Ancestral Vascular Lumen Formation via Basal Cell Surfaces

The cardiovascular system of bilaterians developed from a common ancestor. However, no endothelial cells exist in invertebrates demonstrating that primitive cardiovascular tubes do not require this vertebrate-specific cell type in order to form. This raises the question of how cardiovascular tubes form in invertebrates? Here we discovered that in the invertebrate cephalochordate amphioxus, the basement membranes of endoderm and mesoderm line the lumen of the major vessels, namely aorta and heart. During amphioxus development a laminin-containing extracellular matrix (ECM) was found to fill the space between the basal cell surfaces of endoderm and mesoderm along their anterior-posterior (A-P) axes. Blood cells appear in this ECM-filled tubular space, coincident with the development of a vascular lumen. To get insight into the underlying cellular mechanism, we induced vessels in vitro with a cell polarity similar to the vessels of amphioxus. We show that basal cell surfaces can form a vascular lumen filled with ECM, and that phagocytotic blood cells can clear this luminal ECM to generate a patent vascular lumen. Therefore, our experiments suggest a mechanism of blood vessel formation via basal cell surfaces in amphioxus and possibly in other invertebrates that do not have any endothelial cells. In addition, a comparison between amphioxus and mouse shows that endothelial cells physically separate the basement membranes from the vascular lumen, suggesting that endothelial cells create cardiovascular tubes with a cell polarity of epithelial tubes in vertebrates and mammals.     

Cyclosporin A Disrupts Notch Signaling and Vascular Lumen Maintenance

Cyclosporin A (CSA) suppresses immune function by blocking the cyclophilin A and calcineurin/NFAT signaling pathways. In addition to immunosuppression, CSA has also been shown to have a wide range of effects in the cardiovascular system including disruption of heart valve development, smooth muscle cell proliferation, and angiogenesis inhibition. Circumstantial evidence has suggested that CSA might control Notch signaling which is also a potent regulator of cardiovascular function. Therefore, the goal of this project was to determine if CSA controls Notch and to dissect the molecular mechanism(s) by which CSA impacts cardiovascular homeostasis. We found that CSA blocked JAG1, but not Dll4 mediated Notch1 NICD cleavage in transfected 293T cells and decreased Notch signaling in zebrafish embryos. CSA suppression of Notch was linked to cyclophilin A but not calcineurin/NFAT inhibition since N-MeVal-4-CsA but not FK506 decreased Notch1 NICD cleavage. To examine the effect of CSA on vascular development and function, double transgenic Fli1-GFP/Gata1-RFP zebrafish embryos were treated with CSA and monitored for vasculogenesis, angiogenesis, and overall cardiovascular function. Vascular patterning was not obviously impacted by CSA treatment and contrary to the anti-angiogenic activity ascribed to CSA, angiogenic sprouting of ISV vessels was normal in CSA treated embryos. Most strikingly, CSA treated embryos exhibited a progressive decline in blood flow that was associated with eventual collapse of vascular luminal structures. Vascular collapse in zebrafish embryos was partially rescued by global Notch inhibition with DAPT suggesting that disruption of normal Notch signaling by CSA may be linked to vascular collapse. However, multiple signaling pathways likely cause the vascular collapse phenotype since both cyclophilin A and calcineurin/NFAT were required for normal vascular function. Collectively, these results show that CSA is a novel inhibitor of Notch signaling and vascular function in zebrafish embryos.     

