of the IGFBP-4 protease, we treated normal adult human fibroblasts with various hormones and growth regulatory factors, and collected the human fibroblast-conditioned medium (HFCM) for analysis of IGFBP-4 protease activity. The IGFBP-4 protease assay involved incubation of 50 microliters HFCM with or without 5 nM IGF-II for 6 h at 37 C under cell-free conditions; IGF-activated IGFBP-4 hydrolysis was assessed by Western ligand blotting. In HFCM from cells treated with vehicle, GH, insulin, epidermal growth factor, steroid hormones, or forskolin, IGF-II induced the select loss of detectable IGFBP-4 during the assay. In contrast, IGFBP-4 levels were maintained when HFCM from cells treated with phorbol ester tumor promoters was incubated with IGF-II under cell-free conditions. Hydrolysis of [125I]IGFBP-4 to 18,000 and 14,000 mol wt fragments also was prevented in HFCM from cells treated with phorbol esters. Phorbol esters had no effect on endogenous or exogenous IGFBP-4 proteolysis when added directly to HFCM during the assay, however. Treatment of cells with actinomycin-D or cycloheximide could prevent a phorbol ester-induced block of IGF-dependent IGFBP-4 proteolysis. These data suggest that phorbol ester tumor promoters stimulate human fibroblasts to produce and secrete an inhibitor of the IGFBP-4 proteolytic reaction. Alterations in IGFBP-4 protease activity could affect local IGF action through regulation of IGFBP-4 availability.     

Substrates and inhibitors of human T-cell leukemia virus type I protease

HTLV-I is an oncogenic retrovirus that is associated with adult T-cell leukemia. HTLV-I protease and HTLV-I protease fused to a deca-histidine containing leader peptide (His-protease) have been cloned, expressed, and purified. The refolded proteases were active and exhibited nearly identical enzymatic activities. To begin to characterize the specificity of HTLV-I, we measured protease cleavage of peptide substrates and inhibition by protease inhibitors. HTLV-I protease cleavage of a peptide representing the HTLV-I retroviral processing site P19/24 (APQVLPVMHPHG) yielded Km and kcat values of 470 microM and 0.184 s-1 while cleavage of a peptide representing the processing site P24/15 (KTKVLVVQPK) yielded Km and kcat values of 310 microM and 0.0060 s-1. When the P1' proline of P19/24 was replaced with p-nitro-phenylalanine (Nph), the ability of HTLV-I protease to cleave the substrate (APQVLNphVMHPL) was improved. Inhibition of HTLV-I protease and His-protease by a series of protease inhibitors was also tested. It was found that the Ki values for inhibition of HTLV-I protease and His-protease by a series of pepsin inhibitors ranged from 7 nM to 10 microM, while the Ki values of a series of HIV-1 protease inhibitors ranged from 6 nM to 127 microM. In comparison, the Ki values for inhibition of pepsin by the pepsin inhibitors ranged from 0.72 to 19.2 nM, and the Ki values for inhibition of HIV-1 protease by the HIV protease inhibitors ranged from 0.24 nM to 1.0 microM. The data suggested that the substrate binding site of HTLV-I protease is different from the substrate binding sites of pepsin and HIV-1 protease, and that currently employed HIV-1 protease inhibitors would not be effective for the treatment of HTLV-I infections.     

Differential expression and biological effects of insulin-like growth factor-binding protein-4 and -5 in vascular smooth muscle cells

