Organ Size Control Research Articles

Importance of the filamin A-Sav1 interaction in organ size control: evidence from transgenic mice.

The nucleocytoplasmic translocation of yes-associated protein 1 (YAP1) controls the growth of animal tissues and organs. YAP1 binds to transcription factors in the nucleus to activate the transcription of proliferation and anti-apoptotic genes. The Hippo pathway prevents the nuclear translocation of YAP1 by phosphorylating YAP1, while mechanical forces promote it by opening the nuclear pore complex and stimulating other signaling pathways. Recently we found that Protein salvador homolog 1 (SAV1), a component of the Hippo pathway, interacts with filamin A (FLNA) in a force-dependent manner, raising a possibility that the Hippo pathway is regulated by mechanical force through the FLNA-SAV1 axis. To test this hypothesis, we generated conditional knock-in (KI) mice expressing non-Flna-binding mutant Sav1 in hepatocytes by crossing with mice carrying Cre recombinase driven by the serum albumin (alb) gene promoter. Unexpectedly, the insertion of the flox cassette skipped exon 2, resulting in a shorter Sav1 in all the transgenic mice. Since exon 2 encodes a fragment containing a Flna-binding domain, we analyzed both point mutant KI and exon 2-deleted mutant mice. Here we show that disruption of the Flna-Sav1 interaction in the mouse liver promotes apoptosis and suppresses tissue and organ growth without affecting the phosphorylation level of Yap1. These results provide evidence that the growth of animal tissues and organs is regulated by apoptosis downstream of the force-dependent FLNA-SAV1 interaction, at least in part.

Small-Molecule Cyanamide Pan-TEAD·YAP1 Covalent Antagonists.

Transcriptional enhanced associate domains (TEADs) are transcription factors that bind to cotranscriptional activators like the yes-associated protein (YAP) or its paralog transcriptional coactivator with a PDZ-binding motif (TAZ). TEAD·YAP/TAZ target genes are involved in tissue and immune homeostasis, organ size control, tumor growth, and metastasis. Here, we report isoindoline and octahydroisoindole small molecules with a cyanamide electrophile that forms a covalent bond with a conserved cysteine in the TEAD palmitate-binding cavity. Time- and concentration-dependent studies against TEAD1-4 yielded second-order rate constants kinact/KI greater than 100 M-1 s-1. Compounds inhibited YAP1 binding to TEADs with submicromolar IC50 values. Cocrystal structures with TEAD2 enabled structure-activity relationship studies. In mammalian cells, compounds suppressed CTGF mRNA levels and inhibited TEAD1-4 transcriptional activity with submicromolar IC50 values. Inhibition of TEAD binding to YAP1 in mammalian cells was also observed. Several compounds inhibited the cell viability of sarcoma, hepatocellular carcinoma, glioblastoma, and breast cancer cells with single-digit micromolar IC50 values.

A positive feedback loop of the TAZ/β-catenin axis promotes Helicobacter pylori-associated gastric carcinogenesis.

Helicobacter pylori infection is the strongest known risk factor for gastric cancer. The Hippo signaling pathway controls organ size and maintains tissue homeostasis by coordinately regulating cell growth and proliferation. Here, we demonstrate the interactive role of TAZ, the transcriptional coactivator of the Hippo pathway, and beta-catenin in promoting the pathogenesis of H. pylori infection. TAZ expression was evaluated in human gastric tissues and H. pylori-infected insulin-gastrin (INS-GAS) mice. Western blot, immunofluorescence, immunohistochemistry, and RT-PCR assays were performed. Coimmunoprecipitation was performed to examine the interaction between TAZ and β-catenin. TAZ and β-catenin were silenced using small interfering RNAs. HA-β-catenin and Flag-TAZ were constructed. Increased TAZ was noted in human gastric cancer tissues compared to chronic gastritis tissues and in H. pylori-positive gastritis tissues compared to H. pylori-negative gastritis tissues. In addition, H. pylori infection induced TAZ expression and nuclear accumulation in the gastric tissue of INS-GAS mice and cultured gastric epithelial cells, which was dependent on the virulence factor CagA. Moreover, TAZ or β-catenin knockdown significantly suppressed H. pylori infection-induced cell growth, survival, and invasion. Furthermore, the interactive regulation of TAZ and β-catenin activation was revealed. Finally, β-catenin was required for H. pylori-induced TAZ activation. These findings suggest the existence of a positive feedback loop of activation between TAZ and β-catenin that could play an important role in CagA+ H. pylori infection-induced gastric carcinogenesis. TAZ inhibition represents a potential target for the prevention of H. pylori infection-associated gastric cancer.