An Initial Step of GAS-Containing Autophagosome-Like Vacuoles Formation Requires Rab7

Group A streptococcus (GAS; Streptococcus pyogenes) is a common pathogen that invades non-phagocytic human cells via endocytosis. Once taken up by cells, it escapes from the endocytic pathway to the cytoplasm, but here it is contained within a membrane-bound structure termed GAS-containing autophagosome-like vacuoles (GcAVs). The autophagosome marker GFP-LC3 associates with GcAVs, and other components of the autophagosomal pathway are involved in GcAV formation. However, the mechanistic relationship between GcAV and canonical autophagy is largely unknown. Here, we morphologically analyzed GcAV formation in detail. Initially, a small, GFP-LC3-positive GcAV sequesters each streptococcal chain, and these then coalesce into a single, large GcAV. Expression of a dominant-negative form of Rab7 or RNAi-mediated knockdown of Rab7 prevented the initial formation of small GcAV structures. Our results demonstrate that mechanisms of GcAV formation includes not only the common machinery of autophagy, but also Rab7 as an additional component, which is dispensable in canonical autophagosome formation.     

EGFL7与肿瘤研究新进展

EGFL7蛋白为一种内皮细胞特异性分泌因子,它是血管管腔形成所必需的因子,它的缺乏将导致管腔形成受阻,从而影响血管功能的完善.其在早期胚胎的血管中有较强的表达,而在成年人仅在少数器官(如:心脏、肺脏、肾脏)和肿瘤、炎症组织中有高水平表达.在肿瘤的生长转移过程中新生血管的作用十分重大,阻断肿瘤新生血管EGFL7的表达将有助于抑制肿瘤生长和转移,为肿瘤治疗提供一个新的途径.     

Medin Oligomer Membrane Pore Formation: A Potential Mechanism of Vascular Dysfunction

Medin, a 50-amino-acid cleavage product of the milk fat globule-EGF factor 8 protein, is one of the most common forms of localized amyloid found in the vasculature of individuals older than 50 years. Medin induces endothelial dysfunction and vascular inflammation, yet despite its prevalence in the human aorta and multiple arterial beds, little is known about the nature of its pathology. Medin oligomers have been implicated in the pathology of aortic aneurysm, aortic dissection, and more recently, vascular dementia. Recent in vitro biomechanical measurements found increased oligomer levels in aneurysm patients with altered aortic wall integrity. Our results suggest an oligomer-mediated toxicity mechanism for medin pathology. Using lipid bilayer electrophysiology, we show that medin oligomers induce ionic membrane permeability by pore formation. Pore activity was primarily observed for preaggregated medin species from the growth-phase and rarely for lag-phase species. Atomic force microscopy (AFM) imaging of medin aggregates at different stages of aggregation revealed the gradual formation of flat domains resembling the morphology of supported lipid bilayers. Transmission electron microscopy images showed the coexistence of compact oligomers, largely consistent with the AFM data, and larger protofibrillar structures. Circular dichroism spectroscopy revealed the presence of largely disordered species and suggested the presence of β-sheets. This observation and the significantly lower thioflavin T fluorescence emitted by medin aggregates compared to amyloid-β fibrils, along with the absence of amyloid fibers in the AFM and transmission electron microscopy images, suggest that medin aggregation into pores follows a nonamyloidogenic pathway. In silico modeling by molecular dynamics simulations provides atomic-level structural detail of medin pores with the CNpNC barrel topology and diameters comparable to values estimated from experimental pore conductances.     

Distinct origins of adult and embryonic blood in Xenopus

Whether embryonic and adult blood derive from a single (yolk sac) or dual (yolk sac plus intraembryonic) origin is controversial. Here, we show, in Xenopus, that the yolk sac (VBI) and intraembryonic (DLP) blood compartments derive from distinct blastomeres in the 32-cell embryo. The first adult hematopoietic stem cells (HSCs) are thought to form in association with the floor of the dorsal aorta, and we have detected such aortic clusters in Xenopus using hematopoietic markers. Lineage tracing shows that the aortic clusters derive from the blastomere that gives rise to the DLP. These observations indicate that the first adult HSCs arise independently of the embryonic lineage.     