Insulin-like growth factor-I (IGF-I) plays an important role in regulating vascular smooth muscle cell (VSMC) proliferation, migration, and apoptosis. The bioactivity of IGF-I is modulated by a group of high affinity, specific binding proteins (IGF-binding proteins; IGFBPs) that are present in the interstitial fluid. Previously, we have reported that porcine VSMCs synthesize and secrete IGF-I and several forms of IGFBPs, including IGFBP-2, IGFBP-4, and IGFBP-5. In this study, we examined the role of autocrine/paracrine secreted IGF-I in controlling the expression of IGFBP-4 and IGFBP-5 as well as the effects of these IGFBPs in modulating the cellular replication response to IGF-I. The concentrations of IGFBP-4 in the conditioned medium increased significantly from <50 ng/ml to 742 +/- 105 ng/ml. This increase was associated with a decrease in the activity of an IGF-I-regulated IGFBP-4 protease. In contrast, the synthesis of IGFBP-5 was inversely correlated with culture density, and its concentration decreased from 792 +/- 91 to 44 +/- 14 ng/ml. IGFBP-5 mRNA in sparse cultures was 3-fold higher compared with those in confluent cultures. This culture density-dependent change in IGFBP-5 mRNA correlated closely with endogenous IGF-I levels. Since treatment of VSMC with exogenous IGF-I increased IGFBP-5 mRNA levels, we neutralized the effect of endogenously secreted IGF-I with an anti-IGF-I antibody to determine if it would alter IGFBP-5 mRNA abundance. This resulted in a 4.4-fold decrease in IGFBP-5 mRNA levels. When added together with IGF-I, exogenous IGFBP-4 inhibited IGF-I-induced DNA synthesis in a concentration-dependent manner. IGFBP-5, on the other hand, potentiated the effect of IGF-I. Therefore, IGFBP-4 and IGFBP-5 appear to be differentially regulated by autocrine/paracrine IGF-I through distinct mechanisms. These two proteins, in turn, play opposing roles in modulating IGF-I action in stimulating VSMC proliferation.     

Porcine insulin-like growth factor (IGF)-binding protein-3 elicits bi-phasic effects on IGF-I stimulated DNA synthesis in neonatal porcine skin fibroblasts

The present study was undertaken to investigate the effects of porcine IGFBP-3 on IGF-I stimulated DNA synthesis in neonatal porcine skin fibroblasts. IGF-I stimulated DNA synthesis in skin fibroblasts in a concentration dependent manner. DNA synthesis was maximally stimulated by 5 to 20 fold at 5 nM IGF-I; half-maximal stimulation was observed at approximately 1 nM IGF-I. Co-incubation of IGFBP-3 with a maximally effective dose of IGF-I (10 nM) did not inhibit the stimulatory effects of IGF-I on DNA synthesis. In contrast, when IGFBP-3 at concentrations of 0 to 20 nM was co-incubated with 1 nM IGF-I, a bi-phasic dose response was observed with IGFBP-3 being inhibitory only at a 10 to 20 fold molar excess to IGF-I. Based on the approximately equal molar ratio of IGFBP-3:IGF-I present in the circulation of control and pST-treated pigs our results suggest that IGFBP-3 does not inhibit the mitogenic effects of IGF-I. In summary, these results indicate that the combination of IGFBP-3 with IGF-I optimizes mitogenic signalling via the type I IGF receptor and suggest that IGFBP-3 does not inhibit the effects of ST that are mediated by IGF-I.     

Effects of prior exercise on exercise-induced arterial hypoxemia in young women

Insulin-like growth factor binding proteins (IGFBP) proteases have been proposed to be involved in changes of serum IGFBP pattern during pregnancy. IGFBP-4 and -5 are degraded specifically by proteases in pregnancy serum in vitro, whereas IGFBP-3 proteolytic activity was also detected in nonpregnancy serum. To identify and characterize IGFBP proteases, human pregnancy serum was fractionated by size exclusion chromatography revealing IGFBP-4 protease activities in fractions coeluting with proteins of approximately 600-kDa and 50- to 100-kDa molecular mass. In both fractions, a predominant 50-kDa gelatinase was found, suggesting that parts of the gelatinase activity might aggregate or are complexed with other proteins forming a higher molecular complex. Hydroxyapatite chromatography and chromatofocusing of the 50- to 100-kDa serum fraction showed that the IGFBP-4 protease and the 50-kDa gelatinase activity were copurified. When the 50-kDa gelatinase-containing band was excised from the polyacrylamide gel, it exhibited IGFBP-4 proteolytic activity, resulting in the formation of 17- and 10-kDa fragments. [125I] IGFBP substrate zymography combined with fragment blotting showed that the 1,10-phenanthroline-sensitive 50-kDa protease activity purified by chromatofocusing also cleaved IGFBP-3 and -5. Other proteases detected in pregnancy serum fractions with Mr estimates of 79-, 30-, and 22-kDa degraded IGFBP-3 and -5 but not IGFBP-4. [125I] IGFBP-5 substrate zymography revealed that the 30-kDa IGFBP protease was inhibited by serine protease inhibitors. Whereas 1,10-phenanthroline inhibited the IGFBP proteolytic activity in the solution assay, serine protease inhibitors failed to affect proteolysis, indicating the predominant contribution of the metalloproteinase to IGFBP proteolysis. Tissue inhibitors of matrix metalloproteinases-1 and -2 revealed weak or no inhibition of IGFBP-4 and -5 proteolytic activity, whereas a hydroxamic acid-based inhibitor, potentially inhibiting disintegrin metalloproteases, completely prevented the proteolysis of IGFBPs. Whereas no specific immunoreactivity of the 50-kDa protein with antimatrix metalloproteinase-1, -2, -3, -9, or -13 antibodies was observed, antidisintegrin domain-specific antibodies bound to the 50-kDa gelatinase. These studies provide the first direct biochemical evidence that human pregnancy serum contains a 50-kDa IGFBP protease with properties of a soluble disintegrin metalloproteinase that appears to be potentially involved in regulating IGF bioavailability for placental and fetal growth.     

