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B Groner

Publications and source records attributed to B Groner.

At least 127 records · Page 7Linked to original sources
PubMed1992

Mammary gland-specific nuclear factor activity is positively regulated by lactogenic hormones and negatively by milk stasis.

The mammary gland-specific nuclear factor (MGF) is a crucial contributor to the regulation of transcription from the beta-casein gene promoter. The beta-casein gene encodes a major milk protein, which is expressed in mammary epithelial cells during lactation and can be induced by lactogenic hormones in the clonal mammary epithelial cell line HC11. We have investigated the specific DNA-binding activity of MGF in mammary epithelial cells in vivo and in vitro. Comparison of MGF in HC11 cells and mammary gland cells from lactating mice revealed molecules with identical DNA-binding properties. Bandshift and UV cross-linking experiments indicated that MGF in HC11 cells has a higher mol wt than MGF found in mice. Little MGF activity was detected in nuclear extracts from HC11 cells cultured in the absence of lactogenic hormones. Lactogenic hormone treatment of HC11 cells led to a strong induction of MGF activity. The induction of MGF activity as well as utilization of the beta-casein promoter were suppressed when epidermal growth factor was present in the tissue culture medium simultaneously with the lactogenic hormones. In lactating animals, MGF activity is regulated by suckling, milk stasis, and systemic hormone signals. The mammary glands from maximally lactating animals, 16 days postpartum, contain drastically reduced MGF activity after removal of the pups for only 8 h. The down-regulation of MGF by pup withdrawal was slower in early lactation, 6 days postpartum. We also investigated the relative contributions of local signals, generated by milk stasis, and systemic hormone signals to the regulation of MGF activity. The access to one row of mammary glands of lactating mothers was denied to the pups for 24 h. High levels of MGF were found in the accessible mammary glands, and intermediate levels of MGF were found in the inaccessible glands of the same mouse. Very low MGF levels were detected when the pups were removed from the dams for 24 h. We conclude that systemic as well as local signals cooperate in the in vitro regulation of MGF activity.

Animals↗
PubMed1991

Beta-casein gene promoter activity is regulated by the hormone-mediated relief of transcriptional repression and a mammary-gland-specific nuclear factor.

Transcription from the beta-casein milk protein gene promoter is induced by the synergistic action of glucocorticoid and prolactin hormones in the murine mammary epithelial cell line, HC11. We analyzed the binding of nuclear proteins to the promoter and determined their binding sites. Site-directed mutagenesis was used to determine the function of nuclear factor binding. During lactogenic hormone induction of HC11 cells, the binding of two nuclear factors increased. The binding of two other nuclear factors, present in uninduced cells, decreased. The basal activity of the promoter could be increased to and above the level of the induced wild-type promoter when the recognition sequences of the negatively regulated factors were mutated. This suggests that the beta-casein promoter is regulated by the relief of the repression of transcription. An essential tissue-specific factor was also found in nuclear extracts from the mammary glands of mice. Mutation of its recognition sequence in the beta-casein promoter led to the abolition of the induction of transcription by lactogenic hormones. The DNA sequences recognized by all five of these nuclear factors are conserved in the promoters of different casein genes from several species, confirming their importance in the regulation of milk protein gene transcription.

Animals↗
PubMed1991

Lactogenic hormone and cell type-specific control of the whey acidic protein gene promoter in transfected mouse cells.

The whey acidic protein (WAP) is a major milk protein. It is abundantly expressed in mammary epithelial cells, and its gene is controlled by lactogenic hormones. The identification of regulatory cis-acting sequences of the mouse WAP gene was so far dependent on the analysis of transgenic animals. We report here the possibility of analyzing regulatory sequences by gene transfer experiments using the lactogenic hormone-dependent mammary epithelial cell line HC11. A WAP-chloramphenicol acetyltransferase construct containing 2.5 kilobases of the 5'-flanking sequence of the WAP gene was stably transfected into HC11 cells. High chloramphenicol acetyltransferase activity was induced in pools of transfected cells cultured in the presence of the lactogenic hormones glucocorticoid, PRL, and insulin. A lower induction was observed by glucocorticoid hormone alone. PRL by itself was not able to induce the WAP gene promoter above the level observed in the absence of lactogenic hormones. A time course of hormone induction showed a weak initial response with a steady increase over at least 4 days of hormone treatment. Induction was not observable in the mammary epithelial cell line NOG-8 and NIH3T3 fibroblasts, despite the presence of functional glucocorticoid receptor in these cells. This indicates the requirement for a cell type-specific transcription factor present in the mammary epithelial cell line HC11, but not in NOG-8 epithelial cells or NIH3T3 fibroblasts.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Epidermal growth factor receptor, platelet-derived growth factor receptor, and c-erbB-2 receptor activation all promote growth but have distinctive effects upon mouse mammary epithelial cell differentiation.

