Angiogenesis in collagen I requires alpha2beta1 ligation of a GFP*GER sequence and possibly p38 MAPK activation and focal adhesion disassembly

Angiogenesis depends on proper collagen biosynthesis and cross-linking, and type I collagen is an ideal angiogenic scaffold, although its mechanism is unknown. We examined angiogenesis using an assay wherein confluent monolayers of human umbilical vein endothelial cells were overlain with collagen in a serum-free defined medium. Small spaces formed in the cell layer by 2 h, and cells formed net-like arrays by 6-8 h and capillary-like lumens by 24 h. Blocking of alpha2beta1, but not alpha1 or alpha(v)beta3 integrin function halted morphogenesis. We found that a triple-helical, homotrimeric peptide mimetic of a putative alpha2beta1 binding site: alpha1(I)496-507 GARGERGFP*GER (where single-letter amino acid nomenclature is used, P* = hydroxyproline) inhibited tube formation, whereas a peptide carrying another putative site: alpha1(I)127-138 GLP*GERGRP*GAP* or control peptides did not. A chemical inhibitor of p38 mitogen-activated protein kinase (p38 MAPK), SB202190, blocked tube formation, and p38 MAPK activity was increased in collagen-treated cultures, whereas targeting MAPK kinase (MEK), focal adhesion kinase (FAK), or phosphatidylinositol 3-kinase (PI3K) had little effect. Collagen-treated cells had fewer focal adhesions and 3- to 5-fold less activated FAK. Thus capillary morphogenesis requires endothelial alpha2beta1 integrin engagement of a single type I collagen integrin-binding site, possibly signaling via p38 MAPK and focal adhesion disassembly/FAK inactivation.     

Mesenchymal cells potentiate vascular endothelial growth factor-induced angiogenesis in vitro

The role of soluble factors (including angiogenic cytokines) and extracellular matrix components in the regulation of angiogenesis is clearly established. However, the interrelationship between these factors and perivascular mesenchymal cells is not well understood. Here we have used a three-dimensional collagen gel coculture system to assess the effect of mesenchymal C3H10T1/2 cells on vascular endothelial growth factor-A (VEGF-A)- and fibroblast growth factor-2 (FGF-2)-induced angiogenesis in vitro. We found that coculture markedly potentiated the angiogenic activity of VEGF-A, irrespective of whether or not direct cell-to-cell contact occurred. In contrast, under conditions in which cell-to-cell contact was possible, FGF-2-induced angiogenesis was inhibited by cocultured 10T1/2 cells; this effect was not seen when cell-to-cell contact was prevented. Attempts to identify the molecules responsible for this effect allowed us to exclude FGF-2, transforming growth factorbeta1, platelet derived growth factor-BB, angiopoietin-1, and NO as possible mediators of the potentiating effect of coculture on VEGF-A-induced invasion. In the living organism, angiogenesis occurs in a three-dimensional microenvironment. Contrary to the inhibitory effect of 10T1/2 cells previously reported by others in two-dimensional cultures, our data demonstrate that the paracrine interaction between endothelial and mesenchymal cells potentiates angiogenesis in vitro and that this is cytokine-specific, i.e., it occurs with VEGF-A but not with FGF-2.     

p38 Mitogen-activated protein kinase regulation of endothelial cell migration depends on urokinase plasminogen activator expression

The migration of endothelial cells in response to various stimulating factors plays an essential role in angiogenesis. The p38 MAPK pathway has been implicated to play an important role in endothelial cell migration because inhibiting p38 MAPK activity down-regulates vascular endothelial growth factor (VEGF)-stimulated migration. Currently, the signaling components in the p38 MAPK activation pathway and especially the mechanisms responsible for p38 MAPK-regulated endothelial cell migration are not well understood. In the present study, we found that p38 MAPK activity is required for endothelial cell migration stimulated by both VEGF and nongrowth factor stimulants, sphingosine 1-phosphate and soluble vascular cell adhesion molecule. By using dominant negative forms of signaling components in the p38 MAPK pathway, we identified that a regulatory pathway consisting of MKK3-p38alpha/gamma-MAPK-activated protein kinase 2 participated in VEGF-stimulated migration. In further studies, we showed that a minimum of a 10-h treatment with SB203580 (specific p38 MAPK inhibitor) was needed to block VEGF-stimulated migration, suggesting an indirect role of p38 MAPK in this cellular event. Most interestingly, the occurrence of SB203580-induced migratory inhibition coincided with a reduction of urokinase plasminogen activator (uPA) expression. Furthermore, agents disrupting uPA and uPA receptor interaction abrogated VEGF-stimulated cell migration. These results suggest a possible association between cell migration and uPA expression. Indeed, VEGF-stimulated migration was not compromised by SB203580 in endothelial cells expressing the uPA transgene; however, VEGF-stimulated migration was inhibited by agents disrupting uPA-uPA receptor interaction. These results thus suggest that the p38 MAPK pathway participates in endothelial cell migration by regulating uPA expression.     

