Regulation of matrilysin expression in airway epithelial cells by Pseudomonas aeruginosa flagellin.

Matrilysin (matrix metalloproteinase-7) is expressed by mucosal epithelia throughout the body and functions in host defense by activating murine intestinal alpha-defensins. In normal adult human lung, matrilysin is expressed at low levels in the airway epithelium, but is markedly up-regulated in cystic fibrosis (CF). Because CF lungs support a heavy bacterial load, we assessed if relevant CF pathogens regulate matrilysin expression in human lung epithelial cells. Indeed, acute infection with Pseudomonas aeruginosa (but not Staphylococcus aureus, Haemophilus influenzae, or Klebsiella pneumoniae) induced the expression of matrilysin in Calu-3 lung epithelial cells. Increased matrilysin mRNA levels were detectable at 3 h post-infection and peaked at a 25-fold induction between 6 and 8 h. Both P. aeruginosa CF isolates and laboratory strains induced matrilysin expression to similar levels. Flagellin, the monomeric precursor of bacterial flagella, was identified as the inductive factor released by P. aeruginosa that regulated matrilysin expression. In addition, flagellin-null mutants failed to stimulate matrilysin expression in cultured cells or in lungs infected in vivo. These data show that P. aeruginosa (and specifically flagellin) potently stimulates matrilysin expression in lung epithelial cells and may mediate the overexpression of this proteinase in CF lungs.

Matrilysin in epithelial repair and defense.

Repair involves an orderly progression of events to reestablish the integrity of the injured tissue. During each stage in this process, secreted proteinases are needed to remodel extracellular matrix, facilitate cell migration, and process latent proteins, among other functions. In lung epithelium, several of these processes are mediated by matrilysin, a matrix metalloproteinase (MMP). Unlike most MMPs, matrilysin is produced by intact, noninjured airway and peribronchial epithelial cells. In other intact epithelial tissues, namely the small intestine, matrilysin functions in host defense by activating the latent form of defensins, a family of antimicrobial peptides. This metalloproteinase may serve a similar function in the lung. Furthermore, in models of airway injury, matrilysin expression is upregulated in migrating epithelial cells, and the activity of this proteinase is required for repair of airway wounds. These observations indicate that matrilysin serves key functions in both epithelial defense and repair.

Bacterial exposure induces and activates matrilysin in mucosal epithelial cells.

Matrilysin, a matrix metalloproteinase, is expressed and secreted lumenally by intact mucosal and glandular epithelia throughout the body, suggesting that its regulation and function are shared among tissues. Because matrilysin is produced in Paneth cells of the murine small intestine, where it participates in innate host defense by activation of prodefensins, we speculated that its expression would be influenced by bacterial exposure. Indeed, acute infection (10-90 min) of human colon, bladder, and lung carcinoma cells, primary human tracheal epithelial cells, and human tracheal explants with type 1-piliated Escherichia coli mediated a marked (25-50-fold) and sustained (>24 h) induction of matrilysin production. In addition, bacterial infection resulted in activation of the zymogen form of the enzyme, which was selectively released at the apical surface. Induction of matrilysin was mediated by a soluble, non-LPS bacterial factor and correlated with the release of defensin-like bacteriocidal activity. Bacteria did not induce matrilysin in other cell types, and expression of other metalloproteinases by epithelial cells was not affected by bacteria. Matrilysin was not detected in germ-free mice, but the enzyme was induced after colonization with Bacteroides thetaiotaomicron. These findings indicate that bacterial exposure is a potent and physiologically relevant signal regulating matrilysin expression in epithelial cells.

Regulation of intestinal alpha-defensin activation by the metalloproteinase matrilysin in innate host defense.

Precursors of alpha-defensin peptides require activation for bactericidal activity. In mouse small intestine, matrilysin colocalized with alpha-defensins (cryptdins) in Paneth cell granules, and in vitro it cleaved the pro segment from cryptdin precursors. Matrilysin-deficient (MAT-/-) mice lacked mature cryptdins and accumulated precursor molecules. Intestinal peptide preparations from MAT-/- mice had decreased antimicrobial activity. Orally administered bacteria survived in greater numbers and were more virulent in MAT-/- mice than in MAT+/+ mice. Thus, matrilysin functions in intestinal mucosal defense by regulating the activity of defensins, which may be a common role for this metalloproteinase in its numerous epithelial sites of expression.

Induction and evasion of host defenses by type 1-piliated uropathogenic Escherichia coli.