Three different receptor tyrosine kinases, epidermal growth factor (EGF), c-erbB-2/neu, and platelet-derived growth factor (PDGF) receptors, have been found to be present in the mouse mammary epithelial cell line HC11. We have investigated the consequences of receptor activation on the growth and differentiation of HC11 cells. HC11 cells are normal epithelial cells which maintain differentiation-specific functions. Treatment of the cells with the lactogenic hormones glucocorticoids and prolactin leads to the expression of the milk protein beta-casein. Activation of EGF receptor has a positive effect on cell growth and causes the cells to become competent for the lactogenic hormone response. HC11 cells respond optimally to the lactogenic hormone mixture and synthesize high levels of beta-casein only if they have been kept previously in a medium containing EGF. Transfection of HC11 cells with the activated rat neuT receptor results in the acquisition of competence to respond to the lactogenic hormones even if the cells are grown in the absence of EGF. The activation of PDGF receptor, through PDGF-BB, also stimulates the growth of HC11 cells. Cells kept only in PDGF do not become competent for lactogenic hormone induction. The results show that activation of the structurally related EGF and c-erbB-2/neu receptors, but not the PDGF receptor, allows the HC11 cells to subsequently respond optimally to lactogenic hormones.

Animals↗
PubMed1990

Hormone-dependent phosphorylation of the glucocorticoid receptor occurs mainly in the amino-terminal transactivation domain.

Phosphorylation of glucocorticoid receptors is increased by hormone binding and has been implicated in transcriptional regulation. We performed a phosphoamino acid analysis and identified the phosphorylated regions of the glucocorticoid receptor with respect to its functional domains before and after hormone activation. Receptor was isolated by immunoprecipitation from [32P]orthophosphate-labeled FTO 2B rat hepatoma cells grown in the absence or presence of glucocorticoids. The receptor contained mainly phosphoserine, with little phosphothreonine and no phosphotyrosine. Partial proteolysis of receptor from hormone-treated or control cells revealed a similar phosphopeptide pattern. Chemical cleavage with hydroxylamine and cyanogen bromide or digestion with trypsin and chymotrypsin localized the majority of receptor phosphorylation sites to a transactivation domain amino-terminal of the DNA-binding domain. Phosphorylation of this region, termed tau 1/enh2, was increased 2-3-fold by hormone treatment. The DNA-binding domain itself is weakly phosphorylated; no phosphorylation was found in the hormone-binding domain. Phosphorylated regions were also identified in receptor deletion mutants stably transfected into CV-1 monkey kidney cells. Hormone-independent phosphorylation was observed with a strong constitutively active mutant lacking the hormone-binding domain. No phosphorylation was detected in a mutant lacking the amino-terminal region, which showed only weak, hormone-dependent activity. These results support the idea that phosphorylation is important for the strength of the glucocorticoid receptor as a transcriptional regulator.

Animals↗
PubMed1990

Induction of the endogenous whey acidic protein (Wap) gene and a Wap-myc hybrid gene in primary murine mammary organoids.

In rodents, the whey acidic protein (Wap) is the major whey protein expressed in mammary glands in response to lactogenic hormones. The regulation of the Wap gene differs from that of other milk protein genes, with one consequence being that little or no Wap expression is detectable in cell culture. Here we describe the efficient in vitro induction of the Wap gene in mammary organoids isolated from midpregnant mice. Mammary organoids were isolated as intact epithelial subcomponents which retained the glandular microarchitecture. If organoids were cultured in contact with a monolayer of 3T3-L1 adipocytes, significant levels of Wap mRNA were induced upon hormonal stimulation, with the highest level of Wap mRNA being induced by a combination of hydrocortisone, prolactin, and insulin. Dissociation of the three-dimensional organization abrogated Wap inducibility. Organoids cultured on plastic or hydrated type I collagen did not transcribe Wap mRNA even after hormonal stimulation. Addition of hormones was required to maintain low levels of Wap mRNA in organoids cultured on reconstituted basement membrane, however, Wap mRNA was not induced. Organoid-adipocyte interactions as well as cell-cell interactions inherent in the structure of organoids promote hormone-dependent Wap mRNA expression. In order to study the Wap promoter region in vitro, we cocultured organoids from transgenic mice harboring a chimeric Wap-myc gene with 3T3-L1 adipocytes. Lactogenic hormones induced the Wap-myc transgene in vitro. The kinetics of induction were similar for both the transgene and the endogenous Wap gene indicating that the 2.5-kb regulatory Wap region present in the hybrid gene contains the sequence elements required for hormone-induced gene expression in vitro.

