Article |
Address correspondence to Lena Claesson-Welsh, Dept. of Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Uppsala, S-751 85, Sweden. Tel.: 46-18-471-4363. Fax: 46-18-55-89-31. E-mail: lena.welsh{at}genpat.uu.se
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Abstract |
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Key Words: p38 mitogenactivated protein kinase; differentiation; angiogenesis; fibroblast growth factor; vascular endothelial growth factor
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Introduction |
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Recent studies have demonstrated that p38 is also activated by several growth factors including FGF-2 (Ono and Han, 2000). In certain types of cells, FGF-2 activates p38 and its downstream target MAPK-activated protein kinase-2 via a Ras-dependent pathway, which ultimately leads to transcriptional activation of cyclic AMP response element-binding protein and activating transcription factor (ATF)-2 (Tan et al., 1996; Xing et al., 1996). MKK3 and MKK6 are relatively specific upstream regulators of p38 (Ono and Han, 2000). To date, four isoforms of the p38 family, namely p38, p38ß, p38
, and p38
, have been identified; these are encoded by distinct genetic loci but share a common Thr-Gly-Tyr motif in kinase subdomain VIII (Ono and Han, 2000).
In this report, we describe the role of p38 activation in FGF-2induced angiogenesis. We present evidence that p38 activity is induced by FGF-2 in endothelial cells in vivo and that p38 negatively regulates endothelial cell differentiation by decreasing the expression of differentiation-specific proteins such as the Notch ligand Jagged1. Inhibition of p38 function leads to increased vessel formation, but a large proportion of the vessels appeared nonfunctional.
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Results |
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Inhibition of p38 activation enhances FGF-2induced tubular morphogenesis
We examined the consequence of inhibition of p38 activity for FGF-2induced tubular morphogenesis in BCE cells cultured in collagen gels. After a 16-h serum starvation, BCE cells were cultured between collagen gels and treated or not with FGF-2 in the presence or absence of 10 µM SB202190. Tubular morphogenesis was enhanced by treatment with SB202190 in an FGF-2dependent manner (Fig. 3, A and B). Thus, in cultures treated with both FGF-2 and SB202190 for 24 h an increased number of cells were engaged in tube formation, and there was an overall increase in tube length (for quantification see Fig. 3 B). By 72 h, the tubular structures had regressed in the FGF-2treated cultures, whereas cultures treated with FGF-2 together with SB202190 still contained tubular structures of considerable length. The enhancing effect was dose dependent at concentrations from 0.5 to 10 µM of SB202190 (Fig. 3 C). Similar results were obtained when cells were treated with SB203580, another inhibitor of p38 (unpublished data). In contrast, the inactive compound SB202474 had no effect (Fig. 3 B). These findings indicate that inhibition of p38 activation enhances FGF-2induced tubular morphogenesis.
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FGF-2 treatment of BCE cells overexpressing wild-type MKK3/6 slightly (1.3-fold) increased the level of activated p38, whereas cells expressing dominant negative MKK3/6 failed to respond to FGF-2 with increased p38 activity (Fig. 4 D, top). Expression of exogenous MKK3/6 was confirmed by immunoblotting using anti-hemagglutinin (HA) monoclonal antibody; the expression of MKK3 clearly exceeded that of MKK6 (Fig. 4 D, bottom). Taken together, these data strongly support the notion that p38 activation exerts a negative regulatory role in tubular morphogenesis.
SB202190 treatment directly enhances FGF-2induced cell differentiation as indicated by expression of Jagged1 during tubular morphogenesis
Recent studies have shown that p38 plays an essential role in cell differentiation in several different cell types (Engelman et al., 1998; Morooka and Nishida, 1998; Zetser et al., 1999). To examine the effect of p38 inhibition on endothelial cell differentiation, we first compared the cell morphology after 8 h of incubation in collagen gel cultures treated with FGF-2 alone and those treated with a combination of FGF-2 and SB202190. At that stage, cells treated with FGF-2 alone were connected by thin extensions (Fig. 5 A, left). In cultures treated with both FGF-2 and SB202190, cell bodies had fused in a majority of cells in the culture (Fig. 5 A, right, arrowheads). As shown in Fig. 1 C, fusion of endothelial cell bodies was induced by FGF-2 alone only after 16 h.
