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From the Center for Molecular Medicine, Maine Medical Center Research Institute, Scarborough, Maine and Center for the Biophysical Sciences, University of Maine, Orono, Maine
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several of the components of the Jagged/Notch gene families have been described to be expressed in the cardiovasculature system. Notch49 and Dll410 seem to be generally restricted to endothelial cells during embryogenesis and in the adult whereas Jagged1 and Notch1 are also expressed in the heart and vasculature, respectively.11,12 Studies of human tissues demonstrate that Notch3 expression is restricted to vascular smooth muscle cells in adult tissues.7 Murine genetic studies generating null mutations of the Jagged/Notch genes have indicated that the vascular system seems to be developmentally reliant on intact Notch signaling pathways. Jagged1 null mutant mice display profound defects in the vasculature,13 and a Notch1 null or processing-deficient allele,14 as well as Notch1/4 double mutants exhibit defects in vascular remodeling and angiogenesis.15 In addition, a hypomorphic Notch2 mutation causes defects in the myocardium and eye vasculature of homozygous mice.16 These observations in combination with the vascular defects seen in the human conditions in which Notch3 signaling may be impaired suggest that responses to cardiovascular injury may also be regulated by Jagged/Notch gene family members.
We have previously characterized an in vitro system of stably transfected NIH3T3 cells expressing a soluble form of Jagged1.17 The cDNAs for both the transmembrane form of Jagged1 as well as a variant lacking the transmembrane and intracellular regions were cloned from human umbilical vein endothelial cells undergoing in vitro angiogenesis,18 suggesting that cells may be able to produce variants of the Notch ligands. Previous studies have shown that this soluble form of Jagged1 promotes morphological changes including a branching phenotype, inhibits the expression of collagen type I, abolishes contact inhibition of cell growth in vitro, and stimulates angiogenesis in a chick chorioallantoic membrane assay.17 We have further characterized the effects of the soluble Jagged1 protein with regard to characteristics that are important in vascular cell remodeling, namely cell migration and interaction with neighboring cells and the underlying matrix, and report significant differences in cell behavior in the presence of the soluble Jagged1 protein.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Vascular injury using balloon catheter denudation of rat carotid arteries and aortae was performed as described19 with the approval of the Institutional Animal Care and Use Committee. En face specimens were prepared for in situ hybridization as previously described.20
In Situ Hybridization
Full-length cDNA clones for Jagged1, Jagged2, and Notch1 through 4 were a generous gift of G. Weinmaster (UCLA School of Medicine, Los Angeles, CA), and were used for the generation of 35S-UTP-labeled sense and antisense riboprobes. Tissue sections or en face preparations were treated with 20 of µg/ml proteinase K (Sigma Chemical Co., St. Louis, MO) before hybridization with 2 x 104 dpm/µl probe overnight at 50°C. Hybridized slides were treated with 20 µg/ml of RNase A, then washed in a 50% formamide, 2x standard saline citrate, 20 mmol/L 2-mercaptoethanol buffer at 55 to 60°C.
Cell Lines and Tissue Culture
The stable soluble Jagged1 NIH3T3 clones and the vector controls have been characterized previously,17 and were maintained in Dulbecco’s Modified Eagle Medium with 10% calf serum, 2 mmol/L L-glutamine, 50 µg/ml gentamicin, and 0.3 mg/ml G418 at 37°C with 5% CO2. For experiments, monolayers were removed with trypsin, and viable cells determined by the exclusion of trypan blue. Cells were counted and used for assays as described below.
Migration Assays
Cells were harvested by brief trypsin digestion and seeded at a
density of 15,000 cells per cm2
on a six-well
plate, allowed to grow to a confluent monolayer (24 hours), and then a
scratch wound with a Teflon comb (2.2 mm in diameter) was made the
length of the dish as described.21
After the scratch, the
wells were rinsed with phosphate-buffered saline (PBS) to remove
detached cells and then fed with growth medium. For studies of cell
interaction with the matrix, plates were first coated with either PBS
as a control or fibronectin17
at 10 µg/ml for 30 minutes
before cell seeding. The peptide integrin inhibitor SM256 (DuPont
Pharmaceuticals, Wilmington, DE) displays a high affinity and
specificity for the 
vß3 integrin, although it can also inhibit
GPIIb/IIIa, 5ß1, and vß5 at higher
concentrations.22
In assays in which inhibitor was used,
SM256 was added with the cell suspensions at the given concentrations.
Denuded area in µm2
was evaluated using
computer image analysis (NIH Image) at 24-hour intervals until total
closure of the denuded area was accomplished.
