The role of sFRP4, a secreted frizzled-related protein, in ovulation.

Ovulation is a complex, multi-factorial event that involves the degeneration of a specific area of the follicular and ovarian surface via apoptosis. Many apoptosis related genes have been identified in the ovary. Secreted Frizzled Related Protein 4 (sFRP4) is a protein that appears to antagonize a molecular pathway for cell survival. sFRP4 gene expression is known to be upregulated with apoptosis in the ovarian corpus luteum. In this study, ovulation was hormonally induced in immature Wistar rats and their ovaries collected for analysis of apoptosis and sFRP4. TUNEL staining identified a greater amount of dying cells in the thecal layer of treated rat ovaries compared to controls. The results of 3'-end labelling revealed a significant increase (p < 0.01) in apoptosis at 12 hours following treatment compared to other time points and control. In situ hybridization exhibited a visible increase in amounts of sFRP4 mRNA expression in the thecal layers of follicles from treated rats compared to controls. Quantitative RT-PCR revealed no significant difference in sFRP4 expression levels between treated and control tissues although a clear trend towards an increase was observed in the treated group. This study demonstrates an association between sFRP4 and apoptosis in rat ovulation.

Apoptosis-linked in vivo regulation of the tissue transglutaminase gene promoter.

Tissue transglutaminase (tTG) is upregulated in various cells undergoing apoptosis. To investigate the transcriptional regulation of tTG a mouse strain carrying a beta-galactosidase reporter gene under the control of a 3.8 kilobase fragment of the tTG promoter was characterised. The transgene construct was shown to be expressed in the apoptotic regions of the mouse embryo. Here we report that the regulation of the transgene is also apoptosis-linked in adult animals. The transgene is induced in endocrine apoptosis involving mammary gland involution and corpus luteum regression. Induction of the reporter gene is detectable during in vivo but not in vitro apoptosis of thymocytes induced by the glucocorticoid receptor, the nur77, p53 and the retinoid receptor gamma mediated pathways. Additionally, the lacZ expression mimics the activation of the endogenous promoter in tissues characterised by high apoptotic turnover. These results suggest that the apoptosis-specific transcriptional regulation of tTG is mediated through elements of a 3.8 kb promoter and may require cosignals available only in tissue environment. Cell Death and Differentiation (2000) 7, 1225 - 1233.

Physiological apoptosis in hormone-dependent tissues: involvement of caspases.

Physiological apoptosis in mammals is a type of programmed cell death, an important element in the developmental repertoire ensuring tissue homeostasis and proper disposal of cells that are no longer needed, such as milk-producing epithelial cells in the mammary gland after lactation, luteal cells in the post partum Corpus luteum or secretory cells in the prostate after castration. Although incompletely described, apoptosis in hormone-dependent tissues is apparently initiated and executed using common biochemical strategies. These include survival pathways governed by local and systemic factors and hormones, diverse regulatory pathways and caspase-dependent execution pathways. Using an antibody that recognizes processed effector caspases or a fluorogenic caspase substrate, we present for the first time evidence that caspases are activated in the mammary gland, in the prostate and in the ovary at the time when apoptosis occurs. Most likely phagocytosis of apoptotic cells by neighboring cells may represent an important step, since only a modest involvement of professional phagocytes is apparent. Here, we will summarize and discuss recent data and will attempt to draw a generalized picture of how physiological apoptosis may occur in these organs.

Energy metabolism in the involuting mammary gland.

A strong and coordinated upregulation of the glycolytic, glutaminolytic and pentose phosphate pathway enzymes occurs during the onset of lactation in the normal mouse mammary gland. Induction of apoptosis by removing the pups led to an inactivation of the same enzymes with different time courses. While the ATP-consuming glycolytic 6-phosphofructo 1-kinase and mitochondrial bound hexokinase still remained high on days one and two of involution, the ATP-regenerating pyruvate kinase was immediately reduced. The enzymes of the pentose phosphate and glutaminolytic pathway were inactivated on the first two days of involution. In accordance with such an inactivation of the enzymes ATP, GTP, UTP, ADP, NAD NADH and lactate concentrations decreased. The synthetic product of UTP, UDP-N-acetylglucosamine, increased. AMP was found in the milk, not in the epithelial cells. The inactivation of the enzymes was caused by partial proteolysis or by a loss of the intact proteins from the cytosol without signs of proteolysis.

The epithelium-specific ets transcription factor ESX is associated with mammary gland development and involution.

