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J Appl Physiol 101: 375-384, 2006. First published April 27, 2006; doi:10.1152/japplphysiol.01515.2005
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Transforming growth factor-beta1-induced endothelial barrier dysfunction involves Smad2-dependent p38 activation and subsequent RhoA activation

Qing Lu, Elizabeth O. Harrington, Heather Jackson, Nicole Morin, Christopher Shannon, and Sharon Rounds

Vascular Research Laboratory, Providence Veterans Affairs Medical Center, Department of Medicine, Brown Medical School, Providence, Rhode Island

Submitted 1 December 2005 ; accepted in final form 24 April 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Lung edema due to increased vascular permeability is a hallmark of acute lung injury and acute respiratory distress syndrome. Both p38 and RhoA signaling events are involved in transforming growth factor (TGF)-beta1-increased endothelial permeability; however, the mechanism by which these pathways cooperate is not clear. In this study, we hypothesized that TGF-beta1-induced changes in endothelial monolayer permeability and in p38 and RhoA activation are dependent on Smad2 signaling. We assessed the role of Smad2 in p38 activation and the role of p38 in RhoA activation by TGF-beta1. We found that TGF-beta1 caused Smad2 phosphorylation between 0.5 and 1 h of exposure in endothelial cells. Knockdown of Smad2 protein prevented TGF-beta1-induced p38 activation and endothelial barrier dysfunction. Furthermore, TGF-beta1-enhanced RhoA activation was dependent on p38 activation. Inhibition of the RhoA-Rho kinase signaling pathway blunted TGF-beta1-induced adherens junction disruption and focal adhesion complex formation. In addition, depletion of heat shock protein 27, a downstream signaling molecule of p38, did not prevent TGF-beta1-induced endothelial barrier dysfunction. Finally, inhibition of de novo protein expression blunted TGF-beta1-induced RhoA activation and endothelial barrier dysfunction. Our data indicate that TGF-beta1 induces endothelial barrier dysfunction involving Smad2-dependent p38 activation, resulting in RhoA activation by possible transcriptional regulation.

heat shock protein 27; endothelium; cell signaling; endothelial monolayer permeability; small GTPases

LUNG VASCULAR ENDOTHELIAL CELLS form a selective barrier that restricts the flux of blood components into the interstitium and alveolar space. Increased endothelial monolayer permeability is an important early step in the development of acute lung injury and acute respiratory distress syndrome (22), devastating lung disorders with mortality of ~30% despite modern intensive care (54). A number of studies have demonstrated that transforming growth factor (TGF)-beta1 plays a critical role in the development of lung edema during lung injury. TGF-beta1 is elevated in bronchoalveolar lavage fluid of patients with acute respiratory distress syndrome (19) and of rats with acute lung injury caused by overexpression of IL-1beta (31). In addition, expression of several TGF-beta1-inducible genes is enhanced in bleomycin-induced lung edema (29). Inhibition of TGF-beta1 signaling by soluble chimeric TGF-beta1 receptor or by {alpha}vbeta6 integrin deficiency protects mice from bleomycin-induced lung edema (42), whereas overexpression of active TGF-beta1 in rat lungs induces perivascular and peribronchial edema (47). Furthermore, TGF-beta1 directly increases monolayer permeability of cultured pulmonary artery endothelial cells (PAEC) (8, 10, 21, 28) and alveolar epithelial cells (42). Thus understanding the signaling mechanism(s) by which TGF-beta1 induces endothelial barrier dysfunction may facilitate development of an effective therapy for diseases with increased lung vascular permeability. We hypothesized that TGF-beta1-induced changes in endothelial monolayer permeability and in p38 and RhoA activation are dependent on Smad2 signaling.

TGF-beta1, a member of the TGF-beta family, is secreted as a latent, inactive complex composed of TGF-beta1 homodimers, latency-associated peptide, and latent TGF-beta binding protein (3, 4). The latent TGF-beta1 complex is activated on cleavage of the latency-associated peptide subunit by a number of molecules, including plasmin, reactive oxygen species, thrombospondin-1, and {alpha}vbeta6 (3, 11, 41). The activated TGF-beta1 binds to and recruits cell surface TGF-beta type I and type II receptors, triggering phosphorylation, nuclear translocation, and transcriptional activation of Smad2/3 (14, 37, 46). In addition, TGF-beta1 modulates a number of other signaling pathways, including mitogen-activated protein kinases (such as ERK1/2, JNK, and p38), RhoA GTPase, protein kinase C{delta}, phosphatidylinositol-3-kinase, and protein phosphatase 1 and 2A (15, 33).

