EphB4 signaling is capable of mediating ephrinB2-induced inhibition of cell migration

Signaling between the ligand ephrinB2 and the respective receptors of the EphB class is known to play a vital role during vascular morphogenesis and angiogenesis. The relative contribution of each EphB receptor type present on endothelial cells to these processes remains to be determined. It has been shown that ephrinB2-EphB receptor signal transduction leads to a repulsive migratory behavior of endothelial cells. It remained unclear whether this anti-migratory effect can be mediated by EphB4 signaling alone or whether other EphB receptors are necessary. It also remained unclear whether the kinase activity of EphB4 is pivotal to its action. To answer these questions, we developed a cellular migration system solely dependent on ephrinB2-EphB4 signaling. Using this system, we could show that EphB4 activation leads to the inhibition of cell migration. Furthermore we identified PP2, a known inhibitor of kinases of the Src family, and PD 153035, a known inhibitor of EGF receptor kinase, as inhibitors of EphB4 kinase activity. Using PP2, the inhibition of cell migration by ephrinB2 could be relieved, demonstrating that the kinase function of EphB4 is of prominent importance in this process. These results show that EphB4 activation is not only accompanying ephrinB2 induced repulsive behavior of cells, but is capable of directly mediating this effect.     

Thrombin (PAR-1)-induced proliferation in astrocytes via MAPK involves multiple signaling pathways

Protease-activated receptors (PARs), newlyidentified members of G protein-coupled receptors, are widelydistributed in the brain. Thrombin evokes multiple cellular responsesin a large variety of cells by activating PAR-1, -3, and -4. Incultured rat astrocytes we investigated the signaling pathway ofthrombin- and PAR-activating peptide (PAR-AP)-induced cellproliferation. Our results show that PAR activation stimulatesproliferation of astrocytes through the ERK pathway. Thrombinstimulates ERK1/2 phosphorylation in a time- andconcentration-dependent manner. This effect can be fully mimicked by aspecific PAR-1-AP but only to a small degree by PAR-3-AP and PAR-4-AP.PAR-2-AP can induce a moderate ERK1/2 activation as well.Thrombin-stimulated ERK1/2 activation is mainly mediated by PAR-1 viatwo branches: 1) the PTX-sensitive Gprotein/(-subunits)-phosphatidylinositol 3-kinase branch, and2) the G_q-PLC-(InsP₃receptor)/Ca²⁺-PKC pathway. Thrombin- or PAR-1-AP-inducedERK activation is partially blocked by a selective EGF receptorinhibitor, AG1478. Nevertheless, transphosphorylation of EGF receptoris unlikely for ERK1/2 activation and is certainly not involved inPAR-1-induced proliferation. The metalloproteinase mechanism involvingtransactivation of the EGF receptor by released heparin-binding EGF wasexcluded. EGF receptor activation was detected by the receptorautophosphorylation site, tyrosine 1068. Our data suggest thatthrombin-induced mitogenic action in astrocytes occurs independently ofEGF receptor transphosphorylation.

     

Wound-healing defect of CD18(-/-) mice due to a decrease in TGF-beta1 and myofibroblast differentiation

We studied the mechanisms underlying the severely impaired wound healing associated with human leukocyte-adhesion deficiency syndrome-1 (LAD1) using a murine disease model. In CD18(-/-) mice, healing of full-thickness wounds was severely delayed during granulation-tissue contraction, a phase where myofibroblasts play a major role. Interestingly, expression levels of myofibroblast markers alpha-smooth muscle actin and ED-A fibronectin were substantially reduced in wounds of CD18(-/-) mice, suggesting an impaired myofibroblast differentiation. TGF-beta signalling was clearly involved since TGF-beta1 and TGF-beta receptor type-II protein levels were decreased, while TGF-beta(1) injections into wound margins fully re-established wound closure. Since, in CD18(-/-) mice, defective migration leads to a severe reduction of neutrophils in wounds, infiltrating macrophages might not phagocytose apoptotic CD18(-/-) neutrophils. Macrophages would thus be lacking their main stimulus to secrete TGF-beta1. Indeed, in neutrophil-macrophage cocultures, lack of CD18 on either cell type leads to dramatically reduced TGF-beta1 release by macrophages due to defective adhesion to, and subsequent impaired phagocytic clearance of, neutrophils. Our data demonstrates that the paracrine secretion of growth factors is essential for cellular differentiation in wound healing.     

