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Originally published In Press as doi:10.1074/jbc.M702573200 on August 16, 2007

J. Biol. Chem., Vol. 282, Issue 42, 30643-30657, October 19, 2007
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CD44 Regulates Hepatocyte Growth Factor-mediated Vascular Integrity

ROLE OF c-Met, Tiam1/Rac1, DYNAMIN 2, AND CORTACTIN*

Patrick A. Singleton, Ravi Salgia, Liliana Moreno-Vinasco, Jaideep Moitra, Saad Sammani, Tamara Mirzapoiazova, and Joe G. N. Garcia1

From the Department of Medicine, Pritzker School of Medicine, University of Chicago, Chicago, Illinois 60637

Received for publication, March 26, 2007 , and in revised form, August 14, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The preservation of vascular endothelial cell (EC) barrier integrity is critical to normal vessel homeostasis, with barrier dysfunction being a feature of inflammation, tumor angiogenesis, atherosclerosis, and acute lung injury. Therefore, agents that preserve or restore vascular integrity have important therapeutic implications. In this study, we explored the regulation of hepatocyte growth factor (HGF)-mediated enhancement of EC barrier function via CD44 isoforms. We observed that HGF promoted c-Met association with CD44v10 and recruitment of c-Met into caveolin-enriched microdomains (CEM) containing CD44s (standard form). Treatment of EC with CD44v10-blocking antibodies inhibited HGF-mediated c-Met phosphorylation and c-Met recruitment to CEM. Silencing CD44 expression (small interfering RNA) attenuated HGF-induced recruitment of c-Met, Tiam1 (a Rac1 exchange factor), cortactin (an actin cytoskeletal regulator), and dynamin 2 (a vesicular regulator) to CEM as well as HGF-induced trans-EC electrical resistance. In addition, silencing Tiam1 or dynamin 2 reduced HGF-induced Rac1 activation, cortactin recruitment to CEM, and EC barrier regulation. We observed that both HGF- and high molecular weight hyaluronan (CD44 ligand)-mediated protection from lipopolysaccharide-induced pulmonary vascular hyperpermeability was significantly reduced in CD44 knock-out mice, thus validating these in vitro findings in an in vivo murine model of inflammatory lung injury. Taken together, these results suggest that CD44 is an important regulator of HGF/c-Met-mediated in vitro and in vivo barrier enhancement, a process with essential involvement of Tiam1, Rac1, dynamin 2, and cortactin.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Endothelial cells (EC)2 constitute the inner lining of all blood vessels and regulate the interface between the circulating blood and the vessel wall, including vascular barrier regulation, passive diffusion and active transport of blood-borne substances, regulation of vascular smooth muscle tone, and blood coagulation properties (1, 2). Disruption of this semi-selective cellular barrier is a critical feature of inflammation as well as an important contributing factor to atherosclerosis and tumor angiogenesis (3, 4). Our prior study indicated that hepatocyte growth factor (HGF) binding to its receptor tyrosine kinase, c-Met, promotes EC barrier function (5); however, the exact mechanism by which this occurs is incompletely defined.

The HGF receptor tyrosine kinase, c-Met, is a disulfide-linked {alpha}beta-heterodimer that has been identified previously as a proto-oncogene (6, 7). The 170-kDa c-Met precursor is glycosylated and then cleaved into a 50-kDa extracellular {alpha}-chain and a 140-kDa membrane-spanning beta-chain. The c-Met protein serves as the high affinity receptor for its natural ligand, HGF (also called scatter factor), a paracrine factor produced by stromal and mesenchymal cells, acting on c-Met-expressing cells, including EC (7, 8). Activation of the HGF/c-Met signaling pathway, which requires phosphorylation of various specific tyrosine residues on c-Met itself, leads to cellular responses, including increased proliferation, scattering (cell-cell repulsion), increased motility, invasion, and branching morphogenesis (7, 8).

It is interesting that the cell-surface receptor for hyaluronan, CD44, which we have also shown to serve as a barrier regulatory receptor (9), has also been implicated in the regulation of c-Met signaling (10–13), although the precise mechanism of this interaction is unknown. Furthermore, the role of CD44 in HGF/c-Met-mediated EC barrier enhancement is completely unexplored.

There are multiple CD44 isoforms, which result from extensive alternative exon splicing events (14, 15), with the alternative splicing often occurring between exons 5 and 15, leading to a tandem insertion of one or more variant exons (v1–v10) within the membrane-proximal region of the extracellular domain (16, 17). The variable primary amino acid sequence of different CD44 isoforms is further modified by extensive N- and O-glycosylations and glycosaminoglycan additions (17, 18).

In EC, as in many other cell types, there exist specialized plasma membrane microdomains containing a specific scaffolding protein called caveolin-1. We and others have shown previously that hyaluronan recruits CD44 into these caveolin-enriched microdomains (CEM) (9, 19–21) and that, in this locale, CD44 interacts with the underlying actin cytoskeleton, most likely through cytoskeletal binding proteins critical for CEM association and function (9, 21, 22). We have shown previously that high molecular weight hyaluronan (HMW-HA) promotes EC barrier enhancement via CD44-mediated phosphatidylinositol 3-kinase/Rac1 signaling in CEM (9).


