Regulation of vimentin expression in cultured human mammary epithelial cells

Using five different monoclonal antibodies to vimentin, we have examined the expression of vimentin in cryostat sections and serum-free cultures of normal human breast tissue. In cryostat sections, myoepithelial cells as well as stromal cells showed immunoreactivity to vimentin, irrespective of the antibody used. In contrast, luminal epithelial cells were negative for vimentin, but positive for keratin K18. In culture, myoepithelial cells showed immunoreactivity to vimentin from their first appearance in monolayer. Moreover, a fraction of luminal epithelial cells expressed vimentin in addition to keratin K18. We found a clear, reversible correlation between proliferation, determined by incorporation of [3H]-TdR, and induction of vimentin in the luminal epithelial cells. Thus, in growth-stimulated cultures on a medium containing cholera toxin (CT), epidermal growth factor (EGF), transferrin (Tf), hydrocortisone (H) and insulin (I), the fraction of vimentin-positive luminal epithelial cells increased, while it decreased within 14 days from approximately 36% to 3% on a medium containing CT and EGF, only. We therefore conclude: (1) vimentin is constantly expressed in myoepithelial cells in situ and in vitro, and (2) expression of vimentin in luminal epithelial cells in vitro is not a result of monolayer cultivation as such, but rather associated with the increased growth rate seen in culture.     

Modification of transepithelial ion transport in human cultured bronchial epithelial cells by interferon-gamma

Human bronchial epithelial cells were treated in vitro with interferon-gamma or tumor necrosis factor-alpha to assess their effect on transepithelial ion transport. Short-circuit current measurements revealed that Na(+) absorption was markedly inhibited by interferon-gamma (10-1,000 U/ml). The cystic fibrosis transmembrane conductance regulator was also downregulated by interferon-gamma as evident at the protein level and by the decrease in the cAMP-dependent current. On the other hand, interferon-gamma caused an increase of the current elicited by apical UTP application, which is due to the activity of Ca(2+)-dependent Cl(-) channels. Tumor necrosis factor-alpha caused few changes in ion transport. Transepithelial fluid transport was measured in normal and cystic fibrosis cells. At rest, both types of cells showed an amiloride-sensitive fluid absorption that was inhibited by interferon-gamma but not by tumor necrosis factor-alpha. Our results show that interferon-gamma alters the transepithelial ion transport of cultured bronchial cells. This effect may change the ion composition and/or volume of periciliary fluid.     

Regulation of taurine transporter expression by NO in cultured human retinal pigment epithelial cells

Taurine is activelytransported at the retinal pigment epithelial (RPE) apical membrane inan Na⁺- and Cl-dependent manner. Diabetes mayalter the function of the taurine transporter. Because nitric oxide(NO) is a molecule implicated in the pathogenesis of diabetes, we askedwhether NO would alter the activity of the taurine transporter incultured ARPE-19 cells. The activity of the transporter was stimulatedin the presence of the NO donor 3-morpholinosydnonimine. Thestimulatory effects of 3-morpholinosydnonimine were not observed duringthe initial 16-h treatment; however, stimulation of taurine uptake waselevated dramatically above control values with 20- and 24-htreatments. Kinetic analysis revealed that the stimulation wasassociated with an increase in the maximal velocity of the transporterwith no significant change in the substrate affinity. The NO-induced increase in taurine uptake was inhibited by actinomycin D and cycloheximide. RT-PCR analysis and nuclear run-on assays provided evidence for upregulation of the transporter gene. This study providesthe first evidence of an increase in taurine transporter geneexpression in human RPE cells cultured under conditions of elevatedlevels of NO.

     

Regulation by aldosterone of Na+,K+-ATPase mRNAs, protein synthesis, and sodium transport in cultured kidney cells

Transepithelial Na+ reabsorption across tight epithelia is regulated by aldosterone. Mineralocorticoids modulate the expression of a number of proteins. Na+,K+-ATPase has been identified as an aldosterone-induced protein (Geering, K., M. Girardet, C. Bron, J. P. Kraehenbuhl, and B. C. Rossier, 1982, J. Biol. Chem., 257:10338-10343). Using A6 cells (kidney of Xenopus laevis) grown on filters we demonstrated by Northern blot analysis that the induction of Na+,K+-ATPase was mainly mediated by a two- to fourfold accumulation of both alpha- and beta-subunit mRNAs. The specific competitor spironolactone decreased basal Na+ transport, Na+,K+-ATPase mRNA, and the relative rate of protein biosynthesis, and it blocked the response to aldosterone. Cycloheximide inhibited the aldosterone-dependent sodium transport but did not significantly affect the cytoplasmic accumulation of Na+,K+-ATPase mRNA induced by aldosterone.     