One-way cross-talk between p38(MAPK) and p42/44(MAPK). Inhibition of p38(MAPK) induces low density lipoprotein receptor expression through activation of the p42/44(MAPK) cascade.

In this paper, we report that SB202190 alone, a specific inhibitor of p38(MAPK), induces low density lipoprotein (LDL) receptor expression (6-8-fold) in a sterol-sensitive manner in HepG2 cells. Consistent with this finding, selective activation of the p38(MAPK) signaling pathway by expression of MKK6b(E), a constitutive activator of p38(MAPK), significantly reduced LDL receptor promoter activity. Expression of the p38(MAPK) alpha-isoform had a similar effect, whereas expression of the p38(MAPK) betaII-isoform had no significant effect on LDL receptor promoter activity. SB202190-dependent increase in LDL receptor expression was accompanied by induction of p42/44(MAPK), and inhibition of this pathway completely prevented SB202190-induced LDL receptor expression, suggesting that p38(MAPK) negatively regulates the p42/44(MAPK) cascade and the responses mediated by this kinase. Cross-talk between these kinases appears to be one-way because modulation of p42/44(MAPK) activity did not affect p38(MAPK) activation by a variety of stress inducers. Taken together, these findings reveal a hitherto unrecognized one-way communication that exists between p38(MAPK) and p42/44(MAPK) and provide the first evidence that through the p42/44(MAPK) signaling cascade, the p38(MAPK) alpha-isoform negatively regulates LDL receptor expression, thus representing a novel mechanism of fine tuning cellular levels of cholesterol in response to a diverse set of environmental cues.     

Role of focal adhesion formation in migration and morphogenesis of endothelial cells

Cell motility and morphogenesis are regulated by a balance between formation and disassembly of stress fibers and focal adhesions. To understand the mechanisms underlying these cellular responses in angiogenesis, we studied the Rho family protein-driven pathways in FGF-2-induced chemotaxis and capillary morphogenesis of murine brain capillary endothelial cell line, IBE cells. Cells seeded onto fibronectin-coated surface migrated toward FGF-2. Expression of dominant negative Rho A (DNRho) or kinase-dead p21-activated kinase 1 (KDPAK1), or treatment with Y27632 inhibited chemotaxis in association with the lack of FGF-2-induced decrease in focal adhesions. On Matrigel, DNRho and Y27632 induced FGF-2-independent capillary morphogenesis despite loss of stress fiber formation. KDPAK1 cells formed stress fibers and showed capillary morphogenesis in response to FGF-2. Increase in focal adhesions was closely associated with capillary morphogenesis. Our results suggest that formation or disassembly of focal adhesions seems to determine the motility or morphogenesis of endothelial cells.     

Role of the Src homology 2 domain-containing protein Shb in murine brain endothelial cell proliferation and differentiation.

To study the role of the Src homology 2 (SH2) domain-containing protein Shb in angiogenesis, wild-type Shb and SH2 domain-mutated Shb (R522K Shb) were overexpressed in murine immortalized brain endothelial cells. The wild-type Shb cells exhibited an increased rate of apoptosis on serum withdrawal. Both wild-type Shb and R522K Shb cells exhibited enhanced spreading concomitant with cytoskeletal rearrangements that occurred independently of fibroblast growth factor (FGF)-2 stimulation. However, these effects may partly be caused by altered regulation of Rac1 and Rap1 activation in the Shb cells. The Shb-induced cytoskeletal rearrangements were not dependent on phosphatidylinositol 3' kinase activity, but could be reversed by inhibition of Src family kinases. FGF-2 failed to further enhance migration of wild-type Shb and R522K Shb cells. The R522K Shb cells cultured in collagen gels exhibit diminished tubular morphogenesis when treated with FGF-2, implicating the need for a functional Shb molecule in this process. These data suggest that Shb plays a role in the proliferation and differentiation of endothelial cells and, hence, participates in angiogenesis.     

