Intracellular protein degradation in cultured bovine lens epithelial cells

Summary Although several proteases have been identified in homogenates of cultured epithelial cells of the eye lens and in lens tissues, there is little information regarding intracellular protein degradation in intact lens cells in vitro. Cultured lens cells may be useful in the study of intracellular protein degradation in the lens, a tissue with a wide range of protein half-lives. This is of interest because alterations in protein turnover in the lens have been implicated in cataract formation. This study examines intracellular protein degradation in cultured bovine lens epithelial cells (BLEC). Cell cultures were incubated with radiolabeled leucine to label intracellular proteins. Protein degradation was measured by monitoring the release of trichloroacetic-acid-soluble radioactivity into the culture medium. The average half-life of long-lived proteins (half-life >50 h) was typically about 57 h in serum-supplemented medium. Average rates of degradation of long-lived proteins increased by up to 73% when fetal bovine serum was withdrawn from the culture medium. Serum had no effect on the degradation of short-lived proteins (half-life <10 h). Degradation of long-lived proteins in the presence and absence of serum was further studied in cultured BLEC from population doubling level (PDL) 2 to 43. Average half-life of proteins in serum-supplemented medium was 52 to 58 h and did not vary significantly as a function of PDL. Degradation rates in serum-free medium increased approximately twofold up to PDL 7, but returned by PDL 25 to original levels, which were maintained through PDL 43. This work was supported in part by grants from U. S. Department of Agriculture contract 53-3K06-5-10, Massachusetts Lions Eye Research Fund, Inc., and the Daniel and Florence Guggenheim Foundation. D. A. E. is a recipient of a National Eye Institute postdoctoral fellowship.     

Regulation of phenol sulfotransferase expression in cultured bovine bronchial epithelial cells by hydrocortisone

One conjugative pathway for the inactivation of endogenous and exogenous hydroxylated aromatic compounds is catalyzed by phenol (aryl) sulfotransferases (PSTs), which esterify phenolic acceptors with sulfate. The tracheobronchial epithelium is commonly exposed to phenolic drugs and pollutants, and metabolic sulfation and PST activity in this tissue have been previously demonstrated. To determine what factors may control PST expression, extracts of serum-free, growth factor-supplemented cultures of bovine bronchial epithelial cells were assayed for PST activity and PST antigen. The most significant finding was dose-dependent, apparent stimulated expression by hydrocortisone (EC₅₀ = 4 nM, maximal stimulation at 20 nM). Time-course experiments, however, revealed progressive loss of PST in the absence of corticosteroid. After decay of extant PST in steroid-free medium, hydrocortisone reinduced the expression of PST three to fivefold. Western blots using mouse anti-bovine PST revealed corresponding increases in 32 kDa PST protein levels in response to hydrocortisone. Steady state kinetic analyses indicated apparent K_m values of 1—3 μM for 2-naphthol regardless of culture conditions. These results suggest that detoxification of phenolic compounds by sulfation may be regulated by corticosteroids.     

Fas-mediated apoptosis is suppressed by calf serum in cultured bovine luteal cells

Calf serum (CS) is a common supplement used in cell culture. It has been suggested that CS contains substances protecting cells against apoptosis. To examine whether a culture system including CS is appropriate for studying apoptosis in bovine luteal cells, we examined the influence of CS on the expression of Fas, bcl-2 and bax gene. Since progesterone (P(4)) is known to be an anti-apoptotic factor in bovine luteal cells, the present study was carried out to examine the P(4) effect on apoptosis. Bovine mid-luteal cells were exposed to Fas ligand (Fas L) in the presence or in the absence of P(4) antagonist (onapristone, OP) in a basal medium (BM) containing 5% CS (BM-CS) or BM containing 0.1% BSA (BM-BSA). Although Fas L alone, OP alone or Fas L plus OP did not show any cytotoxic effect on the cells cultured in BM-CS, administration of OP or OP in combination with Fas L resulted in the killing of 30% and 55% of the cells cultured in BM-BSA medium, respectively (p<0.05). Concomitantly, CS inhibited bax mRNA expression and stimulated bcl-2 expression in the cells (p<0.05). Moreover, in the cells cultured with BM-CS, Fas mRNA expression was smaller than that of cells incubated in BM-BSA medium (p<0.05). The overall results suggest that CS suppressed Fas-mediated cell death in cultured bovine luteal cells by promoting the ratio of bcl-2 to bax expression and by inhibiting Fas expression. Therefore, it may be suggested that CS contains such anti-apoptotic substances (growth factors) amplifying the cell survival pathways in the bovine corpus luteum (CL) in vitro.     

