1 Pulmonary Center, Boston University School of Medicine, Boston 02118 and 2 Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139; and 3 Amgen Corporation, Thousand Oaks, California 91320
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ABSTRACT |
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We examined Gas 6-Axl interactions in human pulmonary artery endothelial cells (HPAEC) and in Axl-transduced HPAEC to test Gas 6 function during endothelial cell survival. We identified the 5.0-kb Axl, 4.2-kb Rse, and 2.6-kb Gas 6 mRNAs in HPAEC. Immunoprecipitation and Western blotting confirmed the presence of these proteins. Gas 6 is present in cell-associated and secreted fractions of growth-arrested HPAEC, independent of cell density. In addition, the Axl receptor is constitutively phosphorylated in growth-arrested cultures, and exogenous Gas 6 enhanced Axl phosphorylation threefold. Gas 6 added to growth-arrested HPAEC resulted in a significant increase in cell number (1.5 nM Gas 6 increased cell number 35%). Flow cytometry revealed that Gas 6 treatment resulted in 28% fewer apoptosing cells. Transduction of a full-length Axl cDNA into HPAEC resulted in 54% fewer apoptosing cells after Gas 6 treatment. Collectively, the data demonstrate antiapoptotic activities for Gas 6 in HPAEC and suggest that Gas 6 signaling may be relevant to endothelial cell survival in the quiescent environment of the vessel wall.
Rse; apoptosis; signal transduction
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INTRODUCTION |
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THE QUIESCENT, NONTHROMBOGENIC phenotype of the vascular endothelium is essential to hemostasis. Under normal conditions, endothelial cell turnover in the vessel wall is relatively low compared with other somatic cell types. However, under certain pathological conditions (e.g., atherosclerosis, pulmonary hypertension, and thrombotic thrombocytopenic purpura), endothelial cell proliferation occurs (9, 18, 20). Endothelial proliferation is associated with increased apoptosis, which in turn generates a prothrombotic phenotype (5). Dysregulation of the endothelial cell phenotype implies that endogenous signaling pathways exist to control cell survival and thus maintain hemostasis.
Gas 6, the product of the growth arrest-specific gene 6, is a soluble factor implicated in the regulation of multiple cellular functions, including growth, survival, adhesion, and chemotaxis (2, 10, 12, 30, 31). Gas 6 signaling is transduced via ligation with three known receptor tyrosine kinases (RTK), Axl (also UFO and Ark) (33), Rse (also Sky, Brt, Tyro-3) (19), and Mer (29). In addition, Gas 6 function is cell-type specific. For example, Gas 6-Axl interactions result in mitogenic and antiapoptotic responses in NIH/3T3 fibroblasts and vascular smooth muscle cells (12, 13, 30, 31). However, Gas 6-Axl interactions mediate cellular aggregation in the murine myeloid 32D cells but show none of the mitogenic or survival activities found in other cell types (26).
There is increasing evidence to suggest that Gas 6 regulates important aspects of vessel wall function. In vascular smooth muscle cells grown in culture, Gas 6 is a growth-potentiating factor for the G protein-coupled receptor agonists thrombin and angiotensin II (30). Gas 6 also prevents growth arrest-induced death and promotes chemotaxis in vascular smooth muscle cells (10, 31). Gas 6 is expressed in vascular endothelial cells (24, 37) and inhibits granulocyte adhesion to activated endothelial cells in vitro (2). In human umbilical vein endothelial cells (HUVEC), Gas 6 promotes cellular viability in the absence of growth factors (34). In vivo, balloon catheterization of rat carotid arteries induces Gas 6 expression within the neointima (27), indicating that Gas 6 is positioned to regulate the vascular response to injury.
Recent studies have begun to address the function for each of the Gas 6 receptors. For example, mice containing targeted deletions of any one
receptor, Axl, Rse, or Mer, reveal no overt phenotype (22). However, deletion of all three receptors results in
viable animals with multiple abnormalities, the most prominent being male sterility, but noted among the various phenotypes was increased apoptosis in the vessel wall (22). Overexpression
of the Axl receptor in cells of myeloid lineages results in a phenotype
similar to non-insulin-dependent diabetes mellitus, likely the result of alterations in tumor necrosis factor- production
(1). In vivo, the Axl receptor has been identified in
vascular smooth muscle cells and capillary endothelial cells of
synovial tissue obtained from patients with rheumatoid arthritis
(34). Collectively, these results suggest that Gas 6-Axl
interactions may be cell and tissue specific.
