Cytoplasm Of Mammary Epithelial Cells Research Articles

Sulfur Amino Acid Metabolism and the Role of Endogenous Cystathionine-γ-lyase/H2S in Holstein Cows with Clinical Mastitis.

Simple SummaryEndogenous hydrogen sulfide (H2S) has been implicated in many physiological and pathological processes, particularly in inflammatory responses and adaptive immunity. In this study, we identified the candidate differentially expressed proteins (DEPs) associated with H2S metabolism in Holstein cows with clinical mastitis (CM). The results revealed 17 DEPs included in 44 Gene Ontology (GO) terms and five Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways related to sulfide metabolism and indicated the important role of cystathionine-γ-lyase (CTH)/H2S in CM. Our findings can support research into the function and regulatory mechanism of CTH/H2S in Holstein cows and provide a basis for the prevention and treatment of CM.H2S plays an important role in various inflammatory diseases. However, the role of H2S and synthetic enzymes in Holstein cows with CM is unknown. The aim of this study was to identify DEPs associated with sulfide metabolism and further investigate their roles in dairy cows with CM. From 3739 DEPs generated by data-independent acquisition proteomics, we identified a total of 17 DEPs included in 44 GO terms and five KEGG pathways related to sulfide metabolism, including CTH and cystathionine-β-synthase (CBS). Immunohistochemical and immunofluorescence staining results showed that CTH and CBS proteins were present mainly in the cytoplasm of mammary epithelial cells. Endogenous H2S production in the serum of the CM group was significantly lower than that of the healthy Holstein cows. CTH and CBS mRNA and protein levels in the mammary glands of the CM group were significantly downregulated compared to those of the healthy group. These results indicate that CTH and H2S were correlated with the occurrence and development of CM in Holstein cows, which provides important insights into the function and regulatory mechanism of CTH/H2S in Holstein cows.

LKB1 regulates PRMT5 activity in breast cancer.

Protein arginine methyltransferase 5 (PRMT5) is the main enzyme responsible for the symmetrical dimethylation of arginine residues on target proteins in both the cytoplasm and the nucleus. Though its activity has been associated with tumor progression in various cancers, the expression pattern of this oncoprotein has been scarcely studied in breast cancer. In the current work, we analyzed its expression in a large cohort of breast cancer patients, revealing higher nuclear PRMT5 levels in ERα-positive tumors and an association with prolonged disease free and overall survival. Interestingly, high PRMT5 nuclear expression was also associated with higher nuclear liver kinase B1 (LKB1), suggesting that a functional relationship may occur. Consistently, several approaches provided evidence that PRMT5 and LKB1 interact directly in the cytoplasm of mammary epithelial cells. Moreover, although PRMT5 is not able to methylate LKB1, we found that PRMT5 is a bona fade substrate for LKB1. We identified T132, 139 and 144 residues, located in the TIM-Barrel domain of PRMT5, as target sites for LKB1 phosphorylation. The point mutation of PRMT5 T139/144 to A139/144 drastically decreased its methyltransferase activity, due probably to the loss of its interaction with regulatory proteins such as MEP50, pICln and RiOK1. In addition, modulation of LKB1 expression modified PRMT5 activity, highlighting a new regulatory mechanism that could have clinical implications.

Expression and prognostic value of c-met in canine mammary tumours.

C-met is a receptor normally expressed on epithelial cells and dysregulated in human breast cancers. Mammary tumours are the most common tumour in female dogs. The aims of this study were to detect the expression of c-met in canine mammary tumours (CMTs) and evaluate the correlations between c-met expression and clinicopathological features. A total of 240 specimens of canine mammary tissues composed of 30 normal glands, 30 hyperplastic ones, 90 benign tumours and 90 carcinomas obtained from 127 bitches were examined by immunohistochemical staining. Positive c-met immunoreactivity was demonstrated in the cytoplasm of mammary epithelial cells at variable levels, and in malignant CMTs, higher c-met expression was found in carcinomas whose grade, stage and mitotic index were low, and metastasis was absent. The median survival time was shorter in dogs with malignant CMTs with a maximum diameter ≥5 cm, regional lymph node or distant metastasis, and a high histologic grade. However, the 2-year survival rate was higher in dogs with malignant CMTs of higher c-met expression than those of low c-met expression (80.1% vs 57%). C-met expression could be used as a valuable positive prognostic factor for the clinical outcomes of dogs with malignant CMTs.

