Lucette Belair

Detection and regulation of leptin receptor mRNA in ovine mammary epithelial cells during pregnancy and lactation

Adipocyte‐epithelial cell interactions and their secretions are critical determinants of mammary gland development. In this present study, we examined the possible involvement of leptin and its receptors in the process of mammogenesis/lactogenesis. We demonstrated by reverse transcription and polymerase chain reaction analysis that long and short forms of leptin receptors were expressed in the ovine mammary gland during pregnancy and lactation. Furthermore, quantitative determinations, via ribonuclease protection assays, provided evidence that the level of leptin receptor expression was greatest during mid‐pregnancy when active growth of the mammary gland is initiated. Location of the leptin receptors, as determined by in situ hybridization analysis, revealed that leptin receptor transcripts were expressed specifically in mammary epithelial cells. These data provide evidence that leptin, with its receptors, could be an important mediator in regulating mammary gland growth and development.

Identification and characterization of growth hormone receptor mRNA in the mammary gland.

The present report describes the first characterization of growth hormone (GH) receptor (GH-R) mRNA in the rabbit mammary gland. Northern blot analysis of poly(A)+ RNA isolated from several tissues of rabbit probed with a rabbit liver GH-R cDNA fragment revealed hybridization to only one transcript of 4.2 kb. A specific hybridizing signal appears in the mammary gland mRNA during gestation, when three different probes derived from liver GH-R cDNA and encoding respectively for extracellular, transmembrane and intracellular regions, were used. The signal is lower than in the liver but highly significant. These results indicate that the three regions are present and well conserved in the GH-R transcript found in the mammary gland. By S1 nuclease mapping analysis we demonstrated that the extracellular and transmembrane domains of mammary gland GH-R mRNA are strongly homologous to the liver GH-R mRNA. In addition, mammary gland GH-R mRNA is probably generated by mammary epithelial cells as demonstrated by the hybridization signal obtained using mRNA extracted from purified acini. The increase in the concentration of GH-R mRNA occurs during epithelial cell proliferation associated with a decrease in the proportion of adipocytes and connective cells at late gestation. The 4.2 kb GH-R mRNA species was also detected in ovine and porcine mammary glands during gestation, suggesting a probable expression of the related form of GH-R in these species.

Polymeric-Ig receptor gene expression in rabbit mammary gland during pregnancy and lactation: evolution and hormonal regulation.

The polymeric immunoglobulin receptor (poly Ig-R) mediates transcytosis of IgA and IgM antibodies produced by local plasma cells across epithelial cells of mucosal and glandular tissues. Gene expression of the poly-Ig R was analyzed in rabbit mammary gland during pregnancy and lactation. The poly Ig-R was expressed as early as day 8 (G8) of gestation and mRNA accumulation remained low until about G18. From G21, the mRNA abundance increased and reached steady state levels approximately 5-fold higher at day 15 of lactation (L15) when compared to basal levels at G8. The hormonal regulation of poly-Ig receptor gene expression was assessed in mammary organ cultures. Poly-Ig R mRNA accumulation in mammary explants cultured for 24 or 48 h in the presence of ovine prolactin (oPRL) was significantly increased to a maximal 4-fold level at 1 microgram ml-1 of oPRL. Estradiol (100 pg ml-1) or progesterone (1 microgram ml-1) did not further stimulate poly-Ig R expression. In contrast, their combination resulted in a significant 30-50% decrease of poly-Ig-R mRNA levels. The addition of 1 microgram ml-1 of cortisol to medium in the absence or presence of estradiol or progesterone decreased the amount of poly-Ig-R mRNA. The results suggest that until mid-pregnancy, poly-Ig-R expression is inhibited by elevated progesterone-estradiol concentrations and that the subsequent increase is due to the concomitant decrease of the two circulating steroids and the increase of serum prolactin levels.

