Adgrf5 contributes to patterning of the endothelial deep layer in retina.

Neovascularization of the inner retinal space is a major cause of vision loss. In retinal angiomatous proliferation (RAP) syndrome, newly formed vessels originate from the retinal plexus and invade the inner retinal space. However, the molecular pathways preventing subretinal vascularization remain largely unknown. In most murine models of RAP, pathological neovascularization occurs concomitantly with the development of the retinal vasculature. Here, we demonstrate that disturbing the sequence of morphogenetic events that shape the three-layered retinal vascular network leads to subretinal vascularization. Sprouts emerging from the perivenous region after the first postnatal week extended toward the retinal space where they merged into the deep layer. The small GTPase Rac1 was required for the formation of these vascular extensions and the vascular inner plexus is formed coaxially to the overarching veins. The adhesion receptor Adgrf5 was highly expressed in the endothelium of the central nervous system, where it regulates blood-brain barrier formation. The vascular superficial plexus of Adgrf5 mutant mouse retinae exhibited an increased vascular density in the perivenous areas with increased projections toward the inner plexus where they subsequently created hyper-dense endothelial cells (EC) clusters. Disturbing the perivenous pool of EC thus significantly altered the inner plexus formation. These abnormalities culminated in transient vascular protrusions in the inner retinal space. Taken together, these results reveal a previously unobserved vascular morphogenetic defect in Adgrf5 knockout mice, implicating a role for ADGRF5 in the initiation of subretinal vascularization. Our findings also illustrate how vein-derived EC shape the inner retinal layer formation and could control the appearance of angiomatous malformations.

Stabilization of the bioactivity of tumor necrosis factor by its soluble receptors.

The receptors for tumor necrosis factor (TNF) exist in cell-associated as well as soluble forms, both binding specifically to TNF. Since the soluble forms of TNF receptors (sTNF-Rs) can compete with the cell-associated TNF receptors for TNF, it was suggested that they function as inhibitors of TNF activity; at high concentrations, the sTNF-Rs indeed inhibit TNF effects. However, we report here that in the presence of low concentrations of the sTNF-Rs, effects of TNF whose induction depend on prolonged treatment with this cytokine are augmented, reflecting an attenuation by the sTNF-Rs of spontaneous TNF activity decay. Evidence that this stabilization of TNF activity by the sTNF-Rs follows from stabilization of TNF structure within the complexes that TNF forms with the sTNF-Rs is presented here, suggesting that the sTNF-Rs can affect TNF activity not only by interfering with its binding to cells but also by stabilizing its structure and preserving its activity, thus augmenting some of its effects.

Dual role of the p75 tumor necrosis factor (TNF) receptor in TNF cytotoxicity.

Whereas there is ample evidence for involvement of the p55 tumor necrosis factor (TNF) receptor (p55-R) in the cytocidal effect of TNF, the role of the p75 TNF receptor (p75-R) in this effect is a matter of debate. In this study, we probed the function of p75-R in cells sensitive to the cytotoxicity of TNF using a wide panel of antibodies (Abs) against the receptor's extracellular domain. Two distinct Ab effects were observed. The Abs triggered signaling for cytotoxicity. This effect: (a) was correlated with the extent of p75-R expression by the cells; (b) was dependent on receptor cross-linking by the Abs; (c) occurred in HeLa cells, but not in A9 cells transfected with human p75-R or in HeLa cells expressing cytoplasmically truncated p75-R mutants, indicating that it involves cell-specific activities of the intracellular domain of the receptor; (d) was synergistic with the cytocidal effect of Abs against p55-R. Moreover, it seemed to reverse induced desensitization to the cytocidal effect of anti p55-R Abs, suggesting that it involves mechanisms different from those of the signaling by the p55 TNF-R. In addition, the Abs affected the response to TNF in a way that does not involve the signaling activity of p75-R. These effects: (a) could be observed also in cells in which only p55-R signaled for the cytocidal effect; (b) were not dependent on receptor cross-linking by the Abs; (c) varied according to the site at which the Abs bound to the receptor; and (d) were correlated inversely with the effects of the Abs on TNF binding to p75-R. That is, Abs binding to the membrane-distal part of the receptor's extracellular domain displaced TNF from the p75 receptor and enhanced cytocidal effect, whereas Abs that bind to the membrane-proximal part of the extracellular domain--a region at which a conformational change seems to take place upon TNF binding--decreased the dissociation of TNF from p75-R and inhibited its cytocidal effect. The above findings suggest that p75-R contributes to the cytocidal effect of TNF both by its own signaling and by regulating the access of TNF to p55-R.

