Changes of phospholipase A2 inhibitory activity in the K+-sensitive actin gelation factor during the differentiation of myeloid leukemia cells

The phospholipase A2 inhibitory activity of a 38 kDa K+-sensitive actin gelation factor in a murine leukemia cell line (M1) was examined. A specific antibody against 38 kDa protein was found to cross-react with 37 kDa protein (lipocortin) in rat peritoneal exudates. Although the native 38 kDa protein from M1 cells did not block phospholipase A2 activity, pretreatment with alkaline phosphatase produced a form that did inhibit this enzyme. However, a purified 38 kDa protein from differentiated M1 cells blocked phospholipase A2 activity without pretreatment with alkaline phosphatase. Phospholipase A2 inhibitory activity of the 38 kDa protein was not altered by addition of actin. These findings suggest that the phospholipase A2 inhibitory of our 38 kDa protein was induced during differentiation. We also proposed that our 38 kDa protein has the same epitope as lipocortin.     

cDNA-cloning, sequencing and expression in glucocorticoid-stimulated quiescent Swiss 3T3 fibroblasts of mouse lipocortin I

We isolated and sequenced mouse lipocortin I cDNA clones from a lambda gt10 cDNA library prepared from Swiss 3T3 mRNA. The homology with human lipocortin I at the amino acid level is 86%. When confluent layers of Swiss 3T3 cells were stimulated with 10% fetal calf serum, expression of lipocortin I was strongly stimulated. In parallel, DNA synthesis was induced with a peak at 24 hours after glucocorticoid treatment indicating induction of cell proliferation. In the absence of serum glucocorticoid treatment provoked neither induction of DNA synthesis nor expression of lipocortin I. We conclude that serum contains an unidentified factor, which acts synergistically with glucocorticoids on cell proliferation and lipocortin I expression.     

Production of lipocortin-like proteins by cultured human tracheal submucosal gland cells

Evidence is obtained for the presence of lipocortin-like proteins in human tracheal gland cells in culture. Using polyclonal antibodies to lipocortin I, indirect immunofluorescence studies demonstrate that lipocortin I is mainly confined to the tracheal gland cell surface. From cell membranes, four Ca2(+)-dependent proteins (35, 40, 45 and 67 kDa) were identified as lipocortin related proteins by using immunoblotting and fluorography following [35S]methionine metabolic labeling experiments. A strong immunoreactivity for the 35 kDa protein was observed. In addition, lipocortin-like proteins with apparent Mr33, 35, 37 and 67 kDa, respectively, were released in the apical culture medium by tracheal gland cells cultured on microporous membrane of a double chamber culture system.     

Evidence from pulse-chase labeling studies that the antiglucocorticoid hormone RU486 stabilizes the nonactivated form of the glucocorticoid receptor in mouse lymphoma cells

A pulse-chase labeling technique was used to determine the properties of glucocorticoid receptors occupied by the antiglucocorticoid hormone RU486 in S49.1 mouse lymphoma cells. Cells were pulse-labeled with [35S]methionine and then at the beginning of the chase, either no hormone (control), dexamethasone, or RU486 was added to cells. At 4 h into the chase, cytosol was prepared and receptors were immunoadsorbed to protein A-Sepharose using the BuGR2 antireceptor antibody. Immunoadsorbed proteins were resolved by gel electrophoresis and analyzed by autoradiography. The 90 kDa heat shock protein (hsp90) coimmunoadsorbed with receptors from control cells when protein A-Sepharose pellets were washed with 250 mM NaCl but not when protein A-Sepharose pellets were washed with 500 mM NaCl, indicating that hsp90-receptor complexes are disrupted by a high concentration of salt in the absence of molybdate. hsp90 coimmunoadsorbed with receptors from RU486-treated cells even when protein A-Sepharose pellets were washed with 500 mM NaCl, indicating that RU486 stabilizes the association of hsp90 with the glucocorticoid receptor. In contrast, hsp90 did not coimmunoadsorb with receptors from dexamethasone-treated cells, consistent with earlier evidence that hsp90 dissociates from the receptor when the receptor binds glucocorticoid hormone. Dexamethasone induced a rapid quantum decrease in the amount of normal receptor recovered from cytosol but did not induce a decrease in the amount of nuclear transfer deficient receptor recovered from cytosol, consistent with tight nuclear binding of normal receptors occupied by dexamethasone. In contrast, RU486 did not induce a quantum decrease in the recovery of normal receptors from cytosol, indicating that receptors occupied by RU486 are not tightly bound in the nuclear fraction. We conclude that the antiglucocorticoid hormone RU486, in contrast to the glucocorticoid hormone dexamethasone, stabilizes the association between the glucocorticoid receptor and hsp90. The decreased affinity of receptors occupied by RU486 for the nuclear fraction may be due to their association with hsp90 and may account for the failure of RU486 to exert agonist activity.     