Effect of laminin-1 on intestinal cell differentiation involves inhibition of nuclear nucleolin

Intestinal epithelial cells are characterized by continuous renewal and differentiation events, which may be influenced by the basement membrane, and in particular laminins, which are major components of this specialized extracellular matrix. The function and signaling pathways of laminins in these processes are still poorly documented. In this study, we investigated the possible role and the subcellular localization of nucleolin, a nuclear shuttling protein, in relation to differentiation of human intestinal epithelial Caco2/TC7 cells triggered by exogenous laminin-1. Immunofluorescence and Western blot analysis indicated that laminin-1 induced early differentiation of the cells concomitantly to a decrease in nuclear nucleolin and its a cell surface location. We also showed that both effects of laminin-1 on Caco2/TC7 cells--induction of the differentiation marker sucrase-isomaltase and redistribution of nucleolin--could be mediated by a beta1-integrin dependent cascade that implicated activation of the p38 MAPK pathway. In addition, knock-down of nucleolin expression by the small interfering RNA strategy mimicked the effect of laminin-1 as it resulted in the induction of cell polarization and differentiation. Thus, our study suggests that changes in the subcellular distribution and expression level of nucleolin play an important role in intestinal cell differentiation and relay the signaling pathway induced by laminin-1.     

PGE2-induced desensitization of adenylate cyclase of a murine macrophage-like cell line (P388D1)

The mode of PGE2-induced desensitization of the adenylate cyclase of a murine macrophage-like cell line, P388D1 was investigated. The exposure of cells to PGE2 for 60 min induced PGE2-specific desensitization of the adenylate cyclase system which still responded normally to other specific ligand such as isoproterenol, 5'-guanylimidodiphosphate (Gpp(NH)p), or forskolin. The exposure of the cells to PGE2 for 6 hr induced heterologous desensitization, as the responses of adenylate cyclase to PGE2 as well as to isoproterenol or Gpp(NH)p were significantly reduced. The lowest concentration of PGE2 to induce both early homologous and late heterologous desensitization was found to be about two-fold over the KD of the low affinity PGE2-binding sites of P388D1 cells. The early homologous desensitization appeared to be due in part to the reduction in number of PGE2 receptors from the cell surface. The late heterologous desensitization may involve functional and/or structural alteration of Gs proteins, in addition to the reduction of PGE2 receptors from the cell surface.     

Smad3 is essential for TGF-beta 1 to suppress IL-2 production and TCR-induced proliferation, but not IL-2-induced proliferation

Transforming growth factor-beta1 is essential to maintain T cell homeostasis, as illustrated by multiorgan inflammation in mice deficient in TGF-beta1 signaling. Despite the physiological importance, the mechanisms that TGF-beta1 uses to regulate T cell expansion remain poorly understood. TGF-beta1 signals through transmembrane receptor serine/threonine kinases to activate multiple intracellular effector molecules, including the cytosolic signaling transducers of the Smad protein family. We used Smad3(-/-) mice to investigate a role for Smad3 in IL-2 production and proliferation in T cells. Targeted disruption of Smad3 abrogated TGF-beta1-mediated inhibition of anti-CD3 plus anti-CD28-induced steady state IL-2 mRNA and IL-2 protein production. CFSE labeling demonstrated that TGF-beta1 inhibited entry of wild-type anti-CD3 plus anti-CD28-stimulated cells into cycle cell, and this inhibition was greatly attenuated in Smad3(-/-) T cells. In contrast, disruption of Smad3 did not affect TGF-beta1-mediated inhibition of IL-2-induced proliferation. These results demonstrate that TGF-beta1 signals through Smad3-dependent and -independent pathways to inhibit T cell proliferation. The inability of TGF-beta1 to inhibit TCR-induced proliferation of Smad3(-/-) T cells suggests that IL-2 is not the primary stimulus driving expansion of anti-CD3 plus anti-CD28-stimulated T cells. Thus, we establish that TGF-beta1 signals through multiple pathways to suppress T cell proliferation.     