Beta 1 integrin is essential for teratoma growth and angiogenesis.

Teratomas are benign tumors that form after ectopic injection of embryonic stem (ES) cells into mice and contain derivatives of all primitive germ layers. To study the role of beta 1 integrin during teratoma formation, we compared teratomas induced by normal and beta1-null ES cells. Injection of normal ES cells gave rise to large teratomas. In contrast, beta 1-null ES cells either did not grow or formed small teratomas with an average weight of <5% of that of normal teratomas. Histological analysis of beta 1-null teratomas revealed the presence of various differentiated cells, however, a much lower number of host-derived stromal cells than in normal teratomas. Fibronectin, collagen I, and nidogen were expressed but, in contrast to normal teratomas, diffusely deposited in beta1-null teratomas. Basement membranes were present but with irregular shape and detached from the cell surface. Normal teratomas had large blood vessels with a smooth inner surface, containing both host- and ES cell-derived endothelial cells. In contrast, beta 1-null teratomas had small vessels that were loosely embedded into the connective tissue. Furthermore, endothelial cells were always of host-derived origin and formed blood vessels with an irregular inner surface. Although beta 1- deficient endothelial cells were absent in teratomas, beta 1-null ES cells could differentiate in vitro into endothelial cells. The formation of a complex vasculature, however, was significantly delayed and of poor quality in beta1-null embryoid bodies. Moreover, while vascular endothelial growth factor induced proliferation of endothelial cells as well as an extensive branching of blood vessels in normal embryoid bodies, it had no effect in beta 1-null embryoid bodies.

Induction of cell scattering by expression of beta1 integrins in beta1-deficient epithelial cells requires activation of members of the rho family of GTPases and downregulation of cadherin and catenin function.

Adhesion receptors, which connect cells to each other and to the surrounding extracellular matrix (ECM), play a crucial role in the control of tissue structure and of morphogenesis. In this work, we have studied how intercellular adhesion molecules and beta1 integrins influence each other using two different beta1-null cell lines, epithelial GE11 and fibroblast-like GD25 cells. Expression of beta1A or the cytoplasmic splice variant beta1D, induced the disruption of intercellular adherens junctions and cell scattering in both GE11 and GD25 cells. In GE11 cells, the morphological change correlated with the redistribution of zonula occluden (ZO)-1 from tight junctions to adherens junctions at high cell confluency. In addition, the expression of beta1 integrins caused a dramatic reorganization of the actin cytoskeleton and of focal contacts. Interaction of beta1 integrins with their respective ligands was required for a complete morphological transition towards the spindle-shaped fibroblast-like phenotype. The expression of an interleukin-2 receptor (IL2R)-beta1A chimera and its incorporation into focal adhesions also induced the disruption of cadherin-based adhesions and the reorganization of ECM-cell contacts, but failed to promote cell migration on fibronectin, in contrast to full-length beta1A. This indicates that the disruption of cell-cell adhesion is not simply the consequence of the stimulated cell migration. Expression of beta1 integrins in GE11 cells resulted in a decrease in cadherin and alpha-catenin protein levels accompanied by their redistribution from the cytoskeleton-associated fraction to the detergent-soluble fraction. Regulation of alpha-catenin protein levels by beta1 integrins is likely to play a role in the morphological transition, since overexpression of alpha-catenin in GE11 cells before beta1 prevented the disruption of intercellular adhesions and cell scattering. In addition, using biochemical activity assays for Rho-like GTPases, we show that the expression of beta1A, beta1D, or IL2R-beta1A in GE11 or GD25 cells triggers activation of both RhoA and Rac1, but not of Cdc42. Moreover, dominant negative Rac1 (N17Rac1) inhibited the disruption of cell-cell adhesions when expressed before beta1. However, all three GTPases might be involved in the morphological transition, since expression of either N19RhoA, N17Rac1, or N17Cdc42 reversed cell scattering and partially restored cadherin-based adhesions in GE11-beta1A cells. Our results indicate that beta1 integrins regulate the polarity and motility of epithelial cells by the induction of intracellular molecular events involving a downregulation of alpha-catenin function and the activation of the Rho-like G proteins Rac1 and RhoA.