Glucocorticoid and progesterone inhibit involution and programmed cell death in the mouse mammary gland

Milk production during lactation is a consequence of the suckling stimulus and the presence of glucocorticoids, prolactin, and insulin. After weaning the glucocorticoid hormone level drops, secretory mammary epithelial cells die by programmed cell death and the gland is prepared for a new pregnancy. We studied the role of steroid hormones and prolactin on the mammary gland structure, milk protein synthesis, and on programmed cell death. Slow-release plastic pellets containing individual hormones were implanted into a single mammary gland at lactation. At the same time the pups were removed and the consequences of the release of hormones were investigated histologically and biochemically. We found a local inhibition of involution in the vicinity of deoxycorticosterone- and progesterone-release pellets while prolactin-release pellets were ineffective. Dexamethasone, a very stable and potent glucocorticoid hormone analogue, inhibited involution and programmed cell death in all the mammary glands. It led to an accumulation of milk in the glands and was accompanied by an induction of protein kinase A, AP-1 DNA binding activity and elevated c-fos, junB, and junD mRNA levels. Several potential target genes of AP-1 such as stromelysin-1, c-jun, and SGP-2 that are induced during normal involution were strongly inhibited in dexamethasone-treated animals. Our results suggest that the cross-talk between steroid hormone receptors and AP-1 previously described in cells in culture leads to an impairment of AP-1 activity and to an inhibition of involution in the mammary gland implying that programmed cell death in the postlactational mammary gland depends on functional AP-1.     

Purification and characterization of phospholipase A2 inhibitory proteins from pig thyroid gland

A 32 kDa phospholipase A2 inhibitory protein was isolated from pig thyroid gland after calcium precipitation and fast protein liquid anion-exchange chromatography. SDS-polyacrylamide gel electrophoresis revealed the purity of the protein. The protein activity was assessed by the inhibition of pancreatic phospholipase A2 on [3H]oleic acid-labelled Escherichia coli membranes as substrate and on the prostaglandin E2 production of cultured thyroid cells. The amino acid composition and the isoelectric point were quite similar to those of endonexin previously described in other tissues or cells. The cross-reactivity of a polyclonal antibody against a 32 kDa lipocortin from human peripheral blood mononuclear cells with our thyroidal 32 kDa protein confirmed its lipocortin nature. Before the purification by fast protein liquid chromatography, the Ca2+ pellet contained lipocortin I (35 kDa and its core protein 33 kDa) identified by its cross-reactivity with a polyclonal antibody.     

Calcium-independent phospholipases from guinea pig digestive tract as probes to study the mechanism of lipocortin

Two calcium-independent phospholipases isolated from guinea pig pancreas (lipase Ia, 37 kDa) and from guinea pig intestine (phospholipase B, 97 kDa) have been used to probe the mechanism of phospholipase inhibition by lipocortin. In the presence of calcium, both enzymes were inhibited by lipocortin I in a manner very similar to the inhibition of pig pancreas phospholipase A2. By using phospholipases that lack a requirement for calcium, we have for the first time been able to dissociate enzymatic activity from the role of calcium in the inhibitory process. It was found that lipocortin was without effect against phospholipase A1 and phospholipase B in the absence of calcium, under which conditions the inhibitory protein is unable to interact with anionic phospholipid surfaces. The same behavior toward phospholipase A1 was observed with two other related proteins, endonexin II or lipocortin V, and p68/67-kDa calelectrin or lipocortin VI. Together with the observation that lipocortins are active only in the presence of limited amounts of substrate, these data give further support to the "surface depletion model" of lipocortin inhibition, rather than to a mechanism involving a direct interaction between enzyme and inhibitor.     