Effect of insulin and insulin-like growth factor-1 on vascular smooth muscle cells

Non-insulin-dependent diabetes mellitus, obesity, and essential hypertension are associated with hyperinsulinemia that results from insulin resistance and insulin has been reported to accelerate atherosclerosis. We studied the effects of insulin and insulin-like growth factor-1 (IGF-1) on the growth of porcine vascular smooth muscle cells and on the synthesis of extracellular matrix. The cells were cultured 3-8 changes of Dulbecco's modified Eagle's medium (DMEM) with 10% FCS. Subconfulent cells were put in wells 1 x 10(4) or 1 x 10(5) cells/well in DMEM with or without insulin or IGF-1. The number of cells was counted, and protein and DNA synthesis, expression of genes for collagen alpha1(1), and collagen synthesis were measured. Insulin (0, 16, and 160 nM) and IGF-1 (0, 1, 31, and 13.1 nM) increased number of cells by 50% and 40%, in a dose-dependent manner. Protein and DNA synthesis were also increased by insulin (3.8 and 3.0 times) and by IGF-1 (3.9 and 1.8 time). Collaged protein synthesis was increased 2.3-fold by IGF-1 at 13.1 nM, and insulin (16,000 nM) caused a 26.5-fold increase. Levels of collagen alpha1(1) mRNA were also increased by both insulin and IGF-1. These results suggest that insulin and IGF-1 can cause vascular hyperplasia associated with increased collagen synthesis, which indicates that insulin, IGF-1, or both may have an important role in vascular growth.     

Human immunodeficiency virus protease ligand specificity conferred by residues outside of the active site cavity

To gain greater understanding of the structural basis of human immunodeficiency virus (HIV) protease ligand specificity, we have crystallized and determined the structures of the HIV-1 protease (Val32Ile, Ile47Val, Val82Ile) triple mutant and simian immunodeficiency virus (SIV) protease in complex with SB203386, a tripeptide analogue inhibitor containing a C-terminal imidazole substituent as an amide bond isostere. SB203386 is a potent inhibitor of HIV-1 protease (Ki = 18 nM) but shows decreased inhibition of the HIV-1 protease (Val32Ile, Ile47Val, Val82Ile) triple mutant (Ki = 112 nM) and SIV protease (Ki = 960 nM). Although SB203386 binds in the active site cavity of the triple mutant in a similar fashion to its binding to the wild-type HIV-1 protease [Abdel-Meguid et al. (1994) Biochemistry 33, 11671], it binds to SIV protease in an unexpected mode showing two inhibitor molecules each binding to half of the active site. Comparison of these two structures and that of the wild-type HIV-1 protease bound to SB203386 reveals that HIV protease ligand specificity is imparted by residues outside of the catalytic pocket, which causes subtle changes in its shape. Furthermore, this work illustrates the importance of structural studies in order to understand the structure-activity relationship (SAR) between related enzymes.     

