Adhesion Molecule-Mediated Hippo Pathway Modulates Hemangioendothelioma Cell Behavior

Hemangioendotheliomas are categorized as intermediate-grade vascular tumors that are commonly localized in the lungs and livers. The regulation of this tumor cell''s proliferative and apoptotic mechanisms is ill defined. We recently documented an important role for Hippo pathway signaling via endothelial cell adhesion molecules in brain microvascular endothelial cell proliferation and apoptosis. We found that endothelial cells lacking cell adhesion molecules escaped from contact inhibition and exhibited abnormal proliferation and apoptosis. Here we report on the roles of adherens junction molecule modulation of survivin and the Hippo pathway in the proliferation and apoptosis of a murine hemangioendothelioma (EOMA) cell. We demonstrated reduced adherens junction molecule (CD31 and VE-cadherin) expression, increased survivin and Ajuba expression, and a reduction in Hippo pathway signaling resulting in increased proliferation and decreased activation of effector caspase 3 in postconfluent EOMA cell cultures. Furthermore, we confirmed that YM155, an antisurvivin drug that interferes with Sp1-survivin promoter interactions, and survivin small interference RNA (siRNA) transfection elicited induction of VE-cadherin, decreased Ajuba expression, increased Hippo pathway and caspase activation and apoptosis, and decreased cell proliferation. These findings support the importance of the Hippo pathway in hemangioendothelioma cell proliferation and survival and YM155 as a potential therapeutic agent in this category of vascular tumors.     

CD44 acts through RhoA to regulate YAP signaling

The Hippo pathway plays an important role in both physical and pathogenesis processes. As crucial downstream effectors of Hippo pathway, YAP is inhibited by Lats1/2 through phosphorylation. However, upstream signals that regulate the Hippo pathway have been still poorly understood. Here, we found that knockdown of CD44 reduced YAP expression and nuclear localization, but nearly had no effect on its upstream effectors, Mst1 and Lats1. Downregulated CD44 expression also significantly decreased the expression of YAP downstream effectors CTGF, Cyr61 and EDN1 at mRNA level. Our next study showed that knockdown of CD44 inhibited RhoA expression, which was consistent with RhoA knockdown mediated YAP downregulation. Furthermore, we demonstrated that over expression of the constitutively active RhoA (RhoA-V14) could block the YAP expression decrease mediated by CD44 knockdown. Moreover, downregulation of CD44 significantly promoted cell apoptosis and inhibited cell proliferation, cell cycle progression and migration, which were consistent with the effects of RNAi-mediated YAP knockdown in both A549 and HepG2 cells. Overall, data are presented showing that CD44 could act through RhoA signaling to regulate YAP expression and this study also provide new insights into the regulatory mechanisms of the Hippo–YAP pathway.     

CD44: functional relevance to inflammation and malignancy

CD44 is a principal cell surface receptor for hyaluronan, a major component of extracellular matrices. Cells are surrounded by and encounter matrix in vivo, which in turn serves a variety of cell functions through the direct adhesion via their receptors. CD44 communicates cell-matrix interactions into the cell via "outside-in signaling" and has an important role in biological activities. The interaction of CD44 with fragmented hyaluronan on rheumatoid synovial cells induces expression of VCAM-1 and Fas on the cells, which leads to Fas-mediated apoptosis of synovial cells by the interaction of T cells bearing FasL. On the other hand, engagement of CD44 on tumor cells derived from lung cancer reduces Fas expression and Fas-mediated apoptosis, resulting in less susceptibility of the cells to CTL-mediated cytotoxicity through Fas-FasL pathway. Thus, although the CD44-mediated signaling differs among cells and circumstances, we here propose the functional role of CD44 in inflammatory processes and tumor susceptibility and the rational design of future therapeutic strategies including the exploitation of CD44-mediated pathway in vivo.     

Platelet-endothelial cell adhesion molecule-1 (CD31) recycles and induces cell growth inhibition on human tumor cell lines

CD31 (PECAM-1) is a 130-kDa member of the immunoglobulin gene superfamily expressed on endothelial cells, platelets, and most leukocytes. This report demonstrates by Western Blot and immunofluorescence that some human melanoma and adenocarcinoma cell lines express CD31 on the cell surface. The surface expression of CD31 was regulated by cell-cell contact, being higher on sparse and spontaneously detached cells. Indeed, fixing and permeabilizing tumor cells revealed a cytoplasmic pool, which was confirmed by confocal microscopy. Some of the plasma surface molecule is endocytosed following mAb binding. Engagement of CD31 on tumor cells via domain-3 inhibited proliferation by inducing cell apoptosis. On the other hand, apoptosis does not increase CD31 expression. Overall, these results indicate that there is an intracellular pool of CD31 on some tumor cells, which modulates CD31 surface expression and its role in cancer cell growth and metastasis. Thus, the expression of CD31 and its role in cell survival in some tumor cells appears to differ from endothelial cells.     