An Anti-TAZ Monoclonal Antibody Recognizing Cell Surface Expressed TAZ Protein in Human Tumor Cells.

WWTR1 or TAZ is a transcriptional co-activator protein expressed in cytoplasm which functions as the main downstream effector of the Hippo signaling pathway. This pathway is an evolutionally conserved signal cascade, which plays a pivotal role in organ size control and tumorigenesis. Ectopic expression of TAZ has already been observed in many malignancies, while the ectopic localization of TAZ is reported for the first time. The aim of this study was to produce a specific monoclonal antibody (mAb) against a synthetic peptide derived from WWTR1 protein to be used as a research tool in human carcinomas. A 21-mer synthetic peptide (derived from human TAZ protein) was used for immunization of BALB/c mice after conjugation with Keyhole Limpet Haemocyanin (KLH) using hybridoma technology. The generated mAb reacted with the immunizing peptide employing ELISA assay. The reactivity of the antibody with native TAZ protein was assessed through Western blot, immunocytochemistry, and flow cytometry using different cancer cell lines. The produced mAb could recognize the immunizing peptide in ELISA and Kaff was 0.6×10-9 M. The produced anti-TAZ mAb unlike available commercial anti-TAZ antibody, was capable of specifically recognizing cell surface TAZ in human carcinoma cell lines including MCF-7, Raji, and A431 in Western blot, immunocytochemistry, and flow cytometry assays. As expected, no reactivity was observed using normal Peripheral Blood Mononuclear Cell (PBMC) from healthy donors. Based on the results, TAZ is ectopically expressed on the surface of tumor cell lines which is not the case in normal cells. The generated mAb has a potential to be used as a research tool in studying the expression of TAZ in human carcinomas in different applications.

Role of YAP as a Mechanosensing Molecule in Stem Cells and Stem Cell-Derived Hematopoietic Cells.

Yes-associated protein (YAP) and WW domain-containing transcription regulator protein 1 (WWTR1, also known as TAZ) are transcriptional coactivators in the Hippo signaling pathway. Both are well-known regulators of cell proliferation and organ size control, and they have significant roles in promoting cell proliferation and differentiation. The roles of YAP and TAZ in stem cell pluripotency and differentiation have been extensively studied. However, the upstream mediators of YAP and TAZ are not well understood. Recently, a novel role of YAP in mechanosensing and mechanotransduction has been reported. The present review updates information on the regulation of YAP by mechanical cues such as extracellular matrix stiffness, fluid shear stress, and actin cytoskeleton tension in stem cell behaviors and differentiation. The review explores mesenchymal stem cell fate decisions, pluripotent stem cells (PSCs), self-renewal, pluripotency, and differentiation to blood products. Understanding how cells sense their microenvironment or niche and mimic those microenvironments in vitro could improve the efficiency of producing stem cell products and the efficacy of the products.

Kidney tubular transcription co-activator, Yes-associated protein 1 (YAP), controls the expression of collecting duct aquaporins and water homeostasis

Final urine volume and concentration are defined by water reabsorption through the water channel proteins aquaporin (AQP)-2, -3 and -4 in the collecting duct. However, the transcriptional regulation of these AQPs is not well understood. The Hippo/Yes-associated protein 1 (YAP) pathway plays an important role in organ size control and tissue homeostasis. When the Hippo pathway including the Mst1/Mst2 kinases is inhibited, YAP is activated and functions as a transcription co-activator. Our previous work revealed a pathological role of tubular YAP activation in chronic kidney disease, but the physiological role of YAP in the kidney remains to be established. Here, we found that tubule-specific Yap knockout mice showed increased urine output and decreased urinary osmolality. Decreases in Aqp2, -3 and -4 mRNA and protein abundance in the kidney were evident in Yap knockout mice. Analysis of Mst1/Mst2 double knockout and Mst1/Mst2/Yap triple knockout mice showed that expression of Aqp2 and Aqp4 but not Aqp3 was dependent on YAP. Furthermore, YAP was recruited to the promoters of the Aqp2 and Aqp4 genes and stimulated their transcription. Interestingly, YAP was found to interact with transcription factors GATA2, GATA3 and NFATc1. These three factors promoted Aqp2 transcription in a YAP dependent manner in collecting duct cells. These three factors also promoted Aqp4 transcription whereas only GATA2 and GATA3 enhanced Aqp3 transcription. Thus, our results suggest that YAP promotes Aqp2 and Aqp4 transcription, interacts with GATA2, GATA3 and NFATc1 to control Aqp2 expression, while Aqp-2, -3 and -4 exploit overlapping mechanisms for their baseline transcriptional regulation.