Distinct Lipid A Moieties Contribute to Pathogen-Induced Site-Specific Vascular Inflammation

Several successful pathogens have evolved mechanisms to evade host defense, resulting in the establishment of persistent and chronic infections. One such pathogen, Porphyromonas gingivalis, induces chronic low-grade inflammation associated with local inflammatory bone loss and systemic inflammation manifested as atherosclerosis. P. gingivalis expresses an atypical lipopolysaccharide (LPS) structure containing heterogeneous lipid A species, that exhibit Toll-like receptor-4 (TLR4) agonist or antagonist activity, or are non-activating at TLR4. In this study, we utilized a series of P. gingivalis lipid A mutants to demonstrate that antagonistic lipid A structures enable the pathogen to evade TLR4-mediated bactericidal activity in macrophages resulting in systemic inflammation. Production of antagonistic lipid A was associated with the induction of low levels of TLR4-dependent proinflammatory mediators, failed activation of the inflammasome and increased bacterial survival in macrophages. Oral infection of ApoE^−/− mice with the P. gingivalis strain expressing antagonistic lipid A resulted in vascular inflammation, macrophage accumulation and atherosclerosis progression. In contrast, a P. gingivalis strain producing exclusively agonistic lipid A augmented levels of proinflammatory mediators and activated the inflammasome in a caspase-11-dependent manner, resulting in host cell lysis and decreased bacterial survival. ApoE^−/− mice infected with this strain exhibited diminished vascular inflammation, macrophage accumulation, and atherosclerosis progression. Notably, the ability of P. gingivalis to induce local inflammatory bone loss was independent of lipid A expression, indicative of distinct mechanisms for induction of local versus systemic inflammation by this pathogen. Collectively, our results point to a pivotal role for activation of the non-canonical inflammasome in P. gingivalis infection and demonstrate that P. gingivalis evades immune detection at TLR4 facilitating chronic inflammation in the vasculature. These studies support the emerging concept that pathogen-mediated chronic inflammatory disorders result from specific pathogen-mediated evasion strategies resulting in low-grade chronic inflammation.     

Expression of EGFL7 in primordial germ cells and in adult ovaries and testes

We have previously reported the isolation and characterization of a novel endothelial-restricted gene, Egfl7, that encodes a secreted protein of about 30-kDa. We and others demonstrated that Egfl7 is highly expressed by endothelial cells during embryonic development and becomes down-regulated in the adult vasculature. In the present paper, we show that during mouse embryonic development, Egfl7 is also expressed by primordial germ cells (PGC). Expression is down-regulated when PGCs differentiate into pro-spermatogonia and oogonia, and by 15.5 dpc Egfl7 can no longer be detected in the germ line of both sexes. Notably, Egfl7 is again transiently up-regulated in germ cells of the adult testis. In contrast, expression in the ovary remains limited to the vascular endothelium. Our results provide the first evidence of a non-endothelial expression of EGFL7 and suggest distinctive roles for Egfl7 in vascular development and germ cell differentiation.     

EGFL7 基因RNAi慢病毒表达载体的构建及鉴定

目的：构建人类表皮生长因子域7（EGFL7）基因RNA干扰（RNAi）重组慢病毒表达载体。方法：参照小分子干扰RNA 的设 计原则,应用OligoDesigner 3.0 软件设计三条靶向人EGFL7 基因的RNA干扰序列（hEGFL7-RNAi）,并将其分别插入含有绿色 荧光蛋白（GFP）的慢病毒载体pLV3 中,获得重组质粒,与包装质粒pRsv-REV、pMDlg-pRRE 和pMD2G共同转染293T细胞,包 装产生重组慢病毒,培养72 h后,应用qRT-PCR 检测慢病毒感染人脐静脉内皮细胞（HUVEC）后靶基因mRNA 的水平,以评价 其基因沉默效果。结果：筛选出3 条人EGFL7 基因的RNAi 序列,分别包装出重组慢病毒,其滴度分别为1× 108、2× 108和5× 108TU/mL,将其转染入HUVEC 后,EGFL7mRNA表达均受到明显抑制(P<0.05)。结论：成功筛选出三条针对人EGFL7基因的 RNAi有效靶序列,并成功构建重组慢病毒表达载体,证明该序列可沉默HUVECs 中EGFL7 基因的表达。     