Endothelial barrier function is modulated by a balance between endothelial contractile forces and cell-cell and cell-matrix adhesive forces (16). Actin stress fibers contribute to cell contraction, whereas intercellular junctions and focal adhesion complexes contribute to cell attachments and adhesions (16). Rho GTPases are a family of small GTP-binding proteins that regulate the organization of actin cytoskeleton and intercellular junctions (43, 55). RhoA is an important mediator of both basal endothelial barrier function (24) and agonist-induced endothelial barrier dysfunction (36, 50, 56). Recent studies have shown that RhoA activation is required for TGF-beta1-increased endothelial permeability (8, 10). p38 also plays an important role in TGF-beta1-induced endothelial barrier dysfunction (21). TGF-beta1 initiates numerous cellular responses by both Smad-dependent and -independent pathways (15). Recent studies demonstrated that ALK5, a TGF-beta type I receptor, and Smad4 are required for TGF-beta1-increased endothelial permeability (7). In this study, we investigated the signaling mechanism by which TGF-beta1 induced endothelial barrier dysfunction via p38 and RhoA. We demonstrated that TGF-beta1 induced p38 activation via Smad2, resulting in subsequent RhoA activation and endothelial barrier dysfunction. In addition, TGF-beta1-induced RhoA activation and endothelial barrier dysfunction were dependent on de novo protein expression. Thus our findings are the first to demonstrate that TGF-beta1-induced RhoA activation is mediated by Smad2-dependent p38 activation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Cell lines and reagents.   Bovine PAEC were isolated and characterized, as our laboratory previously described (12). Human umbilical vein endothelial cells (HUVEC) were purchased from Clonetics (Walkersville, MD). Recombinant human TGF-beta1 was obtained from R & D Systems, (Minneapolis, MN). Y-27632 was purchased from CalBiochem (San Diego, CA). SB-203580 was obtained from SmithKline Beecham Pharmaceuticals (Philadelphia, PA). Cycloheximide and actinomycin D were purchased from Sigma (St. Louis, MO). Antibodies directed against RhoA, beta-catenin, vinculin, Smad3, p115Rho guanine nucleotide exchange factors (GEF), and Rho guanine nucleotide dissociation inhibitors (GDI)-{alpha} were from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies directed against p38, phospho-p38 (Thr180/Tyr182), heat shock protein (HSP) 27, phospho-HSP27 (Ser82), Smad2, phospho-Smad2 (Ser465/467), Smad4, TGF-activated protein kinase 1 (TAK1), phospho-TAK1 (Thr184), phospho-TAK1 (Thr187), MAP kinase kinase (MKK) 3, MKK6, phospho-MKK3/6 (Ser189/207), MKK4, and phospho-MKK4 (Ser80), and phospho-MKK3/6 control cell extracts were from Cell Signaling Technology (Beverly, MA). Antibody directed against p190Rho GTPase-activating proteins (GAP) was purchased from BD Biosciences (San Jose, CA). Smad2 small-interfering (si)RNA (Smad2 SMARTpool siRNA) (catalog no. M-003561, accession no. NM_005901) was purchased from Upstate (Charlottesville, VA). It contains four pooled siRNA duplexes (21 nucleotides) with "UU" overhangs and 5' phosphate on the antisense strand. The sequences of this siRNA pool are not released from Upstate; HSP27 siRNA (catalog no. sc-29350) was purchased from Santa Cruz Biotechnology. It is a pool of three target-specific 20- to 25-nucleotide siRNA duplexes designed to knockdown HSP27 gene expression. The mRNA sequences for three pool siRNAs are residues 228–246, GAGUGGUCGCAGUGGUUAG; residues 426–444, GACGAGCUGACGGUCAAGA; residues 625–643, CCACGCAGUCCAACGAGAU. Control (nonsilencing) siRNA was purchased from Qiagen (Valencia, CA). pGST-C21 (GST-Rho-binding domain of Rhotekin, GST-Rhotekin-RBD) construct was a generous gift from Dr. J. Collard (Netherlands Cancer Institute, Amsterdam, The Netherlands).

Knockdown of endogenous Smad2 and HSP27.   Smad2 and HSP27 siRNA directly against respective human mRNA was ineffective in suppressing protein expression of either Smad2 or HSP27 in bovine PAEC (data not shown). Thus HUVEC were used for all experiments involving siRNA. Nonspecific, nonsilencing siRNA duplex, designed by Qiagen to not alter the expression level of any proteins, was used as a control treatment. HUVEC were grown to 60% confluence and transfected with 40 nM control siRNA, Smad2 siRNA, or HSP27 siRNA using Lipofectamine 2000 reagent (Invitrogen Life Technologies) according to the manufacturer's protocol. At 48 h posttransfection, cells were used for further analysis.