Nonactivated and activated glucocorticoid receptor complexes from human salivary gland adenocarcinoma cell line

[3H]Triamcinolone acetonide glucocorticoid receptor complexes from human salivary gland adenocarcinoma cells (HSG cells) were shown to be activated with an accompanying decrease in molecular weight in intact cells, as analyzed by gel filtration, DEAE chromatography, the mini-column method and glycerol gradient centrifugation. Glucocorticoid receptor complexes consist of steroid-binding protein (or glucocorticoid receptor) and non-steroid-binding factors such as the heat-shock protein of molecular weight 90,000. To determine whether the steroid-binding protein decreases in molecular weight upon activation, affinity labeling of glucocorticoid receptor in intact cells by incubation with [3H]dexamethasone 21-mesylate, which forms a covalent complex with glucocorticoid receptor, was performed. Analysis by gel filtration and a mini-column method indicated that [3H]dexamethasone 21-mesylate-labeled receptor complexes can be activated under culture conditions at 37 degrees C. SDS-polyacrylamide gel electrophoresis of [3H]dexamethasone 21-mesylate-labeled steroid-binding protein resolved only one specific 92 kDa form. Furthermore, only one specific band at 92 kDa was detected in the nuclear fraction which was extracted from the cells incubated at 37 degrees C. These results suggest that there is no change in the molecular weight of steroid-binding protein of HSG cell glucocorticoid receptor complexes upon activation and that the molecular weight of nuclear-binding receptor does not change, although the molecular weight of activated glucocorticoid receptor complexes does decrease. Triamcinolone acetonide induced an inhibitory effect on DNA synthesis in HSG cells. Dexamethasone 21-mesylate exerted no such effect and blocked the action of triamcinolone acetonide on DNA synthesis. These results suggests that dexamethasone 21-mesylate acts as antagonist of glucocorticoid in HSG cells. The fact that dexamethasone 21-mesylate-labeled receptor complexes could be activated and could bind to DNA or nuclei as well as triamcinolone acetonide-labeled complexes suggests that dexamethasone 21-mesylate-labeled complexes can not induce specific gene expression after their binding to DNA.     

Lipocortins 1 and 2 as substrates for the insulin receptor kinase in rat liver

Lipocortins 1 and 2 are major substrates for the epidermal growth factor receptor and the pp60v-src tyrosine kinases in transformed cells. In the present study, we have characterized the phosphorylation of lipocortins 1 and 2 by the insulin receptor tyrosine kinase in vitro and in vivo. In vitro, the solubilized insulin receptor, partially purified from rat liver, catalyzed phosphorylation of human recombinant lipocortin 1 and purified bovine lipocortin 2. Phosphorylation of lipocortin 1 was increased 15-fold upon stimulation with 10(-7) M insulin. The apparent Km of the reaction was 3.3 microM and was not affected by insulin stimulation. Insulin stimulated phosphate incorporation into lipocortin 2 by 20-fold (apparent Km greater than 20 microM). Both lipocortins were phosphorylated exclusively on tyrosine residues as judged by phosphoamino acid analysis. Based upon peptide mapping, lipocortin 1 was phosphorylated on Tyr-21, a site phosphorylated by other tyrosine kinases. Polyclonal anti-phosphotyrosine antibodies recognized the tyrosine-phosphorylated lipocortin 2, but not lipocortin 1 in its phosphorylated form. In hepatocytes from normal and dexamethasone-treated rats, lipocortin 1 content was less than 50 ng/10(6) cells. Insulin-induced phosphorylation of lipocortin 1 was detected in intact hepatocytes from corticosteroid-treated animals but not in cells from normal rats. No phosphorylation of lipocortin 2 was found, although its content was approximately 100 ng/10(6) cells from normal animals and increased to approximately 1 microgram/10(6) cells following treatment of rats with dexamethasone for 4 days. Thus, although lipocortins 1 and 2 are in vitro substrates of the insulin receptor kinase, only lipocortin 1 is phosphorylated in an insulin-dependent manner in intact hepatocytes, and this is only observed after dexamethasone treatment of the rats.     