Adipose Tissue↗
PubMed1990

c-erbB-2 protein expression in node negative breast cancer.

We investigated the expression of c-erB-2 protein in two matched groups of breast cancer patients, one with and one without relapse. 37 patients with relapse were compared with 42 patients without recurrence for time of observation, adjuvant treatment, age, menopausal status and estrogen receptor content. Paraffin-embedded sections were stained with the polyclonal antibody 21N, raised against a synthetic peptide from the predicted sequence of the c-erbB-2 protein. The staining of c-erbB-2 was measured on a scale of 0 to 3+. C-erbB-2 staining was negative in 16 (38%) patients in the relapse-free group, and in 8 (22%) of the patients with metastases. Neither disease-free survival (DFS) nor overall survival (OS) were dependent upon the extent of c-erbB-2 expression. An analysis by estrogen receptor (ER) status (i.e. positive or negative) and by c-erbB-2 expression (i.e. positive or negative) revealed that patients with ER-positive primaries and negative c-erbB-2 have the longest disease-free survival (DFS) and overall survival (OS). We conclude that c-erbB-2 expression might be clinically useful only if other prognostic variables (e.g. estrogen receptor content in the tumor) are also considered.

Adult↗
PubMed1990

Epidermal growth factor receptor, but not c-erbB-2, activation prevents lactogenic hormone induction of the beta-casein gene in mouse mammary epithelial cells.

The HC11 cell line was isolated from mammary gland cells of pregnant mice. The cells displayed a normal phenotype and retained some characteristics of mammary epithelial cell differentiation. After treatment with the lactogenic hormones prolactin and glucocorticoids, the HC11 cells expressed the milk protein beta-casein. Various oncogenes were transfected and expressed in HC11 cells. The oncogenes were tested for their transformation ability and for their effects upon the differentiation of the HC11 cells. All of the oncogenes tested, including activated human Ha-ras, human transforming growth factor-alpha, activated rat neuT, and human c-erbB-2 activated by a point mutation in the transmembrane domain, caused transformation of the HC11 cells, as shown by tumor formation in nude mice. HC11 cells expressing the neuT and activated c-erbB-2 genes synthesized beta-casein in response to lactogenic hormones, whereas those expressing the Ha-ras or transforming growth factor-alpha oncogenes were no longer able to respond to the lactogenic hormones. This inhibition of beta-casein production occurs at the transcriptional level and in the transforming growth factor-alpha-transformed cells is due to an autocrine mechanism involving the activation of the epidermal growth factor receptor. This suggests that, although the c-erbB-2 and epidermal growth factor receptors are structurally quite similar, their activation has different effects upon mammary epithelial cell differentiation.

Animals↗
PubMed1990

Localization of the vicinal dithiols involved in steroid binding to the rat glucocorticoid receptor.

Our previous studies with the thiol-specific reagent methyl methanethiolsulfonate (MMTS) and the vicinal dithiol-specific reagent sodium arsenite have established that 2 spatially close thiols (i.e. vicinal dithiols) are involved in steroid binding to the intact 98 K rat glucocorticoid receptor. These 2 thiols form an intramolecular disulfide after treatment with low concentrations of MMTS. One of these thiols was proposed to by Cys-656. In an effort to identify both thiols, we have examined the effects of MMTS and arsenite on proteolytic fragments of the receptor, which contain progressively fewer cysteines. MMTS and arsenite are now found to cause the same dithiothreitol-reversible inhibition of steroid binding and affinity labeling of both the 42 K chymotrypsin fragment and the 16 K steroid-binding core fragment of the receptor as was seen for the intact receptor. Characteristic responses include a bimodal inhibition curve for steroid binding after preincubation with MMTS and an inhibition of binding by very low concentrations of arsenite. Low concentrations of MMTS could block steroid binding by forming a disulfide bond between the receptor and a tightly associated, nonreceptor protein. However, no evidence for such cross-linking was observed when intact 98 K receptors, 42 K chymotrypsin fragments, or 16 K trypsin fragments were treated with various concentrations of MMTS, separated on nonreducing sodium dodecyl sulfate-polyacrylamide gels, and visualized by Western blotting with antiheat shock protein 90 or antireceptor antibodies. One of the antireceptor antibodies (aP1) that had been raised against the rat receptor sequence 440-795 was now found to recognize at least 1 epitope in the 16 K core fragment. We conclude that the vicinal dithiols involved in steroid binding are 2 of the 3 cysteines in the sequence of Thr537-Arg673.

