Albumin endocytosis in endothelial cells induces TGF-{beta} receptor II signaling

Shahid S. Siddiqui, Zeba K. Siddiqui, and Asrar B. Malik

Department of Pharmacology, College of Medicine, University of Illinois, Chicago, Illinois 60612

Submitted 9 December 2003 ; accepted in final form 30 December 2003


    ABSTRACT
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Vascular endothelial cells undergo albumin endocytosis using a set of albumin binding proteins. This process is important for maintaining cellular homeostasis. We showed by several criteria that the previously described 73-kDa endothelial cell surface albumin binding protein is the 75-kDa transforming growth factor (TGF)-{beta} receptor type II (T{beta}RII). Albumin coimmunoprecipitated with T{beta}RII from a membrane fraction from rat lung microvascular endothelial cells. Albumin endocytosis-negative COS-7 cells became albumin endocytosis competent when transfected with wild-type T{beta}RII but not when transfected with a domain-negative kinase mutant of T{beta}RII. An antibody specific for T{beta}RII inhibited albumin endocytosis. A mink lung epithelial cell line, which expresses both the TGF-{beta} receptor type I (T{beta}RI) and the T{beta}RII receptor, exhibited albumin binding to the cell surface and endocytosis. In contrast, mutant L-17 and DR-26 cells lacking T{beta}RI or T{beta}RII, respectively, each showed a dramatic reduction in binding and endocytosis. Albumin endocytosis induced Smad2 phosphorylation and Smad4 translocation as well as increased protein expression of the inhibitory Smad, Smad7. We identified regions of significant homology between amino acid sequences of albumin and TGF-{beta}, suggesting a structural basis for the interaction of albumin with the TGF-{beta} receptors and subsequent activation of T{beta}RII signaling. The observed albumin-induced internalization of T{beta}RII signaling may be an important mechanism in the vessel wall for controlling TGF-{beta} responses in endothelial cells.

albumin-transforming growth factor-{beta} sequence identity; transforming growth factor-{beta} receptor II complimentary deoxyribonucleic acid; Smad2; Smad4; Smad7


THE TRANSFORMING GROWTH FACTOR-{beta} (TGF-{beta}) superfamily comprises a diverse group of cytokines that include bone morphogenetic proteins (BMPs) and activins (5, 21, 22). Members of the TGF-{beta} family of proteins activate transcription of genes critical for cell cycle regulation, production of extracellular matrix, cell differentiation, and other related responses (4, 21, 22, 24). TGF-{beta} signaling is triggered by binding of the TGF-{beta} homodimer to a complex consisting of two TGF-{beta} transmembrane serine/threonine kinase receptors, type I and type II, referred to as T{beta}RI and T{beta}RII (56 and 75 kDa, respectively; see Refs. 9 and 19). T{beta}RI and T{beta}RII are each homodimers before ligand binding (11, 15). TGF-{beta} binds directly to T{beta}RII, which in turn induces the formation of the activated heteromeric complex consisting of TGF-{beta}, T{beta}RI, and T{beta}RII (5, 14, 21, 22). As this complex is formed, T{beta}RII phosphorylates T{beta}RI (3, 38, 39), which initiates signaling through the Smad effector proteins (14, 17).

Eight Smad proteins, identified in mammals, have been classified into the following three subgroups: receptor-regulated Smads (R-Smads); a common-partner Smad (co-Smad); and inhibitory Smads (I-Smads). R-Smads directly interact with the activated T{beta}RI and are themselves activated through phosphorylation of their COOH-terminal SSXS motifs. These activated R-Smads have then been shown to form a heteromeric complex with the co-Smad, Smad4. R-Smads have been shown to move into the nucleus where they regulate gene transcription (3, 5, 14, 17, 21, 22, 30). I-Smads, such as Smad7, have been shown to negatively regulate signaling initiated by TGF-{beta}, activin, and BMP (13, 25). Internalization of TGF-{beta} receptor-activated signaling via endocytosis is required for these discrete functional responses (1, 2, 14, 15, 18, 28, 38, 4043). The fate of the internalized receptor was distinct whether it is partitioned in caveolae or clathrin-coated pits (where receptor signaling emanates from early endosomes; see Ref. 7).

Albumin has been shown to induce endocytosis in the endothelial cells lining the vessel wall (23, 36). Albumin endocytosis is extensively studied in endothelial cells, and four albumin-binding proteins, including a 73-kDa protein, are described (10, 3335). In the present study, we addressed the possibility that albumin endocytosis facilitates the internalization of the 75-kDa TGF-{beta} receptor, T{beta}RII, and induces the internalization of T{beta}RII signaling.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Reagents. Drs. Peter ten Dijke and Carl-Henrik Heldin (Ludwig Cancer Institute, Uppsala, Sweden) provided the rabbit polyclonal antibody to Smad2-P. Polyclonal antibodies against Smad2 (sc-6200), Smad4 (sc-7154), Smad7 (sc-7004), T{beta}RII (sc-1700), fluorescein isothiocynate (FITC)- and Texas red isothiocynate (TRITC)-conjugated polyclonal goat and donkey antibodies against mouse and rabbit IgG, horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG, HRP-conjugated goat anti-mouse IgG, normal mouse IgG, normal rabbit IgG, rT{beta}RII protein (sc-4122), and Smad7 peptide (sc-7004p) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). A primary culture of rat lung microvascular endothelial cells (RLMVEC; passage 11) was obtained from American Type Culture Collection (Bethesda, MD). The mink lung epithelial cell line (Mv1.Lu) was a kind gift of Dr. J. Massague (Sloan Kettering Memorial Institute, New York, NY), and mutant lines, L-17 and DR-26, lacking T{beta}RI and T{beta}RII receptors, respectively, were kind gifts of Dr. E. Loeff (Mayo Clinic, Rochester, MN). Antibody to BSA [no. A0433; BSA, fraction V 99% pure, endotoxin-free (BSA, Sigma no. A3059)]; human serum albumin (HSA), A3782; FITC-labeled BSA (FITC-albumin); TRITC-BSA (TRITC-albumin); 4',6-diamidine-2-phenylindole (DAPI); methyl-{beta}-cyclodextrin; and disuccinimidyl suberate were purchased from Sigma (St. Louis, MO). n-Propylgallate was purchased from Molecular Probes. Cell culture reagents were purchased from BioWhittaker (Walkersville, MD). Drs. T. Imamura and K. Miyazono (Cancer Institute, Tokyo University, Tokyo, Japan) provided the wild-type and dominant-negative kinase mutant [K277R (39); cDNA constructs of human T{beta}RII, subcloned into pcDNA3.1]. Protein G-Sepharose and ECL reagents were purchased from Amersham-Pharmacia (Piscataway, NJ). Dr. R. Minshall, University of Illinois at Chicago, provided 125I-albumin (BSA and HSA; see Ref. 23).

