Expression Of Whey Acidic Protein Research Articles

Inguinal White Adipocytes Differentiate to Alveolar Epithelial Cells that Constitute Mammary Cells in Pregnant Rats

IntroductionInguinal adipose tissue has been shown to exhibit high plasticity that exceeds its capacity to differentiate to beige or brown adipocytes ( Giordano et al., 2014, Cinti, 2018). This has led to the concept of pinking whereby adipocytes differentiate to develop an alveolar structure that forms part of the mammary gland.MethodsVirgin and pregnant Sprague Dawley rats were used in this study. Food intake for virgin animals (aged 4 weeks) and body weight for virgin and pregnant animals were measured three times a week. Virgin rats were tissue sampled at 10 weeks of age while pregnant rats, mated at 10 weeks of age, were tissue sampled at either 50% and 90% of pregnancy (10 and 19 days of pregnancy, respectively). Gene expression was studied in the interscapular BAT, omental and inguinal adipose tissue using qPCR. Omental and inguinal sections were stained with H&E for examination of histological appearance.ResultsThe histological sections showed differentiation of inguinal adipocytes to show an alveoli appearance composed of epithelial cells in pregnant but not virgin rats. Gene expression analysis revealed a higher expression of whey acidic protein (WAP) and E74‐Like ETS Transcription Factor 5 (ELF5) genes during pregnancy in inguinal adipocytes of pregnant rats compared to virgin rats. These two genes were absent in the interscapular and omental fat depots of virgin rats. Continuous loss of PPARG with pregnancy appeared in the inguinal adipose tissue. At the gross level, the inguinal fat appeared more compact in pregnant compared to virgin rats.ConclusionsInguinal adipocytes appear to exhibit a pronounced ability to form alveoli which comprises part of the mammary cell structure through gestation. Previous studies have confirmed this from measures of histology and molecular analysis. Further studies designed to examine the mechanism will help to determine factors that may accelerate the formation of neoplastic cells in the mammary glands and possibly breast cancer. Inguinal adipose tissue may no longer be considered a true fat depot in pregnancy.Support or Funding InformationThis work was funded by the EU‐CASCADE fellowship scheme funded by the EU’s 7th FP PCOFUND‐GA‐2012‐600181 (ND), LA was the recipient of a doctoral scholarship from Mutah University.

Isoflavones and their metabolites influence the milk component synthesis ability of mammary epithelial cells through prolactin/STAT5 signaling.

Isoflavones are a class of polyphonic compounds present in legumes and are called phytoestrogens because of their estrogen-like activity. Estrogen influences the behavior of mammary epithelial cells (MECs) during pregnancy and lactation. In this study, we investigated the direct influences of isoflavones and their metabolites in milk production ability of MECs. Mouse MECs were cultured with prolactin and dexamethasone (glucocorticoid analog) to induce milk production ability. Subsequently, lactating MECs were treated with each isoflavone. Coumestrol, biochanin A, genistein, and formononetin decreased the intracellular and secreted β-casein. On the other hand, p-ethylphenol, daidzein, and equol did not significantly influence β-casein production at any concentration. Coumestrol, biochanin A and genistein down-regulated the mRNA expression of whey acidic protein (WAP), lactoferrin and α-lactalbumin. In contrast, p-ethylphenol, daidzein and equol up-regulated β-casein and/or WAP with α-lactalbumin. Furthermore, coumestrol and genistein down-regulated the expression of prolactin receptor and signal transducer and activator of transcription 5 (STAT5) accompanied by a decrease in STAT5 phosphorylation. Isoflavones and their metabolites influence the milk production ability of MECs through different interactions with prolactin/STAT5 signaling. Simultaneous intake of multiple isoflavones by consumption of legumes may induce promotive or adverse effects on lactating MECs.