Effect of progesterone on protein synthesis and secretion by cultured epithelial cells from guinea-pig endometrium

Summary The pattern of proteins synthesized and secreted in response to progesterone by guinea-pig endometrial epithelial cell cultured with estradiol-17 was investigated. Glandular epithelial cells were maintained in culture for 3 days on growth medium, then washed three times with a steroid-free medium. After this period, 2 × 10^-8 M estradiol-17 or 2 × 10^-8 M estradiol-17 plus 5 × 10^-7 M progesterone were added to the medium for 48 h. To study biochemical changes, the proteins were labeled by a 6 h pulse of ³⁵S-methionine. The proteins in medium and in cells were analyzed by one- and two-dimensional polyacrylamide gel electrophoresis, followed by fluorography. The addition of progesterone to estradiol-17 in the culture medium caused a change in the patterns of cellular and secreted proteins: one-dimensional gel electrophoresis showed that variation of 8 cellular proteins and 12 secreted proteins was caused by progesterone. Induction of individual proteins by progesterone treatment was observed by two-dimensional gel electrophoresis: one cellular protein (Mr 49000; pI 5.90) and one secreted protein (Mr 14300; pI 4.80) were specifically induced and might serve as markers of progesterone action.     

Regulation of angiotensin I-converting enzyme in cultured bovine bronchial epithelial cells

The purpose of this study was to determine whether angiotensin I-converting enzyme (ACE) is present in cultured bovine bronchial epithelial cells (BBECs) and whether its activity can be modulated. We found that extracts of confluent monolayers of cultured BBECs degraded [glycine-1-¹⁴C]hippuryl-L -histidyl-L -leucine at a rate of 843 ± 66 pmol/hr/mg protein (mean ± SEM, n = 5). In addition, we found that the enzyme was shed into the culture medium. ACE activity in BBECs was inhibited by three selective, but structurally different, ACE inhibitors (captopril, quinapril, and cisalaprilat) with an IC₅₀ of approximately 2 nM. Increasing chloride concentration in the assay buffer resulted in an increase in BBECs ACE activity of 63%. Enzyme activity was also modulated by the presence of zinc cation in the assay buffer. Addition of dexamethasone to the culture medium was associated with a significant increase in BBECs ACE activity (P < 0.05), which was inhibited by the steroid receptor antagonist RU 38486. Western blot analysis of BBECs, tracheal and bronchial mucosal strips utilizing a cross-reacting rabbit anti-mouse ACE antibody, showed a faint 175 kDa band and additional strong 52 kDa and 47 kDa band. The mechanism of generation of the low M.W. bands is unknown. Our data indicate the presence of ACE in cultured BBECs and that enzyme activity can be modulated.     

Selective maintenance of cultured epithelial cells from DMBA-induced mammary tumours by bovine colostrum supplement

Growth kinetics in cultures of DMBA-induced mammary tumours supplemented with bovine colostrum were compared with the kinetics of cultures maintained with the conventional supplement of foetal calf serum. Although the latter permitted a greater degree of cell proliferation, a substantial amount of the cell growth was due to the fibroblastic proliferation. In the presence of bovine colostrum, epithelial islands surrounded by a few solitary cells became established. Unlike the foetal calf serum supplemented cultures, these cultures frequently did not become completely confluent within 7 days. The absence of fibroblasts in colostrum supplemented cultures was confirmed by electron microscopy. Results from this study suggest that colostrum may be useful in selective maintenance of primary cultures of epithelial origin.     

Lens glycoproteins: biosynthesis in cultured epithelial cells of bovine lens.

1. Radioactivity from [3H]glucosamine is rapidly incorporated into cellular fractions of lens epithelial cells cultured in vitro. The incorporated isotope appears largely in glycoproteins of the cell surface that are exposed to trypsin and are released into a soluble form by proteolysis of intact cells. Glycoproteins are also secreted by cultured cells and can be recovered in the culture fluids. Sodium dodecysulphate-polyacrylamide gell electrophoresis shows that a range of glycoproteins with apparent molecular weights from approximately 14000 to 120000 are present. The relationships of these glycoproteins to collagen and the non-collagenous glycoproteins of lens basement membranes are discussed. 2. A plasma membrane fraction obtained from non-trypsinised lens epithelial cells contains one major glycoprotein of apparent molecular weight 120000. A major non-glycosylated polypeptide of molecular weight about 38000 detectable by Bloemendal et al. (1972) in plasma membranes of differentiated lens fibre cells was not prominent in lens epithelial cell membranes. 3. Examination of lens basement membranes extracted in various ways failed to reveal major glycoproteins of low molecular weight. Higher molecular weight glycoproteins, some of them related to collagen, were present.     