Cell culture. Cells were grown and incubated in phenol red-free DMEM with 100 IU/ml pencillin and 100 µg/ml streptomycin at 37°C, 5% CO2, in the presence or absence of FBS as stated.

FITC/TRITC-albumin binding and internalization assays. Albumin internalization by RLMVEC, Mv1.Lu, DR-26, L-17, and COS-7 cells was visualized using FITC- or TRITC-BSA as a tracer. Cells were grown in monolayers to 80–90% confluence on glass coverslips in six-well culture plates in DMEM made 10% (37°C, 5% CO2). Before the assay, cells were grown for 24 h in serum-free DMEM to starve for albumin. The cells were washed in 4°C DMEM and incubated with DMEM (37°C); the incubation medium concentration was made 100 µg/ml with unlabeled BSA and 10 µg/ml of either FITC-BSA or TRITC-BSA for the times described to determine the albumin uptake. Cells were placed on ice, washed with PBS, pH 7.4, with 100 mM NaOAc, pH 2.5, and 150 mM NaCl (to remove cell surface albumin), and with 100 mM NaHPO4, pH 7.4 (to restore the pH to neutral). All reagents were used at 4°C. The antifade agent, n-propylgallate in glycerol (5% wt/vol), was added to the mounting medium to reduce photobleaching. Fluorescence and differential interference contrast (DIC) images of cells were visualized using appropriate excitation and barrier filters for fluorescein, rhodamine, and DAPI in an Eclipse E800 microscope (Nikon). The images were captured with a CCD [cooled integrating charged-coupled camera (Hamamatsu Photonics, Hamamatsu, Japan)] and analyzed using Metamorph imaging software (Universal West Imaging, West Chester, PA). The fluorescence measurements were reported as average intensity per unit area of three cell fields, normalized to uptake at 30 min.

Competition of 125I-labeled albumin uptake with unlabeled albumin by RLMVEC. Albumin-starved RLMVEC were incubated with 125I-labeled albumin (50,000 cpm) in the absence or presence of increasing concentrations of unlabeled albumin (0.1–2 mg/ml) for 20 min at 37°C. The cells were placed on ice and washed to remove surface BSA, scraped in lysis buffer (50 mM Tris·HCl buffer, pH 7.4, 1% Triton X-100, and 0.5% SDS), and homogenized. The protein concentration of homogenates was determined. Aliquots of cell homogenates were counted using a Beckman gamma counter. The results are reported as counts per minute 125I-albumin per milligram protein.

Methyl {beta}-cyclodextrin treatment of RLMVEC. Albumin-starved RLMVEC were incubated for 2 h in DMEM with 3 mM methyl {beta}-cyclodextrin (MBC). After MBC treatment, cells were washed with DMEM and incubated with 1 mg/ml BSA and 0.1 mg/ml TRITC-BSA for 30 min at 37°C. Fluorescence and DIC images of cells were captured and analyzed as described above.

Immunostaining of RLMVEC. RLMVEC were grown to 80–90% confluence. The cells were washed in 4°C PBS and kept at 4°C. They were fixed in 2% paraformaldehyde for 30 min. The cells were permeabilized with 0.1% Triton X-100 in PBS (15 min) and washed with PBS. The cells were incubated with primary antibody (or normal IgG, as a control) for 1–2 h. Cells were washed with 0.1% Triton X-100-PBS and incubated with FITC-labeled appropriate secondary IgG for 2 h. The cells were washed and visualized as described for FITC/TRITC-albumin binding and internalization assays.

Whole cell lysate and membrane fraction from RLMVEC. To prepare whole cell lysates, RLMVEC were grown to 80–90% confluence in 10-cm dishes. Cells were disrupted by incubation with lysis buffer {100 mM Tris·HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1.0% Nonidet P-40, 0.25% sodium deoxycholate, 0.1% SDS, 1 mM Na3VO4, 1 mM NaF, and protease inhibitor cocktail [2 µg/ml leupeptin, 2 µg/ml pepstatin A, 2 µg/ml aprotonin, and 50 µg/ml phenylmethylsulfonyl fluoride (PMSF)]} at 4°C for 45 min. All manipulations were at 4°C. The lysate was centrifuged at 20,000 g for 15 min, and the supernatant was recovered as whole cell lysate. To isolate a membrane fraction, RLMVEC (1 x 108) were grown in 50-ml flasks overnight. Cells were manipulated at 4°C throughout. Cells were washed with PBS at 4°C, scraped, and suspended in membrane isolation buffer (25 mM HEPES/Tris, pH 7.2, 150 mM NaCl, 0.2 mM PMSF, and 0.5 µg/ml leupeptin, and pepstatin; MIB). The cells were washed in MIB and centrifuged at 500 g for 5 min two times. The cells were homogenized in a glass hand-held homogenizer and then sonicated (30-s pulse 6 times). The cell homogenate was centrifuged at 2,500 g for 10 min to remove cell debris and unbroken cells. The supernatant was centrifuged at 100,000 g for 2 h. The pellet was centrifuged again at 100,000 g for 60 min. The final pellet was dissolved in 1 ml MIB and stored in small aliquots at -70°C. The protein concentration of the whole cell lysate and membrane fraction was determined.

Immunoblotting. Standard procedures were followed. Proteins were subjected to SDS-PAGE and transferred to nitrocellulose. The membrane was blocked in 20 mM Tris·HCl, pH 7.5, and 150 mM NaCl (TBS) with 5% nonfat dried milk (5% Blotto) for 2 h at 20°C. The blocked membrane was washed in TBS with 0.1% Triton X-100 (TBS-Tr), and incubated with relevant primary antibody for 12 h at 4°C. The membrane was washed (TBS-Tr). An HRP-conjugated relevant secondary IgG (1:1,000 in TBS-Tr) was used to detect the primary antibody. The membranes were washed as before and developed using ECL; signals were detected using Kodak X-AR film.

Coimmunoprecipitation of internalized albumin and T{beta}RII. Albumin-starved RLMVEC were incubated with BSA (100 µg/ml) for 30 min. Harvested cells were washed in 100 mM NaOAc, pH 2.5, and 150 mM NaCl to remove surface-bound albumin, washed in DMEM to restore pH to neutral, lysed, immunoprecipitated with an anti-albumin antibody or an anti-T{beta}RII antibody, and subjected to immunoblot analysis, as described above. Antialbumin immunoprecipitates were probed with anti-T{beta}RII and anti-T{beta}RII immunoprecipitates with antialbumin.

Inhibition of albumin endocytosis by anti-T{beta}RII antibody. Albumin-starved RLMVEC cells were preincubated with 25–100 µg/ml rabbit anti-T{beta}RII (or normal rabbit IgG) for 1 h. Cells were incubated for 60 min with 100 µg/ml FITC-BSA and 10 µg/ml FITC-BSA, and internal FITC-BSA was visualized as described above.

