SHARPIN regulates collagen architecture and ductal outgrowth in the developing mouse mammary gland

Abstract

SHARPIN is a widely expressed multifunctional protein implicated in cancer, inflammation, linear ubiquitination and integrin activity inhibition; however, its contribution to epithelial homeostasis remains poorly understood. Here, we examined the role of SHARPIN in mammary gland development, a process strongly regulated by epithelial-stromal interactions. Mice lacking SHARPIN expression in all cells (Sharpin), and mice with a stromal (S100a4-Cre) deletion of Sharpin, have reduced mammary ductal outgrowth during puberty. In contrast, Sharpin mammary epithelial cells transplanted in vivo into wild-type stroma, fully repopulate the mammary gland fat pad, undergo unperturbed ductal outgrowth and terminal differentiation. Thus, SHARPIN is required in mammary gland stroma during development. Accordingly, stroma adjacent to invading mammary ducts of Sharpin mice displayed reduced collagen arrangement and extracellular matrix (ECM) stiffness. Moreover, Sharpin mammary gland stromal fibroblasts demonstrated defects in collagen fibre assembly, collagen contraction and degradation in vitro Together, these data imply that SHARPIN regulates the normal invasive mammary gland branching morphogenesis in an epithelial cell extrinsic manner by controlling the organisation of the stromal ECM.

Integrin binding dynamics modulate ligand-specific mechanosensing in mammary gland fibroblasts

“…1A). As expected based on the higher integrin β1 activity and faster focal adhesion (FA) turnover compared to wild-type MSFs (Peuhu, Kaukonen et al, 2017, Rantala et al, 2011, Sharpin cpdm MSFs appeared larger compared to wild-type MSFs when seeded on fibronectin-coated hydrogels (Fig. 1A, B).…”

“…Based on previous observations that primary MSFs from SHARPIN-deficient (Sharpin cpdm/cpdm ; Sharpin cpdm ) mice have impaired capacity to contract collagen gels (Peuhu, Kaukonen et al, 2017) we sought to investigate the link between integrin activity and force transduction. We first studied the ability of SHARPIN-expressing (Sharpin +/+ or Sharpin cpdm/+ ; from here on referred to as wild-type) and Sharpin cpdm MSFs to spread in response to ECM stiffness and ligand type.…”

“…We first studied the ability of SHARPIN-expressing (Sharpin +/+ or Sharpin cpdm/+ ; from here on referred to as wild-type) and Sharpin cpdm MSFs to spread in response to ECM stiffness and ligand type. MSFs were seeded on soft (2 kPa) fibronectin or collagen I pre-coated polyacrylamide gels, approximating the stiffness of the mammary tissue in vivo (Lopez et al, 2011, Peuhu, Kaukonen et al, 2017, Plodinec et al, 2012 ( Fig. 1A).…”

“…Given that Sharpin cpdm MSFs have faster FA dynamics (increased assembly and disassembly rates) on collagen-coated rigid glass substrate (Peuhu, Kaukonen et al, 2017), we next evaluated the effect of SHARPIN deficiency on FA maturation (number and average length) on a range of hydrogel rigidities. Vinculin, a mechanosensitive adaptor molecule recruited to mature FA (Chen et al, 2006, was immunolabeled in wild-type and Sharpin cpdm MSFs plated on 0.8-10 kPa collagen I-coated hydrogels (Fig.…”

“…While the role of integrins as cell mechanosensors and transducers is well established, the link between the regulation of integrin activity and the mechanosensing response on different ECM ligands remains poorly understood. SHARPIN is a cytosolic adaptor protein that, among other functions, binds to the intracellular integrin alpha tails and inhibits integrin activity in vitro and in vivo (Peuhu, Kaukonen et al, 2017, Peuhu, Salomaa et al, 2017, Pouwels et al, 2013, Rantala et al, 2011. We have previously demonstrated that stromal SHARPIN deficiency interferes with normal mouse mammary gland development and collagen fiber assembly in vivo (Peuhu, Kaukonen et al, 2017).…”