To study mammary gland expression of the epithelium-restricted Ets factor, ESX, mouse cDNA and genomic sequences were cloned and a approximately 350 bp proximal promoter region with >80% mouse-human homology was identified that mediates ESX induction by serum, heregulin (HRG), or epidermal growth factor (EGF). ESX mRNA expression progressively increases during embryonic mouse development from day 7 and is detectable in virgin mammary glands; it shows little if any change during pregnancy, then declines to barely detectable levels after 3 days of lactation. Similarly, cultured HC11 cells from midpregnant mouse mammary epithelium show an increase in ESX expression upon reaching lactogenic competency (in the presence of EGF or HRG), with a decline to barely detectable levels upon exposure to lactogenic hormones that induce milk protein (beta-casein) expression. In contrast, involuting mouse and rat mammary glands show maximal ESX expression. High ESX levels are also seen in the involuting ventral prostate gland of rats. These findings, including the persistence of up-regulated ESX in fully regressed mammary glands, suggest that ESX expression can be induced by soluble growth factors and is maximally up-regulated in those partially committed epithelial cells destined to survive both the apoptotic and remodeling phases of glandular involution.

Apoptosis in the rat mammary gland and ventral prostate: detection of cell death-associated genes using a coincident-expression cloning approach.

Apoptosis plays a striking role in the hormone-dependent involution of the mammary gland, but it has proved difficult to distinguish between the 'cell death' associated genes and the 'tissue remodelling' genes which are expressed concurrently. To identify cell death-associated genes, we have established a 'coincidence analysis' based on the previously described 'RNA differential display' method of Liang and Pardee (1992). Coincidence analysis allows the detection of genes expressed during related processes in different organs and was employed here to identify transcripts in which expression patterns are seen to be associated with apoptosis during involution of both rat mammary- and the ventral prostate glands. That the coincidence analysis is a promising approach can be seen from the fact that while widely accepted apoptosis markers such as transglutaminase (Fesus et al, 1987; Strange et al, 1992) and sulfated glycoprotein-2 (Buttyan et al, 1989; Strange et al, 1992; Guenette et al 1994) exhibited similar expression in both regressing tissues, transcription of tissue remodelling enzymes was minimal in the involuting prostate. We describe here the characteristics of five clones isolated which show coincident expression during programmed cell death in mammary and prostate tissues. Partial sequence analysis revealed for three clones high homologies with previously described genes; the putative rat homolog of the growth arrest gene gas-1 (Schneider et al, 1988; Del Sal et al, 1992), an homolog of the mouse 'Integrin Associated Protein' (IAP) (Brown et al, 1990; Lindberg et al, 1993) and the sequence encoding for the 'Allograft Inflammatory Factor' AIF-1 (Autieri et al, 1995; Utans et al, 1995). One clone displayed homology with an expressed human sequence tag and one clone unrelated to any known DNA sequence was isolated. The expression of these genes in involuting rat mammary and ventral prostate, was correlated with that in other organs and in situ hybridization was applied to establish that the secretory epithelial cells which undergo programmed cell death are the site of elevated expression during the course of involution. Furthermore, we conclude that the coincidence analysis approach described here could be easily applied to facilitate the characterization of gene expression i.e. for the detection and comparison of hormonally regulated genes in different organs.

Regulation of a physiological apoptosis: mouse mammary involution.

Continuous milk production during lactation is dependent on a complex interplay of lactogenic hormones and the suckling stimulus exerted by the young. Involution can be initiated in the mouse mammary gland at any stage of lactation by removing the pups; involution then remains reversible for about 30 to 36 h. Involution in the mouse mammary gland is characterized by a massive loss of secretory epithelial cells from programmed cell death. The nuclear activation of protein kinase A and transcription factor activator protein 1 precede the irreversible phase of involution that is characterized by internucleosomal DNA fragmentation. Activation of activator protein 1 and fragmentation of chromosomal DNA can be prevented by lactogenic hormone treatment in explant cultures derived from mammary tissue at lactation. The elevation in activator protein 1 coincides with the epithelial expression of sulfated glycoprotein 2, a potential target gene of activator protein 1. Programmed cell death in the mammary gland is associated with the expression of the growth arrest gene, gas-1, and the integrin-associated protein gene, IAP, which codes for a putative Ca2+ channel that is dependent on integrin. Their potential roles during involution are discussed.

Expression and activation of tissue transglutaminase in apoptotic cells of involuting rodent mammary tissue.