Figure 1
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  		FIGURE 1.
Analysis of HGF-induced c-Met recruitment to human EC CEM. A, EC were grown to confluency. Lysates were obtained; run on SDS-polyacrylamide gels; transferred to nitrocellulose; and immunoblotted with anti-CD44 (IM-7, common domain), anti-CD44 variant (CD44v; v3–v10), anti-CD44v3, anti-CD44v6, or anti-CD44v10 antibody. Experiments were performed in triplicate with highly reproducible findings; representative data are shown. B, EC were grown to confluency, serum-starved for 1 h, and either left untreated (control) or treated with 25 ng/ml HGF for 5 min or with the lipid raft-abolishing, cholesterol-depleting agent MbetaCD (5 mM) for 1 h prior to 25 ng/ml HGF treatment (5 min). Cellular material was solubilized at 4 °C in 1% Triton X-100, and soluble and insoluble fractions were obtained. The Triton X-100-insoluble fraction was overlaid with 60, 40, 30, and 20% OptiPrepTM and centrifuged at 35,000 rpm in an SW 60 rotor for 12 h at 4 °C. The Triton X-100-soluble material and OptiPrepTM fractions were run on SDS-polyacrylamide gels; transferred to nitrocellulose; and immunoblotted with anti-caveolin-1 (panel a), anti-c-Met (panel b), anti-CD44 (IM-7, common domain; panel c), anti-CD44v10 (panel d), or anti-vascular endothelial growth factor receptor-2 (Anti-VEGF R.; panel e) antibody. The 20% OptiPrepTM (*) fraction is the CEM fraction. Experiments were performed in triplicate with highly reproducible findings; representative data are shown. C, the immunoreactive bands from the experiments depicted in B were analyzed using ImageQuantTM software (see "Experimental Procedures") and quantitated. % Total Protein in CEM on the y axis refers to the following: (SAGV for 20% OptiPrepTM immunoreactive band/(SAGV for 20 + 30 + 40 + 60% OptiPrepTM immunoreactive bands of interest + SAGV for Triton X-100-insoluble material immunoreactive band of interest)) x 100.

 


Figure 2
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  		FIGURE 2.
Effect of CD44v10 on HGF-induced c-Met activation and recruitment to CEM. EC were grown to confluency; serum-starved for 1 h; and either left untreated (control) or treated with normal rabbit IgG (preimmune, 10 µg/ml) or anti-CD44v10 antibody (10 µg/ml), followed by no treatment or treatment with 25 ng/ml HGF for 5 min and EC lysates or CEM (lipid raft) fractions (20% OptiPrepTM layer) prepared as described under "Experimental Procedures." A, EC lysates were run on SDS-polyacrylamide gels; transferred to nitrocellulose; and immunoblotted with anti-phospho-Tyr1234/Tyr1235 c-Met (panel a), anti-c-Met (panel b), or anti-actin (panel c) antibody. Experiments were performed in triplicate with highly reproducible findings; representative data are shown. B, the immunoreactive bands from the experiments depicted in A were analyzed using ImageQuantTM software (see "Experimental Procedures") and quantitated. % c-Met Phosphorylation on the y axis refers to the following: (SAGV for phospho-Tyr1234/Tyr1235 c-Met immunoreactive band/SAGV for c-Met immunoreactive band) x 100. C, CEM (lipid raft) fractions (20% OptiPrepTM layer) were run on SDS-polyacrylamide gels; transferred to nitrocellulose; and immunoblotted with anti-c-Met (panel a), anti-CD44 (IM-7, common domain; panel b), anti-CD44v10 (panel c), anti-vascular endothelial growth factor receptor-2 (Anti-VEGF R. 2; panel d), or anti-caveolin-1 (panel e) antibody. Experiments were performed in triplicate with highly reproducible findings; representative data are shown.

 
Rac1-mediated cytoskeletal reorganization is involved in a number of models of EC barrier enhancement (3, 4, 9, 22, 23), with Rac1 activation being the consequence of specific Rho family guanine nucleotide exchange factors that catalyze the exchange of GDP-bound Rac1 (inactive) for GTP-bound Rac1 (active), including Tiam1 (T-lymphoma invasion and metastasis gene 1) (24, 25). Tiam1 is recruited to CEM and is essentially involved in hyaluronan-induced Rac1 activation and EC barrier function (22).

Cortactin is an ~80–85-kDa actin-binding protein that is critically involved in EC cortical cytoskeletal rearrangements (26–28) and agonist-mediated EC barrier enhancement (29, 30). Furthermore, cortactin is an important downstream effector of CD44 signaling (31, 32), with hyaluronan inducing cortactin translocation to the plasma membrane, a process that requires Rac1 activation (31).

Dynamin 2 is an ~96-kDa GTPase implicated in lipid raft internalization, modulation of cell shape, and regulation of podosomal adhesion (33). Dynamin 2 regulates cortical actin dynamics through its interaction with cortactin (33) and has been implicated in Rac1 cellular localization and activation (34). It has been shown recently that dynamin 2 can directly bind to caveolin-1 (35).

In this study, we extend our earlier work to explore CD44 as an important regulator of HGF/c-Met-mediated EC barrier enhancement both in vitro and in vivo. Our novel findings indicate that HGF/c-Met-mediated, CD44-regulated CEM signaling promotes Tiam1/dynamin 2-dependent Rac1 activation and peripheral recruitment of cortactin, processes essential for EC barrier integrity. Understanding the mechanism(s) by which HGF promotes increased EC barrier function may lead to novel treatments for diseases involving vascular barrier disruption, including inflammation, tumor angiogenesis, atherosclerosis, and acute lung injury.


Figure 3
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  		FIGURE 3.
Analysis of HGF-induced c-Met/CD44 interactions. EC were grown to confluency, serum-starved for 1 h, and either left untreated (control) or treated with 25 ng/ml HGF (5, 15, or 30 min) and CEM (lipid raft) fractions (20% OptiPrepTM layer) prepared as described under "Experimental Procedures." A, the CEM fractions were run on SDS-polyacrylamide gels; transferred to nitrocellulose; and immunoblotted with anti-phospho-Tyr1234/Tyr1235 c-Met (panel a), anti-phospho-Tyr1349 c-Met (panel b), anti-c-Met (panel c), anti-CD44 (IM-7, common domain; panel d), anti-CD44v10 (panel e), anti-vascular endothelial growth factor receptor-2 (Anti-VEGF R. 2; panel f), or anti-caveolin-1 (panel g) antibody. Experiments were performed in triplicate with highly reproducible findings; representative data are shown. B, EC lysates were solubilized in immunoprecipitation buffer and immunoprecipitated (Ippt) with anti-c-Met antibody. The resulting Immunobeads were run on SDS-polyacrylamide gels; transferred to nitrocellulose; and immunoblotted with anti-CD44 (IM-7, common domain; panel a), anti-phosphoserine (panel b), or anti-c-Met (panel c) antibody. Experiments were performed in triplicate with highly reproducible findings; representative data are shown. C, the CEM fractions were solubilized in immunoprecipitation buffer and immunoprecipitated with anti-CD44 (IM-7, common domain) antibody. The resulting Immunobeads were run on SDS-polyacrylamide gels; transferred to nitrocellulose; and immunoblotted with anti-c-Met (panel a), anti-phosphoserine (panel b), or anti-CD44 (IM-7, common domain; panel c) antibody. Experiments were performed in triplicate with highly reproducible findings; representative data are shown.