Identification and characterization of phospholipase A2 inhibitory proteins in human mononuclear cells

Calcium-dependent phospholipid binding and phospholipase A2 inhibitory proteins were isolated from human mononuclear cells. Lipocortins I and II were present whereas lipocortin IV (endonexin I) was not. The other proteins were purified to homogeneity and shown to have molecular masses of 35, 36, 32 and 73 kDa. The 36-kDa and 73-kDa proteins are related, the smaller appears to be part of the larger. The 73-kDa protein is related to the 67-kDa calelectrin and to lipocortin VI; the 32-kDa protein is different from endonexin I but related to chromobindin 7 and to lipocortin V. The 35-kDa protein has been identified by tryptic peptide sequencing as lipocortin III. All these proteins inhibit phospholipase A2 activity in vitro and the three smaller ones inhibit the [3H]arachidonic acid release from prelabelled monocytes induced by the calcium ionophore A23187 in a dose-dependent manner.     

Expression of lipocortins in human bronchial epithelial cells: effects of IL-1beta , TNF-alpha, LPS and dexamethasone

In this study, we investigated the expression of lipocortin I and II (annexin I and I in the human bronchial epithelium, both in vivo and in vitro. A clear expression of lipocortin I and II protein was found in the epithelium in sections of bronchial tissue. In cultured human bronchial epithelial cells we demonstrated the expression of lipocortin I and II mRNA and protein using Northern blotting, FACScan analysis and ELISA. No induction of lipocortin I or II mRNA or protein was observed after incubation with dexamethasone. Stimulation of bronchial epithelial cells with IL-1beta, TNF-alpha or LPS for 24 h did not affect the lipocortin I or II mRNA or protein expression, although PGE(2) and 6-keto-PGF(1alpha) production was significantly increased. This IL-1beta- and LPS-mediated increase in eicosanoids could be reduced by dexamethasone, but was not accompanied by an increase in lipocortin I or II expression. In human bronchial epithelial cells this particular glucocorticoid action is not mediated through lipocortin I or II induction.     

Nonactivated and activated glucocorticoid receptor complexes from human salivary gland adenocarcinoma cell line

[³H]Triamcinolone acetonide glucocorticoid receptor complexes from human salivary gland adenocarcinoma cells (HSG cells) were shown to be activated with an accompanying decrease in molecular weight in intact cells, as analyzed by gel filtration, DEAE chromatography, the mini-column method and glycerol gradient centrifugation. Glucocorticoid receptor complexes consist of steroid-binding protein (or glucocorticoid receptor) and non-steroid-binding factors such as the heat-shock protein of molecular weight 90 000. To determine whether the steroid-binding protein decreases in molecular weight upon activation, affinity labeling of glucocorticoid receptor in intact cells by incubation with [³H]dexamethasone 21-mesylate, which forms a covalent complex with glucocorticoid receptor, was performed. Analysis by gel filtration and a mini-column method indicated that [³H]dexamethasone 21-mesylate-labeled receptor complexes can be activated under culture conditions at 37°C. SDS-polyacrylamide gel electrophoresis of [³H]dexamethasone 21-mesylate-labeled steroid-binding protein resolved only one specific 92 kDa form. Furthermore, only one specific band at 92 kDa was detected in the nuclear fraction which was extracted from the cells incubated at 37°C. These results suggest that there is no change in the molecular weight of steroid-binding protein of HSG cell glucocorticoid receptor complexes upon activation and that the molecular weight of nuclear-binding receptor does not change, although the molecular weight of activated glucocorticoid receptor complexes does decrease. Triamcinolone acetonide induced an inhibitory effect on DNA synthesis in HSG cells. Dexamethasone 21-mesylate exerted no such effect and blocked the action of triamcinolone acetonide on DNA synthesis. These results suggests that dexamethasone 21-mesylate acts as antagonist of glucocorticoid in HSG cells. The fact that dexamethasone 21-mesylate-labeled receptor complexes could be activated and could bind to DNA or nuclei aas well as triamcinolone acetonide-labeled complexes suggests that dexamethasone 21-mesylate-labeled complexes can not induce specific gene expression after their binding to DNA.     

