A mammary-derived growth inhibitor (MDGI) related 70 kDa antigen identified in nuclei of mammary epithelial cells

The aim of the present study was to investigate the expression of the mammary-derived growth inhibitor (MDGI) and the subcellular localization of MDGI-related antigens in bovine mammary glands. Cell-free translation of poly(A+) = RNA, immunoprecipitation with rabbit anti-MDGI-antibodies, and estimation of the relative contents of MDGI by a radioimmunoassay in mammary tissue of different functional states revealed that the 13 kDa MDGI was dramatically increased in terminally differentiated mammary tissue compared with the proliferating tissue from pregnant animals. To address the question of tissue localization, polyclonal anti-MDGI antibodies and antibodies directed against a synthetic peptide corresponding to residues 69 to 78 of MDGI were used. Western blotting of tissue fractions revealed the cytosolic and microsomal localization of MDGI. Additionally, both types of antibodies detected a 70-kDa antigen in the nuclear fraction of differentiated mammary glands. Salt extraction and DNase I digestion of isolated nuclei, as well as chromatin purification, indicated an association of the 70-kDa antigen with the chromatin. By means of the immunogold technique, MDGI-related antigens were localized within euchromatic nuclear regions of epithelial cells in the intact differentiated mammary gland. The immunostaining was markedly diminished in the proliferating tissue. This finding raises the possibility that MDGI and the 70-kDa antigen influence cell proliferation by acting on gene expression within the nuclei of mammary glands.     

Irregular distribution of mammary-derived growth inhibitor in the bovine mammary epithelium

Localization of a mammary-derived growth inhibitor (MDGI) in the bovine mammary gland was verified by light-and electron-microscopic methods. Expression of MDGI, which is known to inhibit the growth of mammary epithelial cell lines in vitro, was found to be highest in the late pregnant and in the lactating state. A combination of immunohistochemical and immunocytochemical methods with semi- and ultrathin resin sections revealed marked variations in MDGI staining. High MDGI levels were predominantly detectable in epithelial cells with large milk fat droplets. Distinct cell types that were almost free of label could be identified among bovine mammary epithelial cells that always exhibited high MDGI levels. Similar results were obtained when using a serum-free organ culture system in which MDGI was hormonally induced in cell types of comparable differentiation state. The specific occurrence of the growth inhibitor in developing alveoli and certain cell types points to the association between MDGI expression and functional differentiation in the normal mammary gland.     

Histochemical localization of heart-type fatty-acid binding protein in human and murine tissues

Cellular fatty acid-binding proteins (FABP) are a highly conserved family of proteins consisting of several subtypes, among them the mammary-derived growth inhibitor (MDGI) which is quite homologous to or even identical with the heart-type FABP (H-FABP). The FABPs and MDGI have been suggested to be involved in intracellular fatty acid metabolism and trafficking. Recently, evidence for growth and differentiation regulating properties of MDGI and H-FABP was provided. Using four affinity-purified polyclonal antibodies against bovine and human antigen preparations, the cellular localization of MDGI/H-FABP in human and mouse tissues and organs was studied. The antibodies were weakly cross-reactive with adipose tissue extracts known to lack H-FABP, but failed to react by Western blot analysis with liver-type FABP (L-FABP) and intestinal-type FABP (I-FABP). MDGI/H-FABP protein was mainly detected in myocardium, skeletal and smooth muscle fibres, lipid and/or steroid synthesising cells (adrenals, Leydig cells, sebaceous glands, lactating mammary gland) and terminally differentiated epithelia of the respiratory, intestinal and urogenital tracts. The results provide evidence that expression of H-FABP is associated with an irreversibly postmitotic and terminally differentiated status of cells. Since all the antisera employed showed spatially identical and qualitatively equal immunostaining, it is suggested that human, bovine and mouse MDGI/H-FABP proteins share highly homologous epitopes.     

Epithelial proliferation and differentiation in the mammary gland do not correlate with cFABP gene expression during early pregnancy

Cardiac fatty acid binding protein (cFABP) is abundantly expressed in the nondividing, functionally differentiated mammary ephithelium. It is very closely related, if not identical to, a previously described protein termed mammary derived growth inhibitor (MDGI). In vitro studies suggest that low concentrations of diffusible cFABP/MDGI may play a hormone-like role in limiting proliferative activity and promoting functional differentiation of this tissue, but no in vivo data to support this idea have been published. To test this hypothesis, we compared the levels of cFABP mRNA with both the epithelial DNA labelling index and levels of β-casein mRNA in wild-type mice. We also investigated the effect of a precocious experimental increase of cFABP levels in the mammary gland of transgenic mice on the labelling index and β-casein mRNA levels. This was accomplished by expressing a bovine cFABP cDNA under the control of the ovine β-lactoglobulin (BLG) gene promoter. We found that although both the DNA labelling index, β-casein mRNA levels, and cFABP mRNA levels in wild-type mice are developmentally regulated, they do not correlate with each other during early pregnancy in individual mice. Moreover, a three- to fourfold increase of total cFABP mRNA in two transgenic lines did not affect the DNA labelling index or the levels of β-casein mRNA, an established marker of differentiation of the mammary epithelium, at this developmental stage. These data suggest that epithelial DNA synthesis, β-casein gene expression, and expression of the cFABP gene are regulated independently in the proliferatively active mammary gland and that the rapidly dividing mammary epithelial cells are not susceptible to the action of cFABP during early pregnancy. © 1995 Wiley-Liss, Inc.     