Developmental expression of pIgR gene in sheep mammary gland and hormonal regulation

Secretory IgA found in external secretions are constituted by polymeric IgA (pIgA) bound to the extra-cellular part of the polymeric immunoglobulin receptor (pIgR). The receptor mediates transcytosis of pIgA across epithelial cells. The aim of the present study was to analyse the evolution of pIgR expression in the sheep mammary gland during the development of the mammary gland and to analyse its hormonal regulation. Gene expression of the pIgR was analysed in sheep mammary gland during pregnancy and lactation. By Northern Blot analysis, we observed that low levels of pIgR mRNA are expressed until day 70 of pregnancy. Accumulation of pIgR mRNA started during the third part of pregnancy and intensified 3 d after parturition to reach highest levels during established lactation (day 70). In situ hybridization analysis was used to confirm the increase in pIgR gene expression per mammary epithelial cell. In order to examine the hormonal regulation of the pIgR expression, virgin ewes were hormonally treated. Treatment with oestradiol and progesterone increased pIgR mRNA levels slightly. Subsequent addition of gluocotricoids induced a significant accumulation of pIgR mRNA in the mammary gland of the treated animals. Immunohistochemical analysis was performed to verify that the increase of pIgR mRNA level was associated with enhancement of the pIgR protein in mammary cells. No increase of pIgR mRNA levels were observed if PRL secretion was blocked by bromocryptine injections throughout the hormonal procedure. In conclusion, the present experiments suggest that the enhancement of pIgR levels during lactation result from combined effects of both prolactin and glucocorticoids.

Increase in prolactin receptor (PRL-R) mRNA level in the mammary gland after hormonal induction of lactation in virgin ewes☆

In order to examine the hormonal regulation of the prolactin-receptor (PRL-R) gene expression during mammary gland development, ewes were treated to induce lactation via an estrogen-progesterone-hydrocortisone and ovine growth hormone treatment. In situ hybridization analysis was used and revealed that sex steroids increased PRL-R mRNA levels in the mammary gland. Using RNase protection assay we showed that the estradiol + progesterone treatment increased both the levels of the long and the short forms of PRL-R mRNA. Addition of hydrocortisone increased the level of alphaS1-casein transcripts and the level of the ratio of the long to the short form of the PRL-R mRNA. This ratio can be further enhanced by addition of ovine growth hormone to the latter treatment. This suggests a role of hydrocortisone and ovine growth hormone in the alternative splicing that leads to the preferential expression of the long form of the PRL-R mRNA. In conclusion, the present experiments suggest that estrogen, progesterone and hydrocortisone are the major regulators of the PRL-R gene expression during pregnancy and prepare the mammary gland for its differentiation.

Characterization and modulation of a prolactin receptor mRNA isoform in normal and tumoral human breast tissues.

The role of prolactin (PRL) and its specific receptor (R‐PRL) in human breast tumorigenesis remains unclear. We have investigated here the presence of extracellular‐deleted hPRL‐R isoforms in normal human breast, fibrocystic disease, primary breast carcinoma (ductal carcinoma, ductulo‐lobular and lobular) and breast cancer cell lines (T47‐D and MCF‐7). RT‐PCR and Southern blot analysis demonstrated the expression of full‐length hPRL‐R transcript in all samples tested. We also detected a hPRL‐R transcript generated by alternative exon 6 splicing. This isoform has a 170 bp deletion in its extracellular sub‐domain that induces a frameshift. Thus, the predicted amino‐acid sequence should encode a putative soluble protein with the N‐terminal sub‐domain of the hPRL‐R and 10 additional carboxy‐terminal residues. This isoform should not bind PRL as previously demonstrated by other experiments. Moreover, the ratio of full‐length to deleted form of hPRL‐R transcripts differs from normal to tumoral breast tissue. This ratio is higher in tumoral mammary gland than in normal tissue. Our data suggest that the alternative splicing of the hPRL‐R gene towards the deleted transcript may be a mechanism to down‐ or up‐regulate the expression of the native transcript of hPRL‐R in accordance to the physiological or pathological state of the mammary gland. Int. J. Cancer 85:771–776, 2000.

Up-regulation of polymeric immunoglobulin receptor mRNA in mammary epithelial cells by IFN-γ

As shown in previous in vivo experiment, the amount of polymeric immunoglobulin receptor (pIgR), which mediates the transcytosis of pIgA across epithelial cells, is regulated by lactogenic hormones (PRL and cortisol) during the development of the mammary gland. In the present in vitro study, it appeared that these hormones were insufficient to induce the strong expression of the gene that we observed in vivo. Several papers have shown that IFN-gamma is a strong stimulator of pIgR gene expression in different models. In contrast, nothing is known of the effects of IFN-gamma on pIgR gene expression in the mammary gland. We report here that IFN-gamma strongly increased pIgR mRNA levels through a direct effect on mammary epithelial cells. We show that IFN-gamma activated not only Stat1 but also Stat5 and that expression of the pIgR and IRF-1 genes was strongly correlated following IFN-gamma stimulation in mammary epithelial cells. In conclusion, these experiments enabled the analysis of different types of regulation of pIgR gene expression in the mammary gland and suggest possible co-operation between circulating hormones and locally produced cytokines, leading to pIgR gene expression in the mammary gland.