Apoptosis is a form of cell death in which cellular integrity is maintained and neither cytoplasmic nor nuclear content is released. The Ca(2+)-dependent tissue transglutaminase (tTG) is an enzyme that forms protein cross-links between specific glutamyl and lysyl side-chains of intra- and extracellular proteins, therefore it may be responsible for the structural stabilization observed during the death process. In this study, the expression of tTG was investigated following the physiological process of forced weaning which results in an almost complete elimination of secretory epithelium by apoptosis and remodelling of the tissue structure. A dramatic induction of tTG was detected by immunoblotting of total mammary gland homogenates prepared from the involuting glands. The concentration of epsilon(gamma-glutamyl)-lysine crosslinks was also elevated in these samples, showing that the enzyme is activated during apoptosis. To determine the distribution of tTG and its relationship to apoptotic cells, paraffin-embedded specimens were studied by RNA in situ hybridization and immunohistochemical methods as well as using in situ labeling for nuclear fragmentation. All of these approaches indicated that mammary secretory epithelium expressed tissue transglutaminase coincident with the onset of apoptosis. The apoptotic and tTG-expressing cells were found to be identical as demonstrated by a histological double-labeling technique.

Release of a mitogenic factor by adult rat lung slices in culture.

In an effort to develop a suitable bioassay for testing lung growth factors that might be operative during compensatory lung growth following partial pneumonectomy, a simple and inexpensive lung organ culture system was characterized. The culture employs lung tissue slices obtained by means of a device allowing thicknesses of 500 microns to be cut reproducibly. To avoid the collapse of the organ, the alveolar spaces were filled prior to culture with Noble agar-containing Eagle's-Dulbecco's modified medium. Lung tissue sections could be maintained ultrastructurally intact for at least one week. The results showed that upon culture, a part of the type II pneumocytes undergo differentiation into type I pneumocytes, thus demonstrating that the culture system may be suited for differentiation studies. One surprising feature of this culture system was the mitogenic impulse associated with culture. Radioactively labeled thymidine incorporation was strongly stimulated in the culture, mainly affecting the epithelial cells, as could be established by "back-to-back" autoradiography. With a reconstruction experiment, it was possible to demonstrate the local release of a mitogenic factor following slicing, mincing, or dissection of the lung tissue, which could be assayed by its ability to induce serum-starved Balb/c 3T3 cells to synthesize DNA in culture.

Isolation of cell death-associated cDNAs from involuting mouse mammary epithelium.

Although apoptosis is important in determining cell fate and maintaining tissue homeostasis, the initiation and control of apoptotic cell death in epithelium is not well understood. Post-lactationai involution of the mammary gland provides both an important developmental process and a normal physiological setting for studying apoptosis of epithelium. We used a differential screening strategy, based on previous studies correlating morphology with gene expression and nucleic acid integrity during mammary gland involution, to isolate genes involved in the regulation and execution of apoptotic cell death in regressing mammary epithelium. This screening strategy yielded a large number of genes the expression of which is significantly altered during mammary gland involution. These include genes associated with cell death processes, tissue remodelling and mesenchymal differentiation. In addition, a number of novel genes have been isolated. We have used Northern analysis and in situ hybridisation to study the expression of a selection of these putative death-associated genes during post-lactational mouse mammary gland involution.

Programmed cell death during mammary gland involution.

Understanding the cascade of gene expression and subsequent protein interactions that result both in the death of secretory mammary epithelium and the remodeling and renewal of the mammary gland for another cycle of lactation poses significant challenges (see Chapters 7 and 8, this volume). The complexity of mammary gland involution warrants caution in sorting through the various potential regulators and executors of apoptotic cell death in the mammary gland. As demonstrated by the number of remodeling enzymes expressed during involution, the relationship between mammary epithelium and its related mesenchyme is important for maintenance of differentiated function (Barcellos-Hoff et al., 1989; Streuli et al., 1991). Components of the extracellular matrix may play the role of survival factors, or may provide a source of factors, as a reserve of matrix-bound growth factors, necessary for survival of the secretory epithelium. Perturbation of this interaction alters mammary-specific differentiation gene expression, for example, production of milk proteins (Parry et al., 1987; Strange et al., 1991; Talhouk et al., 1992). Thus, alteration of the interaction between epithelium and its associated mesenchyme, which is an integral part of mammary involution, may also play a role in epithelial cell death. However, the epithelial-mesenchymal interactions that are the determining features in either mediating or modulating this cell death are just beginning to be defined. Stimuli that alter differentiated function may also induce apoptotic cell death of the epithelium but may have no physiological correlate. They may, however, have significant application in prevention or control of breast neoplasia.

