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Unlocking the Secrets of the Secretome
Proteins secreted from HMEC upon treatment with PMA primarily belong to three functional categories: proteolysis, cell signaling, and extracellular matrix.
The secretome—defined as the family of proteins secreted by cells—of human mammary epithelial cells (HMEC) was identified by researchers at Pacific Northwest National Laboratory (PNNL) and the Environmental Molecular Sciences Laboratory (EMSL), a national scientific user facility located at PNNL. Opening new doors for the study of this poorly characterized subset of the proteome, the researchers’ method is applicable to other cell lines. Because the secretome is important for tissue function and homeostasis as well as cell communication, proliferation, and organization, studying these proteins may lead to therapeutic applications and biomarker discovery.
For their studies, the team used PMA (phorbol 12-myristate 13-acetate)—a secretagogue and tumor promoter—to stimulate the release of the secretome from HMEC. In addition to causing proteins to be secreted through general exocytic pathways and pathways involving metalloproteases, PMA specifically causes epidermal growth factor receptor (EGFR) transactivation, in part, by stimulating EGFR autocrine ligands to shed. Autocrine activation of EGFR is critical for normal HMEC function, including proliferation and motility. Therefore, PMA is a general tool for studying the secretome and has the added specific benefit of enabling examination of secreted proteins involved in HMEC function.
Using mass spectrometry-based analysis and bioinformatics comparisons, the team identified 889 proteins in conditioned HMEC medium and compared the medium proteins to those identified in whole cell lysates as well as to bovine proteins present in the bovine serum added to the cell growth medium. Narrowing the potential secretome with these comparisons, the team’s analysis yielded a conservative estimate of 151 proteins belonging to the secretome. Of these, 36 proteins were specifically induced by PMA.
Interestingly, the HMEC PMA-stimulated secreted proteins primarily belong to three functional categories: proteolysis, cell signaling, and extracellular matrix. Furthermore, many PMA-stimulated secreted proteins are expressed in a range of human cancers—an interesting finding, considering the HMEC line used for the studies is nontumorigenic.
The team also indentified a relationship between EGFR and matrix metalloprotease (MMP) regulation. PMA transactivation of EGFR is involved in the regulation of MMP secretion, in particular of MMP-1, MMP-9, and MPP-10. Two of these MMPs (MMP-1 and MMP-9) are known to regulate tumor invasion and angiogenesis. These findings may help researchers understand the relationship between EGFR signaling and mammary carcinogenesis.
The team included Jon Jacobs, Katrina Waters, Loel Kathmann, Dave Camp, Dick Smith, and Brian Thrall from PNNL and Steve Wiley from PNNL and EMSL. Their work was published online in the January 2008 edition of the Journal of Proteome Research.
Learn more about this and other biological research being conducted by scientists in PNNL's Biological Sciences Division.