Transforming growth factor-beta1 stimulates vascular endothelial growth factor 164 via mitogen-activated protein kinase kinase 3-p38alpha and p38delta mitogen-activated protein kinase-dependent pathway in murine mesangial cells

Transforming growth factor-beta1 (TGF-beta1) is a potent inducer of extracellular matrix synthesis leading to progressive glomerular fibrosis. The intracellular signaling mechanisms involved in this process remain incompletely understood. The p38 mitogen-activated protein kinase (MAPK) is a major stress signal transducing pathway that is rapidly activated by TGF-beta1 in mesangial cells. We have previously demonstrated MKK3 as the immediate upstream MAPK kinase required for selective activation of p38 MAPK isoforms, p38alpha and p38delta, and stimulation of pro-alpha1(I) collagen by TGF-beta1 in murine mesangial cells. In this study, we further sought to determine MAPK kinase 3 (MKK3)-dependent TGF-beta1 responses by gene expression profiling analysis utilizing mesangial cells isolated from Mkk3-/- mice compared with Mkk3+/+ controls. Interestingly, vascular endothelial growth factor (VEGF) was identified as a TGF-beta1-induced gene affected by deletion of Mkk3. VEGF is a well known endothelial mitogen, whose actions in nonendothelial cell types are still not well understood. We confirmed that TGF-beta1 increased VEGF mRNA and protein synthesis of VEGF164 and VEGF188 isoforms in wild-type mesangial cells. However, in the Mkk3-/- mesangial cells, both TGF-beta1-induced VEGF mRNA and VEGF164 protein expression were inhibited, whereas TGF-beta1-induced VEGF188 protein expression was unaffected. Furthermore, transfection of dominant negative mutants of p38alpha and p38delta resulted in marked inhibition of TGF-beta1-induced VEGF164 expression but not VEGF188, and treatment with recombinant mouse VEGF164 increased collagen and fibronectin mRNA expression in mesangial cells. Taken together, our findings suggest a critical role for the MKK3-p38alpha and p38delta MAPK pathway in mediating VEGF164 isoform-specific stimulation by TGF-beta1 in mesangial cells. Further, VEGF164 stimulates collagen and fibronectin expression in mesangial cells and thus in turn enhances TGF-beta1-induced extracellular matrix and may play an important role in progressive glomerular fibrosis.     

Fibroblast growth factor-2-mediated capillary morphogenesis of endothelial cells requires signals via Flt-1/vascular endothelial growth factor receptor-1: possible involvement of c-Akt

Capillary morphogenesis is a crucial angiogenic response of endothelial cells. Although fibroblast growth factor-2 (FGF-2) potently induces capillary morphogenesis, the contribution of vascular endothelial growth factor-A (VEGF-A) in this response has not been clarified well. Here we examined the role of VEGF signaling in FGF-2-induced capillary morphogenesis by murine brain capillary endothelial cells (IBE cells) and human umbilical vein endothelial cells. FGF-2-treated IBE cells rapidly extended on Matrigel in association with actin reorganization. Chimeric protein, of which the extracellular domain of VEGF receptor-1 (VEGFR-1) fused to immunoglobulin Fc, inhibited FGF-2-induced cell extension, resulting in decreased capillary morphogenesis. Blocking antibody against VEGFR-1 inhibited FGF-2-induced capillary formation. Also, anti-VEGF-A antibody inhibited FGF-2-induced capillary morphogenesis, which was restored by the addition of placental growth factor-1. Similar results were obtained by the experiments with human umbilical vein endothelial cells. Expression of kinase-inactive c-Akt in IBE cells showed impaired capillary morphogenesis promoted by FGF-2. Conversely, stable cell lines expressing activated c-Akt demonstrated ligand-independent capillaries, which were resistant to the treatment with anti-VEGFR-1 blocking antibody. Upstream of c-Akt, calmodulin-dependent signals seemed to be involved. Taken together, signals via VEGFR-1 were required for FGF-2-induced capillary morphogenesis by endothelial cells, and c-Akt activity seemed to be involved in this process.     