Endothelial monolayer permeability assay.   Changes in endothelial monolayer permeability were assessed by measuring electrical resistance across monolayers using the electrical cell impedance sensor technique (Applied Biophysics), as our laboratory previously described (36). Briefly, equivalent numbers of endothelial cells were plated on collagen-coated gold electrode arrays (8W10E) and grown to confluence in growth medium. Cells were then treated as described, and electrical resistance across monolayers was recorded over time.

RhoA GTPase activity assay.   RhoA GTPase activity was measured using pull-down assay, as our laboratory previously described (36). GTP-bound RhoA (GTP-RhoA) was purified from lysates containing equal amounts of proteins with GST-Rhotekin-RBD agarose beads. The levels of precipitated RhoA (GTP-RhoA) and total RhoA in corresponding crude lysates were assessed by immunoblot analysis. RhoA activity was presented as the GTP-RhoA relative to the total RhoA.

Gel electrophoresis and immunoblot analysis.   Lysates were solubilized in Laemmli buffer, and proteins were resolved using SDS-PAGE and then transferred to nitrocellulose membranes and immunoblotted with indicated antibodies, as previously described (5).

Immunofluorescence confocal microscopy.   Confluent PAEC on coverslips were treated as described and fixed with 4% paraformaldehyde and rendered permeable with 0.1% Triton X-100. Adherens junctions and focal adhesion complexes were stained with antibodies directly against the junctional protein beta-catenin and focal adhesion complex component vinculin, respectively. The primary antibody was detected by subsequent staining of the slides with species-specific Texas red-conjugated secondary antibody. Images were visualized by fluorescence laser scanning confocal microscopy at x630 magnification and recorded, as our laboratory previously described (36).

Data analysis.   All experiments were performed at least in triplicate. Data are presented as means ± SE. ANOVA tests were used to analyze differences among groups. Differences among means were considered significant when P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Smad2 is required for TGF-beta1-induced endothelial barrier dysfunction.   TGF-beta1 initiates numerous cellular responses through both Smad-dependent and -independent pathways (15). Recently, it has been shown that ALK5 and Smad4 are involved in TGF-beta1-increased endothelial monolayer permeability (7). Because ALK5 activation can cause Smad2/3 activation (35), we first tested whether Smad2 was activated by TGF-beta1 in endothelial cells. We noted that TGF-beta1 dose-dependently increased endothelial monolayer permeability in PAEC with a maximal effect at 1 ng/ml (data not shown); thus this dose was used throughout the study. PAEC treated with TGF-beta1 demonstrated an elevated level of phosphorylated, activated Smad2 at 0.5 and 1 h of exposure (Fig. 1A). The level of phosphorylated Smad2 was significantly diminished by 4 h and returned to basal level by 24 h following exposure to TGF-beta1 (Fig. 1A). Next, we examined whether Smad2 was required for TGF-beta1-induced endothelial barrier dysfunction by knocking down Smad2 protein expression using siRNA. Equivalent numbers of HUVEC were transfected with control siRNA or Smad2 siRNA for 48 h, and depletion of Smad2 protein was confirmed by immunoblot analysis (Fig. 1B). Neither Smad3 nor Smad4 protein levels were altered by Smad2 siRNA (Fig. 1B). As shown in Fig. 1C, TGF-beta1 significantly decreased electrical resistance across endothelial monolayers transiently transfected with control siRNA. This effect was prevented by depletion of Smad2 protein, suggesting a requirement of Smad2 in TGF-beta1-induced increases in endothelial monolayer permeability.


Figure 1
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  		Fig. 1. Role of Smad2 in transforming growth factor (TGF)-beta1-induced endothelial barrier dysfunction. A: PAEC were treated with TGF-beta1 (1 ng/ml) for the indicated times, and the level of phosphorylated Smad2 (P) was detected by immunoblot analysis using antibody directly against phospho-Smad2 (Ser465/467). The immunoblots were stripped and reprobed with anti-Smad2 antibody. Representative immunoblots are shown, n = 3. ', minutes. B and C: equivalent numbers of HUVEC were transfected with control siRNA or Smad2 small-interfering (si)RNA for 48 h and then treated with vehicle or TGF-beta1 (1 ng/ml) for 20 h. The level of Smad2 was detected by immunoblot analysis using anti-Smad2 antibody (B). The immunoblots were stripped and reprobed for Smad3, Smad4, and vinculin using respective antibodies (B). Representative immunoblots are shown. Endothelial monolayer permeability was assayed by measuring electrical resistance across the monolayers over time by electrical cell impedance sensor (C). Arrows indicate the time of addition of treatments. Data are means ± SE in resistance relative to time 0; n = 6. *P < 0.05 vs. Smad2 siRNA with TGF-beta1.