Localization of mammary-derived growth inhibitor in capillary endothelial cells of the bovine mammary gland

Mammary-derived growth inhibitor (MDGI) has previously been localized in the mammary parencyma, dependent on the stage of differentiation of the mammary gland. Here, we have elucidated the distribution of MDGI in the mammary stroma by a combined immunohisto-and cytochemical analysis with antibodies raised against MDGI. Distinct staining of capillary endothelial cells has been revealed. Although its subcellular distribution resembles former observations in secretory epithelial cells, the expression of MDGI in capillary endothelial cells clearly precedes that in secretory epithelial cells. On the other hand, no endothelial MDGI staining has been detected in bovine heart, which contains a fatty acid-binding protein almost identical to MDGI. The localization of MDGI in the mammary capillary endothelium is discussed in terms of its possible involvement in the intracellular transport of hydrophobic ligands or in the regulation of endothelial cell proliferation.     

Isolation and Sequence Characterization of Mammary Derived Growth Inhibitor Gene of Riverine Buffalo (Bubalus Bubalis)

In this study, attempts have been made to identify and characterize water buffalo (Bubalus bubalis) mammary derived growth inhibitor (MDGI) gene, isolated from a mammary gland cDNA library of lactating buffalo. The complete MDGI cDNA was of 698 nucleotides, consisting 61 nucleotides in 5′ UTR, coding region of 402 nucleotides, and 235 nucleotides representing the 3′ UTR. Comparison of nucleotide and deduced amino acid sequence data with that of MDGI//fatty acid binding protein (FABP) of other species shows three buffalo specific nucleotide changes while seven nucleotide changes were common to cattle and buffalo. Buffalo and cattle MDGI had 100% amino acid sequence similarity, which also shared three amino acid changes: 34 (Ala-Gly), 109 (Leu-Met), and 132 (Glu-Gln) as compared to other species. Comparison with FABPs reported from other cattle tissues revealed highest amino acid sequence similarity with FABP-heart (100%) and least with FABP-liver (20.5%). Phylogenetic analysis revealed cattle MDGI to be closest to buffalo, while mouse MDGI was distantly placed, whereas different tissue derived FABPs of cattle showed FABP-heart closest and FABP-epidermis most distantly placed from buffalo MDGI. This report also differs from the earlier findings that MDGI is intermediate of FABP-heart and adipose.     

Isolation and sequence characterization of mammary derived growth inhibitor gene of riverine buffalo (Bubalus bubalis)

In this study, attempts have been made to identify and characterize water buffalo (Bubalus bubalis) mammary derived growth inhibitor (MDGI) gene, isolated from a mammary gland cDNA library of lactating buffalo. The complete MDGI cDNA was of 698 nucleotides, consisting 61 nucleotides in 5' UTR, coding region of 402 nucleotides, and 235 nucleotides representing the 3' UTR. Comparison of nucleotide and deduced amino acid sequence data with that of MDGI/fatty acid binding protein (FABP) of other species shows three buffalo specific nucleotide changes while seven nucleotide changes were common to cattle and buffalo. Buffalo and cattle MDGI had 100% amino acid sequence similarity, which also shared three amino acid changes: 34 (Ala-Gly), 109 (Leu-Met), and 132 (Glu-Gln) as compared to other species. Comparison with FABPs reported from other cattle tissues revealed highest amino acid sequence similarity with FABP-heart (100%) and least with FABP-liver (20.5%). Phylogenetic analysis revealed cattle MDGI to be closest to buffalo, while mouse MDGI was distantly placed, whereas different tissue derived FABPs of cattle showed FABP-heart closest and FABP-epidermis most distantly placed from buffalo MDGI. This report also differs from the earlier findings that MDGI is intermediate of FABP-heart and adipose.     

