Myoepithelial cell contraction and milk ejection are impaired in mammary glands of mice lacking smooth muscle alpha-actin

Mammary myoepithelial cells are specialized smooth musclelike epithelial cells that express the smooth muscle actin isoform: smooth muscle alpha-actin (ACTA2). These cells contract in response to oxytocin to generate the contractile force required for milk ejection during lactation. It is believed that ACTA2 contributes to myoepithelial contractile force generation; however, this hypothesis has not been directly tested. To evaluate the contribution of ACTA2 to mammary myoepithelial cell contraction, Acta2 null mice were utilized and milk ejection and myoepithelial cell contractile force generation were evaluated. Pups suckling on Acta2 null dams had a significant reduction in weight gain starting immediately postbirth. Cross-fostering demonstrated the lactation defect is with the Acta2 null dams. Carmine alum whole mounts and conventional histology revealed no underlying structural defects in Acta2 null mammary glands that could account for the lactation defect. In addition, myoepithelial cell formation and organization appeared normal in Acta2 null lactating mammary glands as evaluated using an Acta2 promoter-GFP transgene or phalloidin staining to visualize myoepithelial cells. However, mammary myoepithelial cell contraction in response to oxytocin was significantly reduced in isolated Acta2 null lactating mammary glands and in in vivo studies using Acta2 null lactating dams. These results demonstrate that lack of ACTA2 expression impairs mammary myoepithelial cell contraction and milk ejection and suggests that ACTA2 expression in mammary myoepithelial cells has the functional consequence of enhancing contractile force generation required for milk ejection.     

The mammary myoepithelial cell

Over the last few years, the discovery of basal-type mammary carcinomas and the association of the regenerative potential of the mammary epithelium with the basal myoepithelial cell population have attracted considerable attention to this second major mammary lineage. However, many questions concerning the role of basal myoepithelial cells in mammary morphogenesis, functional differentiation and disease remain unanswered. Here, we discuss the mechanisms that control the myoepithelial cell differentiation essential for their contractile function, summarize new data concerning the roles played by cell-extracellular matrix (ECM), intercellular and paracrine interactions in the regulation of various aspects of the mammary basal myoepithelial cell functional activity. Finally, we analyze the contribution of the basal myoepithelial cells to the regenerative potential of the mammary epithelium and tumorigenesis.     

Heparin affin regulatory peptide in milk: its involvement in mammary gland homeostasis

HARP (heparin affin regulatory peptide) is a heparin binding growth factor implicated in cellular growth and differentiation. Previously, HARP had been localized in the human mammary, in both alveolar epithelial and myoepithelial cells although HARP mRNAs were only expressed by myoepithelial cells [J. Histochem. Cytochem. 45 (1997) 1]. In the present study, we demonstrate that HARP is secreted in human mature milk with concentrations ranging from 17.68+/-6.4ng/ml in mature milk to 59.9+/-11.22ng/ml in colostrum. In vitro, HARP was found to be mitogenic on human mammary epithelial and myoepithelial cell lines and correlated with the expression of its high affinity receptor tyrosine kinase ALK (anaplastic lymphoma kinase). In vivo, ALK is expressed in both mammary epithelial and myoepithelial cells, suggesting that HARP could act in vivo as a paracrine and autocrine growth factor in the regulation of the mammary gland development and its homeostatic maintenance during pregnancy and lactation.     

Autocrine regulation of mammary cell differentiation

Summary Milk secretion and mammary function in dairy animals are regulated by local mechanisms sensitive to the frequency or efficiency of milking. Acute local control of milk secretion occurs through autocrine feedback inhibition by a milk protein. Sustained changes in milking frequency and milk secretion are associated with longer-term adaptations in the degree of differentiation and, ultimately, the number of mammary epithelial cells. Differentiation of cultured mammary cells is suppressed by a milk fraction containing the inhibitor, suggesting that intra-mammary regulation of differentiation in vivo is elicited by the same autocrine regulator subsequent to its acute effect on milk secretion. The autocrine factor may affect mammary cell differentiation by modulating the number of cell surface hormone receptors for prolactin, thereby changing their sensitivity to circulating hormones.Dedicated to Professor Stuart Patton on the occasion of his 70th birthday.     