CD44 promotes resistance to apoptosis in murine colonic epithelium

Dysregulated expression of CD44 isoforms occurs consistently in colon carcinogenesis, and this change occurs also in most other types of cancer. One of the basic features of malignant transformation is the acquisition of resistance to apoptosis. We previously found that the colonic epithelium of mice, deficient in CD44 is predisposed to apoptosis. In this study, we asked whether the expression of CD44 alters the response of the colon to an apoptotic stimulus, and what are the mechanisms involved. For this, we assessed the susceptibility of the murine colon to apoptosis by total body irradiation to induce apoptosis. Apoptotic and concomitant changes relevant to the mechanisms of apoptosis were monitored by molecular markers of apoptosis. We found enhanced susceptibility to apoptosis in CD44 deficient colonic epithelium based on an increase in the number of apoptotic bodies, and activation of caspase 3. This was not associated with alterations in proliferations as shown by comparable Ki-67 expression and BrdU labeling. Furthermore, upregulated active caspase 3 in CD44 deficient colon was accompanied by concomitant molecular alterations in caspase 9 and not caspase 8, and this indicated the involvement of the mitochondrial pathway in apoptosis execution. Overall, this is the first report demonstrating CD44 mediated resistance to apoptosis in the colonic epithelium in vivo. This implicates CD44 in promoting cell transformation into a malignant phenotype, in conjunction with other anti-apoptotic factors.     

Evidence for the involvement of CD44 in endothelial cell injury and induction of vascular leak syndrome by IL-2.

At sites of chronic inflammation seen during infections, autoimmunity, graft-vs-host response, and cytokine therapy, endothelial cell injury is known to occur, the exact mechanism of which is unknown. In the current study we used IL-2-induced vascular leak syndrome (VLS) as a model to investigate whether cytotoxic lymphocytes use CD44 in mediating endothelial cell injury. Administration of IL-2 to wild-type mice triggered significant VLS in the lungs and liver. In contrast, in CD44 knockout (KO) mice, IL-2-induced VLS was markedly reduced in the lungs and liver. IL-2-treated wild-type and CD44 KO mice had similar levels of perivascular infiltration with lymphocytes in the lungs and liver. This suggested that the decrease in VLS seen in CD44 KO mice was not due to the inability of lymphocytes to migrate to these organs. Ultrastructural studies demonstrated extensive endothelial cell damage in the lungs and liver of IL-2-treated wild-type, but not CD44 KO, mice. Moreover, CD44-KO mice exhibited a marked decrease in IL-2-induced lymphokine-activated killer cell activity. The induction of VLS was dependent on the expression of CD44 on immune cells rather than endothelial cells because adoptive transfer of CD44+, but not CD44- spleen cells along with IL-2 into CD44 KO mice triggered VLS. The IL-2-induced VLS was blocked by administration of F(ab')2 of Abs against CD44. The current study demonstrates that CD44 plays a key role in endothelial cell injury. Blocking CD44 in vivo may offer a novel therapeutic approach to prevent endothelial cell injury by cytotoxic lymphocytes in a variety of clinical disease models.     

CD44-deficient mice exhibit enhanced hepatitis after concanavalin A injection: evidence for involvement of CD44 in activation-induced cell death