Methionine protects against hyperthermia-induced cell injury in cultured bovine mammary epithelial cells

The aim of this study was to investigate the effects of methionine on cell proliferation, antioxidant activity, apoptosis, the expression levels of related genes (HSF-1, HSP70, Bax and Bcl-2) and the expression levels of protein (HSP70) in mammary epithelial cells, after heat treatment. Methionine (60 mg/L) increased the viability and attenuated morphological damage in hyperthermia-treated bovine mammary epithelial cells (BMECs). Additionally, methionine significantly reduced lactate dehydrogenase leakage, malondialdehyde formation, nitric oxide, and nitric oxide synthase activity. Superoxide dismutase, catalase, and glutathione peroxidase enzymatic activity was increased significantly in the presence of methionine. Bovine mammary epithelial cells also exhibited a certain amount of HSP70 reserve after methionine pretreatment for 24 h, and the expression level of the HSP70 gene and protein further increased with incubation at 42 °C for 30 min. Compared to the control, the expression of HSF-1 mRNA increased, and there was a significantly reduced expression of Bax/Bcl-2 mRNA and a reduced activity of caspase-3 against heat stress. Methionine also increased survival and decreased early apoptosis of hyperthermia-treated BMECs. Thus, methionine has cytoprotective effects on hyperthermia-induced damage in BMECs.     

Sexual dimorphism in interferon-tau production by in vivo-derived bovine embryos

Interferon-tau (IFN-tau) is an anti-luteolytic factor responsible for preventing regression of the maternal corpus luteum (CL) during early pregnancy of cattle. In vitro-produced (IVP) bovine embryos first produce IFN-tau as blastocysts. In the present study, we have examined whether sexually dimorphic production of IFN-tau, which is observed among IVP blastocysts, also occurs among in vivo-produced blastocysts, and whether this difference between the sexes persists to day 14 when silencing of one of the X-chromosomes in the trophectoderm is complete. Embryos were flushed from cattle that had been superovulated and bred by AI. Blastocysts (63 male, 62 female) recovered between days 8.5 and 9.5 of pregnancy, were cultured individually. No differences were observed between males and females in either their developmental stage or quality at the beginning, during, and at the end of culture. Female embryos produced more IFN-tau than males by 24 hr (mean values, males: 16.6 +/- 3.7, females: 49.4 +/- 9.0 pg per embryo; P < 0.05) and 48 hr (male: 189.8 +/- 37.1, female: 410.9 +/- 66.6 pg per embryo; P < 0.05). However, the variability in IFN-tau production between individual blastocysts was so great that IFN-tau secretion is unlikely to be of value as a non-invasive means to predict embryo sex. When conceptuses were recovered at day 14, elongating males (n = 25) and females (n = 24) were similar in dimension and did not differ in their IFN-tau production after 4.5 hr (male: 2,550 +/- 607, female: 2,376 +/- 772 ng per conceptus) and 24 hr (male: 12,056 +/- 2,438, female: 8,447 +/- 1,630 ng per conceptus) of culture. Thus, sexual dimorphism in IFN-tau production is observed in both IVP and in vivo-produced expanded blastocysts, but is lost by day 14 of in vivo development.     

A comparison of DNA repair in cultured bovine lens epithelial cells and lung fibroblast cells

The effect of X-irradiation has been assessed on cultivated bovine lens epithelial cells by analysis of the DNA sedimentation in alkaline sucrose gradient and compared with bovine fibroblasts. (1) It can be concluded that for the same irradiation, DNA from both sources were broken to the same extent; (2) the rate of DNA repair was similar for both types of cells. The relation between these results and the fact that X-ray induces cataract very easily in the lens is discussed.     

Interferon-gamma induction of major histocompatibility complex antigens on cultured bovine luteal cells

To investigate immunological mechanisms that may be involved in luteal function, the presence of Class I and Class II major histocompatibility complex (MHC) antigens on cultured bovine luteal cells was examined. After 72 h in serum-free culture, Class I antigens were markedly expressed on luteal cells, as determined by indirect immunofluorescence, whereas expression of Class II antigens was limited. The expression of MHC antigens on luteal cells was increased by treatment with the T-lymphocyte factor, interferon-gamma (IFN-gamma). Class I and II antigens were elevated 25% and 370% above controls, respectively, after IFN-gamma exposure. Since the corpus luteum is regulated by luteinizing hormone (LH), luteal cells were treated with either hormone alone or hormone in addition to IFN-gamma, and antigen expression was determined. LH treatment attenuated IFN-gamma-induction of Class II antigens on bovine luteal cells. These observations are the first to demonstrate the presence of MHC antigens on bovine luteal cells and the modulation of antigen expression by the lymphokine IFN-gamma and by LH.     