Vector constructs and transfection of COS-7 cells. cDNA encoding the entire human T{beta}RII sequence (19) or a T{beta}RII dominant-negative (K277R) kinase mutant (gift from K. Miyazono and Dr. T. Imamura, Cancer Institute, Tokyo University) subcloned into pcDNA3.1 [under control of the cytomegalovirus (CMV) promoter] was used to transfect COS-7 cells. Cells were cotransfected with pcDNA3.1 encoding green fluorescent protein (GFP) to assess transfection efficiency. COS-7 cells were transfected with 5 µg/ml DNA, using the Qiagen Superfect reagent, following the manufacturer's protocol. Cells positive for GFP expression were used in the studies. TRITC-BSA uptake was measured 48 h after transfection. In control experiments, the GFP-encoding vector was transfected alone or with empty vector.

Surface binding and uptake of 125I-albumin. Mv1.Lu and two related mutant cell lines that lacked either the T{beta}RII (DR-26) or the T{beta}RI (L-17; see Ref. 39) were grown as described. HSA (1 mg/ml) was added to each well, together with 5 µl 125I-labeled albumin HSA (50,000 cpm). Cells were incubated 10 min at 4°C, when they were placed on ice and washed one time with PBS (pH 7.4). In the internalization assays, cell surface albumin was removed by washing the cells two times with 100 mM NaOAc, pH 2.5, and 150 mM NaCl before a PBS wash. Cells were then scraped in lysis buffer (50 mM Tris·HCl, pH 7.4, with 1% Triton X-100 and 0.5% SDS) and homogenized. The cell homogenates were transferred to tubes, and the 125I was counted using a Beckman gamma counter. The radioactive counts were normalized to protein concentration. Duplicate samples were counted from three independent experiments.

TGF-{beta}-induced translocation of Smad2-P. RLMVEC were grown in serum-free DMEM to 80–90% confluence and stimulated with TGF-{beta} (10 ng/ml) for 16 h. In control cells, no TGF-{beta} was added. Postincubation, the cells were fixed in formaldehyde (2% in PBS), permeabilized with 0.1% Triton X-100 in PBS, and incubated for 1 h with a polyclonal rabbit antibody against Smad2-P (1:200), a gift from Dr. ten Dijke and Dr. C. Heldin. Secondary antibody staining, washing, and cell observations were as described above.

Data analysis. All experiments were performed a minimum of three times, and in all cases data from three separate experiments are reported. Results are reported as means ± SE.

Sequence analysis. Sequence homology was computed using the Huang and Miller Alignment Algorithm (16).


    RESULTS
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Albumin endocytosis in endothelial cells. We monitored endocytosis of albumin by RLMVEC by incubating albumin-starved cells with FITC-albumin. Albumin internalization was observed by fluorescence microscopy of cells incubated, for different periods of time, with 100 µg/ml albumin and 10 µg/ml FITC-BSA, used as a tracer. At each of the time points, endocytosis was stopped by placing the cells on ice. Surface-bound albumin was removed by acid wash, followed by washes in DMEM to restore the pH to neutral. Maximum fluorescence units, defined as FITC-albumin uptake at 30 min, were considered as 100%. Albumin uptake was evident in RLMVEC within 5–10 min, with the maximal value attained between 15 and 30 min (Fig. 1A, f–j). Albumin uptake reached saturation at 30 min as it did not increase significantly beyond 30 min (Fig. 1Ak).



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Fig. 1. A: albumin binds to endothelial cells in a saturable manner. Albumin-starved rat lung microvascular endothelial cells (RLMVEC) were induced with albumin: 100 µg/ml unlabeled BSA with 10 µg/ml fluorescein isothiocynate (FITC)-albumin, used as a tracer. At specific time points (0–30 min), plates were removed to ice and washed at 4°C. To visualize the internalized albumin selectively, the external FITC-albumin was removed by washing the cells first with 100 mM NaOAc, pH 2.5, 150 mM NaCl, and then with 100 mM NaHPO4, pH 7.4. Cells accumulated albumin within 5–10 min and reached a peak over 15–30 min. f–i show the punctate staining of internalized FITC-albumin, and a–e show 4',6-diamidine-2-phenylindole (DAPI) staining of the same fields. Results are representative of 3 experiments. Bar represents 10 µm. k shows quantitative analysis of endocytosis of FITC-albumin. The fluorescence intensity was measured using Metamorph imaging software. The data are reported as %control (incubated with FITC-albumin, 30 min). Uptake of albumin was evident within 5 min and showed a rapid increase up to 15 min, and the uptake reached a maximum at 30 min. Bars are mean values ± SE. B: albumin competes with 125I-labeled albumin for uptake by endothelial cells. Albumin-starved RLMVEC were incubated with 125I-albumin (50,000 cpm), in the absence or presence of increasing concentrations (0.1–2 mg/ml) of nonradio-labeled albumin. After 20 min, the cells were placed on ice, and cell surface 125I-albumin was removed as described in A. The washed cells were lysed (50 mM Tris·HCl, pH 7.4, with 1% Triton X-100 and 0.5% SDS) and homogenized. The aliquots of the lysates were transferred to tubes for counting (Beckman gamma counter). The quantitative data show that unlabeled albumin successfully competed with 125I-albumin for uptake by endothelial cells.

 

We next tested the ability of unlabeled BSA to compete with 125I-albumin for uptake by RLMVEC (Fig. 1B). Unlabeled albumin was found to successfully compete with 125I-albumin for uptake by RLMVEC, as shown in Fig. 1B. Additionally, to determine whether endocytosis depended on the native conformation of albumin, we carried out uptake assays using albumin that had been denatured by heat or reduction with dithiothreitol. We found that denatured albumin was not internalized by RLMVEC (data not shown). From these data, we conclude that the binding of albumin to RLMVEC is saturable, subject to competition, and dependent on the native conformation of the ligand. These data support the conclusion that albumin binding to RLMVEC occurred via receptor-mediated endocytosis.

MBC inhibits albumin endocytosis. Because caveolae have been shown to have a role in albumin endocytosis in endothelial cells (27), we examined the role of caveolae in albumin uptake by RLMVEC. We evaluated the effect of MBC, known to disrupt the integrity of cholesterol-rich caveolae, on albumin uptake in RLMVEC. RLMVEC, preincubated with 3 mM MBC for 2 h, exhibited a marked reduction in uptake of TRITC-albumin compared with untreated controls (Fig. 2A, a and b). We conclude that the effect of cyclodextrin on albumin endocytosis suggested a role for caveolae in albumin endocytosis in RLMVEC.