Expression of stromelysin-1 and TIMP-1 in the involuting mammary gland and in early invasive tumors of the mouse.

The mammary gland, during post-lactational involution, is subjected to extensive tissue reconstruction. This process is governed by the concerted expression of extracellular-matrix-degrading enzymes and their inhibitors. During carcinogenesis, the invasive growth of tumor cells is characterized by the penetration of the basement membrane and stromal invasion. We compared the expression of the tissue-remodeling enzymes stromelysin-1, a matrix metalloproteinase, and its inhibitor, tissue inhibitor of metalloproteinase-1 (TIMP-1), during mammary gland involution and carcinogenesis in mouse. In involuting mammary glands, stromelysin-1 was expressed in myoepithelial cells, whereas TIMP-1 was confined to the stromal tissue. To analyze the involvement of these tissue-remodeling genes in tumor development, we examined mammary tumors of transgenic mice expressing either the activated Ha-ras or c-myc oncogene under the control of a milk-protein gene promoter. In the undifferentiated and metastasizing Ha-ras-induced tumors, stromelysin-1 expression was comparable to that seen in involution, whereas TIMP-1 expression was greatly elevated. During Ha-ras-induced carcinogenesis, stromelysin-1 expression was first detected in the myo-epithelial cells surrounding preneoplastic lesions. In contrast, in the well-differentiated and non-metastatic mammary tumors induced by c-myc, no expression of either gene was observed. Thus, expression of stromelysin-1 and TIMP-1 is confined to the aggressively growing tumors and is induced in the earliest stages of carcinogenesis.

Overexpression of Mos, Ras, Src, and Fos inhibits mouse mammary epithelial cell differentiation.

Mammary epithelial cells terminally differentiate in response to lactogenic hormones. We present evidence that oncoprotein overexpression is incompatible with this hormone-inducible differentiation and results in striking cellular morphological changes. In mammary epithelial cells in culture, lactogenic hormones (glucocorticoid and prolactin) activated a transfected beta-casein promoter and endogenous beta-casein gene expression. This response to lactogenic hormone treatment was paralleled by a decrease in cellular AP-1 DNA-binding activity. Expression of the mos, ras, or src (but not myc) oncogene blocked the activation of the beta-casein promoter induced by the lactogenic hormones and was associated with the maintenance of high levels of AP-1. Mos expression also increased c-fos and c-jun mRNA levels. Overexpression of Fos and Jun from transiently transfected constructs resulted in a functional inhibition of the glucocorticoid receptor in these mouse mammary epithelial cells. This finding clearly suggests that glucocorticoid receptor inhibition arising from oncogene expression will contribute to the block in hormonally induced mammary epithelial cell differentiation. Expression of Src resulted in the loss of the normal organization and morphological phenotype of mammary epithelial cells in the epithelial/fibroblastic line IM-2. Activation of a conditional c-fos/estrogen receptor gene encoding an estrogen-dependent Fos/estrogen receptor fusion protein also morphologically transformed mammary epithelial cells and inhibited initiation of mammary epithelial differentiation-associated expression of the beta-casein and WDNM 1 genes. In response to estrogen treatment, the cells displayed a high level of AP-1 DNA-binding activity. Our results demonstrate that high cellular AP-1 levels contribute to blocking the ability of mammary epithelial cells in culture to respond to lactogenic hormones. This and other studies indicate that the oncogene products Mos, Ras, and Src exert their effects, at least in part, by stimulating cellular Fos and probably cellular Jun activity.

Lyn, a src-like tyrosine-specific protein kinase, is expressed in HL60 cells induced to monocyte-like or granulocyte-like cells.

During the in vitro differentiation of HL60 cells tyrosine-specific kinases are activated. The expression of lyn, a src-related tyrosine kinase, was studied by analysis of the steady-state levels of its transcript during the cell differentiation process induced by retinoic acid, phorbol 12-myristate 13-acetate and 1,25-dihydroxyvitamin D3. In contrast to an earlier report we observe only a small induction of the lyn-RNA levels compared to uninduced control cells. In unstimulated HL60 cells, the level for the lyn-transcript was comparatively high. A second, minor human lyn-transcript with an estimated size of 3.7 kb which has not been previously described, was identified.

Apoptotic cell death and tissue remodelling during mouse mammary gland involution.

