Myoepithelial Cells Of Mammary Glands Research Articles

MIOEPITHELIAL CELLS: STRUCTURE, FUNCTION, AND THEIR ROLE IN NORMAL AND NEOPLASTIC TRANSFORMATION OF THE BREAST

Abstract. Myoepithelial cells (MECs) are characteristic of exocrine glands and are responsible for synthesizing the extracellular matrix, particularly the structural components of the basal membrane. MECs are of epithelial origin and exhibit contractile function, which aids in milk ejection from the ducts during lactation. Scientific literature suggests that MECs act as suppressors of tumors and play an important diagnostic role in mammary gland pathology, especially in distinguishing diff erent neoplastic processes.Research objective: T o elucidate the histological features and immunohistochemical verifi cation of myoepithelial cells in normal breast tissue and their role in neoplastic transformation of the mammary gland.Materials and Methods. The histological examination of myoepithelial cells of the mammary gland was conducted on archival slides from the Department of Histology, Cytology, and Embryology, Danylo Halytsky Lviv National Medical University. Histological sections, stained with hematoxylin and eosin, as well as immunohistochemical markers p63 (Clone 4A4, Masterdiagnostica) and cytokeratin 5/6Ab-2 (D5/16 B4, Thermoscientifi c) for confi rming the myoepithelial origin of the cells, were analyzed under a light microscope (UlabXSP-137TLED) with 10x and 40x objectives and a 10x eyepiece. Photographs were taken using an XCAM 1080P camera.Research Results. The structural units of the mammary gland (MG) are ductal-l obular formations, the terminal portions of which have an alveolar shape and are formed by secretory exocrine cells with underlying myoepithelial cells – basket-like cells. Identifi cation of myoepithelial cells using conventional histological staining techniques such as hematoxylin and eosin does not always provide a clear understanding of their topography. All myoepithelial cells are located on the basal membrane and contribute to the synthesis of its components (laminin, entactin, etc.). The myoepithelial cell layer, which is consistently present in the normal mammary gland, can appear diff erently: resembling smooth muscle or epithelioid cells, having a stellate or spindle- shaped form, or not being visualized at all. It has been shown that p63 is a sensitive and specifi c nuclear myoepithelial marker, expressed in myoepithelial cells of the normal mammary gland, in residual lobular acini, sclerosing adenosis, carcinoma in situ, and not detected in invasive carcinomas. It can be included in immunohistochemical panels aimed at identifying myoepithelial cells in diagnostically challenging breast pathologies.Conclusions. Myoepithelial cells in the mammary glands play a key role in their function, facilitating the process of milk secretion. However, in certain cases, they also play an important role in the development of neoplastic transformations. Intercellular connections between secretory and myoepithelial cells indicate that the last ones suppress both the progression of carcinoma in situ to invasive breast cancer and the carcinomainduced angiogenesis. The use of the immunohistochemical marker p63 is mandatory in the panel of antibodies to identify myoepithelial cells in the mammary gland and diff erentiate between benign and malignant lesions.

Cell-specific expression of functional glucose transporter 8 in mammary gland

Differentiated mammary epithelial cells are responsible for milk synthesis during lactation, supporting early postnatal life in mammals. These cells are found in the terminal alveoli of a secretory epithelium, which is surrounded by myoepithelial cells and a stroma rich in fatty tissue. The aim of this study was to explore the cell-specific expression of the glucose transporter GLUT8 in mammary gland and evaluate its functionality for glucose transport, in order to confirm its role in lactose synthesis. Our histological results revealed that GLUT8 is expressed in adipocytes and the epithelial and myoepithelial cells in mammary gland, with a predominant intracellular granular pattern. Colocalization studies of endogenous and green fluorescent protein fused GLUT8 revealed their expressions in lysosome and Golgi, respectively, with Pearson's coefficient correlations of 0.82 ± 0.05 and 0.68 ± 0.16. Functional studies of dileucine to dialanine mutant of GLUT8 showed a fructose-sensitive 2-deoxy glucose uptake at a rate of 83.3 pmoles/(min∗106 cells), 7 folds over empty vector, with a 60 ± 4 and 72 ± 6% decline in 2-deoxy glucose in the presence of 20 and 50 mM fructose, respectively.We concluded that functional GLUT8 is expressed in mammary gland, localizing in mammary epithelial and myoepithelial cells, and adipocytes. In lactation, GLUT8 is expressed mainly in luminal epithelial cells, at the compartments of the endomembrane system. It is necessary to explore the physiological/pathological functions of GLUT8 in mammary gland, including its role in lactation.