Ptp61F integrates Hippo, TOR, and actomyosin pathways to control three-dimensional organ size.

Precise organ size control is fundamental for all metazoans, but how organ size is controlled in a three-dimensional (3D) way remains largely unexplored at the molecular level. Here, we screen and identify Drosophila Ptp61F as a pivotal regulator of organ size that integrates the Hippo pathway, TOR pathway, and actomyosin machinery. Pathologically, Ptp61F loss synergizes with RasV12 to induce tumorigenesis. Physiologically, Ptp61F depletion increases body size and drives neoplastic intestinal tumor formation and stem cell proliferation. Ptp61F also regulates cell contractility and myosin activation and controls 3D cell shape by reducing cell height and horizontal cell size. Mechanistically, Ptp61F forms a complex with Expanded (Ex) and increases endosomal localization of Ex and Yki. Furthermore, we demonstrate that PTPN2, the conserved human ortholog of Ptp61F, can functionally substitute for Ptp61F in Drosophila. Our work defines Ptp61F as an essential determinant that controls 3D organ size under both physiological and pathological conditions.

Myotubularin functions through actomyosin to interact with the Hippo pathway.

The Hippo pathway is an evolutionarily conserved developmental pathway that controls organ size by integrating diverse regulatory inputs, including actomyosin-mediated cytoskeletal tension. Despite established connections between the actomyosin cytoskeleton and the Hippo pathway, the upstream regulation of actomyosin in the Hippo pathway is less defined. Here, we identify the phosphoinositide-3-phosphatase Myotubularin (Mtm) as a novel upstream regulator of actomyosin that functions synergistically with the Hippo pathway during growth control. Mechanistically, Mtm regulates membrane phospholipid PI(3)P dynamics, which, in turn, modulates actomyosin activity through Rab11-mediated vesicular trafficking. We reveal PI(3)P dynamics as a novel mode of upstream regulation of actomyosin and establish Rab11-mediated vesicular trafficking as a functional link between membrane lipid dynamics and actomyosin activation in the context of growth control. Our study also shows that MTMR2, the human counterpart of Drosophila Mtm, has conserved functions in regulating actomyosin activity and tissue growth, providing new insights into the molecular basis of MTMR2-related peripheral nerve myelination and human disorders.

Immune function analysis of LsSd, a transcription factor of the Hippo signaling pathway, in the cigarette beetle Lasioderma serricorne.

The Scalloped (Sd) is a transcription factor that regulates organ size control in the Hippo-signaling pathway. Recent studies have showed that Hippo signaling also functions in the innate immune response. Although the Sd gene has been reported in many insects, their immune functions remain unexplored. In this study, the LsSd gene of Lasioderma serricorne, with a complete open reading frame that encodes a protein composed of 402 amino acids was identified. LsSd was predominantly expressed in early pupae. Tissue-specific analyses revealed that the highest concentrations of LsSd were detected in the midgut and brain. At 1–24 h after Escherichia coli infection, LsSd expression increased substantially. However, LsSd expression was downregulated 3–12 h after Staphylococcus aureus infection. RNA interference-mediated silencing of the LsSd transcript resulted in deformed, considerably smaller, and degenerated wings. Meanwhile, LsCycE, LsDiap1, and LsVg, which are involved in cell proliferation and wing development, were drastically reduced when LsSd was depleted. In a survival assay, the LsSd knockdown considerably decreased the susceptibility to S. aureus, a gram-positive bacterium. In addition, knockdown of LsSd remarkably downregulated the transcription of LsCact in response to S. aureus stimulation, while upregulating the expression of five immune-related genes. Our results provide conclusive proof for the important roles of LsSd in the immune response of L. serricorne.