Administration of Con A induces severe injury to hepatocytes in mice and is considered to be a model for human hepatitis. In the current study, we investigated the role of CD44 in Con A-induced hepatitis. Intravenous administration of Con A (20 mg/kg) caused 100% mortality in C57BL/6 CD44-knockout (KO) mice, although it was not lethal in C57BL/6 CD44 wild-type (WT) mice. Administration of lower doses of Con A (12 mg/kg body weight) into CD44 WT mice induced hepatitis as evident from increased plasma aspartate aminotransferase levels accompanied by active infiltration of mononuclear cells and neutrophils, and significant induction of apoptosis in the liver. Interestingly, CD44 KO mice injected with similar doses of Con A exhibited more severe acute suppurative hepatitis. Transfer of spleen cells from Con A-injected CD44 KO mice into CD44 WT mice induced higher levels of hepatitis when compared with transfer of similar cells from CD44 WT mice into CD44 WT mice. The increased hepatitis seen in CD44 KO mice was accompanied by increased production of cytokines such as TNF-alpha, IL-2 and IFN-gamma, but not Fas or Fas ligand. The increased susceptibility of CD44 KO mice to hepatitis correlated with the observation that T cells from CD44 KO mice were more resistant to activation-induced cell death when compared with the CD44 WT mice. Together, these data demonstrate that activated T cells use CD44 to undergo apoptosis, and dysregulation in this pathway could lead to increased pathogenesis in a number of diseases, including hepatitis.     

CRISPR/Cas9介导的LATS1/2敲除抑制卵巢癌细胞ES-2增殖

Hippo信号通路在哺乳动物肝脏发育、动态平衡、再生和疾病中发挥非常重要的作用。大肿瘤抑制基因1/2（large tumor suppressor 1/2, LATS1/2）激酶是Hippo信号通路的关键激酶,可以磷酸化YES相关蛋白（yes-associated protein,YAP）,从而调节YAP的核质定位和降解。本文采用CRISPR/Cas9方法构建慢病毒介导的Last1/2基因敲除的载体,通过包装、感染和嘌呤霉素筛选,获得LATS1/2部分敲除的人卵巢癌ES-2和H08910细胞,免疫印迹方法检测LATS1/2表达明显减少。细胞增殖实验检测LATS1/2缺失明显抑制ES-2和HO8910细胞增殖。软琼脂克隆形成实验表明,LATS1/2缺失抑制卵巢癌ES-2细胞的克隆形成能力。细胞划痕和Transwell实验证明,LATS1/2缺失明显抑制卵巢癌ES-2细胞迁移。流式细胞检测发现,LATS1/2敲除促进卵巢癌ES-2细胞凋亡并影响细胞周期。裸鼠成瘤实验表明,LATS1/2缺失明显抑制体内肿瘤组织增殖。分子机制研究表明, LATS1/2敲除促进卵巢癌ES-2细胞中胶原I型α1（collagen type I α1,ColIα1）基因表达量增加,在卵巢癌ES-2细胞中同时敲除LATS1/2和COL1A1,可以促进细胞克隆形成。综上结果,人卵巢癌ES-2细胞中LATS1/2缺失能促进COL1A1表达增加, 从而抑制细胞增殖、转移和克隆形成,并影响细胞周期和促进细胞凋亡。     

哺乳动物Hippo信号通路:肿瘤治疗的新标靶

Hippo信号通路是首次在果蝇中发现具有调节细胞增殖与凋亡作用的信号通路。最近发现果蝇Hippo信号通路的组成、分子作用机制和生物学功能在进化过程中高度保守。Hippo信号通路在胚胎发育中对细胞的生长分化、组织器官形成以及成体干细胞的维持和自稳态的保持等方面具有重要作用。同时,Hippo信号通路与Wnt信号通路、Notch信号通路等相互作用、密切联系,在肿瘤的发生、发展过程中也起到关键作用。文章综述了哺乳动物Hippo信号通路的作用机理、与其他信号通路和蛋白质因子的相互联系及与肿瘤的关系,对于肿瘤的诊断、预防和治疗具有一定的参考价值。     

CD31 is required on CD4+ T cells to promote T cell survival during Salmonella infection

Hematopoietic cells constitutively express CD31/PECAM1, a signaling adhesion receptor associated with controlling responses to inflammatory stimuli. Although expressed on CD4(+) T cells, its function on these cells is unclear. To address this, we have used a model of systemic Salmonella infection that induces high levels of T cell activation and depends on CD4(+) T cells for resolution. Infection of CD31-deficient (CD31KO) mice demonstrates that these mice fail to control infection effectively. During infection, CD31KO mice have diminished numbers of total CD4(+) T cells and IFN-γ-secreting Th1 cells. This is despite a higher proportion of CD31KO CD4(+) T cells exhibiting an activated phenotype and an undiminished capacity to prime normally and polarize to Th1. Reduced numbers of T cells reflected the increased propensity of naive and activated CD31KO T cells to undergo apoptosis postinfection compared with wild-type T cells. Using adoptive transfer experiments, we show that loss of CD31 on CD4(+) T cells alone is sufficient to account for the defective CD31KO T cell accumulation. These data are consistent with CD31 helping to control T cell activation, because in its absence, T cells have a greater propensity to become activated, resulting in increased susceptibility to become apoptotic. The impact of CD31 loss on T cell homeostasis becomes most pronounced during severe, inflammatory, and immunological stresses such as those caused by systemic Salmonella infection. This identifies a novel role for CD31 in regulating CD4 T cell homeostasis.     