Regulation of the reduced-folate transporter by nitric oxide in cultured human retinal pigment epithelial cells

The regulation of the reduced-folate transporter (RFT) by nitric oxide (NO) was analyzed in human retinal pigment epithelial (HRPE) cells. NO inhibited specifically and reversibly the uptake of N5-methyltetrahydrofolate by a cGMP-independent mechanism. The inhibition was associated with a decrease in substrate affinity. The NO-induced inhibition was prevented by antioxidants and NO scavengers. Agents capable of modifying thiol groups in proteins inhibited RFT, indicating that the likely mechanism of NO-induced inhibition is via modification of essential thiol groups in this protein. These studies suggest that NO produced during retinal disease may affect the function of RFT in adjacent RPE cells.     

Sodium-dependent, concentrative nucleoside transport in cultured intestinal epithelial cells

In a simple salts medium, monolayers of IEC-6 intestinal cells achieved concentrations of unmetabolized formycin B (an analog of inosine) about 6-fold higher than in the medium. Rates of formycin B influx were a saturable function of Na+ concentrations in the medium. Although IEC-6 cells possess sites with high affinity for nitrobenzylthioinosine, a potent inhibitor of equilibrative (facilitated diffusion) nucleoside transport systems in certain cell types, the inhibitor had only minor effects on formycin B uptake in IEC-6 cells, but reduced efflux of the analog from these cells. These findings indicate the joint presence in IEC-6 cells of nucleoside transporters of two types, one that is concentrative and Na+-dependent, and another that is sensitive to nitrobenzylthioinosine and apparently equilibrative.     

Regulation of inositol 1,4,5-trisphosphate receptor-mediated calcium release by the Na/K-ATPase in cultured renal epithelial cells

It is known that the Na/K-ATPase alpha1 subunit interacts directly with inositol 1,4,5-triphosphate (IP(3)) receptors. In this study we tested whether this interaction is required for extracellular stimuli to efficiently regulate endoplasmic reticulum (ER) Ca(2+) release. Using cultured pig kidney LLC-PK1 cells as a model, we demonstrated that graded knockdown of the cellular Na/K-ATPase alpha1 subunit resulted in a parallel attenuation of ATP-induced ER Ca(2+) release. When the knockdown cells were rescued by knocking in a rat alpha1, the expression of rat alpha1 restored not only the cellular Na/K-ATPase but also ATP-induced ER Ca(2+) release. Mechanistically, this defect in ATP-induced ER Ca(2+) release was neither due to the changes in the amount or the function of cellular IP(3) and P2Y receptors nor the ER Ca(2+) content. However, the alpha1 knockdown did redistribute cellular IP(3) receptors. The pool of IP(3) receptors that resided close to the plasma membrane was abolished. Because changes in the plasma membrane proximity could reduce the efficiency of signal transmission from P2Y receptors to the ER, we further determined the dose-dependent effects of ATP on protein kinase Cepsilon activation and ER Ca(2+) release. The data showed that the alpha1 knockdown de-sensitized the ATP-induced ER Ca(2+) release but not PKCepsilon activation. Moreover, expression of the N terminus of Na/K-ATPase alpha1 subunit not only disrupted the formation of the Na/K-ATPase-IP(3) receptor complex but also abolished the ATP-induced Ca(2+) release. Finally, we observed that the alpha1 knockdown was also effective in attenuating ER Ca(2+) release provoked by angiotensin II and epidermal growth factor.     

Regulation of 22Na+ transport by calcium in an established kidney epithelial cell line.

