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EGFR Signaling Regulates Maspin/SerpinB5 Phosphorylation and Nuclear Localization in Mammary Epithelial Cells.

ABSTRACT: Maspin (SerpinB5) is a non-inhibitory serpin (serine protease inhibitor) with very diverse biological activities including regulation of cell adhesion, migration, death, control of gene expression and oxidative stress response. Initially described as a tumor and metastasis suppressor, clinical data brought controversies to the field, as some studies reported no correlation between SerpinB5 expression and prognosis value. These data underscore the importance of understanding SerpinB5 function in a normal physiological context and the molecular mechanism involved. Several SerpinB5 phosphoforms have been detected in different cell lines, but the signaling pathways involved and the biological significance of this post-translational modification in vivo remains to be explored. In this study we investigated SerpinB5 expression, subcellular localization and phosphorylation in different stages of the mouse mammary gland development and the signaling pathway involved. Here we show that SerpinB5 is first detected in late pregnancy, reaches its highest levels in lactation and remains at constant levels during post-lactational regression (involution). Using high resolution isoelectric focusing followed but immunoblot, we found at least 8 different phosphoforms of SerpinB5 during lactation, which decreases steadily at the onset of involution. In order to investigate the signaling pathway involved in SerpinB5 phosphorylation, we took advantage of the non-transformed MCF-10A model system, as we have previously observed SerpinB5 phosphorylation in these cells. We detected basal levels of SerpinB5 phosphorylation in serum- and growth factor-starved cells, which is due to amphiregulin autocrine activity on MCF-10A cells. EGF and TGF alpha, two other EGFR ligands, promote important SerpinB5 phosphorylation. Interestingly, EGF treatment is followed by SerpinB5 nuclear accumulation. Altogether, these data indicate that SerpinB5 expression and phosphorylation are developmentally regulated. In vitro analyses indicate that SerpinB5 phosphorylation is regulated by EGFR ligands, but EGF appears to be the only able to induce SerpinB5 nuclear localization.

SUBMITTER: Tamazato Longhi M

PROVIDER: S-EPMC4957797 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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EGFR Signaling Regulates Maspin/SerpinB5 Phosphorylation and Nuclear Localization in Mammary Epithelial Cells.

Tamazato Longhi Mariana M Magalhães Magna M Reina Jeffrey J Morais Freitas Vanessa V Cella Nathalie N

PloS one 20160722 7

Maspin (SerpinB5) is a non-inhibitory serpin (serine protease inhibitor) with very diverse biological activities including regulation of cell adhesion, migration, death, control of gene expression and oxidative stress response. Initially described as a tumor and metastasis suppressor, clinical data brought controversies to the field, as some studies reported no correlation between SerpinB5 expression and prognosis value. These data underscore the importance of understanding SerpinB5 function in ...[more]

PMID: 27447178

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Project description:Background: Maspin (SERPINB5) is a potential tumor suppressor gene with pleiotropic biological activities, including regulation of cell proliferation, death, adhesion, migration and gene expression. Several studies indicate that nuclear localization is essential for maspin tumor suppression activity. We have previously shown that the EGFR activation leads to maspin nuclear localization in MCF-10A cells. The present study investigated which EGFR downstream signaling molecules are involved in maspin nuclear localization and explored a possible role of cell-cell contact in this process. Methods: MCF-10A cells were treated with pharmacological inhibitors against EGFR downstream pathways followed by EGF treatment. Maspin subcellular localization was determined by immunofluorescence. To investigate the role of cell-cell contact these cells were either treated with chelating agents or plated on different cell densities. Maspin and E-cadherin subcellular localization was determined by immunofluorescence. In addition, proteomic and interactome analyses were conducted in order to identify molecular partners which interact with maspin in EGF-treated cells only. Results: EGFR activation regulates cell behavior via de extracellular signal–related kinase (ERK)/MAP kinase, phosphatidyl-inositol-3 (PI 3) kinase- AKT and Jak-STAT pathways. Using pharmacological inhibitors against key components of these signaling pathways we found that PI3K-AKT and Jak-STAT, but not MAP kinase pathway, regulate EGF-induced maspin nuclear accumulation in MCF-10A cells. Interestingly, we observed that maspin is predominantly nuclear in sparse cell culture, but it is redistributed to the cytoplasm in confluent cells even in the presence of EGF. Proteomic and interactome results suggest a role of maspin on post-transcriptional and translation regulation, protein folding and cell-cell adhesion.Conclusions: Maspin nuclear accumulation is determined by an interplay between EGFR (via PI3K-AKT and Jak-STAT pathways) and cell-to-cell contact.

Dataset Information

EGFR Signaling Regulates Maspin/SerpinB5 Phosphorylation and Nuclear Localization in Mammary Epithelial Cells.

Publications

EGFR Signaling Regulates Maspin/SerpinB5 Phosphorylation and Nuclear Localization in Mammary Epithelial Cells.

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