We asked whether Gas 6 regulates endothelial cell survival at growth arrest. To address this question, we characterized Axl, Rse, and Gas 6 expression in human pulmonary artery endothelial cells (HPAEC). We identified Axl and Rse expression in HPAEC and found that Axl is autophosphorylated in growth-arrested cells and exogenous Gas 6 enhances Axl phosphorylation 3.5-fold. We examined cell-associated and secreted Gas 6 expression and found that growth arrest induces Gas 6 secretion into the medium independent of cell density. Our data also demonstrate that exogenous Gas 6 promotes cellular viability but is not a growth-potentiating factor for the G protein-coupled agonist thrombin. Finally, Gas 6 inhibits programmed cell death in endothelial cells, and this response is enhanced in HPAEC that overexpress the Axl receptor. Thus the results of our studies suggest that Gas 6 functions as an antiapoptotic factor in pulmonary vascular endothelial cells through ligation with the high-affinity Axl receptor tyrosine kinase.
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METHODS |
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Cells and culture conditions. HPAEC (Clonetics) were grown to confluence in growth medium (EBM, Clonetics) containing 1.38 µM hydrocortisone, 3 pM recombinant human epidermal growth factor (both from Clonetics), 10 µg/ml endothelial cell growth supplement (Sigma), 10,000 U/ml penicillin and streptomycin (Sigma) and supplemented with 10% fetal bovine serum (FBS, HyClone). At confluence, HPAEC were growth arrested by replacing serum-containing medium with serum-free medium supplemented with hydrocortisone, penicillin, and streptomycin and with or without recombinant human Gas 6 (Amgen), or human protein S (Enzyme Research Laboratories, South Bend, IN).
For proliferation assays, HPAEC were plated at 25 × 103 cells/cm2 and incubated for 24 h in growth medium. Growth medium was removed, cells were washed 2× with PBS, and test medium containing 0.5% FBS supplemented with recombinant human Gas 6 (1-6 nM), human thrombin (0.1-10.0 U/ml, Enzyme Research Laboratories), or Gas 6 (1.5 nM) plus thrombin (0.1-10.0 U/ml) was added. Medium was exchanged daily, cells were trypsinized, and cell number was determined electronically (Coulter Counter, Hialeigh, FL).PCR and Northern blot analysis. Total RNA was isolated from HPAEC and C57/Black mouse lung using the TRIzol reagent (GIBCO BRL) or the guanidinium thiocyanate method (7) and measured by optical density (260- to 280-nm absorbance ratio). RNA integrity was checked by electrophoresis through formaldehyde-agarose gels stained with 25 µM ethidium bromide. Poly(A)+ RNA was purified from total RNA by oligo(dT) cellulose column chromatography (GIBCO BRL).
cDNA probes for Northern blot analysis were generated using RT-PCR with total RNA isolated from either U937 cells (for Axl sequences) or HPAEC. The primers were 5'-GCAGGCTGAAGAAAGTCCCTTCG and 3'-GCTGGCTGACCACTATCCAGTC for Axl; 5'-CTGCAGTGTGGAGGGGATGGAGG and 3'-GCCACACTGGCTGGGAGATCTCGG for Rse; 5'-CAATCTCTGTTGAGGAGCTGG and 3'-GACCACGTGCTCTTGGCCGTC for Gas 6, and 5'-CCTTCCTGGGCATGGAGTCCTG and 3'-GGAGCAATGATCTTGATCTTC forHPAEC metabolic labeling and Rse immunoprecipitation. Rse receptor biosynthesis was determined in HPAEC by metabolically labeling confluent cultures of cells with [35S]methionine (ICN) for 4 h after a 1-h incubation in methionine-free medium (GIBCO BRL). HPAEC extracts were prepared to enrich for membrane and cytoplasmic proteins and exclude cell nuclei from the preparation. HPAEC extracts were prepared by standard techniques. Protein concentration was determined by the Bio-Rad protein assay, and equal concentrations of cellular extracts were used for immunoprecipitation. Rse was immunoprecipitated from extracts with a polyclonal antibody raised against the amino terminus of Rse and defined here as anti-Rse IgG (originally anti-Sky IgG, the generous gift of Dr. Kensako Mizuno, Kyushu University, Fukuoka, Japan) (35). Rabbit anti-Rse or normal rabbit serum immunoprecipitates were collected on protein A (A/G) agarose (Santa Cruz Biotechnology), and bound proteins, eluted with SDS buffer, were electrophoresed on 7.5% polyacrylamide gels and prepared for fluorography.