HcToll3 was involved in anti-Vibrio defense in freshwater pearl mussel, Hyriopsis cumingii

Toll-like receptors (TLRs) play an important role in the activation of innate immune response but their functions in bivalves remain largely unknown. In this study, we identified a TLR from the freshwater pearl mussel Hyriopsis cumingii (HcToll3) and investigated its functions in immunity. The full-length cDNA of HcToll3 is 3852 bp and includes an open reading frame (ORF) of 3228 bp that encodes a polypeptide of 1075 amino acids. The predicted HcToll3 protein shares similar structural characteristics with other known Toll family proteins. Quantitative real-time PCR analysis revealed that HcToll3 mRNA is broadly expressed in all of the examined tissues; its transcript level was significantly up-regulated by challenge with gram-negative bacteria Vibrio parahaemolyticus or lipopolysaccharide, but not gram-positive Staphylococcus aureus or peptidoglycan. RNA interference by siRNA results showed that HcToll3 regulated expression of whey acidic protein (HcWAP) and lysozymes (HcLyso1 and HcLyso2) in vivo and knockdown of HcToll3 suppressed the elimination of V. parahaemolyticus. These findings suggest that HcToll3 might be involved in anti-Vibrio defense in H. cumingii.

Mammary Epithelial-specific Deletion of the Focal Adhesion Kinase Gene Leads to Severe Lobulo-Alveolar Hypoplasia and Secretory Immaturity of the Murine Mammary Gland

Integrin-mediated cell adhesion and signaling is required for mammary gland development and functions. As a major mediator of integrin signaling, focal adhesion kinase (FAK) has been implicated to play a role in the survival, proliferation, and differentiation of mammary epithelial cells in previously studies in vitro. To assess the role of FAK in vivo, we created mice in which FAK is selectively deleted in mammary epithelial cells. The mammary gland FAK conditional knock-out (MFCKO) mice are viable, fertile, and macroscopically indistinguishable from the control littermates. In virgin MFCKO mice, mammary ductal elongation is retarded at 5 weeks of age but reaches the full extent by 8 weeks of age compared with the control mice. However, the MFCKO females are unable to nurse their pups due to severe lobulo-alveolar hypoplasia and secretory immaturity during pregnancy and lactation. Analysis of the mammary epithelial cells in MFCKO mice showed reduced Erk phosphorylation, expression of cyclin D1, and a corresponding decrease in proliferative capability compared with the littermate controls. In addition, phosphorylation of STAT5 and expression of whey acidic protein are significantly reduced in the mammary glands of MFCKO mice, suggesting defective secretory maturation in these mice. Therefore, the combination of the severe lobulo-alveolar hypoplasia and defective secretory differentiation is responsible for the inability of the MFCKO females to nurse their pups. Together, these results provide strong support for a role of FAK in the mammary gland development and function in vivo.

Reduction of tumorigenesis and invasion of human breast cancer cells by whey acidic protein (WAP)

Whey acidic protein (WAP) is a major component of whey, which has two or three WAP motif domains characterized by a four-disulfide core (4-DSC) structure similar to the serine protease inhibitor. We have previously found that WAP inhibits the proliferation of mammary epithelial cells in vitro and in vivo [N. Nukumi, K. Ikeda, M. Osawa, T. Iwamori, K. Naito, H. Tojo, Regulatory function of whey acidic protein in the proliferation of mouse mammary epithelial cells in vivo and in vitro, Dev. Biol. 274 (2004) 31–44]. We report herein that WAP also reduces the progression of human breast cancer cells (MCF-7 and MDA-MB-453 cells). We have demonstrated that the forced expression of WAP in MCF-7 cells reduces the proliferation in either the presence or absence of estrogen. The tumor progression of WAP-expressing MCF-7 cells in nude mice is significantly suppressed more than that of mock-MCF-7 cells following the reduced expression of angiopoietin-2 gene. We have confirmed that the invasive activity of breast cancer cells is reduced to ∼30% of that of mock cells by the forced expression of exogenous WAP through its inhibition of degradation of laminin. These data suggest that WAP has a protease-inhibitory function on the progression of breast cancer cells. It is therefore possible to utilize WAP as therapeutic protein against tumorigenesis of breast cancer.