Virtually all uropathogenic strains of Escherichia coli encode filamentous surface adhesive organelles called type 1 pili. High-resolution electron microscopy of infected mouse bladders revealed that type 1 pilus tips interacted directly with the lumenal surface of the bladder, which is embedded with hexagonal arrays of integral membrane glycoproteins known as uroplakins. Attached pili were shortened and facilitated intimate contact of the bacteria with the uroplakin-coated host cells. Bacterial attachment resulted in exfoliation of host bladder epithelial cells as part of an innate host defense system. Exfoliation occurred through a rapid apoptosis-like mechanism involving caspase activation and host DNA fragmentation. Bacteria resisted clearance in the face of host defenses within the bladder by invading into the epithelium.

Growth inhibition of human breast cancer cells by 1,25-dihydroxyvitamin D3 is accompanied by induction of apolipoprotein D expression.

We have analyzed the effect of 1,25-dihydroxyvitamin D3 on the expression of the gene encoding apolipoprotein D (apoD), a protein component of the human plasma lipid transport system that is overproduced by a specific subset of breast carcinomas. Northern blot analysis revealed that 1,25-dihydroxyvitamin D3 strongly up-regulated apoD mRNA levels in T-47D human breast cancer cells in a time- and dose-dependent manner. The potency of this vitamin as an inducer of apoD expression was stronger than the effect observed for such steroid hormones as androgens and progesterone, described previously as hormonal up-regulators of apoD expression in these cells. A time course study demonstrated that the induction of apoD mRNA reached a level of 5-fold over the untreated cells after 48 h of incubation in the presence of 10(-7) M 1,25-dihydroxyvitamin D3. A dose-response analysis showed that a 10(-6) M concentration of this vitamin consistently induced a maximal accumulation of 7-fold over the control cells. Similar up-regulatory effects on the apoD gene expression were obtained by treatment of T-47D cells with 1,25-dihydroxyvitamin D3 analogues, including MC 903, which is relatively devoid of hypercalcemic side effects in clinical applications. Western blot analysis revealed that the inductive effect of 1,25-dihydroxyvitamin D3 was also reflected at the protein level as an increase of immunoreactive protein in the conditioned media of vitamin-treated cells. This increased expression of apoD was accompanied by an inhibition of cell growth and morphological changes in T-47D cells. By contrast, we did not detect any inductive effect of 1,25-dihydroxyvitamin D3 on apoD gene expression in MDA-MB-231 cells, which are refractory to the growth-inhibitory effects of this compound. On the basis of these results, we propose 1,25-dihydroxyvitamin D3 as an important regulator of the expression of the apoD gene in breast carcinomas. We also suggest that apoD may be of interest as a biochemical marker of the action of 1,25-dihydroxyvitamin D3 derivatives in current studies using these compounds as inhibitors of breast cancer cell growth or as chemotherapeutic agents in the prevention of breast cancer.

Retinoic acid-induced expression of apolipoprotein D and concomitant growth arrest in human breast cancer cells are mediated through a retinoic acid receptor RARalpha-dependent signaling pathway.

Apolipoprotein D (apoD) is a human plasma protein, belonging to the lipocalin superfamily, that is produced by a specific subtype of highly differentiated breast carcinomas and that is strongly up-regulated by retinoic acid (RA) in breast cancer cells. In this work, we have examined the molecular mechanisms mediating the induction of apoD gene expression by retinoids in T-47D human breast cancer cells. Northern blot analysis revealed that Ro40-6055, a synthetic retinoid that selectively binds and activates the retinoic acid receptor RARalpha, induced the accumulation of apoD mRNA in breast cancer cells in a time- and dose-dependent manner. The time course analysis demonstrated that apoD mRNA was induced 14-fold over control cells after 48 h of incubation with 10(-8) M Ro40-6055. As little as 10(-11) M of this retinoid induced apoD mRNA 5-fold over the control, whereas incubation with 10(-7) M Ro40-6055 induced maximally 15-fold over control cells. RARalpha-selective antagonists counteracted the inductive effects of all-trans-RA, 9-cis-RA, and Ro40-6055 on the expression of apoD, when present at the same concentration as the retinoid agonists. By contrast, RARbeta-, RARgamma-, and RXR-selective retinoids did not affect apoD gene expression. The retinoid agonist Ro40-6055 had an antiproliferative effect on T-47D cells, with maximal growth inhibition of approximately 60% obtained after 7 days of incubation with 10(-7) M. This antiproliferative effect could be counteracted by a 100-fold excess of the antagonist Ro41-5253. Treatment of the cells with retinoids that do not bind the nuclear retinoic acid receptors did not affect apoD expression, despite the fact that they did have a strong antiproliferative effect on T-47D cells. On the basis of these results, a role for RARalpha on apoD gene expression induction by retinoids in breast cancer cells is proposed.

Arachidonic acid binds to apolipoprotein D: implications for the protein's function.