Immunoprecipitation and Western blot analysis of Axl and Gas 6. Cell lysates for cell-associated Axl and Gas 6 were prepared as described for biosynthetic labeling but without radioisotope. Gas 6 was identified in HPAEC-conditioned medium by collecting medium after 2, 4, and 5 days of serum depletion. Conditioned medium was concentrated 40-fold by centrifugation in a concentrator fitted with a YM-30 membrane (Amicon). Concentrated medium and cell lysates were prepared for electrophoresis through 10% polyacrylamide gels, transferred to nitrocellulose membranes (Schleicher and Schuell), and detected by successive incubations with anti-Gas 6 antibody (Amgen), anti-rabbit IgG labeled with horseradish peroxidase (Santa Cruz Biotechnology), and enhanced chemiluminescence (Pierce).
Western blot analysis for Axl detection was as described for Gas 6 except that the primary antibody was an affinity-purified, rabbit anti-Axl IgG (Amgen). Axl was also immunoprecipitated from cell lysates using a goat anti-Axl IgG (Santa Cruz Biotechnology). Alternatively, tyrosine-phosphorylated Axl receptor was immunoprecipitated from confluent HPAEC cultures serum deprived for 24 h and then left untreated or treated with Gas 6, protein S, or FBS for 5 min. Tyrosine-phosphorylated Axl was immunoprecipitated from cell lysates with the monoclonal anti-phosphotyrosine IgG clone 4G10 (Upstate Biotechnology). Blotted proteins were detected using either the rabbit anti-Axl IgG from Amgen or a second rabbit anti-Axl IgG (the generous gift of Dr. E. Liu, University of North Carolina, Chapel Hill, NC).Apoptosis assays. DNA fragmentation was determined as described (16). Briefly, cells were grown to confluence in serum-containing medium, at which time serum was removed and cells were cultured for 72-120 h in serum-free medium with one medium exchange at 48 h. In all three apoptosis assays described below, each experiment included a negative and a positive control. Cells maintained in serum-containing medium were used as a negative control, and cells maintained in serum-containing medium supplemented with 1 µM staurosporine (Sigma), a protein kinase inhibitor that induces apoptosis, were used as a positive control (16). DNA was isolated from both floating cells, which were pelleted from test medium, and attached cells. The samples were electrophoresed through a 1.8% agarose gel containing 25 µM ethidium bromide.
For Hoechst staining, cells were grown to confluence on glass coverslips, serum deprived, and treated with various factors. Cells were fixed on days 3 and 4 and stained simultaneously by inverting glass coverslips onto a drop of staining solution containing 4% formaldehyde, 0.6% Nonidet P-40, and 18.7 µM Hoechst 33258 (Sigma) in PBS at room temperature for 30 min (28). Fifty cells from three fields were counted for each condition in duplicate. Cells were scored as apoptotic if they displayed a highly condensed and fragmented nucleus. Annexin V-positive- and propidium iodide-negative-stained HPAEC were detected by flow cytometry. HPAEC were grown as described previously except that cells were harvested on days 2 and 3 of serum-free culture. Cells were trypsinized, counted, and costained with fluorescein-conjugated annexin V and propidium iodide (R&D Systems). Cells were analyzed by flow cytometry (Becton Dickinson) and quantitated using Cell Quest software.Retroviral transduction of HPAEC with Axl constructs. A full-length cDNA encoding the Axl gene (gift of Dr. E. T. Liu) was subcloned into the EcoRI site of pMSCVpac (15). The Axl-retroviral construct was transfected into Phoenix cells to generate retroviral supernatants as previously described (6). Transduced HPAEC were selected by puromycin resistance and analyzed between passages 6 and 9.