The cysteine-rich domain of human ADAM 12 supports cell adhesion through syndecans and triggers signaling events that lead to beta1 integrin-dependent cell spreading.

The ADAMs (a disintegrin and metalloprotease) family of proteins is involved in a variety of cellular interactions, including cell adhesion and ecto- domain shedding. Here we show that ADAM 12 binds to cell surface syndecans. Three forms of recombinant ADAM 12 were used in these experiments: the cys-teine-rich domain made in Escherichia coli (rADAM 12-cys), the disintegrin-like and cysteine-rich domain made in insect cells (rADAM 12-DC), and full-length human ADAM 12-S tagged with green fluorescent protein made in mammalian cells (rADAM 12-GFP). Mesenchymal cells specifically and in a dose-dependent manner attach to ADAM 12 via members of the syndecan family. After binding to syndecans, mesenchymal cells spread and form focal adhesions and actin stress fibers. Integrin beta1 was responsible for cell spreading because function-blocking monoclonal antibodies completely inhibited cell spreading, and chondroblasts lacking beta1 integrin attached but did not spread. These data suggest that mesenchymal cells use syndecans as the initial receptor for the ADAM 12 cysteine-rich domain-mediated cell adhesion, and then the beta1 integrin to induce cell spreading. Interestingly, carcinoma cells attached but did not spread on ADAM 12. However, spreading could be efficiently induced by the addition of either 1 mM Mn(2+) or the beta1 integrin-activating monoclonal antibody 12G10, suggesting that in these carcinoma cells, the ADAM 12-syndecan complex fails to modulate the function of beta1 integrin.

Mice lacking desmocollin 1 show epidermal fragility accompanied by barrier defects and abnormal differentiation.

The desmosomal cadherin desmocollin (Dsc)1 is expressed in upper epidermis where strong adhesion is required. To investigate its role in vivo, we have genetically engineered mice with a targeted disruption in the Dsc1 gene. Soon after birth, null mice exhibit flaky skin and a striking punctate epidermal barrier defect. The epidermis is fragile, and acantholysis in the granular layer generates localized lesions, compromising skin barrier function. Neutrophils accumulate in the lesions and further degrade the tissue, causing sloughing (flaking) of lesional epidermis, but rapid wound healing prevents the formation of overt lesions. Null epidermis is hyperproliferative and overexpresses keratins 6 and 16, indicating abnormal differentiation. From 6 wk, null mice develop ulcerating lesions resembling chronic dermatitis. We speculate that ulceration occurs after acantholysis in the fragile epidermis because environmental insults are more stringent and wound healing is less rapid than in neonatal mice. This dermatitis is accompanied by localized hair loss associated with formation of utriculi and dermal cysts, denoting hair follicle degeneration. Possible resemblance of the lesions to human blistering diseases is discussed. These results show that Dsc1 is required for strong adhesion and barrier maintenance in epidermis and contributes to epidermal differentiation.

Loss of RhoA promotes skin tumor formation and invasion by upregulation of RhoB.

Cellular movement is controlled by small GTPases, such as RhoA. Although migration is crucial for cancer cell invasion, the specific role of RhoA in tumor formation is unclear. Inducing skin tumors in mice with a keratinocyte-restricted loss of RhoA, we observed increased tumor frequency, growth and invasion. In vitro invasion assays revealed that in the absence of RhoA cell invasiveness is increased in a Rho-associated protein kinase (ROCK) activation and cell contraction-dependent manner. Surprisingly, loss of RhoA causes increased Rho signaling via overcompensation by RhoB because of reduced lysosomal degradation of RhoB in Gamma-aminobutyric acid receptor-associated protein (GABARAP)+ autophagosomes and endosomes. In the absence of RhoA, RhoB relocalized to the plasma membrane and functionally replaced RhoA with respect to invasion, clonogenic growth and survival. Our data demonstrate for the first time that RhoA is a tumor suppressor in 7,12-dimethylbenz[a]anthracene/12-O-tetradecanoylphorbol 13-acetate skin carcinogenesis and identify Rho signaling dependent on RhoA and RhoB as a potent driver of tumor progression.

beta1-integrin-mediated signaling essentially contributes to cell survival after radiation-induced genotoxic injury.