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SB202190 treatment affects apoptosis, DNA synthesis, and proliferation during tubular morphogenesis
It is known that p38 regulates apoptotic cell death in response to cellular stress or certain inflammatory cytokines (Kyriakis and Avruch, 1996). We determined the extent of apoptosis in FGF-2treated BCE cell cultures in collagen gels in the presence or absence of SB202190 by staining with Hoechst 33342. Cells released from dissolved tubes displayed hypercondensed and fragmented nuclei, hallmarks of apoptosis (Fig. 1 H). Quantitation of apoptotic cells showed that endothelial cell cultures underwent massive apoptosis in the absence of growth factors during the first 24 h of culture (Fig. 6 A); in the presence of FGF-2, 20% of the cells were apoptotic. There was no effect of the FGF-2 plus SB202190 treatment compared with FGF-2 alone at 24 h, but at 48 h cells treated with FGF-2 in combination with SB202190 were protected from apoptosis. Treatment with SB202190 alone had a marginal anti-apoptotic effect, probably depending on the endogenous production of growth factors. The exposure of phosphatidylserine on the outer leaflet of the plasma membrane serves as a sensitive marker for early stages of apoptosis (Martin et al., 1995). To determine whether decreased apoptosis contributed to the early effect of SB202190 in promotion of tubular morphogenesis, exposed phosphatidylserine was detected by use of fluorescein-conjugated annexin V. As shown in Fig. 6 B, the percentage of annexin Vpositive cells was not affected by FGF-2 plus SB202190 treatment compared with FGF-2 alone or FGF-2 plus SB202474 treatment at 8 and 24 h when FGF-2induced tubular morphogenesis was modulated by SB202190 treatment. In contrast, treatment with FGF-2 and SB202190 significantly inhibited an increase in annexin Vpositive cells at 48 h. These results indicate that FGF-2 regulates cell survival through activation of p38 during the late phase of tubular morphogenesis but that early effects of SB202190 treatment leading to increased tube length are independent of inhibition of apoptosis.
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Discussion |
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The mechanistic explanation for the induction of p38 activation in the differentiating but not the proliferating endothelial cells could be based on specific gene regulation, since differentiation of endothelial cells is accompanied by induction of specific gene transcription (Kahn et al., 2000). On the other hand, we cannot exclude that p38 activation was dependent on the collagen matrix, since integrin signaling pathways are known to synergize with growth factor receptor pathways to regulate cell proliferation, adhesion, and migration (Clark and Brugge, 1995). The 2ß1 integrin, which binds type 1 collagen, has been shown to mediate p38 activation in the osteosarcoma cell line Saos-2, and the signal transduction molecules Cdc42, MKK3, and MKK4 were implicated in this pathway (Ivaska et al., 1999). Activation of p38 by collagenous matrix has also been shown in human dermal fibroblasts (Ravanti et al., 1999). These results suggest that collagen matrixendothelial cell interaction via integrins such as
2ß1 may modulate FGF-2induced p38 activation.
Recent studies have demonstrated that p38 activation is required for differentiation in several different cell types including neuronal cells (Morooka and Nishida, 1998), adipocytes (Engelman et al., 1998), and myoblasts (Zetser et al., 1999). We provide experimental evidence that activation of p38 directly modulates endothelial cell differentiation during tubular morphogenesis based on the following observations. First, inhibition of p38 in FGF-2stimulated BCE cell collagen gel cultures enhanced FGF-2induced tubular morphogenesis but did not affect apoptosis in the 8- and 24-h cultures (Fig. 3 B and Fig. 6). Second, treatment with SB212190 enhanced fusion of endothelial cells, which is an early step in the organization of the cells into tube-like structures (Fig. 5 A). Third, we found that expression of Jagged1, a ligand for Notch family transmembrane receptors, was increased during the early stage of tubular morphogenesis and that this expression was enhanced by treatment with a p38 inhibitor (Fig. 5 B). Recent studies have shown that Notch signaling is essential for endothelial cell differentiation during tubular morphogenesis. Rat brain microvascular endothelial cells, overexpressing Jagged1 or Notch4/int-3, a constitutive active form of Notch4, form tubular structures spontaneously (Uyttendaele et al., 2000). Targeted gene inactivation of Jagged1 and Notch1 and double inactivation of Notch1/Notch4 results in embryonic lethality with severe defects in vascular remodeling (Xue et al., 1999; Krebs et al., 2000). Expression of an activated form of Notch4 in the mouse embryonic endothelium causes disorganization of the vasculature and embryonic lethality (Uyttendaele et al., 2001). Notch4 is expressed specifically in endothelial cells during embryonic and adult life (Uyttendaele et al., 1996). Although the specificity of Jagged1 for binding to the various Notch receptors is still unclear, these findings imply that the interaction of endothelial Jagged1 with Notch4 regulates vascular morphogenesis. Our data support that induction of Jagged1 is coupled to tubular morphogenesis and that p38 may be a potent regulator of Jagged1 expression.