Bip-Yorkie interaction determines oncogenic and tumor-suppressive roles of Ire1/Xbp1s activation

Unfolded protein response (UPR) is the mechanism by which cells control endoplasmic reticulum (ER) protein homeostasis. ER proteostasis is essential to adapt to cell proliferation and regeneration in development and tumorigenesis, but mechanisms linking UPR, growth control, and cancer progression remain unclear. Here, we report that the Ire1/Xbp1s pathway has surprisingly oncogenic and tumor-suppressive roles in a context-dependent manner. Activation of Ire1/Xbp1s up-regulates their downstream target Bip, which sequesters Yorkie (Yki), a Hippo pathway transducer, in the cytoplasm to restrict Yki transcriptional output. This regulation provides an endogenous defensive mechanism in organ size control, intestinal homeostasis, and regeneration. Unexpectedly, Xbp1 ablation promotes tumor overgrowth but suppresses invasiveness in a Drosophila cancer model. Mechanistically, hyperactivated Ire1/Xbp1s signaling in turn induces JNK-dependent developmental and oncogenic cell migration and epithelial-mesenchymal transition (EMT) via repression of Yki. In humans, a negative correlation between XBP1 and YAP (Yki ortholog) target gene expression specifically exists in triple-negative breast cancers (TNBCs), and those with high XBP1 or HSPA5 (Bip ortholog) expression have better clinical outcomes. In human TNBC cell lines and xenograft models, ectopic XBP1s or HSPA5 expression alleviates tumor growth but aggravates cell migration and invasion. These findings uncover a conserved crosstalk between the Ire1/Xbp1s and Hippo signaling pathways under physiological settings, as well as a crucial role of Bip-Yki interaction in tumorigenesis that is shared from Drosophila to humans.

Targeting the Hippo Pathway in Gastric Cancer and Other Malignancies in the Digestive System: From Bench to Bedside.

The Hippo pathway is an evolutionally conserved signaling cascade that controls organ size and tissue regeneration under physiological conditions, and its aberrations have been well studied to promote tumor initiation and progression. Dysregulation of the Hippo tumor suppressor signaling frequently occurs in gastric cancer (GC) and other solid tumors and contributes to cancer development through modulating multiple aspects, including cell proliferation, survival, metastasis, and oncotherapy resistance. In the clinic, Hippo components also possess diagnostic and prognostic values for cancer patients. Considering its crucial role in driving tumorigenesis, targeting the Hippo pathway may greatly benefit developing novel cancer therapies. This review summarizes the current research progress regarding the core components and regulation of the Hippo pathway, as well as the mechanism and functional roles of their dysregulation in gastrointestinal malignancies, especially in GC, and discusses the therapeutic potential of targeting the Hippo pathway against cancers.

A Dilp8-dependent time window ensures tissue size adjustment in Drosophila.

The control of organ size mainly relies on precise autonomous growth programs. However, organ development is subject to random variations, called developmental noise, best revealed by the fluctuating asymmetry observed between bilateral organs. The developmental mechanisms ensuring bilateral symmetry in organ size are mostly unknown. In Drosophila, null mutations for the relaxin-like hormone Dilp8 increase wing fluctuating asymmetry, suggesting that Dilp8 plays a role in buffering developmental noise. Here we show that size adjustment of the wing primordia involves a peak of dilp8 expression that takes place sharply at the end of juvenile growth. Wing size adjustment relies on a cross-organ communication involving the epidermis as the source of Dilp8. We identify ecdysone signaling as both the trigger for epidermal dilp8 expression and its downstream target in the wing primordia, thereby establishing reciprocal hormonal feedback as a systemic mechanism, which controls organ size and bilateral symmetry in a narrow developmental time window.

LATS1/2 control TGFB-directed epithelial-to-mesenchymal transition in the murine dorsal cranial neuroepithelium through YAP regulation.