 

   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Reagents—Human pulmonary microvascular EC were obtained from Cambrex Corp. (Walkersville, MD) and cultured as described previously (4) in endothelial cell basal medium-2 complete medium (Cambrex Corp.) at 37 °C in a humidified atmosphere of 5% CO2 and 95% air, with passages 6–10 used for experimentation. Unless otherwise specified, reagents were obtained from Sigma. Reagents for SDS-PAGE were purchased from Bio-Rad, Immobilon-P transfer membrane from Millipore Corp. (Bedford, MA), and gold microelectrodes from Applied Biophysics (Troy, NY). Recombinant human HGF, rabbit anti-von Willebrand factor (vWF; Factor VIII) antibody, goat anti-CD44 variant (v3–v10) antibody, and mouse anti-KDR (vascular endothelial growth factor receptor-2) antibody were purchased from Chemicon International (Temecula, CA). Rat anti-CD44 (IM-7, common domain) antibody was purchased from BD Biosciences. Rabbit anti-phospho-Tyr1234/Tyr1235 c-Met, rabbit anti-phospho-Tyr1349 c-Met, and mouse anti-c-Met antibodies were purchased from Cell Signaling Technology (Boston, MA). Rabbit anti-CD44v3, anti-CD44v6, and anti-CD44v10 antibodies were purchased from Calbiochem. Fluorescein isothiocyanate-conjugated anti-CD44 (homing cell adhesion molecule) antibody was purchased from Abcam (Cambridge, MA). Rabbit anti-phosphoserine antibody was purchased from Zymed Laboratories Inc. (South San Francisco, CA). Rabbit anti-dynamin 2, rabbit anti-Tiam1, and rabbit anti-caveolin-1 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse anti-Rac1 and mouse anti-cortactin antibodies were obtained from Upstate%20Biotechnology">Upstate Biotechnology (Lake Placid, NY). Mouse anti-beta-actin antibody, lipopolysaccharide (LPS), and OptiPrepTM were purchased from Sigma. Horseradish peroxidase-labeled secondary antibodies were purchased from Amersham Biosciences.

CEM Isolation—CEM known as detergent-resistant membranes or lipid rafts were isolated from human pulmonary microvascular EC as we described previously (9, 22). Triton X-100-insoluble materials were mixed with 0.6 ml of cold 60% OptiPrepTM and overlaid with 0.6 ml of 40–20% OptiPrepTM; the gradients were centrifuged at 35,000 rpm using an SW 60 rotor for 12 h at 4 °C; and different fractions were collected and analyzed.

Immunoprecipitation and Immunoblotting—Cellular materials from treated or untreated human pulmonary microvascular EC were incubated in immunoprecipitation buffer (50 mM HEPES (pH 7.5), 150 mM NaCl, 20 mM MgCl2, 1% Nonidet P-40, 0.4 mM Na3VO4, 40 mM NaF, 50 µM okadaic acid, 0.2 mM phenylmethylsulfonyl fluoride, and 1:250 dilution of Calbiochem protease inhibitor mixture III). The samples were then immunoprecipitated with either anti-CD44 or anti-dynamin 2 IgG, followed by SDS-PAGE on 4–15% polyacrylamide gels, transfer onto Immobilon-P membranes, and development with specific primary and secondary antibodies. Visualization of immunoreactive bands was achieved by enhanced chemiluminescence (Amersham Biosciences). In some cases, standardized average gray values (SAGV) processed from ImageQuantTM software (Amersham Biosciences) were obtained for immunoreactive bands for quantification.

Construction and Transfection of Small Interfering RNA (siRNA) against c-Met, CD44, Tiam1, Cortactin, Dynamin 2, Rac1, and Sphingosine 1-Phosphate (S1P) Receptor-1—The siRNA sequence(s) targeting human c-Met, CD44, Tiam1, cortactin, dynamin 2, Rac1, and S1P receptor-1 were generated using mRNA sequences from the GenBankTM Data Bank (gi: 42741654, gi:30353932, gi:897556, gi:20357555, gi:32451864, gi:29792301, and gi:13027635 respectively). For each mRNA (or scramble), two targets were identified. Specifically, c-Met target sequence 1 (5'-AAAGATAAACCTCTCATAATG-3'), c-Met target sequence 2 (5'-AAACCTCTCATAATGAAGGCC-3'), CD44 target sequence 1 (5'-AATATAACCTGCCGCTTTGCA-3'), CD44 target sequence 2 (5'-AAAAATGGTCGCTACAGCATC-3'), Tiam1 target sequence 1 (5'-AAACAGCTTCAGAAGCCTGAC-3'), Tiam1 target sequence 2 (5'-AATGCTCTGAATCCTAGTCTC-3'), cortactin target sequence 1 (5'-AATGCCTGGAAATTCCTCATT-3'), cortactin target sequence 2 (5'-AAACAGAATTTCGTGAACAGC-3'), dynamin 2 target sequence 1 (5'-AACATGCCGAGTTTTTGCACT-3'), dynamin 2 target sequence 2 (5'-AAACAGAACATGCCGAGTTTT-3'), Rac1 target sequence 1 (5'-AAAACTTGCCTACTGATCAGT-3'), Rac1 target sequence 2 (5'-AACTTGCCTACTGATCAGTTA-3'), S1P receptor-1 target sequence 1 (5'-AAGCTACACAAAAAGCCTGGA-3'), S1P receptor-1 target sequence 2 (5'-AAAAAGCCTGGATCACTCATC-3'), scrambled sequence 1 (5'-AAGAGAAATCGAAACCGAAAA-3'), and scrambled sequence 2 (5'-AAGAACCCAATTAAGCGCAAG-3') were utilized. Sense and antisense oligonucleotides were purchased from Integrated DNA Technologies (Coralville, IA). For construction of the siRNA, a transcription-based kit (SilencerTM siRNA construction kit, Ambion) was used. Human lung EC were then transfected with siRNA using siPORTTM Amine (Ambion) as the transfection reagent according to the manufacturer's protocol. Cells (~ 40% confluent) were serum-starved for 1 h, followed by incubation with 3 µM (1.5 µM each siRNA) target siRNA (or scrambled siRNA or no siRNA) for 6 h in serum-free medium. Serum-containing medium was then added (10% serum final concentration) for 42 h before biochemical experiments and/or functional assays were conducted.