Vascular homeostasis, which prevails, for example, in the healthy male is thought to depend on a balance between progression and regression, regulated by the microenvironment (Folkman and Haudenschild, 1980). Recent studies indicate that apoptosis of the endothelium plays a central role in the regression of the vasculature (Dimmeler and Zeiher, 2000). Apoptosis-mediated cell deletion during tubular morphogenesis has been demonstrated in human umbilical vein endothelial (HUVE) cells (Pollman et al., 1999) and bovine aortic endothelial cells (Kuzuya et al., 1999). Activation of p38 is likely to be important in the induction of apoptosis and regression of angiogenesis in accordance with the well-established notion that apoptosis induced by several stress stimuli involves the action of p38 (Kyriakis and Avruch, 1996). In line with this view, our data showed that p38 inhibition reduced the degree of apoptosis in the differentiating primary endothelial cells (Fig. 6). Regulation of growth may be achieved in part by induction of apoptosis/regression but also partly by direct inhibition of cell cycle progression. The p38 pathway has been shown to play a negative regulatory role in cell cycle progression (Molnar et al., 1997; Takenaka et al., 1998; Yu and Sato, 1999). Yu and Sato (Yu and Sato, 1999) reported that inhibition of p38 correlated with decreased expression of cdk inhibitor p27Kip1 and hyperphosphorylation of the Rb tumor suppressor protein in HUVE cells. Therefore, growth arrest of endothelial cells during tubular morphogenesis may be mediated via a p38-mediated block in cell cycle progression (Fig. 7, A and B).
Gene inactivation of upstream regulators of p38 or individual p38 isoforms has demonstrated a role for the p38 pathway in vascular development and regulation of angiogenesis. Disruption of the p38 gene results in embryonic lethality around E12.5 due to severe defects in placental development (Adams et al., 2000; Mudgett et al., 2000; Tamura et al., 2000). The p38
-null placentas display a severe reduction in the labyrinthine layer and lack of intermingling embryonic and maternal blood vessels, consistent with a defect in vascularization. The p38
-null mice also display defects in cardiovascularization. However, this defect is likely to be secondary to the impairment in placental function (Adams et al., 2000). A similar phenotype with vascular defects in the placenta was noted in mice lacking expression of MEKK3, a p38 MAPK kinase kinase (Yang et al., 2000). On the other hand, inactivation of MKK3, a p38 MAPK kinase, did not reveal vascular abnormalities (Lu et al., 1999), indicating the complexity of the p38 pathway. Several different downstream targets of p38 have been identified, including protein kinases such as MAPK-activated protein kinase-2/3 and transcription factors such as ATF-2, Elk-1, CHOP, and myocyte enhance factor (MEF)2C (Ono and Han, 2000). For example, MEF2C is expressed in endothelial cells in the embryo and plays a crucial role in cardiovascular development (Lin et al., 1997, 1998). In embryos lacking expression of MEF2C, endothelial cells fail to organize properly and form vascular anomalies characterized by extreme variability in lumen size and defects in remodeling. Thus, MEF2C may be involved downstream of the p38-dependent signaling pathway for regulating vascular morphogenesis.