Role of proteolysis in apoptosis: involvement of serine proteases in internucleosomal DNA fragmentation in immature thymocytes

Three chemically distinct serine, but not cysteine, protease inhibitors (phenylmethylsulphonyl fluoride, N-tosyl-L-phenylalanylchloromethyl ketone and 3,4-dichloroisocoumarin) prevented, in a dose-dependent manner, the characteristic apoptotic internucleosomal DNA cleavage (DNA ladder) typically observed in thymocytes in response to dexamethasone and teniposide VM-26. This effect was not the result of a direct inhibition of the Ca2+,Mg(2+)-dependent endonuclease, since oligonucleosomal DNA cleavage occurred in the presence of these inhibitors in isolated nuclei. The proteolytic step occurred at a very early stage of apoptosis, and preincubation of thymocytes with the inhibitors before dexamethasone or teniposide VM-26 were added irreversibly suppressed ladder formation. This implied that the cellular effector(s) of these compounds preexisted and were not resynthesized in response to the inducers of apoptosis. Serine protease inhibitors also suppressed apoptotic cell shrinkage and complete nuclear collapse, suggesting that these morphological changes were directly related to internucleosomal fragmentation of DNA. However, the serine protease inhibitors did not prevent high molecular weight DNA cleavage (> 50 kilobases) that preceded the ladder formation and thymocytes still died by apoptosis. This supported the view that internucleosomal DNA cleavage, considered to be the biochemical marker of apoptosis, might in fact be a late and dispensable step and that the newly described high molecular weight DNA cleavage might be a better indicator of apoptosis.     

Proteolysis of Saccharomyces cerevisiae RNase H1 in E coli

Expression of S cerevisiae RNase H1 in E coli leads to the formation of a proteolytic product with a molecular mass of 30 kDa that is derived from the 39-kDa full length protein. The 30-kDa form retains RNase H1 activity, as determined by renaturation gel assay. The amount of proteolysis observed depends on the procedure used in preparing the cell extracts for protein analysis. The cleavage site on the amino acid sequence of the 39-kDa RNase H1 was determined by N-terminal sequence analysis of the 30-kDa proteolytic form. The cut occurs between two arginines located at the amino terminus region of the protein. The pattern of proteolysis was examined for both the wild-type RNase H1 and a mutant RNase H1 that was constructed in this work. In the mutant the second arginine of the cleavage site was changed to a lysine. Comparisons of the expression of the wild-type and altered protein in two different E coli strains demonstrate that the protease responsible for the degradation has a specificity very similar to that of the OmpT protease. However, the proteolysis observed in an OmpT background in extracts, prepared by boiling the cells in SDS containing buffer, indicates that the protease may, unlike OH108.     

Effects of treatment with megestrol acetate, aminoglutethimide, or formestane on insulin-like growth factor (IGF) I and II, IGF-binding proteins (IGFBPs), and IGFBP-3 protease status in patients with advanced breast cancer

The effects of treatment with the aromatase inhibitors aminoglutethimide (AG) and formestane or the synthetic progestin megestrol acetate (MA) on plasma levels of insulin-like growth factor I (IGF-1), IGF-II, IGF-binding proteins (IGFBPs), and IGFBP-3 protease status were investigated in 39 patients suffering from advanced breast cancer. Treatment with AG and MA elevated plasma levels of IGF-I by mean values of 27% (n = 15; P < 0.025) and 81% (n = 7; P < 0.025), respectively, whereas treatment with formestane had no effect (n = 13). Treatment with AG increased plasma levels of IGFBP-2, as evaluated by Western blotting (P < 0.01). MA caused a significant reduction in IGFBP-3 protease activity (mean reduction, 69%; P < 0.05). These alterations in plasma IGF-I and IGFBP-3 protease activity were reversed 4 weeks after terminating MA therapy (n = 8; P < 0.025). Taken together, 13 of 15 patients had reduced IGFBP-3 protease activity during treatment with MA compared to the control situation (P < 0.0025). Total levels of IGFBP-3 as measured by RIA were moderately elevated by treatment with MA (mean increase, 19%; P < 0.05), and Western immunoblotting revealed an increase in the amount of intact IGFBP-3 and reduced amounts of IGFBP-3 in the modified form. None of the treatment modalities had any influence on plasma levels of IGF-II. The increase in the plasma IGF-I concentration seen during treatment with MA may be secondary to an increased level of intact IGFBP-3. This could reflect an alteration in IGF availability that contributes to the antitumor effect of MA.     

