We show in rat lung microvessel endothelial cells (RLMVEC) that endocytosis is a critical determinant of activation of mitogen-activated protein kinase (MAPK) and thereby regulates endothelial monolayer integrity. In RLMVEC grown in serum-free medium, we observed that albumin supplementation induced the phosphorylation of p38 MAPK within 30 min, which persisted for up to 2 h. Engagement of the endocytic machinery regulated the activation of p38 MAPK that contributed to endothelial cell proliferation and reduction of apoptosis. We also observed an interaction between the caveolar protein caveolin-1 and p38 MAPK with reciprocal coimmunoprecipitation assays and colocalization using double-label immunofluorescence staining. Knockdown of caveolin-1 expression with small interfering RNA significantly reduced endocytosis and activation of p38 MAPK and interfered with the ability of endothelial cells to form a confluent monolayer. Thus caveolae-mediated endocytosis and concomitant activation of p38 MAPK may help to maintain endothelial monolayer integrity by signaling proliferation and survival of endothelial cells.
- stress mitogen-activated protein kinase
- transforming growth factor-β signaling
- cell proliferation
the basic function of endothelial cells lining the vessel wall is their ability to form a continuous confluent monolayer and serve as a semipermeable barrier (11). Endothelial cells also have a large number of vesicles comprising 15–20% of cell volume (20). There is a constitutive process of endocytosis mediated by the internalization of the plasmalemmal invaginations, caveolae (20). Although caveolar internalization is responsible for transcytosis of albumin and other plasma proteins across the endothelial barrier, endocytosis may also have key functions in cell signaling (4, 5, 7, 25). In the present study, we investigated the possibility that caveolae-mediated endocytosis is a determinant of endothelial cell proliferation and cell survival and thus regulates the integrity of the endothelial monolayer.
We have shown (24) that albumin activation of transforming growth factor (TGF)-β receptor (TβR)II stimulates endocytosis in rat lung microvessel endothelial cell (RLMVEC) monolayers. TGF-β signaling is triggered by its binding to a heteromeric complex consisting of two transmembrane serine/threonine kinase receptors, type I and type II (TβRI and TβRII) (8, 16, 26). In serum-deprived RLMVEC, we observed that supplementation with albumin induced activation of caveolae-mediated endocytosis (11) and subsequent TβRII signaling that resulted in Smad2 phosphorylation and Smad4 translocation (21, 24). These studies did not examine the downstream consequences of activation of endocytosis in promoting endothelial cell proliferation and survival.
Mammalian p38 mitogen-activated protein kinase (MAPK) is activated in response to extracellular stimuli such as UV light, heat, osmotic shock, inflammatory cytokines (TNF-α and IL-1β), and growth factors (e.g., colony-stimulating factor-1) (28). TGF-β, through activation of TGF-β-activated protein kinase-1-binding protein mediates p38 MAPK activation, thus linking TGF-β signaling to p38 MAPK activation (23, 27). In the present study, we showed that induction of caveolae-mediated endocytosis in endothelial cells resulted in the activation of p38 MAPK enzyme activity and phosphorylation of p38 MAPK. The activation of p38 MAPK activation induced endothelial cell proliferation and reduced apoptosis. Moreover, depletion of caveolin-1 expression with small interfering RNA (siRNA) significantly reduced endocytosis as well as activation of p38 MAPK and resulted in the impairment of proliferation and apoptosis responses in endothelial cells. These results point to a crucial role of p38 MAPK activation occurring as the result of caveolae-mediated endocytosis in regulating endothelial cell proliferation and survival and thereby controlling the integrity of the endothelial monolayer.
Polyclonal antibodies directed against caveolin-1, p38 MAPK, and the phosphorylated form of p38 MAPK (p38-P MAPK), fluorescein isothiocyanate (FITC)- and Texas Red isothiocyanate (TRITC)-conjugated polyclonal goat and donkey antibodies against mouse and rabbit IgG, goat anti-mouse IgG, mouse IgG, and rabbit preimmune serum IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit anti-phospho-activating transcription factor (ATF)-2 antibody was purchased from Cell Signaling (Beverly, MA). RLMVEC (VEC Technologies, Rensselaer, NY) were cultured as described previously (11). Bovine serum albumin fraction V (BSA; 99% pure, endotoxin free), FITC-labeled BSA, TRITC-BSA, and bovine IgG were purchased from Sigma (St. Louis, MO). DMEM, PBS, fetal bovine serum (FBS), and antibiotics (ampicillin, penicillin) were purchased from Biowhittaker (Walkersville, MD). Sepharose IgG-conjugated beads were purchased from Amersham Pharmacia (Piscataway, NJ). Carboxyfluorescein diacetate succinimidyl ester (CFSE), an intracellular dye to monitor cell proliferation, was a gift of Dr. Ronald Hoffman, University of Illinois at Chicago Cancer Center. Experiments were performed at least three times, and in specific instances representative data are reported.