Figure 4
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  		FIGURE 4.
Effect of CEM, c-Met, and CD44 on HGF-induced human EC barrier enhancement. A, shown are the results from immunoblot analysis of untreated and siRNA-treated human EC. Untransfected cellular lysates (control, no siRNA) or cellular lysates from scrambled siRNA (siRNA that does not target any known human mRNA), c-Met siRNA, or CD44 siRNA transfection were analyzed by immunoblotting with anti-c-Met (panel a), anti-CD44 (IM-7; panel b), or anti-actin (panel c) antibody as described under "Experimental Procedures." Experiments were performed in triplicate, each with similar results; representative data are shown. B, EC were plated on gold microelectrodes, serum-starved for 1 h, and treated with either phosphate-buffered saline (pH 7.4; control) or 5 mM MbetaCD (a cholesterol-depleting agent that abolishes CEM formation) 30 min prior to addition of phosphate-buffered saline (pH 7.4) or 25 ng/ml HGF. The arrows indicate the times of MbetaCD and HGF addition. The TER tracing represents pooled data ± S.E. from three independent experiments as described under "Experimental Procedures." C, EC were plated on gold microelectrodes and treated with scrambled siRNA (control) or c-Met siRNA for 48 h. EC were then serum-starved for 1 h, followed by addition of 25 ng/ml HGF. The arrow indicates the time of HGF addition. The TER tracing represents pooled data ± S.E. from three independent experiments as described under "Experimental Procedures." D, EC were plated on gold microelectrodes and treated with scrambled siRNA (control) or CD44 siRNA for 48 h. EC were then serum-starved for 1 h, followed by addition of 25 ng/ml HGF. The arrow indicates the time of HGF addition. The TER tracing represents pooled data ± S.E. from three independent experiments as described under "Experimental Procedures." E, the graph shows the percent maximal S1P-induced change in EC permeability. EC were plated on gold microelectrodes and treated with no siRNA, scrambled siRNA, CD44 siRNA, or S1P receptor-1 (S1P1) siRNA for 48 h. EC were then serum-starved for 1 h, followed by addition of 1 µM S1P. The bars represent pooled TER data ± S.E. at 30 min after addition of agonist from three independent experiments as described under "Experimental Procedures."

 
Preparation and Quantitation of HMW-HA—The method of preparation was similar to that described previously (9, 36). Briefly, 500 mg of rooster comb HA (~106-Da polymers) (37) was dissolved in distilled water and centrifuged in a Millipore Ultrafree-MCTM 100,000-Da molecular weight cutoff filter, and the flow-through fraction (<100,000 Da) was discarded. HMW-HA was quantitated using an enzyme-linked immunosorbent assay-like competitive binding assay with a known amount of fixed HA and biotinylated HA-binding peptide as the indicator (38). HMW-HA and DNA standards were run on 4–20% SDS-polyacrylamide gels and stained with combined Alcian blue and silver to further determine HA purity and size (39).

Determination of Serine Phosphorylation of CD44—Solubilized CEM proteins in immunoprecipitation buffer were immunoprecipitated with rat anti-CD44 antibody, followed by SDS-PAGE on 4–15% polyacrylamide gels and transfer onto Immobilon-P membranes. After blocking nonspecific sites with 5% bovine serum albumin, the blots were incubated with either rat anti-CD44 or rabbit anti-phosphoserine antibody, followed by incubation with horseradish peroxidase-labeled goat anti-rabbit or goat anti-rat IgG. Visualization of immunoreactive bands was achieved enhanced chemiluminescence.

Determination of Complex Formation between CD44 and c-Met—Solubilized CEM proteins in immunoprecipitation buffer were immunoprecipitated with rat anti-CD44 or anti-c-Met antibody, followed by SDS-PAGE on 4–15% polyacrylamide gels and transfer onto Immobilon-P membranes. After blocking nonspecific sites with 5% bovine serum albumin, the blots were incubated with either rat anti-CD44 or mouse anti-c-Met antibody, followed by incubation with horseradish peroxidase-labeled goat anti-mouse or goat anti-rat IgG. Visualization of immunoreactive bands was achieved by enhanced chemiluminescence.


Figure 5
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  		FIGURE 5.
Role of CD44 in HGF-induced recruitment of c-Met, Tiam1, cortactin, and dynamin 2 to human EC CEM. EC were treated with scrambled siRNA (control) or CD44 siRNA for 48 h. EC were then grown to confluency; serum-starved for 1 h; and either left untreated (control) or treated with 25 ng/ml HGF for 5, 15, or 30 min. A, EC lysates were run on SDS-polyacrylamide gels, transferred to nitrocellulose, and immunoblotted with anti-phospho-Tyr1234/Tyr1235 c-Met (panels a and c) or anti-c-Met (panels b and d) antibody. Experiments were performed in triplicate with highly reproducible findings; representative data are shown. B, CEM (lipid raft) fractions (20% OptiPrepTM layer) prepared as described under "Experimental Procedures" were run on SDS-polyacrylamide gels; transferred to nitrocellulose; and immunoblotted with anti-c-Met (panels a and f), anti-Tiam1 (panels b and g), anti-cortactin (panels c and h), anti-dynamin 2 (panels d and i), or anti-caveolin-1 (panels e and j) antibody. Experiments were performed in triplicate with highly reproducible findings; representative data are shown.

 
Measurement of EC Electrical Resistance—EC were grown to confluence in polycarbonate wells containing evaporated gold microelectrodes, and trans-EC electrical resistance (TER) measurements were performed using an electrical cell substrate impedance sensing system obtained from Applied Biophysics as described previously in detail (4). TER values from each microelectrode were pooled at discrete time points and plotted versus time as the means ± S.E.


