©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Inhibition of Capillary Morphogenesis and Associated Apoptosis by Dominant Negative Mutant Transforming Growth Factor- Receptors (*)

(Received for publication, June 1, 1995)

Mary E. Choi (§) Barbara J. Ballermann

From the Division of Nephrology and Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Transforming growth factor-beta1 (TGF-beta1) induces angiogenesis in vivo and capillary morphogenesis in vitro. Two receptor serine/threonine kinases (types I and II) have been identified as signal transducing TGF-beta receptors. We explored the possibility of inhibiting TGF-beta- mediated events in glomerular capillary endothelial cells using a TGF-beta type II receptor (TbetaR-II) transdominant negative mutant.

A mutant TGF-beta type II receptor (TbetaR-II(M)), lacking the cytoplasmic serine/threonine kinase domain, was produced by polymerase chain reaction using rat TbetaR-II cDNA as template. Since TbetaR-II and TGF-beta type I receptor (TbetaR-I) heterodimerize for signal transduction, the mutant receptor competes for binding to wild-type TbetaR-I, hence acting in a dominant negative fashion. Glomerular capillary endothelial cells were stably transfected with TbetaR-II(M), and four independent clones were expanded. That the TbetaR-II(M) mRNA was expressed was shown by reverse transcriptase-polymerase chain reaction, RNase protection assay, and Northern analysis. Presence of cell surface TbetaR-II(M) protein was shown by affinity cross-linking with I-TGF-beta1. In wild-type endothelial cells, TGF-beta1 (2 ng/ml) significantly inhibited [^3H]thymidine incorporation to 63 ± 10% of control (n = 4). In transfected endothelial cells carrying TbetaR-II(M), TGF-beta1 stimulated [^3H]thymidine incorporation to 131 ± 9% of control (n = 4, p < 0.005). Also, in wild-type endothelial cells, endogenous and exogenous TGF-beta1 induced apoptosis and associated capillary formation. Both apoptosis and capillary formation were uniformly and entirely absent in transfected endothelial cells carrying TbetaR-II(M).

This represents the first demonstration that capillary morphogenesis in vitro is associated with apoptosis, and that interference with TbetaR-II signaling inhibits this process in glomerular capillary endothelial cells.


INTRODUCTION

Angiogenesis, the process of new blood vessel formation, is an integral part of development, wound repair, and tumor growth. The formation of capillary networks requires a complex series of cellular events, in which endothelial cells locally degrade their basement membrane, migrate into the connective tissue stroma, proliferate at the migrating tip, elongate and organize into capillary loops(1) . In response to angiogenic stimuli, endothelial cells in culture develop networks of capillary-like tubes.

Transforming growth factor-beta1 (TGF-beta1) (^1)is a 25-kDa homodimeric polypeptide that belongs to a family of homologous multifunctional cytokines. TGF-beta1 regulates diverse cellular functions including proliferation and differentiation. TGF-beta1 is strongly expressed during embryogenesis (2, 3) and in sites undergoing intense development and morphogenesis(4, 5) . Moreover, TGF-beta1 induces angiogenesis in vivo(6, 7) and capillary morphogenesis in vitro(8) . The mechanism by which TGF-beta1 induces angiogenesis is not yet well defined.

In the early stages of angiogenesis, proteases are required for extracellular matrix (ECM) proteolysis to facilitate endothelial cell migration(9) . TGF-beta1 induces endothelial cell secretion of plasminogen activator (PA) which activates plasmin, a protease that degrades ECM proteins(10, 11) . Increased production of PA has been associated with the invasive properties of cultured endothelial cells in response to angiogenic stimuli(10, 11) . In addition, plasmin activates latent TGF-beta1(12) , in an autocrine fashion. Furthermore, TGF-beta1 is a potent chemoattractant for macrophages and fibroblasts (13, 14) , which are postulated to release angiogenic peptides in vivo, such as basic fibroblast growth factor (bFGF), platelet-derived growth factor, or tumor necrosis factor-alpha(15) .

Two transmembrane serine/threonine kinases, types I and II, have been identified as signal transducing TGF-beta receptors. TGF-beta type II receptor (TbetaR-II), a constitutively active kinase, directly binds TGF-beta1, and this ligand binding results in the recruitment and phosphorylation of TGF-beta type I receptor (TbetaR-I) to produce a heteromeric signaling complex(16) . TbetaR-I alone is unable to bind TGF-beta1, and TbetaR-II is unable to signal without TbetaR-I(17) .