A Distinct Pathway for Polar Exocytosis in Plant Cell Wall Formation

Post-Golgi protein sorting and trafficking to the plasma membrane (PM) is generally believed to occur via the trans-Golgi network (TGN). In this study using Nicotiana tabacum pectin methylesterase (NtPPME1) as a marker, we have identified a TGN-independent polar exocytosis pathway that mediates cell wall formation during cell expansion and cytokinesis. Confocal immunofluorescence and immunogold electron microscopy studies demonstrated that Golgi-derived secretory vesicles (GDSVs) labeled by NtPPME1-GFP are distinct from those organelles belonging to the conventional post-Golgi exocytosis pathway. In addition, pharmaceutical treatments, superresolution imaging, and dynamic studies suggest that NtPPME1 follows a polar exocytic process from Golgi-GDSV-PM/cell plate (CP), which is distinct from the conventional Golgi-TGN-PM/CP secretion pathway. Further studies show that ROP1 regulates this specific polar exocytic pathway. Taken together, we have demonstrated an alternative TGN-independent Golgi-to-PM polar exocytic route, which mediates secretion of NtPPME1 for cell wall formation during cell expansion and cytokinesis and is ROP1-dependent.Plant development and growth require coordinated tissue and cell polarization. Two of the most essential cellular processes involved in polarization are cell expansion and cytokinesis, which determines cell morphology and functions (Jaillais and Gaude, 2008; Dettmer and Friml, 2011; Li et al., 2012). Pollen tube and root hair growth require highly polarized membrane trafficking (Libault et al., 2010; Kroeger and Geitmann, 2012). Cytokinesis, by which new cells are formed, separates daughter cells by forming a new structure within the cytoplasm termed the cell plate (CP). Made up of a cell wall (CW), surrounded by new plasma membrane (PM), the cell plate is generally considered to be an example of internal cell polarity in a nonpolarized plant cell (Bednarek and Falbel, 2002; Baluska et al., 2006).The conventional view of pollen tube tip growth and cell plate formation is supported by polar exocytic secretion of numerous vesicles (diameter of 60–100 nm) to the pollen tube tip and phragmoplast areas during cytokinesis. These polar exocytic vesicles, which are generally believed to originate from the Golgi apparatus, are delivered to the site of secretion via the cytoskeleton and fuse with the target membrane with the aid of fusion factors (Jurgens, 2005; Backues et al., 2007). However, whether these polar exocytic vesicles undergoing post-Golgi trafficking are part of the conventional Golgi-trans-Golgi network (TGN)-PM/CP exocytosis or are derived from some other unidentified exocytic secretion pathway remain unclear.Polar exocytosis is regulated and controlled by a conserved Rho GTPase signaling network in fungi, animals, and plants (Burkel et al., 2012; Ridley, 2013). Rho of plant (ROP), the sole subfamily of Rho GTPases in plant, participate in signaling pathways that regulate cytoskeleton organization and endomembrane trafficking, consequently determining cell polarization, polar growth and cell morphogenesis (Gu et al., 2005; Lee et al., 2008). In growing pollen tubes, ROP1 participates in regulating polar exocytosis in the tip region via two downstream pathways to regulate apical F-actin dynamics: RIC4-mediated F-actin polymerization and RIC3-mediated apical actin depolymerization. A constitutively