The enzymatic activity of lysyl oxidas-like-2 (LOXL2) is not required for LOXL2-induced inhibition of keratinocyte differentiation

Lysyl oxidase-like-2 (LOXL2) induces tumor progression and fibrosis. It also inhibits the differentiation of keratinocytes promoting development of squamous cell carcinomas. Stimulation of HaCaT skin keratinocytes with exogenous LOXL2 or overexpression of LOXL2 in these cells inhibits their differentiation as manifested by inhibition of calcium or vitamin D-induced involucrin expression. The inhibition was abrogated by the LOXL2 function-blocking monoclonal antibody AB0023 as well as by an anti-LOXL2 polyclonal antibody. Surprisingly, a point-mutated form of LOXL2 (LOXL2(Y689F)) lacking enzymatic activity, as well as a LOXL2 deletion mutant lacking the entire catalytic domain, also inhibited calcium or vitamin D-induced up-regulation of involucrin expression, suggesting that the enzymatic activity of LOXL2 is not required for this activity. This conclusion was supported by experiments that showed that β-aminoproprionitrile, an irreversible competitive inhibitor of the enzymatic activity of all lysyl oxidases, is unable to abolish the LOXL2-induced inhibition of HaCaT cell differentiation. The activity of LOXL2(Y689F) required the presence of the fourth scavenger receptor-cysteine-rich (SRCR) domain of LOXL2, which is also the binding target of AB0023. Epitope-tagged LOXL2(Y689F) was internalized at 37 °C by HaCaT cells. The internalization was inhibited by AB0023 and by competition with unlabeled LOXL2, suggesting that these cells may express a LOXL2 receptor. Our results suggest that agents that inhibit the enzymatic activity of LOXL2 may not suffice to inhibit completely the effects of LOXL2 on complex processes that involve altered states of cellular differentiation.     

Effect of TGF-beta 1, TGF-beta 2, and bFGF on chick cartilage and muscle cell differentiation

In insulin containing defined medium TGF-beta 1, TGF-beta 2, and bFGF all stimulate chondrogenic differentiation in high-density micromass cultures of distal limb bud mesenchyme cells of chick embryos. In addition bFGF inhibits myogenic differentiation, while TGF-beta 1 and TGF-beta 2 appear to have no effect. TGF-beta 1 and bFGF together act additively to enhance chondrogenesis, while TGF-beta blocks the bFGF inhibitory action on myoblasts, thus allowing them to differentiate. In the absence of insulin, the inhibitory effect of bFGF on muscle cell differentiation is reduced; cartilage differentiation in the presence of the above growth factors is also slightly reduced.     

RLIP76 (RalBP1) is an R-Ras effector that mediates adhesion-dependent Rac activation and cell migration

The Ras family of small GTPases regulates cell proliferation, spreading, migration and apoptosis, and malignant transformation by binding to several protein effectors. One such GTPase, R-Ras, plays distinct roles in each of these processes, but to date, identified R-Ras effectors were shared with other Ras family members (e.g., H-Ras). We utilized a new database of Ras-interacting proteins to identify RLIP76 (RalBP1) as a novel R-Ras effector. RLIP76 binds directly to R-Ras in a GTP-dependent manner, but does not physically associate with the closely related paralogues H-Ras and Rap1A. RLIP76 is required for adhesion-induced Rac activation and the resulting cell spreading and migration, as well as for the ability of R-Ras to enhance these functions. RLIP76 regulates Rac activity through the adhesion-induced activation of Arf6 GTPase and activation of Arf6 bypasses the requirement for RLIP76 in Rac activation and cell spreading. Thus, we identify a novel R-Ras effector, RLIP76, which links R-Ras to adhesion-induced Rac activation through a GTPase cascade that mediates cell spreading and migration.     

Regulation of TXB(2) and PGE(2) production by TGF-beta(1) in in vitro silica dust-exposed rat alveolar macrophage

We investigated the effect of transforming factor factor-beta(1) (TGF-beta(1)) on thromboxane B(2) (TXB(2)) and prostaglandin E(2) (PGE(2)) production in in vitro silica dust-exposed rat alveolar macrophages (AM). In the presence of 5 mug of anti-TGF-beta(1) antibodies, TXB(2) production decreased, but PGE(2) production increased. Addition of 2 ng of TGF-beta(1) to the culture medium potentiated TXB(2) production, but PGE(2) production apparently did not change. At 50 ng of TGF-beta(1), TXB(2) production decreased, and PGE(2) production varied. Our data suggest that in rat AM: (1) both endogenous and exogenous TGF-beta(1) regulate TXB(2) production; and (2) in the absence of endogenous TGF-beta(1) the liberation of PGE(2) increases; however, exogenous TGF-beta(1) does not have a regulatory effect on PGE(2).     