We explored the possibility of inhibiting TGF-beta1-mediated events in renal glomerular capillary endothelial cells using a TbetaR-II transdominant negative mutant. A mutant TbetaR-II construct (TbetaR-II(M)), lacking the cytoplasmic serine/threonine kinase domain, but with full transmembrane spanning and extracellular domains, was produced by polymerase chain reaction (PCR) using rat TbetaR-II cDNA (18) as template. Since TbetaR-II and TbetaR-I heterodimerize for signal transduction, the mutant receptor competes for binding to wild-type TbetaR-I, hence acting in a dominant negative fashion(19, 20, 21) . When the transdominant negative mutant construct was stably expressed in glomerular capillary endothelial cells, capillary morphogenesis and associated apoptosis were entirely blocked in these cells.


EXPERIMENTAL PROCEDURES

Cell Culture

Glomerular capillary endothelial cells were isolated from bovine kidney cortex as described previously(22) , with the following modifications. After collagenase digestion, the cells were plated at low density on gelatin-coated plates, in RPMI 1640 medium containing 15% fetal bovine serum (FBS) to which 8 ng/ml acidic fibroblast growth factor (aFGF) (R & D Systems), 0.1 µg/ml heparin, and 5 units/ml penicillin, and 5 µg/ml streptomycin were added. The aFGF was used to stimulate endothelial cell proliferation, and the heparin both to increase the affinity of aFGF for endothelial cell FGF receptors and to inhibit mesangial cell growth. Colonies of endothelial cells were subjected to two rounds of cloning to establish cell lines free of contaminating mesangial cells. Once the cells were established in culture, they were maintained in RPMI 1640 with 15% FBS and 5 units/ml penicillin, and 5 µg/ml streptomycin. Cells between passages 5 and 15 were used for experiments described herein. That the cells are endothelial cells was documented for each isolation by labeling with fluorescent acetylated low density lipoprotein (Biomedical Technologies Inc.).

To induce capillary tube formation, cells grown to confluence were placed in RPMI medium containing 0.5% FBS, in the presence or absence of exogenous 2 ng/ml TGF-beta1 (Collaborative Biomedical Products). For transmission electron microscopy, the cells were fixed in 3% glutaraldehyde in phosphate-buffered saline, postfixed in 1% osmium tetroxide, dehydrated in a graded series of ethanol, and embedded in epon directly on the culture dish, in order to preserve morphology of the capillary tubes. Thin sections were cut, stained with uranyl acetate and lead citrate, and subjected to electron microscopy (Paragon Biotech Inc., Baltimore, MD). In experiments with neutralizing antibody to TGF-beta1, cells grown to confluence were placed in RPMI medium containing 0.5% FBS in the presence or absence of 10 ng/ml turkey anti-human TGF-beta1 IgG (Collaborative Biomedical Products).

Mutant TbetaR-II Construct

A truncated TGF-beta type II receptor construct (TbetaR-II(M)), lacking the cytoplasmic serine/threonine kinase domain, but with full transmembrane spanning and extracellular domains, was generated by PCR using a rat TbetaR-II cDNA as the template (Fig. 1). Primer sequences were as follows: sense primer 5`-GTTAAGGCTAGCGACGGGGGCTGCCATG-3`; antisense primer 5`-GGCGGTCGACTAGACACGGTAACAGTAGAAG-3`; and contained the sequences for the restriction enzymes NheI and SalI, respectively (underlined), for directional cloning, and a stop codon in the antisense primer. Each 100-µl PCR reaction mixture contained 1 ng of template DNA, 0.25 µM primers, 0.05 mM dNTPs, 0.75 mM MgCl(2), 1 PCR buffer II (Perkin-Elmer), and 2 units of Taq polymerase (Perkin-Elmer). Amplification consisted of initial denaturation at 95 °C for 1 min, followed by 25 cycles (15 s at 95 °C, 15 s at 50 °C, and 15 s at 72 °C) in GeneAmp PCR System 9600 (Perkin-Elmer). This reaction product was gel-purified and cloned with NheI and SalI into pMAMneo (CLONTECH), a glucocorticoid-inducible mammalian expression vector. That the clone contained correct directionality and in-frame sequences of the PCR product were verified by restriction mapping with EcoRI, BamHI, and HindIII, and sequencing by dideoxy chain termination technique using Sequenase 2.0 (U. S. Biochemical).


Figure 1: Mutant TbetaR-II construct. Schematic diagram of wild-type TbetaR-II and mutant construct (TbetaR-II(M)), lacking the cytoplasmic serine/threonine kinase domain, but with full transmembrane spanning and extracellular domains, as indicated. The rat wild-type TbetaR-II cDNA contains an open reading frame of 1701 nt long, corresponding to a protein of 567 amino acid residues. TbetaR-II(M) cDNA is 573 nt long, corresponding to 191 amino acids. The horizontal arrows indicate the positions of the PCR primers.



