Characterization of human mucin gene MUC4 promoter: importance of growth factors and proinflammatory cytokines for its regulation in pancreatic cancer cells.

The human mucin gene MUC4 encodes a large transmembrane mucin that is thought to play important roles in tumor cell biology and that is overexpressed in human pancreatic carcinomas. In this report, we describe the structure and functional activity of the 5'-flanking region, including 1.0 kilobase of the promoter. The long 5'-untranslated region (2.7 kilobases) is characterized by a high content of GC in its 3'-end. The first TATA box was located at -2672/-2668. Multiple transcription start sites and a high density of putative binding sites for Sp1 (GC and CACCC boxes), AP-1/-2/-4, cAMP-responsive element-binding protein, GATA, GR, and STAT transcription factors were found within the 5'-flanking region. Transcriptional activity of the promoter was assessed using pGL3-luciferase deletion mutants in two MUC4-expressing (CAPAN-1 and CAPAN-2) and one nonexpressing (PANC-1) pancreatic cancer cell line. Two highly active fragments (-219/-1 and -2781/-2572) that drive MUC4 transcription in CAPAN-1 and CAPAN-2 cells were identified. Gel retardation assays indicated that Sp1 and Sp3 bind to cognate cis-elements found in the 5'-flanking region and that Sp1 transactivates, whereas Sp3 inhibits the GC-rich region (-464/-1) in CAPAN-2 cells. Activation of protein kinase C with phorbol ester and treatment of cells with epidermal growth factor and transforming growth factor-alpha resulted in up-regulation of the promoter. Tumor necrosis factor-alpha and interferon (IFN)-gamma inflammatory cytokines had no or mild effect on MUC4 transcriptional activity when used alone. However, a very strong synergistic effect (10-12-fold activation) between IFN-gamma and tumor necrosis factor-alpha or IFN-gamma and transforming growth factor-alpha was obtained in CAPAN-2 cells. Altogether these results demonstrate that the 5'-flanking region of MUC4 contains epithelial cell-specific, positive, and negative regulatory cis-elements, that Sp1/Sp3 are important regulators of MUC4 basal expression, and that its regulation in pancreatic cancer cells involves complex interplay between several signaling pathways.

Sequential biosynthesis of sulfated and/or sialylated Lewis x determinants by transferases of the human bronchial mucosa.

The structural determination of sulfated carbohydrate chains from a cystic fibrosis patient respiratory mucins has shown that sulfation may occur either on the C-3 of the terminal Gal, or on the C-6 of the GlcNAc residue of a terminal N -acetyllactosamine unit. The two enzymes responsible for the transfer of sulfate from PAPS to the C-3 of Gal or to the C-6 of GlcNAc residues have been characterized in human respiratory mucosa. These two enzymes, in conjunction with fucosyl- and sialyltransferases, allow the synthesis of different sulfated epitopes such as 3-sulfo Lewis x (with a 3- O -sulfated Gal), 6-sulfo Lewis x and 6-sulfo-sialyl Lewis x (with a 6- O -sulfated GlcNAc). In the present study, the sequential biosynthesis of these epitopes has been investigated using microsomal fractions from human respiratory mucosa incubated with radiolabeled nucleotide-sugars or PAPS, and oligosaccharide acceptors, mostly prepared from human respiratory mucins. The structures of the radiolabeled products have been determined by their coelution in HPAEC with known oligosaccharidic standards. In the biosynthesis of 6- O -sulfated carbohydrate chains by the human respiratory mucosa, the 6- O -sulfation of a terminal nonreducing GlcNAc residue precedes beta1-4-galactosylation, alpha2-3-sialylation (to generate 6-sulfo-sialyl- N -acetyllactosamine), and alpha1-3-fucosylation (to generate the 6-sulfo-sialyl Lewis x determinant). The 3- O -sulfation of a terminal N -acetyllactosamine may occur if this carbohydrate unit is not substituted. Once an N -acetyllactosamine unit is synthesized, alpha1-3-fucosylation of the GlcNAc residue to generate a Lewis x structure blocks any further substitution. Therefore, the present study defines the pathways for the biosynthesis of Lewis x, sialyl Lewis x, sulfo Lewis x, and 6-sulfo-sialyl Lewis x determinants in the human bronchial mucosa.

Characterization of an N-acetylglucosamine-6-O-sulfotransferase from human respiratory mucosa active on mucin carbohydrate chains.