active mutant of ROP1 (CA-rop1) prevents fusion of these vesicles with the PM and enhances the accumulation of exocytic vesicles in the apical cortex of pollen tubes (Lee et al., 2008). Although ROP GTPases have been extensively researched, their roles in polar membrane expansion in pollen tubes and epidermal pavement cells remains unclear (Xu et al., 2010; Yang and Lavagi, 2012), and there have been insufficient studies on the functions of ROPs in controlling cell plate formation during cytokinesis. Cell division requires precise regulation and spatial organization of the cytoskeleton for delivery of secretion vesicles to the expanding cell plate (Molendijk et al., 2001).In addition, newly made cell walls during cell expansion and cell plate formation require sufficient plasticity in order to integrate new membrane materials to support the polarized membrane extension. They also should be strong enough to withstand the internal turgor pressure and thereby maintain the shape of the cell (Zonia and Munnik, 2011; Hepler et al., 2013). Recent studies have demonstrated that pectins are important for both cytokinesis and cell expansion (Moore and Staehelin, 1988; Bosch et al., 2005; Chebli et al., 2012; Altartouri and Geitmann, 2015; Bidhendi and Geitmann, 2016). Pectins are one of the major cell wall components of the middle lamella and primary cell wall. They are polymerized and methylesterified in the Golgi and subsequently released into the apoplastic space as “soft” methylesterified polymers. The homogalacturonan components of pectin are later de-methylesterified by pectin methylesterases (PMEs). The demethylesterified pectins can be cross-linked, interact with Ca²⁺, and finally form the “hard” pectin matrix of the cell wall. Therefore, the enzymatic activity of PMEs determines the rigidity of the cell wall (Micheli, 2001; Peaucelle et al., 2011).In Arabidopsis (Arabidopsis thaliana) and tobacco (Nicotiana tabacum) pollen tubes, PMEs are found predominantly polar localized in the tip region and determine the rigidity of the apical cell wall (Bosch et al., 2005; Jiang et al., 2005; Fayant et al., 2010; Chebli et al., 2012; Wang et al., 2013). PME isoform knockout mutants in Arabidopsis (AtPPME1 or vanguard1) produce unstable pollen tubes which burst when germinated in vitro and have reduced fertilization abilities (Jiang et al., 2005; Rockel et al., 2008). Recent studies have shown that in growing tobacco pollen tubes, polar targeting of NtPPME1 to the pollen tube apex depends on an apical F-actin mesh network (Wang et al., 2013). Although the functions of PME in cell wall constriction are well documented, the intracellular secretion and regulation mechanism of the exocytic process of PME still remain largely unexplored. In addition, pectins are also found to be abundant in the forming cell plate, raising the possibility that PMEs may also function during cell plate formation (Moore and Staehelin, 1988; Dhonukshe et al., 2006).In our study, we have used NtPPME1 as a marker to identify a polar exocytic process which is distinct from the conventional Golgi-TGN-PM exocytosis pathway in both pollen tube tip growth and cell plate formation. We have identified a Golgi-derived secretory vesicle (GDSV) for the polar secretion and targeting of NtPPME1 to the cell wall that bypasses the TGN during cell polarization. Further investigations using ROP1 mutants have shown that this polar exocytosis is ROP1 dependent.     