 
TGF-beta1 induces p38 activation via Smad2.   TGF-beta1 has been shown to activate p38, and this activation is required for TGF-beta1-induced endothelial barrier dysfunction (21). Because Smad2 was required for TGF-beta1-induced endothelial barrier dysfunction, we next assessed whether p38 activation was dependent on Smad2 in response to TGF-beta1. HUVEC transfected with control siRNA or Smad2 siRNA for 48 h were treated with vehicle or TGF-beta1 for 1 h. As shown in Fig. 2A, the levels of active p38 were not significantly different between control siRNA and Smad2 siRNA transfected cells exposed to vehicle. p38 activation upon TGF-beta1 exposure, however, was significantly attenuated in endothelial cells transfected with Smad2 siRNA, demonstrating a requirement for Smad2 in TGF-beta1-induced p38 activation.


Figure 2
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  		Fig. 2. Effect of Smad2 on TGF-beta1-induced p38 activation. A: human umbilical vein endothelial cells (HUVEC) were transfected with control siRNA or Smad2 siRNA for 48 h and then treated with vehicle or TGF-beta1 (1 ng/ml) for 1 h. The level of phosphorylated p38 was detected by immunoblot analysis using antibody directly against phospho-p38 (Thr180/Tyr182). The immunoblots were stripped and reprobed with anti-p38 antibody. Phosphorylation of p38 is presented as the ratio of phospho-p38 to total p38. Representative immunoblots are shown (n = 4). *P < 0.05. B: pulmonary artery endothelial cells (PAEC) were treated with TGF-beta1 (1 ng/ml) for the indicated times and the level of phosphorylated MKK3/6 was detected by immunoblot analysis using antibody directly against phospho-MKK3/6 (Ser189/207). The immunoblots were stripped and reprobed with antibody directly against MKK3. Representative immunoblots are shown (n = 3). Total cell extracts from NIH/3T3 cells treated with ultraviolet light serve as the positive control.

 
TAK1 activates p38 through MKK3 and MKK6 (40). MKK4 also activates p38 (13). TAK1 is activated in response to TGF-beta1 and bone morphogenetic protein 4 in yeast (57). To test whether TAK1/MKK3/6 pathway and MKK4 were involved in TGF-beta1-induced p38 activation, we assessed activation of TAK1, MKK3/6, and MKK4 in response to TGF-beta1 in endothelial cells by measuring phosphorylation of these proteins. As shown in Fig. 2B, MKK3/6 was not phosphorylated by TGF-beta1. Similarly, neither TAK1 nor MKK4 was phosphorylated by TGF-beta1 in either PAEC or HUVEC (data not shown), suggesting that these signaling pathways are not involved.

p38 mediates TGF-beta1 activation of RhoA.   RhoA is a key factor involved in TGF-beta1-increased endothelial monolayer permeability (8, 10). Because both p38 and RhoA were activated by TGF-beta1, we next analyzed whether there was any cross talk between these pathways. To test whether inhibition of p38 altered RhoA activity in response to TGF-beta1, PAEC were preincubated with vehicle or the p38 inhibitor SB-203580 for 1 h and then exposed to TGF-beta1 in the presence or absence of SB-203580 for 2 h. We found that p38 inhibition prevented TGF-beta1 activation of RhoA (Fig. 3A), suggesting that TGF-beta1-induced-RhoA activation is dependent on p38 activity. To determine whether p38 activation by TGF-beta1 was dependent on RhoA signaling through Rho kinase, PAEC were preincubated with vehicle or the Rho kinase inhibitor Y-27632 for 1 h and then exposed to TGF-beta1 in the presence or absence of Y-27632 for 0.5, 1, and 2 h. Inhibition of Rho kinase did not significantly affect TGF-beta1 activation of p38 at 0.5 h (data not shown), 1 h (Fig. 3B), and 2 h (data not shown). Taken together, these results suggest that RhoA activation by TGF-beta1 is dependent on p38 activity.


