Transforming growth factor-beta induces apoptosis in activated murine T cells through the activation of caspase 1-like protease.

Transforming growth factor-beta (TGF-beta) has been known as a potent immunosuppressive cytokine that can induce apoptosis in lymphoid cells. We established an IL-2-independent cell line, CTLL-2A, from murine T cell line CTLL-2. CTLL-2A expressed higher levels of CD95, CD69, and CD18 molecules than CTLL-2 did, suggesting a more activated state in CTLL-2A than in the CTLL-2 by phenotype. Exposing both CTLL-2 and CTLL-2A to TGF-beta results in differential apoptosis patterns defined by DNA fragmentation and plasma membrane alteration. Among the bcl-2 family members, bcl-2, bcl-w, and bcl-x(L) were also differently expressed in these two cell lines. In CTLL-2A, bcl-x(L) was amplified as a major anti-apoptotic molecule, and TGF-beta-induced cell death was more enhanced than in the original cell line. Caspase 1-like protease was activated by TGF-beta treatment and consequently it cleaved bcl-x(L) in CTLL-2A. TGF-beta-induced DNA fragmentation and cleavage of bcl-x(L) were inhibited by pretreatment with tetra peptide caspase 1 inhibitor, YVAD.cmk. These findings suggest that TGF-beta induces cell death in activated murine T cells through cleavage of bcl-x(L) via activated caspase 1-like protease, which may act as an important executor in that process.

Potentiation of early necrotic death of glucose-starved pheochromocytoma 12 cells by nerve growth factor.

Recently suggested is an arguable hypothesis that neurotrophins can induce necrosis but suppress apoptosis of target cells in some pathological conditions. We examined this hypothesis by tracing the type of NGF-promoted cell death occurring in a hypoglycemic condition at various angles, such as kinetic analyses, histological examinations of membrane alterations, morphological observations in ultra-structural changes, and determinations of DNA fragmentation. Glucose-starved cell death consisted of two kinetically different stages, suggesting that it be mixed with early and delayed death. Several lines of evidence revealed that NGF prominently enhanced the early death with necrotic characters. By contrast, apoptotic characters of glucose-starved delayed death were not much affected by NGF. Nifedipine, a voltage-gated calcium channel blocker, could completely compensate for the enhancement of the early glucose-starved death by NGF. Interestingly, the NGF-promoted cell death was also blocked by cycloheximide that did not keep PC12 cells alive from glucose starvation. Therefore, all the data in this study suggest that NGF accelerates the early necrosis of glucose-starved cell death probably through the alterations of intracellular calcium ions and protein syntheses.

Silica-induced nuclear factor-kappaB activation: involvement of reactive oxygen species and protein tyrosine kinase activation.

Nuclear factor-kappaB (NF-kappaB) is a multiprotein complex that may regulate a variety of inflammatory cytokines involved in the initiation and progression of silicosis. The present study documents the ability of in vitro silica exposure to induce DNA-binding activity of NF-kappaB in a mouse peritoneal macrophage cell line (RAW264.7 cells) and investigates the role of reactive oxygen species (ROS) and/or protein tyrosine kinase in this activation. In vitro exposure of mouse macrophages to silica (100 microg/ml) resulted in a twofold increase in ROS production, measured as the generation of chemiluminescence (CL), and caused activation of NF-kappaB. Silica-induced CL was inhibited 100% by superoxide dismutase (SOD) and 75% by catalase, while NF-kappaB activation was inhibited by a variety of antioxidants (catalase, superoxide dismutase, alpha-tocopherol, pyrrolidine dithiocarbamate, or N-acetylcysteine). Further evidence for the involvement of ROS in NF-kappaB activation is that 1 mM H2O2 enhanced NF-kappaB/DNA binding and that this activation was inhibited by catalase. Specific inhibitors of protein tyrosine kinase, such as herbimycin A, genistein, and AG-494, prevented NF-kappaB activation in silica-treated cells. Genistein and AG-494 also reduced NF-kappaB activation in H2O2-treated cells. Results confirm that tyrosine phosphorylation of several cellular proteins (approximate molecular mass of 39, 58-70, and 103 kD) was increased in silica-exposed macrophages and that genistein inhibited this silica-induced phosphorylation. In contrast, inhibitors of protein kinase A or C, such as H89, staurosporin, calphostin C, and H7, had no marked inhibitory effect on silica-induced NF-kappaB activation. The results suggest that ROS may play a role in silica-induced NF-kappaB activation in macrophages and that phosphorylation events mediated by tyrosine kinase may be involved in this activation.