Immunolocalization of estrogen alpha and progesterone beta receptors in goat mammary gland

Steroid receptors particularly estrogen receptor alpha and progesterone receptor beta are essential for mammary gland development. Objective of this study was to explore transcript and protein expression profile of steroid receptors in goat mammary glands. A varied expression of ER-alpha and PR-B was observed during lactation, nonlactating/ dry, mastitic and mammary pre-cancer/cancer in goats. During lactation, immunopositivity of ER-alpha was observed only in the nuclei of mammary epithelial cells (MEC) and stromal cells. However, in non-lactating stage, ER-alpha immunopositivity was observed both in nucleus and cytoplasm of MEC. In mammary pre-cancer (based on aberrant expression of CD10, FNDC3B and MUC1) immunoreactivity of ER-alpha (38±12.5%) varied from non-lactating (14.8±3.1%) and lactating (7.9±2.6%) glands. During naturally infected mastitis, a reduction in the expression of ER-alpha and PR-B was observed.We observed similar expression patterns of ER-alpha and PR-B as that of their protein expression. Transcripts of these receptors were highest in mammary precancer. In comparison to lactating glands, expressions of ER-alpha and PR-B was upregulated in mammary precancers by 17- folds and 9.2-folds, respectively. These results showed a reduction in expression of steroid receptors in mastitic glands and upregulation in mammary precancer indicating role of these receptors in cell proliferation.

Increased Expression of Follistatin in Breast Cancer Reduces Invasiveness and Clinically Correlates with Better Survival.

Activin and its antagonist follistatin (FST) have been implicated in several solid tumours. This study investigated the role of FST in breast cancer. FST expression was examined using reverse transcription polymerase chain reaction (RT-PCR), real-time quantitative polymerase chain reaction (qPCR) and immunohistochemistry in a cohort of breast cancer samples. Expression was correlated to pathological and prognostic parameters in our patient cohort. FST was overexpressed in MCF-7 cells and assays for growth and invasion were performed. FST is expressed in breast tissue, in the cytoplasm of mammary epithelial cells. Expression was decreased in breast cancer tissue in comparison to normal mammary tissue. Over-expression of FST in vitro led to significantly increased growth rate and reduced invasion. Higher FST associates with lower-grade tumours and better survival. Our results suggest a role for FST as a suppressor of invasion and metastasis in breast cancer.

Survival and Differentiation of Mammary Epithelial Cells in Mammary Gland Development Require Nuclear Retention of Id2 Due to RANK Signaling

RANKL plays an essential role in mammary gland development during pregnancy. However, the molecular mechanism by which RANK signaling leads to mammary gland development is largely unknown. We report here that RANKL stimulation induces phosphorylation of Id2 at serine 5, which leads to nuclear retention of Id2. In lactating Id2Tg; RANKL(-/-) mice, Id2 was not phosphorylated and was localized in the cytoplasm. In addition, in lactating Id2(S5A)Tg mice, Id2(S5A) (with serine 5 mutated to alanine) was exclusively localized in the cytoplasm of mammary epithelial cells (MECs), while endogenous Id2 was localized in the nucleus. Intriguingly, nuclear expression of Id2(S5A) rescued increased apoptosis and defective differentiation of MECs in RANKL(-/-) mice. Our results demonstrate that nuclear retention of Id2 due to RANK signaling plays a decisive role in the survival and differentiation of MECs during mammary gland development.