Statistics. The data are expressed as means ± SD. Analysis of variance was carried out using the two-factor ANOVA. Statistical analysis comparing cells maintained in serum-free medium in the presence or absence of Gas 6 was conducted using Student's t-test. Differences were significant at P < 0.05.
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RESULTS |
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Vascular endothelial cells express the RTK Axl and Rse.
We amplified a 193-bp Axl fragment, a 208-bp Rse fragment, and a 589-bp
Gas 6 fragment from HPAEC by RT-PCR. Axl and Rse expression were
confirmed by Northern blot analysis of HPAEC RNA (Fig.
1A). Northern blotting
revealed the presence of a major band migrating at 4.2 kb for Rse mRNA.
The Axl probe identified a single major transcript at 5 kb and a second
transcript just visible at 3.4 kb. It is noteworthy that in transformed
and tumorigenic cells, both the 5.0- and 3.4-kb Axl transcripts are
represented equally (33). Quantitation of the 5.0-kb Axl
and 4.2-kb Rse mRNAs compared with -actin from the same cell sample
demonstrates that Rse mRNA is 2.2 times more abundant than Axl mRNA in
HPAEC.
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Gas 6 is expressed in pulmonary endothelial cells in culture and in whole lung. Previous investigations identified the ligand Gas 6 in many cells and organs, but particularly high levels were identified in HUVEC and bovine aortic endothelial cells and in human and murine lungs (2, 24, 37). We examined Gas 6 expression in endothelial cells isolated from human pulmonary artery and tested whether our human cDNA Gas 6 probe also hybridizes to murine Gas 6. Northern blot analysis of Gas 6 transcripts (Fig. 1B) revealed the presence of a single major band migrating at ~2.6 kb in HPAEC and in whole lung extracts from C57/Black mice.
Immunoprecipitation and Western blot analysis of Axl and Rse.
We examined cell lysates for the presence of the Axl and Rse receptors.
Using three different Axl antibodies, we detected several forms of the
immunoreactive Axl RTK. For example, with a rabbit polyclonal Axl
antibody, the Axl receptor appears as a single major band with a
relative mobility of 125 kDa and a second minor band with a relative
mobility of 104 kDa (Fig. 2, lane
1). When Axl was immunoprecipitated using a goat polyclonal Axl
antibody and then blotted with a second rabbit Axl antibody, the Axl
receptor is seen as a doublet with a relative mobility of 140 and 110 kDa (Fig. 2, lane 3). A similar pattern was observed when cells were metabolically labeled and immunoprecipitated with these
same two immunoreagents (not shown). Anti-Axl antibody premixed with a
fivefold molar excess of an Axl-Fc fusion molecule failed to recognize
all forms of the Axl receptor. Several forms of the Axl receptor, which
correspond in relative mobility to those shown in Fig. 2, have been
described in other cell types as the precursor (p104) Axl polypeptide
and partial (p120) and fully glycosylated (p140) forms of Axl
(32).
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Growth arrest induces Gas 6 secretion independent of cell density.
Because Gas 6 expression is associated with growth arrest, we asked
whether cell density affects Gas 6 expression and secretion. Therefore,
we examined HPAEC cultures under sparse (8 × 103
cells/cm2) and confluent (32 × 103
cells/cm2) cell densities for cell-associated and soluble
forms of Gas 6 (Fig. 4). We found that
HPAEC maintained in culture under either serum-free or low-serum
conditions (0.5% FBS) expressed Gas 6 regardless of cell density.