Platelet-derived growth factor-induced H(2)O(2) production requires the activation of phosphatidylinositol 3-kinase.

Autophosphorylation of the platelet-derived growth factor (PDGF) receptor triggers intracellular signaling cascades as a result of recruitment of Src homology 2 domain-containing enzymes, including phosphatidylinositol 3-kinase (PI3K), the GTPase-activating protein of Ras (GAP), the protein-tyrosine phosphatase SHP-2, and phospholipase C-gamma1 (PLC-gamma1), to specific phosphotyrosine residues. The roles of these various effectors in PDGF-induced generation of H(2)O(2) have now been investigated in HepG2 cells expressing various PDGF receptor mutants. These mutants included a kinase-deficient receptor and receptors in which various combinations of the tyrosine residues required for the binding of PI3K (Tyr(740) and Tyr(751)), GAP (Tyr(771)), SHP-2 (Tyr(1009)), or PLC-gamma1 (Tyr(1021)) were mutated to Phe. PDGF failed to increase H(2)O(2) production in cells expressing either the kinase-deficient mutant or a receptor in which the two Tyr residues required for the binding of PI3K were replaced by Phe. In contrast, PDGF-induced H(2)O(2) production in cells expressing a receptor in which the binding sites for GAP, SHP-2, and PLC-gamma1 were all mutated was slightly greater than that in cells expressing the wild-type receptor. Only the PI3K binding site was alone sufficient for PDGF-induced H(2)O(2) production. The effect of PDGF on H(2)O(2) generation was blocked by the PI3K inhibitors LY294002 and wortmannin or by overexpression of a dominant negative mutant of Rac1. These results suggest that a product of PI3K is required for PDGF-induced production of H(2)O(2) in nonphagocytic cells, and that Rac1 mediates signaling between the PI3K product and the putative NADPH oxidase.

Reduced activity of topoisomerase II in an Adriamycin-resistant human stomach-adenocarcinoma cell line.

A human stomach-adenocarcinoma cell line (MKN-45) was selected for resistance to Adriamycin by stepwise exposure to increasing concentrations of this agent. The resulting cell line (MKN/ADR) exhibited a high level of cross-resistance to topoisomerase II (topo II)-targeted drugs such as Adriamycin, mitoxantrone, and etoposide but showed no cross-resistance to other chemotherapeutic agents such as cisplatin, carboplatin, 5-fluorouracil, or mitomycin-C. P-glycoprotein encoded by the mdr-1 gene was not overexpressed in the MKN/ADR cell line. The doubling time of the MKN/ADR cell line (2.1 days) increased only slightly as compared with that of the MKN cell line (1.7 days). The patterns of cross-resistance to various chemotherapeutic agents led us to examine the cellular contents of topo II in both the drug-sensitive and the drug-resistant cells. Extractable topo II enzyme activity was 3-fold lower in MKN/ADR cells as compared with the parental MKN cells. Levels of topoisomerase I (topo I) catalytic activity were similar in both wild-type MKN and drug-resistant MKN/ADR cells. Southern-blot analysis of genomic DNA probed with topo IIalpha or IIbeta showed no sign of either gene rearrangement or hypermethylation. Northern-blot analysis revealed that both topo IIalpha and topo IIbeta mRNA transcripts were essentially identical in the MKN and MKN/ADR cells. In contrast, Western-blot analysis revealed an approximately 20-fold lower level of topo IIalpha in drug-resistant cells as compared with drug-sensitive cells, whereas topo IIbeta levels were similar in both lines. Moreover, the amount of in vivo topo IIalpha-DNA covalent complexes formed in the presence of etoposide was also approximately 20-fold lower in drug-resistant cells. No mutation was detected in the promoter region of the topo IIalpha gene in resistant cells as compared with sensitive cells. Thus, low levels of topo IIalpha polypeptide cannot be ascribed to changes in the mRNA levels. Collectively, the data suggest that a quantitative reduction in topo IIalpha may contribute to the resistance of MKN cells to Adriamycin and other topo II-targeted drugs.