Stable Transfection of Glomerular Capillary Endothelial Cells

To generate clones that stably expressed TbetaR-II(M), glomerular capillary endothelial cells were transfected by using Lipofectin (Life Technologies, Inc.) as follows. Cells grown to approximately 50% confluency on 6-well plates were washed with RPMI, then incubated with 1-5 µg of DNA (TbetaR-II(M) ligated in pMAMneo) in RPMI and 5-10 µl of Lipofectin suspension for 5 h at 37 °C in a 5% CO(2) atmosphere. Control cells were incubated with pMAMneo vector (not containing TbetaR-II(M)) and Lipofectin. Following a 5-h incubation, medium containing 20% FBS in RPMI was added to each well to make a final concentration of 10% FBS, and incubated further for 48 h. Then the medium was changed to 10% FBS in RPMI (no antibiotics) and incubated for another 24 h. To select for stable transfectants, cells were treated with 400 µg/ml Geneticin (Life Technologies, Inc.) in RPMI medium containing 15% FBS, and the medium was changed every 2-3 days. Clones emerged at approximately 14 days after lipofection. Stably transfected clones were subcloned using ring cylinders, expanded, and maintained in RPMI medium containing 15% FBS, 200 µg/ml Geneticin, 5 units/ml penicillin, and 5 µg/ml streptomycin. Four independent, stably transfected clones containing TbetaR-II(M) and 4 clones containing empty vector were expanded. Non-transfected glomerular endothelial cells similarly treated with Lipofectin served as additional controls.

Solution Hybridization/RNase Protection

RNase protection analysis was done using the RPA II kit (Ambion) according to the manufacturer's instructions. The P-labeled antisense RNA probe was prepared from the linearized plasmid containing a fragment of the rat TbetaR-II cDNA using T7 RNA polymerase, yielding a probe 488 nucleotides (nt) long. 20 µg of total RNA from wild-type and transfected cells were hybridized with the P-labeled probe. Hybridization was for 16-18 h at 42 °C in 50% formamide, 5 SSPE, 0.1 M Tris, pH 7.4, and 50 µg/ml salmon sperm DNA. The samples were then digested with RNase A/T1, and resolved on a 6% acrylamide, 7.7 M urea sequencing gel. A sample of P-labeled 1-kb ladder DNA was loaded in adjacent lanes as the molecular size marker.

Northern Blot Analysis

Total RNA from cells grown in the absence or presence of 1 µM dexamethasone (Sigma) was isolated by lysis with TRI reagent (Molecular Research Center, Inc.) according to the manufacturer's instructions, and size fractionated (30 µg/lane) on a 1% agarose, 2% formaldehyde gel in 20 mM MOPS, 5 mM sodium acetate, and 1 mM EDTA, pH 7.2. Messenger RNA was transferred to a nylon membrane (Nytran, Schleicher & Schuell) and UV linked to the membrane. The blot was prehybridized at 65 °C using 1% bovine serum albumin (Sigma), 7% SDS, 0.5 M phosphate buffer, 1 mM EDTA, pH 8.0, and 100 µg/ml heat-denatured salmon sperm DNA for 2 h, hybridized in the same solution containing the appropriate P-labeled cDNA at 65 °C overnight, followed by two 30-min washes at 65 °C with 0.5% bovine serum albumin, 5% SDS, 40 mM phosphate buffer, 1 mM EDTA, pH 8.0, then four 15-min washes with 1% SDS, 40 mM phosphate buffer, 1 mM EDTA, pH 8.0, at 65 °C. The membrane was then exposed to Kodak X-AR 5 film for 25-48 h. The TbetaR-II probe is a 2.8-kb rat TbetaR-II full-length cDNA (17) which was labeled with [P]dCTP using random primer labeling system (Life Technologies, Inc.).

Covalent Labeling of TGF-beta Receptors

Cells on 100-mm plates (Corning) were washed twice with cold 40 mM HEPES, pH 7.4, in Hanks' balanced salt solution (HBSS), then incubated in binding assay buffer (HBSS, 40 mM HEPES, pH 7.4, and 1 mM bacitracin) with 400 pMI-TGF-beta1 (DuPont) in the presence or absence of 100 nM unlabeled TGF-beta1 (Collaborative Biomedical Products), at room temperature for 90 min. The cells were then washed twice with 40 mM HEPES, pH 7.4, in HBSS, followed by incubation for 30 min at 4 °C with covalent cross-linking reagent disuccinimidyl suberate (Pierce) in dimethyl sulfoxide at a final concentration of 0.3 mM in 40 mM HEPES, pH 7.4, HBSS. The cross-linking reaction was quenched by washing three times with cold 250 mM sucrose, 10 mM Tris, pH 7.4, 1 mM EDTA. The cells were lysed with 100 µl of 1% Triton X-100, 10 mM Tris, pH 7.4, 1 mM EDTA, 10 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 50 µg/ml aprotinin, 10 µg/ml pepstatin, and subjected to centrifugation at 13,000 g for 30 min to remove particulate matter. Sample loading buffer (sucrose, 0.01% bromphenol blue, 2% beta-mercaptoethanol, 5 mM EDTA) was added (1:1, v/v) and boiled for 5 min, followed by 10% SDS-polyacrylamide gel electrophoresis. The gel was stained with Coomassie Brilliant Blue (Bio-Rad) to visualize equivalence in protein loading, and destained prior to autoradiography.