Densitometric analysis from four independent experiments revealed that
1.1 ± 0.3 ng Gas 6 per 1 × 106 cells
accumulates in the conditioned medium of confluent cultures. Densitometric analysis from two experiments revealed that 1.4 ± 0.1 ng Gas 6 per 1 × 106 cells accumulates in the
conditioned medium of sparse cultures. The cell-associated forms of Gas
6 that were present at 2 and 4 days of serum deprivation correspond to
the mature polypeptide, with a relative mobility of ~70 kDa, a higher
molecular mass form at 110 kDa (probably a dimer), and a third
immunoreactive species at 50 kDa (likely an intracellular precursor or
degradation product) (Fig. 4). The 70-kDa form is the predominant form
present in the conditioned medium of both sparse and confluent
serum-deprived HPAEC at both 2 (data not shown) and 4 days of culture.
The anti-Gas 6 antibody readily detects between 0.2 and 2 ng of the
recombinant human Gas 6 (Fig. 4, lanes 5-7) but does
not cross-react with 20 ng of recombinant human protein S (data not
shown).
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Axl receptor is constitutively phosphorylated in HPAEC.
The expression and secretion of Gas 6 in HPAEC led us to ask whether
endogenous Gas 6 binds and activates its receptors. We found that the
Axl receptor is phosphorylated in untreated cells (Fig.
5, lane 1). Moreover, the
addition of exogenous Gas 6 (Fig. 5, lane 2) but not of
serum (Fig. 5, lane 3) or protein S (data not shown)
enhances Axl phosphorylation 3.5-fold. Phosphorylated Rse receptor was
not detected (data not shown).
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Gas 6 effects on HPAEC proliferation.
In cell types that express Gas 6 plus both the Axl and Rse receptors, a
mitogenic and/or antiapoptotic function for Gas 6 has been
identified (10, 12, 21, 30, 31). Thus the presence of both
the ligand Gas 6 and the two receptors Axl and Rse suggested that Gas 6 has proliferative and antiapoptotic properties in HPAEC. Our data
show that the addition of recombinant human Gas 6 to HPAEC cultures
results in a statistically significant increase in cell number (Fig.
6). The maximal increase in cell number
occurred with exposure to 1.5 nM Gas 6 (100 ng/ml), resulting in a 36% increase in cell number. Higher concentrations of Gas 6, i.e., 3.0 and
6.0 nM (200 and 400 ng/ml) did not enhance the proliferative response
further. In contrast, exposure to 10% FBS caused a 180% increase in
cell number. The HPAEC response to Gas 6 stimulation is similar to
previous findings by other investigators analyzing nonendothelial cell
types (12, 21, 30).
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Apoptosis in HPAEC. Previous studies have identified antiapoptotic functions for Gas 6 in nonendothelial cells maintained under serum-free conditions (3, 12, 25, 31). HPAEC, like other endothelial cells, will apoptose if deprived of serum and growth factors. Therefore, we optimized the culture conditions to promote apoptosis in HPAEC before testing whether Gas 6 affects HPAEC survival.
In confluent cultures of HPAEC, DNA fragmentation is easily detected in cells maintained under serum-free culture conditions (Fig. 7, lanes 4 and 5) but not in cells grown in serum-containing medium (Fig. 7, lane 3). DNA fragmentation induced by staurosporine treatment is shown for comparison (Fig. 7, lane 2). These data confirm that serum-free culture conditions induce programmed cell death in HPAEC.
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Axl mediates Gas 6 antiapoptotic function.
The Axl receptor exhibits the highest affinity for Gas 6 compared with
Rse and Mer (29). Therefore, to test Gas 6-receptor interactions during HPAEC survival, we generated Axl-transduced HPAEC
using a full-length Axl cDNA (Axlwt). We quantified Axl
expression in transduced and nontransduced HPAEC by Western blot
analysis and found a twofold increase in ectopic Axl expression (a
representative blot is shown in Fig. 9).
We used the Axlwt HPAEC to test the effect of Gas 6 on
cellular survival. We found that Gas 6 decreases the number of
apoptotic Axlwt HPAEC by 54% (Gas 6 5%, control 11%,
P < 0.05) as shown in Fig. 10.
