Transcriptome analysis of mammary epithelial cell gene expression reveals novel roles of the extracellular matrix.

BACKGROUND: The unique lactation strategy of the tammar wallaby (Macropus eugeni) has been invaluable in evaluating the role of lactogenic hormones and the extracellular matrix (ECM) in the local control of mammary gland function. However molecular pathways through which hormones and ECM exert their effect on wallaby mammary gland function remain unclear. This study undertakes transcriptome analysis of wallaby mammary epithelial cells (WallMEC) following treatment with mammary ECM from two distinct stages of lactation. METHODS: WallMEC from MID lactation mammary glands were cultured on ECM from MID or LATE lactation and treated for 5 days with 1 µg/ml cortisol, 1 µg/ml insulin, 0.2 µg/ml prolactin, 650 pg/ml triodothyronine and 1 pg/ml estradiol to induce lactation. WallMEC RNA from triplicate ECM treatments was used to perform RNAseq. RESULTS: ECM from MID and LATE lactation differentially regulated key genes in sugar and lipid metabolism. Seven pathways including galactose metabolism, lysosome, cell adhesion molecules (CAM), p53 signaling, the complement and coagulation and Nod-like receptor signaling pathways were only significantly responsive to ECM in the presence of hormones. The raw RNA-seq data for this project are available on the NCBI Gene Expression Omnibus (GEO) browser (accession number GSE81210). CONCLUSIONS: A potential role of ECM in regulation of the caloric content of milk, among other functions including apoptosis, cell proliferation and differentiation has been identified. GENERAL SIGNIFICANCE: This study has used a non-eutherian lactation model to demonstrate the synergy between ECM and hormones in the local regulation of mammary function.

The extracellular matrix locally regulates asynchronous concurrent lactation in tammar wallaby (Macropus eugenii).

Asynchronous concurrent lactation (ACL) is an extreme lactation strategy in macropod marsupials including the tammar wallaby, that may hold the key to understanding local control of mammary epithelial cell function. Marsupials have a short gestation and a long lactation consisting of three phases; P2A, P2B and P3, representing early, mid and late lactation respectively and characterised by profound changes in milk composition. A lactating tammar is able to concurrently produce phase 2A and 3 milk from adjacent glands in order to feed a young newborn and an older sibling at heel. Physiological effectors of ACL remain unknown and in this study the extracellular matrix (ECM) is investigated for its role in switching mammary phenotypes between phases of tammar wallaby lactation. Using the level of expression of the genes for the phase specific markers tELP, tWAP, and tLLP-B representing phases 2A, 2B and 3 respectively we show for the first time that tammar wallaby mammary epithelial cells (WallMECs) extracted from P2B acquire P3 phenotype when cultured on P3 ECM. Similarly P2A cells acquire P2B phenotype when cultured on P2B ECM. We further demonstrate that changes in phase phenotype correlate with phase-specific changes in ECM composition. This study shows that progressive changes in ECM composition in individual mammary glands provide a local regulatory mechanism for milk protein gene expression thereby enabling the mammary glands to lactate independently.

The extracellular matrix regulates MaeuCath1a gene expression.

We have previously shown that the gene for MaeuCath1, a cathelicidin secreted in wallaby milk is alternately spliced into two variants, MaeuCath1a and MaeuCath1b which are temporally regulated in order to provide antimicrobial protection to the newborn and stimulate mammary growth, respectively. The current study investigated the extracellular matrix (ECM) for its regulatory role in MaeuCath1 gene expression. Reverse transcription qPCR using RNA isolated from mammary epithelial cells (WallMEC) cultured on ECM showed that ECM regulates MaeuCath1a gene expression in a lactation phase-dependent manner. Luciferase reporter-based assays and in silico analysis of deletion fragments of the 2245bp sequence upstream of the translation start site identified ECM-dependent positive regulatory activity in the -709 to -15 region and repressor activity in the -919 to -710 region. Electrophoretic Gel Mobility Shift Assays (EMSA) using nuclear extract from ECM-treated WallMEC showed differential band shift in the -839 to -710 region.

Tammar wallaby mammary cathelicidins are differentially expressed during lactation and exhibit antimicrobial and cell proliferative activity.

Cathelicidins secreted in milk may be central to autocrine feedback in the mammary gland for optimal development in addition to conferring innate immunity to both the mammary gland and the neonate. This study exploits the unique reproductive strategy of the tammar wallaby (Macropus eugenii) model to analyse differential splicing of cathelicidin genes and to evaluate the bactericidal activity and effect of the protein on mammary epithelial cell proliferation. Two linear peptides, Con73 and Con218, derived from the heterogeneous carboxyl end of cathelicidin transcripts, MaeuCath1 and MaeuCath7 respectively, were evaluated for antimicrobial activity. Both Con73 and Con218 significantly inhibited the growth of Staphylococcus aureus, Pseudomonas aureginosa, Enterococcus faecalis and Salmonella enterica. In addition both MaeuCath1 and MaeuCath7 stimulated proliferation of primary tammar wallaby mammary epithelial cells (WallMEC). Lactation-phase specific alternate spliced transcripts were determined for MaeuCath1 showing utilisation of both antimicrobial and proliferative functions are required by the mammary gland and the suckled young. The study has shown for the first time that temporal regulation of milk cathelicidins may be crucial in antimicrobial protection of the mammary gland and suckled young and mammary cell proliferation.