Retinoic acid down-regulates VPAC(1) receptors and TGF-beta 3 but up-regulates TGF-beta 2 in lung cancer cells

The effects of retinoic acid (RA) on lung cancer cells were investigated. Both all-trans (t-RA) and 13-cis RA (c-RA) decreased specific ¹²⁵I-VIP binding to NCI-H1299 cells in a time- and concentration-dependent manner. After 20 hr, 30 μM t-RA decreased specific ¹²⁵I-VIP binding by 60%. By Scatchard analysis, the density of VIP binding sites but not the affinity was reduced by 42%. NCI-H1299 VPAC₁ receptor mRNA was reduced by 48%. VIP caused a 3-fold elevation in the NCI-H1299 cAMP, and the increase in cAMP caused by VIP was reduced by 38% if the NCI-H1299 cells were treated with t-RA. Using the MTT assay, 3 μM t-RA and 3 μM c-RA inhibited NCI-H1299 proliferation by 60 and 23% respectively. Also, transforming growth factor (TGF)-β2 increased after treatment of NCI-H1299 cells with t-RA whereas TGF-β1 mRNA was unaffected and TGF-β3 mRNA was decreased. These results suggest that RA may inhibit lung cancer growth by down-regulating VPAC₁ receptor and TGF-β3 mRNA but up-regulating TGF-β2 mRNA.     

Alveolar epithelial cell inhibition of fibroblast proliferation is regulated by MCP-1/CCR2 and mediated by PGE2

CC chemokine receptor 2 (CCR2) -/- mice are protected from experimental pulmonary fibrosis, a disease increasingly recognized as being mediated by dysfunctional interactions between epithelial cells and fibroblasts. We have sought to investigate the interactions between alveolar epithelial cells (AECs) and fibroblasts in these fibrosis-resistant (CCR2 -/-) and fibrosis-sensitive (CCR2 +/+) mice. AECs from CCR2 -/- mice suppress fibroblast proliferation more than AECs from CCR2 +/+ mice (77 vs. 43%). Exogenous administration of the CCR2 ligand monocyte chemoattractant protein-1 (MCP-1) to the fibroblast-AEC cocultures reverses the suppression mediated by CCR2 +/+ AECs but has no effect with CCR2 -/- AECs. MCP-1 regulates AEC function but not fibroblast function. AEC inhibition of fibroblast proliferation was mediated by a soluble, aspirin-sensitive factor. Accordingly, AECs from CCR2 -/- mice produce greater quantities of PGE(2) than do AECs from CCR2 +/+ mice, and MCP-1 inhibits AEC-derived PGE(2) synthesis. Diminished PGE(2) production by AECs results in enhanced fibroproliferation. Thus an important profibrotic mechanism of MCP-1/CCR2 interactions is to limit PGE(2) production in AECs after injury, thus promoting fibrogenesis.     

Terminal differentiation of nail matrix keratinocytes involves up-regulation of DNase1L2 but is independent of caspase-14 expression

Abstract Terminal differentiation of keratinocytes in the epidermis and in epidermal appendages results in specialized forms of cell death. Keratinocytes of the nail matrix differentiate into nail corneocytes, the building blocks of the nail plate. Here, we show that, in contrast to the abrupt breakdown of the nucleus during corneocyte formation of epidermal keratinocytes, chromatin undergoes progressive condensation over several nail matrix cell layers below the transition zone to the nail plate, where nuclear DNA disappears. Virtually all keratinocytes in the cell layer immediately beneath the nail plate contained terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end labeling-positive DNA fragments. Nail matrix keratinocytes lacked processed caspase-3, a marker of apoptosis, and did not express caspase-14, a protease up-regulated during terminal differentiation of epidermal keratinocytes. By contrast, DNase1L2, which is also up-regulated during the differentiation of epidermal keratinocytes and plays an essential role in differentiation-associated degradation of nuclear DNA in epidermal keratinocytes, was strongly expressed in the nail matrix–nail plate transition layer. Our results show that caspase-14 is not strictly, if at all, required for differentiation-associated keratinocyte cell death and implicates DNase1L2 in terminal differentiation of nail matrix keratinocytes.     

TGF-beta 1-induced inhibition of mouse mammary ductal growth: developmental specificity and characterization

