Hiroshi Nojima

The Hippo Signaling Pathway Components Lats and Yap Pattern Tead4 Activity to Distinguish Mouse Trophectoderm from Inner Cell Mass

Outside cells of the preimplantation mouse embryo form the trophectoderm (TE), a process requiring the transcription factor Tead4. Here, we show that transcriptionally active Tead4 can induce Cdx2 and other trophoblast genes in parallel in embryonic stem cells. In embryos, the Tead4 coactivator protein Yap localizes to nuclei of outside cells, and modulation of Tead4 or Yap activity leads to changes in Cdx2 expression. In inside cells, Yap is phosphorylated and cytoplasmic, and this involves the Hippo signaling pathway component Lats. We propose that active Tead4 promotes TE development in outside cells, whereas Tead4 activity is suppressed in inside cells by cell contact- and Lats-mediated inhibition of nuclear Yap localization. Thus, differential signaling between inside and outside cell populations leads to changes in cell fate specification during TE formation.

A functional interaction between Hippo-YAP signalling and FoxO1 mediates the oxidative stress response

The Hippo pathway is an evolutionarily conserved regulator of organ size and tumorigenesis that negatively regulates cell growth and survival. Here we report that YAP, the terminal effector of the Hippo pathway, interacts with FoxO1 in the nucleus of cardiomyocytes, thereby promoting survival. YAP and FoxO1 form a functional complex on the promoters of the catalase and MnSOD antioxidant genes and stimulate their transcription. Inactivation of YAP, induced by Hippo activation, suppresses FoxO1 activity and decreases antioxidant gene expression, suggesting that Hippo signaling modulates the FoxO1-mediated antioxidant response. In the setting of ischemia/reperfusion (I/R) in the heart, activation of Hippo antagonizes YAP-FoxO1, leading to enhanced oxidative stress-induced cell death through downregulation of catalase and MnSOD. Conversely, restoration of YAP activity protects against I/R injury. These results suggest that YAP is a nuclear co-factor of FoxO1 and that the Hippo pathway negatively affects cardiomyocyte survival by inhibiting the function of YAP-FoxO1.

Lats2 is a negative regulator of myocyte size in the heart

Mammalian sterile 20–like kinase (Mst)1 plays an important role in mediating apoptosis and inhibiting hypertrophy in the heart. Because Hippo, a Drosophila homolog of Mst1, forms a signaling complex with Warts, a serine/threonine kinase, which in turn stimulates cell death and inhibits cell proliferation, mammalian homologs of Warts, termed Lats1 and Lats2, may mediate the function of Mst1. We here show that Lats2, but not Lats1, dose-dependently increased apoptosis in cultured cardiac myocytes. Lats2 also dose-dependently reduced [3H]phenylalanine incorporation and cardiac myocyte size, whereas dominant negative Lats2 (DN-Lats2) increased them, suggesting that endogenous Lats2 negatively regulates myocyte growth. DN-Lats2 significantly attenuated induction of apoptosis and inhibition of hypertrophy by Mst1, indicating that Lats2 mediates the function of Mst1 in cardiac myocytes. Cardiac specific overexpression of Lats2 in transgenic mice significantly reduced the size of left and right ventricles, whereas that of DN-Lats2 caused hypertrophy in both ventricles. Overexpression of Lats2 reduced left ventricular systolic and diastolic function without affecting baseline levels of myocardial apoptosis. Expression of endogenous Lats2 was significantly upregulated in response to transverse aortic constriction. Overexpression of DN-Lats2 significantly enhanced cardiac hypertrophy and inhibited cardiac myocyte apoptosis induced by transverse aortic constriction. These results suggest that Lats2 is necessary and sufficient for negatively regulating ventricular mass in the heart. Although Lats2 is required for cardiac myocyte apoptosis in response to pressure overload, it was not sufficient to induce apoptosis at baseline. In conclusion, Lats2 affects both growth and death of cardiac myocytes, but it primarily regulates the size of the heart and acts as an endogenous negative regulator of cardiac hypertrophy.

