Identification of Wnt responsive genes using a murine mammary epithelial cell line model system

Background  

The Wnt/Wg pathway plays an important role in the developmental program of many cells and tissues in a variety of organisms. In addition, many Wnts and components of their downstream signaling pathways, such as β-catenin and APC, have been implicated in tumorigenesis. Over the past years, several genes have been identified as Wnt responsive, including c-myc, siamois, and cyclin D1.     

SHARPIN regulates collagen architecture and ductal outgrowth in the developing mouse mammary gland

SHARPIN is a widely expressed multifunctional protein implicated in cancer, inflammation, linear ubiquitination and integrin activity inhibition; however, its contribution to epithelial homeostasis remains poorly understood. Here, we examined the role of SHARPIN in mammary gland development, a process strongly regulated by epithelial–stromal interactions. Mice lacking SHARPIN expression in all cells (Sharpin^cpdm), and mice with a stromal (S100a4‐Cre) deletion of Sharpin, have reduced mammary ductal outgrowth during puberty. In contrast, Sharpin^cpdm mammary epithelial cells transplanted in vivo into wild‐type stroma, fully repopulate the mammary gland fat pad, undergo unperturbed ductal outgrowth and terminal differentiation. Thus, SHARPIN is required in mammary gland stroma during development. Accordingly, stroma adjacent to invading mammary ducts of Sharpin^cpdm mice displayed reduced collagen arrangement and extracellular matrix (ECM) stiffness. Moreover, Sharpin^cpdm mammary gland stromal fibroblasts demonstrated defects in collagen fibre assembly, collagen contraction and degradation in vitro. Together, these data imply that SHARPIN regulates the normal invasive mammary gland branching morphogenesis in an epithelial cell extrinsic manner by controlling the organisation of the stromal ECM.     

Monocarboxylate transporter genes in the mammary gland of lactating cows

This study is the first to examine the expression of the 14 monocarboxylate transporter genes (MCT1–MCT14) in the mammary gland of mammals. RT-PCR, Western blot, immunohistochemistry, and immunofluorescence confocal laser microscopy were applied in a comprehensive approach to assess the expression and cellular localization of MCTs in the mammary gland of lactating cattle. RT-PCR revealed the existence of nine MCT isoforms, namely MCT1, MCT2, MCT3, MCT4, MCT5, MCT8, MCT10, MCT13, and MCT14 in cow mammary gland. The amplified cDNA segments were confirmed by sequence analysis and deposited in the GenBank. Using the commercially available antibodies against MCT1–MCT8, Western blotting verified the protein expression of MCT1, MCT2, MCT3, MCT4, MCT5, and MCT8 in the cow mammary gland. The precise cellular localization of the identified MCT proteins showed that both MCT1 and MCT2 were basolaterally localized on the cow mammary alveolar epithelial cells. In contrast, MCT4 protein signal was expressed on the apical membrane of these alveolar epithelia. MCT8, however, was predominantly localized on the basolateral membranes of the lactocytes, along with its weak labeling on the apical membrane of the same cells. No immunoreactive staining for MCT3 and MCT5 proteins could be detected histochemically in lactating bovine mammary tissue. Additionally, we proved the colocalization of CD147 with both MCT1 and MCT4 on the boundaries of the cow mammary alveolar epithelia. The existence and localization pattern of MCT genes in the mammary gland of lactating cows suggest their possible involvement in the transport of essential elements required for milk synthesis and secretion.     