Preeclampsia-Like Features and Partial Lactation Failure in Mice Lacking Cystathionine γ-Lyase—An Animal Model of Cystathioninuria

Elevated plasma homocysteine levels are considered as a risk factor for cardiovascular diseases as well as preeclampsia—a pregnancy disorder characterized by hypertension and proteinuria. We previously generated mice lacking cystathionine γ-lyase (Cth) as cystathioninuria models and found them to be with cystathioninemia/homocysteinemia. We investigated whether Cth-deficient (Cth−/−) pregnant mice display any features of preeclampsia. Cth−/− females developed normally but showed mild hypertension (~10 mmHg systolic blood pressure elevation) in late pregnancy and mild proteinuria throughout development/pregnancy. Cth−/− dams had normal numbers of pups and exhibited normal maternal behavior except slightly lower breastfeeding activity. However, half of them could not raise their pups owing to defective lactation; they could produce/store the first milk in their mammary glands but not often provide milk to their pups after the first ejection. The serum oxytocin levels and oxytocin receptor expression in the mammary glands were comparable between wild-type and Cth−/− dams, but the contraction responses of mammary gland myoepithelial cells to oxytocin were significantly lower in Cth−/− dams. The contraction responses to oxytocin were lower in uteruses isolated from Cth−/− mice. Our results suggest that elevated homocysteine or other unknown factors in preeclampsia-like Cth−/− dams interfere with oxytocin that regulates milk ejection reflex.

Immunohistochemical Expression of p53 and p63 in Adenomas and Carcinomas of Canine Mammary Glands

Mammary tumors are very common in female dogs and its prognosis varies significantly according to the tumor invasion degree. Myoepithelial layer destruction is considered a differential diagnosis parameter of in situ from invasive lesions in human breast cancer. Aiming to evaluate the immunohistochemical expression of p63, a protein specifically expressed by myoepithelial cells of mammary gland, and its correlation with p53 expression, 10 adenomas and 20 carcinomas with and without metastasis of canine mammary gland were studied. There was no significant difference between adenomas and carcinomas for both proteins staining, however, the percentage and continuity of staining was lower in metastatic carcinomas. There was no correlation between p53 and p63 expression, but the higher values of p53 staining were found in carcinomas, according to previous studies. Results showed that p63 is specifically expressed also in myoepithelial cells of canine mammary gland. However, in order to ensure its use as a prognostic marker in canine mammary tumors, further studies involving a higher number of samples should be conducted.

Broad tissue expression of membrane progesterone receptor Alpha in normal mice

The broad tissue distribution of membrane progesterone receptor alpha (mPRalpha) in vertebrates suggests multiple physiological functions of the receptor. Current knowledge regarding the receptor distribution, however, is largely obtained via non-histological assays. In this study, the tissue distribution of mPRalpha in mice of both sexes was described using both histological and non-histological methods. Immunohistochemical analysis revealed that abundant expression of mPRalpha was consistently detected in the cytoplasm and membrane of smooth muscles in vasculatures, gastro-intestines, and uterus. It was also observed in myoepithelial cells of mammary gland and intra-ovarian myofibroblasts. These findings suggest that mPRalpha may function as a mediator of P4 in regulating function of smooth muscles or smooth muscle-like cells in numerous physiological processes such as vasodilation, transportation of contents within luminary organs, relaxation of the uterine myometrium during pregnancy, release of oocytes, and milk secretion. In addition, strong mPRalpha expression was identified in the parietal cells of gastric glands, indicating the potential roles of P4/mPRalpha signaling in the modulation of gastric acid secretion. Surprisingly, in the testis of male mice mPRalpha was mainly seen in the nuclei, rather than cytoplasm and/or membrane, of the primary and secondary spermatocytes, suggesting a direct role of the receptor in gene regulation. Our results indicate that mPRalpha may function as a key modulator of P4 in the modulation of multiple physiological functions in normal mice.

