Characterization of the tammar wallaby (Macropus eugenii) whey acidic protein gene: new insights into the function of the protein.

Whey acidic protein (WAP) belongs to a family of four disulfide core (4-DSC) proteins rich in cysteine residues and is the principal whey protein found in milk of a number of mammalian species. Eutherian WAPs have two 4-DSC domains, whereas marsupial WAPs are characterized by the presence of an additional domain at the amino terminus. Structural and expression differences between marsupial and eutherian WAPs have presented challenges to identifying physiological functions of the WAP protein. We have characterized the genomic structure of tammar WAP (tWAP) gene, identified its chromosomal localization and investigated the potential function of tWAP. We have demonstrated that tWAP and domain III (DIII) of the protein alone stimulate proliferation of a mouse mammary epithelial cell line (HC11) and primary cultures of tammar mammary epithelial cells (Wall-MEC), whereas deletion of DIII from tWAP abolishes this proliferative effect. However, tWAP does not induce proliferation of human embryonic kidney (HEK293) cells. DNA synthesis and expression of cyclin D1 and cyclin-dependent kinase-4 genes were significantly up-regulated when Wall-MEC and HC11 cells were grown in the presence of either tWAP or DIII. These data suggest that DIII is the functional domain of the tWAP protein and that evolutionary pressure has led to the loss of this domain in eutherians, most likely as a consequence of adopting a reproductive strategy that relies on greater investment in development of the newborn during pregnancy.

Molecular analysis of tammar (Macropus eugenii) mammary epithelial cells stimulated with lipopolysaccharide and lipoteichoic acid.

The immunological function of the metatherian mammary gland plays a crucial part in neonatal survival of the marsupial young. Marsupial pouch young do not develop adult like immune responses until just prior to leaving the pouch. The immune components of the maternal milk secretions are important during this vulnerable early post-partum period. In addition, infection of the mammary gland has not been recognized in metatherians, despite the ready availability of pathogens in the pouch. Regardless of which, little is known about the immunobiology of the mammary gland and the immune responses of mammary epithelial cells in metatherians. In this study, a molecular approach was utilized to examine the response of tammar (Macropus eugenii) mammary epithelial cells to Escherichia coli derived lipopolysaccharide (LPS) and Staphylococcus aureus derived lipoteichoic acid (LTA). Using custom-made cDNA microarrays, candidate genes were identified in the transciptome, which were involved in antigen presentation, inflammation, cell growth and proliferation, cellular damage and apoptosis. Quantification of mRNA expression of several of these candidate genes, along with seven other genes (TLR4, CD14, TNF-alpha, cathelicidin, PRDX1, IL-5 and ABCG2) associated with innate immunity in LPS and LTA challenged mammary epithelial cells and leukocytes, was assessed for up to 24 h. Differences in genes associated with cellular damage and pro-inflammatory cytokine production were seen between stimulated mammary epithelial cells and leukocytes. LTA challenge tended to result in lower level induction of pro-inflammatory cytokines, increased PRDX1 mRNA levels, suggesting increased oxidative stress, and increased CD14 expression, but in a non-TLR4-dependent manner. The use of functional genomic tools in the tammar identified differences in the response of tammar mammary epithelial cells (MEC) and leukocytes to challenge with LPS and LTA, and validates the utility of the approach. The results of this study are consistent with a model in which tammar mammary epithelial cells have the capacity to elicit a complex and robust immune response to pathogens.

The endocrine regulation of milk lipid synthesis and secretion in tammar wallaby (Macropus eugenii).

Lipids in tammar milk are predominantly triacylglycerols, and the fatty acid composition varies during the lactation cycle. Little is known about the regulation of their synthesis. This study investigates the endocrine regulation of lipid synthesis in mammary explants from pregnant tammars. Treatment of mammary explants with insulin resulted in a high level of lipid synthesis, but the lipids accumulated in the cytosol. Culture with prolactin resulted in a small increase in lipid synthesis, but electron microscopy showed lipid globules were synthesized in the mammary epithelial cells and secreted into the lumen. Culture with both insulin and prolactin demonstrated elevated levels of synthesis and secretion of lipid. Analysis of the type of fatty acids synthesized in these mammary explants showed that the initiation of synthesis of C(16:0), which also occurs in the first week of lactation, could be reproduced in the pregnant explants cultured with prolactin alone. However, treatment of mammary explants with hydrocortisone did not show a significant effect on lipid synthesis, secretion or the fatty acid synthesized. These results provide new information identifying the role of insulin and prolactin in regulating milk lipid synthesis and secretion in the tammar.

Changes in milk protein composition during acute involution at different phases of tammar wallaby (Macropus eugenii) lactation.

