Cost-effective method for growing three-dimensional cell cultures in extracellular matrix extract.

Three-dimensional (3-D) cell cultures are advantageous for modeling complex cellular behavior. A major issue with 3-D cultures grown in Engelbreth-Holm-Swarm (EHS) extracellular matrix extract, however, is the expense. We describe here a simple method using a rubber gasket laid over a coverslip, which provides sufficient 3-D area filled with a minimal amount of EHS, resulting in cost-effective 3-D cellular cultures that are easy to manipulate.

Phosphatidic acid regulates tyrosine phosphorylating activity in human neutrophils: enhancement of Fgr activity.

In human neutrophils, the activation of phospholipase D and the Tyr phosphorylation of proteins are early signaling events upon cell stimulation. We found that the pretreatment of neutrophils with ethanol (0.8%) or 1-butanol (0.3%), which results in the accumulation of phosphatidylalcohol at the expense of phosphatidic acid (PA), decreased the phorbol myristate acetate-stimulated Tyr phosphorylation of endogenous proteins (42, 115 kDa). When neutrophil cytosol was incubated in the presence or absence of PA, these and other endogenous proteins became Tyr-phosphorylated in a PA-dependent manner. In contrast, phosphatidylalcohols exhibited only 25% (phosphatidylethanol) or 5% (phosphatidylbutanol) of the ability of PA to stimulate Tyr phosphorylation in the cell-free assay. Similarly, other phospholipids (phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidylinositol, polyphosphoinositides, and sphingosine 1-phosphate) showed little ability to stimulate Tyr phosphorylation. These data suggest that PA can function as an intracellular regulator of Tyr phosphorylating activity. Gel filtration chromatography of leukocyte cytosol revealed a peak of PA-dependent Tyr phosphorylating activity distinct from a previously described PA-dependent phosphorylating activity (Waite, K. A., Wallin, R., Qualliotine-Mann, D., and McPhail, L. C. (1997) J. Biol. Chem. 272, 15569-15578). Among the protein Tyr kinases expressed in neutrophils, only Fgr eluted exclusively in the peak of PA-dependent Tyr phosphorylating activity. Importantly, Fgr isolated from unstimulated neutrophil lysates showed increased activity in the presence of PA but not phosphatidylbutanol. Moreover, the pretreatment of neutrophils with 1-butanol decreased Fgr activity in cells stimulated with formyl-methionyl-leucyl phenylalanine plus dihydrocytochalasin B. Together, these results suggest a new second messenger role for PA in the regulation of Tyr phosphorylation.

Why expression of phosphatidylethanolamine N-methyltransferase does not rescue Chinese hamster ovary cells that have an impaired CDP-choline pathway.

The mutant Chinese hamster ovary cell line (CHO), MT58, has a temperature-sensitive mutation in CTP:phosphocholine cytidylyltransferase (CT), preventing phosphatidylcholine (PC) synthesis at 40 degrees C which results in apoptosis. Previous studies (Houweling, M., Cui, Z., and Vance, D. E. (1995) J. Biol. Chem. 270, 16277-16282) showed that expression of wild-type CT-alpha rescued the cells at 40 degrees C, whereas expression of phosphatidylethanolamine N-methyltransferase-2 (PEMT2) did not, even though PC levels appeared to be maintained at wild-type levels after 24 h at the restrictive temperature. We report that the failure of PEMT2 to rescue the MT58 cell line is due to inadequate long term PC synthesis. We found that changing the medium every 24 h rescued the PEMT2-expressing MT58 cells grown at 40 degrees C. This was due to the uptake and utilization of lipids in the serum. At 40 degrees C, PC levels in the wild-type CHO cells and CT-expressing MT58 cells increased over time whereas PC levels did not change in both the MT58 and PEMT2-expressing MT58 cell lines. Further investigation found that both the PEMT2-expressing MT58 and MT58 cell lines accumulated triacylglycerol at 40 degrees C. Pulse-chase experiments indicated that lyso-PC accumulated to a higher degree at 40 degrees C in the PEMT2-expressing MT58 cells compared with CT-expressing MT58 cells. Transfection of the PEMT-expressing MT58 cells with additional PEMT2 cDNA partially rescued the growth of these cells at 40 degrees C. Inhibition of PC degradation, by inhibitors of phospholipases, also stimulated PEMT-expressing MT58 cell growth at 40 degrees C. Best results were observed using a calcium-independent phospholipase A(2) inhibitor, methyl arachidonyl fluorophosphonate. This inhibitor also increased PC mass in the PEMT2-expressing MT58 cells. When the cells are shifted to 40 degrees C, PC degradation by enzymes such as phospholipases is greater than PC synthesis in the mutant PEMT2-expressing MT58 cells. Taken together, these results indicate that PEMT2 expression fails to rescue the mutant cell line at 40 degrees C because it does not maintain PC levels required for cellular replication.