Recombinant Extracellular Domain of Rabbit Growth Hormone Receptor (rbGHR-ECD): Preparation and use for Comparing Binding Capacity and Biological Activity of Somatogenic Hormones

ABSTRACT The cDNA of the extracellular domain of rabbit growth hormone receptor (rbGHR-ECD) was cloned in the prokaryotic expression vector pMON, to enable its expression in Escherichia coli after induction with nalidixic acid. The bacterially expressed rbPRLR-ECD protein, contained within the refractile-body pellet, was solubilized in 4.5 M urea, refolded, and purified on a Q-Sepharose column, pH 8, by stepwise elution with NaCl. The bioactive monomeric 28-kDa fraction was eluted in 0.15 M NaCl, yielding 50 mg/2.51 of induced culture.

Effect of DMBA on the expression of prolactin receptors and IGF1 genes in rat mammary gland

Prolactin receptor and IGF1 gene expression were measured in mammary glands from Sprague-Dawley rats at different times (10, 30; and 58 d) after administration of a single dose of 15 mg dimethylbenz(a)-anthracene (DMBA) per os at 55 d of age, and in DMBA-induced mammary tumors appearing in these rats at approximately 2 months after DMBA administration; The relative gene expression of prolactin receptor and insulin-like growth factor (IGF1) mRNAs was measured by hybridization to Northern blots prepared from pools of tissue. The probes used were 32P-labelled cDNAs specific to the extracellular domain of the receptor (E probe), common to all forms, and a probe specific to the intracellular position of the long form of the receptor (I probe), a human IGF1 probe, and chicken beta-actin probe, to correct for loss of tissue and different metabolic activity of the tissues. Hybridization with the prolactin receptor probes revealed bands at 2.5, 3; and 5.5 kb hybridizing with the long form of the receptor and a more intense band at 1.8 kb that corresponded to the short form of the receptor. There were no changes in the relative expression of prolactin receptor mRNAs in the mammary gland of control (oil-treated) or DMBA-treated rats, although there was a gradual diminution of expression with increasing age of the animals. In contrast, in DMBA-induced mammary tumors, there was a marked increase in the relative expression of prolactin receptor mRNAs with, however, no modification in the relative proportion of short and long forms.(ABSTRACT TRUNCATED AT 250 WORDS)

Transduction pathways of GH in ovine mammary acini involving regulated and functional growth hormone receptors.

We have investigated the localization and regulation of growth hormone (GH) receptor-related proteins in the ovine mammary gland. Using a new rabbit polyclonal antibody (7122A) directed against the recombinant extracellular domain of GH receptor (GHR-ECD) for western blot assays, we found two bands with apparent molecular weights of 70,000 and 50–60,000 Da in ovine mammary gland solubilized proteins. The 70,000-protein was consistent with a membrane GH receptor form deprived of post-translational modifications such as phosphorylation, glycosylation or ubiquitin binding. The 50–60,000 Da was consistent with soluble GH binding protein, generated by the cleavage of membrane GH receptor. The intensity of related GHR proteins increased slightly throughout mammary gland development and was correlated with the amount of GHR immunoreactivity observed in the mammary gland sections. Moreover, a temporal and spatial regulation of GHR immunoreactivity was found in alveolar epithelial cells. Clearly, marked GHR immunoreactivity was associated with the apical membranes of alveolar epithelial cells at lactation. The up-regulation of related GHR proteins during the differentiation of mammary tissue supports the hypothesis that GH may act specifically via its own receptors. In ovine mammary cells, GH was able to promote a time-dependent activation of MAP kinases such as prolactin (Prl) and placental lactogen (PL). GH was also able to promote slight and transient Stat5 DNA-binding activity. Differences in the time dependence of Stat5 DNA-binding activation by the three different ligands, GH, Prl and PL, were found. All these results emphasize the direct action of GH on ovine mammary cells and highlight the specificity of action of this ligand.