The lipocalin apolipoprotein D (ApoD) is associated in human plasma with lecithin-cholesterol acyl transferase. It has also been found in high concentration in the fluid of gross cystic disease of the mammary gland. Using protein fluorescence quenching, it is shown that ApoD binds arachidonic acid (Ka of 1.6 x 10(8) M-1) and as previously known progesterone (Ka of 2.5 x 10(6) M-1), but neither cholesterol nor any of the other prostanoid molecules examined had measurable affinity. This specific binding of arachidonate, also observable directly, suggests a role for ApoD in the mobilisation of arachidonic acid, and hence prostaglandin synthesis.

Apolipoprotein D gene induction by retinoic acid is concomitant with growth arrest and cell differentiation in human breast cancer cells.

We have examined the regulation by retinoic acid of the gene encoding apolipoprotein D (apoD), a human plasma protein belonging to the superfamily of the lipocalins that is produced by a specific subtype of highly differentiated breast carcinomas. Northern blot analysis revealed that all-trans-retinoic acid (RA) strongly induced the accumulation of apoD mRNA in T-47D and ZR-75-1 estrogen receptor-positive human breast cancer cells in a time- and dose-dependent manner, while no inductive effect was observed in estrogen receptor-negative cell lines, including MDA-MB-231 and MDA-MB-435. The effect of RA on apoD expression by T-47D cells was at least 12-fold more potent than the effect of the steroids dihydrotestosterone and dexamethasone, which had been previously described as hormonal up-regulators of apoD expression in these cells. A time course study demonstrated that the induction of apoD mRNA reached a level of 15-fold over the untreated control cells after 48 h of incubation in the presence of a 10(-7) M concentration of RA. A dose-response analysis showed that as little as 10(-13) M RA produced an accumulation of 5-fold over the control, while incubation of the cells in the presence of 10(-5) M RA induced a maximal accumulation of 24-fold over the control untreated cells. The induction of apoD mRNA was independent of the synthesis of proteins de novo, as demonstrated by the fact that the induction was also detected in the presence of cycloheximide. The incubation of the cells in the presence of RA did not affect significantly the stability of apoD mRNA. By contrast, treatment of the T-47D cells with RA produced an increase of approximately 8-fold in the rate of transcription of the apoD gene. Furthermore, treatment of the T-47D cells with RA induced the synthesis and secretion to the culture medium of apoD. This increased expression of apoD was accompanied by an inhibition of cell proliferation and a progression through a more differentiated phenotype, suggesting that the mechanisms controlling RA-induced growth arrest, cell differentiation, and apoD synthesis may be directly coordinated in human breast cancer cells.

Glucocorticoids and androgens up-regulate the Zn-alpha 2-glycoprotein messenger RNA in human breast cancer cells.

We have studied the hormonal regulation of the gene encoding Zn-alpha 2-glycoprotein (Zn-alpha 2-gp), a human protein with a high degree of amino acid sequence similarity to class I histocompatibility antigens that is produced by a specific subset of breast carcinomas. Northern blot analysis revealed that dexamethasone and 5 alpha-dihydrotestosterone strongly induced the accumulation of Zn-alpha 2-gp mRNA in T-47D human breast cancer cells. Furthermore, the effect of these two hormones was shown to be additive, since the combination of both hormones produced a stimulation of Zn-alpha 2-gp mRNA of at least 3-fold over that produced by either hormone alone. By contrast, the addition of 5 beta-dihydrotestosterone, 17 beta-estradiol, or progesterone failed to induce the expression of Zn-alpha 2-gp. The stimulatory effect of glucocorticoids and androgens on Zn-alpha 2-gp expression was produced in a time and dose dependent manner, without significantly affecting the cell proliferation rate. A time-course study demonstrated that the induction of Zn-alpha 2-gp mRNA by androgens and glucocorticoids reached a level of 4 or 3.2-fold over the untreated control after seven days of incubation in the presence of a 10(-7) M concentration of 5 alpha-dihydrotestosterone or dexamethasone, respectively. A dose-response study showed that as little as 10(-11) M of 5 alpha-dihydrotestosterone or dexamethasone produced an accumulation of Zn-alpha 2-gp mRNA of 2.4 or 2.1-fold over the control, respectively. On the basis of these results, we propose that Zn-alpha2-gp may be useful as a biochemical marker of breast carcinomas with a specific pattern of hormone responsiveness in whose development glucocorticoids and/or androgens may play a significant role.

Human Zn-alpha 2-glycoprotein: complete genomic sequence, identification of a related pseudogene and relationship to class I major histocompatibility complex genes.