Figure 3
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  		Fig. 3. Analysis of cross talk between p38 and RhoA pathways on TGF-beta1 exposure. A: PAEC were preincubated with vehicle or SB-203580 (20 µM) for 1 h and then treated with vehicle, SB-203580 (20 µM), TGF-beta1 (1 ng/ml) alone, or TGF-beta1 plus SB-203580 for 2 h. Cell lysates were used to detect RhoA activity by pull-down assay. Representative immunoblots are shown (n = 3). B: PAEC were preincubated with vehicle or Y-27632 (20 µM) for 1 h and then treated with vehicle, Y-27632 (20 µM), TGF-beta1 (1 ng/ml) alone, or TGF-beta1 plus Y-27632 for 1 h. The levels of phosphorylated p38 and total p38 were detected using anti-phospho-p38 (Thr180/Tyr182) and anti-p38 antibodies, respectively. Phosphorylation of p38 is presented as the ratio of phospho-p38 to total p38. Representative immunoblots are shown (n = 3). *P < 0.05 vs. vehicle.

 
RhoA signaling through Rho kinase is essential for TGF-beta1-induced reorganization of adherens junctions and focal adhesion complexes.   RhoA/Rho kinase signaling pathway has been shown to be important in TGF-beta1-induced endothelial barrier dysfunction (8, 10). Endothelial barrier integrity is maintained by the opposing forces generated by stress fibers, intercellular junctions, and focal adhesion complexes (16). RhoA/Rho kinase signaling is important for TGF-beta1-induced stress fiber formation (10). In this study, we assessed the role of RhoA/Rho kinase signaling in TGF-beta1-mediated reorganization of adherens junctions and focal adhesion complexes. Confluent PAEC were preincubated with vehicle or Y-27632 for 1 h and then exposed to TGF-beta1 in the presence or absence of Y-27632 for 3 h. We found that Rho kinase inhibition blunted TGF-beta1-induced disruption of adherens junctions (Fig. 4A) and formation of focal adhesion complexes (Fig. 4B). These results suggest that RhoA/Rho kinase signaling pathway activation is important for TGF-beta1-induced reorganization of adherens junctions and focal adhesion complexes.


Figure 4
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  		Fig. 4. Role of Rho kinase on TGF-beta1-induced reorganization of adherens junctions and focal adhesion complexes. PAEC were preincubated with vehicle or Y-27632 (20 µM) for 1 h and then incubated with vehicle, Y-27632 (20 µM), TGF-beta1 (1 ng/ml) alone, or TGF-beta1 plus Y-27632 for 3 h. beta-Catenin (A) and vinculin (B) localization were assessed by immunofluorescence analysis. Arrows indicate the intercellular gaps. Representative pictures are shown (n = 3).

 
TGF-beta1-induced endothelial barrier dysfunction is independent of HSP27.   HSP27, a downstream target of p38, has been implicated in stress fiber formation and focal adhesion assembly in airway smooth muscle cells and endothelial cells (1, 2, 27, 34, 38, 44). We next tested the effects of TGF-beta1 on HSP27 activation by measuring changes in phosphorylation of HSP27 at serine 82. PAEC treated with TGF-beta1 demonstrated a significant increase in HSP27 phosphorylation following 1, 4, and 24 h of exposure (Fig. 5A). We further found that TGF-beta1-increased HSP27 phosphorylation was completely abolished upon p38 inhibition (Fig. 5B), suggesting that TGF-beta1-induced HSP27 activation is dependent on p38. To determine whether HSP27 was required for TGF-beta1-induced endothelial barrier dysfunction, equivalent numbers of HUVEC were transfected with control siRNA or HSP27 siRNA for 48 h, and depletion of HSP27 protein was confirmed by immunoblot analysis (Fig. 6A). As shown in Fig. 6B, TGF-beta1 increased endothelial monolayer permeability to a similar extent in control siRNA and HSP27 siRNA transfected cells, suggesting that HSP27 is not involved in TGF-beta1-increased endothelial permeability.


Figure 5
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  		Fig. 5. Effect of p38 on TGF-beta1-induced heat shock protein (HSP) 27 activation. PAEC were treated with TGF-beta1 (1 ng/ml) for the indicated times (A) or were preincubated with vehicle or SB-203580 (20 µM) for 1 h and then treated with vehicle, SB-203580 (20 µM), TGF-beta1 (1 ng/ml) alone, or TGF-beta1 plus SB-203580 for 1 h (B). The levels of phosphorylated HSP27 and total HSP27 were detected by immunoblot analysis using antibodies directly against phospho-HSP27 (Ser82) and HSP27, respectively. Representative immunoblots are shown (n = 3).