In the present study, treatment with the p38 inhibitorenhanced FGF-2 induced the CAM neovascularization, but 60% of the vessels displayed features indicative of hyperplasia of the endothelial cells with partially or completely closed lumen. These in vivo results correlate with the in vitro data, since the p38 inhibitor treatment induced cell proliferation and survival in BCE cells collagen gel culture. The morphological features of the vascular defect in p38
-null placentas are different from that in the CAMs treated with the p38 inhibitor. The cellular response to p38 activation may be dependent on the expression pattern of the p38 isoforms and the stimulating agent. Placentas express high level of p38
and p38
but not p38ß and p38
(Wang et al., 1997), whereas vascular endothelial cells coexpress p38
, p38ß, and p38
(Hale et al., 1999). VEGF and placenta growth factor, which are implicated as major regulators of placenta angiogenesis, also activate p38 (Rousseau et al., 1997; Desai et al., 1999). Interestingly, VEGF-induced p38 activity has been shown to positively regulate migration and actin reorganization in HUVE cells (Rousseau et al., 1997). In this study, activation of p38 by VEGF was weaker and more transient in the differentiating primary microcapillary endothelial cell compared with treatment with FGF-2.
In conclusion, our data indicate that FGF-2stimulated p38 signaling pathway has a critical role during angiogenesis in negative regulation of different aspects of endothelial cell function. This regulation may be required for the normal assembly of endothelial cells during vascular morphogenesis and the maintenance of established vessels.
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Materials and methods |
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p38 assays
Serum-starved BCE cells were untreated or treated with 50 ng/ml FGF-2 or 100 ng/ml VEGF (Peprotech) for 560 min or with 300 mM NaCl for 30 min. Alternatively, BCE cells were seeded on collagen gels, incubated for 8 h, and then serum starved and stimulated. Cells were lysed in a Triton X-100 containing phosphatase and protease inhibitors, and immunoprecipitation was performed using an anti-p38 antibody (Gerwins et al., 1997). Samples were washed in kinase buffer (20 mM Hepes, pH 8.0, 20 mM MgCl2, 2 mM MnCl2, 1 mM DTT) and incubated for 30 min at 30°C in kinase buffer, 5 µCi of [-32P]ATP (Amersham Pharmacia Biotech), and 5 µg recombinant NH2-terminal fragment of ATF-2. Samples were resolved by SDS-PAGE in 12% gels and analyzed using a Bio-Imager BAS-1800II (Fuji).
For in vitro kinase inhibition assays, p38 was immunoprecipitated and preincubated or not with 10 µM SB202190 (Calbiochem-Novabiochem">Calbiochem-Novabiochem), a specific inhibitor of p38, or 10 µM SB202474 (Calbiochem-Novabiochem">Calbiochem-Novabiochem), an inactive compound, before the kinase assay. The p38 assays in the CAM was performed essentially according to Eliceiri et al. (1998). Briefly, 0.8 x 0.8 cm filter disks (Whatman) were saturated with 3 mg/ml cortisone acetate (Sigma-Aldrich) and soaked in PBS (40 µl for each filter) with or without FGF-2 (0.5 µg for each filter) on the CAMs. CAM tissue was harvested, snap frozen, and homogenized in a modified RIPA buffer containing 1% deoxycholic acid, 1% Triton X-100, and 0.1% SDS. Equivalent amounts (700 µg) of protein were analyzed as described above.
Tube formation assay
Serum-starved BCE cells were inoculated onto the collagen gels in 6-well dishes at 5 x 104 cells/cm2 and incubated for 3 h at 37°C, and then a second layer of collagen was added. After gelling, cultures were treated with or without 10 ng/ml FGF-2 in DME, 1% NCS. In indicated experiments, SB202190, SB202474, or 0.1% DMSO was added to cells 1 h before addition of FGF-2. The cultures were incubated for 72 h without readdition of growth factors or inhibitors. The cells were examined using a phasecontrast microscope (Nikon Eclipse TE 300) at indicated time points. To quantify the length of tubular structures, three random phasecontrast photomicrographs (x10 objective) per well were taken, and the tubule length was measured using NIH image software (version 1.56). Tubes shorter than 100 µm were excluded from the measurements.
Detection of apoptotic cells
BCE cells treated or not with 10 ng/ml FGF-2 and 10 µM SB202190 or 10 µM SB202474 were fixed in 3% paraformaldehyde (Sigma-Aldrich) in PBS and stained with 5 µg/ml Hoechst 33342 (Molecular Probes) for 30 min. Nuclei with condensed chromatin and fragmented nuclei from five fields (x20 objective) per well were counted. For early detection of apoptosis, BCE cells were stained with fluorescein-conjugated annexin V using Annexin-V-FLUOS staining kit (Roche Molecular Biochemicals). Nuclei were counterstained with 5 µg/ml Hoechst 33342, and the number of annexin Vpositive cells was counted from three fields (x20 objective) per well. Necrotic cells identified through propidium iodide were excluded from the estimation.