[^3H]Thymidine Incorporation

10^4 cells were plated in 24-well dishes and incubated in medium containing 15% FBS and grown to subconfluence. Medium was then changed to serum-free RPMI for 24 h, followed by incubation in 0.5% FBS in the presence or absence of TGF-beta1 (2 ng/ml). After 45 h, the medium was removed, and cells were exposed for 3 h to 1 µCi/ml [^3H]thymidine in RPMI 1640 medium containing 2% bovine platelet-poor plasma derived serum, at 37 °C. The cells were washed three times with RPMI, and then extracted three times with ice-cold 6% trichloroacetic acid, followed by solubilization in 1 N NaOH and counted in a Packard Liquid scintillation counter. For determination of the time course of [^3H]thymidine incorporation, similar methods were utilized and the cells were extracted at the various time points, with 3-h [^3H]thymidine exposure prior to each period.

Genomic DNA Isolation and Analysis

Cells were plated on 150-mm plates, and grown to confluence in medium containing 15% FBS, then incubated in the presence or absence of 1 µM dexamethasone for 24 h. The medium was then changed to 0.5% FBS and incubated in the presence or absence of TGF-beta1 (2 ng/ml) at 37 °C for 5 days. Genomic DNA isolation was performed using Puregene (Gentra) according to the manufacturer's instructions. Briefly, cells were lysed directly on the plate by removing the culture medium and adding the cell lysis solution, followed by incubation with RNase A, then protein precipitation solution. The samples were centrifuged at 2,000 g for 10 min and supernatant transferred to new tubes. Precipitated DNA was resuspended in DNA hydration solution, and quantitated by UV spectrophotometer. 20 µg of DNA was analyzed with a 1.5% agarose gel electrophoresis.


RESULTS

Expression of TbetaR-II(M)mRNA

To demonstrate that the transfected glomerular endothelial cells expressed TbetaR-II(M) mRNA, RNase protection assay was performed using antisense RNA prepared from the rat TbetaR-II cDNA with some flanking vector sequence. The rat TbetaR-II probe contained 488 nt of authentic rat TbetaR-II sequence, which included only 211 nt of TbetaR-II(M), and predicted to hybridize fully with rat (but not bovine) TbetaR-II mRNA. Based on the size of the TbetaR-II cDNA probe, the expected size of the protected fragment produced from transfected rat TbetaR-II(M) cDNA sequence was 211 nt. As shown in Fig. 2A, hybridization with the rat TbetaR-II antisense riboprobe protected a fragment 211 nt in length from RNase digestion in transfected cells. No protected fragment was seen in the wild-type bovine glomerular endothelial cells or mock transfected cells with vector alone. Expression of TbetaR-II(M) mRNA in glomerular endothelial cells was also demonstrated by RT-PCR utilizing the sense and antisense primers to rat cDNA sequence used in producing the mutant construct (data not shown), and four independent transfected clones expressing TbetaR-II(M) mRNA were propagated for all subsequent experiments.


Figure 2: Expression of TbetaR-II mRNA in wild-type and transfected glomerular endothelial cells. A, RNase protection assay. Total RNA from wild-type and transfected glomerular endothelial cells, treated with 1 µM dexamethasone, was hybridized in solution with a P-labeled rat TbetaR-II cDNA probe followed by RNase digestion. As expected, no protected fragment was observed in the wild-type cells (lane 1). In the transfected cells (lane 2), a 211-nt long protected fragment was seen, demonstrating that mRNA for rat TbetaR-II(M) was expressed. Controls represent P-labeled rat TbetaR-II cDNA probe hybridized with yeast total RNA, followed by digestion with RNase (lane 3), or without RNase treatment (lane 4). No protected fragment was seen with RNase treatment (lane 3). The undigested probe was 488 nt long (lane 4). Molecular size was determined by P-labeled 1-kb ladder DNA (not shown). B, Northern analysis. Total RNA (30 µg/lane) from wild-type glomerular endothelial cells incubated in the absence (lane 1) or presence (lane 2) of 1 µM dexamethasone was subjected to Northern blot hybridization with a P-labeled rat TbetaR-II cDNA probe. Only a 5.5-kb mRNA was detected, corresponding to wild-type TbetaR-II. Lanes 3 and 4 represent total RNA from transfected endothelial cells incubated in the absence or presence of 1 µM dexamethasone, respectively. Both the 5.5- and a 1.8-kb mRNA are observed, reflecting wild-type TbetaR-II and TbetaR-II(M), respectively. The 1.8-kb mRNA was strongly induced by dexamethasone (lane 4). Also, a modest induction of the 5.5-kb wild-type TbetaR-II mRNA was seen with dexamethasone (lanes 2 and 4). Similar densities for the 18 S signals indicate approximate equivalence of RNA loading.