Fluorescence measurements in live cells (RLMVEC) were performed as described previously (24) with a Nikon Eclipse 800 fluorescence microscope equipped with differential interference contrast (DIC). Fluorescence and DIC images were acquired with a charge-coupled device camera (Hamamatsu Photonics, Hamamatsu, Japan) and processed with Metamorph imaging analysis software (Universal Imaging, West Chester PA).
Endocytosis was assayed by the uptake of albumin with FITC-BSA or TRITC-BSA as the tracer (24). In RLMVEC treated with the MAPK p38 inhibitor SB-203580 (6, 7, 25) (Calbiochem, San Diego, CA), the drug (1–20 μM) was incubated in increasing concentrations in confluent cell monolayers for 4 h at 37°C. Care was taken to avoid exposure to light during the uptake assay. Endocytosis following caveolin-1 siRNA treatment was similarly assayed.
Terminal deoxynucleotidyltransferase nick end labeling (TUNEL) was used to determine the number of cells undergoing apoptosis. RLMVEC monolayers were fixed with 4% formaldehyde in PBS for 30 min and washed with chilled PBS. Cells were treated with terminal transferase together with FITC-dATP and dATP. In controls, no terminal transferase was included. Cells were observed with a FITC filter set and a Nikon E600 fluorescence microscope. Apoptotic nuclei looked fragmented and stained intensely with fluorescein-conjugated substrate. Nonapoptotic cells showed large vesicular nuclei and had regular chromatin staining. Cells (100) were randomly selected in a given microscopic field, and the percentage of apoptotic cells was determined. At least five such microscopic fields were selected from each experimental condition.
Apoptosis and cell survival assay.
Cells undergoing apoptosis were stained with the nuclear dye 4′,6′-diamidino-2-phenylindole (DAPI) and counted in UV images acquired with a Nikon E600 microscope. Apoptotic cells showed nuclei that appeared fragmented and stained brightly with DAPI, whereas normal live cells had nuclei with rounded vesicular morphology that stained lightly with DAPI and showed finely stippled chromosomal threads. In each randomly chosen field of RLMVEC, 100–200 cells per field were counted. For the cell survival index, a similar assessment using DAPI staining of live cells was used to determine the fraction of live cells. Three experiments were conducted in each condition, and the results are reported as means ± SD.
Fluorescence-activated cell sorting analysis of apoptosis.
RLMVEC were grown in serum-free or DMEM medium supplemented with albumin and various treatments such as taxol, TGF-β, p38 inhibitor SB-20385, or caveolin-1 siRNA as indicated. After treatment, cells were exposed to trypsin, washed twice in 2% FBS in PBS (pH 7.4), and filtered through a 60-μm nylon mesh. Cells were stained with Vybrant Apoptosis Assay Kit no. 5 (Molecular Probes, Eugene, OR), and fluorescence-activated cell sorting (FACS) analysis was performed. Populations of viable, apoptotic, and dead cells were easily distinguished by flow cytometry using the 488-nm line of the Ar laser. Dead cells were labeled with propidium iodide (PI), and the blue fluorescent Hoechst 33342 stain labeled condensed chromatin of apoptotic cells more brightly than the chromatin of normal cells. Before data acquisition, 1 μl of PI per milliliter of cell suspension was added on ice, followed by 1 μl of Hoechst 33342. Cells were incubated on ice for 2 min, and PI and Hoechst 33342 fluorescence was read at 575 and 480 nm, respectively. For each experimental condition three independent experiments were conducted, and the data are presented as means ± SD.
Immunostaining and confocal microscopy.