Figure 6
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  		FIGURE 6.
Effect of Tiam1, cortactin, and dynamin 2 on HGF-induced human EC barrier enhancement. A, shown are the results of immunoblot analysis of untreated and siRNA-treated human EC. Untransfected cellular lysates (control, no siRNA) or cellular lysates from scrambled siRNA (siRNA that does not target any known human mRNA), Tiam1 siRNA, dynamin 2 siRNA, or cortactin siRNA transfection were analyzed by immunoblotting with anti-Tiam1 (panel a), anti-dynamin 2 (panel b), anti-cortactin (panel c), or anti-actin (panel d) antibody as described under "Experimental Procedures." Experiments were performed in triplicate, each with similar results; representative data are shown. For B and C, EC were grown to confluency; serum-starved for 1 h; and either left untreated (control) or treated with 25 ng/ml HGF for 5, 15, or 30 min. CEM (lipid raft) fractions (20% OptiPrepTM layer) were then prepared as described under "Experimental Procedures." B, EC were treated with scrambled siRNA (control), dynamin 2 siRNA, or Tiam1 siRNA for 48 h. The CEM fractions were run on SDS-polyacrylamide gels, transferred to nitrocellulose, and immunoblotted with anti-cortactin (panels a, c, and e) or anti-caveolin-1 (panels b, d, and f) antibody. Experiments were performed in triplicate with highly reproducible findings; representative data are shown. C, CEM fractions were solubilized in immunoprecipitation buffer and immunoprecipitated (Ippt) with anti-dynamin 2 antibody. The resulting Immunobeads were run on SDS-polyacrylamide gels; transferred to nitrocellulose; and immunoblotted with anti-Tiam1 (panel a), anti-cortactin (panel b), anti-caveolin-1 (panel c), or anti-dynamin 2 (panel d) antibody. Experiments were performed in triplicate with highly reproducible findings; representative data are shown. D, the immunoreactive bands from the experiments depicted in C were analyzed using ImageQuantTM software (see "Experimental Procedures") and quantitated. % Protein Association with Dynamin-2 on the y axis refers to the following: (SAGV for immunoreactive band of interest/SAGV for dynamin 2 immunoreactive band) x 100. E, the percent maximal HGF-induced change in EC permeability is represented graphically. EC were plated on gold microelectrodes and treated with scrambled siRNA (control), Tiam1 siRNA, dynamin 2 siRNA, or cortactin siRNA for 48 h. EC were then serum-starved for 1 h, followed by addition of 25 ng/ml HGF. The bars represent pooled TER data ± S.E. at 30 min after addition of agonist from three independent experiments as described under "Experimental Procedures."

 
Rac1 Activation Assay—Rac1 activity assays in human lung EC were performed as described previously (40).

Determination of Complex Formation between Dynamin 2 and Cortactin/Caveolin-1—Solubilized CEM proteins in immunoprecipitation buffer were immunoprecipitated with rabbit anti-dynamin 2 antibody, followed by SDS-PAGE on 4–15% polyacrylamide gels and transfer onto Immobilon-P membranes. After blocking nonspecific sites with 5% bovine serum albumin, the blots were incubated with rabbit anti-dynamin 2, mouse anti-cortactin, rabbit anti-caveolin-1, or rabbit anti-Tiam1 antibody, followed by incubation with horseradish peroxidase-labeled goat anti-rabbit or goat anti-mouse IgG. Visualization of immunoreactive bands was achieved by enhanced chemiluminescence.

Animal Preparation and Treatment—Male C57BL/6J and CD44 knock-out mice (8–10 weeks old; The Jackson Laboratory, Bar Harbor, ME) were anesthetized with intraperitoneal ketamine (150 mg/kg) and acetylpromazine (15 mg/kg) according to approved protocols. LPS (2.5 mg/kg) or saline (control) was instilled intratracheally, and 4 h later, HGF (50 µg/kg) or saline control was delivered intravenously through the internal jugular vein. The animals were allowed to recover for 24 h, followed by bronchoalveolar lavage protein analysis and/or lung immunohistochemistry.

Murine Lung Immunohistochemistry—To characterize protein expression in mouse lung vascular EC, lungs from C57BL/6J control (untreated) mice were formalin-fixed; 5-µm paraffin sections were obtained and hydrated; and epitope retrieval was performed (DakoCytomation target retrieval solution, pH 6.0). The sections were then histologically evaluated by either fluorescein isothiocyanate-conjugated anti-CD44 antibody or anti-c-Met or anti-vWF (Factor VIII) antibody and fluorescent secondary antibody (Alexa FluorTM 610 for vWF and Alexa FluorTM 350 for c-Met; Molecular Probes). Negative controls for immunohistochemical analysis were performed by the same method as described above but without primary antibody. Immunofluorescent stained sections were photographed (magnification x100) using a Leica Axioscope.

Determination of Bronchoalveolar Lavage Protein Concentration—Bronchoalveolar lavage was performed by an intratracheal injection of 1 ml of Hanks' balanced salt solution, followed by gentle aspiration. The recovered fluid was processed for protein concentration (BCA protein assay kit, Pierce) as described previously (41).

Statistical Analysis—Student's t test was used to compare the means of data from two or more different experimental groups. Results are expressed as the means ± S.E.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Role of CD44 in HGF/c-Met-mediated Human EC Barrier Enhancement—HGF binding to its plasma membrane receptor tyrosine kinase, c-Met, induces a variety of cell functions (7, 8, 42), including EC barrier enhancement (5). However, the mechanism by which this occurs remains poorly defined. CD44, a major HA receptor localized in CEM, has been implicated in regulating HGF/c-Met signaling (10–13). We therefore examined the role of CD44 in HGF-induced EC barrier regulation.

Our data indicate that there are two main CD44 isoforms expressed in human pulmonary EC, CD44v10 (~120 kDa) and CD44s (standard form, ~85 kDa) (Fig. 1A). In the absence of HGF (control), CD44s, but not CD44v10 or c-Met, was localized to CEM (also termed detergent-resistant membranes or lipid rafts). HGF (25 ng/ml) treatment of human EC induced recruitment of ~70% of total CD44v10 and ~55% of total c-Met into CD44s-containing CEM (Fig. 1, B and C).