Northern blot analysis of total RNA isolated from wild-type and transfected glomerular endothelial cells, probed with TbetaR-II cDNA, showed a 5.5-kb band in all cells (Fig. 2B), corresponding to wild-type TbetaR-II. A new 1.8-kb band, corresponding to TbetaR-II(M), was observed only in cells transfected with TbetaR-II(M), and not present in wild-type or mock transfected cells, and was strongly induced by dexamethasone. Also, a modest induction of the 5.5-kb wild-type TbetaR-II mRNA was seen with dexamethasone.

Cell Surface Expression of TbetaR-II(M)

Affinity cross-linking with I-TGF-beta1 in wild-type glomerular endothelial cells detected two distinct bands with molecular masses of approximately 89 and 70 kDa corresponding with TbetaR-II and TbetaR-I, respectively (Fig. 3). Only in the transfected glomerular endothelial cells expressing TbetaR-II(M), two labeled bands of approximately 48 and 36 kDa were observed, both induced by dexamethasone. Labeling of TbetaR-I in transfected glomerular endothelial cells appeared diminished in three separate experiments.


Figure 3: Affinity cross-linking of I-labeled TGF-beta1 to cell surface receptors. Lanes 1 and 2 represent wild-type glomerular endothelial cells cross-linked in the presence or absence of unlabeled TGF-beta1, respectively. Lanes 3 and 4 represent transfected glomerular endothelial cells in the presence or absence of unlabeled TGF-beta1, respectively. Specifically labeled bands are observed at approximately 89 kDa and approximately 70 kDa corresponding to wild-type TbetaR-II and TbetaR-I, respectively. Bands of approximately 48 and 36 kDa are seen only in the transfected glomerular endothelial cells.



Effect of TGF-beta1 on Endothelial Cell [^3H]Thymidine Incorporation

Treatment of wild-type glomerular endothelial cells in culture for 48 h with exogenous TGF-beta1 significantly inhibited [^3H]thymidine incorporation to 63 ± 10% of control (Fig. 4A). Inhibition of [^3H]thymidine incorporation was similarly observed in empty vector transfected cells treated with TGF-beta1. In contrast, treatment of transfected cells carrying TbetaR-II(M) with exogenous TGF-beta1 stimulated [^3H]thymidine incorporation to 131 ± 9% of control (*, p < 0.005, Student's t test, n = 4). The time course of [^3H]thymidine incorporation in transfected cells grown in 0.5% FBS and in the presence or absence of TGF-beta1 (2 ng/ml) is shown in Fig. 4B. The results are mean values of triplicate determinations ± S.E. Exogenous TGF-beta1 stimulated [^3H]thymidine incorporation significantly above the basal rate in 0.5% FBS at 36 and 48 h (p < 0.005, analysis of variance).


Figure 4: Effect of TGF-beta1 on [^3H]thymidine incorporation. A, cells were incubated in the presence or absence of TGF-beta1 (2 ng/ml) for 45 h, followed by [^3H]thymidine incorporation into the cells for 3 h. Lane 1, TGF-beta1 inhibited [^3H]thymidine incorporation in wild-type glomerular endothelial cells to 63 ± 10%. Lane 2, in transfected glomerular endothelial cells carrying TbetaR-II(M), TGF-beta1 stimulated [^3H]thymidine incorporation to 131 ± 9% (*, p < 0.005, Student's t test, n = 4). B, transfected glomerular endothelial cells were incubated in 0.5% FBS and in the presence (bullet) or absence () of TGF-beta1 (2 ng/ml), and [^3H]thymidine incorporation was determined at various time points. Data represent means of triplicate determinations ± S.E. (p < 0.005, analysis of variance). A second experiment gave essentially the same results.



Effect of Serum Deprivation and TGF-beta1 on Capillary Morphogenesis

In cultured wild-type glomerular endothelial cells, with serum deprivation in the presence or absence of dexamethasone pretreatment, many of the cells detached from their substratum while remaining cells organized into capillary-like structures (Fig. 5A). Furthermore, treatment with exogenous TGF-beta1, in the presence or absence of dexamethasone, also induced cell detachment and formation of capillary-like structures (Fig. 5B). These events were accelerated by approximately 48 h when compared to those cells under serum deprivation alone. Both cell detachment and formation of capillary-like structures were observed with serum deprivation in mock transfected cells carrying empty vector. In contrast, cell detachment and formation of capillary-like structures were uniformly and entirely absent in transfected cells carrying TbetaR-II(M) treated either with serum deprivation (Fig. 5C) or exogenous TGF-beta1 (Fig. 5D). When examined with a high power phase-contrast objective, after 5 days of culture the cellular cords formed by wild-type glomerular endothelial cells appeared as tubes that contained a central translucent lumen-like space along their length (Fig. 6A), similar to the in vitro angiogenesis models described by Ingber and Folkman (23) and Montesano et al.(11) . Lumen formation was confirmed by transmission electron microscopy, which revealed groups of endothelial cells that were joined by interdigitated cell processes and enclosed a central lumenal space, as shown in Fig. 6B. Amorphous material within the lumen, also previously described by Ingber and Folkman(23) , likely represents matrix and debris. Clathrin-coated pits and vesicles, as well as cell junctional complex are also observed.