Members of the fatty acid binding protein family are differentiation factors for the mammary gland

Mammary gland development is controlled by systemic hormones and by growth factors that might complement or mediate hormonal action. Peptides that locally signal growth cessation and stimulate differentiation of the developing epithelium have not been described. Here, we report that recombinant and wild-type forms of mammary-derived growth inhibitor (MDGI) and heart-fatty acid binding protein (FABP), which belong to the FABP family, specifically inhibit growth of normal mouse mammary epithelial cells (MEC), while growth of stromal cells is not suppressed. In mammary gland organ culture, inhibition of ductal growth is associated with the appearance of bulbous alveolar end buds and formation of fully developed lobuloalveolar structures. In parallel, MDGI stimulates its own expression and promotes milk protein synthesis. Selective inhibition of endogenous MDGI expression in MEC by antisense phosphorothioate oligonucleotides suppresses appearance of alveolar end buds and lowers the beta-casein level in organ cultures. Furthermore, MDGI suppresses the mitogenic effects of epidermal growth factor, and epidermal growth factor antagonizes the activities of MDGI. Finally, the regulatory properties of MDGI can be fully mimicked by an 11-amino acid sequence, represented in the COOH terminus of MDGI and a subfamily of structurally related FABPs. This peptide does not bind fatty acids. To our knowledge, this is the first report about a growth inhibitor promoting mammary gland differentiation.     

Nucleotide and primary amino acid sequence of porcine lactoferrin.

A cDNA encoding porcine lactoferrin (pLF) was isolated from a porcine mammary gland lambda gt11 cDNA library using human lactoferrin cDNA as the hybridization probe. Nucleotide sequence analysis indicates that pLF is 686 amino acids in length and shares 72.6%, 70.7% and 62.2% overall amino acid sequence identity with bovine, human and murine lactoferrin, respectively.     

Characteristics of fatty acid-binding proteins and their relation to mammary-derived growth inhibitor

Summary Based on sequence relationships the cytoplasmic fatty acid-binding proteins (FABPs) of mammalian origin are divided into at least three distinct types, namely the hepatic-, intestinal- and cardiac-type. Highly conserved sequences of FABPs within the same type correlate with immunological crossreactivities. Isoforms of hepatic-type FABP are found in several mammalian species and for bovine liver FABP specific shifts in isoelectric points upon lipidation with fatty acids are observed. Isoforms of intestinal-type FABP are not known and the occurrence of cardiac-type isoforms so far is confined to bovine heart tissue. A bovine mammary-derived growth inhibitor (MDGI) is 95% homologous to the cardiac-type FABP from bovine heart. Dissociation constants of FABP/fatty acid complexes are in the range of 1 M and 1:1 stoichiometries are usually found, but the neutral isoform of hepatic FABP from bovine liver binds 2 fatty acids. On subcellular levels hepatic- and cardiac-type FABPs are differently distributed. Though mainly cytosolic in either case, immunoelectron microscopy as well as a gelchromatographic immunofluorescence assay demonstrate the association of hepatic FABP in liver cells with microsomal and outer mitochondrial membranes and with nuclei, whereas in heart cells cardiac FABP is confined to mitochondria' matrix and nuclei. In mammary epithelial cells MDGI is associated with neither mitochondria nor endoplasmic reticulum, and is expressed in a strictly developmental-dependent spatial and temporal pattern. The specific role proposed for MDGI is to arrest growth of mammary epithelial cells when they become committed to differentiation in the mammary gland.     

Nuclear coactivator protein p100 is present in endoplasmic reticulum and lipid droplets of milk secreting cells

We have identified the p100 protein, previously known as a novel cellular coactivator, as a constituent of endoplasmic reticulum and cytosolic lipid droplets from milk secreting cells. Cytosolic lipid droplets of terminally differentiated mammary epithelial cells are secreted as milk lipid globules. However, milk lipid globules did not have detectable amounts of p100 protein. The p100 protein was found also in cytosol from lactating mammary gland, in storage lipid droplets from mouse adipocytes, and in endoplasmic reticulum from liver. Immunofluorescence microscopy of mammary epithelial cells confirmed the presence of p100 in non-nuclear regions of these cells. Partial sequence analysis of tryptic peptides from p100 from cow mammary gland showed extensive homology with the reported sequence of p100 determined from a human cDNA. Antibodies against a peptide synthesized to duplicate a sequence in human p100 recognized a protein of the size of p100 in cow, mouse and rat cell fractions.     

Bovine lactoferrin mRNA: sequence, analysis, and expression in the mammary gland.

The mRNA sequence for bovine lactoferrin expressed in the mammary gland was determined by sequencing three over lapping cDNA clones and by direct sequencing of the mRNA. The mRNA (2351 bases) codes for a 708 amino acid protein with a 19 amino acid signal peptide immediately preceding a sequence identical to the N-terminal 40 amino acids reported for bovine lactoferrin. A putative destabilizing sequence (AUUUA) was identified in the 3'-untranslated region. The nucleic acid sequence and deduced amino acid sequence are highly homologous with other transferrin family members. Lactoferrin mRNA concentrations in bovine mammary tissue were quite low two days before parturition and during lactation but were high three days after the cessation of milking, a sharp contrast from the pattern of regulation of the other milk proteins.     

