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Epithelial cell adhesion to the surrounding extracellular matrix is necessary for their proper behavior and function. During pregnancy and lactation, mammary epithelial cells (MECs) receive signals from their interaction with laminin via β1‐integrin (β1‐itg) to establish apico‐basal polarity and to differentiate in response to prolactin. Downstream of β1‐itg, the scaffold protein Integrin Linked Kinase (ILK) has been identified as the key signal transducer that is required for both lactational differentiation and the establishment of apico‐basal polarity. ILK is an adaptor protein that forms the IPP complex with PINCH and Parvins, which are central to its adaptor functions. However, it is not known how ILK and its interacting partners control tissue‐specific gene expression. Expression of ILK mutants, which weaken the interaction between ILK and Parvin, revealed that Parvins have a role in mammary epithelial differentiation. This conclusion was supported by shRNA‐mediated knockdown of the Parvins. In addition, shRNA knockdown of the Parvin‐binding guanine nucleotide exchange factor αPix prevented prolactin‐induced differentiation. αPix depletion did not disrupt focal adhesions, MEC proliferation, or polarity. This suggests that αPix represents a differentiation‐specific bifurcation point in β1‐itg‐ILK adhesive signaling. In summary, this study has identified a new role for Parvin and αPix downstream of the integrin‐ILK signaling axis for MEC differentiation. J. Cell. Physiol. 231: 2408–2417, 2016. © 2016 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.

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.

Circadian clocks drive ∼24 h rhythms in tissue physiology. They rely on transcriptional/translational feedback loops driven by interacting networks of clock complexes. However, little is known about how cell-intrinsic circadian clocks sense and respond to their microenvironment. Here, we reveal that the breast epithelial clock is regulated by the mechano-chemical stiffness of the cellular microenvironment in primary cell culture. Moreover, the mammary clock is controlled by the periductal extracellular matrix in vivo, which contributes to a dampened circadian rhythm during ageing. Mechanistically, the tension sensing cell-matrix adhesion molecule, vinculin, and the Rho/ROCK pathway, which transduces signals provided by extracellular stiffness into cells, regulate the activity of the core circadian clock complex. We also show that genetic perturbation, or age-associated disruption of self-sustained clocks, compromises the self-renewal capacity of mammary epithelia. Thus, circadian clocks are mechano-sensitive, providing a potential mechanism to explain how ageing influences their amplitude and function., Circadian clocks regulate physiological and behavioural rhythms. Here, the authors show that the stiffness of the extracellular environment regulates circadian clocks in murine breast epithelium via Rho signalling, and explain how extracellular matrix stiffening in aging affects circadian rhythm, with implication in disease.