This study exploited the unusual lactation cycle of the tammar wallaby (Macropus eugenii) to characterise milk composition during acute involution, a time when the mammary gland is subjected to increased risk of infection. In early-lactation, tammar milk contains elevated levels of complex oligosaccharides and low protein and lipid content. Later in lactation, protein and lipid concentrations increase significantly, whereas carbohydrate content is reduced dramatically and changes to monosaccharides. Following initiation of involution at early-lactation, the carbohydrate concentration greatly decreased, while lipid and protein concentrations were elevated, suggesting that complex oligosaccharides are the major osmole in milk at this time. In contrast, involution at late lactation, when carbohydrate concentration was very low, led to an increase in the lipid concentration, but the concentration of protein was not significantly altered. This indicates that protein synthesis during acute involution at late lactation in the tammar may be down-regulated much more rapidly than during early-lactation. Analysis of milk at day 3 after the onset of involution at early-lactation identified a number of potential antimicrobials secreted at high concentrations, including lysozyme, dermcidin, polymeric immunoglobulin receptor and fragments of beta-lactoglobulin. These proteins may protect the mammary gland by minimising the risk of potential infection during involution.

Identification, characterization and expression of cathelicidin in the pouch young of tammar wallaby (Macropus eugenii).

Antimicrobial peptides, such as cathelicidin, are an evolutionarily old defense system. However they have more complex actions than just simply their antimicrobial effects, including immunoregulation and interaction with the adaptive immune system. In this study we have characterized several novel cathelicidin-like peptides from the tammar wallaby (Macropus eugenii). The tammar cathelicidin-like (MaeuCath) mRNA were isolated based on the conservation of the cathelin-like amino terminus. Mature MaeuCath peptides were positively charged with hydrophobic carboxyl tails, features that are fundamental for antimicrobial function. MaeuCath1 was induced in tammar leukocytes in response to pathogen-associated molecular patterns from both gram positive and negative bacteria. In addition, we also examined the expression of MaeuCath1 in the primary and secondary lymphoid organs of the tammar neonate throughout early pouch life. The results from this study demonstrate the importance that MaeuCath1 may play in innate defense of the marsupial young, especially in the mucosal organs. Such expression of antimicrobial peptides may form part of the immune strategies of marsupials for neonatal survival during their post-partum development.

Lactation transcriptomics in the Australian marsupial, Macropus eugenii: transcript sequencing and quantification.

BACKGROUND: Lactation is an important aspect of mammalian biology and, amongst mammals, marsupials show one of the most complex lactation cycles. Marsupials, such as the tammar wallaby (Macropus eugenii) give birth to a relatively immature newborn and progressive changes in milk composition and milk production regulate early stage development of the young. RESULTS: In order to investigate gene expression in the marsupial mammary gland during lactation, a comprehensive set of cDNA libraries was derived from lactating tissues throughout the lactation cycle of the tammar wallaby. A total of 14,837 express sequence tags were produced by cDNA sequencing. Sequence analysis and sequence assembly were used to construct a comprehensive catalogue of mammary transcripts. Sequence data from pregnant and early or late lactating specific cDNA libraries and, data from early or late lactation massively parallel sequencing strategies were combined to analyse the variation of milk protein gene expression during the lactation cycle. CONCLUSION: Results show a steady increase in expression of genes coding for secreted protein during the lactation cycle that is associated with high proportion of transcripts coding for milk proteins. In addition, genes involved in immune function, translation and energy or anabolic metabolism are expressed across the lactation cycle. A number of potential new milk proteins or mammary gland remodelling markers, including noncoding RNAs have been identified.

Generation and analysis of Siah2 mutant mice.

Siah proteins function as E3 ubiquitin ligase enzymes to target the degradation of diverse protein substrates. To characterize the physiological roles of Siah2, we have generated and analyzed Siah2 mutant mice. In contrast to Siah1a knockout mice, which are growth retarded and exhibit defects in spermatogenesis, Siah2 mutant mice are fertile and largely phenotypically normal. While previous studies implicate Siah2 in the regulation of TRAF2, Vav1, OBF-1, and DCC, we find that a variety of responses mediated by these proteins are unaffected by loss of Siah2. However, we have identified an expansion of myeloid progenitor cells in the bone marrow of Siah2 mutant mice. Consistent with this, we show that Siah2 mutant bone marrow produces more osteoclasts in vitro than wild-type bone marrow. The observation that combined Siah2 and Siah1a mutation causes embryonic and neonatal lethality demonstrates that the highly homologous Siah proteins have partially overlapping functions in vivo.

Cross-fostering of the tammar wallaby (Macropus eugenii) pouch young accelerates fore-stomach maturation.