Stem Cell Research: A Novel Boulevard towards Improved Bovine Mastitis Management

The dairy industry is a multi-billion dollar industry catering the nutritional needs of all age groups globally through the supply of milk. Clinical mastitis has a severe impact on udder tissue and is also an animal welfare issue. Moreover, it significantly reduces animal value and milk production. Mammary tissue damage reduces the number and activity of epithelial cells and consequently contributes to decreased milk production. The high incidence, low cure rate of this highly economic and sometimes deadly disease is an alarming for dairy sector as well as policy makers. Bovine mammary epithelial cells (MECs) and their stem cells are very important in milk production and bioengineering. The adult mammary epithelium consists of two main cell types; an inner layer of luminal epithelial cells, which produce the milk during lactation, and an outer layer of myoepithelial cells resting on a basement membrane, which are responsible for pushing the milk through the ductal network to the teat cistern. Inner layer of columner/luminal cells of bovine MECs, is characterized by cytokeratin18, 19 (CK18, CK19) and outer layer such as myoepithelial cells which are characterized by CK14, α-smooth muscle actin (α-SMA) and p63. Much work has been done in mouse and human, on mammary gland stem cell research, particularly in cancer therapy, but stem cell research in bovine is still in its infancy. Such stem/progenitor cell discoveries in human and mouse mammary gland bring some hope for application in bovines. These progenitors may be therapeutically adopted to correct the structural/cytological defects in the bovine udder due to mastitis. In the present review we focused on various kinds of stem/progenitor cells which can have therapeutic utility and their possibilities to use as a potential stem cell therapy in the management of bovine post-mastitis damage in orders to restore milk production. The possibilities of bovine mammary stem cell therapy offers significant potential for regeneration of tissues that can potentially replace/repair diseased and damaged tissue through differentiation into epithelial, myoepithelial and/or cuboidal/columnar cells in the udder with minimal risk of rejection and side effects.     

Interaction of Wnt3a, Msgn1 and Tbx6 in neural versus paraxial mesoderm lineage commitment and paraxial mesoderm differentiation in the mouse embryo

Smooth muscle α-actin (Acta2) is one of six highly conserved mammalian actin isoforms that appear to exhibit functional redundancy. Nonetheless, we have postulated a specific functional role for the smooth muscle specific isoform. Here, we show that Acta2 deficient mice have a remarkable mammary phenotype such that dams lacking Acta2 are unable to nurse their offspring effectively. The phenotype was rescued in cross fostering experiments with wild type mice, excluding a developmental defect in Acta2 null pups. The mechanism for the underlying phenotype is due to myoepithelial dysfunction postpartum resulting in precocious involution. Further, we demonstrate a specific defect in myoepithelial cell contractility in Acta2 null mammary glands, despite normal expression of cytoplasmic actins. We conclude that Acta2 specifically mediates myoepithelial cell contraction during lactation and that this actin isoform therefore exhibits functional specificity.     

A mouse bone marrow stromal cell line, TBR-B, shows inducible expression of smooth muscle-specific genes

We established an in vitro culture system which mimicked the differentiation pathway of smooth muscle cell, using TBR-B, a bone marrow stromal cell line derived from transgenic mice harboring temperature-sensitive SV40 large T-antigen gene. TBR-B cells have the potential to express smooth muscle-specific genes including h1-calponin, h-caldesmon, SM22alpha and alpha-actin, only after cultured in the differentiation medium for 2 weeks. The differentiation state of TBR-B was well controlled by using different culture medium. Using this cell line, we also found that ascorbic acid is a potent factor inducing the expression of h1-calponin and alpha-actin. TBR-B cells will serve as a useful tool for elucidating the regulatory mechanisms of smooth muscle-specific gene expression, and for identifying compounds that regulate the differentiation state of vascular smooth muscle cells.     