Nuclear Localization of STAT5A Modified with O-Linked N-Acetylglucosamine and Early Involution in the Mammary Gland of Hirosaki Hairless Rat

Hirosaki hairless rat (HHR) is a mutant strain spontaneously derived from Sprague-Dawley rats (SDR), and its inheritance is autosomal recessive. In addition to hair loss, female HHRs show involution of the mammary gland at an early stage of lactation. In the present study we investigated the mammary gland development in HHR. Morphological examinations revealed that HHR mammary glands are underdeveloped in virgins and exhibit distended alveoli on day 1 of lactation (L1), followed by involution. Milk secretion was observed on L1 in HHR. Whey acidic protein and other proteins were increased in milk of HHR and heterozygous rats on SDS-polyacrylamide gel electrophoresis. Terminal deoxynucleotide transferase-mediated dUTP nick-end labeling assay revealed apoptosis induction in HHRs at an early stage of lactation. By Western blotting, signal transducer and activator of transcription (STAT) 5A levels in cytoplasmic and nuclear fractions of the mammary glands were not different between HHR and SDR on L1 and L7. Nuclear localization of STAT5A in HHR and SDR was confirmed by immunohistochemistry. Tyr-phosphorylated STAT5A was not detected in HHR but was detected in SDR nuclear fractions. Several proteins modified with O-linked N-acetylglucosamine (O-GlcNAc) were detected in HHR nuclear extract on L1, although not in SDR or heterozygous rats by Western blotting. When HHR nuclear extract was applied to wheat germ agglutinin-agarose, a part of STAT5A was recovered in bound fractions. STAT5A of SDR or heterozygous rat nuclei were not bound to the lectin. Electrophoretic mobility shift assay revealed that STAT5A modified with O-GlcNAc is bound to the STAT5-responsive element. These results indicate that the mammary glands of HHR showed terminal differentiation for a short period, followed immediately by involution. In HHR, STAT5A is modified with O-GlcNAc but is not Tyr-phosphorylated. This type of glycosylation is suggested to be involved in the transient activation of STAT5A in HHR.

Whey Acidic Protein (WAP) Depresses the Proliferation of Mouse (MMT) and Human (MCF-7) Mammary Tumor Cells

We previously reported that the enforced expression of exogenous whey acidic protein (WAP) significantly inhibited the proliferation of mouse mammary epithelial cells (HC11 and EpH4/H6 cells). This paper presents the first evidence that WAP also depresses the proliferation of mammary tumor cells from mouse (MMT cells) and human (MCF-7 cells). We established WAP-clonal MMT and MCF-7 cell lines, and confirmed the secretion of WAP from the WAP-clonal cells into culture medium. The enforced expression of WAP significantly inhibited the proliferation of MMT and MCF-7 cells in in vitro culture. FACScan analyses revealed that G0/G1 phase cell-cycle progression was disordered and elongated in the WAP-clonal MMT and MCF-7 cells compared to that of the control cells. The expression of cyclin D1 was significantly decreased in the WAP-clonal MMT and MCF-7 cells, suggesting that progression from the G1 to the S phase was delayed in the WAP-clonal cells. The present results indicate that WAP plays a negative regulatory role in the cell-cycle progression of mammary tumor cells via a paracrine mechanism.

Aberrant Development of Mammary Glands, but Precocious Expression of .BETA.-casein in Transgenic Females Ubiquitously Expressing Whey Acidic Protein Transgene

It has been suggested that whey acidic protein (WAP) may function as a protease inhibitor. However, the actual function of WAP remains obscure. We investigated the histological development of the mammary glands of transgenic mice ubiquitously expressing WAP and CAG/WAP transgene. Ubiquitous expression of WAP induced aberrant development of the lobular alveoli of the mammary glands: mammary alveoli that were either aberrantly large or small in size increased in number in the developing mammary glands of these transgenic females during pregnancy and lactation. The expression of beta-casein was precociously induced in the mammary glands of the transgenic females during early pregnancy and accompanying this was a histological observation that abnormally developed lobular alveoli filled with milk proteins appeared in the mammary glands of transgenic females during early pregnancy. However, during lactation, the development of mammary glands was impaired in transgenic females. To investigate the possible paracrine action of WAP associated with mammary gland aberration, we transplanted the mammary tissue of CAG/EGFP transgenic females into the fat pad of virgin CAG/WAP transgenic females and initiated pregnancy by mating. The development of mammary tissue transplanted to the recipient was histologically examined on day 3 of lactation. The results revealed that the development of grafted mammary tissues was impaired in a manner similar to that of the mammary glands of CAG/WAP transgenic females, indicating that the inhibitory effect of WAP acts via a paracrine mechanism. In vitro experiments using HC11 cells with forced expression of exogenous WAP demonstrated the inhibitory function of WAP on proliferation of mammary epithelial cells.