A microsomal GlcNAc-6-O-sulfotransferase activity from human bronchial mucosa, able to transfer a sulfate group from adenosine 3'-phosphate 5'-phosphosulfate onto methyl-N-acetylglucosaminides or terminal N-acetylglucosamine residues of carbohydrate chains from human respiratory mucins, has been characterized. The reaction products containing a terminal HO3S-6GlcNAc were identified by high performance anion-exchange chromatography. Using methyl-beta-N-acetylglucosaminide as a substrate, the optimal activity was obtained with 0.1% Triton X-100, 30 mM NaF, 20 mM Mn2+, 5 mM AMP in a 30 mM MOPS (3-(N-morpholino) propanesulfonic acid) buffer at pH 6.7. The apparent Km values for adenosine 3'-phosphate 5'-phosphosulfate and methyl-beta-N-acetylglucosaminide were observed at 9.1 x 10(-6) M and 0.54 x 10(-3) M, respectively. The enzyme had more affinity for carbohydrate chains with a terminal GlcNAc residue than for methyl-beta-N-acetylglucosaminide; it was unable to catalyze the transfer of sulfate to position 6 of the GlcNAc residue contained in a terminal Galbeta1-4GlcNAc sequence. However, oligosaccharides with a nonreducing terminal HO3S-6GlcNAc were substrates for a beta1-4 galactosyltransferase from human bronchial mucosa. These data point out that GlcNAc-6-O-sulfotransferase must act before beta1-4 galactosylation in mucin-type oligosaccharide biosynthesis.

Characterization of a sulfotransferase from human airways responsible for the 3-O-sulfation of terminal galactose in N-acetyllactosamine-containing mucin carbohydrate chains.

A galactose 3-O-sulfotransferase activity able to transfer a sulfate group from adenosine 3'-phosphate 5'-phosphosulfate to methyl galactosides or terminal N-acetyllactosamine-containing carbohydrate chains from human respiratory mucins was characterized in microsomal fractions prepared from human respiratory mucosa. The reaction products, methyl alpha- or beta-galactose 3-sulfate and three oligosaccharide alditols containing the sequence HSO3-3Gal beta 1-4GlcNAc beta 1-6GalNAc-itol were identified by high performance anion-exchange chromatography. Using methyl beta-galactoside as a substrate, the optimum activity was obtained with 0.1% Triton X-100, 30 mM NaF, 20 mM Mn2+, and 10 mM AMP in a 30 mM 2-(N-morpholino)ethanesulfonic acid buffer at pH 6.1. The apparent Km for methyl beta-galactoside and for adenosine 3'-phosphate 5'-phosphosulfate were observed at 0.69 x 10(-3) M and at 4 x 10(-6) M respectively. This sulfotransferase is different from that responsible for sulfatide synthesis.

Western blotting of basic proteins after nondenaturing electrophoresis in acid conditions using the PhastSystem.

Electroblotting of basic proteins was performed from minigels after electrophoresis, under nondenaturing acidic conditions, by using the automated PhastSystem. Depending on the molecular masses of the proteins to be studied, various precast gel media were chosen. The transfer membranes with various types (nitrocellulose and polyvinylidene difluoride) and pore sizes (0.45 and 0.2 micron) were chosen accordingly. For the semidry electric transfer, a simple, discontinuous two-buffer system was used. The anode solution contained 0.3 M Tris, pH 10.4, and the cathode solution, 40 mM 6-amino-n-hexanoic acid, pH 7.6, with 20% v/v methanol each. The addition of 0.1% sodium dodecyl sulfate (SDS) in the cathode solution facilitated the elution of proteins from the gels and directed the migration of the negative SDS-protein complexes towards the anode membranes. The transfer conditions following native polyacrylamide gel electrophoresis allowed the visualization of basic proteins, with molecular weights ranging from 29,000 to 5,000, for which isoforms could be resolved and which retained their biological properties.

Identification of proteins by crossed immunoelectrophoresis with a trap-gel.

Simple improvements of the crossed immunoelectrophoresis method are described. A trap-gel was prepared with a small quantity of monospecific antiserum and was submitted to preelectrophoresis. It blocked, during the first dimension, an individual protein as a rocket. The corresponding peak was reduced or disappeared in the second dimension. Identification of precipitation peaks in human or Macacus cynomolgus bronchoalveolar lavage fluids is illustrated and unequivocal recognition of some antigens is shown.

"In vivo" and "in vitro" inhibition of human pancreatic chymotrypsin A by serum inhibitors.

The interaction of human pancreatic chymotrypsin A with serum inhibitors was assessed by enzyme immunoassay, enzymatic activity and inhibitory capacity measurements and electrophoretic analyses. In normal serum, chymotrypsin A was detected in four forms: one form (Mr approximately equal to 25,000) which might be chymotrypsinogen A and three forms complexed to the main inhibitors present in serum, alpha 2-macroglobulin (alpha 2-M), alpha 1-proteinase inhibitor (alpha 1-PI) and alpha 1-antichymotrypsin (alpha 1-Achy). As chymotrypsin A remains to 90% active when bound to alpha 2-M, the chymotrypsin A/alpha 2-M complex was quantified by an enzymatic assay. The kinetic parameters of the interaction of chymotrypsin A with alpha 1-PI and alpha 1-Achy were determined. Using these data the partition of chymotrypsin A between the different inhibitors in serum was calculated. In acute pancreatitis, the chymotrypsin A plasma level follows the progression of the disease and in this case as well as in normal serum alpha 1-PI is the major antagonist of chymotrypsin A.