There are two phases of fore-stomach development during the first 200 days of pouch life in tammar wallaby. For the first 170 days, the mucosa displays an immature gastric glandular phenotype that changes to a cardia glandular phenotype, which remains for the rest of the animal's life. During this 200-day period after birth, the pouch young (PY) is dependent on maternal milk, which progressively changes in composition. We showed previously that PY cross-fostered to host mothers at a later stage of lactation accelerated development. In this study, we investigated whether cross-fostering and exposure to late lactation stage milk affected the transition to cardia glandular phenotype. In fostered PY fore-stomach, there was increased apoptosis, but no change in cell proliferation. The parietal cell population was significantly reduced, and expression of gastric glandular phenotype marker genes (ATP4A, GKN2, GHRL and NDRG2) was down-regulated, suggesting down-regulation of gastric phenotype in fostered PY fore-stomach. The expression of cardia glandular phenotype genes (MUC4, KRT20, CSTB, ITLN2 and LPLUNC1) was not changed in fostered PY. These data suggest that fore-stomach maturation proceeds via two temporally distinct processes: down-regulation of gastric glandular phenotype and initiation of cardia glandular phenotype. In fostered PY, these two processes appear uncoupled, as gastric glandular phenotype was down-regulated but cardia glandular phenotype was not initiated. We propose that milk from later stages of lactation and/or herbage consumed by the PY may play independent roles in regulating these two processes.

A population of mammary epithelial cells do not require hormones or growth factors to survive.

Hormonal stimulation of mammary explants mimics many of the biochemical changes observed during lactogenesis. Previous studies using eutherian species conclude that mammary explants require addition of exogenous macromolecules to remain hormone responsive in culture. The present study examines the survival of mammary explants from the wallaby and mouse using milk protein gene expression as a functional marker of lactation and cell viability. Mammary explants from pregnant tammars and mice showed that milk protein gene expression was significantly elevated after 3 days of culture with lactogenic hormones. The subsequent removal of exogenous hormones from the media for 10 days resulted in the down-regulation of milk protein genes. Surprisingly, mammary explants remained hormone responsive and expression of milk protein genes was re-induced after a second challenge with lactogenic hormones. Furthermore, the alveolar architecture was maintained. Global functional microarray analysis showed that classic involution markers were not differentially expressed, although two stress-induced survival genes were significantly up-regulated. We report that a population of mammary epithelial cells have an intrinsic capacity to remain viable and hormone responsive for extended periods in chemically defined media without any exogenous macromolecules. We propose that the mammary explant culture model uncouples the first phase of involution, as milk accumulation that normally provides involution stimuli is absent in this culture model allowing a population of cells to survive.

A novel transcript from the KLKP1 gene is androgen regulated, down-regulated during prostate cancer progression and encodes the first non-serine protease identified from the human kallikrein gene locus.

BACKGROUND: The kallikrein-related (KLK) serine protease, prostate specific antigen is the current marker for prostate cancer (PCa). Other members of the KLK family are also emerging as potential adjunct biomarkers for this disease. Our aim was to identify and characterize novel KLK-related genes with potential as PCa bio-markers. METHODS: Low stringency DNA screening was coupled with amplification techniques to identify novel sequences. Transcripts were examined by Northern blot, RT-PCR, and in situ hybridization analysis and in silico bioinformatics approaches. Protein characterization was performed by Western blot and confocal microscopy analysis. Gene regulation studies were performed by quantitative PCR and promoter reporter assays. RESULTS: We identified a novel kallikrein-related mRNA designated KRIP1 (kallikrein-related, expressed in prostate 1) which, together with the recently reported PsiKLK1 and KLK31P transcripts, is transcribed from KLKP1; a gene evolved from, and located within, the KLK locus. Significantly, in contrast to these other non-coding KLKP1 transcripts, the KRIP1 mRNA generates an approximately 18 kDa intracellular protein-the first non-serine protease identified from the KLK locus. KRIP1 mRNA is abundant only in normal prostate and is restricted to cells of epithelial origin in normal and diseased glands. Ligand binding of the androgen receptor increases transcription from the KLKP1 gene. Consistently, KRIP1 mRNA levels are lower in PCa samples compared to benign prostatic hyperplasia. CONCLUSIONS: Transcription from KLKP1 is reduced as cells de-differentiate on the pathway to malignancy. KLKP1/KRIP1 has potential as a marker of both PCa progression and recent evolutionary events within the KLK locus.

The tammar wallaby: a model to examine endocrine and local control of lactation.

Marsupials, such as the tammar wallaby (Macropus eugenii), have adopted a reproductive strategy that is very different to eutherians. Both the rate of production and the composition of milk changes progressively during the lactation cycle to meet the nutritional demands of an altricial young. The tammar therefore provides a valuable model to study changes in milk composition, and in particular the genes that code for proteins secreted in the milk, to more accurately assess the role of gene products regulating either development of the young or mammary function.