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Fig. 2. Cyclodextrin impairs albumin endocytosis: albumin-starved RLMVEC were incubated with or without the caveola-disrupting agent cyclodextrin (3 mM) for 2 h, washed, and incubated with DMEM containing albumin (1 mg/ml) trace labeled with Texas red isothiocynate (TRITC)-albumin (0.1 mg/ml). Cells incubated with 3 mM cyclodextrin showed a reduction in TRITC-albumin uptake compared with untreated control cells. Bar is 2 µm. Results are representative of 3 experiments.

 

Expression of T{beta}RII in endothelial cells. Because T{beta}RII has a reported molecular mass, 75 kDa, similar to that of one of the described albumin binding proteins (10), we determined if albumin bound T{beta}RII on the surface of RLMVECs. We first assessed the subcellular distribution of T{beta}RII in RLMVEC by carrying out indirect immunofluorescence on permeabilized cells. Incubation with an anti-T{beta}RII antibody resulted in punctate staining of RLMVEC (Fig. 3A). Analysis of different focal planes with confocal microscopy demonstrated that the immunostaining was present both within the cell and cell surface. Immunoblot analysis of RLMVEC whole cell lysates and of a membrane fraction (obtained by differential high-speed centrifugation) with an anti-T{beta}RII antibody revealed an ~75-kDa (range 70–80) species (Fig. 3B, lanes 3 and 5) that corresponded to the reported size of T{beta}RII, 75 kDa.



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Fig. 3. A: intracellular and cell surface transforming growth factor (TGF)-{beta} receptor type II (T{beta}RII) in RLMVEC. RLMVEC monolayers were grown on coverslips in DMEM with 10% FBS. Cells were washed with PBS, fixed in chilled paraformaldehyde (2%) for 30 min, permeabilized with 0.1% Triton in PBS, washed with PBS, and incubated with anti-T{beta}RII polyclonal antibody (1:250) at 4°C for 1–2 h. Cells were washed with PBS-Triton X-100 (0.1%) and incubated with an FITC-labeled goat anti-rabbit IgG secondary antibody for 2 h at 4°C. In control experiments, normal rabbit IgG replaced the anti-T{beta}RII IgG, and no staining was seen (data not shown). Results are representative of 3 experiments. B: T{beta}RII is present as a 75-kDa protein in RLMVEC. Proteins (50 µg) from a whole cell lysate of RLMVEC were subjected to SDS-PAGE and transferred to nitrocellulose. Lane 1, staining of total protein with India ink (I.I.). The remainder of the membrane was blocked in 5% nonfat dry milk in TBS, probed with anti-T{beta}RII antibody (lane 3) or with anti-T{beta}RII antibody preincubated with the cognate peptide against which the serum was raised and affinity purified (lane 2). The immunoblot was then incubated with horseradish peroxidase-conjugated anti-rabbit IgG, developed by ECL, and exposed to film. Arrow points to T{beta}RII. Additionally, a membrane fraction was prepared from cells as described in EXPERIMENTAL PROCEDURES. Briefly, a membrane fraction was isolated by differential centrifugation and subjected to immunoblot analysis as described above except that, as a control, the membrane was probed with normal rabbit IgG (N IgG) in place of the T{beta}RII antibody. The T{beta}RII-specific band (arrow) has a molecular mass of ~75 kDa (lane 3). Protein size was estimated using prestained molecular mass protein standards. Results are representative of 3 experiments.

 

To assess the specificity of the anti-T{beta}RII antibody binding, we preincubated the antibody with the cognate full-length T{beta}RII recombinant protein against which the antibody had been made and affinity purified. As shown in Fig. 3B, lane 2, no band was seen, indicating that the antibody is specific for T{beta}RII. Additionally, normal rabbit IgG did not recognize this band (Fig. 3B, lane 4). These data show that RLMVEC expresses a 75-kDa protein that was found on the endothelial cell surface and that is recognized by an affinity-purified anti-T{beta}RII antibody. The cytosolic fraction also showed the presence of T{beta}RII (data not shown); however, the membrane fraction consistently showed a somewhat greater amount of T{beta}RII than the cytosolic fraction (i.e., densitometry analysis showed ~45% in the cytosolic pool and ~55% in the membrane pool). From these data, we conclude that the 75-kDa T{beta}RII was expressed on the cell surface of RLMVEC and therefore available for binding.

Coimmunoprecipitation of albumin and T{beta}RII. To determine whether albumin binds to the 75-kDa T{beta}RII, we immunoprecipitated T{beta}RII from albumin-starved RLMVEC incubated with albumin for 30 min. The cells were harvested, washed in low-pH buffer (pH 2.5) to remove surface-bound albumin, washed in DMEM to restore pH to neutral (pH 7.4), lysed, and immunoprecipitated with an anti-T{beta}RII antibody, and the eluate was subjected to immunoblot analysis as previously described in EXPERIMENTAL PROCEDURES, but probing with an antialbumin antibody. Immunoblot results showed that the internalized albumin specifically coprecipitated with T{beta}RII (Fig. 4A, lane 2). This ~66-kDa band was not seen when the membrane was probed with normal rabbit IgG (Fig. 4A, lane 1) or when the anti-albumin antibody was preblocked with albumin (Fig. 4A, lane 3). The reverse experiment was also performed. An anti-albumin immunoprecipitate probed with an anti-T{beta}RII antibody demonstrated an ~75-kDa T{beta}RII-specific band (Fig. 4B, lane 2). This band was not seen when the membrane was probed with normal rabbit IgG (Fig. 4B, lane 1) or when the anti-T{beta}RII antibody was preblocked with rT{beta}RII (Fig. 4B, lane 3). Thus the activation of albumin endocytosis (by adding albumin to albumin-starved cells) induces the internalization of albumin-associated T{beta}RII in endothelial cells.



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Fig. 4. Coimmunoprecipitation of T{beta}RII and albumin. RLMVEC were grown in monolayers in DMEM and 10% FBS. After 24-h albumin starvation, endocytosis was induced by incubation with 100 µg/ml albumin for 30 min. Cells were washed with PBS, and with 100 mM NaOAc, pH 2.5, and 150 mM NaCl, followed by washes in PBS. All reagents were at 4°C. The cells were lysed and incubated with anti-T{beta}RII antibody (Fig. 4A, lane 2). The antibodies were concentrated with protein G-Sepharose and immunoblotted, as described above. The nitrocellulose membranes containing proteins immunoprecipitated with anti-T{beta}RII were probed with antialbumin (A, lane 2) and show a diffused band corresponding to 63 kDa (marked as Alb). Alternatively, membranes containing proteins immunoprecipitated (IP) with antialbumin were probed with anti-T{beta}RII. The membranes were incubated with normal rabbit IgG (lane 1 in A and B) or immunoprecipitated with anti-T{beta}RII antibodies that identified a 75-kDa band corresponding to T{beta}RII (B, lane 2). When anti-T{beta}RII is preblocked with cognate T{beta}RII protein (B, lane 3) no staining is observed.