Animals↗
PubMed1990

Prolactin and glucocorticoid hormones control transcription of the beta-casein gene by kinetically distinct mechanisms.

Transcription of the beta-casein milk protein gene in the HC11 mouse mammary epithelial cell line is induced synergistically by the hormones glucocorticoid and PRL. Sequential treatment of HC11 cells with glucocorticoid and PRL demonstrated that the two hormones had different modes of action on beta-casein transcription. Pretreatment with dexamethasone enhanced the response to subsequent induction by PRL, but not vice versa. Dexamethasone increased the sensitivity of the cells to respond to PRL. The increase in sensitivity was slow, extended for 16 days, and could be rapidly reversed by withdrawal of dexamethasone. The dexamethasone-induced sensitivity for the rapid transcriptional regulation by PRL could be observed with transfected rat beta-casein promoter-chloramphenicol acetyltransferase constructs retaining only 175 basepairs upstream from the transcription initiation site. Expression of the endogenous mouse beta-casein gene was regulated identically to that of the promoter constructs with respect to the synergy of the hormones and their different kinetics of action. In contrast to the slow induction of sensitivity toward PRL, dexamethasone rapidly induced the transcription of a mouse mammary tumor virus long terminal repeat controlled gene in HC11. This demonstrated a normal transcriptional activation of the glucocorticoid receptor in this cell line. Thus, glucocorticoid may regulate beta-casein gene transcription indirectly, inducing or repressing other glucocorticoid-regulated genes, whereas the interaction of PRL with its receptor causes a rapid induction of the beta-casein gene promoter.

Animals↗
PubMed1990

Transforming growth factor-beta inhibits lactogenic hormone induction of beta-casein expression in HC11 mouse mammary epithelial cells.

HC11 mouse mammary epithelial cells can undergo a limited functional differentiation in terms of beta-casein synthesis in response to the combined action of dexamethasone and prolactin. Transforming growth factor-beta (TGF-beta) can inhibit beta-casein expression in HC11 cells in a dose-dependent manner. This effect is reversible and specific as shown by comparison with the effect of other growth factors. TGF-beta also inhibits DNA synthesis of HC11 cells. These findings suggest a possible role of TGF-beta as an inhibitor of functional differentiation in the mammary gland.

Animals↗
PubMed1989

Characterization of a membrane-associated phosphotyrosyl protein phosphatase from the A431 human epidermoid carcinoma cell line.

The A431 human epidermoid carcinoma cell line exhibits a 30-100-fold overexpression of the epidermal growth factor (EGF) receptor. We have characterized a membrane-associated phosphotyrosyl-protein phosphatase (PTPase) in these cells since it seemed reasonable that overexpression of the EGF-receptor tyrosine kinase will be matched by high PTPase activity. Indeed, of 12 cell lines tested, the A431 cells had the highest specific PTPase activity. About 70% of the total cellular PTPase activity was found associated with membranes after cell fractionation. The membrane-associated PTPase was hydrophobic as judged by its behaviour in Triton X-114 phase partitioning. High-performance liquid chromatography (HPLC) on a DEAE column revealed a single, homogeneous species of membrane-associated PTPase with an apparent molecular mass of 43 kDa as determined by HPLC on a gel permeation column in the presence of Triton X-100. Comparison of this PTPase with the membrane-associated PTPase activities present in rat spleen and in the human chronic myelogenous leukemia cell line K562 revealed additional species resolvable by DEAE-HPLC. These findings suggest that cells may possess different PTPase activities depending on their growth and differentiation states.

Animals↗
PubMed1989

Down-regulation and phosphorylation of glucocorticoid receptors in cultured cells. Investigations with a monospecific antiserum against a bacterially expressed receptor fragment.