Constitutive Coexpression of Nitric Oxide Synthase-1 and Soluble Guanylyl Cyclase in Myoepithelial Cells of Mammary Glands in Mice

An impact of nitric oxide (NO) on lactation and milk secretion in mammary glands has previously been documented, but the underlying molecular mechanisms for this modulatory effect remain unclear. Therefore, we investigated the expression patterns of NO synthase (NOS)-1, NOS-3 and the NO receptor soluble guanylyl cyclase (sGC) in mammary glands of lactating and non-lactating female C57/Bl6 mice. RT-PCR demonstrated the existence of NOS-1-mRNA and NOS-3-mRNA in both lactating and resting mammary tissue. Immunoblots loaded with equal amounts of homogenate proteins from lactating and resting mammary tissues revealed comparable intensities of NOS-1 and sGC bands. Performing catalytic NADPH diaphorase histochemistry and immunohistochemistry, NOS-1 was only detected in myoepithelial cells (MEC), while sGC was localized in alveolar epithelial cells (lactocytes) and MEC in both lactating and non-lactating mammary glands. The non-modulated co-expression of both enzymes suggests that NOS-1 and sGC contribute to the constitutive regulation of tone in MEC.

Clinical significance of Caveolin-1, Caveolin-2 and HER2/neu mRNA expression in human breast cancer

Caveolin-1 and -2 (CAV1, CAV2) are closely linked genes localised to the fragile region of 7q31 (FRA7G), and loss of heterozygosity involving this region has been reported in breast cancer. Several studies have suggested that CAV1 is a negative regulator of HER2/neu signal transduction in vitro. However, the clinical significance of CAV1 in breast cancer has not yet been clarified. We examined quantitatively the mRNA levels of CAV1, CAV2 and HER2/neu in 162 cases of breast cancer using real-time PCR. Caveolin-1 and -2 protein expression was also examined by Western blotting and immunohistochemistry. We then evaluated for correlations between CAV1, CAV2 and HER2/neu gene expression and clinicopathologic factors in the 162 breast cancer cases. Results showed higher HER2/neu mRMA levels and lower CAV1 and CAV2 mRMA levels in breast cancer tissues than in corresponding normal tissues (P<0.001). Caveolin-1 and -2 protein expression levels were also suppressed in cancer tissues compared to normal tissues by Western blotting. Immunohistochemistry revealed that CAV1 and CAV2 proteins were abundantly expressed in mammary gland myoepithelial cells, but only weakly in ductalepithelial cells. Reduced CAV1 mRNA level was significantly associated with increasing tumour size (P=0.041), and negative oestrogen receptor status (P=0.021). There was also a significant association between low CAV2 mRNA level and negative progesterone receptor status (P=0.013), and between high HER2/neu mRNA level and negative hormonal receptor status (ER, P=0.029, PgR, P=0.019). While there was no relationship between HER2/neu and CAV1 mRNA levels, a significant association between CAV1 and CAV2 mRNA levels was observed (P<0.001). Our results indicated that CAV1 suppression correlated closely with that of CAV2 in breast cancer, that CAV1 level was inversely correlated with tumour size, and that CAV1 and CAV2 levels were correlated with hormonal receptor status. Therefore, CAV1 and CAV2 play an important role in tumour progression in breast cancer patients.

Immunolocalization of the smooth muscle-specific protein calponin in complex and mixed tumors of the mammary gland of the dog: assessment of the morphogenetic role of the myoepithelium.