Dual secretory and myoepithelial differentiation in the transplantable R3230AC rat mammary carcinoma

The hormone-responsive R3230AC mammary carcinoma, serially transplantable in Fisher rats, shows striking functional and morphological similarities to the normal mammary gland. We have studied its cellular composition by both light and electron microscopy, employing markers of myoepithelial and epithelial cells. We identified two cell types: the major cellular component corresponded to epithelial milk-protein secreting cells, while a second component showed immunocytochemical and ultrastructural characteristics of the myoepithelial cells. These cells were positive with a monoclonal antibody detecting alpha smooth muscle actin. The dual differentiation which normally occurs in breast ducts is therefore reproduced in a malignant experimental tumor. The coexistence of neoplastic cell populations, divergent in morphology and function, that persist in a tumor despite many transplant generations, leads to reconsideration of the relationship between cellular differentiation and malignant transformation.     

Precocious Mammary Gland Development in P-Cadherin–deficient Mice

To investigate the functions of P-cadherin in vivo, we have mutated the gene encoding this cell adhesion receptor in mice. In contrast to E- and N-cadherin– deficient mice, mice homozygous for the P-cadherin mutation are viable. Although P-cadherin is expressed at high levels in the placenta, P-cadherin–null females are fertile. P-cadherin expression is localized to the myoepithelial cells surrounding the lumenal epithelial cells of the mammary gland. The role of the myoepithelium as a contractile tissue necessary for milk secretion is clear, but its function in the nonpregnant animal is unknown. The ability of the P-cadherin mutant female to nurse and maintain her litter indicates that the contractile function of the myoepithelium is not dependent on the cell adhesion molecule P-cadherin. The virgin P-cadherin–null females display precocious differentiation of the mammary gland. The alveolar-like buds in virgins resemble the glands of an early pregnant animal morphologically and biochemically (i.e., milk protein synthesis). The P-cadherin mutant mice develop hyperplasia and dysplasia of the mammary epithelium with age. In addition, abnormal lymphocyte infiltration was observed in the mammary glands of the mutant animals. These results indicate that P-cadherin–mediated adhesion and/or signals derived from cell–cell interactions are important determinants in negative growth control in the mammary gland. Furthermore, the loss of P-cadherin from the myoepithelium has uncovered a novel function for this tissue in maintaining the undifferentiated state of the underlying secretory epithelium.     

A monoclonal antibody against alpha-smooth muscle actin: a new probe for smooth muscle differentiation

A monoclonal antibody (anti-alpha sm-1) recognizing exclusively alpha- smooth muscle actin was selected and characterized after immunization of BALB/c mice with the NH2-terminal synthetic decapeptide of alpha- smooth muscle actin coupled to keyhole limpet hemocyanin. Anti-alpha sm- 1 helped in distinguishing smooth muscle cells from fibroblasts in mixed cultures such as rat dermal fibroblasts and chicken embryo fibroblasts. In the aortic media, it recognized a hitherto unknown population of cells negative for alpha-smooth muscle actin and for desmin. In 5-d-old rats, this population is about half of the medial cells and becomes only 8 +/- 5% in 6-wk-old animals. In cultures of rat aortic media SMCs, there is a progressive increase of this cell population together with a progressive decrease in the number of alpha- smooth muscle actin-containing stress fibers per cell. Double immunofluorescent studies carried out with anti-alpha sm-1 and anti- desmin antibodies in several organs revealed a heterogeneity of stromal cells. Desmin-negative, alpha-smooth muscle actin-positive cells were found in the rat intestinal muscularis mucosae and in the dermis around hair follicles. Moreover, desmin-positive, alpha-smooth muscle actin- negative cells were identified in the intestinal submucosa, rat testis interstitium, and uterine stroma. alpha-Smooth muscle actin was also found in myoepithelial cells of mammary and salivary glands, which are known to express cytokeratins. Finally, alpha-smooth muscle actin is present in stromal cells of mammary carcinomas, previously considered fibroblastic in nature. Thus, anti-alpha sm-1 antibody appears to be a powerful probe in the study of smooth muscle differentiation in normal and pathological conditions.