Integrin-mediated adhesion to extracellular matrix proteins confers resistance to radiation- or drug-induced genotoxic injury. To analyse the underlying mechanisms specific for beta1-integrins, wild-type beta1A-integrin-expressing GD25beta1A cells were compared to GD25beta1B cells, which express signaling-incompetent beta1B variants. Cells grown on fibronectin, collagen-III, beta1-integrin-IgG or poly-l-lysine were exposed to 0-6 Gy X-rays in presence or depletion of growth factors and phosphatidylinositol-3 kinase (PI3K) inhibitors (LY294002, wortmannin). In order to test the relevance of these findings in tumor cells, human A-172 glioma cells were examined under the same conditions after siRNA-mediated silencing of beta1-integrins. We found that beta1A-integrin-mediated adhesion to fibronectin, collagen-III or beta1-IgG was essential for cell survival after radiation-induced genotoxic injury. Mediated by PI3K, pro-survival beta1A-integrin/Akt signaling was critically involved in this process. Additionally, the beta1-integrin downstream targets p130Cas and paxillin-impaired survival-regulating PI3K-dependent JNK. In A-172 glioma cells, beta1-integrin knockdown and PI3K inhibition confirmed the central role of beta1-integrins in Akt- and p130Cas/paxillin-mediated prosurvival signaling. These findings suggest beta1-integrins as critical regulators of cell survival after radiation-induced genotoxic injury. Elucidation of the molecular circuitry of prosurvival beta1-integrin-mediated signaling in tumor cells may promote the development of innovative molecular-targeted therapeutic antitumor strategies.

Neurocan is dispensable for brain development.

Neurocan is a component of the extracellular matrix in brain. Due to its inhibition of neuronal adhesion and outgrowth in vitro and its expression pattern in vivo it was suggested to play an important role in axon guidance and neurite growth. To study the role of neurocan in brain development we generated neurocan-deficient mice by targeted disruption of the neurocan gene. These mice are viable and fertile and have no obvious deficits in reproduction and general performance. Brain anatomy, morphology, and ultrastructure are similar to those of wild-type mice. Perineuronal nets surrounding neurons appear largely normal. Mild deficits in synaptic plasticity may exist, as maintenance of late-phase hippocampal long-term potentiation is reduced. These data indicate that neurocan has either a redundant or a more subtle function in the development of the brain.

Functional consequences of integrin gene mutations in mice.

Integrins are cell-surface receptors responsible for cell attachment to extracellular matrices and to other cells. The application of mouse genetics has significantly increased our understanding of integrin function in vivo. In this review, we summarize the phenotypes of mice carrying mutant integrin genes and compare them with phenotypes of mice lacking the integrin ligands.

Suppression of tumor growth in vivo by local and systemic 90K level increase.

Expression levels of the immunostimulatory 90K antigen in mammary carcinoma, glioblastoma, and other tumor-derived cell lines inversely correlate with their tumorigenicity in athymic mice. Engineered enhancement of 90K expression results in significant (> 80%) tumor growth inhibition, not by direct action on the tumor cell, but by stimulation of the residual cell-mediated immune defense of the nude mouse. Enhanced 90K level effects are both localized and systemic and involve induction of ICAM-1 and VCAM-1 in the tumor endothelium. The findings presented suggest a role for 90K as a molecular alarm signal for the body's cellular defense against pathogens, which in a subset of tumors is suppressed to allow cancer progression.

Beta1 integrin promotes but is not essential for metastasis of ras-myc transformed fibroblasts.