The study of individual differences as a method for dividing into stages the acquisition of a complex reflex

Cultured hepatic stellate cells (HSCs), the cell type primarily involved in the progression of liver fibrosis, secrete insulin-like growth factor-I (IGF-I) and IGF binding protein (IGFBP) activity. IGF-I exerts a mitogenic effect on HSCs, thus potentially contributing to the fibrogenic process in an autocrine fashion. However, IGF-I action is modulated by the presence of specific IGFBPs that may inhibit and/or enhance its biologic effects. Therefore, we examined IGFBP-1 through IGFBP-6 mRNA and protein expression in HSCs isolated from human liver and activated in culture. Regulation of IGFBPs in response to IGF-I and other polypeptide growth factors involved in the hepatic fibrogenic process was also assessed. RNase protection assays and ligand blot analysis demonstrated that HSCs express IGFBP-2 through IGFBP-6 mRNAs and release detectable levels of IGFBP-2 through IGFBP-5. Because IGF-I, platelet-derived growth factor-BB (PDGF-BB), and transforming growth factor-beta (TGF-beta) stimulate HSC proliferation and/or matrix production, we tested their effect on IGFBPs released by HSCs. IGF-I induced IGFBP-3 and IGFBP-5 proteins in a time-dependent manner without an increase in the corresponding mRNAs. IGFBP-4 protein levels decreased in response to IGF-I. TGF-beta stimulated IGFBP-3 mRNA and protein but decreased IGFBP-5 mRNA and protein. In contrast, PDGF-BB failed to regulate IGFBPs compared with controls. Recombinant human IGFBP-3 (rhIGFBP-3) was then tested for its effect on IGF-I-induced mitogenesis in HSCs. rhIGFBP-3 inhibited IGF-I-stimulated DNA synthesis in a dose-dependent manner, with a peak effect observed at 25 nM IGFBP-3. Because TGF-beta is highly expressed in cirrhotic liver tissue, we determined whether IGFBP-3 mRNA expression is increased in liver biopsies obtained from patients with an active fibroproliferative response due to viral-induced chronic active hepatitis. In the majority of these samples, IGFBP-3 mRNA was increased compared with normal controls. These findings indicate that human HSCs, in their activated phenotype, constitutively produce IGFBPs. IGF-I and TGF-beta differentially regulate IGFBP-3, IGFBP-4, and IGFBP-5 expression, which, in turn, may modulate the in vitro and in vivo action of IGF-I.     

Rabeprazole in treatment of acid peptic diseases: results of three placebo-controlled dose-response clinical trials in duodenal ulcer, gastric ulcer, and gastroesophageal reflux disease (GERD). The Rabeprazole Study Group

The binding of 125I-factor Xa to human aortic smooth muscle cell (SMC) monolayers was studied. At 4 degreesC, 125I-factor Xa bound to a single class of binding sites with a dissociation constant value of 3.6+/-0.7 nM and a binding site density of 11,720+/-1,240 sites/cell (n = 9). 125I-factor Xa binding was not affected by factor X, thrombin, or by DX9065, a direct inhibitor of factor Xa, but was inhibited by factor Xa (IC50 = 5.4+/-0.2 nM; n = 9) and by antibodies specific for the effector cell protease receptor 1 (EPR-1), a well-known receptor of factor Xa on various cell types. A factor X peptide duplicating the inter-EGF sequence Leu83-Leu88-(Gly) blocked the binding of 125I-factor Xa to these cells in a dose-dependent manner (IC50 = 110+/-21 nM). Factor Xa increased phosphoinositide turnover in SMCs and when added to SMCs in culture was a potent mitogen. These effects were inhibited by DX9065 and by antibodies directed against EPR-1 and PDGF. Increased expression of EPR-1 was identified immunohistochemically on SMCs growing in culture and in SMCs from the rabbit carotid artery after vascular injury. When applied locally to air-injured rabbit carotid arteries, antibodies directed against EPR-1 (100 mug/ artery) strongly reduced myointimal proliferation 14 d after vascular injury (65-71% inhibition, P < 0.01). DX9065 (10 mg/kg, subcutaneous) inhibited myointimal proliferation significantly (43% inhibition, P < 0.05). These findings indicate that SMCs express functional high affinity receptors for factor Xa related to EPR-1, which may be of importance in the regulation of homeostasis of the vascular wall and after vascular injury.     