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DISCUSSION |
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The vascular endothelium is a monolayer of contact-inhibited, growth-arrested cells lining the luminal surface of the mature blood vessel wall. The molecular mechanisms contributing to the unique longevity of endothelial cells remain undefined. Gas 6 is a mitogen and survival factor for various cell types, transducing signals through its receptors Axl and Rse. To determine whether the Gas 6 signaling pathway is a potential mediator of endothelial cell survival at growth arrest, we examined the expression of Gas 6 and the receptors Axl and Rse and characterized the proliferative and antiapoptotic activities for Gas 6 in pulmonary endothelial cells in vitro.
We found that HPAEC simultaneously express both the Axl and Rse RTK. Axl and Rse receptors are detected from either total RNA or poly(A)+ RNA, with a twofold higher steady-state level of Rse mRNA. Conversely, Western blot analysis indicates the Axl receptor is more highly expressed than the Rse receptor; this is most likely a reflection of differing antibody affinities rather than true differences in protein expression levels.
Gas 6 was originally identified as one of several molecules whose expression negatively correlates with cellular proliferation and serum depletion (24, 36). We questioned whether growth arrest by serum deprivation differs from growth arrest by contact inhibition in regard to Gas 6 expression and secretion. Measurement of Gas 6 levels in sparse vs. confluent cell cultures under serum-free conditions demonstrated no significant difference in cell-associated or soluble Gas 6 between the two cell densities, indicating that serum deprivation induces Gas 6 expression in vitro and contact-inhibited growth does not further augment Gas 6 expression. These results are in contrast to a recent study in which soluble Gas 6 was detected in the cell-associated fraction but not in the conditioned medium of HUVEC, suggesting that secreted Gas 6 may be completely bound to cell surface receptors (2). The difference between our findings and those of Avanzi et al. (2) may be due to the detection assays (i.e., Western blot vs. ELISA, respectively) or to the heterogeneity of endothelial cells isolated from different vascular beds. However, our data confirm a previous report demonstrating that Gas 6 is released into the conditioned medium from bovine aortic endothelial cells (37). Our results demonstrate that HPAEC growth arrested by either contact inhibition or serum depletion secrete Gas 6, which remains in a soluble form in the conditioned medium.
Previous studies revealed that Gas 6 is a growth-potentiating factor for the G protein-coupled receptor agonists such as thrombin and angiotensin II (21, 27, 30). Furthermore, it was shown that Gas 6 mitogenic activity is separable from Gas 6 antiapoptotic function; Gas 6 induces entry into the S phase of the cell cycle in the presence of low serum but is an antiapoptotic factor in the complete absence of serum (and growth factors) (3, 12). We found that at sparse cell densities in low-serum-containing medium, there is a statistically significant increase in HPAEC cell number in the presence of increasing concentrations of exogenous Gas 6. However, this proliferative response observed after 5 days of Gas 6 treatment may represent increased cell viability and not entry into S phase. The small increase in cell number (~5-7% per day) makes it difficult to test this hypothesis by standard techniques (e.g., measurement of [3H]thymidine incorporation or 5-bromo-2'-deoxyuridine). In addition, we were unable to detect a Gas 6 growth-potentiating effect in the presence of thrombin. This finding supports the results of a previous study demonstrating that thrombin has a differential effect on endothelial cells isolated from distinct vascular beds and that long exposures to thrombin inhibit endothelial cell mitogenesis regardless of endothelial cell type (39). Our findings support the supposition that Gas 6 increases cell viability rather than stimulating mitosis in HPAEC.
Our data demonstrate that Gas 6 has an antiapoptotic function for HPAEC. Although the total population of HPAEC undergoing apoptosis on day 2 (or day 3) of serum-free culture is relatively small (14% of total cells), the small number of apoptotic endothelial cells is in agreement with studies conducted on NIH/3T3 cells in which Gas 6 treatment decreased the number of apoptotic cells from ~11 to 4% (3). Hoechst staining revealed similar numbers of apoptosing HPAEC on days 3 and 4 of serum-free culture. Furthermore, overexpression of the full-length Axl cDNA results in over a twofold increase in Axl protein levels and a corresponding decrease in the percentage of apoptotic cells. The results of studies examining apoptosis in the vessel wall in atherosclerotic lesions and regions of restenosis show a similar percentage of apoptotic cells, 2-30%, as detected by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) and Hoechst staining (11, 14, 17, 23). Moreover, in a single study addressing tumor angiogenesis, the complete removal of vascular endothelial growth factor resulted in the detachment of endothelial cells and subsequent tumor regression; however, only occasional TUNEL-positive endothelial cells could be identified within the blood vessel wall at any single time point (4). Thus the low numbers of HPAEC undergoing apoptosis in our studies are consistent with the results of studies performed on whole vessels. Collectively, our functional studies reveal that Gas 6 causes an increase in viability and a decrease in apoptosis, suggesting that Gas 6 is a survival factor for HPAEC.
