Th1- and Th2-dependent endothelial progenitor cell recruitment and angiogenic switch in asthma

Increased numbers of submucosal vessels are a consistent pathologic component of asthmatic airway remodeling. However, the relationship between new vessel formation and asthmatic inflammatory response is unknown. We hypothesized that angiogenesis is a primary event during the initiation of airway inflammation and is linked to the recruitment of bone marrow-derived endothelial progenitor cells (EPC). To test this hypothesis, circulating EPC and EPC-derived endothelial cell colony formation of individuals with asthma or allergic rhinitis and health controls was evaluated. Circulating EPC were increased in asthma, highly proliferative, and exhibited enhanced incorporation into endothelial cell tubes as compared with controls. In an acute allergen challenge murine asthma model, EPC mobilization occurred within hours of challenge and mobilized EPC were selectively recruited into the challenged lungs of sensitized animals, but not into other organs. EPC recruitment was Th1 and Th2 dependent and was temporally associated with an increased microvessel density that was noted within 48 h of allergen challenge, indicating an early switch to an angiogenic lung environment. A chronic allergen challenge model provided evidence that EPC recruitment to the lung persisted and was associated with increasing microvessel density over time. Thus, a Th1- and Th2-dependent angiogenic switch with EPC mobilization, recruitment, and increased lung vessel formation occurs early but becomes a sustained and cumulative component of the allergen-induced asthmatic response.     

The early phase change gene in maize

Recessive mutations of the early phase change (epc) gene in maize affect several aspects of plant development. These mutations were identified initially because of their striking effect on vegetative phase change. In certain genetic backgrounds, epc mutations reduce the duration of the juvenile vegetative phase of development and cause early flowering, but they have little or no effect on the number of adult leaves. Except for a transient delay in leaf production during germination, mutant plants initiate leaves at a normal rate both during and after embryogenesis. Thus, the early flowering phenotype of epc mutations is explained completely by their effect on the expression of the juvenile phase. The observation that epc mutations block the rejuvenation of leaf primordia in excised shoot apices supports the conclusion that epc is required for the expression of juvenile traits. This phenotype suggests that epc functions normally to promote the expression of the juvenile phase of shoot development and to suppress the expression of the adult phase and that floral induction is initiated by the transition to the adult phase. epc mutations are epistatic to the gibberellin-deficient mutation dwarf1 and interact additively with the dominant gain-of-function mutations Teopod1, Teopod2, and Teopod3. Genetic backgrounds that enhance the mutant phenotype of epc demonstrate that, in addition to its role in phase change, epc is required for the maintenance of the shoot apical meristem, leaf initiation, and root initiation.     

The accumulation of dendritic cells in the lung is impaired in CD18-/- but not in ICAM-1-/- mutant mice

Bone marrow-derived dendritic cell (DC) precursors migrate via the blood stream to peripheral tissues to adopt their sentinel function. To identify factors facilitating their emigration to the lung, mutant mice deficient in E-selectin, P-selectin, E/P-selectin, ICAM-1, or CD18 and their respective controls were examined. DCs and monocytes/macrophages were immunolabeled with M5/114 and MOMA-2 mAbs, respectively, and quantified morphometrically. Of these genotypes, the numbers of DC and MOMA-2+ cells were significantly less only in the lungs of CD18-/- mice by 68 and 35% in alveolar walls and by 28 and 26% in venous walls, respectively. DCs were reduced by 30 and 41% around large and small airways, respectively, but the number of MOMA-2+ cells in these locations was not significantly different from controls. Ablation of a single gene may be associated with augmented expression of other, related gene products. Therefore, we examined the expression of VCAM-1. Increased numbers of arteries exhibited continuous luminal VCAM-1 staining in both CD18-/- and ICAM-1-/- mutants. VCAM-1 expression was absent in pulmonary capillaries and unchanged in veins. These data suggest that under nonperturbing conditions, CD18-mediated adhesion is required for the full complement of DC precursors to accumulate in the lungs. However, the defect in CD18-/- mice is partial, suggesting that CD18-independent adhesion occurs. The alternative pathway may involve VLA-4/VCAM-1 in arteries and venules but not in capillaries. The smaller defect in ICAM-1-/- mice suggests that the CD11/CD18 complex recognizes ligands other than ICAM-1 at some sites.     