Growth factor regulation of insulin-like growth factor (IGF) binding proteins (IGFBP) and preadipocyte differentiation in porcine stromal-vascular cell cultures

The influence of anti-IGF-1 and anti-transforming growth factor beta (TGF-beta) neutralizing antibodies on preadipocyte differentiation and secretion of IGFBPs was examined in serum free porcine stromal-vascular cultures. Cultures were stained for morphological analysis and conditioned media were collected for: TGF-beta determination by ELISA, IGF-1 by RIA, and IGFBP analysis by ligand blotting. After 6 d of treatment, anti-TGF-beta increased fat proportions by 2.7 fold compared to controls. Anti-IGF-1 decreased fat cell proportions by 14-fold. Anti-TGF-beta increased concentrations of IGF-1 5.8-fold and IGFBP-2 and IGFBP-3 by 8- and 7-fold in conditioned media whereas IGFBP-4 decreased 5-fold. Anti-IGF-1 increased concentrations of IGFBP-2 and 3 by 9- and 35-fold, respectively. TGF-beta increased concentrations of IGFBP-1, 2 and 3 by 3-fold, 18-fold and 3-fold, respectively, after 9 d in culture (6 d of treatment). There was no change in TGF-beta levels in anti-IGF-1 treated cultures compared to controls. Control antibodies and negative controls had no effect. These results provide evidence that endogenously produced IGF-1 and TGF-beta has a major influence on preadipocyte differentiation in serum free media by modulating IGFBP production/secretion.     

Stimulation of protein kinase C redistribution and inhibition of leukotriene B4-induced inositol 1,4,5-trisphosphate generation in human neutrophils by lipoxin A4

1. To test the hypothesis that protein kinase C (PKC) is involved in the inhibitory actions of lipoxin A4 (LXA4) on second messenger generation, we studied the effects of LXA4 on PKC in human neutrophils and on leukotriene B4 (LTB4)-stimulated inositol-1,4,5-trisphosphate (Ins(1,4,5)P3) generation. 2. LXA4, 1 microM, caused a fall in cytosolic PKC-dependent histone phosphorylating activity to 23.5% of basal levels. 3. LXA4, caused an increase in particulate PKC-dependent histone phosphorylating activity with a bell-shaped dose-response fashion; maximal stimulation was observed at 10 nM LXA4. 4. Western blot analysis with affinity-purified antibodies to alpha- and beta-PKC showed that only the beta-PKC isotype was translocated by LXA4. 5. LXA4 inhibited LTB4-stimulated Ins(1,4,5)P3 generation in a bell-shaped fashion with maximal inhibition at 1 nM LXA4. The observed inhibition was dose-dependently removed by pre-incubation with a PKC inhibitor (Ro-31-8220). 6. These results show that LXA4 activates PKC in whole cells and supports a role for PKC activation in the inhibitory action of LXA4 on LTB4-induced Ins(1,4,5)P3 generation. 7. LXA4 (1-1000 nM) pre-incubation did not affect specific binding of [3H]-LTB4 to neutrophils. Thus, the inhibitory effect of LXA4 on LTB4-stimulated Ins(1,4,5)P3 generation could not be attributed to an effect on LTB4 receptors.     