Trafficking of Crumbs3 during Cytokinesis Is Crucial for Lumen Formation

Although lumen generation has been extensively studied through so-called cyst-formation assays in Madin-Darby canine kidney (MDCK) cells, an underlying mechanism that leads to the initial appearance of a solitary lumen remains elusive. Lumen formation is thought to take place at early stages in aggregates containing only a few cells. Evolutionarily conserved polarity protein complexes, namely the Crumbs, Par, and Scribble complexes, establish apicobasal polarity in epithelial cells, and interference with their function impairs the regulated formation of solitary epithelial lumina. Here, we demonstrate that MDCK cells form solitary lumina during their first cell division. Before mitosis, Crumbs3a becomes internalized and concentrated in Rab11-positive recycling endosomes. These compartments become partitioned in both daughter cells and are delivered to the site of cytokinesis, thus forming the first apical membrane, which will eventually form a lumen. Endosome trafficking in this context appears to depend on the mitotic spindle apparatus and midzone microtubules. Furthermore, we show that this early lumen formation is regulated by the apical polarity complexes because Crumbs3 assists in the recruitment of aPKC to the forming apical membrane and interference with their function can lead to the formation of a no-lumen or multiple-lumen phenotype at the two-cell stage.     

Microparticle-Induced Activation of the Vascular Endothelium Requires Caveolin-1/Caveolae

Microparticles (MPs) are small membrane fragments shed from normal as well as activated, apoptotic or injured cells. Emerging evidence implicates MPs as a causal and/or contributing factor in altering normal vascular cell phenotype through initiation of proinflammatory signal transduction events and paracrine delivery of proteins, mRNA and miRNA. However, little is known regarding the mechanism by which MPs influence these events. Caveolae are important membrane microdomains that function as centers of signal transduction and endocytosis. Here, we tested the concept that the MP-induced pro-inflammatory phenotype shift in endothelial cells (ECs) depends on caveolae. Consistent with previous reports, MP challenge activated ECs as evidenced by upregulation of intracellular adhesion molecule-1 (ICAM-1) expression. ICAM-1 upregulation was mediated by activation of NF-κB, Poly [ADP-ribose] polymerase 1 (PARP-1) and the epidermal growth factor receptor (EGFR). This response was absent in ECs lacking caveolin-1/caveolae. To test whether caveolae-mediated endocytosis, a dynamin-2 dependent process, is a feature of the proinflammatory response, EC’s were pretreated with the dynamin-2 inhibitor dynasore. Similar to observations in cells lacking caveolin-1, inhibition of endocytosis significantly attenuated MPs effects including, EGFR phosphorylation, activation of NF-κB and upregulation of ICAM-1 expression. Thus, our results indicate that caveolae play a role in mediating the pro-inflammatory signaling pathways which lead to EC activation in response to MPs.     

Mechanism of chromatin assembly in Xenopus oocytes

We have analyzed the chromatin assembly reaction catalyzed by the Xenopus oocyte extract (S-150). A 50 S complex is formed upon mixing the 17 S pUC DNA and the S-150. Mature histones are not detected in this complex, which contains relaxed DNA and protein, and generates subnucleosomal 7 S particles upon digestion with micrococcal nuclease. The relaxed nucleoprotein is gradually supercoiled into nucleosomal chromatin in the S-150, via a pathway that requires ATP and is blocked by novobiocin, and this process is accompanied by the appearance of mature histones H3 and H4. Isolated complexes also supercoil in vitro, which implies the complex is a kit that contains histone precursors, as well as topoisomerases and other enzymes required for assembly. We discuss the biological implications of these findings.     

Sporophyte Formation and Life Cycle Completion in Moss Requires Heterotrimeric G-Proteins