SB202190 调节蚕豆保卫细胞中SA 诱导H2O2 产生

运用激光共聚焦扫描技术, 在p38 MAP激酶专一抑制剂SB202190处理下, 探索植物促分裂原活化蛋白激酶(mitogenactivated protein kinase, MAP激酶)介导蚕豆(Vicia faba)保卫细胞中H2O2为代表的活性氧(reactive oxygen species, ROS)信号机制, 发现: p38 MAP激酶专一抑制剂SB202190处理没有导致蚕豆保卫细胞中H2O2和Ca2+探针荧光强度增强, 与水杨酸 (salicylic acid, SA) 或脱落酸 (abscisic acid, ABA) 迅速加强2种探针荧光强度形成鲜明对比; 而该抑制剂分别与SA和ABA共同处理, 前者H2O2探针荧光强度没有增加, 而后者荧光强度仍然能够增加; 而进一步使用Ca2+螯合剂BAPTA和SB202190 ＋SA共同处理, H2O2探针荧光强度没有增加。这些结果初步表明: 无论胞质Ca2+浓度高低, SB202190调节蚕豆保卫细胞中SA诱导H2O2产生, 但是不调节植物逆境信使分子ABA 此类的反应。因此推测, 植物细胞中可能有类似动物和酵母细胞中的p38MAP激酶类, 并可能专一调节植物保卫细胞中H2O2信号通路。据我们所知, 这是首次报道SB202190和SA共同调节植物保卫细胞中ROS信号过程。     

SB202190调节蚕豆保卫细胞中SA诱导H2O2产生

运用激光共聚焦扫描技术,在p38MAP激酶专一抑制剂SB202190处理下,探索植物促分裂原活化蛋白激酶（mitogen-activated protein kinase,MAP激酶）介导蚕豆（Vicia faba）保卫细胞中H2O2为代表的活性氧（reactive oxygen species,ROS）信号机制,发现：p38MAP激酶专一抑制剂SB202190处理没有导致蚕豆保卫细胞中H2O2和Ca^2＋探针荧光强度增强,与水杨酸（salicylic acid,SA）或脱落酸（abscisic acid,ABA）迅速加强2种探针荧光强度形成鲜明对比;而该抑制剂分别与SA和ABA共同处理,前者H2O2探针荧光强度没有增加,而后者荧光强度仍然能够增加;而进一步使用Ca^2＋螯合剂BAPTA和SB202190＋SA共同处理,H2O2探针荧光强度没有增加。这些结果初步表明：无论胞质Ca^2＋浓度高低,SB202190调节蚕豆保卫细胞中SA诱导H2O2产生,但是不调节植物逆境信使分子ABA此类的反应。因此推测,植物细胞中可能有类似动物和酵母细胞中的p38MAP激酶类,并可能专一调节植物保卫细胞中H2O2信号通路。据我们所知,这是首次报道SB202190和SA共同调节植物保卫细胞中ROS信号过程。     

HIV-1 gp120-induced tubular epithelial cell apoptosis is mediated through p38-MAPK phosphorylation

BACKGROUND: HIV-associated nephropathy is accompanied by significant tubular alterations in the form of tubular cell proliferation, apoptosis, and microcystic dilatation. In the present study we evaluated the role of CD4 receptors in HIV-1-induced tubular cell injury. METHODS: To confirm the presence of CD4 receptors in tubular cells, immunocytochemical, Western and Northern blot studies were carried out. To determine the downstream effect of CD4 and gp120 interaction, we evaluated the effect of gp120 on tubular cell p38 mitogen-activated protein kinase (MAPK) activity and phosphorylation. To establish causal relationships between gp120, CD4, and p38 MAPK pathways, we studied the effect of anti-CD4 antibody and SB 202190 (an inhibitor of p38 MAPK) on gp120-induced tubular cell apoptosis. RESULTS: Proximal tubular cells in culture as well as in intact tissue showed expression of CD4 (immunocytochemical and Western blot studies). Cultured tubular cells also showed mRNA expression for CD4 (Northern blot studies). Gp120, at concentrations of 10-100 ng/ ml, triggered tubular cell apoptosis; however, this effect of gp120 was inhibited by anti-CD4 antibody. SB 202190 also inhibited gp120-induced tubular cell apoptosis. In addition, gp120 promoted tubular cell p38 MAPK phosphorylation in a time- and dose- dependent manner. CONCLUSION: Gp120 through interaction with CD4 triggers tubular cell apoptosis. This effect of gp120 on tubular cells is mediated through phosphorylation of p38 MAPK.     

p38 Mitogen-activated protein kinase mediates cell death and p21-activated kinase mediates cell survival during chemotherapeutic drug-induced mitotic arrest