Plasmid constructions and transient transfections
Human cDNAs encoding MKK3 (sequence data available from GenBank/EMBL/DDBJ under accession no. L36719) and MKK6 (sequence data available from GenBank/EMBL/DDBJ under accession no. U39657) were generated by PCR from HL60 cells using primers where the 5' primer contained the HA tag sequence YPYDVPDYA. The dominant negative versions of MKK3 and MKK6 (Raingeaud et al., 1996) were created by site-directed mutagenesis where S189/T193 in MKK3 and S207/T211 in MKK6 were changed to alanines. All constructs were verified by DNA sequencing. Transient transfections of BCE cells with the different constructs were performed using LipofectAMINETM (Life Technologies).
Cell proliferation assay
Serum-starved BCE cells were seeded at 9 x 104 cells/well on collagen gelcontaining 24-well plates. After 3 h, cells were stimulated with 10 ng/ml FGF-2. In indicated experiments, 10 µM SB202190, 10 µM SB202474, or 0.1% DMSO was added to cells 1 h before addition of FGF-2. At indicated time points, the gel was solubilized with 20 µl collagenase (2.5 U/µl in PBS; Sigma-Aldrich) for 2 h at 37°C, after which time 230 µl trypsin/EDTA per well was added. Changes in the cell count were determined with a Beckman Coulter counter.
Detection of S-phase cells
Serum-starved BCE cells were inoculated onto collagen gelcontaining coverslips at a density of 105 cells/cm2 and incubated at 37°C for 2 h. Cultures were treated with 10 µM SB202190, 10 µM SB202474, or 0.1% DMSO for 1 h followed by addition of 10 ng/ml FGF-2. After 24 h incubation at 37°C, 10 µM BrdU (Sigma-Aldrich) was added, and incubation continued for 4 h. Cells were fixed in 70% ethanol for 30 min, washed with PBS, and incubated in 0.02 M NaOH for 2 min followed by five washes with PBS. FITC-conjugated anti-BrdU mAb (2:5 dilution; Becton Dickinson) was added, and after 1 h the cells were stained with 5 µg/ml Hoechst 33342 for counter staining of whole nuclei. The labeling index was expressed as the percentage of labeled nuclei/total nuclei from five fields (x20 objective) per well.
Immunoblotting
Serum-starved BCE cells were pretreated for 1 h with 10 µM SB202190 or 0.1% DMSO and then treated with 10 ng/ml FGF-2. The cells were lysed in 500 µl boiling SDS sample buffer at the indicated time points, and samples were analyzed by SDS-PAGE and immunoblotting using nitrocellulose filter (Hybond-ECL; Amersham Pharmacia Biotech). The blots were incubated with anti-Jagged1 antibody (1:200 dilution; Santa Cruz Biotechnology, Inc.) overnight at 4°C, washed, and then incubated with HRP-conjugated antirabbit Ig antibody (1:2,500 dilution; Amersham Pharmacia Biotech) followed by ECL detection (Amersham Pharmacia Biotech). For immunoblotting of p38 and HA-tagged constructs, one part of the cell lysates (80 µl) was separated by SDS-PAGE, transferred, and probed with anti-p38 antibody (1:1,000 dilution) or anti-HA mAb (1:1,000 dilution; Boehringer).
Angiogenesis assay in chicken embryos
The angiogenesis assay in the CAM was performed as described (Sasaki et al., 1999) using fertilized 10-d chick eggs in which the CAM was exposed by removing a 1 x 1 cm piece of the shell to allow application of Whatman filter disks saturated with 3 mg/ml cortisone acetate soaked in PBS (50 µl for each filter) with or without FGF-2 (0.1 µg for each filter) and 10 µM SB202190 or 10 µM SB202474. After 3 d of incubation, the CAM was cut around the filter and inspected in a double-blind procedure to determine the number of vessel branches in the area of the filter disk as described previously (Brooks et al., 1994).