Functional significance of Sp1, Sp2, and Sp3 transcription factors in regulation of the murine CTP:phosphocholine cytidylyltransferase alpha promoter.

The transcription factor Sp1 has been implicated in regulation of the expression of the murine CTP:phosphocholine cytidylyltransferase alpha (CTalpha) gene, Ctpct (M. Bakovic, K. Waite, W. Tang, I. Tabas, and D. E. Vance. 1999. Biochim. Biophys. Acta. 1438: 147;-165). We have utilized transient transfections, mutation analysis, electromobility gel-shifts, and immunoblot analysis to test the hypothesis that expression of the CTalpha gene is controlled in part by the binding of three trans-acting nuclear factors, Sp1, Sp2, and Sp3. Sp1 and Sp3 activate CTalpha gene transcription through sequence specific binding within three promoter domains. In Sp1-mediated transcription, Sp3 acts as an activator in a dose-dependent manner and vice versa. Sp2 represses Sp1- and Sp3-driven transcription in Drosophila SL2 cells, but stimulates transcription in C3H10T1/2 mammalian cells. Our results suggest that the predominant action of Sp proteins is a direct function of local organization of three cis-acting elements in the regions A (-31/-9), B (-88/-50), and C (-148/-128). The ability of distal C (-148/-128) and proximal A (-31/-9) regions to activate or repress transcription depends upon the cellular background. The multiple binding elements at position B (-88/-50) confer a positive regulation independent of the cell context. However, the effectiveness of Sp proteins at this site is strongly governed by neighboring sites A and C. The results suggest that the level of expression of the CTalpha gene will depend on the cell type, the availability of Sp proteins, and the structure and organization of three cis-acting elements.

A phosphatidic acid-activated protein kinase and conventional protein kinase C isoforms phosphorylate p22(phox), an NADPH oxidase component.

Using a phosphorylation-dependent cell-free system to study NADPH oxidase activation (McPhail, L. C., Qualliotine-Mann, D., and Waite, K. A. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7931-7935), we previously showed that p47(phox), a cytosolic NADPH oxidase component, is phosphorylated. Now, we show that p22(phox), a subunit of the NADPH oxidase component flavocytochrome b(558), also is phosphorylated. Phosphorylation is selectively activated by phosphatidic acid (PA) versus other lipids and occurs on a threonine residue in p22(phox). We identified two protein kinase families capable of phosphorylating p22(phox): 1) a potentially novel, partially purified PA-activated protein kinase(s) known to phosphorylate p47(phox) and postulated to mediate the phosphorylation-dependent activation of NADPH oxidase by PA and 2) conventional, but not novel or atypical, isoforms of protein kinase C (PKC). In contrast, all classes of PKC isoforms could phosphorylate p47(phox). In a gel retardation assay both the phosphatidic acid-dependent kinase and conventional PKC isoforms phosphorylated all molecules of p22(phox). These findings suggest that phosphorylation of p22(phox) by conventional PKC and/or a novel PA-activated protein kinase regulates the activation/assembly of NADPH oxidase.

A novel protein kinase target for the lipid second messenger phosphatidic acid.