 

Figure 6
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  		Fig. 6. Effect of HSP27 on TGF-beta1-induced endothelial barrier dysfunction. Equivalent numbers of HUVEC were transfected with control siRNA or HSP27 siRNA for 48 h and then treated with vehicle or TGF-beta1 (1 ng/ml) for 20 h. A: level of HSP27 was detected by immunoblot analysis. The immunoblots were stripped and reprobled for vinculin to show equal protein loading. B: endothelial monolayer permeability was measured. Data are means ± SE in resistance at 20 h of incubation with vehicle or TGF-beta1; n = 5. *P < 0.05 vs. respective vehicle.

 
TGF-beta1-induced RhoA activation and endothelial barrier dysfunction requires de novo protein synthesis.   Protein synthesis has been shown to be required for TGF-beta1-enhanced myosin light chain phosphorylation in endothelial cells (21). To directly address whether gene transcription and protein translation were important for TGF-beta1-induced endothelial barrier dysfunction, equivalent numbers of PAEC were preincubated with vehicle or an inhibitor of transcription, actinomycin D, or an inhibitor of translation, cycloheximide, for 1 h and then exposed to TGF-beta1 in the presence or absence of either inhibitor for 20 h. The effect of actinomycin D or cycloheximide on basal endothelial monolayer permeability was dependent on the dosage used and treatment time. High concentrations of actinomycin D (0.5 µg/ml) and cycloheximide (10 µg/ml) significantly increased monolayer permeability of endothelial cells exposed for 6 h and longer (data not shown). Low concentrations of actinomycin D (25 ng/ml) (data not shown) and cycloheximide (0.5 µg/ml) (Fig. 7A), however, did not significantly alter basal endothelial monolayer permeability up to 20 h of exposure. TGF-beta1-increased endothelial monolayer permeability was blunted by both 0.5 µg/ml cycloheximide (Fig. 7A) and 25 ng/ml actinomycin D (data not shown). These results suggest that de novo protein expression is required for this process.


Figure 7
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  		Fig. 7. Role of de novo protein synthesis in TGF-beta1-induced endothelial barrier dysfunction and RhoA and p38 activation. Equivalent numbers of PAEC were preincubated with vehicle, cycloheximide (CHX; 0.5 µg/ml), or actinomycin D (25 ng/ml) for 1 h and then incubated with vehicle, cycloheximide (0.5 µg/ml), actinomycin D (ACD; 25 ng/ml), TGF-beta1 (1 ng/ml) alone, or TGF-beta1 plus either cycloheximide or actinomycin D for 20 h (A) or 2 h (B and C). A: changes in endothelial monolayer permeability were assayed over time. Arrow indicates the time of addition of treatments (n = 5). *P < 0.05 vs. vehicle. {Psi}P < 0.05 vs. TGF-beta1. B: RhoA activity was assessed by pull-down assay. RhoA activity is presented as the ratio of GTP-RhoA to total RhoA (n = 3). *P < 0.05 vs. vehicle. Representative immunoblots are shown. C: levels of phosphorylated p38 and total p38 were detected using anti-phospho-p38 (Thr180/Tyr182) and anti-p38 antibodies, respectively. Representative immunoblots are shown (n = 3).

 
We next examined whether de novo protein expression was required for TGF-beta1-induced p38 activation and RhoA activation. PAEC were preincubated with vehicle, actinomycin D, or cycloheximide for 1 h and then exposed to TGF-beta1 in the presence or absence of either inhibitor for 2 h, and the activities of p38 and RhoA were assessed. As shown in Fig. 7B, neither inhibitor altered basal RhoA activity; yet both significantly blunted TGF-beta1-elevated RhoA activity, suggesting that TGF-beta1-induced RhoA activation requires newly synthesized protein(s). However, to our surprise, inhibition of protein synthesis with cycloheximide dramatically enhanced both basal and TGF-beta1-stimulated p38 activities (Fig. 7C). Similar results were observed when cells were treated with actinomycin D (data not shown). These results suggest that TGF-beta1 activation of RhoA requires de novo protein synthesis downstream from p38.