Figure 5: Effect of serum deprivation and exogenous TGF-beta1 on capillary morphogenesis. Formation of capillary-like structures were observed in wild-type glomerular endothelial cells incubated in 0.5% FBS medium, in the absence (panel A) or presence (panel B) of exogenous TGF-beta1 (2 ng/ml). Whereas, capillary-like structures were not observed in transfected glomerular endothelial cells carrying TbetaR-II(M), incubated in 0.5% FBS medium, both in the absence (panel C) or presence (panel D) of exogenous TGF-beta1 (2 ng/ml).




Figure 6: Details of capillary-like structures formed by wild-type glomerular capillary endothelial cells. A, phase-contrast view of an endothelial cell cord consisting of both solid and hollow segments. The arrows indicate lumen-like translucent space. B, transmission electron micrograph demonstrating the presence of a central lumenal space (L). Clathrin-coated pits and vesicles are observed as well as cell junctional complex (arrow).



Induction of Apoptosis by Serum Deprivation and Exogenous TGF-beta1

Fig. 7shows genomic DNA size analysis. In wild-type glomerular endothelial cells treated either with serum deprivation or exogenous TGF-beta1, DNA fragmentation was observed. This occurred both with and without dexamethasone pretreatment. Genomic DNA fragmentation was absent in transfected glomerular endothelial cells carrying TbetaR-II(M).


Figure 7: Agarose gel electrophoresis of genomic DNA. A, in wild-type glomerular endothelial cells, serum deprivation with (+) or without(-) exogenous TGF-beta1 induced apoptosis as shown by genomic DNA fragmentation. This was observed both with (+) and without(-) 24-h incubation with 1 µM dexamethasone. B, genomic DNA fragmentation was uniformly and entirely absent in serum deprived transfected glomerular endothelial cells carrying TbetaR-II(M), with (+) or without(-) exogenous TGF-beta1, both with (+) and without(-) 24-h incubation with 1 µM dexamethasone.



Inhibition of Capillary Morphogenesis by Anti-TGF-beta1 Antibody

Fig. 8shows serum-deprived wild-type glomerular endothelial cells, incubated in the absence (panel A) or presence (panel B) of neutralizing antibody to TGF-beta1. Cell detachment and formation of capillary-like structures were observed with serum deprivation alone. However, with the addition of neutralizing antibody to TGF-beta1, both cell detachment and formation of capillary-like structures were not observed. Additionally, neutralizing antibody to TGF-beta1 inhibited DNA fragmentation in serum-deprived wild-type glomerular capillary endothelial cells (data not shown).


Figure 8: Inhibition of capillary morphogenesis by neutralizing antibody to TGF-beta1. Panel A shows capillary-like structures formed by wild-type glomerular endothelial cells with serum deprivation. This formation of capillary-like structures was inhibited by the addition of neutralizing antibody to TGF-beta1, as shown in panel B.




DISCUSSION

This study sought to explore the role of TGF-beta receptors in capillary morphogenesis, using renal glomerular capillary endothelial cells stably transfected with a TbetaR-II construct designed to inhibit TbetaR-II dependent signals through a transdominant negative action. TGF-beta receptors types I and II are co-expressed by most cells(24) , and heterodimerize upon ligand binding(16, 17) . Heterodimer formation was recently shown to induce phosphorylation of TbetaR-I at the GS domain, an effect dependent on TbetaR-II kinase activity. Phosphorylation of TbetaR-I by TbetaR-II is thought to be essential for the propagation of TGF-beta1 signals(16) . Mutant TbetaR-II lacking the cytoplasmic signaling domain can be predicted to inhibit TbetaR-II dependent signals by virtue of competition for TbetaR-I binding, as long as the ability to heterodimerize is conserved. Chen et al.(19) previously showed that mutant TbetaR-II lacking the cytoplasmic kinase domain overexpressed in a dominant negative fashion selectively blocked TbetaR-II signaling for inhibition of cell proliferation. Wieser et al.(20) demonstrated that truncated TbetaR-II lacking the cytoplasmic domain was able to bind TGF-beta, and form a complex with TbetaR-I, but failed to inhibit cell proliferation, activate extracellular matrix synthesis, or activate transcription from a promoter containing TGF-beta-responsive elements. Moreover, mutant TbetaR-II with kinase domain deleted was shown to confer resistance to TGF-beta control of developmentally regulated cardiac genes(21) . In this study, essentially the same construct was utilized, and stably expressed in cultured renal glomerular capillary endothelial cells. Expression of TbetaR-II(M) mRNA in transfected glomerular capillary endothelial cells was demonstrated by reverse transcriptase-PCR, RNase protection assay, and Northern blot analysis. Although the TbetaR-II(M) construct was under a glucocorticoid-regulated promoter, expression was observed even in uninduced conditions, although at lower levels than that in the presence of dexamethasone. That such promoters ``leak'' during uninduced conditions has previously been observed by others(25) .