Intracellular killing of mastitis pathogens by penethamate hydriodide following internalization into mammary epithelial cells

Penethamate hydriodide was highly effective in killing Streptococcus uberis, Streptococcus dysgalactiae subsp. dysgalactiae and Staphylococcus aureus that internalized mammary epithelial cells. At higher concentrations (32 microg/mL to 32 mg/mL), killing rates ranged from 85% to 100%. At lower concentrations (0.032 microg/mL to 3.2 microg/mL), killing rates ranged from 0 to 80%. Results of this proof-of-concept study demonstrated that: (1) penethamate hydriodide is capable of entering mammary epithelial cells and killing intracellular mastitis pathogens without affecting mammary epithelial cell viability, (2) the in vitro model used is capable of quantifying the fate of mastitis pathogens internalized into mammary epithelial cells, and (3) this in vitro model can be used to determine the effectiveness of antibiotics at killing bacteria within the cytoplasm of mammary epithelial cells.

Expression and localisation of GLUT1 and GLUT12 glucose transporters in the pregnant and lactating rat mammary gland.

Glucose plays a major role in mammary gland function during lactation as it is used both as a fuel and as a precursor of milk components. In rats, previous studies have shown that the facilitative glucose transporter GLUT1 is expressed in mammary epithelial cells. We have used confocal immunofluorescence to localise GLUT1 and GLUT12, a recently identified member of the sugar transporter family, in pregnant and lactating rat mammary gland. GLUT12 staining was observed in the cytoplasm of mammary epithelial cells at day 20 of pregnancy, and at 1 and 6 days postpartum. Furthermore, GLUT12 staining was present at the apical plasma membrane of epithelial cells during lactation. In contrast, GLUT1 protein localised to the cytoplasm and basolateral surface of mammary epithelial cells. Forced weaning resulted in decreased cytoplasmic GLUT1 staining intensity, but no change in GLUT12 staining. The results suggest a possible role for GLUT12 in the metabolism of mammary epithelial cells during pregnancy and lactation.

Cloning and Expression of Bovine BRCA1

Breast ovarian cancer susceptibility (BRCA) proteins appear to be involved in cell cycle regulation, DNA repair or genome integrity and induction of apoptosis in a variety of cells from humans and laboratory animals. The BRCA gene and protein have not been identified in cattle. The pattern of BRCA1 gene expression during normal mammary gland development and involution has not been examined in detail in any mammalian species. Therefore, the purpose of the present study was to clone the BRCA1 gene in Holstein dairy cattle and determine if the BRCA1 gene is differentially expressed through various stages of mammary gland development. We also localized immunoreactive BRCA1 protein in bovine mammary cells and milk fat globule membrane. Bovine BRCA1 cDNA was highly conserved to the human. Five hundred base pairs of exon 11 (+3384/ + 3866, human BRCA1 cDNA position) and the C-terminus 1 kb were identical to the human. Seven hundred base pairs of the N-terminus, which contain two ring domains, showed 90 % homology to human BRCA1. In bovine tissues, the degree of BRCA1 gene expression, from highest to lowest , was as follows: liver, spleen, mammary tissues and kidney. The mammary tissues of early pregnancy heifers (3 months) showed much higher mammary gland BRCA1 mRNA expression than other mammary developmental stages. BRCA1 expression declined during the remainder of pregnancy and remained low for lactating cows. However, its expression increased when involution proceeded. Immunohistochemical studies showed that immunoreactive BRCA1 was localized in the nucleus and cytoplasm of mammary epithelial cells from lactating cows. It was not present in myoepithelial cells. The protein was also localized in the milk fat globule membranes. Our data suggest that BRCA1 is involved in bovine mammary gland development and/or differentiation, is specifically localized in secretary epithelial cells and is likely a secreted protein during normal lactation.

Specific DNA Binding of Stat5, but Not of Glucocorticoid Receptor, Is Required for Their Functional Cooperation in the Regulation of Gene Transcription