Survival pathways regulating the apoptosis induced by tumour necrosis factor-alpha in primary cultured bovine endothelial cells

The aim of the present study was to identify biochemical pathways driving the resistance of endothelial cells to apoptosis induced by tumour necrosis factor-alpha (TNF). (1) Although nuclear factor-kappa B (NF-kappaB) was activated by TNF, its inhibition by MG-132 failed to sensitize these cells. (2) The activation of protein kinase C (PKC) by phorbol ester completely abolished the TNF-induced cell death. (3) The phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin (Wo) triggered apoptosis and enhanced the TNF-induced cell death. (4) The MEK inhibitor PD98059 did not affect the TNF-induced apoptotic process. (5) The p38 is activated by TNF and its inhibition by SB203580 sensitized the cells to TNF. This is correlated with the inhibition of phosphorylation of heat-shock protein of 27 kDa (HSP27).These results indicate that TNF activates NF-kappaB, which does not drive any anti-apoptotic response, and p38, which plays an anti-apoptotic function probably through HSP27 phosphorylation. Moreover, PKC and PI3K are involved in the control of survival pathways.     

Down-regulation of oxytocin-induced cyclooxygenase-2 and prostaglandin F synthase expression by interferon-tau in bovine endometrial cells

Oxytocin (OT) is responsible for the episodic release of luteolytic prostaglandin (PG) F2alpha from the uterus in ruminants. The attenuation of OT-stimulated uterine PGF2alpha secretion by interferon-tau (IFN-tau) is essential for prevention of luteolysis during pregnancy in cows. To better understand the mechanisms involved, the effect of recombinant bovine IFN-tau (rbIFN-tau) on OT-induced PG production and cyclooxygenase-2 (COX-2) and PGF synthase (PGFS) expression in cultured endometrial epithelial cells was investigated. Cells were obtained from cows at Days 1-3 of the estrous cycle and cultured to confluence in RPMI medium supplemented with 5% steroid-free fetal calf serum. The cells were then incubated in the presence or absence of either 100 ng/ml OT or OT+100 ng/ml rbIFN-tau for 3, 6, 12, and 24 h. OT significantly increased PGF2alpha and PGE2 secretion at all time points (p < 0.01), while rbIFN-tau inhibited the OT-induced PG production and reduced OT receptor binding in a time-dependent manner. OT increased the steady-state level of COX-2 mRNA, measured by Northern blot, which was maximal at 3 h (9-fold increase) and then decreased with time (p < 0.01). OT also caused an increase in COX-2 protein, which peaked at 12 h (11-fold increase), as measured by Western blot. Addition of rbIFN-tau suppressed the induction of COX-2 mRNA (89%, p < 0.01) and COX-2 protein (50%, p < 0.01) by OT. OT also increased PGFS mRNA, and this stimulation was attenuated by rbIFN-tau (p < 0.01). To ensure that the decrease in COX-2 was not solely due to down-regulation of the OT receptor, cells were stimulated with a phorbol ester (phorbol 12-myristate 13-acetate; PMA) in the presence and absence of rbIFN-tau. The results showed that rbIFN-tau also decreased PMA-stimulated PG production and COX-2 protein. It can be concluded that rbIFN-tau inhibition of OT-stimulated PG production is due to down-regulation of OT receptor, COX-2, and PGFS.     

MicroRNA-216b inhibits heat stress-induced cell apoptosis by targeting Fas in bovine mammary epithelial cells

Heat stress affects milk yield and quality in lactating dairy cows in summer. Bovine mammary epithelial cells (bMECs) play a key role in milk secretion, and microRNAs (miRNAs) regulate numerous functions of bMEC. Previous reports have verified that miR-216b regulated cell apoptosis through repressing target genes in several cancer cells. So, our purpose was to explore the potential involvement of miR-216b in heat stress-induced cell apoptosis in bMECs. Firstly, the heat stress model was constructed and we found that apoptotic rates of bMECs significantly increased under heat stress. The expression of miR-216b, Bax mRNA, and caspase-3 mRNA was upregulated. However, Bcl-2 mRNA level was detected to differentially downregulated. Overexpression of miR-216b remarkably downregulated the expression of caspase-3 and Bax mRNA and protein, and the mRNA and protein level of Bcl-2 was increased. Inhibition of miR-216b increased the activity of caspase-3 and Bax, and the level of Bcl-2 was inhibited. Moreover, Fas was identified as a target gene of miR-216b through bioinformatic analysis and dual-luciferase reporter assay. Fas activity was significantly inhibited and enhanced respectively after transfecting miRNA mimics and inhibitor. Finally, inhibition of Fas via the small interfering RNA (siRNA) also inhibited cell apoptosis induced by heat stress. Taken together, our results indicated that miR-216b exerted as an anti-apoptotic effect under heat stress in bMECs by targeting Fas.