The Mammary Epithelial Cell Secretome and Its Regulation by Signal Transduction Pathways

Abstract

Extracellular proteins released by mammary epithelial cells are critical mediators of cell communication, proliferation, and organization, yet the actual spectrum of proteins released by any given cell (the secretome) is poorly characterized. To define the set of proteins secreted by human mammary epithelial cells (HMEC), we combined analytical and computational approaches to define a secretome protein set based upon probable biological significance. Analysis of HMEC-conditioned medium by liquid chromatography-mass spectrometry resulted in identification of 889 unique proteins, of which 151 were found to be specifically enriched in the extracellular compartment when compared with a database of proteins expressed in whole HMEC lysates. Additional high mass accuracy analysis revealed 36 proteins whose extracellular abundance increased after treatment with phorbol ester (PMA), a protein kinase C agonist and general secretagogue. Many of the PMA stimulated proteins have been reported to be aberrantly expressed in human cancers and appear to be coregulated as multigene clusters. By inhibiting PMA-mediated transactivation of the epidermal growth factor receptor (EGFR), a pathway critically required for normal HMEC function, we found that the secretion of specific matrix metalloproteases was also coordinately regulated through EGFR transactivation. This study demonstrates a tiered strategy by which extracellular proteins can be identified and progressively assigned to classes of increasing confidence and regulatory importance.

ERBBs in the gastrointestinal tract: Recent progress and new perspectives

“…Finally, it should be recalled that there can be GPCR-independent, ligand-dependent transactivation of the EGFR. One example is the phorbol ester TPA, presumably acting as a general cell surface sheddase [24]. However, this action of TPA may be counter-balanced by a PKC-mediated phosphorylation of threonine (T)654 within the cytoplasmic tail of the EGFR that results in receptor internalization and downregulation.…”

Glucose induces rapid changes in the secretome of Saccharomyces cerevisiae

“…Secretory proteins include growth factors, inflammatory cytokines, coagulation factors, extracellular matrix proteins, proteases, and protease inhibitors [1-9]. As such, they participate in various physiological processes such as immune defense, blood coagulation, cell growth, cell differentiation, and proliferation [1-5,7-9]. For fungi that have thick cell walls, secretory proteins are involved in the formation and maintenance of cell walls, cell separation, and nutrient scavenging [10-17].…”

“…In the classical pathway, proteins that contain a specific N-terminal signal sequence are translocated into the ER, transported to the Golgi and then secreted by secretory vesicles [30,32,33]. Accumulating evidence indicates that a large number of signal-less proteins can also be secreted through the non-classical pathway [1,2,4,5,10,21,23,31], [34], including metabolic enzymes, chaperones, translation factors, and transcriptional regulators [5,8,9,11,20-24,31,34-37]. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a glycolytic enzyme and has been shown by immuno-transmission electron microscopy (immuno-TEM) to be present on the surface of Candida albicans and Saccharomyces cerevisiae [14].…”

An Extensive Survey of Tyrosine Phosphorylation Revealing New Sites in Human Mammary Epithelial Cells

“…These cells have been extensively used as a model system for studying the mechanisms controlling proliferation and differentiation in normal human cells 17, especially for understanding the role of the epidermal growth factor receptor (EGFR) signaling pathways 18–20. More recently, several studies have been focused on studying the dynamics of tyrosine phosphorylation involved in the EGFR signaling pathways by applying iTRAQ14 labeling and multiple reaction monitoring10, 21 coupled with anti-pTyr IP and LC-MS/MS.…”