Insulin activates Ras in the PC12 cell line.

To address whether Ras can be activated by insulin in the PC12 cell line, proteins interacting with insulin receptor and IRS-1 molecules and their tyrosine phosphorylation were analyzed by immunoblotting following immunoprecipitation with antibodies. Tyrosine phosphorylation of the insulin receptor and IRS-1 was increased by insulin. Grb2 and Ras-GAP appeared in the immunoprecipitates by anti-insulin receptor and anti-IRS-1 from insulin-treated cells. In addition, PI 3-kinase was activated by insulin treatment in this cell line and Grb2, Ras-GAP, and MAP kinase were coprecipitated with Ras from both insulin-treated and NGF-treated cells. Analysis of MAP kinases from insulin-treated cells revealed that insulin, like NGF, increased tyrosine phosphorylation. However, activation of the MAP kinase by NGF lasted longer than activation by insulin. These results indicate that Ras can be activated by insulin in the PC12 cell line and that Ras activation is neither an accurate nor a plausible method of discriminating signals between proliferation and differentiation.

Calcipotriol (MC 903), a synthetic derivative of vitamin D3 stimulates differentiation of squamous carcinoma cell line in the raft culture.

We investigated whether calcipotriol, a synthetic derivative of vitamin D3 has the ability to correct defects in the control of proliferation and differentiation of human squamous carcinoma cells using the raft culture of SCC 13 cell line. Calcipotriol treatment at concentrations of 10(-8)-10(-6) M considerably enhanced terminal differentiation of SCC 13 cells, as shown by the appearance of enucleated-eosinophilic cells as well as granular cells in their upper cell layers. Immunohistochemical staining showed marked increases in the differentiation of marker proteins such as keratin 1, involucrin, or filaggrin expressing cells in their upper layers. The elevated expression at protein level was confirmed by immunoblotting analysis. Furthermore, calcipotriol also stimulated basal cell marker proteins such as keratin 14 and EGF receptor. However, the numbers of basal marker expressing cells within the architecture of SCC 13 raft culture were markedly reduced upon calcipotriol treatment, and their localization was mainly restricted in the innermost cell layer. In addition, calcipotriol stimulated EGF receptor biosynthesis for the first 16 hours post treatment and subsequently inhibited [3H]-thymidine incorporation of SCC 13 cells at 24 hours. In this study, we have clearly demonstrated that the long term application of calcipotriol considerably improves the complex defects in the regulation of proliferation and differentiation of SCC 13 cells, as supported by morphological and biochemical observations. This provides an evidence that calcipotriol can be applied clinically as a potent differentiation inducer in the treatment of human squamous cell carcinoma.

Identification of an 18,000-dalton protein in mammalian lens fiber cell membranes.

Monoclonal antibodies have been produced against electrophoretically purified MP18, a major calf lens membrane Mr = 18,000 substrate for cAMP-dependent protein kinase. One of these antibodies (monoclonal antibody 2D10) cross-reacted with both native MP18 in lens membranes, and sodium dodecyl sulfate-denatured, electrophoretically purified MP18. In immunoblots, this antibody recognized MP18 in pig, sheep, rat, human, but not chicken lens membranes, indicating the similarity of this protein in mammalian lenses. Amino acid sequencing revealed that the N-terminal sequence of MP18 is identical in these five different mammalian species and is unrelated to any previously sequenced lens or junctional proteins. Electron microscopic examination of monoclonal antibody 2D10-labeled bovine, pig and rat lens membranes indicated that MP18 is localized exclusively to the thicker 16-17 nm junctions in isolated preparations of lens fiber cell membranes. These results provide evidence of a role for MP18 in mammalian lens fiber cell junctional organization.

Structural organization of the lens fiber cell plasma membrane protein MP18.