TGF-beta 1, implanted into growing mouse mammary glands, was previously shown to inhibit ductal growth in an apparently normal and fully reversible manner. In this report we extend these findings to show that TGF-beta 1 inhibition is highly specific. In pregnant or hormone-treated mice, doses of TGF-beta 1 that were capable of fully inhibiting ductal elongation had little effect on the proliferation of lobuloalveolar structures. Additionally, the inhibitory action of TGF-beta 1 on ducts is epithelium-specific, resulting in cessation of DNA synthesis in the rapidly proliferating epithelium of mammary end buds, but does not inhibit DNA synthesis in the stroma surrounding the end buds. At the cellular level, transplant studies showed that TGF-beta 1 inhibited the regeneration of mammary ductal cells when implanted into mammary gland-free fat pads by suppressing the formation of new end buds, without inhibiting maintenance DNA synthesis in ductal lumenal epithelium; this observation indicates the potential of TGF-beta 1 to maintain patterning by suppressing adventitious lateral branching. The time-course of TGF-beta 1 inhibition of end buds was rapid, with cessation of DNA synthesis by 12 hr, followed by loss of the stem cell (cap cell) layer. The question of glandular exposure to TGF-beta 1 administered in EVAc implants was also investigated. Incorporation of TGF-beta 1 into EVAc was found not to degrade the hormone, while the release kinetics of the ligand from implants, its retention in the gland, and the demonstrable zone of exposure were consistent with observed inhibitory effects. These results support the hypothesis that TGF-beta 1 is a natural regulator of mammary ductal growth.     

PGE2-mediated inhibition of T cell p59fyn is independent of cAMP

We recently observed that prostaglandinE₂(PGE₂)-mediated suppression of Tcell functions could result from an attenuation ofp59^fyn protein tyrosine kinaseactivity. The present study evaluated the effects of an adenylatecyclase agonist (forskolin) and antagonist (SQ-22536), as well as thoseof cAMP analogues (dibutyryl cAMP and 8-bromo- cAMP), on T cellp59^fyn kinase activity. The studyallowed us to assess whetherPGE₂-mediated activation ofadenylate cyclase by itself or the elevation in intracellular cAMPlevels is an integral event in the modulation of anti-CD3-linkedp59^fyn activation in T cells. Theexperiments were carried out with splenic T cells from maleSprague-Dawley rats. A 30-50% suppression in theautophosphorylation and the kinase activity ofp59^fyn in T cells incubated withPGE₂ or forskolin was observed.Pretreatment of T cells with SQ-22536 prevented significantPGE₂-mediated inhibition of T cellp59^fyn kinase activity. Incontrast, no change in p59^fynautophosphorylation and kinase activity in T cells treated with cAMPanalogues was observed. These data suggest thatPGE₂-mediated suppression ofp59^fyn autophosphorylation andkinase activity in T cells is dependent on the activation of adenylatecyclase and independent of the elevation in cAMP levels.

     

Cdc42 is required for PIP(2)-induced actin polymerization and early development but not for cell viability

BACKGROUND: Cdc42 and other Rho GTPases are conserved from yeast to humans and are thought to regulate multiple cellular functions by inducing coordinated changes in actin reorganization and by activating signaling pathways leading to specific gene expression. Direct evidence implicating upstream signals and components that regulate Cdc42 activity or for required roles of Cdc42 in activation of downstream protein kinase signaling cascades is minimal, however. Also, whereas genetic analyses have shown that Cdc42 is essential for cell viability in yeast, its potential roles in the growth and development of mammalian cells have not been directly assessed. RESULTS: To elucidate potential functions of Cdc42 mammalian cells, we used gene-targeted mutation to inactivate Cdc42 in mouse embryonic stem (ES) cells and in the mouse germline. Surprisingly, Cdc42-deficient ES cells exhibited normal proliferation and phosphorylation of mitogen- and stress-activated protein kinases. Yet Cdc42 deficiency caused very early embryonic lethality in mice and led to aberrant actin cytoskeletal organization in ES cells. Moreover, extracts from Cdc42-deficient cells failed to support phosphatidylinositol 4,5-bisphosphate (PIP(2))-induced actin polymerization. CONCLUSIONS: Our studies clearly demonstrate that Cdc42 mediates PIP(2)-induced actin assembly, and document a critical and unique role for Cdc42 in this process. Moreover, we conclude that, unexpectedly, Cdc42 is not necessary for viability or proliferation of mammalian early embryonic cells. Cdc42 is, however, absolutely required for early mammalian development.     

Involvement of MAPK signaling molecules and Runx2 in the NELL1-induced osteoblastic differentiation

NELL1 is an extracellular protein inducing osteogenic differentiation and bone formation of osteoblastic cells. To elucidate the intracellular signaling cascade evoked by NELL1, we have shown that NELL1 protein transiently activates the MAPK signaling cascade, induces the phosphorylation of Runx2, and promotes the rapid intracellular accumulation of Tyr-phosphorylated proteins. Unlike BMP2, NELL1 protein does not activate the Smad signaling cascade. These findings suggest that upon binding to a specific receptor NELL1 transduces an osteogenic signal through activation of certain Tyr-kinases associated with the Ras-MAPK cascade, and finally leads to the osteogenic differentiation.