Prior studies of CD44 involvement in regulating HGF-induced c-Met signaling (10–13) demonstrated that CD44 variant isoforms can bind HGF (10) and regulate c-Met autophosphorylation (Tyr1234/Tyr1235) (12, 13), suggesting that CD44 can act as a coreceptor for c-Met. Using CD44v10-blocking antibody treatment of human EC showed that CD44v10 regulated HGF-mediated c-Met phosphorylation at Tyr1234/Tyr1235 by ~50% (Fig. 2, A and B) and recruitment of c-Met into CEM (Fig. 2C). As shown in Fig. 3A, the c-Met recruited to CEM was active (tyrosine-phosphorylated). Fig. 3 (B and C) shows that HGF induced a time-dependent association of c-Met with CD44v10, followed by CD44s as well as CD44 activation (defined by CD44 serine phosphorylation) in CEM (20, 43–45). Abolishing the potential for CEM formation with methyl-beta-cyclodextrin (MbetaCD), a plasma membrane cholesterol-depleting agent, or reducing the expression of c-Met or CD44 (via siRNA) attenuated HGF-induced increases in human EC barrier function (Fig. 4). These results appear to be specific for HGF, as silencing CD44 expression did not alter the barrier-enhancing effects of another CEM-regulated agonist, S1P (Fig. 4E) (32). Furthermore, silencing CD44 expression blocked c-Met autophosphorylation (Fig. 5A). These results demonstrate an essential role for CD44 and CEM in HGF-induced c-Met activation and EC barrier regulation.

Role of Tiam1, Cortactin, and Dynamin 2 in HGF/c-Met-mediated Human EC Barrier Enhancement—Considering our results indicating that CD44 regulated HGF-induced EC barrier enhancement (Fig. 4), we examined whether Tiam1, cortactin and/or dynamin 2 is involved in HGF-induced increases in EC barrier integrity. Fig. 5B indicates that Tiam1, cortactin, and dynamin 2 were present in modest amounts within CEM in control EC, with increased recruitment to these caveolin-enriched plasma membrane microdomain structures following HGF (25 ng/ml) treatment. Silencing CD44 expression (siRNA) attenuated the HGF-induced recruitment of these molecules to CEM (Fig. 5B), with silencing either Tiam1 or dynamin 2 expression abolishing cortactin localization to CEM (Fig. 6, A and B). Immunoprecipitation of dynamin 2 from CEM showed that cortactin and caveolin-1, but not Tiam1, were complexed with dynamin 2 and that HGF treatment of human EC enhanced this association (Fig. 6B). Finally, silencing Tiam1, cortactin, or dynamin 2 expression attenuated the EC barrier-enhancing effects of HGF (Fig. 6E), indicating the critical involvement of these molecules in this response.

Role of Rac1 in HGF/c-Met-mediated Human EC Barrier Enhancement—Rac1 activation is required for the barrier-enhancing effects of S1P (30), simvastatin (46), hyaluronan (9), ATP (47), and HGF (5). However, the mechanism of HGF-induced Rac1 activation in human EC remains poorly defined. Fig. 7 shows that HGF (25 ng/ml) induced Rac1 activation, which is required for HGF-induced human EC barrier enhancement. Preventing CEM formation with MbetaCD, a plasma membrane cholesterol-depleting agent, or silencing c-Met, CD44, Tiam1, or dynamin 2 expression (siRNA) inhibited HGF-induced Rac1 activation. In contrast, silencing cortactin expression did not affect HGF-mediated Rac1 activation.

Role of CD44 in HGF-mediated Regulation of Lung Vascular Integrity in Vivo—Consistent with our in vitro results in human pulmonary microvascular EC, immunohistochemical studies revealed the colocalized expression of CD44 and c-Met in C57BL/6J wild-type murine lung endothelium (Fig. 8A). We next examined whether HGF is an effective barrier protective agent in an in vivo model of LPS-induced murine lung vascular permeability. LPS administered via an intratracheal route induced murine inflammation and increased vascular leakiness as measured by the protein concentration in bronchoalveolar lavage fluid (Fig. 8, B and C) (48). Intravenous injection of either the CD44 ligand HMW-HA (1.5 mg/kg) (Fig. 8B) or HGF (50 µg/kg) (Fig. 8C) 4 h after LPS delivery attenuated C57BL/6J wild-type mouse pulmonary hyperpermeability. In contrast, this potent protective effect of both HMW-HA and HGF on LPS-induced inflammatory lung injury was markedly attenuated in the CD44 knock-out mouse, indicating that the protective effect of HGF in LPS-induced pulmonary hyperpermeability is dependent upon regulation by CD44.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we have presented several novel observations, including the finding that CD44 isoforms temporally regulate HGF/c-Met-mediated EC barrier integrity in vitro. Furthermore, CD44 regulates HGF-mediated EC barrier integrity in vivo. HGF binding to c-Met induces CD44v10-dependent c-Met translocation to CEM and temporal association of activated (tyrosine-phosphorylated) c-Met with CD44s (serine-phosphorylated). CD44 activation is required for HGF-induced recruitment of the Rac1 guanine nucleotide exchange factor Tiam1 and the vesicular regulator dynamin 2 to CEM. Both Tiam1 and dynamin 2 contribute to HGF-induced Rac1 activation and recruitment of the cortical actin cytoskeletal regulatory protein cortactin to CEM. Furthermore, dynamin 2 forms a complex with caveolin-1 and cortactin within these CEM structures (Fig. 9), an event that appears to contribute to HGF/c-Met-mediated CD44 regulation of EC barrier function.