RLMVEC were fixed with 4% paraformaldehyde and stained with mouse anti-caveolin-1 monoclonal antibody and polyclonal rabbit anti-p38-P MAPK antibody as described previously (24). Cells were washed with PBS-Triton (0.1%) and labeled with Alexa 488 or Alexa 594 secondary antibodies (1:200 in PBS) for 1 h at room temperature, washed, and mounted with Prolong antifade mounting medium (Molecular Probes). Images were collected with a Zeiss LSM 510 META confocal microscope with the pinhole set to achieve 1 Airy unit.
Lysate and membrane-enriched fraction isolation from RLMVEC.
Detergent-soluble membrane fractions were isolated from RLMVEC as described previously (24).
SDS-PAGE and immunoblotting.
RLMVEC were grown to confluence in albumin-free or albumin-supplemented medium in 10-cm or six-well dishes. SDS-PAGE, immunoblotting, and signal detection were performed as described previously (24).
Inhibition of p38 MAPK.
RLMVEC were incubated for 4 or 8 h with the p38 MAPK inhibitor SB-203580 (10–20 μM). Confluent cells were grown either in the absence of serum or in medium supplemented with albumin. Hyperosmotic shock was provided with 0.5 M NaCl.
p38 MAPK assay.
RLMVEC were grown to confluence in 10-cm culture plates in DMEM supplemented with 10% FBS and antibiotics and then grown for 24 h in either serum-free or FBS-supplemented medium for 24 h. The p38 MAPK assay was carried out as described previously (1). Briefly, cells were incubated in the presence or absence of albumin or FBS for 1 h or with 0.5 M NaCl for 30 min at 37°C. Cells were then lysed as described previously (24). Cell lysate was immunoprecipitated with rabbit anti-p38 polyclonal antibody together with protein G-Sepharose. The immunocomplex was subsequently washed with phosphorylation lysis buffer containing 0.1% Triton X-100 and twice with kinase buffer [mM: 25 HEPES, 25 MgCl2, 25 α-glycerophosphate, 0.5 ATP, 2 dithiothreitol (DTT), and 0.1 Na3VO4, with 20 μg of glutathione S-transferase-ATF-2 fusion protein] for 20 min at 30°C. Proteins were analyzed by SDS-PAGE, and the phosphorylated protein substrate was detected by immunoblotting with anti-phospho-ATF-2 antibody diluted 1:100 in PBS. The blot from the kinase assay was stripped and probed with an antibody against p38 MAPK to normalize protein loading. We similarly studied the induction of p38 MAPK phosphorylation after treatment of RLMVEC with TGF-β. Cells were incubated with the optimum dose of 2 ng/ml TGF-β (R&D Systems, Minneapolis, MN) for different time periods. Cell lysates were prepared, and protein fractions were immunoblotted with antibodies against p38-P MAPK. The blots were stripped and reblotted with rabbit anti-p38 MAPK as described above.
CFSE dye dilution assay and FACS analysis.
RLMVEC were grown to confluence in 10-cm dishes in DMEM supplemented with 10% FBS and antibiotics. Confluent monolayers were incubated in serum-free medium for 24 h. In one group 100 μg/ml albumin-supplemented DMEM was used, whereas in the other group RLMVEC were maintained in albumin-free medium. Cells were detached with trypsin, suspended and washed with chilled PBS twice, and stained with 5 μg/ml of CFSE dye on ice for 10 min. At the concentration of CFSE dye used, cytotoxic effects were not observed. The cell-permeant fluorescein-based dye attaches to cytoplasmic components of the cells, resulting in uniform brightness. On cell division, the dye is distributed equally between daughter cells, allowing the resolution of up to eight cycles of cell divisions by flow cytometry (10, 15). Cells were suspended at a final concentration of 5 × 107 cells/ml, washed with DMEM three times to remove nonspecific binding of the dye, and analyzed with a Beckman cell sorter. FITC-labeled beads were used to calibrate the input. Samples from the cells were collected on day 0 from albumin-free and albumin-supplemented cultures. The stained cells were activated by incubating in medium containing albumin and grown at 37°C in the dark. RLMVEC proliferation was checked in cells grown in the presence of the p38 MAPK inhibitor SB-203580 at concentrations of 10 and 20 μg/ml. In indicated experiments, cells were treated with 2 mM taxol or transfected with caveolin-1-specific siRNA or control siRNA (see Knockdown of caveolin-1 expression by siRNA) and starved for 24 h after 2 days of siRNA transfection. In one group of cells transfected with caveolin-1 siRNA albumin was supplemented in serum-free medium for 24 h, whereas the other group of cells were simply grown in serum-free medium. After 24 h, the CFSE fluorescence was measured and analyzed with a Beckman Coulter counter (Chaska, MN).