QUASIMODO1 encodes a putative membrane-bound glycosyltransferase required for normal pectin synthesis and cell adhesion in Arabidopsis

Pectins are a highly complex family of cell wall polysaccharides. As a result of a lack of specific mutants, it has been difficult to study the biosynthesis of pectins and their role in vivo. We have isolated two allelic mutants, named quasimodo1 (qua1-1 and qua1-2), that are dwarfed and show reduced cell adhesion. Mutant cell walls showed a 25% reduction in galacturonic acid levels compared with the wild type, indicating reduced pectin content, whereas neutral sugars remained unchanged. Immersion immunofluorescence with the JIM5 and JIM7 monoclonal antibodies that recognize homogalacturonan epitopes revealed less labeling of mutant roots compared with the wild type. Both mutants carry a T-DNA insertion in a gene (QUA1) that encodes a putative membrane-bound glycosyltransferase of family 8. We present evidence for the possible involvement of a glycosyltransferase of this family in the synthesis of pectic polysaccharides, suggesting that other members of this large multigene family in Arabidopsis also may be important for pectin biosynthesis. The mutant phenotype is consistent with a central role for pectins in cell adhesion.     

Deletion of GEL2 encoding for a beta(1-3)glucanosyltransferase affects morphogenesis and virulence in Aspergillus fumigatus

The first fungal glycosylphosphatidylinositol anchored beta(1-3)glucanosyltranferase (Gel1p) has been described in Aspergillus fumigatus and its encoding gene GEL1 identified. Glycosylphosphatidylinositol-anchored glucanosyltransferases play an active role in the biosynthesis of the fungal cell wall. We characterize here GEL2, a homologue of GEL1. Both homologues share common characteristics: (i) GEL1 and GEL2 are constitutively expressed during over a range of growth conditions; (ii) Gel2p is also a putative GPI-anchored protein and shares the same beta(1-3)glucanosyltransferase activity as Gel1p and (iii) GEL2, like GEL1, is able to complement the Deltagas1 deletion in Saccharomyces cerevisiae confirming that Gelp and Gasp have the same enzymatic activity. However, disruption of GEL1 did not result in a phenotype whereas a Deltagel2 mutant and the double mutant Deltagel1Deltagel2 exhibit slower growth, abnormal conidiogenesis, and an altered cell wall composition. In addition, the Deltagel2 and the Deltagel1Deltagel2 mutant have reduced virulence in a murine model of invasive aspergillosis. These data suggest for the first time that beta(1-3)glucanosyltransferase activity is required for both morphogenesis and virulence in A. fumigatus.     

The small GTPase Rap1 is required for Mn(2+)- and antibody-induced LFA-1- and VLA-4-mediated cell adhesion

In T-lymphocytes the Ras-like small GTPase Rap1 plays an essential role in stimulus-induced inside-out activation of integrin LFA-1 (alpha(L)beta(2)) and VLA-4 (alpha(4)beta(1)). Here we show that Rap1 is also involved in the direct activation of these integrins by divalent cations or activating antibodies. Inhibition of Rap1 either by Rap GTPase-activating protein (RapGAP) or the Rap1 binding domain of RalGDS abolished both Mn(2+)- and KIM185 (anti-LFA-1)-induced LFA-1-mediated cell adhesion to intercellular adhesion molecule 1. Mn(2+)- and TS2/16 (anti-VLA-4)-induced VLA-4-mediated adhesion were inhibited as well. Interestingly, both Mn(2+), KIM185 and TS2/16 failed to induce elevated levels of Rap1GTP. These findings indicate that available levels of GTP-bound Rap1 are required for the direct activation of LFA-1 and VLA-4. Pharmacological inhibition studies demonstrated that both Mn(2+)- and KIM185-induced adhesion as well as Rap1-induced adhesion require intracellular calcium but not signaling activity of the MEK-ERK pathway. Moreover, functional calmodulin signaling was shown to be a prerequisite for Rap1-induced adhesion. From these results we conclude that in addition to stimulus-induced inside-out activation of integrins, active Rap1 is required for cell adhesion induced by direct activation of integrins LFA-1 and VLA-4. We suggest that Rap1 determines the functional availability of integrins for productive binding to integrin ligands.     