Regulatory function of whey acidic protein in the proliferation of mouse mammary epithelial cells in vivo and in vitro

Although possible biological functions of whey acidic protein (WAP) have been suggested, few studies have focused on investigating the function of WAP. This paper describes evidence for WAP function in lobulo-alveolar development in mammary glands in vivo and in the cell cycle progression of mammary epithelial cells in vitro. Ubiquitous overexpression of WAP transgene impaired only lobulo-alveolar development in the mammary glands of transgenic female mice but not other physiological functions, indicating that the inhibitory function of WAP is specific to mammary alveolar cells. The forced expression of WAP significantly inhibited the proliferation of mouse mammary epithelial cells (HC11 cells and EpH4/K6 cells), whereas it did not affect that of NIH3T3 cells. Co-culturing of WAP-clonal cells and control cells using a transwell insert demonstrated that WAP inhibited the proliferation of HC11 cells through a paracrine action but not that of NIH3T3 cells, and that WAP was able to bind to HC11 cells but not to NIH3T3 cells. Apoptosis was not enhanced in the HC11 cells with stable WAP expression (WAP-clonal HC11 cells). BrdU incorporation and FACScan analyses revealed that cell cycle progression from the G0/G1 to the S phase was inhibited in the WAP-clonal HC11 cells. Among G1 cyclins, the expression of cyclin D1 and D3 was significantly decreased in the WAP-clonal HC11 cells. The present results provide the first documented evidence that WAP plays a negative regulatory role in the cell cycle progression of mammary epithelial cells through an autocrine or paracrine mechanism in vivo.

A Novel Effect of Dioxin: Exposure during Pregnancy Severely Impairs Mammary Gland Differentiation

Many ligands for the aryl hydrocarbon receptor (AhR) are considered endocrine disruptors and carcinogens, and assessment of adverse health effects in humans exposed to such chemicals has often focused on malignancies, including breast cancer. Mammary tissue contains the AhR, and inappropriate activation of the AhR during fetal development causes defects in mammary development that persist into adulthood. However, it is not known whether the extensive differentiation of mammary tissue that occurs during pregnancy is also sensitive to disruption by AhR activation. To examine this, we exposed pregnant C57Bl/6 mice to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on days 0, 7, and 14 of pregnancy. Examination of mammary glands on days 9, 12, and 17 of pregnancy and on the day of parturition showed severe defects in development, including stunted growth, decreased branching, and poor formation of lobular alveolar structures. This impaired differentiation was biologically significant, as expression of whey acidic protein in the gland was suppressed, and all pups born to TCDD-treated dams died within 24 h of birth. Analysis of circulating progesterone, prolactin, and estradiol suggest that hormone production was slightly impaired by inappropriate activation of the AhR. However, hormone levels were affected only very late in pregnancy. Given that the observed defects in gland development preceded these hormonal effects, altered hormone levels are an unlikely mechanistic explanation for impaired mammary development. This novel finding that AhR activation during pregnancy disrupts mammary gland differentiation raises questions about the susceptibility of mammary tissue to direct injury by endocrine disrupting agents and the potential for AhR-mediated signaling to adversely affect lactation and breast tissue development in human populations.

Inhibitory function of whey acidic protein in the cell-cycle progression of mouse mammary epithelial cells (EpH4/K6 cells).