The expression of the glucocorticoid receptor (GR) gene and the phosphorylation of the GR protein has been studied as a function of time after hormone addition to NIH 3T3 cells. We detected a ligand-induced decrease of GR gene expression at both the level of RNA and protein. GR mRNA declined to 25% of the control within 3 h of dexamethasone treatment and remained at this level for at least 24 h. GR protein was analyzed with a monospecific antiserum directed against the DNA- and hormone-binding domains of the rat GR synthesized in Escherichia coli. Pulse-chase experiments revealed a decrease in GR half-life from 8 h in the absence of hormone to 3 h following hormone treatment. Both effects resulted in a reduction of total GR protein to 20% as determined by quantitative immunoblotting. The level of the GR protein returned to that of untreated cells within 24 h after withdrawal of hormone. Furthermore, dexamethasone treatment led to a 3-4-fold increase in GR phosphorylation within 60 min. The glucocorticoid antagonist 17 beta-hydroxy-11 beta-(4-dimethylamino-phenyl)-17 alpha-(1-propynyl)-oestra-4,9-dien-3-one (RU 486) did not change the phosphorylation state of the receptor, but a down-regulation of the GR was still observed. These results suggest that ligand-dependent receptor phosphorylation is not involved in down-regulation of the GR but may be important in the transcriptional activation of hormone responsive genes.

Animals↗
PubMed1989

Ha-ras activates the Na+/H+ antiporter by a protein kinase C-independent mechanism.

In quiescent Ha-ras-transfected NIH 3T3 cells, addition of serum growth factors, bombesin or 12-O-tetradecanoylphorbol-13-acetate (TPA) leads to a dimethylamiloride-sensitive intracellular alkalinization which can be inhibited by staurosporine, a potent inhibitor of protein kinase C. Expression of the transforming Ha-ras gene causes a growth factor-independent increase in cytoplasmic pH. This Ha-ras-induced alkalinization is sensitive to dimethylamiloride but is not affected by staurosporine concentrations which prevent the pH response after addition of growth factors or TPA. Protein kinase C depletion by long term exposure to TPA eliminates the pH response to bombesin and phorbol ester but does not effect the Ha-ras-induced intracellular alkalinization. It is concluded that expression of Ha-ras causes an activation of the Na+/H+ antiporter by an as yet unknown protein kinase C-independent mechanism.

Alkaloids↗
PubMed1989

Oncogene mediated repression of glucocorticoid hormone response elements and glucocorticoid receptor levels.

We have previously described the inhibition of glucocorticoid-dependent transcription from the mouse mammary tumor virus long terminal repeat promoter by products of the H-ras and v-mos oncogenes. We have studied the effects of conditional oncogenes on expression of glucocorticoid-dependent indicator genes. Expression of the glucocorticoid-dependent transcription of the tyrosine aminotransferase gene was monitored in FTO-2B rat hepatoma cells during Mr 21,000 protein (p21) H-ras induction. A strong transcriptional repression of the tyrosine aminotransferase gene followed p21 H-ras expression. The sequences in a glucocorticoid-dependent promoter which are responsible for the oncogene-mediated repression could be localized to the glucocorticoid response element; a construct in which a 15-base pair glucocorticoid response element was inserted 5' of the thymidine kinase promoter exhibited the oncogene-mediated repression of transcription. We observed a strong repression of glucocorticoid-dependent promoters and promoter constructs not only in the presence of p21 H-ras and p37 v-mos but also with p60 v-src. p57 v-myc, however, had no effect. Oncogene expression is not a sufficient prerequisite for an initial repression of glucocorticoid hormone-dependent gene transcription, since even in the presence of constitutively high levels of oncogene product a transient stimulation of glucocorticoid-dependent gene expression was found. Protein synthesis inhibition experiments revealed that no hormonally induced cellular protein is needed for the oncogene-mediated repression. It seemed reasonable that this phenomenon might reflect oncogene effects on the glucocorticoid receptor. We, therefore, made measurements of the glucocorticoid receptor protein. In the presence of glucocorticoid hormone the receptor translocated rapidly from the cytoplasm to the nucleus. In normal NIH 3T3 cells, after 24-h treatments the nuclear receptor levels had declined to about 50% of those determined at 2 h and in the presence of p21 H-ras they declined to 15%. The levels of cytoplasmic receptor were not affected by p21 H-ras expression.

Animals↗