Coimmunoprecipitation of caveolin-1 and p38 MAPK.
Immunoprecipitation and Western blotting were used to determine the molecular association between p38 MAPK and caveolin-1. Cell lysate preparation from RLMVEC, immunoprecipitation, Western blotting, and signal detection were carried out as described previously (24). About 250 μl of the total cell lysate containing an equal amount of total protein was incubated with 4 μg of rabbit polyclonal anti-p38 MAPK antibody. The immunoprecipitated proteins were separated by SDS-PAGE and Western blotted with anti-caveolin-1 antibody. In the first set of experiments, rabbit polyclonal affinity-purified anti-caveolin-1 antibody (sc-894, Santa Cruz Biotechnology) was used. Control rabbit IgG resulted in negative staining, whereas the anti-p38 MAPK antibody (sc-535) stained a band of 38 kDa. In controls, control IgG rabbit or primary antibody preadsorbed with the antigenic peptide did not stain the protein band corresponding to p38 MAPK. Experiments were also performed in reverse in which anti-p38 MAPK (sc-535) antibody was used for immunoprecipitation and anti-caveolin-1 antibody was used for Western blotting. When specific peptides were used to block the primary anti-caveolin-1 antibody, no staining was observed by immunoblot, indicating the specificity of the affinity-purified primary antibodies. The experiments were repeated three times and gave similar results.
Knockdown of caveolin-1 expression by siRNA.
RLMVEC were grown to confluence on gelatin-coated cover glass and placed in six-well culture plates in 10% FBS-supplemented DMEM. The target sequences for caveolin-1 (GenBank accession no. BC009685) double-stranded (ds)RNA correspond to the 21 nucleotides 403–423 (AAGAGCTTCCTGATTGAGATT) of the coding sequence of human caveolin-1 (2). We determined by a BLAST search that this 21-nucleotide sequence is completely conserved in the human, mouse, and rat caveolin-1 genes. Cells were treated with either siRNA specific for caveolin-1 or sham siRNA with scrambled sequence that was checked with the NCBI Blast program. dsRNA corresponding to the caveolin-1 and sham dsRNA were purchased from Dharmacon (Lafayette, CO) and used according to the manufacturer's instructions. RLMVEC were seeded at a density of 5 × 104 cells on gelatin-coated glass coverslips 24 h before siRNA transfection. For each well, 200 μl of serum-free DMEM was mixed with 5 μl of Neophectin (Deerfield, IL) liposome-based transfection reagent and incubated at room temperature (RT) for 10 min. To this mixture, caveolin-1-specific or sham dsRNA was added and incubated for 10 min at RT. Cells were washed with 1 ml of serum-free medium and incubated with 30 ng of dsRNA or only the Neophectin-containing medium. Complete serum-containing medium (800 μl) was added to each well, and cells were incubated at 37°C in a humidified 5% CO2 chamber for 24 or 48 h. Downregulation of caveolin-1 expression was ascertained by both Western blotting and immunostaining using caveolin-1-specific antibody. The experiments were repeated three times and gave reproducible results.
Activation of endocytosis prevents loss of endothelial monolayer integrity.
Figure 1Aa shows that RLMVEC grown in minimal medium (DMEM) in the absence of serum and other growth factors for 24 h lose their integrity as a monolayer, begin to detach from the surface, and show signs of cell degeneration. Cells also assumed a rounded phenotype (see arrow pointing to a cell beginning to detach, Fig. 1Aa). In contrast, cells grown in DMEM in the presence of 10% normal serum grew in a monolayer of typical cobblestone pattern that did not show gaps, cell rounding, or detachment (Fig. 1Ab). When serum-deprived cells were supplemented with physiological concentrations of albumin (BSA), the monolayer showed the same phenotype as cells grown in the presence of serum (Fig. 1Ad). Denatured albumin (treated with 50 mM reducing agent DTT) for 1 h and made free of DTT by dialysis of denatured DTT-albumin with three changes of 1 liter of PBS, failed to preserve RLMVEC monolayer integrity (Fig. 1Ac). We further tested the effects of denaturation of albumin by boiling albumin solution for 10 min, which also failed to preserve monolayer integrity of RLMVEC in serum-free medium (data not shown). There was no significant difference in the ability of recombinant human albumin, human serum albumin, or rat serum albumin (data not shown) to preserve monolayer integrity. We found that, in contrast, bovine immunoglobulin (10–200 μg/ml) or ovalbumin (100 μg/ml) could not be substituted for albumin (data not shown).