Prior studies of CD44 involvement in regulating HGF-induced c-Met signaling (10–13) demonstrated that the CD44v3 isoform can bind HGF (10), suggesting that CD44 can act as a coreceptor for c-Met. Like CD44v3, CD44v10 contains glycosaminoglycan attachment sites that are involved in recognition of ligands other than HA (49), indicating that CD44v10 can directly or indirectly bind to HGF. Furthermore, the extracellular domain of the CD44 isoform CD44v6 regulates c-Met autophosphorylation (12, 13), whereas the cytoplasmic domain of CD44v6 regulates HGF-induced Sos, Ras, MEK (mitogen-activated protein kinase/extracellular signal-regulated kinase kinase), and ERK (extracellular signal-regulated kinase) MAPK (mitogen-activated protein kinase) activation through involvement of ERM (ezrin/radixin/moesin) proteins (12, 13). We have extended these observations to show that CD44v10 regulates c-Met CEM localization and that CEM-localized CD44 regulates Tiam1/dynamin 2-mediated cortactin recruitment and Rac1 activation. Interestingly, we also observed an HGF-induced time-dependent association of c-Met with CD44s (standard form) within CEM. Whether CD44v10 and/or CD44s can also regulate HGF-induced Ras pathway signaling in human EC is currently being investigated in our laboratory.

We observed evidence for a complex formed between activated (tyrosine-phosphorylated) c-Met and activated (serine-phosphorylated) CD44v10 and CD44s in CEM fractions following HGF treatment. It remains unknown whether these interactions are direct or mediated through an adaptor protein. Furthermore, the mechanism of differential association of c-Met with CD44 isoforms is currently being investigated in our laboratory. Using phospho site-specific antibodies, we observed HGF-induced c-Met phosphorylation at Tyr1234/Tyr1235 and Tyr1349. Phosphorylation at Tyr1234/Tyr1235 within the catalytic domain of c-Met is critical for c-Met kinase activity, whereas phosphorylation at Tyr1349 induces direct binding of Gab1 (Grb2-associated binder-1) (6–8, 42). CD44 induces serine/threonine phosphorylation and plasma membrane recruitment of Gab1 (50). Whether Gab1 is involved in the c-Met·CD44 complex formation is currently being investigated in our laboratory. The CD44 cytoplasmic domain can be phosphorylated at Ser291, Ser316, Ser323, and Ser325 by various serine/ threonine kinases, including ROCK (Rho kinase) and protein kinase C, which regulates CD44 association with the cytoskeletal protein ankyrin and ERM proteins (18, 20, 43–45). We are also investigating these proteins as potential linkers between CD44 isoforms and c-Met.


Figure 7
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  		FIGURE 7.
Effect of Tiam1, cortactin, and dynamin 2 on HGF-induced Rac1 activation. A, EC were either left untreated or treated with scrambled siRNA, c-Met siRNA, CD44 siRNA, dynamin 2 siRNA, Tiam1 siRNA, or cortactin siRNA for 48 h. EC were grown to confluency, serum-starved for 1 h, and either left untreated (control) or treated with 5 mM MbetaCD (a cholesterol-depleting agent that abolishes CEM formation) 30 min prior to addition of phosphate-buffered saline (pH 7.4) or 25 ng/ml HGF. EC were then solubilized in immunoprecipitation buffer and incubated with p21-binding domain-conjugated beads to bind activated (GTP-bound) Rac1. The p21-binding domain bead-associated material was run on SDS-polyacrylamide gels, transferred to nitrocellulose, and immunoblotted with anti-Rac1 antibody. Experiments were performed in triplicate with highly reproducible findings; representative data are shown. B, the immunoreactive bands from the experiments depicted in A were analyzed using ImageQuantTM software (see "Experimental Procedures") and quantitated. % Rac1 Activation on the y axis refers to the following: (SAGV for activated Rac1 immunoreactive band/SAGV for total Rac1 immunoreactive band) x 100. C, shown are the results from immunoblot analysis of untreated and siRNA-treated human EC. Untransfected cellular lysates (control, no siRNA) or cellular lysates from scrambled siRNA (siRNA that does not target any known human mRNA) or Rac1 siRNA transfection were analyzed by immunoblotting with anti-Rac1 (panel a) or anti-actin (panel b) antibody as described under "Experimental Procedures." Experiments were performed in triplicate, each with similar results; representative data are shown. D, the percent maximal HGF-induced change in EC permeability is represented graphically. EC were plated on gold microelectrodes and treated with scrambled siRNA (control) or Rac1 siRNA for 48 h. EC were then serum-starved for 1 h, followed by addition of 25 ng/ml HGF. The bars represent pooled TER data ± S.E. at 30 min after addition of agonist from three independent experiments as described under "Experimental Procedures."

 


Figure 8
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  		FIGURE 8.
Role of CD44 in HGF-induced protection from LPS-induced vascular hyperpermeability in vivo. A, shown are immunohistochemical fluorescent stained images of control (untreated) mouse lung using either bright-field (differential interference contrast (DIC)) imaging (panel a) or treatment with anti-vWF (Factor VIII) antibody (panel b), anti-c-Met antibody (panel c), or fluorescein isothiocyanate-conjugated anti-CD44 antibody (panel d) and fluorescent secondary antibody (Alexa FluorTM 610 for vWF and Alexa FluorTM 350 for c-Met) as described under "Experimental Procedures." Images are shown at magnification x100. Arrows indicate immunostaining of EC, with panel e being an overlay of panels b–d. Insets, negative controls for immunohistochemical analysis that were done by the same method as described above but without primary antibody. B and C, male C57BL/6J and CD44 knock-out mice were anesthetized and given either saline (control) or LPS (2.5 mg/kg) intratracheally. After 4 h, mice were given intravenous injections through the internal jugular vein of saline (control), HMW-HA (1.5 mg/kg; B), or HGF (50 µg/kg; C). The treated mice were allowed to recover for 24 h; bronchoalveolar lavage (BAL) fluids were obtained; and protein concentrations were determined (see "Experimental Procedures"). *, significant difference (p < 0.05) between control and LPS treatment. There was also a significant difference (p < 0.05) between LPS and HMW-HA + LPS treatment in the wild-type mouse, but not in the CD44 knock-out mouse. **, significant difference (p < 0.05) between LPS and HGF + LPS treatment. There was also a significant difference (p < 0.05) upon HGF + LPS treatment between the wild-type and CD44 knock-out mice.