 

Anti-T{beta}RII antibody incubation inhibits endocytosis. To ascertain the functional role of T{beta}RII in the mechanism of albumin endocytosis in RLMVEC monolayers, we tested the effect of affinity-purified polyclonal anti-T{beta}RII antibody on albumin endocytosis. Preincubation of RLMVEC with the anti-T{beta}RII antibody inhibited endocytosis of TRITC-albumin by RLMVEC (Fig. 5); thus, access to T{beta}RII contributed to albumin endocytosis. Reduction in endocytosis (~35%) was observed with the anti-T{beta}RII antibody at 25 µg/ml, and there was a further reduction (80%) at 100 µg/ml (data not shown). However, because endocytosis was not completely blocked, it is likely that other mechanisms also contribute to albumin endocytosis (23). In control experiments, incubation of cells with the normal rabbit IgG failed to inhibit albumin endocytosis (Fig. 5). When the anti-T{beta}RII antibody was preincubated with rT{beta}RII, comprising the entire coding region of T{beta}RII, the antibody no longer blocked albumin endocytosis (data not show).



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Fig. 5. Preincubation of RLMVEC with anti-T{beta}RII antibody results in marked reduction of TRITC-albumin uptake. Cells were preincubated with the rabbit polyclonal anti-T{beta}RII antibody. RLMVEC show TRITC-albumin endocytosis of cells preincubated with normal rabbit IgG (a). b shows TRITC-albumin uptake after preincubation (1 h) with anti-T{beta}RII antibody. The anti-T{beta}RII antibody resulted in a marked reduction of albumin endocytosis by RLMVEC. Preincubation of the anti-T{beta}RII antibody with its cognate protein (rT{beta}RII) prevented the block in endocytosis seen with the unblocked antibody (data not shown). Bar represents 2 µm. Results are representative of 3 experiments.

 

Ectopic expression of T{beta}RII in COS-7 cells results in albumin endocytosis and T{beta}RII signaling. To address whether the T{beta}RII was sufficient to induce endocytosis of albumin, we evaluated the effects of transient, ectopic expression of a vector containing human T{beta}RII cDNA in COS-7 cells and cells that lack both T{beta}RI and T{beta}RII. A human cDNA clone encoding the full-length transcript of T{beta}RII (19), subcloned in the pcDNA 3.1 vector under the CMV promoter, was cotransfected into COS-7 cells together with gfp subcloned in pcDNA 3.1. COS-7 cells normally do not exhibit albumin endocytosis. However, transient expression of T{beta}RII cDNA in COS-7 cells resulted in significant uptake of TRITC-BSA (Fig. 6C). In a control experiment, there was no increase in albumin endocytosis in COS-7 cells transfected with pcDNA 3.1, which expressed only GFP (Fig. 6A). We further tested the role of T{beta}RII by transfecting COS-7 cells with a dominant-negative kinase mutant (K277R) of T{beta}RII. As shown in Fig. 6E, this T{beta}RII kinase mutant markedly reduced albumin endocytosis seen with transfected wild-type T{beta}RII, suggesting that albumin endocytosis is greatly facilitated by the functional kinase domain of T{beta}RII. GFP marker shows the transfected cells with the wild-type and kinase mutant transfected cells (Fig. 6, D and E). From these data, we conclude that albumin endocytosis is mediated by T{beta}RII in a T{beta}RI transfected cell line that is otherwise incompetent in the endocytosis of albumin.



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Fig. 6. Reconstitution of endocytosis in COS-7 cells by ectopic expression of T{beta}RII. COS-7 cells were transiently transfected with a plasmid encoding green fluorescent protein (GFP) and a cDNA clone encoding human T{beta}RII, both in pcDNA 3.1 under control of the cytomegalovirus (CMV) promoter. A: in control experiments, pcDNA 3.1 vector (carrying no insert) that cotransfected with the GFP-expressing vector albumin endocytosis was markedly lower. B: GFP expression of the control pcDNA 3.1 empty vector that does not carry any T{beta}RII receptor cDNA. C shows TRITC-albumin endocytosis in COS-7 cells transiently transfected with wild-type T{beta}RII. Transfected cells in C and F are identified by green fluorescence of the transfection GFP marker (D and E). E shows a marked reduction in TRITC-albumin endocytosis in cells transfected with cDNA encoding a dominant-negative K277R kinase mutant of T{beta}RII. Bar represents 2 µm. Results are representative of 3 experiments. WT, wild type.

 

Inhibition of albumin binding and endocytosis in cells expressing mutant TGF-{beta} receptors. To see whether both TGF-{beta} receptors are required for albumin endocytosis, we took advantage of two mutant mink lung epithelial cell lines, L-17 and DR-26, defective in T{beta}RI and T{beta}RII, respectively. These mutant cells were derived from the Mv1.Lu cell line, which expresses both wild-type T{beta}RI and T{beta}RII and thereby provides a positive control for albumin endocytosis. Figure 7Aa shows that, although the wild-type Mv1.Lu cells accumulated TRITC-albumin, DR-26 (Fig. 7Ab), L-17 (Fig. 7Ac) and cells that lacked functional TGF-{beta} receptors each showed a marked inhibition in albumin endocytosis. These data strongly implicate a role for both TGF-{beta} receptors in albumin endocytosis. However, as shown in Fig. 7B, the role for T{beta}RII is greater than that of T{beta}RI. We further investigated the binding of 125I-albumin tracer to the surface of Mv1.Lu, L-17, and DR-26 cells by incubating the cells at 0°C for 1 h with radiolabeled albumin.



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Fig. 7. A: reduction of albumin endocytosis in cells lacking functional TGF-{beta} receptor type I (T{beta}RI) or T{beta}RII. TRITC-albumin endocytosis in Mv1.Lu [T{beta}RI(+/+); T{beta}RII(+/+)] lung epithelial cells and the L-17 [T{beta}RII(+/+); T{beta}RI(-/-)] and DR-26 [T{beta}RII(-/-), T{beta}RI(+/+)] cells. Cells were treated as described in Fig. 1A. TRITC-albumin endocytosis is seen in the wild-type Mv1.Lu cells [T{beta}RI(+/+); T{beta}RII(+/+), a]; however, there is marked reduction in endocytosis in DR-26 cells (b) and in L-17 cells (c). Bar is 2 µm. Results are representative of 3 experiments. B: reduced surface binding of 125I-albumin by L-17 and DR-26 cells. Cells were incubated as described in EXPERIMENTAL PROCEDURES. Mink lung epithelial cells lacking functional T{beta}RI (L-17) and T{beta}RII (DR-26) receptors showed reduced cell surface 125I-albumin binding compared with control cells (Mv1.Lu). Radioactive counts per minute were normalized to Mv1.Lu uptake under identical conditions at 20 min (100%). Values are means ± SE of 3 experiments. C: reduced uptake of 125I-albumin by L-17 and DR-26 cells: mink lung epithelial cells, L-17 cells (lacking functional T{beta}RI), and DR-26 cells (lacking T{beta}RII) showed a reduction in 125I-albumin compared with the uptake in Mv1.Lu cells that expressed both functional T{beta}RII and T{beta}RI. Results are reported as cpm/mg protein. Values are means ± SE of 3 experiments.