The abi1-1 mutation blocks ABA signaling downstream of cADPR action

Arabidopsis thaliana abscisic acid insensitive 1-1 (abi1-1) is a dominant mutant that is insensitive to the inhibition of germination and growth by the plant hormone, abscisic acid (ABA). The mutation severely decreases the catalytic activity of the ABI1 type 2C protein phosphatase (PP2C). However, the site of action of the abi1-1/ABI1 in the ABA signal transduction pathway has not yet been determined. Using single cell assays, we showed that microinjecting mutant abi1-1 protein inhibited the activation of RD29A-GUS and KIN2-GUS in response to ABA, cyclic ADP-ribose (cADPR), and Ca2+. The inhibitory effect of the mutant protein, however, was reversed by co-microinjection of an excess amount of the ABI1 protein. In transgenic Arabidopsis plants, overexpression of abi1-1 rendered the plants insensitive to ABA during germination, whereas overexpression of ABI1 did not have any apparent effect. Moreover, transgenic plants overexpressing abi1-1 were blocked in the induction of ABA-responsive genes; however, overexpression of ABI1 did not affect gene expression. Taken together, our results demonstrate that abi1-1 is likely to be a dominant negative mutation and ABI1 likely acts downstream of cADPR in the ABA-signaling pathway. Our results on ABI1 overexpression in Arabidopsis are not compatible with a negative regulatory role of this phosphatase in ABA responses.     

Genetic deletion of the Pten tumor suppressor gene promotes cell motility by activation of Rac1 and Cdc42 GTPases

Pten (Phosphatase and tensin homolog deleted on chromosome 10) is a recently identified tumor suppressor gene which is deleted or mutated in a variety of primary human cancers and in three cancer predisposition syndromes [1]. Pten regulates apoptosis and cell cycle progression through its phosphatase activity on phosphatidylinositol (PI) 3,4,5-trisphosphate (PI(3,4,5)P(3)), a product of PI 3-kinase [2-5]. Pten has also been implicated in controlling cell migration [6], but the exact mechanism is not very clear. Using the isogenic Pten(+/+) and Pten(-/-) mouse fibroblast lines, here we show that Pten deficiency led to increased cell motility. Reintroducing the wild-type Pten, but not the catalytically inactive Pten C124S or lipid-phosphatase-deficient Pten G129E mutant, reduced the enhanced cell motility of Pten-deficient cells. Moreover, phosphorylation of the focal adhesion kinase p125(FAK) was not changed in Pten(-/-) cells. Instead, significant increases in the endogenous activities of Rac1 and Cdc42, two small GTPases involved in regulating the actin cytoskeleton [7], were observed in Pten(-/-) cells. Overexpression of dominant-negative mutant forms of Rac1 and Cdc42 reversed the cell migration phenotype of Pten(-/-) cells. Thus, our studies suggest that Pten negatively controls cell motility through its lipid phosphatase activity by down-regulating Rac1 and Cdc42.     

Role of the I-domain in collagen binding specificity and activation of the integrins α1β1 and α2β1

Adhesion to collagens by most cell types is mediated by the integrins α1β1 and α2β1. Both integrin α subunits belong to a group which is characterized by the presence of an I domain in the N-terminal half of the molecule, and this domain has been implicated in the ligand recognition. Since purified α1β1 and α2β1 differ in their binding to collagens I and IV and recognize different sites within the major cell binding domain of collagen IV, we investigated the potential role of the α1 and α2 I domains in specific collagen adhesion. We find that introducing the α2 I domain into α1 results in surface expression of a functional collagen receptor. The adhesion mediated by this chimeric receptor (α1-2-1β1) is similar to the adhesion profile conferred by α2β1, not α1β1. The presence of α2 or α1-2-1 results in preferential binding to collagen I, whereas α1 expressing cells bind better to collagen IV. In addition, α1 containing cells bind to low amounts of a tryptic fragment of collagen IV, whereas α2 or α1-2-1 bearing cells adhere only to high concentrations of this substrate. We also find that collagen adhesion of NIH-3T3 mediated by α2β1 or α1-2-1β1, but not by α1, requires the presence of Mn²⁺ ions. This ion requirement was not found in CHO cells, implicating the I domain in cell type-specific activation of integrins. J. Cell. Physiol. 176:634–641, 1998. © 1998 Wiley-Liss, Inc.     

BAG3 directly associates with guanine nucleotide exchange factor of Rap1, PDZGEF2, and regulates cell adhesion

BAG3, a member of the Hsc70 binding co-chaperone BAG-family proteins, has critical roles in regulating actin organization, cell adhesion, cell motility and tumor metastasis. The PDZ domain containing guanine nucleotide exchange factor 2 (PDZGEF2) was cloned as a BAG3-interacting protein. PDZGEF2 induces activation of Rap1 and increases integrin-mediated cell adhesion. The PPDY motif at the C-terminus of PDZGEF2 binds to the WW domain of BAG3 in vitro and in vivo. BAG3 deletion mutant lacking the WW domain lose its cell adhesion and motility activity. Gene knockdown of PDZGEF2 leads to the loss of cell adhesion on fibronectin-coated plates while BAG3 overexpression increases cell adhesion in Cos7 cells, but not in PDZGEF2 gene knockdown cells indicating that PDZGEF2 is a critical partner for BAG3 in regulating cell adhesion.     