Hippo signaling, an evolutionarily conserved kinase cascade involved in organ size control, plays key roles in various tissue developmental processes, but its role in craniofacial development remains poorly understood. Using the transgenic Wnt1-Cre2 driver, we inactivated the Hippo signaling components Lats1 and Lats2 in the cranial neuroepithelium of mouse embryos and found that the double conditional knockout (DCKO) of Lats1/2 resulted in neural tube and craniofacial defects. Lats1/2 DCKO mutant embryos had microcephaly with delayed and defective neural tube closure. Furthermore, neuroepithelial cell shape and architecture were disrupted within the cranial neural tube in Lats1/2 DCKO mutants. RNA sequencing of embryonic neural tubes revealed increased TGFB signaling in Lats1/2 DCKO mutants. Moreover, markers of epithelial-to-mesenchymal transition (EMT) were upregulated in the cranial neural tube. Inactivation of Hippo signaling downstream effectors, Yap and Taz, suppressed neuroepithelial defects, aberrant EMT and TGFB upregulation in Lats1/2 DCKO embryos, indicating that LATS1/2 function via YAP and TAZ. Our findings reveal important roles for Hippo signaling in modulating TGFB signaling during neural crest EMT.

Identification of a Yorkie homolog from Litopenaeus vannamei as a negative regulator in anti-WSSV immune response

Hippo signaling pathway is a serine threonine kinase cascade that is evolutionary conserved with well-established roles in organ size control, development, tumorigenesis and immunity. As its core molecule, Yorkie also plays an important role against pathogen. In this study, we cloned and characterized a Yorkie homolog from Litopenaeus vannamei, designed as LvYKI, which has a 1650 bp open reading frame. It has the characterized domains of Yokie family, and displayed to be close to the insects and crustacean. Quantitative Real-time PCR showed that LvYKI had different regulatory mechanisms in different tissues. The transcriptional level of Lvyki was down-regulated in gill, while up-regulated in hepatopancreas post white spot syndrome virus (WSSV) infection. Moreover, the expression and phosphorylation of LvYKI was reduced upon WSSV infection, which indicated that LvYKI was involved in WSSV infection. Furthermore, RNAi was performed to evaluate the role of LvYKI in shrimp immune responses. Knocking down of Lvyki resulted in inhibition of the transcription of WSSV gene ie1 and vp28, and delayed mortality of shrimp post WSSV infection. Meanwhile, the apoptosis of hemocyte was increased as well. All results suggested that shrimp can promote apoptosis to resist WSSV infection mediated by down-regulation of LvYKI. In addition, it was found that LvYKI could interact with Lvβ-catenin, which cross-linked the Wnt and Hippo signaling pathway in innate immunity. Conclusively, our study provided clues that LvYKI plays an important role in the interaction between shrimp and virus. It will promote our understanding of the molecular mechanism in innate immunity.

135 Transcription coactivators YAP and TAZ are essential for postnatal maturation of the dermal extracellular matrix in mouse skin

YAP and TAZ are critical downstream effectors of the hippo signaling pathway, which play pivotal roles in controlling organ size and maintaining tissue homeostasis. Although dysregulation of YAP/TAZ has been linked to various human diseases their role in dermal extracellular matrix (ECM) regulation remains largely unknown. Here, we describe the role of YAP/TAZ in mouse dermal ECM maturation during early postnatal development We find that from birth (P0) to postnatal day 20 (P20), skin surface area increases 6-fold (N=6).

Hippo pathway regulation by phosphatidylinositol transfer protein and phosphoinositides.

The Hippo pathway plays a key role in development, organ size control and tissue homeostasis, and its dysregulation contributes to cancer. The LATS tumor suppressor kinases phosphorylate and inhibit the YAP/TAZ transcriptional co-activators to suppress gene expression and cell growth. Through a screen of marine natural products, we identified microcolin B (MCB) as a Hippo activator that preferentially kills YAP-dependent cancer cells. Structure-activity optimization yielded more potent MCB analogs, which led to the identification of phosphatidylinositol transfer proteins α and β (PITPα/β) as the direct molecular targets. We established a critical role of PITPα/β in regulating LATS and YAP. Moreover, we showed that PITPα/β influence the Hippo pathway via plasma membrane phosphatidylinositol-4-phosphate. This study uncovers a previously unrecognized role of PITPα/β in Hippo pathway regulation and as potential cancer therapeutic targets.

TEAD4 as an Oncogene and a Mitochondrial Modulator.