 

Figure 7B shows the relative binding of 125I-albumin to the cell surface of the three cell lines. We observed a reduction in the surface binding of 125I-albumin to cells lacking either the T{beta}RI (~12%) or T{beta}RII (~20%) when compared with wild-type Mv1.Lu cells. A similar inhibition of endocytosis was found in the T{beta}RI and T{beta}RII mutant cell lines, although the requirement of T{beta}RII appears to be higher than that of the T{beta}RI (Fig. 7C). These results indicate that these two TGF-{beta} receptors play a crucial role both in the binding and endocytosis of albumin.

Endocytosis activates TGF-{beta} receptor signaling. To ascertain the involvement of albumin endocytosis in TGF-{beta} receptor-specific signaling in endothelial cells, we investigated the downstream signaling pathways of this receptor. On ligand binding, phosphorylation of T{beta}RI by the constitutively phosphorylated T{beta}RII has been shown to phosphorylate Smad-2 and Smad-3, thereby promoting their association with Smad-4 (14, 17, 21, 22, 30). We observed a basal level of cell surface expression of T{beta}RII in RLMVEC grown in albumin-free medium (Fig. 8Aa). However, after albumin endocytosis, T{beta}RII expression was induced, as evidenced by a marked increase in the cell surface-immunoreactive T{beta}RII punctate staining seen using confocal microscopy (Fig. 8Ab). This analysis is based on the visualization of specific cellular focal planes, determined by deconvolution microscope settings that allow one to distinguish the cell surface from the cytosolic and perinuclear focal planes. We also examined levels of Smad2 phosphorylation (Smad2-P; using a Smad2-P-specific antibody) in RLMVEC. We observed an increase in the amount of Smad2-P after albumin endocytosis compared with the basal level of Smad2-P found in cells grown under albumin-free conditions. We also observed an albumin-induced increase in Smad2-P-specific staining in the membrane, cytosolic, and perinuclear regions (Fig. 8Ad). Albumin endocytosis also increased the phosphorylation of Smad2, as detected by immunoblotting (Fig. 8Ai). An increase in the level of Smad2-P was observed at a low concentration of albumin (1 mg/ml) and was found to increase up to 3 mg/ml albumin. Interestingly, at albumin concentrations >3 mg/ml, we observed inhibition of Smad2 activation, as measured by immunoblot of Smad2-P (data not shown). Under similar conditions when TGF-{beta}1 (10 ng/ml) was used to stimulate RLMVEC, we observed that Smad2-P moved primarily into the nucleus (Fig. 8Bb), as previously reported (11, 12).



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Fig. 8. Albumin induces T{beta}RII expression, increased Smad2 phosphorylation (Smad2-P) and translocation, and Smad4 translocation. A: RLMVEC were grown up as previously described. RLMVEC were incubated in the absence (a) or presence (b) of albumin (100 µg/ml) and stained for T{beta}RII. b shows an increase in immunoreactive T{beta}RII after 30 min of incubation with albumin. Note the highly punctate nature of the pattern detected by T{beta}RII staining of the albumin-induced cells. Bar represents 2 µm. Results are representative of 3 experiments. To detect intracellular Smad2-P, RLMVEC, incubated in the absence (c) or (d) of albumin (100 µg/ml), were fixed with 2% paraformaldehyde, washed, and incubated with anti-Smad2-P antibody. FITC-labeled goat anti-rabbit IgG was used to visualize Smad2-P staining. We observed increased Smad2-P in the perinuclear region and plasmalemma of the albumin-induced cells (c vs. d). Results are representative of 3 experiments. To assess the response of Smad4 to albumin, RLMVEC were incubated in the absence (e) or presence (f) of albumin, as described in EXPERIMENTAL PROCEDURES. Cells were washed with DMEM and PBS, fixed in 4% formaldehyde, and incubated with anti-Smad4 antibody for 2 h and with FITC-conjugated goat anti-rabbit antibody. Albumin endocytosis induced Smad4 expression in the perinuclear area and nucleus. For each Smad2-P and Smad4 antibody, negative controls were performed in which the absence of primary antibody did not show any staining of cells (data not shown). Results are representative of 3 experiments. Immunoblot analysis of Smad2-P was assessed in RLMVEC after albumin endocytosis as described in EXPERIMENTAL PROCEDURES. Smad2-P was increased in albumin-induced cells (i, lane 2). Similar induction for Smad-2P was observed when RLMVEC were exposed to the ligand TGF-{beta} (data not shown). For loading controls, nitrocellulose membrane was stripped and reprobed with anti-Smad2 antibody (i, lanes 1 and 2). Results are representative of 3 experiments. B: TGF-{beta} induction of Smad2-P. As a control, TGF-{beta} was incubated with RLMVEC to assess its effect on the localization of Smad2-P. RLMVEC were untreated (a) or incubated with TGF-{beta} (10 ng/ml; b). TGF-{beta}, as previously reported, resulted in Smad2-P translocation from the cytosol to nucleus. Results are representative of 3 experiments. C: time course of Smad2-P levels. Densitometric analysis of immunoblots probed for Smad2-P expression in RLMVEC cells incubated with 0.5 mg/ml albumin.

 

The TGF-{beta} family members are know to activate discrete regulatory Smads (e.g., Smad1, Smad2, Smad3, and Smad5), but all identified regulatory Smads share Smad4 as a key partner (4, 5, 17, 30). As a result of stimulation by TGF-{beta}, the coactivator Smad4 forms a complex with activated Smad1-P or Smad2-P, and together the heteromeric complexes have been shown to move into the nucleus. In light of this, we investigated the distribution of Smad4 after albumin endocytosis. Smad4 was predominantly found in the perinuclear region of the cells grown in albumin-free medium (Fig. 8Ae) with a distribution similar to that of Smad2-P. However, after albumin endocytosis for 30 min to 2 h, Smad4 was distributed throughout the cell, in the cytosol, perinuclear region, and nucleus (Fig. 8Af). TGF-{beta}1 also induced the translocation of Smad4 from the cytosol to the nucleus in RLMVEC (data not shown), as reported in Mv1.Lu cells (26). This finding is in contrast to the translocation of Smad2-P to the perinuclear region observed after albumin endocytosis (Fig. 8Ad). Although both TGF-{beta} and albumin induced translocation of Smad2-P from the cytosol to the nuclear region of the cell, TGF-{beta}1 stimulation resulted in Smad2-P movement in the nucleus, whereas albumin endocytosis resulted in Smad2-P accumulation in the perinuclear area. Thus the two stimuli, TGF-{beta} and albumin, resulted in different subcellular localizations of this R-Smad.