 


Figure 9
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  		FIGURE 9.
Proposed model of HGF-induced vascular integrity. HGF induces CD44v10-regulated c-Met (Step 1) translocation to CEM and association of activated (tyrosine-phosphorylated) c-Met with activated (serine-phosphorylated) CD44v10 and CD44s (Step 2). CD44 isoforms are required for HGF-induced recruitment of the Rac1 guanine nucleotide exchange factor Tiam1 and the vesicular regulator dynamin 2 to CEM to join pre-existing Tiam1 and dynamin 2 within these structures (Step 3). Both Tiam1 and dynamin 2 contribute to HGF-induced Rac1 activation (Step 4) and recruitment of the cortical actin cytoskeletal regulatory protein cortactin to CEM (Step 5). Furthermore, dynamin 2 forms a complex with caveolin-1 and cortactin within these CEM structures. These events contribute to HGF/c-Met-mediated CD44 regulation of EC barrier function (Step 6).

 
Our results demonstrate that HGF promotes CD44-dependent Tiam1/Rac1 activation in CEM fractions. Previous reports that Tiam1 is involved in cell-cell adhesion (51, 52) are consistent with our findings that Tiam1 expression is required for HGF/c-Met-induced Rac1 activation and EC barrier enhancement. HGF induced significant recruitment of Tiam1 into CEM fractions and increased Tiam1-dependent total Rac1 cellular activation, indicating either post-translational modifications of Tiam1 or enhancement of Tiam1 activity by localization within CEM. Tiam1 is phosphorylated by serine/threonine kinases, including Ca2+/calmodulin-dependent protein kinase II, and protein kinase C-dependent mechanisms (53, 54). Studies examining HGF-induced Tiam1 post-translational modification are currently in progress.

We have demonstrated that dynamin 2 is complexed with caveolin-1 and cortactin, which is augmented in HGF-challenged EC. Furthermore, dynamin 2 is required for HGF-induced c-Met- and CD44-dependent Rac1 activation. These results are in agreement with previous reports indicating that dynamin 2 regulates cortical actin dynamics through its interaction with cortactin (33), regulates Rac1 cellular localization and activation (34), and binds to caveolin-1 (35). In this study, we have provided the novel observation that the dynamin 2·caveolin-1·cortactin complex can regulate HGF-induced EC barrier enhancement.

Our recent data indicate that HMW-HA promotes cortical actin rearrangement and increased barrier function in EC through a process dependent on Rac1 activation (9). Actin cytoskeletal rearrangement is required for EC barrier function because actin-depolymerizing and microfilament-disrupting agents (i.e. cytochalasin B and latrunculin A) abolish this effect (4, 30). Overexpression of a dominant-active form of Rac1 in EC promotes cortical actin "ring" formation associated with barrier enhancement, whereas silencing Rac1 expression (siRNA) inhibits EC barrier enhancement (4, 30). Our results indicate that CD44 is required for HGF-induced Rac1 activation, thus implicating CD44 as a crucial mediator between c-Met signaling and cytoskeletal reorganization.

In this study, we use intratracheal administration of LPS to induced vascular hyperpermeability. LPS induces a delayed EC barrier-disrupting response by activating a receptor complex of TLR4, CD14, and MD2, with consequent Rho-dependent NF-{kappa}B activation, cytokine production, and generation of low molecular weight HA degradation fragments (55–61). In particular, RhoA is important in LPS-mediated regulation of interleukin-8 production (62). Furthermore, LPS induces RhoA/ROCK-mediated myosin light chain phosphorylation (63, 64), with inhibition of ROCK attenuating LPS-induced acute lung injury (65).

The CEM protein caveolin-1 inhibits the pro-inflammatory effects of LPS (66). Two downstream targets of ROCK in CEM that can potentially regulate LPS-induced EC barrier disruption include the actin- and phospholipid-binding protein MARCKS (myristoylated alanine-rich C kinase substrate) (67–69) and the sodium-hydrogen exchanger NHE1 (37). ROCK can also act cooperatively with protein kinase C to induce MARCKS phosphorylation (67). LPS induces MARCKS phosphorylation (70), which inhibits its association with the plasma membrane and promotes cytosolic localization (71–73). LPS can also regulate NHE1 (74), a protein implicated in cell stress responses through regulation of intracellular pH and actin cytoskeletal dynamics (75, 76).

In vivo, CD44 is expressed in a variety of cells, including endothelial, epithelial, and immune cells (18, 20, 77–79). CD44 knock-out mice develop lung fibrosis, inflammatory cell recruitment, and accumulation of hyaluronan fragments at sites of lung injury (78). In addition to being an important regulator of immune cell function, CD44 is involved in regulating epithelial barrier function (79). Therefore, the reduction in HGF protection from LPS-induced injury in the CD44 knock-out mouse can potentially involve immune and epithelial cell involvement in addition to endothelial regulation.

In summary, utilizing both in vitro and in vivo models of pulmonary vascular permeability, we have demonstrated that CD44 regulates HGF-induced vascular integrity via a mechanism we speculate to involve scaffolding of key CEM components (Tiam1, cortactin, dynamin 2, and Rac1) by CD44 isoforms essential to the HGF response. These results further indicate that HGF may serve as a potentially useful therapeutic treatment for diseases characterized by high permeability states.


   FOOTNOTES
 
* This work was supported in part by Ruth L. Kirschstein National Research Award F32 HL68472 and Program Project Grant HL58064 from the National Institutes of Health and by National Scientist Development Grant 0730277N from the American Heart Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Dept. of Medicine, Pritzker School of Medicine, University of Chicago, 5841 S. Maryland Ave., W604, Chicago, IL 60637. Tel.: 773-834-3163; Fax: 773-702-4427; E-mail: jgarcia{at}medicine.bsd.uchicago.edu.

2 The abbreviations used are: EC, endothelial cell(s); HGF, hepatocyte growth factor; CEM, caveolin-enriched microdomain(s); HMW-HA, high molecular weight hyaluronan; vWF, von Willebrand factor; LPS, lipopolysaccharide; SAGV, standardized average gray value(s); siRNA, small interfering RNA; S1P, sphingosine 1-phosphate; TER, transendothelial cell electrical resistance; MbetaCD, methyl-beta-cyclodextrin. Back



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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