Our studies indicate that the Axl receptor is constitutively phosphorylated and the addition of exogenous Gas 6, but not of serum or protein S, increases Axl phosphorylation 3.5-fold. These data indicate that Axl phosphorylation occurs via Gas 6 ligation. We also detected a 54-kDa protein that coprecipitates with the metabolically labeled Rse, which may be a member of the Src family of kinases (Fig. 3) (38), and a higher molecular mass band that may be a Rse-Src complex or a Rse-Gas 6 complex. These results support the supposition that Gas 6 promotes HPAEC survival through constitutive ligation with Axl and/or the Rse RTK.
It remains unknown whether Gas 6 interacts with both receptors or whether Axl and Rse can form heterodimers following ligand binding. The cell types identified in which Gas 6 is a growth-potentiating and a survival factor express one or both receptors (Axl and Rse) in addition to the ligand (Gas 6) (12, 25, 31). These data support the hypothesis that the complex biology of the Gas 6 signaling pathway is regulated by cell type-specific expression of the Gas 6 receptors.
Our measurements indicate that picomolar concentrations of Gas 6 are synthesized by HPAEC under serum-free conditions. However, nanomolar concentrations are required for a cellular response in vitro, both in our studies and in independent studies of several cell types (3, 10, 12, 21, 25, 29). There are at least two possibilities that could explain this difference. Endogenous Gas 6-Axl interactions may not promote HPAEC survival. We think this is unlikely because gene deletion studies indicate that Axl-deficient embryonic fibroblasts are more susceptible to apoptosis after serum withdrawal and are refractory to exogenous Gas 6 treatment compared with Axl wild-type embryonic fibroblasts (3). Moreover, mice null mutant for all three Gas 6 receptors display increased TUNEL-positive cells in the vessel wall (22). We favor the supposition that the amount of endogenous Gas 6 may be limiting under our defined experimental conditions, and, therefore, endogenous Gas 6 cannot completely protect from apoptosis after serum withdrawal. This scenario would explain why we do not observe an increased cell survival in the Axlwt HPAEC on serum withdrawal but do observe a twofold increase in survival after addition of exogenous Gas 6.
Programmed cell death is an integral component of the vascular response to injury. On the one hand, apoptosis in vascular smooth muscle cells counters the exuberant cellular proliferation that leads to intimal thickening (8, 18). On the other hand, apoptosis in vascular endothelium contributes to pathogenesis by promoting intravascular coagulation activation (5). Apoptosis also has a role in the vascular remodeling associated with tumor angiogenesis (4). Thus a balance between cell growth and cell death may be required for vascular remodeling. In this report, we characterized the expression and function of the Gas 6 signaling pathway in pulmonary endothelium in vitro. Further elucidation of this pathway will reveal whether Gas 6 functions in maintaining the equilibrium between cell growth and survival in lung endothelium in vivo.
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ACKNOWLEDGEMENTS |
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We thank Dr. S. M. Notarnicola for a critical reading of the manuscript, Dr. R. D. Rosenberg for the transduction technology, and Laura Morgenthau for expert technical assistance.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grant HL-45537 to H. W. Farber.
Address for reprint requests and other correspondence: A. M. Healy, Pulmonary Center R-3, Boston Univ. School of Medicine, 715 Albany St., Boston, MA 02118 (ahealy{at}bupula.bu.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.
Received 11 October 2000; accepted in final form 26 December 2000.
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