We assessed the time course of Smad2-P in RLMVEC after albumin endocytosis by densitometric analysis of immunoblots. Phosphorylation of Smad2, in response to albumin endocytosis (0.5 mg/ml), peaked at 1 h (Fig. 8C). From 1 h through 5 h the level of Smad2-P dropped, returning to baseline by 5 h.

Albumin endocytosis induces Smad7 expression. Because albumin is the major protein in plasma (29), we addressed the possibility that TGF-{beta} receptor signaling was subject to negative feedback regulation in response to a sustained stimulus. It has been shown that Smad7 forms a stable association with the ligand-stimulated TGF-{beta} receptor complex, thereby inhibiting phosphorylation of Smad2 and Smad3, precluding their association with Smad4, translocation into the nucleus, and subsequent transcriptional activation of target genes (13, 26). Indirect immunostaining of RLMVEC with anti-Smad7 antibodies after 4-h incubation in the absence (Fig. 9Aa) or in the presence (Fig. 9Ab) of albumin demonstrated that albumin endocytosis induced Smad7 protein expression. In control experiments, we tested the specificity of anti-Smad7 antibody by preincubating it with a Smad7 blocking peptide. Preincubation with the blocking peptide prevented Smad7 immunostaining (data not shown). We observed by immunoblotting that protein expression of Smad7 was increased in 4 h after starved RLMVEC exposure to albumin (Fig. 9B) at the point when Smad2-P levels were close to the baseline. We conclude that Smad7 expression was induced by albumin in a time-dependent manner at a time consistent with downregulation of albumin-induced signaling.



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Fig. 9. A: endocytosis induces Smad7 expression. Smad7 expression was assessed by immunostaining RLMVEC after albumin endocytosis (4 h), as described in EXPERIMENTAL PROCEDURES. Cells were washed with DMEM and PBS, fixed with 4% formaldehyde, and incubated with an anti-Smad7 antibody for 2 h followed by FITC-goat anti-rabbit IgG. Increased expression of Smad7 was evident within 2 h of albumin induction. Cells were counterstained with DAPI. Results are representative of 3 experiments. B: endocytosis induces Smad7 expression. Immunoblot analysis of RLMVEC incubated for 4 h with albumin from 0 to 2 mg/ml showed a significant increase in Smad7 protein. The nitrocellulose membrane was stripped and reprobed for actin as shown.

 

Structural homology between albumin and TGF-{beta}. Because albumin endocytosis activated TGF-{beta} receptor signaling (as measured by an increase in the level of Smad2-P and induction of Smad7 protein expression), we examined the possibility of structural homology between human albumin and human TGF-{beta} precursor and mature protein using a protein alignment algorithm (16). We observed that the mature TGF-{beta} 112-residue protein has marked homology with the human albumin amino acid sequence (Fig. 10A). The first region, one of 53 amino acids from residue 59–112 in the mature TGF-{beta}1, is 26.4% identical to a region (residues 367–420) in HSA (Fig. 10A). The second region of homology is 12 amino acids (nos. 10–21) in TGF-{beta}1 that share 50% identity with residues 297–309 of albumin (Fig. 10A). Although in most of the experiments described above we have used BSA, we confirmed the results with HSA. Additionally, the regions of homology described here are highly conserved (90%) between HSA and BSA.



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Fig. 10. Sequence homology between human TGF-{beta}1 and human serum albumin (HSA). A: sequence homology between processed active form of 112-residue human TGF-{beta}1 and HSA. The first region, one of 53 amino acids from residue 59–112 in the mature TGF-{beta}1, is 26.4% identical to a region (residues 367–420) in HSA. The second region of homology is 12 amino acids (aa; nos. 10–21) in TGF-{beta}1 that share 50% identity with residues 297–309 of albumin. The sequence homology was computed using the Huang and Miller (16) Alignment Algorithm. B: sequence homology between the finger II region of TGF-{beta}1 and albumin. Based on X-ray crystal structure of the TGF-{beta}1 ligand and the receptor T{beta}RII complex, a receptor binding domain called the finger II region is present in TGF-{beta}1 (45). HSA has 38.4% identical amino acid residues and 65% sequence similarity with HSA.

 

We further identified a highly conserved region in the human TGF-{beta}1 finger II region (residues 88–101) known to bind the T{beta}RII, based on X-ray crystal structure of the ligand and the receptor (12), and HSA (Fig. 10B), which has 38.4% identical amino acids (residues 555–567) and 65% sequence similarity with HSA. These observations point to a structural basis for the interaction of albumin with the T{beta}RII.


    DISCUSSION
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Endocytosis of cell surface receptors is a means of internalizing and compartmentalizing cell signaling and acts, additionally, as a mechanism for receptor degradation and desensitization (1, 2, 7, 28, 32, 40, 42, 43). We studied endothelial cells because these vessel wall lining cells actively internalize albumin by endocytosis of caveolae in an Src-dependent manner (23, 36, 37). In this report, we focused specifically on the previously identified ~73-kDa albumin binding protein present in the endothelial cell membrane (10). On the basis of several criteria, we identified this protein as T{beta}RII. Analysis by cross-linking showed that native albumin binds to an ~75-kDa protein, which is immunoprecipitated by an anti-T{beta}RII antibody. We also showed that the membrane fraction of endothelial cells was enriched for an ~75-kDa T{beta}RII immunoreactive protein. Uptake of 125I-labeled albumin by RLMVEC appears to be receptor-mediated, since the uptake is competed by unlabeled albumin and reaches saturation at ~2 mg/ml albumin. In addition, an anti-T{beta}RII antibody specifically inhibited albumin endocytosis, thus linking this endocytic event with T{beta}RII. Importantly, transient expression in COS-7 cells of a vector containing a cDNA encoding human T{beta}RII was sufficient to induce albumin endocytosis in these cells. In contrast, expression of a T{beta}RII dominant-negative kinase mutant failed to support endocytosis. We demonstrated that a mink lung epithelial cell line, Mv1.Lu, endogenously expressing both T{beta}RI and T{beta}RII, was capable of albumin endocytosis, whereas two mutant-related cell lines, L-17 and DR-26, lacking functional T{beta}RI or T{beta}RII, respectively, showed inhibition of cell surface albumin binding, as well as albumin endocytosis. DR-26 cells show higher inhibition compared with the L-17 cells in albumin binding to the surface and in albumin uptake. Thus these data indicate an important role for the TGF-{beta} receptors in the mechanism of albumin endocytosis.