Activation of phospholipase D occurs in response to a wide variety of hormones, growth factors, and other extracellular signals. The initial product of phospholipase D, phosphatidic acid (PA), is thought to serve a signaling function, but the intracellular targets for this lipid second messenger are not clearly identified. The production of PA in human neutrophils is closely correlated with the activation of NADPH oxidase, the enzyme responsible for the respiratory burst. We have developed a cell-free system, in which the activation of NADPH oxidase is induced by the addition of PA. Characterization of this system revealed that a multi-functional cytosolic protein kinase was a target for PA, and that two NADPH oxidase components were substrates for the enzyme. Partial purification of the PA-activated protein kinase separated the enzyme from known protein kinase targets of PA. The partially purified enzyme was selectively activated by PA, compared to other phospholipids, and phosphorylated the oxidase component p47-phox on both serine and tyrosine residues. PA-activated protein kinase activity was present in a variety of hematopoietic cells and cell lines and in rat brain, suggesting it has widespread distribution. We conclude that this protein kinase may be a novel target for the second messenger function of PA.

Phosphatidic acid-mediated phosphorylation of the NADPH oxidase component p47-phox. Evidence that phosphatidic acid may activate a novel protein kinase.

Phosphatidic acid (PA), generated by phospholipase D activation, has been linked to the activation of the neutrophil respiratory burst enzyme, NADPH oxidase; however, the intracellular enzyme targets for PA remain unclear. We have recently shown (McPhail, L. C., Qualliotine-Mann, D., and Waite, K. A. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7931-7935) that a PA-activated protein kinase is involved in the activation of NADPH oxidase in a cell-free system. This protein kinase phosphorylates numerous endogenous proteins, including p47-phox, a component of the NADPH oxidase complex. Phospholipids other than PA were less effective at inducing endogenous protein phosphorylation. Several of these endogenous substrates were also phosphorylated during stimulation of intact cells by opsonized zymosan, an agonist that induces phospholipase D activation. We sought to identify the PA-activated protein kinase that phosphorylates p47-phox. The PA-dependent protein kinase was shown to be cytosolic. cis-Unsaturated fatty acids were poor inducers of protein kinase activity, suggesting that the PA-activated protein kinase is not a fatty acid-regulated protein kinase (e.g. protein kinase N). Chromatographic techniques separated the PA-activated protein kinase from a number of other protein kinases known to be activated by PA or to phosphorylate p47-phox. These included isoforms of protein kinase C, p21 (Cdc42/Rac)-activated protein kinase, and mitogen-activated protein kinase. Gel filtration chromatography indicated that the protein kinase has an apparent molecular size of 125 kDa. Screening of cytosolic fractions from several cell types and rat brain suggested the enzyme has widespread cell and tissue distribution. The partially purified protein kinase was sensitive to the same protein kinase inhibitors that diminished NADPH oxidase activation and was independent of guanosine 5'-3-O-(thio)triphosphate and Ca2+. Phosphoamino acid analysis showed that serine and tyrosine residues were phosphorylated on p47-phox by this kinase(s). These data indicate that one or more potentially novel protein kinases are targets for PA in neutrophils and other cell types. Furthermore, a PA-activated protein kinase is likely to be an important regulator of the neutrophil respiratory burst by phosphorylation of the NADPH oxidase component p47-phox.

Cell-free activation of neutrophil NADPH oxidase by a phosphatidic acid-regulated protein kinase.

The phosphorylation-dependent mechanisms regulating activation of the human neutrophil respiratory-burst enzyme, NADPH oxidase, have not been elucidated. We have shown that phosphatidic acid (PA) and diacylglycerol (DG), products of phospholipase activation, synergize to activate NADPH oxidase in a cell-free system. We now report that activation by PA plus DG involves protein kinase activity, unlike other cell-free system activators. NADPH oxidase activation by PA plus DG is reduced approximately 70% by several protein kinase inhibitors [1-(5-isoquinolinesulfonyl)piperazine, staurosporine, GF-109203X]. Similarly, depletion of ATP by dialysis reduces PA plus DG-mediated NADPH oxidase activation by approximately 70%. Addition of ATP, but not a nonhydrolyzable ATP analog, to the dialyzed system restores activation levels to normal. In contrast, these treatments have little effect on NADPH oxidase activation by arachidonic acid or SDS plus DG. PA plus DG induces the phosphorylation of a number of endogenous proteins. Phosphorylation is largely mediated by PA, not DG. A predominant substrate is p47-phox, a phosphoprotein component of NADPH oxidase. Phosphorylation of p47-phox precedes activation of NADPH oxidase and is markedly reduced by the protein kinase inhibitors. In contrast, arachidonic acid alone or SDS plus DG is a poor activator of protein phosphorylation in the cell-free system. Thus, PA induces activation of one or more protein kinases that regulate NADPH oxidase activation in a cell-free system. This cell-free system will be useful for identifying a functionally important PA-activated protein kinase(s) and for dissecting the phosphorylation-dependent mechanisms responsible for NADPH oxidase activation.

