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.

Figure 1. Cyclic stretch increases YAP activity through down-regulation of Hippo signaling

Comparisons of MCF10A cells subject to cyclic stretch (CS) and non-stretched controls (NS) are shown. A) Cell proliferation, visualized by Edu labeling (red) after 6 h CS, with nuclei labeled by Hoechst (blue). Histogram at right shows quantitation of the mean percent labeled cells, from three biological replicates. B) Quantitation of cell numbers after 6 h CS compared to NS controls, from three biological replicates. C) Histogram shows result of quantitative RT-PCR on BIRC3 and CTGF mRNA level in cells after 6 h CS compared to NS controls, from three biological replicates. The mRNA over GAPDH ratio in all samples were normalized to the ratio in NS control cells. D) Immunolocalization of YAP (green) after 6 h CS compared to NS controls. Histogram at right shows quantitation of the mean percent cells with predominantly nuclear YAP, from three biological replicates. E) Cells co-transfected with TBS-mCherry (red) and GFP (green) after 6 h CS compared to NS controls. Graph indicates the percentage of GFP-expressing cells that are positive for TBS-mCherry, from three biological replicates. F) Western blot on lysates of cells after 2h, 4h, and 6 h CS, compared to NS controls, blotted with anti-YAP, anti-pYAP, anti-pLATS, anti-LATS, anti-pMST, anti-MST and anti-GAPDH, as indicated. Histogram shows average (from three biological replicates) of relative levels of YAP normalized to GAPDH (loading control), together with ratio of pYAP/YAP. In all panels, error bars indicate standard error. See also Fig. S1.

Figure 2. Activation of YAP by cyclic stretch is JNK-dependent

Influence of JNK inhibition on the response of MCF10A cells to cyclic stretch (CS). A) Western blot on lysates of cells after 2h, 4h, and 6 h CS, compared to non-stretched (NS) controls, blotted with anti-JNK, anti-pJNK, anti-pcJun, anti-cJun and anti-GAPDH. Histogram shows average ratio of pJNK/JNK from three biological replicates. B) Western Blot on lysates of cells treated with 50 μM SP600125 (JNK inhibitor) and subject to CS for 6 hours, blotted with anti- YAP, pYAP, LATS1, pLATS1, pcJun, cJun and GAPDH. Histogram shows average ratio of pYAP/YAP, and total YAP normalized to GAPDH from four biological replicates. C) Immunolocalization of YAP (green) and nuclei (DNA, blue) in cells treated with 50 μM SP600125 and subject to 6 h CS. Histogram at right shows quantitation of the mean percent cells (from three biological replicates) of cells with predominantly nuclear YAP (green), similar cytoplasm and nuclear staining (purple) and predominantly cytoplasmatic YAP (pink). D) Cell proliferation, visualized by Edu labeling of cells treated with 50 μM SP600125 or vehicle control, and then subject to 6 h CS. Histogram shows quantitation of the mean percent labeled cells, from three biological replicates. E) Mean percentage of transfected (GFP-expressing) cells that are positive for TBS-mCherry, treated with 50 μM SP600125 or vehicle control, and then subject to 6 h CS, from three biological replicates. F) Histogram shows result of quantitative RTPCR on BIRC3 mRNA level in cells after 6 h CS compared to NS controls three biological replicates. The BIRC3 over GAPDH ratio in all samples were normalized to the ratio in CS non-treated cells. In all panels, error bars indicate standard error of the mean. See also Fig. S2.