Immunocytochemical detection of Ki‐67 in Diff‐Quik‐stained cytological smears of canine mammary gland tumours

U.S. Choi and D.Y. Kim Immunocytochemical detection of Ki‐67 in Diff‐Quik‐stained cytological smears of canine mammary gland tumours Objective: To investigate whether Diff‐Quik stained fine needle aspirate smears can be used to evaluate Ki‐67 expression by immunocytochemistry. Methods: Both cytological and histological samples were obtained from 24 dogs with spontaneously developed mammary gland tumours. The cytological and histological specimens were examined by Diff‐Quik and H&E stains, respectively. After examination, both samples were immunostained using the same Ki‐67 antibody. The % Ki‐67 values were calculated based on the percentage of positively stained tumour cells per 500 and 1000 tumour cells in cytology and histology specimens, respectively. Results: Ki‐67 staining was successful in 17/24 smears (71%) and 19/23 sections (83%). The correlation coefficient between the percentage of Ki‐67‐positive cells in cytological smears and in the histological sections was 0.677 (P < 0.01). These values were significantly different between histologically benign and malignant tumour groups both in cytology and histology samples (P < 0.001). The threshold value of the percentage of Ki‐67‐positive cells for distinguishing benign from malignant tumours was set at 4.85% with 90.9% sensitivity and 92.3% specificity by Receiver Operating Characteristic (ROC) curve using histopathology as the gold standard. Conclusion: Diff‐Quik‐stained cytology smears can be used to detect the presence of Ki‐67 antigen when histology sections are not available.     

Estrogen increases calcitonin gene‐related peptide‐immunoreactive sensory innervation of rat mammary gland

Estrogen plays important roles in preparing mammary tissue for lactation. However, estrogen also influences innervation in some tissues. We examined the effect of estrogen on peripheral innervation of mammary tissues of ovariectomized adult virgin female rats. Seven days after ovariectomy, 17β‐estradiol or placebo pellets were implanted subcutaneously, and tissues were harvested 1 week later. Estrogen treatment decreased mammary gland mass and adipocyte content, while ductal content increased and vascular composition was unaffected. Estrogen increased total areas occupied by nerves in mammary gland sections immunostained for the pan‐neuronal marker protein gene product 9.5, and this increase persisted after normalizing for treatment‐induced differences in gland mass. Although a significant increase in tyrosine hydroxylase‐immunoreactive sympathetic nerve area was observed, no difference was detected following correction for differences in gland size, implying a conserved number of sympathetic nerves in the face of reduced gland volume. Calcitonin gene‐related peptide‐immunoreactive sensory nerve sectional area was also increased, and corrected nerve area remained 88% greater, indicating nerve proliferation during estrogen treatment. Total, sensory, and sympathetic innervation of the nipple and adjacent dermal tissue were unaffected by estrogen. We conclude that chronic estrogen elevation induces selective proliferation of rat mammary gland calcitonin gene‐related peptide‐containing nerves, which are associated primarily with blood vessels and are probably nociceptors. Because they are likely to subserve a vasodilatory function, increased innervation may promote increased blood flow necessary for milk formation during suckling. Moreover, these findings may help explain abundant anecdotal reports of increased breast sensitivity in humans under high estrogen conditions. © 2004 Wiley Periodicals, Inc. J Neurobiol 59: 192–204, 2004     

Nandrolone and stanozolol induce leydig cell tumor proliferation through an estrogen‐dependent mechanism involving IGF‐I system

Several substances such as anabolic androgenic steroids (AAS), peptide hormones like insulin‐like growth factor‐I (IGF‐I), aromatase inhibitors and estrogen antagonists are offered via the Internet, and are assumed without considering the potential deleterious effects that can be caused by their administration. In this study we aimed to determine if nandrolone and stanozolol, two commonly used AAS, could have an effect on Leydig cell tumor proliferation and if their effects could be potentiated by the concomitant use of IGF‐I. Using a rat Leydig tumor cell line, R2C cells, as experimental model we found that nandrolone and stanozolol caused a dose‐dependent induction of aromatase expression and estradiol (E2) production. When used in combination with IGF‐I they were more effective than single molecules in inducing aromatase expression. AAS exhibited estrogenic activity and induced rapid estrogen receptor (ER)‐dependent pathways involving IGF1R, AKT, and ERK1/2 phosphorylation. Inhibitors for these kinases decreased AAS‐dependent aromatase expression. Up‐regulated aromatase levels and related E2 production increased cell proliferation as a consequence of increased cyclin E expression. The observation that ER antagonist ICI182,780 was also able to significantly reduce ASS‐ and AAS + IGF‐induced cell proliferation, confirmed a role for estrogens in AAS‐dependent proliferative effects. Taken together these data clearly indicate that the use of high doses of AAS, as it occurs in doping practice, enhances Leydig cell proliferation, increasing the risk of tumor development. This risk is higher when AAS are used in association with IGF‐I. To our knowledge this is the first report directly associating AAS and testicular cancer. J. Cell. Physiol. 227: 2079–2088, 2012. © 2011 Wiley Periodicals, Inc.     