The role of calcium in regulating the Na+ channel in an established kidney epithelial cell line has been examined. Extracellular calcium was inhibitory to Na+ uptake, and a Dixon plot of the initial Na+ uptake rate in the presence of Ca2+ was nonlinear, suggesting a mixed pattern of inhibition. Similar patterns of inhibition were also observed for other divalent cations, including Ba2+, Mg2+, and Mn2+. In contrast elevated concentrations of intracellular calcium resulted in a stimulation of Na+ entry. This intracellular effect was specific to calcium, with Mg2+ and Mn2+ appearing much less effective. Lineweaver-Burk plots of Na+ influx in calcium-loaded and unloaded cells were linear, suggesting that under both conditions a single system transported Na+. Although Na+ entry was stimulated by intracellular Ca2+, the cells did not exhibit other counter transport phenomena reported with cell types in which a Na+/Ca2+ exchange system is operative. Thus, the results indicate that calcium acts as an allosteric regulator of Na+ transport by the Na+ channel.     

Regulation of growth of cultured hepatic epithelial cells by transferrin

Late-passage cells of a nontumorigenic and anchorage-dependent hepatic epithelial line (WB-F344), which produce insulinlike growth factor II and transforming growth factor beta constitutively, grow in serum-free medium supplemented only with transferrin. In the presence of transferrin, epidermal growth factor further augments population growth, although epidermal growth factor alone is without effect. Insulin, platelet-derived growth factor, and several inorganic iron salts are also ineffective in supporting cell growth in the absence of transferrin; furthermore, these factors do not augment the action of transferrin. The population growth-promoting effect of transferrin occurs at concentrations of 0.5 nM or greater and the maximal effect is reached with a concentration of approximately 6 nM. A lipophilic iron chelator, ferric pyridoxal isonicotinoyl hydrazone (FePIH), can fully mimic the effect of transferrin on the proliferation of WB-F344 cells, but the molar concentration of transferrin. These results suggest that the critical function of transferrin in the proliferation of WB-F344 cells may be in the delivery of iron to the cells. In the absence of transferrin the proliferation of WB-F344 cells is arrested in serum-free medium in the G0/G1 phase, and a period of protein synthesis after the addition of transferrin is necessary before the cells can proceed to S phase and initiate DNA synthesis. Replacement of transferrin causes quiescent WB-F344 cells to cycle parasynchronously. Epidermal growth factor does not alter the length of the latency period prior to S phase but appears to stimulate the uptake of [3H]thymidine subsequently. Transferrin may act as a "competence" and/or "progression" factor, allowing the replication of these epithelial cell in vitro.     

Interleukin-10 is up-regulated by prolactin and serum-starvation in cultured mammary epithelial cells

The epithelial cells of the mammary gland go through a cycle of growth, differentiation, and involution during pregnancy. Recently, we found that interleukin-10 (IL-10) was induced at the involution stage and contributed to apoptosis in the mammary gland. To elucidate the role of the epithelial cells in involution, we examined IL-10 expression in an in vitro model of HC11 cells, in various culture conditions. IL-10 was weakly expressed early in growth but when the cells were induced to differentiate by insulin and dexamethasone expression increased slightly. Prolactin in combination with insulin and dexamethasone caused a further increase. To mimic apoptosis the culture was deprived of serum as well as hormones, and this resulted in a gradual increase in IL-10. In contrast with its ligand, the IL-10 receptor itself was not expressed in any conditions. We speculate that release of IL-10 from the epithelial cells recruits lymphocytes, which have IL-10 receptors on their cell membranes, and they in turn secrete death factors inducing apoptosis of the epithelial cells.     

Regulation of migration of primary prostate epithelial cells by secreted factors from prostate stromal cells

Stromal-epithelial interactions, which regulate the migration of prostate epithelial cells, play an important role in prostate development, prostatic hyperplasia, and prostate cancer. The objective of this study was to determine how the prostate stroma stimulates the migration of primary prostate epithelial cells (PECs). In the Boyden chamber assay, PEC migration was strongly induced by the conditioned medium of primary prostate stromal cells (PSC-CM). Stimulation of PEC migration depended on the concerted action of adhesion and motility factors in the PSC-CM. Immobilized proteins from PSC-CM mediated adhesion, spreading, and head-to-tail polarization of PECs. Migration induced by immobilized PSC-CM proteins was significantly increased by hepatocyte growth factor/scatter factor (HGF/SF). Inhibition of P13-kinase or Src-family kinases, but not MEK or PLCchi, abolished migration in the Boyden chamber assay. Consistent with their concerted activity in migration assays, the combination of adhesion and motility factors was required for efficient activation of the P13-kinase/Akt pathway. HGF/SF in the PSC-CM was the principal stimulator of the P13-kinase/Akt pathway and an important mediator of PSC-CM-induced PEC migration. In conclusion, our data show that the migration of primary PECs is regulated by the P13-kinase and Src-family kinase signaling pathways and that the activation of the P13-kinase pathway requires adhesion and motility factors from the prostate stroma.     