To demonstrate cell-surface expression and ligand-binding by the mutant receptor, intact cells were incubated with I-TGF-beta1 followed by affinity cross-linking and analysis of labeled proteins by SDS-polyacrylamide gel electrophoresis. In untransfected or mock transfected cells, only the wild-type TbetaR-I and TbetaR-II, approximately 70 and 89 kDa, respectively, were observed. In transfected cells, two additional bands of 48 and 36 kDa in size were also observed. Mutant receptor has a predicted molecular mass of 23 kDa. The 48-kDa band is interpreted to represent the mutant receptor with TGF-beta dimer bound to it, the 36-kDa band could represent the same mutant receptor with TGF-beta1 monomer or possibly a degradation product of the same receptor. These data are interpreted to show that the mutant receptor is expressed at the cell surface and that it can bind TGF-beta1.

Cross-linking to wild-type TbetaR-I was less in the transfected cells carrying TbetaR-II(M) (Fig. 3), when compared to untransfected or mock transfected cells. A plausible explanation is that the mutant receptor competes and heterodimerizes with wild-type TbetaR-I and is then quickly degraded, internalized, or secreted. Alternatively, the mutant TbetaR-II could interfere with TGF-beta binding to TbetaR-I. Inhibition of binding to wild-type TbetaR-II was not observed under these conditions. This is not unexpected since wild-type TbetaR-II can bind TGF-beta1 in the absence of dimerization with TbetaR-I(16, 17) .

Previous in vitro studies have demonstrated that TGF-beta1 inhibits proliferation of many cell types(26, 27) , including endothelial cells(28) . In wild-type or mock transfected glomerular endothelial cells, TGF-beta1 (2 ng/ml) significantly inhibited [^3H]thymidine incorporation, whereas in the transfected endothelial cells carrying TbetaR-II(M), TGF-beta1 stimulated [^3H]thymidine incorporation. This stimulation of [^3H]thymidine incorporation by TGF-beta1 in TbetaR-II(M) transfected endothelial cells could reflect an alternate receptor signaling pathway such as a TbetaR-I-mediated response, not dependent on phosphorylation of TbetaR-I by TbetaR-II. Time course experiments revealed that in transfected cells, exogenous TGF-beta1 stimulated [^3H]thymidine incorporation significantly above the basal rate in 0.5% FBS at 36 and 48 h (Fig. 4B). In comparison, 5% FBS maximally stimulated [^3H]thymidine incorporation between 12 and 24 h (data not shown). These findings suggest that the stimulation may be a secondary rather than a direct mitogenic effect. Our results of stimulation of DNA synthesis are consistent with previous observations that when cells are released from the negative growth regulatory control of TGF-beta1, cell proliferation occurs and the potential for tumorigenesis can emerge(27, 29) .

TGF-beta1 inhibits proliferation by arresting cells in the G(1) phase and thus interrupting progression through the cell cycle. Laiho et al.(30) observed that TGF-beta1 prevented phosphorylation of the retinoblastoma gene product and arrested cells in late G(1) phase of the cell cycle. The underphosphorylated retinoblastoma gene product has growth-suppressive function. When progression through the cell cycle is prevented, cells may remain quiescent or withdraw from the cell cycle and undergo terminal differentiation. Indeed, Zentella et al.(31) showed that TGF-beta1 inhibited cell cycle progression of skeletal myoblasts through the G(1) phase and induced terminal differentiation.

In addition to the inhibition of [^3H]thymidine incorporation in wild-type glomerular endothelial cells, cell detachment was observed. Cells that remained on the plate tended to organize into capillary-like structures, a process previously described by others(22, 32, 33) . A possible explanation for the observed decreased [^3H]thymidine incorporation and cell detachment may be cell cycle arrest and withdrawal, and entry into a suicide program, or apoptosis. In support of this, proto-oncogene c-myc, an important positive regulator of cell growth induced during the G(0)/G(1) phase of cell cycle, can induce apoptosis under conditions of growth arrest, such as presence of a negative growth regulator, TGF-beta1(34) .