Although the biological role for whey acidic protein (WAP) in milk has been suggested, its true function is not known. This paper describes evidence for WAP function in the cell-cycle progression of EpH4/K6 (EpH4), mammary epithelial cells in vitro. The forced expression of exogenous WAP significantly impaired the proliferation of EpH4 cells, whereas it did not affect that of NIH3T3 cells. Apoptosis was not enhanced in the EpH4 cells with stable expression of WAP (WAP-clonal EpH4 cells). The analyses of BrdU incorporation revealed that forced WAP expression significantly reduced incorporation of BrdU in WAP-clonal EpH4 cells compared with control cells transfected with empty plasmid. Among G1 cyclins, the level expression of cyclins D1 was significantly lower in the WAP-clonal EpH4 cells than in control cells. The inhibitory action of WAP on the proliferation of EpH4 cells was enhanced by the presence of extracellular matrix (ECM), but not by the presence of a single component comprising ECM. The cultured medium of WAP-clonal EpH4 cells inhibited the proliferation of control cells without WAP expression. The present results indicate that WAP plays a negative regulatory role in the cell-cycle progression of mammary epithelial cells through an autocrine/paracrine mechanism.

Essential functions of p21-activated kinase 1 in morphogenesis and differentiation of mammary glands.

Although growth factors have been shown to influence mammary gland development, the nature of downstream effectors remains elusive. In this study, we show that the expression of p21-activated kinase (Pak)1, a serine/threonine protein kinase, is activated in mammary glands during pregnancy and lactation. By targeting an ectopic expression of a kinase-dead Pak1 mutant under the control of ovine β-lactoglobulin promoter, we found that the mammary glands of female mice expressing kinase-dead Pak1 transgene revealed incomplete lobuloalveolar development and impaired functional differentiation. The expression of whey acidic protein and β-casein and the amount of activated Stat5 in the nuclei of epithelial cells in transgenic mice were drastically reduced. Further analysis of the underlying mechanisms revealed that Pak1 stimulated β-casein promoter activity in normal mouse mammary epithelial cells and also cooperated with Stat5a. Pak1 directly interacted with and phosphorylated Stat5a at Ser 779, and both COOH-terminal deletion containing Ser 779 of Stat5a and the Ser 779 to Ala mutation completely prevented the ability of Pak1 to stimulate β-casein promoter. Mammary glands expressing inactive Pak1 exhibited a reduction of Stat5a Ser 779 phosphorylation. These findings suggest that Pak1 is required for alveolar morphogenesis and lactation function, and thus, identify novel functions of Pak1 in the mammary gland development.

Transgenic mice expressing recombinant human protein C exhibit defects in lactation and impaired mammary gland development.

To determine if the production of recombinant human protein C (rHPC) could be increased in milk, we created two lines of mice homozygous for the mouse whey acidic protein (WAP)/human protein C (HPC) transgene. Females of both lines had normal growth, activity and fertility, but failed to lactate normally and were unable to raise litters. Histological analyses of mammary glands from lactating homozygous females showed barely distended alveoli filled with dense-staining milk. Epithelial cells within these alveoli had distinct, centrally located nuclei and contained intracellular lipid droplets. Hemizygous animals derived from these lines were able to lactate and raised normal sized litters. Northern blot analysis showed that the 6.4 homozygous (6.4H) line expressed the transgene at higher levels then corresponding hemizygous (6.4) animals, but the 4.2 homozygous (4.2H) line expressed the transgene at lower levels than the 4.2 hemizygous line. The 6.4H line also had increased rHPC levels in the milk as revealed by western blot analysis. The 4.2H, 6.4, and 6.4H lines showed decreased and/or delayed expression of WAP, beta-casein, and alpha-lactalbumin mRNA's compared to wild type animals during lactogenesis. The 4.2 line showed decreased mRNA expression for beta-casein and alpha-lactalbumin, but normal or higher expression of WAP during lactogenesis. Elevated levels of some proteins were detected in the milk of transgenic mice. From these results, it is concluded that expression of rHPC induced a lactational phenotype that involves abnormal morphological, biochemical, and functional differentiation of mammary epithelial cells. However, the induction of this phenotype does not appear to be directly related to the level of rHPC mRNA expression, thus suggesting that the basis of this phenotype may involve secondary, rather than primary, effects of rHPC on mammary gland development.

Mammary gland specific hEGF receptor transgene expression induces neoplasia and inhibits differentiation.

