Regulation of YAP by Mechanical Strain through Jnk and Hippo Signaling

Abstract

Mechanical forces affect all the tissues of our bodies. Experiments conducted mainly on cultured cells have established that altering these forces influences cell behaviors, including migration, differentiation, apoptosis, and proliferation [1, 2]. The transcriptional coactivator YAP has been identified as a nuclear relay of mechanical signals, but the molecular mechanisms that lead to YAP activation were not identified [3]. YAP is the main transcriptional effector of the Hippo signaling pathway, a major growth regulatory pathway within metazoa [4], but at least in some instances, the influence of mechanical strain on YAP was reported to be independent of Hippo signaling [5, 6]. Here, we identify a molecular pathway that can promote the proliferation of cultured mammary epithelial cells in response to cyclic or static stretch. These mechanical stimuli are associated with increased activity of the transcriptional coactivator YAP, which is due at least in part to inhibition of Hippo pathway activity. Much of this influence on Hippo signaling can be accounted for by the activation of c-Jun N-terminal kinase (JNK) activity by mechanical strain and subsequent inhibition of Hippo signaling by JNK. LATS1 is a key negative regulator of YAP within the Hippo pathway, and we further show that cyclic stretch is associated with a JNK-dependent increase in binding of a LATS inhibitor, LIMD1, to the LATS1 kinase and that reduction of LIMD1 expression suppresses the activation of YAP by cyclic stretch. Together, these observations establish a pathway for mechanical regulation of cell proliferation via JNK-mediated inhibition of Hippo signaling.

YAP Nuclear Localization in the Absence of Cell-Cell Contact Is Mediated by a Filamentous Actin-dependent, Myosin II- and Phospho-YAP-independent Pathway during Extracellular Matrix Mechanosensing

“…We plated cells sparsely on ECMs made of polyacrylamide substrates of defined stiffness with fibronectin coupled to their surface, and performed immunofluorescence and Western blot analysis of YAP localization and phosphorylation, and analysis of F-actin integrity. In agreement with previous reports (8,9), in MEFs grown on stiff substrates (50 kPa), cells were well spread and exhibited stress fibers and strong nuclear localization of YAP, whereas on soft substrates (0.5 kPa), cells were poorly spread and YAP was excluded from the nucleus (Fig. 4, A and B).…”

“…In contact inhibited Drosophila epithelial tissue, myosin II contractility regulates the formation of the Wts kinase complex at AJs to suppress Hippo signaling, and in the absence of contractility this complex is disrupted and Hippo signaling is activated (15). Furthermore, in dense cultures in vitro, YAP nuclear localization is rescued by ECM stretching (9,10). Mechanoregulation of YAP in the absence of cell-cell contact has also been shown to be dependent on ECM stiffness sensation, myosin II motor activity, and cell spreading area (8,19).…”

Oscillatory Strain Promotes Vessel Stabilization and Alignment through Fibroblast YAP‐Mediated Mechanosensitivity

“…A recent study has shown that 6 h of oscillatory stretch of human mammary epithelial cells resulted in increased nuclear YAP levels, which was shown to correlate with cell proliferation 9. Another work has shown that culturing cells on stiff substrates resulted in a flat‐spread morphology with higher proliferation rates and an increase in nuclear YAP, as opposed to soft substrates, which resulted in a rounded cell shape, decreased proliferation rates, and more cytoplasmic YAP 27.…”

“…YAP can be localized in the cytoplasm or translocated to the nucleus, where it associates with TEAD transcription factors 7. Previous studies have shown that various forms of mechanical stimulation, such as substrate stiffness, strain, and shear stress, affected YAP localization by actin fiber remodeling 6, 8, 9, 10. YAP was shown to be involved in the effect of shear stress on ECs and blood vessels,10 however its role in the effect of mechanical strain on vascularization is yet to be established.…”

“…Moreover, distinct alignment and migration of the fibroblasts cells, alongside vessel development, stabilization, and alignment and a subsequent increase in cell secretion of pro‐angiogenesis cytokines, were observed. Since oscillatory strain was previously shown to affect YAP expression,9 we hypothesized that it has a role in transmitting the mechanical stretch signal and affects vessel network development. YAP inhibition, by the addition of verteporfin to the culture media, caused higher damage to the vessels in the mechanically stimulated constructs compared to the vessels in the static control.…”