The immunohistochemical expression of the smooth muscle-specific protein calponin was studied to assess the contribution of myoepithelial cells to the histogenesis of spindle cells of complex and mixed tumors of the mammary gland of the dog and the origin of cartilage and bone in mixed tumors. Formalin-fixed tissues from 55 benign and malignant tumors (49 also containing surrounding normal mammary gland) were evaluated. Periacinar and periductal myoepithelial cells of all the 49 normal mammary glands were diffusely stained by the anti-human calponin monoclonal antibody. Calponin was found in 53 (98%) of the tumors studied, reacting with the myoepithelium-like cells of 86% of benign tumors and their remnants in 85% of malignant tumors. Five different types of calponin-immunoreactive myoepithelial cells were identified: hypertrophic myoepithelial cells. fusiform cells, stellate myoepithelial cells, rounded (myoepithelial) cells, and chondroblasts. Differences in staining intensity and staining pattern among these five types of cells suggested a transition of myoepithelial cells to chondroblasts. Stromal myofibroblasts also showed calponin immunoreactivity, but they did not react with a cytokeratin 14 monoclonal antibody, which recognizes myoepithelial cells in mammary gland. Calponin appears to be a very sensitive marker of normal and neoplastic myoepithelium in the canine mammary gland, and its identification in different cell types of complex and mixed tumors of the mammary gland of the dog suggests a major histogenetic role for myoepithelial cells.

Alterations of myoepithelial cells in the rat mammary gland during pregnancy, lactation and involution, and after estradiol treatment.

Hormone-induced alterations of myoepithelial cells in the mammary gland have not been fully investigated. The aim of the present study was to examine whether myoepithelial cells are altered in response to hormonal conditions. The immunohistochemical findings of smooth muscle actin for myoepithelial cells were studied during pregnancy, lactation and involution, and after estradiol dipropionate (ED) treatment (50, 500, 1000 microg/kg per week for 1-4 weeks) using a total of 71 Wistar female rats. Myoepithelial cells showed a stratified appearance around ducts during pregnancy, extended cytoplasmic processes with wider distance during lactation, and vacuolated cytoplasm after weaning. ED treatment (50-1000 microg/kg per week) for 1 week increased myoepithelial cells to a variable degree, achieving a level similar to that in pregnancy, but ED treatment for 4 weeks reduced them as the dose elevated. The present study showed that the myoepithelial cells became hyperplastic or hypertrophic by low-dose ED treatment within the physiological range, while weaning pups, and excess high-dose ED treatment beyond the physiological range or prolonged ED treatment induced reduction of the myoepithelial cells. Results indicate that myoepithelial cells themselves are also altered by hormonal conditions coordinating the mammary gland development.

Immunohistochemical studies on expression of human vascular smooth muscle myosin heavy chain isoforms in normal mammary glands, benign mammary disorders and mammary carcinomas.

The expression of myosin in normal and diseased mammary glands of 199 Japanese women was evaluated immunohistochemically by the avidin-biotin peroxidase complex method using antibodies to three human smooth muscle myosin heavy chain isoforms derived from the vascular smooth muscle: myosin SM1 is expressed consistently from fetal stage to adulthood, myosin SM2 appears only in well-differentiated smooth muscle after birth, and myosin SMemb is more abundant in embryonic aortas. SM1 was expressed in myoepithelial cells of normal mammary glands and fibrocystic diseases and in myoepithelial-like tumor cells in the basal layer of fibroadenomas and phyllodes tumors. SM2 was expressed only in the myoepithelial cells of mammary glands in breastfeeding women. SMemb was expressed more intensely in the cytoplasm of luminal epithelial cells in larger fibroadenomas (P< 0.01), or in the cytoplasm of carcinoma cells in invasive ductal carcinomas with metastasized lymph nodes (P< 0.001) and in those of higher histological grade (P<0.0001). Multivariate logistic analysis showed a significant correlation only between the expression of SMemb and histological grade (P< 0.0001), which is a prognostic factor of mammary carcinomas. These findings suggested the possible prognostic value of SMemb.