The role of an autoantigen, histidyl-tRNA synthetase, in the induction and maintenance of autoimmunity.

Patients with systemic autoimmune diseases make specific autoantibodies that are directed against self structures. According to one view, these autoantibodies arise as a result of an immune response to foreign antigens such as infectious agents that share, by molecular mimicry, common structures with host proteins. An alternative view is that the target autoantigen itself initiates, selects, and sustains autoantibody synthesis. We show here that anti-Jo-1 autoantibodies directed against histidyl-tRNA synthetase in the human autoimmune muscle disease polymyositis undergo, in addition to spectrotype broadening and class switching, the sine qua non of an immune response to the target antigen--affinity maturation to that antigen. We demonstrate further that these autoantibodies, unlike anti-synthetase antibodies induced in mice immunized with heterologous antigen, bind only nonlinear epitopes on the native human synthetase that remain exposed when the enzyme is complexed to tRNA(His). These data suggest that the native target autoantigen itself has played a direct role in selecting and sustaining the autoantibody response and sharply restrict the time and the way in which a molecular mimic might act to provoke autoantibodies.

A second cell-binding domain on fibronectin (RGDS-independent) for neurite extension of human neuroblastoma cells.

Human neuroblastoma cells (Platt and La-N1) have previously been shown to adhere and extend neurites on tissue-culture substrata coated with a 120K chymotryptic cell-binding fragment (CBF) of plasma fibronectin (pFN), a fragment which lacks heparan sulfate- and collagen-binding activities, and to adhere to--but not extend neurites on--substrata coated with the heparan sulfate (HS)-binding protein, platelet factor-4 (PF4) (Tobey et al., Exp Cell Res 158 (1985) 395 [3]). The mechanisms of these processes on CBF, on the intact pFN molecule, or on heparin-binding fragments of pFN have been tested using a heptapeptide (peptide A) containing the Arg-Gly-Asp-Ser (RGDS) sequence which recognizes a specific 'receptor' on the surface of a variety of cells or a control peptide with a single amino acid substitution. Adherence and neurite extension were completely inhibited on the 120K CBF by peptide A but not by control peptide; these results indicate that the RGDS-dependent 'receptor' is solely responsible for adhesive responses to the 120K CBF-containing region of the pFN molecule. When peptide A was added to cells on CBF which had already formed neurites to test reversibility, retraction of all neurite processes was induced by 1 h and cells eventually detached. In contrast, on intact pFN, peptide A had very limited effects on either initial adherence or neurite extension, revealing a second 'cell-binding' domain on the fibronectin molecule outside of the 120K region competent for neurite differentiation; addition of peptide A at later times to pFN-adherent, neurite-containing cells could induce only a small subset of neurites to retract, thus supporting evidence for the presence of this second domain. A second 'cell-binding' domain was further confirmed by quantitation of neurite outgrowth on these substrata and by analyses of cells on substrata coated with mixtures of CBF/PF4. When substrata coated with chymotrypsin-liberated HBF were tested in a similar fashion, adherence was rapid but neurite outgrowth required much longer times and was completely sensitive to RGDS peptides; supplementation of cells with the complex ganglioside GT1b could not induce RGDS-resistant neurites on heparin-binding fragments (HBF). These latter results indicate that neurite extension on HBF is a consequence of a low concentration of RGDS-dependent activity in HBF (but not to HS-binding activity as characterized by Tobey et al. [3]) and that the second 'cell-binding' domain is sensitive to chymotrypsin digestion of pFN during the liberation of HBF.(ABSTRACT TRUNCATED AT 400 WORDS)