Immunohistochemistry and immunofluorescence
For immunohistochemical analysis of endothelia in the CAM, treated CAM fragments were fixed with formalin and embedded in paraffin. Paraffin-embedded sections were deparaffinized and incubated with antihuman vWF antibody (1:100 dilution; Dako) followed by biotinylated antirabbit Ig (1:100 dilution; Dako). A positive reaction was detected by the ABC method (Dako) and visualized by the DAB reaction. Sections were counterstained with hematoxylin.
Immunofluorescence analysis of phosphorylated p38 in the CAM was performed essentially as described previously (Eliceiri et al., 1998). Treated CAM fragments were embedded in Tissue-Tek OCT compound (Sakura Fintek), frozen, and 4-µm cryostat sections were cut, fixed in acetone for 15 min, blocked in 1% BSA in PBS, incubated with antiphospho-p38 antibody (1:50 dilution) or antihuman vWF antibody (1:100 dilution) overnight, washed, and incubated with TRITC-conjugated goat antirabbit IgG (1:50 dilution; Zymed Laboratories) for 30 min.
Statistical analysis
Results are expressed as mean ± SD. Statistical analyses were performed using StatView software (Version 4.5; SAS Institute, Inc.). Student's t test and Mann-Whitney's U test were used for intergroup comparisons.
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Footnotes |
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Acknowledgments |
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This study was supported by grants from the Swedish Cancer Society, the Novo Nordisk Foundation, and the Göran Gustafsson Foundation.
Submitted: 21 March 2001
Revised: 26 November 2001
Accepted: 30 November 2001
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References |
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---|
Brooks, P.C., R.A. Clark, and D.A. Cheresh. 1994. Requirement of vascular integrin vß3 for angiogenesis. Science. 264:569571.[Medline]
Cuenda, A., J. Rouse, Y.N. Doza, R. Meier, P. Cohen, T.F. Gallagher, P.R. Young, and J.C. Lee. 1995. SB 203580 is a specific inhibitor of a MAP kinase homologue which is stimulated by cellular stresses and interleukin-1. FEBS Lett. 364:229233.[CrossRef][Medline]
Desai, J., V. Holt-Shore, R.J. Torry, M.R. Caudle, and D.S. Torry. 1999. Signal transduction and biological function of placenta growth factor in primary human trophoblast. Biol. Reprod. 60:887892.
Dimmeler, S., and A.M. Zeiher. 2000. Endothelial cell apoptosis in angiogenesis and vessel regression. Circ. Res. 87:434439.
Eliceiri, B.P., R. Klemke, S. Stromblad, and D.A. Cheresh. 1998. Integrin vß3 requirement for sustained mitogen-activated protein kinase activity during angiogenesis. J. Cell Biol. 140:12551263.
Engelman, J.A., M.P. Lisanti, and P.E. Scherer. 1998. Specific inhibitors of p38 mitogen-activated protein kinase block 3T3-L1 adipogenesis. J. Biol. Chem. 273:3211132120.
Folkman, J., and C. Haudenschild. 1980. Angiogenesis in vitro. Nature. 288:551556.[Medline]
Gerwins, P., J.L. Blank, and G.L. Johnson. 1997. Cloning of a novel mitogen-activated protein kinase kinase kinase, MEKK4, that selectively regulates the c-Jun amino terminal kinase pathway. J. Biol. Chem. 272:82888295.
Hale, K.K., D. Trollinger, M. Rihanek, and C.L. Manthey. 1999. Differential expression and activation of p38 mitogen-activated protein kinase , ß,
, and
in inflammatory cell lineages. J. Immunol. 162:42464252.
Ingber, D.E., and J. Folkman. 1989. Mechanochemical switching between growth and differentiation during fibroblast growth factor-stimulated angiogenesis in vitro: role of extracellular matrix. J. Cell Biol. 109:317330.[Abstract]
Ivaska, J., H. Reunanen, J. Westermarck, L. Koivisto, V.M. Kahari, and J. Heino. 1999. Integrin 2ß1 mediates isoform-specific activation of p38 and upregulation of collagen gene transcription by a mechanism involving the
2 cytoplasmic tail. J. Cell Biol. 147:401416.
Jiang, Y., C. Chen, Z. Li, W. Guo, J.A. Gegner, S. Lin, and J. Han. 1996. Characterization of the structure and function of a new mitogen-activated protein kinase (p38ß). J. Biol. Chem. 271:1792017926.