To investigate the role of beta1 integrin during tumor metastasis, we established a ras-myc transformed fibroblastoid cell line with a disrupted beta1 integrin gene on both alleles (GERM 11). Stable transfection of this cell line with an expression vector encoding beta1A integrin resulted in beta1A integrin-expressing sublines. Tumors were induced by subcutaneous injection of GERM 11 cells and 3 independent beta1 integrin expressing sublines (GERM 116, 1A10, 2F2) into syngeneic mice. After 10 days tumors were surgically removed. While average weights of GERM 11 and GERM 116 tumors were similar, tumors induced by the high expressing clones 1A10 and 2F2 were markedly smaller, suggesting an inverse correlation of tumor growth and beta1 integrin expression. The metastasis potential of all three beta1 integrin-expressing GERM 11 sublines tested was significantly higher than that of the beta1-deficient GERM 11 cells. GERM 116 tumors led in all animals to severe metastasis in lung and liver, while GERM 11 tumors induced only a few metastatic foci in the lung. Stroma of both tumors contained nidogen and high amounts of tenascin C, but only a few very low levels of fibronectin, laminin-1, and collagen type I. Beta1 integrin, therefore, increases but is not essential for metastasis of ras-myc transformed fibroblasts.

Short arm region of laminin-5 gamma2 chain: structure, mechanism of processing and binding to heparin and proteins.

Laminin-5 is a typical component of several epithelial tissues and contains a unique gamma2 chain which can be proteolytically processed by BMP-1. This occurs in the N-terminal half of the gamma2 chain (606 residues), which consists of two rod-like tandem arrays of LE modules, LE1-3 and LE4-6, that flank a globular L4m module containing the cleavage site. Recombinant analysis of L4m, which includes an additional imperfect LE module essential for proper folding, demonstrated an unusual pattern of disulfide bonding. These connectivities prevented the release of gamma2LE1-3L4 m after BMP-1 cleavage which required in addition disulfide reshuffling by isomerases. The liberated segment bound through its L4 m module to heparin, nidogen-1, fibulin-1 and fibulin-2. A further heparin/sulfatide-binding site could be attributed to some arginine residues in module LE1. The gamma2LE4-6 segment remaining in processed laminin-5 showed only a strong binding to fibulin-2. Immunological studies showed a similar partial processing in cell culture and tissues and the persistence of the released fragment in tissues. This indicated that both N-terminal regions of the gamma2 chain may have a function in vivo.

Critical off-target effects of the widely used Rac1 inhibitors NSC23766 and EHT1864 in mouse platelets.

BACKGROUND: Platelet aggregation at sites of vascular injury is essential for normal hemostasis, but may also cause pathologic vessel occlusion. Rho GTPases are molecular switches that regulate essential cellular processes, and they have pivotal functions in the cardiovascular system. Rac1 is an important regulator of platelet cytoskeletal reorganization, and contributes to platelet activation. Rac1 inhibitors are thought to be beneficial in a wide range of therapeutic settings, and have therefore been tested in vivo for a variety of disorders. Two small-molecule inhibitors, NSC23766 and EHT1864, have been characterized in different cell types, demonstrating high specificity for Rac1 and Rac, respectively. OBJECTIVES: To analyze the specificity of NSC23766 and EHT1864. METHODS: Platelet function was assessed in mouse wild-type and Rac1-deficient platelets by the use of flow cytometric analysis of cellular activation and aggregometry. Platelet spreading was analyzed with differential interference contrast microscopy, and activation of effector molecules was analyzed with biochemical approaches. RESULTS: NSC23766 and EHT1864 showed strong and distinct Rac1-independent effects at 100 µm in platelet function tests. Both inhibitors induced Rac1-specific inhibition of platelet spreading, but also markedly impaired agonist-induced activation of Rac1(-/-) platelets. Furthermore, glycoprotein Ib-mediated signaling was dramatically inhibited by NSC23766 in both wild-type and Rac1-deficient platelets. Importantly, these inhibitors directly affected the activation of the Rac1 effectors p21-activated kinase (PAK)1 and PAK2. CONCLUSIONS: Our results reveal critical off-target effects of NSC23766 and EHT1864 at 100 µm in mammalian cells, raising questions about their utility as specific Rac1/Rac inhibitors in biochemical studies at these concentrations and possibly as therapeutic agents.

Rac1 is crucial for Ras-dependent skin tumor formation by controlling Pak1-Mek-Erk hyperactivation and hyperproliferation in vivo.