小鼠不同泌乳期乳腺脂肪合成相关基因的mRNA表达

为了研究小鼠不同泌乳期乳脂肪合成相关基因的表达规律, 文章采用荧光定量PCR检测了小鼠乳腺中与脂肪合成和分泌相关20个基因的mRNA相对表达丰度和表达差异。结果表明, 在乳腺中脂蛋白脂酶(LPL)、乙酰辅酶A羧化酶(ACACA)、硬脂酰辅酶A去饱和酶(SCD)、黄嘌呤脱氢酶(XDH)、嗜乳脂蛋白(BTN)、脂肪酸分化蛋白(ADFP)基因都具有高mRNA表达丰度 (表达丰度>5%), 脂肪酸转运体(CD36)、脂肪酸合成酶(FASN)、1-酰基甘油磷酸酰基转移酶(AGPAT6)和甘油酰基转移酶(DGAT)基因具有中等mRNA表达丰度(5%>表达丰度>1%), 与妊娠期乳腺基因的mRNA表达相比, 在泌乳期这些基因的mRNA表达均有显著上调(P<0.05), 并且ACACA、SCD、FASN、AGPAT6和DGAT等脂肪合成酶基因的表达在泌乳中期(12 d)最高, 而在泌乳初期(6 d)和泌乳末期(18 d)较低, 呈现低-高-低的表达模式。转录因子固醇调节元件结合蛋白(SREBF)基因在泌乳开始时mRNA表达增加, 在泌乳中期(12 d)表达有10倍上调, 其变化规律与脂肪合成酶基因的表达模式相同, 说明SREBF基因在小鼠乳腺脂肪合成酶基因的表达调控中发挥重要调节作用。     

小鼠不同泌乳期乳腺脂肪合成相关基因的mRNA表达

为了研究小鼠不同泌乳期乳脂肪合成相关基因的表达规律,文章采用荧光定量PCR检测了小鼠乳腺中与脂肪合成和分泌相关20个基因的mRNA相对表达丰度和表达差异。结果表明,在乳腺中脂蛋白脂酶(LPL)、乙酰辅酶A羧化酶(ACACA)、硬脂酰辅酶A去饱和酶(SCD)、黄嘌呤脱氢酶(XDH)、嗜乳脂蛋白(BTN)、脂肪酸分化蛋白(ADFP)基因都具有高mRNA表达丰度(表达丰度>5%),脂肪酸转运体(CD36)、脂肪酸合成酶(FASN)、1-酰基甘油磷酸酰基转移酶(AGPAT6)和甘油酰基转移酶(DGAT)基因具有中等mRNA表达丰度(5%>表达丰度>1%),与妊娠期乳腺基因的mRNA表达相比,在泌乳期这些基因的mRNA表达均有显著上调(P<0.05),并且ACACA、SCD、FASN、AGPAT6和DGAT等脂肪合成酶基因的表达在泌乳中期(12 d)最高,而在泌乳初期(6 d)和泌乳末期(18 d)较低,呈现低-高-低的表达模式。转录因子固醇调节元件结合蛋白(SREBF)基因在泌乳开始时mRNA表达增加,在泌乳中期(12 d)表达有10倍上调,其变化规律与脂肪合成酶基因的表达模式相同,说明SREBF基因在小鼠乳腺脂肪合成酶基因的表达调控中发挥重要调节作用。