In this study, we report the functional characterization of heterotrimeric G-proteins from a nonvascular plant, the moss Physcomitrella patens. In plants, G-proteins have been characterized from only a few angiosperms to date, where their involvement has been shown during regulation of multiple signaling and developmental pathways affecting overall plant fitness. In addition to its unparalleled evolutionary position in the plant lineages, the P. patens genome also codes for a unique assortment of G-protein components, which includes two copies of Gβ and Gγ genes, but no canonical Gα. Instead, a single gene encoding an extra-large Gα (XLG) protein exists in the P. patens genome. Here, we demonstrate that in P. patens the canonical Gα is biochemically and functionally replaced by an XLG protein, which works in the same genetic pathway as one of the Gβ proteins to control its development. Furthermore, the specific G-protein subunits in P. patens are essential for its life cycle completion. Deletion of the genomic locus of PpXLG or PpGβ2 results in smaller, slower growing gametophores. Normal reproductive structures develop on these gametophores, but they are unable to form any sporophyte, the only diploid stage in the moss life cycle. Finally, the mutant phenotypes of ΔPpXLG and ΔPpGβ2 can be complemented by the homologous genes from Arabidopsis, AtXLG2 and AtAGB1, respectively, suggesting an overall conservation of their function throughout the plant evolution.In all known eukaryotes, cellular signaling involves heterotrimeric GTP-binding proteins (G-proteins), which consist of Gα, Gβ, and Gγ subunits (Cabrera-Vera et al., 2003). According to the established paradigm, when Gα is GDP-bound, it forms a trimeric complex with the Gβγ dimer and remains associated with a G-protein coupled receptor. Signal perception by the receptor facilitates GDP to GTP exchange on Gα. GTP-Gα dissociates from the Gβγ dimer, and both these entities can transduce the signal by interacting with different effectors. The duration of the active state is determined by the intrinsic GTPase activity of Gα, which hydrolyzes bound GTP into GDP and inorganic phosphate (Pi), followed by the reassociation of the inactive, trimeric complex (Siderovski and Willard, 2005).In plants, G-protein signaling has been studied in only a few angiosperms to date at the functional level, although the proteins exist in the entire plant lineage (Hackenberg and Pandey, 2014; Urano and Jones, 2014; Hackenberg et al., 2016). Interestingly, while the overall biochemistry of the individual G-protein components and the interactions between them are conserved between plant and metazoan systems, deviations from the established norm are also obvious. For example, the repertoire of canonical G-proteins is significantly limited in plants; the human genome codes for 23 Gα, 5 Gβ, and 12 Gγ proteins, whereas most plant genomes, including those of basal plants, typically encode 1 canonical Gα, 1 Gβ, and three to five Gγ proteins (Urano and Jones, 2014). The only exceptions are some polyploid species, such as soybean, which have retained most of the duplicated G-protein genes (Bisht et al., 2011; Choudhury et al., 2011). Moreover, even in plants that possess only a single canonical Gα and Gβ protein, for example Arabidopsis (Arabidopsis thaliana) and rice, the phenotypes of plants lacking either one or both proteins are relatively subtle. The mutant plants exhibit multiple developmental and signaling defects but are able to complete the life cycle without any major consequences. These observations have questioned the significance of G-protein mediated signaling pathways in plants.Interestingly, plants also possess certain unique variants of the classical G-protein components such as the type III Cys-rich Gγ proteins and extra-large GTP-binding (XLG) proteins, which add to the diversity and expanse of the G-protein signaling networks (Roy Choudhury et al., 2011; Chakravorty et al., 2015; Maruta et al., 2015). The XLG proteins are almost twice the size of typical Gα proteins, with the C-terminal region that codes for Gα-like domain and an extended N-terminal region without any distinctive features. Plant XLGs are encoded by entirely independent genes and therefore are different from the mammalian extra-long versions of Gα proteins such as XLαs and XXLαs, which are expressed due to the use of alternate exons (Abramowitz et al., 2004). Three to five copies of XLG proteins are present in the genome of most angiosperms. At the functional level, the XLG proteins have been characterized only from Arabidopsis, to date, where recent studies suggest that the proteins compete with canonical Gα for binding with the Gβγ dimers and may form functional trimeric complexes (Chakravorty et al., 2015; Maruta et al., 2015). The XLG and Gβγ mutants of Arabidopsis seem to function in the same pathways during the regulation of a subset of plant