The epidermal growth factor receptor (EGFR) is overexpressed in about 48% of human breast cancer tissues. To analyse the role of the EGFR in mammary tumor development we generated transgenic mice expressing the human EGFR under the control of either the MMTV-LTR (MHERc) or the beta-lactoglobulin promoter (BLGHERc). The BLGHERc-transgene was expressed exclusively in the female mammary gland, whereas the MHERc transgene was expressed more promiscuously in other organs, such as ovary, salivary gland and testis. Female virgin and lactating transgenic mice of both strains have impaired mammary gland development. Virgin EGFR transgenic mice developed mammary epithelial hyperplasias, whereas in lactating animals progression to dysplasias and tubular adenocarcinomas was observed. In both strains the number of dysplasias increased after multiple pregnancies. The transgene expression pattern was heterogeneous, but generally restricted to regions of impaired mammary gland development. Highest EGFR transgene expression was observed in adenocarcinomas. By using a whole mount organ culture system to study the differentiation potential of the mammary epithelium, we observed a reduced number of fully developed alveoli and a decrease in whey acidic protein expression. Taken together, EGFR overexpression results in a dramatic effect of impaired mammary gland development in vitro as well as in vivo, reducing the differentiation potential of the mammary epithelium and inducing epithelial cell transformation.

Cripto-1 induces apoptosis in HC-11 mouse mammary epithelial cells.

Cripto-1 (CR-1) is an epidermal growth factor (EGF)-related protein. CR-1 can inhibit beta-casein and whey acidic protein expression in mouse mammary epithelial cells. The present study demonstrates that CR-1 can induce apoptosis in HC-11 mouse mammary epithelial cells, as measured by bis-benzimide stained nuclei, TUNEL assay and cell death ELISA. Apoptosis could be observed after 2 days of exposure of confluent HC-11 cells to CR-1 in the absence of the survival factors EGF and insulin, with maximum apoptosis occurring at 3 days. A reduction in poly(ADP-ribose) polymerase (PARP) expression and an increase in beta-catenin cleavage was found after 18 h of exposure to CR-1 suggesting that apoptosis was preceded by the induction of a caspase activity since the caspase inhibitor ZFAD.FMK could block the CR-1-induced reduction in PARP expression and CR-1-induced apoptosis. CR-1 was found to increase the expression of caspase-3-like protease. Although, the levels of p27kip1 and p21Bax did not change after exposure to CR-1 for 18 h, the levels of Bcl-xL became undetectable. These studies suggest that CR-1 promotes apoptosis by mediating the induction of caspase-3-like protease and downregulating the expression of Bcl-xL.

Transgenic Mice Overexpressing Foreign Whey Acidic Protein (WAP) Genes Driven by Metallothionein Promoter.

Our previous studies demonstrated that the gene coding for whey acidic protein (WAP) is expressed in a variety of tissues other than mammary glands in lactating mice (Wen et al., 1995). It was suspected, therefore, that WAP plays important physiological roles in tissues of rodents. To clarify biological functions of mouse WAP (mWAP), we produced transgenic mice introducing a 6.4 kb metallothionein-I (mMT-I)/mWAP construct where 1.8 kb mouse MT-I promoter was linked to 4.6 kb WAP structural gene. Three male mice were confirmed to be transgenic by Southern blot and PCR analyses, and served as the founders transmitting the genes to their offspring. The founders, their offspring and normal mice (control) were given drinking-water containing 25 mM ZnSO4 for 4 weeks. The expression levels of WAP genes induced by Zn2+ were about 3 times higher in the liver of transgenic mice than those of normal mice. Unexpectedly, it turned out that the expression of the endogenous WAP was also enhanced by the prolonged exposure to Zn2+. Despite the enhanced expression of WAP, no alteration in phenotypes, e.g., morphological characteristics, growth rates, reproductive performance and/or behavior have so far been detected in all the transgenic mice examined.