Purification of a mammary-derived growth inhibitor (MDGI) related polypeptide expressed during pregnancy.

The present study was undertaken to screen immunochemically for MDGI-related proteins in the mammary gland. A new form, MDGI 2, not present in lactation could be detected in the bovine gland during pregnancy. It was further distinguished from MDGI by its lower molecular weight, its association with a complex binding to WGA, and by lacking immunoreactivity to an anti-MDGI antibody directed against the C-terminus of MDGI. MDGI 2 was purified by chromatography over DEAE-Sepharose, Bio-Gel P-30 in 1% acetic acid, Sephacryl S-200 in 6 M urea and Mono Q. Final purification included HPLC on TSK G-3000 SW and electroelution from SDS-gels. Cell-free translation of poly (A+)mRNA from glands of pregnant animal yielded one form identical with MDGI. We assume that posttranslational processing of MDGI is involved in its activities.     

A 13-kilodalton protein purified from milk fat globule membranes is closely related to a mammary-derived growth inhibitor

With the use of specific antibodies against a previously purified [Boehmer, F.-D., Lehmann, W., Schmidt, H., Lange, P., & Grosse, R. (1984) Exp. Cell Res. 150, 466-477] and sequenced mammary-derived growth inhibitor (MDGI) [Boehmer, F.-D., Kraft, R., Otto, A., Wernstedt, C., Hellmann, U., Kurtz, A., Mueller, T., Rohde, K., Etzold, G., Lehmann, W., Langen, P., Heldin, C.-H., & Grosse, R. (1987) J. Biol. Chem. 262, 15137-15143], the localization and relative amount of immunoreactive 13-kilodalton (kDa) antigen in different fractions of bovine milk were determined. The highest amount of antigen was found to be associated with the milk fat globule membranes (MFGM). As revealed by a dot immunobinding assay, the amount of immunoreactive bovine and human MFGM-associated antigen increased dramatically with the onset of lactation after delivery. This finding corresponds to earlier data obtained for MDGI and indicates a relationship between the proliferative state of mammary epithelial cells and the amount of immunoreactive antigen. The 13-kDa antigen has been purified from MFGM to homogeneity by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroelution. The MFGM-derived 13-kDa polypeptide was found to be almost identical with MDGI as demonstrated by tryptic digestion and partial amino acid sequence analysis of tryptic fragments of both proteins. The results clearly show the presence of a membrane-bound MDGI-related 13-kDa protein, thus supporting the possible involvement of membrane-associated growth inhibitors in growth regulation of mammary epithelial cells.     

Molecular cloning of a bovine immunoglobulin lambda chain cDNA

A cDNA library of the bovine mammary gland constructed in pBR322 was screened by mRNA hybrid-selected translation and by differential hybridization. Several immunoglobulin (Ig) lambda light-chain clones were identified and sequenced. Nucleotide sequence comparison of bovine and human Ig lambda chains showed a high degree of homology for constant regions and for J regions. The amino acid (aa) sequence encoded by the constant region of the bovine Ig lambda chain cDNA contains 107 aa with differences at 24 aa positions from the human Ig lambda chain. Three complementarity-determining regions (CDR1,2,3) characteristic of the variable region of bovine Ig lambda chain cDNA can be distinguished. The bovine and human sequences display good homology in the framework region 3 (FR3) but only patches of homology throughout the FR2 region. The 5' end of the bovine Ig lambda chain cDNA fragment of clone 1-14E contains five stop codons: two in CDR1, one in FR1 and two in the hydrophobic prepeptide region. These data suggest that the Ig lambda mRNA of clone 1-14E is transcribed from the V lambda pseudogene.     

Identification of bacterial clones encoding bovine caseins by direct immunological screening of the cDNA library

A sensitive immunoassay was used to identify recombinant plasmids carrying cDNA fragments of bovine caseins in the cDNA library from bovine mammary gland mRNA. Colonies grown on nitrocellulose filters were lysed in situ and proteins from the lysates were blotted onto CNBr-activated cellulose filter paper. Antigens covalently bound to CNBr-activated paper or bound to nitrocellulose filters were detected by reaction with antiserum to caseins, followed by ¹²⁵I-labelled Staphylococcus aureus protein A and autoradiography. Six clones were found positive among 5400 of the cDNA library: 3-A1, 3-B2, 3-B5, 3-H7, 2-A5 and 2-C9. The molecular weights of chimeric pre-β-lactamase: casein proteins synthesized in Escherichia coli were estimated by immunoblotting. Colony hybridization and nucleotide sequence analysis showed that clone 3-B5 contained a cDNA fragment of bovine χ-casein, clone 3-H7 contained a cDNA fragment of β-casein, while clones 2-A5 and 2-C9 carried cDNA fragments of α_si-casein.