responses, for example primary root length and its regulation by abscisic acid (ABA); the root waving and skewing responses; sensitivity to Glc, salt, and tunicamycin; and sensitivity to certain bacterial and fungal pathogens (Ding et al., 2008; Pandey et al., 2008; Chakravorty et al., 2015; Maruta et al., 2015). However, many of the phenotypes of Arabidopsis Gα and Gβγ mutants are also distinct from that of the xlg triple mutants. For example, compared to the wild-type plants, the canonical G-protein mutants exhibit altered response to gibberellic acid, brassinosteroids, and auxin and show changes in leaf shape, branching, flowering time, and stomatal densities (Ullah et al., 2003; Chen et al., 2004; Pandey et al., 2006; Zhang et al., 2008; Nilson and Assmann, 2010). The xlg triple mutants behave similarly to wild-type plants in all these aspects of development and signaling. Moreover, whether the XLG proteins are authentic GTP-binding and -hydrolyzing proteins and the extent to which they directly participate in G-protein-mediated signaling pathways remains confounding (Chakravorty et al., 2015; Maruta et al., 2015). Even in plants with a limited number of G-protein subunits such as Arabidopsis, one Gα and three XLGs potentially compete for a single Gβ protein, and the analysis of null mutants is not straightforward, that is, it is not possible to delineate whether the phenotypes seen in the Gα null mutants are truly due to the lack of Gα and/or because of an altered stoichiometry or availability of Gβ for the XLG proteins.As a bryophyte, Physcomitrella patens occupies a unique position in the evolutionary history of plants. It lacks vasculature but exhibits alteration between generations, which is dominated by a gametophytic (haploid) phase and a short sporophytic (diploid) phase (Cove et al., 2009). Many of the pathways related to hormone signaling, stress responses, and development are conserved between angiosperms and P. patens (Cove et al., 2009; Sun, 2011; Komatsu et al., 2013; Yasumura et al., 2015). It is also an intriguing example in the context of the G-protein signaling, because its fully sequenced genome does not encode a canonical Gα gene, although genes coding for the Gβ and Gγ proteins exist. A single gene for a potential XLG homolog also exists in the P. patens genome. This unique assortment of proteins predicts several alternative scenarios for G-protein signaling in P. patens. For example, the P. patens Gβγ proteins might be nonfunctional due to the loss of canonical Gα and are left in the genome as evolutionary artifacts. Alternatively, the Gβγ proteins of P. patens might maintain functionality regardless of the existence of a canonical Gα protein in pathways not regulated via classic G-protein signaling modes. Finally, a more likely scenario could be that the potential XLG protein can substitute for the Gα function in P. patens.To explore these possibilities and understand better the conserved and unique mechanisms of G-protein signaling pathways in plants and their significance, we examined the role of G-protein subunits in P. patens. We provide unambiguous evidence for the genetic coupling of XLG and Gβ proteins in controlling P. patens development. In contrast to all other plant species analyzed to date, where G-proteins are not essential for growth and survival, the XLG or one of the Gβ proteins is required for the sporophyte formation and life cycle completion in P. patens. Furthermore, one of the Arabidopsis XLG proteins, XLG2, and the canonical Gβ protein AGB1 can functionally complement the P. patens mutant phenotypes. These data provide new insights in the evolutionary breadth and the spectrum of signaling pathways regulated by G-proteins in plants.     

Role of EGFL7/EGFR-signaling pathway in migration and invasion of growth hormone-producing pituitary adenomas

Currently, the primary therapeutic strategy for most growth hormone-producing pituitary adenomas (GHPA) is surgery. Due to the invasiveness of GHPA, high recurrence has limited the benefit of complete adenoma removal surgery. Epidermal growth factor-like domain 7 (EGFL7) is a secreted factor implicated in tumor angiogenesis, growth, invasiveness and metastasis in GHPA. Herein, we observed that the expression level of EGFL7 and p-EGFR in invasive GHPA was much higher than that of non-invasive GHPA. The overexpression of EGFL7 was positively correlated with activation of EGFR (p-EGFR). Noticeably, EGFL7 knockdown significantly inhibited activation of EGFR signaling cascades, including p-ERGR, p-AKT and p-ERK. Further studies showed that EGFL7 knockdown or pharmacological inhibition of EGFR-pathway, using EGFR inhibitor Tyrphostin AG-1478, significantly suppressed migration and invasion of GH3 and GT1-1 cells. In summary, our findings suggest that EGFL7 is a key factor for regulation of EGFR signaling pathway and plays an important role in migration and invasion of invasive GHPA.