Targeting BCL1 lymphoma with anti-idiotype antibodies: biodistribution kinetics of directly labeled antibodies and bispecific antibody-targeted bivalent haptens

The effects of the non-tumor-promoting protein kinase C (PKC) activator bryostatin 1 and the PKC inhibitors staurosporine and UCN-01 were examined with respect to modulation of 1-[beta-D-arabinofuranosyl]cytosine (ara-C)-induced apoptosis in human myeloid leukemia cells (HL-60) overexpressing the antiapoptotic protein Bcl-2. HL-60/Bcl-2 cells displayed a 5-fold increase in Bcl-2 protein compared with empty-vector counter-parts (HL-60/pCEP4) but comparable levels of Bax, Mcl-1, and Bcl-xL. After exposure to an equimolar concentration of ara-C (10 microM for 6 hr), HL-60/Bcl-2 cells were significantly less susceptible to apoptosis, DNA fragmentation, and loss of clonogenicity than HL-60/pCEP4 cells. The protective effect of increased Bcl-2 expression was manifested by a failure of ara-C to induce activation/cleavage of the Yama protease (CPP32; caspase-3) and degradation of one of its substrates, poly(ADP-ribose)polymerase to an 85-kDa cleavage product. When HL-60/Bcl-2 cells were preincubated with bryostatin 1 (10 nM; 24 hr) or coincubated with either staurosporine (50 nM; 6 hr) or UCN-01 (300 nM; 6 hr) after a 1-hr preincubation, exposures that exerted minimal effects alone, ara-C-induced apoptosis and DNA fragmentation were restored to levels equivalent to, or greater than, those observed in empty-vector controls. These events were accompanied by restoration of the ability of ara-C to induce CPP32 cleavage and activation, poly(ADP-ribose) polymerase degradation, and inhibition of colony formation. Western analysis of Bcl-2 protein obtained from overexpressing cells treated with bryostatin 1, staurosporine, or UCN-01 revealed the appearance of a slowly migrating species and a general broadening of the protein band, effects that were insensitive to the protein synthesis inhibitor cycloheximide. Alterations in Bcl-2 protein mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis were reversed by treatment of lysates with alkaline phosphatase or protein phosphatase 2A; actions of the latter were blocked by the specific phosphatase inhibitor okadaic acid. In vivo labeling studies of Bcl-2 protein demonstrated increased incorporation of [32PO4]orthophosphate in drug-treated cells. Last, phosphorylated Bcl-2 failed to display decreased binding to the proapoptotic protein Bax. Collectively, these findings indicate that bryostatin 1, which down-regulates PKC, and staurosporine and UCN-01, which directly inhibit the enzyme, circumvent resistance of Bcl-2-overexpressing leukemic cells to ara-C-induced apoptosis and activation of the protease cascade. They also raise the possibility that modulation of Bcl-2 phosphorylation status contributes to this effect.     

Rapid dissociation of human apurinic endonuclease (Ape1) from incised DNA induced by magnesium

We investigated the interaction dynamics of human abasic endonuclease, the Ape1 protein (also called Ref1, Hap1, or Apex), with its DNA substrate and incised product using electrophoretic assays and site-specific amino acid substitutions. Changing aspartate 283 to alanine (D283A) left 10% residual activity, contrary to a previous report, but complementation of repair-deficient bacteria by the D283A Ape1 protein was consistent with its activity in vitro. The D308A, D283/D308A double mutant, and histidine 309 to asparagine proteins had 22, 1, and approximately 0. 02% of wild-type Ape1 activity, respectively. Despite this range of enzymatic activities, all the mutant proteins had near-wild-type binding affinity specific for DNA containing a synthetic abasic site. Thus, substrate recognition and cleavage are genetically separable steps. Both the wild-type and mutant Ape1 proteins bound strongly to the enzyme incision product, an incised abasic site, which suggested that Ape1 might exhibit product inhibition. The use of human DNA polymerase beta to increase Ape1 activity by eliminating the incision product supports this conclusion. Notably, the complexes of the D283A, D308A, and D283A/D308A double mutant proteins with both intact and incised abasic DNA were significantly more stable than complexes containing wild-type Ape1, which may contribute to the lower turnover numbers of the mutant enzymes. Wild-type Ape1 protein bound tightly to DNA containing a one-nucleotide gap but not to DNA with a nick, consistent with the proposal that substrate recognition by Ape1 involves a space bracketed by duplex DNA, rather than mere flexibility of the DNA.