Two-dimensional protein profiles of cultured stromal and epithelial cells from hyperplastic human prostate

Studies were undertaken to compare and contrast the two-dimensional protein profiles of epithelial and stromal cells from hyperplastic human prostate to establish the protein composition of the two major cellular components of the prostate. Epithelial and stromal cells were isolated from human prostate obtained from patients undergoing open prostatectomy for benign prostatic hyperplasia (BPH). Proteins, isolated from the two cell populations and separated by two-dimensional (2D) electrophoresis, were analyzed by silver staining, fluorography of [35S]-methionine-labeled proteins, and immunoprotein blotting. Isolated prostatic epithelial cells, but not stromal cells, contained cytokeratin polypeptides 5, 6, 7, 8, 13, 14, 15, 16, 17, 18, and 19. Although vimentin could not be identified in silver stained 2D gels and fluorographs of cultured prostatic epithelial cells, a low level of immunoreactivity was noted following immunoblot analysis of epithelial cells proteins by the use of an anti-vimentin polyclonal. Vimentin was prominently expressed in cultured prostatic stromal cells and could be identified on silver stained 2D gels, fluorographs, and immunoblots of stroma-derived proteins. In addition, stromal marker proteins SM1, SM2, and SM3 were identified in 2D gels of stromal cells to distinguish them from epithelial cells. These studies demonstrate (1) the two-dimensional protein profile and cytokeratin polypeptide composition of cultured epithelial cells from hyperplastic human prostate and (2) the 2D protein profile of cultured prostatic stromal cells and identification of specific stromal marker proteins.     

Na+-linked active transport of ascorbate into cultured bovine retinal pigment epithelial cells: heterologous inhibition by glucose

The transport of ascorbate into cultured bovine retinal pigment epithelial (RPE) cells is reported. Primary or subcultured RPE cells were incubated in the presence of 10-500 microM L-[carboxyl-14C]-ascorbate for various periods of time. Accumulation of ascorbate into RPE cells followed a saturable active transport with a Km of 125 microM and a Vmax of 28 pmole/micrograms DNA/min. RPE intracellular water was calculated to be 0.8 pL/cell, and the transported cellular ascorbate concentration was 7.5 +/- 0.8 mM. Replacement of 150 mM NaCl in the incubation media with choline-Cl strongly inhibited (80 +/- 8%) ascorbate uptake into cultured RPE cells. Although the depletion of cellular ATP by 2,4-dinitrophenol and the inhibition of Na+-K+-ATPase by ouabain reduced ascorbate transport into RPE significantly, active transport of ascorbate was not entirely inhibited by these metabolic inhibitors. The ascorbate analogue, D-isoascorbate, competitively inhibited ascorbate transport into cultured RPE with a Ki of 12.5 mM. Cells grown in the presence of 5 to 50 mM alpha-D-glucose in the growth media did not differ in their ability to transport ascorbate. In contrast, the presence of alpha-D-glucose or its nonmetabolizable analogues, 3-0-methyl-glucose, alpha-methyl-glucose, and 2-deoxy-glucose, but not L-glucose or beta-D-fructose, in the incubation media inhibited ascorbate transport. myo-Inositol (10 or 20 mM) also inhibited ascorbate transport into RPE cells. The active uptake of ascorbate into cultured RPE cells was primarily coupled to the movement of sodium ion down its electrochemical gradient. A bifunctional, cotransport carrier possessing an ascorbate-binding site and a sodium-binding site may be involved in the ascorbate uptake system. The inhibition of ascorbate uptake by sugars appeared to be heterologous in nature, occurring between two distinct carrier systems, both of which were dependent on the sodium ions.