Extracellular matrix promotes clathrin-dependent endocytosis of prolactin and STAT5 activation in differentiating mammary epithelial cells

Rebecca E Bridgewater,Charles H Streuli,Patrick T Caswell

doi:10.1038/s41598-017-04783-6

Abstract

The hormone prolactin promotes lactational differentiation of mammary epithelial cells (MECs) via its cognate receptor and the downstream JAK2-STAT5a signalling pathway. In turn this regulates transcription of milk protein genes. Prolactin signalling depends on a cross-talk with basement membrane extracellular matrix (ECM) via β1 integrins which activate both ILK and Rac1 and are required for STAT5a activation and lactational differentiation. Endocytosis is an important regulator of signalling. It can both enhance and suppress cytokine signalling, although the role of endocytosis for prolactin signalling is not known. Here we show that clathrin-mediated endocytosis is required for ECM-dependent STAT5 activation. In the presence of ECM, prolactin is internalised via a clathrin-dependent, but caveolin-independent, route. This occurs independently from JAK2 and Rac signalling, but is required for full phosphorylation and activation of STAT5. Prolactin is internalised into early endosomes, where the master early endosome regulator Rab5b promotes STAT5 phosphorylation. These data reveal a novel role for ECM-driven endocytosis in the positive regulation of cytokine signalling.

Highlights

Cells within the tissues of multicellular organisms respond to positional signals from the extracellular matrix (ECM) and cell-cell adhesions, and to temporal signals from hormones, cytokines and growth factors[1]
laminin-rich basement membrane (LrBM) ECM is required for Prl/signal transducer and activator of transcription 5a (STAT5a) signalling in lactational differentiation
Eph[4] cells, a mammary epithelial cells (MECs) cell line derived from the mammary epithelium of pregnant mice, express β-casein when cultured in 3D LrBM extract[27], and this is the only cell line model which robustly responds to ECM engagement and Prl in the way that primary cells do

Summary

Introduction

Cells within the tissues of multicellular organisms respond to positional signals from the extracellular matrix (ECM) and cell-cell adhesions, and to temporal signals from hormones, cytokines and growth factors[1]. There, the STAT5a dimer causes transcription of milk protein genes, such as that encoding β-casein Despite this detailed understanding to the molecular signalling pathway, the mechanism through which class I cytokine receptors are activated by ligand (dimerization versus conformational change), and the localisation of downstream signalling within the cell (plasma membrane versus endosomal platforms), remain enigmatic[6, 7]. Caveolar endocytosis of growth hormone receptor (GHR, closely related to PrlR) is implicated in STAT-mediated transcription, and JAK2 associates with GHR following internalisation, suggesting that signalling continues on endosomes[17, 18]. CME has been implicated in the endocytosis and degradation of PrlR20, and in breast cancer cells can enhance downstream signalling to ERK but not STATs21

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