Synthesis and secretion of the mouse whey acidic protein in transgenic sheep

The synthesis of foreign proteins can be targeted to the mammary gland of transgenic animals, thus permitting commercial purification of otherwise unavailable proteins from milk. Genetic regulatory elements from the mouse whey acidic protein (WAP) gene have been used successfully to direct expression of transgenes to the mammary gland of mice, goats and pigs. To extend the practical usefulness of WAP promoter-driven fusion genes and further characterize WAP expression in heterologous species, we introduced a 6.8 kb DNA fragment containing the genomic form of the mouse WAP gene into sheep zygotes. Two lines of transgenic sheep were produced. The transgene was expressed in mammary tissue of both lines and intact WAP was secreted into milk at concentrations estimated to range from 100 to 500 mg/litre. Ectopic WAP gene expression was found in salivary gland, spleen, liver, lung, heart muscle, kidney and bone marrow of one founder ewe. WAP RNA was not detected in skeletal muscle and intestine. These data suggest that unlike pigs, sheep may possess nuclear factors in a variety of tissues that interact with WAP regulatory sequences. Though the data presented are based on only two lines, these findings suggest WAP regulatory sequences may not be suitable as control elements for transgenes in sheep bioreactors.

Characterization of a Protein That Binds a Negative Regulatory Element in the Mammary-Specific Whey Acidic Protein Promoter

Whey Acidic Protein (WAP) gene expression is restricted to the pregnant and lactating mammary gland. We have recently defined a negative regulatory element (NRE) in the WAP promoter which interacts with a factor (NBF) present in all nonWAP expressing cells (Kolb et al., 1994; J. Cell. Biochem. 56:245-261). Here we characterise this factor and show that although it is not related to a number of known transcription factors, including AP-1, NF-1 and SP-1, it may also be involved in controlling the expression from the mouse mammary tumour vints promoter. Three proteins that bind to the WAP-NRE have been identified, one of which is a 53 kDa nuclear protein. This protein is present in nonWAP expressing cells, suggesting that it is responsible for limiting WAP expression to the pregnant and lactating mammary gland. This protein has been partially purified and its binding to the WAP-NRE is not appreciably affected by high salt concentrations.

Expression of whey acidic protein (WAP) genes in tissues other than the mammary gland in normal and transgenic mice expressing mWAP/hGH fusion gene.

Whey acidic protein (WAP) is a major whey protein secreted in rodents' milk. Murine WAP (mWAP) genes have been assumed to be expressed solely in the mammary gland. However, several heterologous genes fused with the mWAP promoter and artificially introduced into animal genomes as transgene were expressed not only in the mammary gland but also in other tissues as well. In the present study, we investigated, by means of the reverse transcription polymerase chain reaction (RT-PCR), the patterns of expression of endogenous WAP genes in tissues of normal mice and in transgenic mice carrying hGH gene coupled to the mWAP promoter sequence. The results revealed that the genes driven by the mWAP promoter, regardless of whether they are endogenous genes or transgenes, were transcribed in a variety of tissues other than the mammary gland of lactating normal female mice, although the expression levels are generally low. The expression of WAP genes in the cerebrum and the liver is regulated, as in the mammary gland, according to the reproductive stages. However, the tissue distribution of endogenous WAP gene expression in mature virgin transgenic female mice was the same as that in lactating normal female mice.

Production of human surfactant protein C in milk of transgenic mice.

Respiratory distress syndrome (RDS), caused by lack of pulmonary surfactant, affects 65 000 infants annually in the USA. Surfactant replacement therapy reduces the morbidity and mortality associated with RDS. Human surfactant protein C (SP-C) is an important component of pulmonary surfactant. To produce human SP-C, a construct using the rat whey acidic protein (WAP) promoter and 3' untranslated regions to target expression of the human SP-C gene to the mammary gland of transgenic mice was created. WAP/SP-C mRNA expression was detected in all transgenic lines analysed. SP-C was expressed in a copy-number-dependent and integration-site-independent fashion, with levels of expression ranging from 0.01% to 36.0% of the endogenous mouse WAP mRNA, and WAP/SP-C mRNA expression levels were greater than those of of the endogenous mouse lung SP-C mRNA. Expression at the RNA level was specific to the mammary gland and paralleled the endogenous WAP expression pattern during mammary gland development. Expression and secretion of the SP-C protein in the lactating mammary gland was demonstrated by western blots performed on whole milk using an anti-SP-C polyclonal antibody. Immunoreactive proteins of MW 22 and 12-14 kDa appeared only in transgenic milk. The 22 kDa protein represents the proprotein, and the 12-14 kDa is a processed form of SP-C.