A number of putative albumin binding proteins in addition to p73 (i.e., gp60, p30, and p16; see Ref. 10) have been partially characterized, but the true molecular identity of the albumin receptor proteins has remained elusive. Here, for the first time, we have demonstrated a novel role for T{beta}RII in regulating albumin endocytosis both in RLMVEC and mink lung epithelial cells.

We also demonstrated that induction of albumin endocytosis was accompanied by activation of T{beta}RII-mediated signaling. We observed increased T{beta}RII expression on the cell surface, increased Smad2-P and translocation to the perinuclear region, and translocation of Smad4 from the cytosol to the perinuclear region. Taken together, these data show that the interaction of albumin with T{beta}RII induces endocytosis and intracellular TGF-{beta} receptor signaling in endothelial cells.

We identified the ~73-kDa albumin binding protein present in the endothelial cell membrane (10) as T{beta}RII. We showed that albumin was capable of increasing the cell surface pool of T{beta}RII, as evidenced by the increase in punctate T{beta}RII, detected by immunofluoresence after exposure to albumin. Because this response was seen within minutes, an explanation is that a population of preexisting T{beta}RII may redistribute from the cytosol to the cell surface. Another explanation is that, since the cells (RLMVEC) were serum starved for >24 h, albumin exposure may produce a membrane topology change that could make T{beta}RII more accessible to immunostaining. It is also possible that the antibody used for detecting the T{beta}RII staining in RLMVEC may work better in the presence of albumin in recognizing the epitope on the cell surface than serum-starved cells.

We examined the profile of effector Smads to address whether albumin endocytosis is capable of activating the T{beta}RII signaling pathway. The three classes of Smad proteins in mammals include the R-Smads, co-Smad (i.e., Smad4), and I-Smad (e.g., Smad6 and Smad7; see Refs. 5, 14, 17, 24, 25). We showed an increase in Smad2 (an R-Smad) phosphorylation and its translocation to the perinuclear region after albumin endocytosis. Thus albumin endocytosis is capable of inducing the internalization of T{beta}RII and activating the canonical T{beta}RII signaling pathway in endothelial cells. However, albumin endocytosis did not mimic the translocation of Smad2-P to the nucleus as in the case of the canonical ligand TGF-{beta}. This disparity suggests a role for albumin endocytosis that is distinct from the TGF-{beta}-induced signal transduction (21, 23). We also observed that Smad7, which serves to negatively regulate TGF-{beta} signaling by blocking the phosphorylation of regulatory Smads (e.g., Smad2) by T{beta}RI kinase (13, 26), was upregulated by albumin.

It has been shown recently that TGF-{beta} receptors can be internalized by two different endocytic mechanisms (26). One mode of internalization is mediated through the clathrin pathway, and the other operates via caveolin-positive vesicles (7). In the present study, T{beta}RII internalization is likely the result of association with caveolae since endothelial cell vesicles are mostly derived from caveolae (20). Also, we observed that endocytosis was inhibited by pretreating the cells with cyclodextrin, a cholesterol binding agent that disrupts caveolae (27, 31). Additionally, we showed that the internalized albumin associated with caveolin-1 (unpublished data), lending further support to the dependence of T{beta}RII internalization on caveolae. The fate of the TGF-{beta} receptors is determined by their association with either clathrin-coated vesicles or caveolae and their subsequent internalization pathways (7). Receptors that partition into caveolae interact with Smad7 and its partner Smurf2 and are destined for degradation (7). Thus it is possible that the albumin-induced internalization of TGF-{beta} receptor signaling via caveolae is a mechanism for downmodulating the TGF-{beta} responses.

A possible explanation for our finding that albumin can activate T{beta}RII is the homology between albumin and the TGF-{beta} precursor protein sequences [Kolmogorov-Simirov statistics (16)]. Two regions show significant homology between albumin and TGF-{beta}1 (Fig. 10). Further examination of the sequence homology between the mature, processed 112-residue TGF-{beta}1 produced by protease cleavage (6, 8) and HSA also reveals regions of identity (Fig. 10A). The first region, one of 53 amino acids from residue 59–112 in the mature TGF-{beta}1, is 26.4% identical to a region (residues 367–420) in HSA (Fig. 10A). The second region of homology is 12 amino acids (nos. 10–21) in TGF-{beta}1 that share 50% identity with residues 297–309 of albumin (Fig. 10A). Thus both the precursor peptide and protease-cleaved active peptide of TGF-{beta}1 share at least three regions with significant sequence homology with albumin. Significant homology also exists between the finger II region of the human TGF-{beta}1 amino acid sequence; that finger II motif has been previously shown to be critical for binding of the ligand TGF-{beta}1 with the receptor T{beta}RII based on the X-crystal structure of the TGF-{beta}1 and T{beta}RII ligand-receptor complex (12). HSA shares 38.4% identity and 65% similarity with the finger II region of the human TGF-{beta}1 (Fig. 10B), suggesting a region of albumin that may interact with the T{beta}RII receptor. On the basis of these homology data, we propose a mechanism in which albumin binds to T{beta}RII, activates endocytosis in endothelial cells, and provides a means for internalizing T{beta}RII signaling. Internalization of T{beta}RII signaling activates Smad2 by phosphorylation, and Smad2-P associates with Smad4 to form a complex. Because of the sequence homology between albumin and TGF-{beta}, both ligands may compete for T{beta}RII, and plasma albumin may continuously modulate and fine-tune TGF-{beta}-activated signaling by shuttling it to a caveolar degradation pathway (26). Because albumin-induced activation of T{beta}RII also activates Smad7, this suggests another mechanism of downregulation of TGF-{beta} signaling. Thus the competition between albumin and TGF-{beta} for T{beta}RII may provide an exquisite mechanism for the regulation of TGF-{beta}-activated signaling in endothelial cells.


    ACKNOWLEDGMENTS
 
We thank Peter ten-Dijke and Carl Henrik-Heldin for Smad2-P specific antibodies, E. Leof, J. Massague, N. Ueno, K. Miyazono, and C. P. Wright for cDNA clones and cell lines, and O. Colamonici, R. Frey, D. Mehta, R. Minshall, G. Patterson, D. Predescu, S. Predescu, D. Riddle, A. Rahman, C. Tiruppathi, S. Uddin, T. Voyno-Yasentskaya, S. Vogel, D. Yau, and R. Ye for reagents and suggestions. S. Uddin is gratefully acknowledged for helping in the initial stages of the project. We thank L. Price for editing the manuscript.

GRANTS

This work was supported by National Heart, Lung, and Blood Institute Grant HL-46350.


    FOOTNOTES
 

Address for reprint requests and other correspondence: S. S. Siddiqui, Dept. of Pharmacology, College of Medicine, Univ. of Illinois, 835 South Wolcott Ave. (M/C 868), Chicago, IL 60612 (E-mail: SSIDDIQU{at}UIC.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.


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