Regulation of Urokinase-Type Plasminogen Activator Production by Rat Mammary Myoepithelial Cells

Addition of basic fibroblast growth factor (bFGF) to a rat mammary gland myoepithelial cell line (25.5−G4.2.3) resulted in a six- to eightfold increase in cellular and secreted urokinase-type plasminogen activator (uPA) activity after a lag phase of 5–8 h. bFGF had no effect on the uPA activity of mammary epithelial cells. bFGF was active on myoepithelial cells over a narrow concentration range (0.5–2 ng/ml). The bFGF-induced increase in uPA activity was inhibited in a dose-dependent manner by hydrocortisone and transforming growth factor-β1 (TGF-β1). Hydrocortisone also inhibited the basal secretion of uPA, as did interleukin-1β and phorbol myristate acetate, both of which increase uPA levels in other cell systems. The effects of bFGF could also be inhibited by factors which bind bFGF, e.g., heparin and methylamine a2-macroglobulin. TGF-β1, but not bFGF, induced the synthesis of plasminogen activator inhibitor-1 in the myoepithelial cell line. Mammary gland myoepithelial cells contribute to the synthesis of and are located next to the basement membrane. Myoepithelial-derived uPA is probably associated with basement membrane turnover. The mammary gland basement membrane undergoes many cycles of remodeling and multiple mechanisms may be required to regulate uPA activity.

Growth hormone gene expression in myoepithelial cells directed by various eucaryotic transcriptional regulatory sequences

Mammary gland myoepithelial cells were isolated from cattle and cell lines were established. Cells were plated onto tissue culture dishes with or without collagen. Cells were transfected with bovine growth hormone rDNA containing one of the following eucaryotic transcriptional regulatory sequences: human cytomegalovirus immediate early promoter, simian virus 40 early promoter, mouse metallothionein I promoter and the Rous sarcoma virus long terminal repeat. These sequences were evaluated for their ability to direct recombinant bovine growth hormone DNA expression in myoepithelial cells. The most effective transcriptional regulatory sequences were the cytomegalovirus immediate early and simian virus 40 early promoters.

Scanning electron microscopic observations of the mammary gland myoepithelial cells of the rat under normal and experimental conditions.

The stromal surface of the mammary gland myoepithelial cells of rats under normal and experimental conditions, which were treated with HCl-collagenase method introduced by Evan et al. (1976), have been studied by scanning electron microscopy. Treatment of the mammary gland with HCl and collagenase removed almost completely the extracellular materials such as collagens and the basal lamina and revealed the myoepithelial cells enclosing the glandular alveolus. Myoepithelial cells consisting of an elongated cell body and long, bifurcating processes grasped the glandular alveolus in the form of a delicate network. With the application of oxytocin, the myoepithelial cells were involved in the morphological changes suggestive of contraction. They had a row of prominent folds arranged perpendicularly to the long axis of the cell body and its processes.

Estrogen-induced alterations in the myoepithelial cells of the rat mammary gland.

Estradiol benzoate was injected into virgin rats, at a concentration of 50 microgram/day, for 7 days. The animals were fixed by perfusion and the mammary glands prepared for electron microscopy. Many myoepithelial cells in the mammary glands were altered as a result of the estradiol treatment. The affected cells had a markedly reduced number of myofilaments that were restricted to a specific region of the cytoplasm. Large foot-like projections with many vesicles extended from the myoepithelial cells into the surrounding connective tissue. The altered cells also had more organelles, suggesting that their metabolic activity was increased. Nuclei in the altered cells had deep infoldings that may reflect an increase in nuclear/cytoplasmic exchanges. The results of this study indicate that estradiol causes specific morphological alterations in myoepithelial cells of the mammary gland.

EFFECT OF SODIUM CITRATE ON MILK YIELD IN RATS.

SummaryThe milk yield of rats was determined on days 14, 16, 18 and 20 by separating the litters from their dams for 10 hours, then injecting 2 USP units of oxytocin and allowing the young to nurse for 30 minutes. Immediately prior to test milking on days 16 and 20 an experimental group of 15 rats was injected with 1.25 ml of 25% sodium citrate to cause convulsions (tetany) due to the decrease in ionized plasma calcium. The milk yield of these rats was greatly reduced in comparison with their own previous production and in comparison with the milk yield of the control group on the same day. It is suggested that the contractility of the mammary gland myoepithelial cells by oxy-tocin is greatly reduced by the lack of plasma calcium. Thus the decreased milk yield of parathyroidectomized rats is due to a reduction in the synthesis of milk and interference with the removal of the milk secreted.