Activation of the mitotic checkpoint by chemotherapeutic drugs such as taxol causes mammalian cells to arrest in mitosis and then undergo apoptosis. However, the biochemical basis of chemotherapeutic drug-induced cell death is unclear. Herein, we provide new evidence that both cell survival and cell death-signaling pathways are concomitantly activated during mitotic arrest by microtubule-interfering drugs. Treatment of HeLa cells with chemotherapeutic drugs activated both p38 mitogen-activated protein kinase (MAPK) and p21-activated kinase (PAK). p38 MAPK was necessary for chemotherapeutic drug-induced cell death because the p38 MAPK inhibitors SB203580 or SB202190 suppressed cell death. Dominant-active MKK6, a direct activator of p38 MAPK, also induced cell death by stimulating translocation of Bax from the cytosol to the mitochondria in a p38 MAPK-dependent manner. Dominant active PAK suppressed this MKK6-induced cell death. PAK seems to mediate cell survival by phosphorylating Bad, and inhibition of PAK in mitotically arrested cells reduced Bad phosphorylation and increased apoptosis. Our results suggest that therapeutic strategies that suppress PAK-mediated survival signals may improve the efficacy of current cancer chemotherapies by enhancing p38 MAPK-mediated cell death.     

Requirement of mitogen-activated protein kinase kinase 3 (MKK3) for activation of p38alpha and p38delta MAPK isoforms by TGF-beta 1 in murine mesangial cells

Transforming growth factor-beta1 (TGF-beta1) is a potent inducer of extracellular matrix (ECM) synthesis that leads to renal fibrosis. Intracellular signaling mechanisms involved in this process remain incompletely understood. Mitogen-activated protein kinase (MAPK) is a major stress signal-transducing pathway, and we have previously reported activation of p38 MAPK by TGF-beta1 in rat mesangial cells and its role in the stimulation of pro-alpha1(I) collagen. In this study, we further investigated the mechanism of p38 MAPK activation by TGF-beta1 and the role of MKK3, an upstream MAPK kinase of p38 MAPK, by examining the effect of targeted disruption of the Mkk3 gene. We first isolated glomerular mesangial cells from MKK3-null (Mkk3-/-) and wild-type (Mkk3+/+) control mice. Treatment with TGF-beta1 induced rapid phosphorylation of MKK3 as well as p38 MAPK within 15 min in cultured wild-type (Mkk3+/+) mouse mesangial cells. In contrast, TGF-beta1 failed to induce phosphorylation of either MKK3 or p38 MAPK in MKK3-deficient (Mkk3-/-) mouse mesangial cells, indicating that MKK3 is required for TGF-beta1-induced p38 MAPK activation. TGF-beta1 selectively activated the p38 MAPK isoforms p38alpha and p38delta in wild-type (Mkk3+/+) mesangial cells, but not in MKK3-deficient (Mkk3-/-) mesangial cells. Thus, activation of p38alpha and p38delta is dependent on the activation of upstream MKK3 by TGF-beta1. Furthermore, MKK3 deficiency resulted in a selective disruption of TGF-beta1-stimulated up-regulation of pro-alpha1(I) collagen expression but not TGF-beta1 induction of fibronectin and PAI-1. These data demonstrate that the MKK3 is a critical component of the TGF-beta1 signaling pathway, and its activation is required for subsequent p38alpha and p38delta MAPK activation and collagen stimulation by TGF-beta1.     

p38丝裂原素激活的蛋白激酶在调节低氧诱导人内皮细胞分泌血管内皮生长因子过程中的作用

血管内皮细胞中血管内皮生长因子(vascular endothelial growthfactor,VEGF)的合成增加在促进血管新生的过程中起着非常重要的作用.然而低氧诱导VEGF分泌的细胞内信号转导机制还不是很清楚.人脐静脉内皮细胞系(ECV304)在低氧或常氧的状态下培养12～24 h后分别用实时定量PCR和Western blot的方法来检测VEGF mRNA的表达及ERK1/2和p38激酶的磷酸化水平.分泌到培养液中的VEGF蛋白用酶联免疫吸附(ELISA)的方法来检测.业已报道,ERK的抑制剂PD98059能够抑制低氧诱导的VEGF基因的表达,根据这个报道,我们发现在低氧情况下,ECV304细胞的ERK1/2磷酸化水平增高以及VEGF的合成增加等这些变化也能被PD98059所抑制.本次实验的新发现是p38激酶的激活在低氧诱导VEGF合成增加中的作用.p38激酶的抑制剂SB202190能抑制低氧诱导的VEGF合成增加.这些数据首次直接证实了p38激酶在低氧诱导人内皮细胞分泌VEGF增加过程中的作用.