Rac1 has a role in proliferation and survival of tumor cells in vitro. The exact effects of Rac1 on growth, apoptosis and corresponding signaling pathways during tumorigenesis in vivo, however, have not been explored yet. Using mice with a keratinocyte-restricted deletion of the Rac1 gene, we found that Rac1 is essential for DMBA/TPA-induced skin tumor formation. This corresponded to a decreased keratinocyte hyperproliferation, although apoptosis was not detectably altered. Activated Rac1 promoted Erk-dependent hyperproliferation by Pak1-mediated Mek activation independent of Mek1 phosporylation at serine 298. Rac1 was furthermore required for Pak2-dependent hyperactivation of Akt, which under in vivo condition was restricted to the suprabasal cell layers corresponding to a suprabasal-specific expression of Pak2. It is surprising that none of these signaling pathways was altered in untreated Rac1-deficient skin, indicating a hyperproliferation-specific function of Rac1 in vivo. These data suggest that blocking of Rac1 function might allow tumor-specific growth repression, as Rac1 is not required for normal growth and growth signaling controlling pathways in skin in vivo.

Leukocyte extravasation as a target for anti-inflammatory therapy - Which molecule to choose?

In view of the central pathogenic importance of leukocyte extravasation in inflammatory skin diseases, therapeutic interference with this - surprisingly complex - process is clearly a promising new approach for treating these dermatoses. Despite some disappointments during the clinical use of these agents and despite their crippling price tag, the recent incorporation of biologicals that target defined molecular controls of leukocyte extravasation into dermatological and rheumatological practise, consequently, has greatly enriched our therapeutic options for battling major, chronic, inflammatory dermatoses such as psoriasis. However, the - as yet unresolved and still rather controversially discussed - critical question is: Which of the multiple steps that control leukocyte extravasation in the human system really offer the most promising, most pragmatic, and safest molecular targets for therapeutic intervention for which disease entity? The current debate intends to stimulate public and rational debate of this crucial issue, beyond the evident commercial interests that are touched by whatever stand one takes.

Fetal and adult hematopoietic stem cells require beta1 integrin function for colonizing fetal liver, spleen, and bone marrow.

Homing of hematopoietic stem cells (HSCs) into hematopoietic organs is a prerequisite for the establishment of hematopoiesis during embryogenesis and after bone marrow transplantation. We show that beta1 integrin-deficient HSCs from the para-aortic splanchnopleura and the fetal blood had hematolymphoid differentiation potential in vitro and in fetal organ cultures but were unable to seed fetal and adult hematopoietic tissues. Adult beta1 integrin null HSCs isolated from mice carrying loxP-tagged beta1 integrin alleles and ablated for beta1 integrin expression by retroviral cre transduction failed to engraft irradiated recipient mice. Moreover, absence of beta1 integrin resulted in sequestration of HSCs in the circulation and their reduced adhesion to endothelioma cells. These findings define beta1 integrin as an essential adhesion receptor for the homing of HSCs.

Increased levels of apoptosis in the prefusion neural folds underlie the craniofacial disorder, Treacher Collins syndrome.

Treacher Collins syndrome (TCS) is an autosomal dominant disorder of human craniofacial development that results from loss-of-function mutations in the gene TCOF1. Although this gene has been demonstrated to encode the nucleolar phosphoprotein treacle, the developmental mechanism underlying TCS remains elusive, particularly as expression studies have shown that the murine orthologue, Tcof1, is widely expressed. To investigate the molecular pathogenesis of TCS, we replaced exon 1 of Tcof1 with a neomycin-resistance cassette via homologous recombination in embryonic stem cells. Tcof1 heterozygous mice die perinatally as a result of severe craniofacial anomalies that include agenesis of the nasal passages, abnormal development of the maxilla, exencephaly and anophthalmia. These defects arise due to a massive increase in the levels of apoptosis in the prefusion neural folds, which are the site of the highest levels of Tcof1 expression. Our results demonstrate that TCS arises from haploinsufficiency of a protein that plays a crucial role in craniofacial development and indicate that correct dosage of treacle is essential for survival of cephalic neural crest cells.