Hormonal regulation of the polarized function and distribution of Na/H exchange and Na/HCO3 cotransport in cultured mammary epithelial cells.

The time course for development of polarized function and morphological distribution of pH regulatory mechanisms has been examined in a mouse mammary epithelial cell line (31EG4). Monolayers grown on permeable supports had tight junctions when grown 3-4 days in the presence of the lactogenic hormones dexamethasone (D, a synthetic glucocorticoid) and insulin (I), or in D, I, and prolactin (P), but there were no tight junctions in the absence of D. Microspectrofluorimetry of the pH-sensitive dye BCECF was used to measure pH (pHi) in cells mounted in a two-sided perfusion chamber to distinguish pH regulatory activity at the apical and basolateral membranes. Na/H exchange was assayed as the Na-dependent, amiloride-sensitive component of pHi recovery from an acid load induced by a pulse of NH3/NH4-containing solution. When monolayers were grown 3-4 d in the presence of P, D, and I, Na/H exchange was restricted to the basolateral membrane. In contrast, in the absence of P, Na/H exchange was present on both the apical and basolateral membranes. After 5-6 days, in the presence or absence of P, Na/H exchange was present only on the basolateral membrane. An antibody to the NHE-1 isoform of the Na/H exchanger was used to determine its morphological distribution. In all hormone conditions the antibody recognized a protein of approximately 110 kD (Western blot), and confocal immunofluorescence microscopy of this antibody and of an anti-ZO-1 (the marker of the tight junctions) antibody showed that the morphological distribution of the Na/H exchanger was similar to the functional distribution under all hormonal treatments. In addition, a putative Na/HCO3 cotransport system was monitored as a Na-dependent, amiloride-insensitive pHi recovery mechanisms that was inhibited by 200 microM H2DIDS. After treatment with D+I (but not with I alone) cotransport appeared exclusively on the basolateral membrane, and the polarized expression of this transporter was not altered by P. We conclude that when mammary cells are grown in D+I-containing media, the Na/H exchanger is expressed initially (i.e., after 3-4 d) on both the apical and basolateral membranes and later (5-6 d) on only the basolateral membrane. P (in the presence of D+I) selectively speeds this polarization, which is determined by polarized distribution of the exchanger to the apical and/or basal membrane and not by the activation and/or inactivation of transporters.(ABSTRACT TRUNCATED AT 400 WORDS)

Glucocorticoid-induced formation of tight junctions in mouse mammary epithelial cells in vitro.

Phenotypically stable cultures of untransformed mouse mammary epithelial cells (denoted 31EG4) were established and utilized to investigate the lactogenic hormone (glucocorticoids, insulin, and prolactin) regulation of tight junction formation. When 31EG4 cells were grown on permeable supports for 4 days in medium containing the synthetic glucocorticoid dexamethasone and insulin, confluent cell monolayers obtained a transepithelial electrical resistance (TER) of 1000-3000 omega.cm2. In contrast, over the same time period, confluent monolayers treated with insulin or insulin and prolactin maintained a low TER (35-150 omega.cm2). Consistent with the formation of tight junctions, apical to basolateral paracellular permeability was decreased from 12% to 1% for [14C]mannitol and 3.3% to 0.3% for [3H]inulin when cells were cultured in dexamethasone. This effect of dexamethasone on TER required extracellular calcium, de novo protein synthesis, dose-dependently correlated with glucocorticoid receptor occupancy, and was not due to an increase in cell density. As shown by direct and indirect immunofluorescence microscopy, dexamethasone treatment did not modulate the production or location of filamentous actin, the tight junction protein ZO-1, or the cell adhesion protein E-cadherin. Our results suggest that glucocorticoids play a fundamental role in the function and maintenance of cell-cell contact in the mammary epithelia by inducing the formation of tight junctions.

Altered effects of glucocorticoids on the trafficking and processing of mouse mammary tumor virus glycoproteins constitutively expressed in rat hepatoma cells in the absence of nonglycosylated viral components.

We have documented previously that glucocorticoid hormones modulate the posttranslational localization of cell surface mouse mammary tumor virus (MMTV) glycoproteins in the viral-infected M1.54 rat HTC hepatoma cell line. To determine whether glucocorticoids affect the trafficking of individually synthesized MMTV glycoproteins, HTC cells were transfected with a constitutively expressed MMTV glycoprotein gene lacking the viral phosphoprotein and polymerase genes. This construct also allows equivalent levels of MMTV glycoproteins to be compared in the presence or absence of glucocorticoids. Indirect immunofluorescence and immunoprecipitation of radiolabeled cells revealed that in transfected cells the transmembrane MMTV glycoproteins are efficiently expressed, transported to the cell surface, and proteolytically cleaved in the presence or in the absence of the synthetic glucocorticoid dexamethasone. Cell surface immunoprecipitation of [35S]methionine-labeled cells showed that the level of plasma membrane gp78 appeared to be stimulated 2-fold after dexamethasone treatment, even though fluorescence-activated cell sorting revealed no discernible change in the total concentration of cell surface MMTV glycoproteins. Analysis of oligosaccharide side chain maturation through a pulse-chase radiolabeling revealed that the rate of rough endoplasmic reticulum-Golgi transport was essentially identical in dexamethasone-treated and untreated transfected cells and was similar to that observed in dexamethasone-treated M1.54 cells. Thus, in contrast to viral-infected hepatoma cells, mostly constitutive cellular machinery mediates the trafficking and maturation of cell surface MMTV glycoproteins expressed outside of the proviral context. Taken together, our results suggest that the glucocorticoid-stimulated synthesis of nonglycosylated viral components may contribute to or be responsible for the regulated trafficking of MMTV glycoproteins observed in viral-infected rat hepatoma cells.