Prolactin and glucocorticoid hormone are signals which regulate the transcription of milk protein genes in mammary epithelial cells. We have investigated the molecular mechanisms by which these hormones cooperate in the induction of transcription. Both hormones activate latent transcription factors in the cytoplasm of mammary epithelial cells. Prolactin exerts its effect through binding to the extracellular domain of the prolactin receptor and through receptor dimerization. This leads to the activation of a protein tyrosine kinase (Jak2), which is noncovalently associated with the cytoplasmic domain of the prolactin receptor. Jak2 phosphorylates the signal transducer and transcription activator (Stat5) which causes its dimerization and nuclear translocation where Stat5 specifically binds to sequence elements in the promoter regions of milk protein genes. In comparison, the glucocorticoid receptor is activated by a lipophilic steroid ligand in the cytoplasm which causes allosteric changes in the molecule, dimerization, and nuclear localization. It has been demonstrated that Stat5 and the glucocorticoid receptor form a molecular complex which cooperates in the induction of transcription of the beta-casein gene. We have defined the DNA sequence requirements for this cooperative mechanism and have delimited the functional domains in Stat5 and the glucocorticoid receptor that are necessary for the functional interaction. We find that the Stat5 response element (Stat5RE) within the beta-casein gene promoter is sufficient to elicit the cooperative action of Stat5 and the glucocorticoid receptor on transcription. Activation of Stat5 through phosphorylation of tyrosine 694 is an absolute prerequisite for transcription. Deletion of the transactivation domain of Stat5 results in a molecule which cannot mediate transactivation by itself but can still cooperate with the glucocorticoid receptor. Mutated variants of the glucocorticoid receptor with a nonfunctional DNA binding domain or a DNA binding domain contributed by the estrogen receptor are still able to cooperate with Stat5 in transcriptional induction. Deletion of the ligand binding domain of the glucocorticoid receptor does not impede cooperation with Stat5, whereas deletion of the AF-1 transactivation domain does prevent cooperation. Our results indicate that the glucocorticoid receptor acts as a ligand-dependent coactivator of Stat5 independently of its DNA binding function.

Effect of Bovine Somatotropin on the Distribution of Immunoreactive Insulin-Like Growth Factor-I in Lactating Bovine Mammary Tissue

The distribution pattern of immunoreactive insulin-like growth factor-I in normal lactating bovine mammary tissue and in tissue obtained after bovine somatotropin treatment was determined by indirect immunofluorescence. In normal tissue, insulin-like growth factor-I immunoreactivity was observed almost exclusively associated with stromal elements. Intralobular stromal cells, small blood vessels, and capillaries all expressed moderate to high immunoreactivity. In contrast, mammary epithelial cells displayed only sparse cytoplasmic immunoreactivity. Immunoreactive material was also present in the periductular connective tissue area, possibly associated with the basal plasma membrane of epithelial cells. Somatotropin treatment of animals resulted in elevated serum insulin-like growth factor-I concentrations and altered the distribution of insulin-like growth factor-I-stainable material in mammary tissue. After somatotropin treatment, immunoreactivity was still detected in mammary stroma; however, prominent staining was also observed in the cytoplasm of mammary epithelial cells. Given the possible role of insulin-like growth factor-I in the regulation of bovine mammary epithelial cell growth and function, our findings raise the possibility that somatotropin may induce insulin-like growth factor-I production in mammary tissue, or other tissues, to influence indirectly the growth or function of the epithelial cells. This offers a possible mechanism for bovine somatotropin stimulation of lactation.

Monoclonal antibodies prepared against PAS-I butyrophilin and GP-55 from guinea-pig milk-fat-globule membrane bind specifically to the apical pole of secretory-epithelial cells in lactating mammary tissue

Monoclonal antibodies to the three major glycoproteins of guinea-pig milk-fat-globule membrane were isolated. The specificity of these antibodies was determined by solid-phase immunoassays and by immunoblotting and autoradiographic techniques after one-and two-dimensional gel electrophoresis. The antibodies bound to PAS-I, a sialoglycoprotein of M r ⩾200 000 and the glycoproteins butyrophilin and GP-55, of M r 63 000 and 55 000, respectively. Immunolocalization studies showed that all three proteins were highly concentrated in the apical pole of secretory-epithelial cells in mammary tissue during lactation. PAS-I, butyrophilin or GP-55, were not detected in either the basal cytoplasm of mammary epithelial cells or in myoepithelial cells, capillary endothelial cells or other cells found in the mammary gland. These proteins were either present in small amounts or were absent from mammary tissue taken in late pregnancy. The monoclonal antibodies characterized in this study will therefore be useful as probes for studies of the biogenesis of apical membrane proteins in mammary epithelial cells during lactation.