Jiang, Y., H. Gram, M. Zhao, L. New, J. Gu, L. Feng, F. Di Padova, R.J. Ulevitch, and J. Han. 1997. Characterization of the structure and function of the fourth member of p38 group mitogen-activated protein kinases, p38. J. Biol. Chem. 272:3012230128.
Kahn, J., F. Mehraban, G. Ingle, X. Xin, J.E. Bryant, G. Vehar, J. Schoenfeld, C.J. Grimaldi, F. Peale, A. Draksharapu, D.A. Lewin, and M.E. Gerritsen. 2000. Gene expression profiling in an in vitro model of angiogenesis. Am. J. Pathol. 156:18871900.
Krebs, L.T., Y. Xue, C.R. Norton, J.R. Shutter, M. Maguire, J.P. Sundberg, D. Gallahan, V. Closson, J. Kitajewski, R. Callahan, et al. 2000. Notch signaling is essential for vascular morphogenesis in mice. Genes Dev. 14:13431352.
Kubota, Y., H.K. Kleinman, G.R. Martin, and T.J. Lawley. 1988. Role of laminin and basement membrane in the morphological differentiation of human endothelial cells into capillary-like structures. J. Cell Biol. 107:15891598.[Abstract]
Kyriakis, J.M., and J. Avruch. 1996. Protein kinase cascades activated by stress and inflammatory cytokines. Bioessays. 18:567577.[Medline]
Li, Z., Y. Jiang, R.J. Ulevitch, and J. Han. 1996. The primary structure of p38: a new member of p38 group of MAP kinases. Biochem. Biophys. Res. Commun. 228:334340.[CrossRef][Medline]
Lin, Q., J. Schwarz, C. Bucana, and E.N. Olson. 1997. Control of mouse cardiac morphogenesis and myogenesis by transcription factor MEF2C. Science. 276:14041407.
Lin, Q., J. Lu, H. Yanagisawa, R. Webb, G.E. Lyons, J.A. Richardson, and E.N. Olson. 1998. Requirement of the MADS-box transcription factor MEF2C for vascular development. Development. 125:45654574.
Lu, H.T., D.D. Yang, M. Wysk, E. Gatti, I. Mellman, R.J. Davis, and R.A. Flavell. 1999. Defective IL-12 production in mitogen-activated protein (MAP) kinase kinase 3 (Mkk3)-deficient mice. EMBO J. 18:18451857.
Martin, S.J., C.P. Reutelingsperger, A.J. McGahon, J.A. Rader, R.C. van Schie, D.M. LaFace, and D.R. Green. 1995. Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl. J. Exp. Med. 182:15451556.[Abstract]
Molnar, A., A.M. Theodoras, L.I. Zon, and J.M. Kyriakis. 1997. Cdc42Hs, but not Rac1, inhibits serum-stimulated cell cycle progression at G1/S through a mechanism requiring p38/RK. J. Biol. Chem. 272:1322913235.
Montesano, R., L. Orci, and P. Vassalli. 1983. In vitro rapid organization of endothelial cells into capillary-like networks is promoted by collagen matrices. J. Cell Biol. 97:16481652.[Abstract]
Montesano, R., J.D. Vassalli, A. Baird, R. Guillemin, and L. Orci. 1986. Basic fibroblast growth factor induces angiogenesis in vitro. Proc. Natl. Acad. Sci. USA. 83:72977301.[Abstract]
Morooka, T., and E. Nishida. 1998. Requirement of p38 mitogen-activated protein kinase for neuronal differentiation in PC12 cells. J. Biol. Chem. 273:2428524288.
Mudgett, J.S., J. Ding, L. Guh-Siesel, N.A. Chartrain, L. Yang, S. Gopal, and M.M. Shen. 2000. Essential role for p38 mitogen-activated protein kinase in placental angiogenesis. Proc. Natl. Acad. Sci. USA. 97:1045410459.
Pepper, M.S., N. Ferrara, L. Orci, and R. Montesano. 1992. Potent synergism between vascular endothelial growth factor and basic fibroblast growth factor in the induction of angiogenesis in vitro. Biochem. Biophys. Res. Commun. 189:824831.[Medline]
Qi, J.H., T. Matsumoto, K. Huang, K. Olausson, R. Christofferson, and L. Claesson-Welsh. 1999. Phosphoinositide 3 kinase is critical for survival, mitogenesis and migration but not for differentiation of endothelial cells. Angiogenesis. 3:371380.