Protein expression by Streptococcus uberis in co-culture with bovine mammary epithelial cells

Three strains of Streptococcus uberis isolated from dairy cows with mastitis were co-cultured with a bovine mammary epithelial cell line (MAC-T) in Dulbecco's modified Eagle's medium without fetal bovine serum. Protein profiles from culture supernatants and bacterial pellets among different treatments were compared by electrophoresis. There were proteins induced or having increased expression in both supernatant and surface-associated samples from S. uberis co-cultured with MAC-T cells. Some of these proteins were recognized by antibodies in serum obtained from a cow infected by S. uberis . In supernatant samples, there were two distinct protein bands at 35 and 36.8 kDa for all three strains of S. uberis co-cultured with MAC-T cells. These two bands were absent when bacterial protein synthesis was inhibited by chloramphenicol. This study clearly indicates that bacterial protein expression was regulated in response to co-culture with mammary epithelial cells.     

The role of lipocortin I in macrophage-mediated immunosuppression in tumor-bearing mice

The soluble mediators and/or mechanisms involved in immunosuppression in tumor-bearing hosts are not well characterized, although macrophages have long been recognized as major participants. We have investigated the role of lipocortin I, a phospholipid-binding protein, in macrophage-mediated immunosuppression in tumor-bearing mice. Proliferation of splenic lymphocytes in response to the mitogens (PHA, Con A, LPS, and PWM) was severely suppressed in tumor (Sqc-NH-1 carcinoma)-bearing mice. This immunosuppression was associated with a decrease in T and B lymphocytes and an increase in macrophages in these spleens. Mac-2+ macrophages were found only in spleens from tumor-bearing mice. Splenic macrophages from tumor-bearing, but not normal, mice were responsible for this immunosuppression, as revealed by negative and positive selection experiments. The levels of lipocortin I mRNA expression were markedly increased in peripheral blood cells from tumor-bearing mice as compared with those from normal mice. Lipocortin I mRNA was strongly induced in splenic mononuclear cells from tumor-bearing mice. Furthermore, these cells displayed increased expression of lipocortin I protein, as judged by Western blot analysis with polyclonal anti-lipocortin I serum. Some nonimmune organs such as the heart, submaxillary gland, muscle, and bladder also displayed increased levels of lipocortin I mRNA expression in tumor-bearing mice. Mac-2+ macrophages among the splenic mononuclear cells in tumor-bearing mice expressed lipocortin I mRNA, as judged by negative and positive selection experiments. Most of these Mac-2+ macrophages also had Mac-1 and Mac-3 Ag. Lipocortin I protein was increased in the serum of tumor-bearing mice as compared with normal mice. The culture supernatants of splenic cells from tumor-bearing mice suppressed the mitogenic responses of splenic cells from normal mice, and addition of anti-lipocortin I antiserum inhibited this suppression. Furthermore, recombinant mouse lipocortin I suppressed mitogenic responses of splenic cells from normal mice. In summary, Mac-2+ macrophage-derived lipocortin I was largely involved in immunosuppression in tumor-bearing mice.     

Suppressor cell-inducing factor: a new lymphokine secreted by a natural suppressor cell line with natural cytotoxic activity. I. Purification to apparent homogeneity and initial characterization of suppressor cell-inducing factor

The lymphokine suppressor cell-inducing factor (SIF), obtained from 15 liters of serum-free culture supernatants of the natural suppressor cell line, M1-A5, has been purified to apparent homogeneity by a combination of gel filtration, ion exchange chromatography, and reverse-phase-HPLC. Purity of SIF was assessed by the migration of the factor as a single band on SDS-PAGE, and the elution from reverse-phase-HPLC column as a single and sharp peak. SIF activity was retained after both procedures. Two protein factors with SIF activity were isolated from M1-A5 culture supernatants. The first protein factor (SIF alpha) had a Mr of 43 kDa, and the second protein factor (SIF beta) had a Mr of 6 kDa. Final purification of SIF alpha yielded 5 micrograms protein with specific activity of 4 x 10(6) U/mg protein. Final purification of SIF beta yielded 40 micrograms protein with specific activity of 7.5 x 10(7) U/mg protein. The relationship between SIF alpha and SIF beta, as well as the relationship with other suppressor factors, will be addressed.