Yes-associated Protein Isoform 1 (Yap1) Promotes Cardiomyocyte Survival and Growth to Protect against Myocardial Ischemic Injury

Background: Yap1 regulates cardiac development, yet the function of Yap1 in the adult heart remains unknown. Results: Yap1 promotes cardiomyocyte survival, hypertrophy, and proliferation and protects against chronic myocardial infarction (MI). Conclusion: Yap1 is critical for basal heart homeostasis, and Yap1 deficiency exacerbates myocardial injury. Significance: Increasing cardiomyocyte survival and proliferation may afford protection in vivo against MI injury. Yap1 is an important regulator of cardiomyocyte proliferation and embryonic heart development, yet the function of endogenous Yap1 in the adult heart remains unknown. We studied the role of Yap1 in maintaining basal cardiac function and in modulating injury after chronic myocardial infarction (MI). Cardiomyocyte-specific homozygous inactivation of Yap1 in the postnatal heart (YapF/FCre) elicited increased myocyte apoptosis and fibrosis, dilated cardiomyopathy, and premature death. Heterozygous deletion (Yap+/FCre) did not cause an overt cardiac phenotype compared with YapF/F control mice at base line. In response to stress (MI), nuclear Yap1 was found selectively in the border zone and not in the remote area of the heart. After chronic MI (28 days), Yap+/FCre mice had significantly increased myocyte apoptosis and fibrosis, with attenuated compensatory cardiomyocyte hypertrophy, and further impaired function versus Yap+/F control mice. Studies in isolated cardiomyocytes demonstrated that Yap1 expression is sufficient to promote increased cell size and hypertrophic gene expression and protected cardiomyocytes against H2O2-induced cell death, whereas Yap1 depletion attenuated phenylephrine-induced hypertrophy and augmented apoptosis. Finally, we observed a significant decrease in cardiomyocyte proliferation in Yap+/FCre hearts compared with Yap+/F controls after MI and demonstrated that Yap1 is sufficient to promote cardiomyocyte proliferation in isolated cardiomyocytes. Our findings suggest that Yap1 is critical for basal heart homeostasis and that Yap1 deficiency exacerbates injury in response to chronic MI.

Cancer susceptibility and embryonic lethality in Mob1a/1b double-mutant mice

Mps one binder 1a (MOB1A) and MOB1B are key components of the Hippo signaling pathway and are mutated or inactivated in many human cancers. Here we show that intact Mob1a or Mob1b is essential for murine embryogenesis and that loss of the remaining WT Mob1 allele in Mob1a(Δ/Δ)1b(tr/+) or Mob1a(Δ/+)1b(tr/tr) mice results in tumor development. Because most of these cancers resembled trichilemmal carcinomas, we generated double-mutant mice bearing tamoxifen-inducible, keratinocyte-specific homozygous-null mutations of Mob1a and Mob1b (kDKO mice). kDKO mice showed hyperplastic keratinocyte progenitors and defective keratinocyte terminal differentiation and soon died of malnutrition. kDKO keratinocytes exhibited hyperproliferation, apoptotic resistance, impaired contact inhibition, enhanced progenitor self renewal, and increased centrosomes. Examination of Hippo pathway signaling in kDKO keratinocytes revealed that loss of Mob1a/b altered the activities of the downstream Hippo mediators LATS and YAP1. Similarly, YAP1 was activated in some human trichilemmal carcinomas, and some of these also exhibited MOB1A/1B inactivation. Our results clearly demonstrate that MOB1A and MOB1B have overlapping functions in skin homeostasis, and exert their roles as tumor suppressors by regulating downstream elements of the Hippo pathway.

The Lats2 tumor suppressor augments p53-mediated apoptosis by promoting the nuclear proapoptotic function of ASPP1

Apoptosis is an important mechanism to eliminate potentially tumorigenic cells. The tumor suppressor p53 plays a pivotal role in this process. Many tumors harbor mutant p53, but others evade its tumor-suppressive effects by altering the expression of proteins that regulate the p53 pathway. ASPP1 (apoptosis-stimulating protein of p53-1) is a key mediator of the nuclear p53 apoptotic response. Under basal conditions, ASPP1 is cytoplasmic. We report that, in response to oncogenic stress, the tumor suppressor Lats2 (large tumor suppressor 2) phosphorylates ASPP1 and drives its translocation into the nucleus. Together, Lats2 and ASPP1 shunt p53 to proapoptotic promoters and promote the death of polyploid cells. These effects are overridden by the Yap1 (Yes-associated protein 1) oncoprotein, which disrupts Lats2-ASPP1 binding and antagonizes the tumor-suppressing function of the Lats2/ASPP1/p53 axis.