The tumor-suppressor gene fat controls tissue growth upstream of expanded in the hippo signaling pathway

BACKGROUND: The tight control of cell proliferation and cell death is essential to normal tissue development, and the loss of this control is a hallmark of cancers. Cell growth and cell death are coordinately regulated during development by the Hippo signaling pathway. The Hippo pathway consists of the Ste20 family kinase Hippo, the WW adaptor protein Salvador, and the NDR kinase Warts. Loss of Hippo signaling in Drosophila leads to enhanced cell proliferation and decreased apoptosis, resulting in massive tissue overgrowth through increased expression of targets such as Cyclin E and Diap1. The cytoskeletal proteins Merlin and Expanded colocalize at apical junctions and function redundantly upstream of Hippo. It is not clear how they regulate growth or how they are localized to apical junctions. RESULTS: We find that another Drosophila tumor-suppressor gene, the atypical cadherin fat, regulates both cell proliferation and cell death in developing imaginal discs. Loss of fat leads to increased Cyclin E and Diap1 expression, phenocopying loss of Hippo signaling. Ft can regulate Hippo phosphorylation, a measure of its activation, in tissue culture. Importantly, fat is needed for normal localization of Expanded at apical junctions in vivo. Genetic-epistasis experiments place fat with expanded in the Hippo pathway. CONCLUSIONS: Together, these data suggest that Fat functions as a cell-surface receptor for the Expanded branch of the conserved Hippo growth control pathway.     

Contact inhibition of VEGF-induced proliferation requires vascular endothelial cadherin,beta-catenin,and the phosphatase DEP-1/CD148

Confluent endothelial cells respond poorly to the proliferative signals of VEGF. Comparing isogenic endothelial cells differing for vascular endothelial cadherin (VE-cadherin) expression only, we found that the presence of this protein attenuates VEGF-induced VEGF receptor (VEGFR) 2 phosphorylation in tyrosine, p44/p42 MAP kinase phosphorylation, and cell proliferation. VE-cadherin truncated in beta-catenin but not p120 binding domain is unable to associate with VEGFR-2 and to induce its inactivation. beta-Catenin-null endothelial cells are not contact inhibited by VE-cadherin and are still responsive to VEGF, indicating that this protein is required to restrain growth factor signaling. A dominant-negative mutant of high cell density-enhanced PTP 1 (DEP-1)//CD148 as well as reduction of its expression by RNA interference partially restore VEGFR-2 phosphorylation and MAP kinase activation. Overall the data indicate that VE-cadherin-beta-catenin complex participates in contact inhibition of VEGF signaling. Upon stimulation with VEGF, VEGFR-2 associates with the complex and concentrates at cell-cell contacts, where it may be inactivated by junctional phosphatases such as DEP-1. In sparse cells or in VE-cadherin-null cells, this phenomenon cannot occur and the receptor is fully activated by the growth factor.     

Transportin Regulates Nuclear Import of CD44

CD44 is a facultative cell surface proteoglycan that serves as the principal cell surface receptor for hyaluronan (HA). Studies have shown that in addition to participating in numerous signaling pathways, CD44 becomes internalized upon engagement by ligand and that a portion of its intracellular domain can translocate to the nucleus where it is believed to play a functional role in cell proliferation and survival. However, the mechanisms whereby fragments of CD44 enter the nucleus have not been elucidated. Here we show that CD44 interacts with two import receptors of the importin β superfamily, importin β itself and transportin. Inhibition of importin β-dependent transport failed to block CD44 accumulation in the nucleus. By contrast, inhibition of the transportin-dependent pathway abrogated CD44 import. Mutagenesis of the intracellular domain of CD44 revealed that the 20 membrane-proximal residues contain sequences required for transportin-mediated nuclear transport. Our observations provide evidence that CD44 interacts with importin family members and identify the transportin-dependent pathway as the mechanism whereby full-length CD44 enters the nucleus.     