The human gene (AZGP1) encoding Zn-alpha 2-glycoprotein (Zn-alpha 2-gp), a protein present in several biological fluids and produced by a subtype of breast carcinomas, has been cloned and its complete nucleotide sequence determined. The gene spans over 9.7 kb, and its overall organization and nucleotide sequence are very similar to those of the first four exons of class I MHC genes. However, the Zn-alpha 2-gp gene differs from these genes in several significant ways. It lacks the coding information for the transmembrane and cytoplasmic domains typical of MHC genes, which is consistent with its presence as a soluble protein in different physiological and pathological fluids. In addition, it contains a high density of repetitive sequences, including Alu, MER, and MIR elements, which are not present at equivalent positions in class I MHC genes. Finally, its 5'-flanking region lacks the class I MHC regulatory complex and the interferon consensus sequence characteristic of class I MHC genes. These findings may explain the different expression pattern of Zn-alpha 2-gp and class I MHC genes in human tissues. Southern blot hybridization of DNA from several species with a cDNA probe indicated that Zn-alpha 2-gp genes are present in a wide variety of animal species, including monkey, rat, mouse, dog, cow, and rabbit. The human genome also contains a putative Zn-alpha 2-gp pseudogene that has been isolated and partially characterized. This pseudogene has an intron-exon organization identical to that of the functional gene, but it presents two deleterious mutations in the third exon that lead to the appearance of premature stop codons. Finally, considering the lack of polymorphism in the Zn-alpha 2-gp gene in comparison with MHC genes, putative roles for this human glycoprotein in the transport of nonpolymorphic substances or in intercellular recognition processes are proposed.

Autophosphorylation of yeast hexokinase PII.

Autophosphorylation of hexokinase PII was studied using an enzyme purified from Saccharomyces cerevisiae. Incubation of this enzyme preparation with [gamma-32P]ATP and Mn2+ or Mg2+ gave a phosphoprotein of molecular mass 58,000 which corresponded to hexokinase PII. D-Xylose stimulated autophosphorylation of hexokinase PII. Dilution of hexokinase PII over a 10-fold concentration range did not change the specific activity of hexokinase PII autophosphorylation suggesting that it may occur by an intramolecular mechanism.

Glucose-stimulated phosphorylation of yeast isocitrate lyase in vivo.

Incorporation of 32P into Saccharomyces cerevisiae isocitrate lyase was observed after addition of glucose to a culture incubated with [32P]orthophosphoric acid. A band of 32P-labelled protein was coincident with the enzyme band when immunoprecipitates were subjected to SDS-PAGE and autoradiography. No label was found in the band corresponding to the isocitrate lyase when immunoprecipitation was done with a control pre-immune serum or in the presence of excess pure unlabelled enzyme. The incorporation of phosphate was associated with a decrease in enzyme activity. Phosphorylated isocitrate lyase was not proteolytically degraded when cells were cultured in mineral medium. The loss of protein antigenicity only took place when the yeast was grown in a complex medium containing glucose.

Purification of isocitrate lyase from Saccharomyces cerevisiae.

Isocitrate lyase purified to homogeneity from Saccharomyces cerevisiae was composed of four identical subunits with a molecular mass of 75 kDa. The enzyme was most active at pH 7.0 in the presence of 5 mM-Mg2+. The Km value for threo-Ds-isocitrate was 1.4 mM. Isocitrate lyase was shown to be thermostable at 50 degrees C for 60 min at a high salt concentration, but rapidly lost activity at -20 degrees C or by dialysis.

Proteolysis of hexokinase PII is not the triggering signal of carbon catabolite derepression in Saccharomyces cerevisiae.

The role of hexokinase PII in mediating carbon catabolite derepression in yeast has been examined. Hexokinase isoenzyme PII (EC 2.7.1.1) was partially degraded when protease inhibitors were omitted from the buffer used for preparation of cell-free extracts. The hexokinase PII inactivation induced by D-xylose was correlated with derepression of maltase (EC 3.2.1.20) in the wild-type strain Saccharomyces cerevisiae G-517 and in D.308.3, a strain that contains the cloned hexokinase PII gene on a multicopy plasmid. This inactivation was not correlated with the loss of hexokinase PII protein as assayed by immunoblotting. We conclude that during the derepression process there is no release of proteolytic peptides from hexokinase PII.

Catabolite inactivation of isocitrate lyase from Saccharomyces cerevisiae.

A reversible carbon catabolite inactivation step is described for isocitrate lyase from Saccharomyces cerevisiae. This reversible inactivation step of isocitrate lyase is similar to that described for fructose 1,6-bisphosphatase. Addition of 2,4-dinitrophenol, nystatin or glucose to cultures, grown in ethanol as carbon source, caused a rapid loss of the isocitrate lyase and fructose 1,6-bisphosphatase activities at pH 5.5 but not at pH 7.5. These results suggest that intracellular acidification and thus a cAMP increase is involved in the catabolite inactivation mechanism of both enzymes. From results obtained by addition of glucose to yeast cultures at pH 7.5 it was concluded that others factors than cAMP can play a role in the catabolite inactivation mechanism of both enzymes.