Raingeaud, J., A.J. Whitmarsh, T. Barrett, B. Derijard, and R.J. Davis. 1996. MKK3- and MKK6-regulated gene expression is mediated by the p38 mitogen-activated protein kinase signal transduction pathway. Mol. Cell. Biol. 16:12471255.[Abstract]
Ravanti, L., J. Heino, C. Lopez-Otin, and V.M. Kahari. 1999. Induction of collagenase-3 (MMP-13) expression in human skin fibroblasts by three-dimensional collagen is mediated by p38 mitogen-activated protein kinase. J. Biol. Chem. 274:24462455.
Rousseau, S., F. Houle, J. Landry, and J. Huot. 1997. p38 MAP kinase activation by vascular endothelial growth factor mediates actin reorganization and cell migration in human endothelial cells. Oncogene. 15:21692177.[CrossRef][Medline]
Sasaki, T., H. Larsson, J. Kreuger, M. Salmivirta, L. Claesson-Welsh, U. Lindahl, E. Hohenester, and R. Timpl. 1999. Structural basis and potential role of heparin/heparan sulfate binding to the angiogenesis inhibitor endostatin. EMBO J. 18:62406248.
Takenaka, K., T. Moriguchi, and E. Nishida. 1998. Activation of the protein kinase p38 in the spindle assembly checkpoint and mitotic arrest. Science. 280:599602.
Tan, Y., J. Rouse, A. Zhang, S. Cariati, P. Cohen, and M.J. Comb. 1996. FGF and stress regulate CREB and ATF-1 via a pathway involving p38 MAP kinase and MAPKAP kinase-2. EMBO J. 15:46294642.[Abstract]
Uyttendaele, H., G. Marazzi, G. Wu, Q. Yan, D. Sassoon, and J. Kitajewski. 1996. Notch4/int-3, a mammary proto-oncogene, is an endothelial cell-specific mammalian Notch gene. Development. 122:22512259.
Uyttendaele, H., J. Ho, J. Rossant, and J. Kitajewski. 2001. Vascular patterning defects associated with expression of activated Notch4 in embryonic endothelium. Proc. Natl. Acad. Sci. USA. 98:56435648.
Wang, X.S., K. Diener, C.L. Manthey, S. Wang, B. Rosenzweig, J. Bray, J. Delaney, C.N. Cole, P.Y. Chan-Hui, N. Mantlo, et al. 1997. Molecular cloning and characterization of a novel p38 mitogen-activated protein kinase. J. Biol. Chem. 272:2366823674.
Xing, J., D.D. Ginty, and M.E. Greenberg. 1996. Coupling of the RAS-MAPK pathway to gene activation by RSK2, a growth factor-regulated CREB kinase. Science. 273:959963.[Abstract]
Xue, Y., X. Gao, C.E. Lindsell, C.R. Norton, B. Chang, C. Hicks, M. Gendron-Maguire, E.B. Rand, G. Weinmaster, and T. Gridley. 1999. Embryonic lethality and vascular defects in mice lacking the Notch ligand Jagged1. Hum. Mol. Genet. 8:723730.
Young, P.R., M.M. McLaughlin, S. Kumar, S. Kassis, M.L. Doyle, D. McNulty, T.F. Gallagher, S. Fisher, P.C. McDonnell, S.A. Carr, et al. 1997. Pyridinyl imidazole inhibitors of p38 mitogen-activated protein kinase bind in the ATP site. J. Biol. Chem. 272:1211612121.
Zetser, A., E. Gredinger, and E. Bengal. 1999. p38 mitogen-activated protein kinase pathway promotes skeletal muscle differentiation. Participation of the Mef2c transcription factor. J. Biol. Chem. 274:51935200.
Zimrin, A.B., M.S. Pepper, G.A. McMahon, F. Nguyen, R. Montesano, and T. Maciag. 1996. An antisense oligonucleotide to the notch ligand Jagged enhances fibroblast growth factor-induced angiogenesis in vitro. J. Biol. Chem. 271:3249932502.