Homeodomain factor Nkx2-3 controls regional expression of leukocyte homing coreceptor MAdCAM-1 in specialized endothelial cells of the viscera

Regulated emigration of blood-borne leukocytes plays a defining role in lymphoid organ development, immune surveillance, and inflammatory responses. We report here that mice deficient in the homeobox gene Nkx2-3, expressed in developing visceral mesoderm, show a complex intestinal malabsorption phenotype and striking abnormalities of gut-associated lymphoid tissue and spleen suggestive of deranged leukocyte homing. Mutant Peyer's patches were reduced in number and size, intestinal villi contained few IgA(+) plasma cells, and mutant spleens were small and often atrophic, showing fused periarterial lymphoid sheaths, partially merged T and B cell zones, an absent marginal zone, and a dearth of macrophages in red pulp. Semiquantitative RT-PCR analysis and immunohistochemistry revealed down-regulation of mucosal addressin cell adhesion molecule-1 (MAdCAM-1) in endothelial cells in which Nkx2-3 is normally expressed. MAdCAM-1 is a member of the immunoglobulin superfamily, acting as an endothelial cell ligand for leukocyte homing receptors L-selectin and alpha4beta7 integrin. Our data suggest a role for a homeodomain factor in establishing the developmental and positional cues in endothelia that regulate leukocyte homing through local control of cellular adhesion and identify MAdCAM-1 as a candidate target gene of Nkx2-3.     

Syndecan-1 and -4 differentially regulate oncogenic K-ras dependent cell invasion into collagen through α2β1 integrin and MT1-MMP

Syndecans function as co-receptors for integrins on different matrixes. Recently, syndecan-1 has been shown to be important for α2β1 integrin-mediated adhesion to collagen in tumor cells by regulating cell adhesion and migration on two-dimensional collagen. However, the function of syndecans in supporting α2β1 integrin interactions with three-dimensional (3D) collagen is less well studied. Using loss-of-function and overexpression experiments we show that in 3D collagen syndecan-4 supports α2β1-mediated collagen matrix contraction. Cell invasion through type I collagen containing 3D extracellular matrix (ECM) is driven by α2β1 integrin and membrane type-1 matrix metalloproteinase (MT1-MMP). Here we show that mutational activation of K-ras correlates with increased expression of α2β1 integrin, MT1-MMP, syndecan-1, and syndecan-4. While K-ras-induced α2β1 integrin and MT1-MMP are positive regulators of invasion, silencing and overexpression of syndecans demonstrate that these proteins inhibit cell invasion into collagen. Taken together, these data demonstrate the existence of a complex interplay between integrin α2β1, MT1-MMP, and syndecans in the invasion of K-ras mutant cells in 3D collagen that may represent a mechanism by which tumor cells become more invasive and metastatic.     

Transfection of the cloned human excision repair gene ERCC-1 to UV-sensitive CHO mutants only corrects the repair defect in complementation group-2 mutants

The human DNA-excision repair gene ERCC-1 is cloned by its ability to correct the excision-repair defect of the ultraviolet light- and mitomycin-C-sensitive CHO mutant cell line 43-3B. This mutant is assigned to complementation group 2 of the excision-repair-deficient CHO mutants. In order to establish whether the correction by ERCC-1 is confined to CHO mutants of one complementation group, the cloned repair gene, present on cosmid 43-34, was transfected to representative cell lines of the 6 complementation groups that have been identified to date. Following transfection, mycophenolic acid was used to select for transferants expressing the dominant marker gene Ecogpt, also present on cosmid 43-34. Cotransfer of the ERCC-1 gene was shown by Southern blot analysis of DNA from pooled (500-2000 independent colonies) transformants of each mutant. UV survival and UV-induced UDS showed that only mutants belonging to complementation group 2 and no mutants of other groups were corrected by the ERCC-1 gene. This demonstrates that ERCC-1 does not provide an aspecific bypass of excision-repair defects in CHO mutants and supports the assumption that the complementation analysis is based on mutations in different repair genes.     