Indeed, TGF-beta1 has been shown to inhibit cell proliferation and induce apoptosis in rat hepatocytes in vivo(35) and in rabbit uterine epithelial cells in vitro(36) . We observed in wild-type glomerular endothelial cells, serum deprivation induced apoptosis as shown by genomic DNA fragmentation and associated capillary formation. Exogenous TGF-beta1 treatment accelerated these events. In contrast, genomic DNA fragmentation and associated capillary formation were not observed in transfected endothelial cells expressing TbetaR-II(M), either with serum deprivation or exogenous TGF-beta1 treatment. Since serum deprivation acted as if exogenous TGF-beta1 had been added, and since effects of serum deprivation were not seen in cells carrying the transdominant negative mutant receptor, it is plausible that endogenous TGF-beta1 might mediate capillary morphogenesis with serum deprivation. In support of this hypothesis, we observed that neutralizing antibody to TGF-beta1 abolished both cell detachment and capillary-like tube formation in serum deprived wild-type endothelial cells as well as DNA fragmentation.

In the process of capillary morphogenesis, our studies show that endothelial cells, in response to TGF-beta1, undergo a programmed cell death and detach from the substratum, a phenomenon called anoikis(37) . The term anoikis is derived from the Greek word for homelessness. Anoikis implies that once cells lose contact with underlying matrix, they undergo programmed cell death, thus preventing these detached cells from establishing themselves in another location. Thus, the phenomenon of anoikis is a mechanism for homeostasis that maintain a certain correct cell number in the body by balancing cell production with cell death. Tumor cells escape this regulation by blocking the apoptotic response. Anoikis may also be important in cell positioning. For instance, in the normal maturation of the skin, the cells that are in contact with the basement membrane proliferate and the cells that migrate away from it into the more superficial layers undergo apoptosis (38) .

Since integrins are primarily responsible for cell adhesion to ECM, integrin-mediated signaling has been implicated in controlling apoptosis. Frisch and Francis (37) demonstrated that apoptosis was induced by disruption of the interactions between normal epithelial cells and ECM. In endothelial cells, Meredith et al.(39) showed that cells incubated in suspension and denied interactions with ECM rapidly underwent apoptosis. Furthermore, when endothelial cells were plated on an integrin beta(1) monoclonal antibody, apoptosis was suppressed. Therefore, regulation of apoptosis may be mediated by disruption of cell-matrix interactions and altered cell-cell interactions. Given this body of evidence, it is not unreasonable to propose that our findings of cell detachment and apoptosis may reflect TGF-beta1-mediated matrix degradation. TGF-beta1 induces endothelial cell secretion of PA, which is an enzyme that cleaves the proenzyme plasminogen to form active plasmin. Plasmin is a protease which degrades ECM proteins. Moreover, plasmin activates latent TGF-beta1(12) , and thus providing a mechanism for autoamplification loop. ECM proteolysis by proteases such as plasmin facilitates endothelial cell migration and angiogenesis in vivo(11) . However, even though apoptosis may be mediated by PA-induced matrix degradation by TGF-beta1, this may not be the sole mechanism, in view of the fact that mitogenic growth factors which can prevent apoptosis, such as bFGF, also induces PA.

Angiogenesis is regulated by a number of cytokines in vivo, including bFGF(40) , vascular endothelial growth factor(41) , and TGF-beta1(6, 7) . In our studies, we were able to isolate and delineate the effects of TGF-beta1 in vitro from other angiogenic growth factors. Our findings raise the intriguing possibility that apoptosis is a phenomenon necessary in the process of capillary morphogenesis and that both are dependent on TGF-beta receptor signaling.


FOOTNOTES

*
This work was done during the tenure of an Established Investigatorship from the American Heart Association (to B. B.) and supported by American Heart Association Grant 92010830. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of Physician Scientist Award 5-K12-DK0129809. To whom correspondence and reprint requests should be addressed: Div. of Nephrology, The Johns Hopkins University School of Medicine, Rm. 943, Ross Research Bldg., 720 Rutland Ave., Baltimore, MD 21205. Tel.: 410-614-0064; Fax: 410-955-0485.

(^1)
The abbreviations used are: TGF, transforming growth factor; ECM, extracellular matrix; PA, plasminogen activator; FGF, fibroblast growth factor; aFGF, acidic FGF; bFGF, basic FGF; TbetaR-II, TGF-beta type II receptor; PCR, polymerase chain reaction; FBS, fetal bovine serum; nt, nucleotide; kb, kilobase(s); MOPS, 3-(N-morpholino)propanesulfonic acid; HBSS, Hanks' balanced salt solution.


ACKNOWLEDGEMENTS

We thank Gula Nourjanova for her technical assistance with cell culture. Transmission electron microscopy was performed by Paragon Biotech Inc.


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