The 18,000-dalton bovine lens fiber cell intrinsic membrane protein MP18 was phosphorylated on a serine residue by both cAMP-dependent protein kinase and protein kinase C. In addition, this protein bound calmodulin and was recognized by a monoclonal antibody (2D10). These different regions were localized using enzymatic and chemical fragmentation of electrophoretically purified MP18 that had been phosphorylated with either cAMP-dependent protein kinase or protein kinase C. Partial digestion of 32P-labeled MP18 with protease V8 resulted in a Mr = 17,000 peptide that bound calmodulin, but neither contained 32P or was recognized by the monoclonal antibody 2D10. Furthermore, the 17-kDa peptide had the same N-terminal amino acid sequence as MP18. Thus, the monoclonal antibody 2D10 recognition site and the protein kinase phosphorylation site(s) are close together and confined to a small region in the C terminus of MP18. This conclusion was confirmed in experiments where MP18 was fragmented with trypsin, endoproteinase Lys-C, or CNBr. The location of the phosphorylation site was confirmed by sequencing the small 32P-labeled, C-terminal peptide that resulted from protease V8 digestion of 32P-labeled MP18. This peptide contained a consensus sequence for cAMP-dependent protein kinase.

Regional distribution of the enzymes and substrates mediating the action of cAMP in the mammalian lens.

Localization of adenylate cyclase activity in the outer cortical regions of the bovine lens correlates with the restriction of the Gs and Gi guanine nucleotide regulatory subunits of this enzyme to these same regions of the lens. In contrast, the major membrane substrates for cAMP-dependent protein kinase (cAMP-PK) (molecular masses of 18, 26 and 28 kDa) were identified in both the inner nuclear and the outer cortical regions of the lens. However, there were differences in the relative amounts of Pi incorporated into the 18 kDa and 28 kDa components in different lens regions. The three major membrane substrates for cAMP-PK were also phosphorylated when homogenates of lens cortex were incubated with [gamma-32P]ATP plus activators of the lens adenylate cyclase. In contrast, there was no incorporation of 32P into these substrates when homogenates of lens nucleus were used. When exogenous cAMP was added to homogenates of lens nucleus or cortex, 32P was incorporated into the membrane substrates for cAMP-PK in both regions of the lens, indicating that cAMP-PK was present in both regions. Interestingly, cAMP phosphodiesterase activity was at least 10-times greater in lens cortex than in the lens nucleus. These results indicate that while the major membrane substrates for cAMP-PK could be phosphorylated in all regions of the lens, there is a restriction of those enzymes that synthesize and degrade cAMP to the outer cortical regions of this organ.

Development of physiological responsiveness to glucagon during embryogenesis of avian heart.

Glucagon increases contractility of the heart muscle by stimulation of adenylate cyclase activity and elevation of cAMP. We have investigated the specific time of onset of glucagon sensitivity of heart muscle during development of the chick embryo. Using both isolated heart preparation and cultured cardiac cells, we have found that the contractile response to glucagon cannot be detected prior to Day 4 of development. Binding studies, carried out with heart cells prepared from 3-, 5-, 7-, and 11-day chick embryos, showed a significant increase in the number of glucagon binding sites between Days 3 and 5. Scatchard analysis showed that for Day 5 cells maximum binding capacity was 0.56 pmole/mg of protein with Kd of 16.0 nM, while for Day 3, maximal binding was only 0.16 pmole/mg with Kd of 15.1 nM. Therefore in this 2-day interval there was a marked increase in the receptor number, without changes in the receptor affinity. Since hormonal stimulation of adenylate cyclase depends on the presence of the regulatory component (Ns), we have used cholera toxin-induced chronotropic effect as an assay for functional Ns. No response to cholera toxin could be detected prior to Day 4 of chick heart development. Therefore, emergence of the cholera toxin sensitivity correlates well with the onset of responsiveness to glucagon. We conclude that as the heart develops it acquires a physiological responsiveness to glucagon. The acquisition of the hormonal sensitivity correlates with the increase in the receptor number and the functional levels of regulatory component.

Regulation of lens cyclic nucleotide metabolism by Ca2+ plus calmodulin.