14-3-3 regulation of cell spreading and migration requires a functional amphipathic groove

The 14-3-3 proteins associate with many cellular proteins that participate in the regulation of various cellular events including apoptosis, the cell cycle, spreading, and migration. We have previously described that 14-3-3beta binds the beta1-integrin and overexpression of 14-3-3beta promoted increased cell spreading and migration (Han et al. [2001] Oncogene 20: 346-357). In this study, we find that mutation of Ser 60 of 14-3-3beta, outside of the amphipathic groove which is involved in 14-3-3 protein interactions with other ligands, abolished its interaction with integrin. Surprisingly, this mutant retained its ability to promote cell spreading, suggesting that 14-3-3beta interaction with the beta1-integrin is not required for its regulation of cell adhesion. We next showed that mutations of several critical residues in the amphipathic groove did not affect 14-3-3beta interaction with the beta1-integrin. As expected, these mutants disrupted their association with the phosphoserine dependent ligands Raf and Cas. Analysis of the groove mutant LF (mutation of Arg129Tyr130 to Leu and Phe) indicated that, unlike wild type 14-3-3beta, it could not stimulate cell spreading or migration, suggesting that a functional amphipathic groove is required for 14-3-3 regulation of cell adhesion and migration. Consistent with this, cells expressing the LF mutant exhibited a delay in F-actin organization compared to cells expressing wild type or the S60A mutant (Ser 60 to Ala mutation) upon cell adhesion to fibronectin (FN). Taken together, these studies identified a novel binding site on 14-3-3 for integrin beta1 and showed that a functional amphipathic groove, rather than its interaction with integrin beta1, is required for 14-3-3 regulation of cell spreading and migration.     

Vortex-mediated mechanical stress induces integrin-dependent cell adhesion mediated by inositol 1,4,5-trisphosphate-sensitive Ca2+ release in THP-1 cells

In the downstream regions of stenotic vessels, cells are subjected to a vortex motion under low shear forces, and atherosclerotic plaques tend to be localized. It has been reported that such a change of shear force on endothelial cells has an atherogenic effect by inducing the expression of adhesion molecules. However, the effect of vortex-induced mechanical stress on leukocytes has not been investigated. In this study, to elucidate whether vortex flow can affect the cell adhesive property, we have examined the effect of vortex-mediated mechanical stress on integrin activation in THP-1 cells, a monocytic cell line, and its signaling mechanisms. When cells are subjected to vortex flow at 400-2,000 rpm, integrin-dependent cell adhesion to vascular cell adhesion molecule-1 or fibronectin increased in a speed- and time-dependent manner. Next, to examine the role of Ca(2+) in this integrin activation, various pharmacological inhibitors involved in Ca(2+) signaling were tested to inhibit the cell adhesion. Pretreatment of cells with BAPTA-AM, thapsigargin +NiCl(2), or U-73122 (a phospholipase C inhibitor) inhibited cell adhesion induced by vortex-mediated mechanical stress. We also found that W7 (a calmodulin inhibitor) blocked the cell adhesion. However, pretreatment of cells with GdCl(3), NiCl(2), or ryanodine did not affect the cell adhesion. These data indicate that vortex-mediated mechanical stress induces integrin activation through calmodulin and inositol 1,4,5-trisphosphate-mediated Ca(2+) releases from intracellular Ca(2+) stores in THP-1 cells.     

Pyk2- and Src-dependent tyrosine phosphorylation of PDK1 regulates focal adhesions

3-Phosphoinositide-dependent protein kinase 1 (PDK1) is a signal integrator that activates the AGC superfamily of serine/threonine kinases. PDK1 is phosphorylated on tyrosine by oxidants, although its regulation by agonists that stimulate G-protein-coupled receptor signaling pathways and the physiological consequences of tyrosine phosphorylation in this setting have not been fully identified. We found that angiotensin II stimulates the tyrosine phosphorylation of PDK1 in vascular smooth muscle in a calcium- and c-Src-dependent manner. The calcium-activated tyrosine kinase Pyk2 acts as a scaffold for Src-dependent phosphorylation of PDK1 on Tyr9, which permits phosphorylation of Tyr373 and -376 by Src. This critical function of Pyk2 is further supported by the observation that Pyk2 and tyrosine-phosphorylated PDK1 colocalize in focal adhesions after angiotensin II stimulation. Importantly, infection of smooth muscle cells with a Tyr9 mutant of PDK1 inhibits angiotensin II-induced tyrosine phosphorylation of paxillin and focal adhesion formation. These observations identify a novel interaction between PDK1 and Pyk2 that regulates the integrity of focal adhesions, which are major compartments for integrating signals for cell growth, apoptosis, and migration.