During post-lactational mammary gland involution, the bulk of mammary epithelium dies and is reabsorbed. This massive cell death and tissue restructuring was found to be accompanied by a specific pattern of gene expression. Northern blot analysis showed that weaning resulted in a dramatic drop in ODC, a gene involved in synthesis of a component of milk, and the nearly simultaneous induction of SGP-2, a gene associated with apoptotic cell death. These changes were followed by decreases in expression of milk protein genes to basal levels and expression of genes associated with regulation of cell proliferation and differentiation, p53, c-myc and TGF-beta 1. Subsequently, additional genes implicated in stress response, tissue remodelling, and apoptotic cell death were transiently expressed, expression peaking at about 6 days post-weaning. A non-random degradation of DNA yielding the oligonucleosomal length fragmentation pattern typical of apoptotic cell death (Wyllie, 1980; Wyllie et al., 1980) was detected in association with morphological changes and gene expression. The correlations between: (a) changes in morphology, (b) pattern of gene expression and (c) changes in DNA integrity suggest that complementary programs for cell death and tissue remodelling direct post-lactational mammary gland involution.

Mammary epithelial differentiation in vitro: minimum requirements for a functional response to hormonal stimulation.

Mammary epithelial differentiation is the culmination of responses to a complex sequence of hormonal stimuli. An in vitro model for this process should retain the basic features of in vivo epithelial differentiation. The IM-2 mouse mammary cell line responds to lactogenic hormone stimulation by synthesizing the milk protein beta-casein. Epithelial and fibroblastic clones derived from IM-2 lack this ability, but cocultures of these clones regain responsiveness to lactogenic hormone stimulation. Studies of the epithelial cell clone 31E under various culture conditions reveal that the role of fibroblastic cells in supporting synthesis and secretion of beta-casein can be supplanted by culture in filter chambers without addition of exogenous extracellular matrix components. Electron microscopic and immunofluorescence studies show that, under these conditions, 31E epithelial cells exhibit the morphology and intercellular organization characteristic of mammary epithelium. Transepithelial electrical resistance measurements indicate that the cells are well polarized. Analysis of glucose metabolism is consistent with this polarization; glucose is utilized from the basal chamber, and lactate is excreted into the basal chamber. Immunoblot analysis demonstrates the vectorial protein secretion expected of polarized mammary epithelium: laminin is secreted into the basal chamber, whereas beta-casein is secreted into the apical chamber in response to lactogenic hormone stimulation from the lower chamber. Thus, the maintenance of a polarized intercellular organization that permits access of the basolateral cell surface to nutrients is sufficient for a pure culture of an established mammary epithelial cell clone to retain differentiated epithelial function in vitro.

Tyrosine kinases: from viral oncogenes to developmental regulators.

Protein tyrosine kinases (PTKs) play a central role in cellular regulation by virtue of their participation in, and control of, signal transduction pathways; they act as a molecular interface between the cell's environment and intercellular metabolism. The mammary gland, unlike most organs, undergoes most of its morphogenesis in juvenile and adult life. The epithelium goes through hormonally controlled cycles of proliferation and regression, the fully differentiated state only being reached at the end of pregnancy. These features make the mammary gland an amenable tissue to study the involvement of PTKs in epithelial cell development and differentiation. We have used a PCR-based molecular cloning strategy to identify PTKs from murine mammary gland cells. Amongst 70 kinase clones characterized we found 3 PTKs previously undescribed in mouse, 4 known PTKs and 5 serine threonine kinases. Expression studies revealed differential tissue specificity and developmental regulation of the 3 previously undescribed PTKs. These results substantiate the view that PTKs are involved in the regulation of cellular differentiation.

Myeloperoxidase is a primary response gene in HL60 cells, directly regulated during hematopoietic differentiation.

The expression of myeloperoxidase was studied in three human myeloid leukemic cell lines. The myeloperoxidase transcript was strongly expressed in promyelocytic HL 60 cells, whereas much lower levels were detected in immature monocytic U 937 cells. Phorbol-12-myristate-13-acetate induction resulted in inhibition of myeloperoxidase expression within 24 hrs. This regulatory event could not be blocked by cycloheximide. Furthermore, cycloheximide did not superinduce myeloperoxidase mRNA levels in KG 1, HL 60 and U 937 cells, arguing against the existence of a negative gene regulator for myeloperoxidase. Therefore, the myeloperoxidase gene can be classified as a primary response gene.