RhoA activity is modulated by RhoGDI{alpha}, p190RhoGAP, and p115RhoGEF (9, 23, 24, 26, 39). To determine whether p38 activates RhoA upon TGF-beta1 stimulation by altering the expression of these modulators, PAEC and HUVEC were treated with vehicle or TGF-beta1 for 0.5, 1, 2, 4, 6, and 24 h, and the protein levels of RhoGDI{alpha}, p190RhoGAP, and p115RhoGEF were assessed. None of these protein levels were altered by TGF-beta1 (data not shown). In addition, the protein levels of RhoGDI{alpha} and p190RhoGAP were also not changed in endothelial cells exposed to 0.5 µg/ml cycloheximide or 25 ng/ml actinomycin D for 2 h (data not shown). These results suggest that p38 activates RhoA upon TGF-beta1 stimulation, probably by acting on the expression of some unidentified protein(s), which regulates activities of RhoA modulators.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Lung vascular endothelial cells form a dynamic barrier, which is critical for the regulation of vascular permeability. TGF-beta1 induces endothelial barrier dysfunction (28) in a manner dependent on p38 (21) and RhoA (8, 10). However, the mechanism(s) by which these signaling pathways cooperate have not been investigated. In this study, we hypothesized that TGF-beta1-induced changes in endothelial monolayer permeability and in p38 and RhoA activation are dependent on Smad2 signaling. We demonstrated that Smad2 activation is required for TGF-beta1-induced p38 activation and endothelial barrier dysfunction. Furthermore, we found that TGF-beta1-induced p38 activation leads to RhoA activation and subsequent endothelial barrier dysfunction. In addition, we demonstrated that TGF-beta1-induced RhoA activation and endothelial barrier dysfunction require de novo protein synthesis. These results suggest that Smad2-dependent p38 activation and subsequent RhoA activation plays a central role in TGF-beta1-induced endothelial barrier dysfunction.

TGF-beta1 initiates numerous cellular responses by both Smad-dependent and -independent pathways (15). It has been suggested that Smad2/3 activation is required for TGF-beta1-induced actin stress fiber formation in epithelial cells (45) and Swiss 3T3 cells (53). Smad2/3 are activated on ALK5 activation by TGF-beta1 in endothelial cells (35). Recently, Birukova et al. (7) have shown that both ALK5 and Smad4 are involved in TGF-beta1-induced endothelial barrier dysfunction. Our study demonstrated that depletion of Smad2 protein prevented TGF-beta1-induced endothelial barrier dysfunction. Taken together, these results suggest that TGF-beta1 regulates endothelial monolayer permeability through a Smad-dependent mechanism.

Mammalian MAP kinases are activated by MKKs via dual phosphorylation on threonine and tyrosine. Among seven identified MKKs, MKK3, 4, and 6 are highly selective for p38 activation (13). TAK1 activates p38 through MKK3 and MKK6 (40). TAK1 is activated in response to TGF-beta1 in yeast (57). We demonstrated that neither TAK1 nor MKK4 nor MKK3/6 was phosphorylated (activated) by TGF-beta1 in either PAEC or HUVEC, suggesting that these signaling pathways are not important for TGF-beta1-induced p38 activation.

Activation of p38 by Rho GTPases has been reported (17, 25). However, others have demonstrated that p38 activation is independent of Rho GTPases (60). In this study, inhibition of Rho kinase did not alter the level of p38 activity upon TGF-beta1 stimulation, suggesting that RhoA/Rho kinase signaling does not regulate TGF-beta1-induced p38 activation in endothelial cells.

The role of Smad proteins in TGF-beta1-induced p38 activation is controversial. Yu et al. (59) have shown that TGF-beta1-induced p38 activation is independent of Smad in mouse mammary epithelial cells. However, others have demonstrated that Smad signaling is critical for TGF-beta1-induced p38 activation in pancreatic cells (49) and murine hepatocytes (58). These results suggest that the role of Smad in TGF-beta1-induced p38 activation may depend on cell type. In this study, we found that depletion of Smad2 protein prevented TGF-beta1-induced p38 activation, demonstrating that Smad2 is required for TGF-beta1-induced p38 activation in endothelial cells.

RhoA regulates endothelial basal barrier function and agonist-induced endothelial barrier dysfunction by increasing stress fiber formation and by disrupting intercellular junctions (24, 36, 50, 51, 56). RhoA has also been implicated in TGF-beta1-induced stress fiber formation and adherens junction disruption in epithelial cells (6, 17, 45). Recent studies have shown that RhoA activation plays an important role in TGF-beta1-induced stress fiber formation and endothelial barrier dysfunction (8, 10). In this study, we demonstrated that RhoA/Rho kinase signaling activation is required for TGF-beta1-induced disruption of adherens junctions and formation of focal adhesion complexes, suggesting that reorganization of these structures contributes to TGF-beta1-induced endothelial barrier dysfunction.