We used TUNEL staining (Fig. 1B, a and b) and DAPI staining (Fig. 1B, c and d) to determine the number of cells undergoing apoptosis in RLMVEC monolayers grown in serum-free medium. We observed that supplementing serum-free medium with albumin reduced endothelial apoptosis (Fig. 1B). The percentage of cells undergoing apoptosis decreased from 22 ± 6% in the absence of albumin to 8 ± 4% in the presence of 4% (wt/vol) albumin, which is the physiological level (Fig. 1Be). There was no significant difference when BSA or rat serum albumin was substituted in these experiments (data not shown). These results were further confirmed by using FACS and TUNEL staining analyses to determine apoptosis. Results showed that albumin supplementation of serum-free medium reduced apoptosis and increased cell survival (Table 1).
Addition of albumin to serum-deprived endothelial cells promotes cell proliferation in a p38 MAPK-dependent manner.
To quantify the ability of albumin to promote cell proliferation, we used the CFSE dye-based dilution assay. This nontoxic cell-permeant fluorescein dye attaches to cytoplasmic components of cells, resulting in uniform brightness. On cell division, the dye is distributed equally (and thus diluted) between daughter cells, allowing resolution of up to eight cell cycles of cell division (15). Figure 2A shows fluorescence micrographs of RLMVEC stained with CFSE at days 0, 1, and 2 in RLMVEC grown in DMEM alone (Fig. 2A, a–c), after supplementation with 0.01% (100 μg/ml) albumin (Fig. 2A, d–f), and after albumin supplementation of cells treated with the p38 MAPK inhibitor SB-203580 (Fig. 2A, g–i). In contrast to serum-deprived cells grown in absence of albumin, cells grown in albumin-supplemented medium showed marked dilution of the dye within 2 days (Fig. 2A, d–f) indicative of cell proliferation. Arrows in Fig. 2A, e and f, indicate dilution of CFSE dye from day 0 to day 2 and formation of a confluent monolayer (Fig. 2Af), i.e., dilution of CFSE dye in albumin-supplemented cells was greater than in cells grown in albumin-free medium (Fig. 2A, a–c). Asterisks in Fig. 2Ac mark intercellular gaps that formed in endothelial monolayers. CFSE dilution similar to that seen in the absence of albumin supplementation was observed when albumin-supplemented RLMVEC were incubated in the presence of the p38 MAPK inhibitor SB-203580 (3, 6, 12) (Fig. 2A, g–i); gaps formed in endothelial monolayers treated with SB-203580 even on day 0 after 2 h of treatment (Fig. 2Ag) and were also evident on days 1 and 2 (asterisks, Fig. 2A, h and i). To address the possibility that CFSE dye could leak out of cells if cell proliferation is blocked, we used the microtubule-binding antiproliferative agent taxol (50 μM). In the taxol-treated cells, CFSE dye did not dilute (data not shown), suggesting that inhibition of proliferation resulted in an absence of dye dilution.