Hippo信号对卵巢生殖干细胞增殖及卵巢功能的影响

已有研究表明,Hippo信号通路对干细胞的自我更新和分化至关重要,且Hippo信号通路在调控卵泡生长中起重要作用,然而,目前关于Hippo通路对卵巢生殖干细胞的增殖和分化以及卵巢功能重塑的影响相关的研究较少。为了明确Hippo信号通路效应因子YAP1与卵巢生殖干细胞体外增殖分化的关系,以及Hippo信号通路对卵巢癌的主要功能。我们采用两步法酶促分离和磁性分离技术分别鉴定卵巢生殖干细胞,通过测定MVH和OCT4标记物的表达,然后选择YAP1作为Hippo信号通路的主要效应分子,作为研究的靶基因。将含有过表达的YAP1或YAP1靶向的shRNA的慢病毒转导入卵巢生殖干细胞中。通过将过表达YAP1或YAP1 shRNA的慢病毒载体微量注射到不育小鼠模型中,观察调节Hippo信号通路对卵巢的增殖、分化和内分泌功能的影响。研究结果表明,在分离的卵巢生殖干细胞中观察到YAP1和MVH的共表达。与对照组相比,过表达YAP1的卵巢生殖干细胞中MVH和OCT4表达水平显著增加。而YAP1敲低后,MVH和OCT4水平显著降低;不育小鼠模型中YAP1过表达15 d后,E2和FSH含量显著升高,而YAP1 shRNA表达后,小鼠血清E2和FSH含量显著降低。YAP1可用于调控卵巢生殖干细胞的增殖和分化以及小鼠的卵巢功能。本研究表明,Hippo信号通路可能是调控卵巢功能重建的一个新的分子靶点。     

Role of the CD95/CD95 ligand system in glucocorticoid-induced monocyte apoptosis

Glucocorticoids (GC) act as potent anti-inflammatory and immunosuppressive agents on a variety of immune cells. However, the exact mechanisms of their action are still unknown. Recently, we demonstrated that GC induce apoptosis in human peripheral blood monocytes. In the present study, we examined the signaling pathway in GC-induced apoptosis. Monocyte apoptosis was demonstrated by annexin V staining, DNA laddering, and electron microscopy. Apoptosis required the activation of caspases, as different caspase inhibitors prevented GC-induced cell death. In addition, the proteolytic activation of caspase-8 and caspase-3 was observed. In additional experiments, we determined the role of the death receptor CD95 in GC-induced apoptosis. CD95 and CD95 ligand (CD95L) were up-regulated in a dose- and time-dependent manner on the cell membrane and also released after treatment with GC. Costimulation with the GC receptor antagonist mifepristone diminished monocyte apoptosis as well as CD95/CD95L expression and subsequent caspase-8 and caspase-3 activation. In contrast, the caspase inhibitor N:-acetyl-Asp-Glu-Val-Asp-aldehyde suppressed caspase-3 activation and apoptosis, but did not down-regulate caspase-8 activation and expression of CD95 and CD95L. Importantly, GC-induced monocyte apoptosis was strongly abolished by a neutralizing CD95L mAb. Therefore, our data suggest that GC-induced monocyte apoptosis is at least partially mediated by an autocrine or paracrine pathway involving the CD95/CD95L system.     

CD34 positive cells isolated from traumatized human skeletal muscle require the CD34 protein for multi-potential differentiation

The CD34 protein is regarded as a marker of stem cells from multiple origins. Recently a mesenchymal progenitor CD34 positive cell identified from traumatized human skeletal muscle demonstrates differentiation capability into vascular endothelial cells, osteoblasts and adipocytes. Here they were treated with a small inhibitory RNA for CD34, which significantly reduced the cellular level of the CD34 protein. These treated cells had a reduced capacity to proliferate, and migrate. They were both unable to differentiation down multiple pathways and to undergo vascular endothelial differentiation as reflected by a lack of expression of VE cadherin, Tie 2 and CD31. Additionally the cells were unable to form tube-like structures in an endothelial tube assay. These treated cells were also unable to undergo osteogenesis, as revealed by lack of alizarin red and alkaline phosphatase staining and were unable to undergo adipogenesis as revealed by lack of oil red O staining. Finally, when CD34 was expressed in cells lacking this protein, the cells were able to undergo vascular endothelial differentiation as revealed by expression of Tie2, VE-cadherin and CD31. These data indicate that in cells derived from traumatized muscle the CD34 protein is required for enhanced proliferation, migration and differentiation down multiple pathways.