Figure 3. Hippo and JNK signaling contribute to YAP activation by ECM stiffness

Comparisons of MCF10A cells plated on soft (0.2 kPa) versus stiff (35 kPa) substrates. A) Immunolocalization of YAP (green) and nuclei (Hoechst, blue). Histogram at right shows quantitation of the mean percent cells (from three biological replicates) with predominantly nuclear YAP (green), similar cytoplasm and nuclear staining (purple) and predominantly cytoplasmic YAP (pink). B) Western Blot on lysates of cells plated on soft and stiff substrates. Cells were lysed 24 h after of plating and then blotted with anti-YAP, pYAP, pJNK, JNK and GAPDH. Histogram shows normalized ratios of pYAP/YAP and YAP/GAPDH, from three biological replicates. C) Cell proliferation, visualized by Edu labeling of cells grown on stiff substrates and treated with 50 μM SP600125 or vehicle control for 4 h. Histogram shows quantitation of the mean percent labeled cells, from three biological replicates. D) Mean percentage of transfected (GFP-positive) cells that are positive for TBS-mCherry, among cells grown on stiff substrates and treated with 50 μM SP600125 or vehicle control for 4 h, from three biological replicates. E) Quantitation of BIRC3 and CTGF mRNA levels by RT-PCR in cells plated on stiff substrates treated with 50 μM SP600125 or vehicle control, from three biological replicates. The mRNA over GAPDH ratio in all samples was normalized to the ratio in stiff vehicle-treated cells F) Immunolocalization of YAP (green) and nuclei (Hoechst, blue) in cells plated on stiff substrates and treated with 50 μM SP600125 or vehicle control for 4 h. Histogram at right shows quantitation of the mean percent cells (from three biological replicates) of cells with predominantly nuclear YAP (green), similar cytoplasmic and nuclear staining (purple) and predominantly cytoplasmic YAP (pink). G) Western Blot on lysates of cells plated on stiff substrates and treated with 50 μM SP600125 or vehicle control for 4 h, blotted with anti- YAP, pYAP, pLATS, LATS and GAPDH. Histogram shows mean YAP levels (normalized to GAPDH) and ratio of pYAP/YAP, from three biological replicates. In all panels, error bars indicate standard error. See also Fig. S3.

Figure 4. Cyclic Stretch increases LIMD1-LATS1 binding to activate YAP

Comparisons of MCF10A cells subject to 6h cyclic stretch (CS) and non-stretched controls (NS) or cells treated with SP600125 or shRNAs, as indicated. A) Western blots showing the results of co-immunoprecipitation experiments on cells subject to CS or NS controls. Upper two blots (input) show relative amounts of endogenous LATS1 and LIMD1 in cell lysates, lower two panels show relative amounts immunoprecipitated using anti-LATS1 sera. Histogram shows average ratio of LIMD1/LATS1 from four biological replicates. B) Western blots showing the results of co-immunoprecipitation experiments on cells subject to CS and treated with SP600125 or vehicle control. Upper two blots (input) show relative amounts of endogenous LATS1 and LIMD1 in cell lysates, lower two panels show relative amounts immunoprecipitated using anti-LATS1 sera. Histogram shows average ratio of LIMD1/LATS1 from four biological replicates, normalized to levels in lysates. C) Western blots on lysates of MCF10A cells treated with DMSO or SP600125, and with or without CS, as indicated. The lower two blots show a standard 4-15% gradient gel, and the upper blot shows a Phos-tag gel. The arrow indicates a band of slow mobility (highly phosphorylated) LIMD1 induced by CS, and suppressed by SP600125. D) YAP activity, visualized by mean percentage of transfected (Flag-expressing) cells that are positive for TBS-mCherry, among cells subject to 6h CS or NS controls, and treated either with a shRNA specific for LIMD1 or a negative control shRNA (scramble), from three biological replicates. Cells expressing shRNAs are marked by expression of a FLAG-epitope from the same plasmid. Inset (upper left), western blot showing the effectiveness of shLIMD1. Lysate of total levels of HEK cells transfected with shLIMD1 for 24 hours and blotted for LIMD1 and GAPDH. E) Model illustrating the proposed mechanism in which cyclical stretch promotes the binding of LIMD1 to LATS1 by activating JNK. This inhibits LATS, and therefore increases YAP activity, which increases cell proliferation. In all panels, error bars indicate standard error. See also Fig. S4.