We further used flow cytometry to measure cell proliferation in the presence and absence of albumin. Confluent cell monolayers were exposed to serum-free medium for 24 h. In one group 100 μg/ml albumin was supplemented, whereas in the other group RLMVEC were grown in albumin-free medium. Cells were detached with trypsin, suspended and washed twice with chilled PBS, and stained with 5 μg/ml of CFSE dye. Figure 2B shows the FACS profile of RLMVEC grown in the presence and absence of 0.01% albumin. The x-axis shows the fluorescence intensity in log units, and the y-axis shows the number of cells. The continuous thin line shows CFSE dye fluorescence at day 0 in cells grown in DMEM, and the thick line shows the cells supplemented with albumin at day 0. The thin dotted line shows CFSE dye distribution at day 1 in cells grown in DMEM, and the thick dotted line shows CFSE dye in cells supplemented with albumin on day 1. There was a greater shift (indicative of dye dilution) toward lower fluorescence intensity in cells grown in the presence of albumin compared with cells grown in DMEM alone. We examined the effects of taxol on RLMVEC survival and apoptosis with FACS analysis in serum-deprived conditions and when cells were supplemented with albumin. Taxol (2 mM) showed an increase in apoptosis in serum-deprived cells, whereas cells supplemented with albumin showed reduced apoptosis (Table 1). No significant increase in apoptosis in RLMVEC was observed when taxol was included in the medium at lower concentrations (100 μM to 1 mM) compared with cells grown in serum-free medium for 24 h (Table 1).
p38 MAPK inhibitor induces endothelial cell survival.
To determine the role of p38 MAPK in RLMVEC survival and induction of apoptosis, we preincubated RLMVEC in serum-free medium or medium supplemented with 4% albumin with the p38 MAPK inhibitor SB-203580. Cells were analyzed with FACS and TUNEL staining as described above. Serum-free conditions reduced cell survival and promoted apoptosis, whereas addition of albumin improved cell survival (Table 1). SB-203580 slightly reduced the effects of serum deprivation on cell death. However, SB-203580 did not significantly alter the effect of albumin in promoting cell survival and reducing apoptosis in serum-deprived cells (Table 1).
TGF-β and albumin supplementation induce p38 MAPK phosphorylation.
We previously demonstrated (24) that engagement of endocytic machinery results in TβRII-activated signaling. Therefore, we investigated whether the TβRII ligand TGF-β induces the phosphorylation of p38 MAPK in RLMVEC. Figure 3, A and B, immunoprecipitation of caveolin-1 using polyclonal anti-caveolin-1 antibody and Western blotting with anti-p38 MAPK antibody reveal that the two proteins are associated. In a reciprocal experiment in which cell lysates were immunoprecipitated with anti-p38 MAPK antibody, we detected caveolin-1 by Western blotting (Fig. 4). As a control, there was absence of caveolin-1 coimmunoprecipitation when normal rabbit IgG was used as the precipitating antibody.
Albumin supplementation induces translocation of p38 MAPK.
To determine the distribution of p38 MAPK and caveolin-1 in the serum-deprived condition and in the presence of albumin, we double-stained RLMVEC with a monoclonal antibody specific for caveolin-1 and a rabbit polyclonal antibody against the phosphorylated form of p38 MAPK (p38-P MAPK). Figure 5A Expression of p38 MAPK in caveolin-1 siRNA-treated cell lysate was not significantly altered (⇓Fig. 7B). We found reduction in the basal p38-P MAPK level in the caveolin-1 depleted cell lysate (Fig. 7A). The requirement of caveolin-1 expression in the induction of p38-P MAPK by albumin endocytosis was further examined by immunostaining. RLMVEC with normal caveolin-1 expression and cells in which caveolin-1 expression was reduced by siRNA were stained after albumin endocytosis (Fig. 7, C and D). Knockdown of caveolin-1 expression resulted in reduced basal expression of p38-P MAPK and inhibition of p38 phosphorylation on albumin supplementation compared with the RLMVEC with normal caveolin-1 expression (Fig. 7C).
Caveolin-1 knockdown reduces cell survival.
We investigated whether caveolin-1 expression affects cell survival in serum-starved and albumin-supplemented RLMVEC. Inhibition of caveolin-1 expression by siRNA reduced apoptosis when RLMVEC were grown in serum-free conditions compared with the RLMVEC transfected with control siRNA (Table 2). There was a decrease in cell survival when caveolin-1 siRNA-treated cells were grown in serum-free medium or after addition of albumin to activate endocytosis (Table 2).
The endocytic machinery in endothelial cells has multiple functions. It is known that endocytosis enables cells to regulate the surface expression of receptors by targeting them for degradation (4, 5, 7). Data presented here show another important function of endocytosis as induced by albumin supplementation of endothelial cells grown in serum-free conditions. We showed that activation of endocytosis was essential in regulating the p38 MAPK activity in endothelial cells. We demonstrated an interaction of p38 MAPK with caveolin-1, the principal marker of caveolae (19). On the basis of these findings, we propose that this interaction is crucial in regulating endothelial cell proliferation and survival and thereby in helping to maintain the integrity of the endothelial monolayer barrier.