Adenylate cyclase activity was identified in membranes isolated from bovine lens fiber cells. Basal activity, in the presence of microM Ca2+ was stimulated by either sodium fluoride, guanosine 5'-[alpha,beta-imido]triphosphate (Gpp(NH)p), or forskolin; ethylene glycolbis(2-aminoethylether) tetraacetic acid (EGTA) markedly inhibited both the basal activity and the extent of stimulation by these agents. Exogenous calmodulin enhanced the Ca2+-dependent stimulation of adenylate cyclase activity. In the presence of optimal concentrations of Ca2+ plus calmodulin, adenylate cyclase activity was approximately 15 times greater than that in the presence of EGTA. Adenylate cyclase activity was not stimulated by a number of potential agonists that included carbachol, serotonin, prostaglandin E1 (PGE1), prostaglandin E2 (PGE2), adenosine, isoproterenol epinephrine, dopamine, and phenylephrine. The presence of the Ns and Ni guanine nucleotide regulatory complexes was indicated by two observations: Cholera toxin catalyzed the adenosine diphosphate (ADP) ribosylation of a number of lens membrane proteins, including a 46,500-dalton component (likely the alpha-subunit of Ns), and Pertussis toxin catalyzed the ADP ribosylation of a single 41,000-dalton lens membrane component (likely the alpha-subunit of Ni). However, that Gpp(NH)p did not inhibit either the forskolin-activated or the calmodulin-activated adenylate cyclase activities does not indicate a role for Ni in regulating this enzyme. Both cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) phosphodiesterase activities were identified in a supernate fraction derived from bovine lens. The cAMP phosphodiesterase activity appeared to be predominantly the low Km form of the enzyme. The cGMP phosphodiesterase activity, which was Ca2+-dependent, was partly inhibited maximally by 7 microM R24571, indicating its probable calmodulin dependence.(ABSTRACT TRUNCATED AT 250 WORDS)

A lens intercellular junction protein, MP26, is a phosphoprotein.

The major protein present in the plasma membrane of the bovine lens fiber cell (MP26), thought to be a component of intercellular junctions, was phosphorylated in an in vivo labeling procedure. After fragments of decapsulated fetal bovine lenses were incubated with [32P]orthophosphate, membranes were isolated and analyzed by SDS PAGE and autoradiography. A number of lens membrane proteins were routinely phosphorylated under these conditions. These proteins included species at Mr 17,000 and 26,000 as well as a series at both 34,000 and 55,000. The label at Mr 26,000 appeared to be associated with MP26, since (a) boiling the membrane sample in SDS led to both an aggregation of MP26 and a loss of label at Mr 26,000, (b) the label at 26,000 was resistant to both urea and nonionic detergents, and (c) two-dimensional gels showed that a phosphorylated Mr 24,000 fragment was derived from MP26 with V8 protease. Studies with proteases also provided for a localization of most label within approximately 20 to 40 residues from the COOH-terminus of MP26. Published work indicates that the phosphorylated portion of MP26 resides on the cytoplasmic side of the membrane, and that this region of MP26 contains a number of serine residues. The same region of MP26 was labeled when isolated lens membranes were reacted with a cAMP-dependent protein kinase prepared from the bovine lens. After the in vivo labeling of lens fragments, phosphoamino acid analysis of MP26 demonstrated primarily labeled serines, with 5-10% threonines and no tyrosines. Treatments that lowered the intracellular calcium levels in the in vivo system led to a selective reduction of MP26 phosphorylation. In addition, forskolin and cAMP stimulated the phosphorylation of MP26 and other proteins in concentrated lens homogenates. These findings are of interest because MP26 appears to serve as a protein of cell-to-cell channels in the lens, perhaps as a lens gap junction protein.

Direct cross-linking of 125I-labeled glucagon to its membrane receptor by UV irradiation.

125I-labeled glucagon was directly crosslinked to its receptor in isolated liver plasma membranes and on the surface of intact hepatocytes, by using a UV irradiation procedure. This investigation resulted in the identification of a glucagon-receptor complex of apparent Mr 62,000. The specificity of labeling was shown by the interference of unlabeled hormone at physiological concentration with incorporation of radioactive glucagon into the 62,000 Mr species. The receptor behaved as a typical integral membrane protein: it was not released by extraction with lithium diiodosalicylate or at basic pH but was solubilized by digitonin treatment. Reduction of the receptor polypeptide with dithiothreitol resulted in a decrease in its electrophoretic mobility, suggesting the presence of intramolecular disulfide bonds. Soluble glucagon-receptor complexes adsorbed to Con A-Sepharose and could be eluted with methyl alpha-D-mannoside, indicating that the receptor molecule is a glycoprotein. Treatment of glucagon-labeled liver plasma membrane with endoglycosidase F resulted in the appearance of four intermediate species, indicating that glucagon receptor contains at least four N-linked oligosaccharide chains.