TEAD4 (TEA Domain Transcription Factor 4) is well recognized as the DNA-anchor protein of YAP transcription complex, which is modulated by Hippo, a highly conserved pathway in Metazoa that controls organ size through regulating cell proliferation and apoptosis. To acquire full transcriptional activity, TEAD4 requires co-activator, YAP (Yes-associated protein) or its homolog TAZ (transcriptional coactivator with PDZ-binding motif) the signaling hub that relays the extracellular stimuli to the transcription of target genes. Growing evidence suggests that TEAD4 also exerts its function in a YAP-independent manner through other signal pathways. Although TEAD4 plays an essential role in determining that differentiation fate of the blastocyst, it also promotes tumorigenesis by enhancing metastasis, cancer stemness, and drug resistance. Upregulation of TEAD4 has been reported in several cancers, including colon cancer, gastric cancer, breast cancer, and prostate cancer and serves as a valuable prognostic marker. Recent studies show that TEAD4, but not other members of the TEAD family, engages in regulating mitochondrial dynamics and cell metabolism by modulating the expression of mitochondrial- and nuclear-encoded electron transport chain genes. TEAD4’s functions including oncogenic activities are tightly controlled by its subcellular localization. As a predominantly nuclear protein, its cytoplasmic translocation is triggered by several signals, such as osmotic stress, cell confluency, and arginine availability. Intriguingly, TEAD4 is also localized in mitochondria, although the translocation mechanism remains unclear. In this report, we describe the current understanding of TEAD4 as an oncogene, epigenetic regulator and mitochondrial modulator. The contributing mechanisms will be discussed.

Modulation of the DA1 pathway in maize shows that translatability of information from Arabidopsis to crops is complex

Modern agriculture is struggling to meet the increasing food, silage and raw material demands due to the rapid growth of population and climate change. In Arabidopsis, DA1 and DAR1 are proteases that negatively regulate cell proliferation and control organ size. DA1 and DAR1 are activated by ubiquitination catalyzed by the E3 ligase BIG BROTHER (BB). Here, we characterized the DA1, DAR1 and BB gene families in maize and analyzed whether perturbation of these genes regulates organ size similar to what was observed in Arabidopsis. We generated da1_dar1a_dar1b triple CRISPR maize mutants and bb1_bb2 double mutants. Detailed phenotypic analysis showed that the size of leaf, stem, cob, and seed was not consistently enlarged in these mutants. Also overexpression of a dominant-negative DA1R333K allele, resembling the da1-1 allele of Arabidopsis which has larger leaves and seeds, did not alter the maize phenotype. The mild negative effects on plant height of the DA1R333K_bb1_bb2 mutant indicate that the genes in the DA1 pathway may control organ size in maize, albeit less obvious than in Arabidopsis.

The unfolding of the Hippo signaling pathway

The development of a functional organ requires not only patterning mechanisms that confer proper identities to individual cells, but also growth-regulatory mechanisms that specify the final size of the organ. At the turn of the 21st century, comprehensive genetic screens in model organisms had successfully uncovered the major signaling pathways that mediate pattern formation in metazoans. In contrast, signaling pathways dedicated to growth control were less explored. The past two decades has witnessed the emergence of the Hippo signaling pathway as a central mediator of organ size control through coordinated regulation of cell proliferation and apoptosis. Here I reflect on the early discoveries in Drosophila that elucidated the core kinase cascade and transcriptional machinery of the Hippo pathway, highlight its deep evolutionary conservation from humans to unicellular relatives of metazoan, and discuss the complex regulation of Hippo signaling by upstream inputs. This historical perspective underscores the importance of model organisms in uncovering fundamental and universal mechanisms of life processes.

Emerging roles of the Hippo signaling pathway in modulating immune response and inflammation-driven tissue repair and remodeling.

Inflammation is an evolutionarily conserved process and part of the body's defense mechanism. Inflammation leads to the activation of immune and non-immune cells that protect the host tissue/organs from injury or intruding pathogens. The Hippo pathway is an evolutionarily conserved kinase cascade with an established role in regulating cell proliferation, survival, and differentiation. It is involved in diverse biological processes, including organ size control and tissue homeostasis. Recent clinical and pre-clinical studies have shown that the Hippo signaling pathway is also associated with injury- and pathogen-induced tissue inflammation and associated immunopathology. In this review, we have summarized the recent findings related to the involvement of the Hippo signaling pathway in modulating the immune response in different acute and chronic inflammatory diseases and its impact on tissue repair and remodeling.