Our findings showed that activation of endocytosis by albumin in serum-deprived endothelial cells was involved in reducing apoptosis and promoting proliferation of endothelial cells. The CFSE dye dilution assay (10, 15) and FACS analysis were used to demonstrate that albumin supplementation of endothelial cells resulted in the activation of endocytosis and thereby signaled endothelial cell proliferation. The TUNEL assay showed that endocytosis also reduced endothelial apoptosis and thereby promoted cell survival. Cell proliferation and survival were impaired in the absence of endocytosis induced by albumin supplementation. Native albumin appeared to be essential for the endocytosis response since albumin denaturation rendered it ineffective in activation of normal levels of endocytosis and promoting cell growth and reducing apoptosis. Recombinant albumin also has the same effect as native albumin (data not shown), suggesting that serum-associated constituents of albumin were not responsible for the effect.
We examined the role of the microtubule-stabilizing drug taxol in the presence and absence of albumin supplementation in regulating the endocytosis-induced endothelial cell survival response. In this experiment, albumin addition to the medium increased the survival of taxol-treated endothelial cells. At lower concentrations of the drug between 100 μM and 1 mM, no significant apoptosis was observed, whereas taxol induced apoptosis at 2 mM. Importantly, apoptosis was reduced when albumin was supplemented in the taxol-treated cells. These results demonstrate that activation of endocytosis plays a key role in promoting cell survival.
We previously reported (24) that TβRII-mediated signaling can activate caveolae-mediated endocytosis in endothelial cells. As p38 MAPK activation is downstream of TβRII activation (9, 27), we examined the possibility that signaling via the TβRII-p38 MAPK pathway is a requirement for endocytosis and the consequent endothelial cell growth and survival responses. We found that TGF-β induced p38 MAPK phosphorylation and that inhibition of p38 MAPK by SB-203580 blocked endocytosis and interfered with endothelial proliferation and survival. These observations suggest an important role of endocytosis induced by the TβRII-p38 MAPK signaling pathway in activating endothelial proliferation and reducing endothelial apoptosis. Our results are in agreement with studies showing that p38 MAPK activation is a crucial determinant of cell proliferation (6, 13).
The mechanism responsible for activation of p38 MAPK following endocytosis is unclear (29, 30). The knockdown of caveolin-1 expression with siRNA resulted in a reduction in p38 MAPK phosphorylation. This finding suggests that caveolin-1-mediated signaling (22, 25) is involved in regulating p38 MAPK activation. Using high-throughput RNA interference and automated image analysis Pelkmans et al. (18) showed that Src and MAP kinases regulate caveolae-mediated endocytosis. The findings of Pelkmans et al. support our observation that activation of endocytosis regulates p38 MAPK activation and thus may control proliferation and survival of endothelial cells and the integrity of the endothelial monolayer. We observed that p38 MAPK phosphorylation did not last beyond 2 h, suggesting a negative-feedback downregulation of p38 MAPK activity. Other studies have demonstrated that MAPK activation via phosphorylation is also short-lived despite the near constant level of MAPK protein during the course of stimulation (28).
Our data show an association of p38 MAPK with caveolin-1 in endothelial cells following albumin supplementation of serum-deprived cells, a condition that activates caveolae-mediated endocytosis (11, 17). This finding raises the possibility that caveolin-1 is involved in p38 MAPK activation during endocytosis. We observed that siRNA-induced knockdown of caveolin-1 reduced endocytosis as well as cell survival. Caveolin-1 association with p38 MAPK secondary to caveolae-mediated endocytosis may be involved in p38 MAPK activation and thereby the engagement of endothelial cell proliferation signaling complex. However, the mechanisms responsible for the association of caveolin-1 and p38 MAPK and regulation of p38 MAPK activity remain unclear. As several kinases are inextricably linked to the endocytic machinery (18), the interaction of caveolin-1 and p38 MAPK may be important in bringing together components of the endocytic machinery and the signaling modules regulating endothelial cell proliferation and apoptosis.
This work was supported by National Heart, Lung, and Blood Institute Grants P01-HL-60678 and R01-HL-71626.
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