The mechanism underlying TGF-beta1-induced RhoA activation has not been well defined. Smad2/3 activation is required for TGF-beta1-induced RhoA activation in epithelial cells (45) and Swiss 3T3 cells (53). In this study, we found that TGF-beta1 activated RhoA via Smad2-dependent p38 activation. In addition, both actinomycin D and cycloheximide attenuated TGF-beta1-induced RhoA activation, suggesting a requirement of de novo protein synthesis in this process. The effect of inhibition of protein expression on TGF-beta1 activation of RhoA indicates two possibilities. First, it is possible that newly synthesized protein(s) is required for TGF-beta1-induced p38 activation. However, to our surprise, our data actually refuted this possibility, due to the exacerbation of TGF-beta1 activation of p38 by actinomycin D and cycloheximide. Second, it is possible that TGF-beta1 activation of RhoA requires de novo protein synthesis downstream from p38. Therefore, although p38 is activated by actinomycin D or cycloheximide, RhoA remains inactive due to inhibition of synthesis of some protein(s) necessary for RhoA activation.

RhoA is activated by GEFs and inhibited by GAPs and GDIs (48). In this study, we found that TGF-beta1 did not alter protein expression of RhoGDI{alpha}, p190RhoGAP, and p115RhoGEF, known modulators of RhoA (9, 23, 24, 26, 39). Thus we speculate that p38 activates RhoA upon TGF-beta1 exposure by controlling the expression of some unidentified protein(s), which regulates activities of RhoA modulators. RhoA activation is also regulated by prenylation and methylation. We have shown that blocking RhoA methylation blunted the level of RhoA activity by increasing RhoGDI{alpha} binding (23). Others have reported that cycloheximide treatment inhibits RhoA prenylation by preventing cyclin E expression, which in turn prevents RhoA activation (20). RhoA is also inactivated by glucosylation. Whether prenylation, methylation, or glucosylation are involved in p38-mediated RhoA activation by TGF-beta1 requires further investigation.

TGF-beta1-induced endothelial barrier dysfunction was completely prevented by p38 inhibition (21) but partially blunted by inhibition of RhoA/Rho kinase (8, 10); thus we speculated that other signaling pathway(s) downstream from p38 contribute to TGF-beta1-increased endothelial permeability. In this study, we have investigated the effect of HSP27, a downstream signaling molecule of p38, in this process and demonstrated that it is not involved in TGF-beta1-induced increase in endothelial permeability. Rac1 and Cdc42 have been reported to regulate endothelial permeability (18, 32, 36, 52, 56). Rac1 is involved in TGF-beta1-induced actin reorganization in hepatoma cells (30). Cdc42 is involved in TGF-beta1-induced mobilization of actin cytoskeleton in human prostate carcinoma cells (17). Further studies are needed to determine the involvement of Rac1 and Cdc42 in TGF-beta1-induced endothelial permeability.

In summary, cooperation among multiple signaling molecules is important in TGF-beta1-induced endothelial barrier dysfunction as described in Fig. 8. Our findings demonstrate a novel signaling pathway by which TGF-beta1 promotes p38 activation and subsequent RhoA/Rho kinase signaling activation via Smad2, resulting in endothelial barrier dysfunction.


Figure 8
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  		Fig. 8. Schematic representation of the intracellular signaling involved in TGF-beta1-induced endothelial barrier dysfunction. F-actin, stress fibers; FAC, focal adhesion complexes; AJ, adherens junctions; TbetaRI, II, TGF-beta type I and type II receptors.

 

   GRANTS
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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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ACKNOWLEDGMENTS
 REFERENCES
 
This material is the result of work supported with resources and the use of facilities at the Providence Veterans Affairs (VA) Medical Center and supported by a Rhode Island Foundation Medical Research Grant, American Lung Association Research Grant RG-1140-N, and a Parker B. Francis Fellowship (Q. Lu); National Heart, Lung, and Blood Institute (NHLBI) Grant HL-64936 and VA Merit Review (S. Rounds); and NHLBI Grant HL-67795 and VA Merit Review (E. O. Harrington). Q. Lu is a Parker B. Francis Fellow.


   ACKNOWLEDGMENTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
REFERENCES
 
The authors thank Dr. John Collard of the Netherlands Cancer Institute for the pGST-C21 construct. We also thank Thomas Kim and Julie Newton for technical assistance.

Some of these results were presented at the American Thoracic Society International Conference, May 21–26, 2004, Orlando, Florida, and were published in abstract form in Am J Respir Crit Care Med 169: A415, 2004.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Qing Lu, Providence VA Medical Center, Research Services, 151, 830 Chalkstone Ave., Providence, RI 02908 (e-mail: Qing_Lu{at}brown.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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