Lats2 kinase potentiates Snail1 activity by promoting nuclear retention upon phosphorylation

Snail1 is a central regulator of epithelial cell adhesion and movement in epithelial‐to‐mesenchymal transitions (EMTs) during embryo development; a process reactivated during cancer metastasis. While induction of Snail1 transcription precedes EMT induction, post‐translational regulation of Snail1 is also critical for determining Snail1s protein level, subcellular localization, and capacity to induce EMT. To identify novel post‐translational regulators of Snail1, we developed a live cell, bioluminescence‐based screen. From a human kinome RNAi screen, we have identified Lats2 kinase as a novel regulator of Snail1 protein level, subcellular localization, and thus, activity. We show that Lats2 interacts with Snail1 and directly phosphorylates Snail1 at residue T203. This occurs in the nucleus and serves to retain Snail1 in the nucleus thereby enhancing its stability. Lats2 was found to positively influence cellular EMT and tumour cell invasion, in a Snail1‐dependent manner. Indeed during TGFβ‐induced EMT Lats2 is activated and Snail1 phosphorylated at T203. Analysis in mouse and zebrafish embryo development confirms that Lats2 acts as a positive modulator of Snail1 protein level and potentiates its in vivo EMT activity.

Silencing of the Lats2 tumor suppressor overrides a p53-dependent oncogenic stress checkpoint and enables mutant H-Ras-driven cell transformation

The Lats2 tumor suppressor protein has been implicated earlier in promoting p53 activation in response to mitotic apparatus stress, by preventing Mdm2-driven p53 degradation. We now report that Lats2 also has a role in an ATR-Chk1-mediated stress check point in response to oncogenic H-Ras. Activated mutant H-Ras triggers the translocation of Lats2 from centrosomes into the nucleus, coupled with an increase in Lats2 protein levels. This leads to the induction of p53 activity, upregulation of proapoptotic genes, downregulation of antiapoptotic genes and eventually apoptotic cell death. Many of the cells that survive apoptosis undergo senescence. However, a fraction of the cells escape this checkpoint mechanism, despite maintaining a high mutant H-Ras expression. These escapers display increased genome instability, as evidenced by a substantial fraction of cells with micronuclei and cells with polyploid genomes. Interestingly, such cells show markedly reduced levels of Lats2, in conjunction with enhanced hypermethylation of the Lats2 gene promoter. Our findings suggest that Lats2 might have an important role in quenching H-Ras-induced transformation, whereas silencing of Lats2 expression might serve as a mechanism to enable tumor progression.

Nucleoredoxin sustains Wnt/β-catenin signaling by retaining a pool of inactive dishevelled protein.

Overexpression of Dishevelled (Dvl), an essential component of the Wnt signaling pathway, is frequently associated with tumors, and thus the Dvl protein level must be tightly controlled to sustain Wnt signaling without causing tumors. Kelch-like 12 (KLHL12) targets Dvl for ubiquitination and degradation, suggesting its potential importance in avoiding aberrant Dvl overexpression. However, the regulatory mechanism of the KLHL12 activity remained elusive. We show here that nucleoredoxin (NRX) determines the Dvl protein level, which is revealed by analyses on NRX(-/-) mice showing skeletal and cardiovascular defects. Consistent with the previously reported Dvl-inhibiting function of NRX, Wnt/β-catenin signaling is hyperactivated in NRX(-/-) osteoblasts. However, the signal activity is suppressed in cardiac cells, where KLHL12 is highly expressed. Biochemical analyses reveal that Dvl is rapidly degraded by accelerated ubiquitination in NRX(-/-) mouse embryonic fibroblasts, and they fail to activate Wnt/β-catenin signaling in response to Wnt ligands. Moreover, experiments utilizing purified proteins show that NRX expels KLHL12 from Dvl and inhibits ubiquitination. These findings reveal an unexpected function of NRX, retaining a pool of inactive Dvl for robust activation of Wnt/β-catenin signaling upon Wnt stimulation.

GAK, a regulator of clathrin-mediated membrane traffic, also controls centrosome integrity and chromosome congression.

Cyclin G-associated kinase (GAK) is an association partner of clathrin heavy chain (CHC) and is essential for clathrin-mediated membrane trafficking. Here, we report two novel functions of GAK: maintenance of proper centrosome maturation and of mitotic chromosome congression. Indeed, GAK knockdown by siRNA caused cell-cycle arrest at metaphase, which indicates that GAK is required for proper mitotic progression. We found that this impaired mitotic progression was due to activation of the spindle-assembly checkpoint, which senses protruded, misaligned or abnormally condensed chromosomes in GAK-siRNA-treated cells. GAK knockdown also caused multi-aster formation, which was due to abnormal fragmentation of pericentriolar material, but not of the centrioles. Moreover, GAK and CHC cooperated in the same pathway and interacted in mitosis to regulate the formation of a functional spindle. Taken together, we conclude that GAK and clathrin function cooperatively not only in endocytosis, but also in mitotic progression.