Immunostaining
For immunofluorescence staining, cells were plated on glass coverslips and fixed 24 hours later with 4% paraformaldehyde in PBS. Fixed cells were blocked for 1 hour in blocking buffer (PBS, 0.1% Tween 20, 0.1% Triton X-100, 5% bovine serum albumin), incubated for 1 hour with primary antibodies (1 µg/ml in blocking buffer), washed with PBS, stained for 30 minutes with secondary fluorochrome-conjugated antibodies (0.1 µg/ml in blocking buffer), washed with PBS, and embedded in 50% glycerol solution. We used monoclonal anti-vinculin antibodies (Sigma Chemical Co.), monoclonal anti-phosphotyrosine antibodies (Upstate Biotechnology, Lake Placid, NY), monoclonal anti-ß-catenin antibodies (Transduction Laboratories, Lexington, KY) and polyclonal anti-pan-cadherin antibodies (Sigma Chemical Co.). As secondary antibodies we used anti-mouse IgG fluorescein isothiocyanate- or CY3-conjugated goat antibodies (Sigma Chemical Co.). Stained cells were examined by fluorescence microscopy and confocal fluorescence microscopy (Leica TCS SP confocal microscope).
Immunoprecipitation and Immunoblotting
For detection of the soluble Jagged1 protein in cells, control or soluble Jagged1 transfectants were metabolically labeled with 35S-met/cys, and immunoprecipitation of cell lysates or conditioned medium to detect the myc tag was performed as previously published.17 For Western blot analysis of Jagged1 protein in tissue lysates, equal amounts of protein from each sample were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the goat polyclonal anti-Jagged antibody (SC-6011; Santa Cruz Biotechnology, Santa Cruz, CA) was used at a 1:500 dilution, and detected as described below. To study the activation of focal adhesion kinase (FAK), soluble Jagged1 transfectants and control vector-transfected cells were scraped in cold PBS containing 1 mmol/L sodium orthovanadate 2 hours and 24 hours after plating on plastic tissue culture dishes, and collected by centrifugation. Cell pellets were lysed in 0.5 ml of cold lysis buffer (20 mmol/L Tris, pH 7.5, containing 300 mmol/L sucrose, 60 mmol/L KCl, 15 mmol/L NaCl, 5% glycerol, 2 mmol/L ethylenediaminetetraacetic acid, 1% Triton X-100, 1 mmol/L phenylmethyl sulfonyl fluoride, 2 µg/ml aprotinin, 2 µg/ml leupeptin, 0.2% deoxycholate, and 1 mmol/L sodium vanadate), and the lysates were clarified by centrifugation at 4°C. Lysates were rotated at 4°C for 1 hour with 1 µg/ml rabbit anti-FAK antibodies (Sigma Chemical Co.) followed by the addition of protein A Sepharose (Pharmacia Biotech, Piscataway, NJ) and further rotation for 1 hour. The antibody complexes were washed three times with lysis buffer, and the immunoprecipitated FAK was eluted in 50 µl SDS-PAGE sample buffer, resolved by 7.5% SDS-PAGE, transferred to Hybond C membrane (Amersham, Arlington Heights, IL) and blotted with the monoclonal anti-phosphotyrosine monoclonal antibody (Upstate Biotechnology, Lake Placid, NY). Phosphorylated FAK was visualized using a horseradish peroxidase-conjugated goat anti-mouse IgG antibody (BioRad, Richmond, CA) and the ECL system (Amersham). The FAK blots were stripped of the anti-phosphotyrosine antibodies using standard stripping buffer,23 reblotted with the anti-FAK antibodies, and FAK visualized using peroxidase-conjugated goat anti-rabbit IgG antibody (BioRad) and the ECL system (Amersham).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Because Jagged/Notch signaling has been implicated in control of
blood vessel morphogenesis during embryogenesis13,24
as
well as during angiogenesis,17,18
we were interested in
analyzing expression of these genes during large vessel repair. Using
the model of endothelial denudation in rat carotid arteries and
aortae25
we performed in situ hybridization to
compare the expression of these genes in normal, uninjured endothelium
versus endothelial cells and smooth muscle cells responding
to injury. Although the ligands Jagged1 and
Jagged2 seemed to exhibit some levels of expression in
normal endothelium, the expression of both genes was dramatically
enhanced after endothelial denudation in the regenerating endothelial
cells. This expression was seen predominantly in the migrating front of
endothelial cells for Jagged1, and more diffusely for
Jagged2 (Figure 1)
. The high
levels of expression of both were maintained during the time period in
which cells were actively migrating and proliferating, but were
diminished at 4 weeks after injury, when cell proliferation and
migration have ceased.19,26,27
Likewise, we also observed
that smooth muscle cells after vascular injury had increased expression
levels of both Jagged1 and 2, in a time course
similar to that of endothelial cells (Figure 1
and Table 1
). However, compared to endothelial
cells, smooth muscle cells exhibited much less Jagged1
expression, whereas levels of the Jagged2 transcript were
high in injured smooth muscle cells (Table 1)
. By Western blot analysis
of tissue lysates from uninjured or denuded carotid arteries, we found
Jagged1 protein present in normal vessels (Figure 1I)
, consistent with
the in situ hybridization showing transcript in normal
endothelium (Figure 1B)
. However, protein levels were increased 7 days
after balloon catheter denudation in carotid arteries (Figure 1I)
. As
the carotid artery lysates were from denuded portions of the injured
vessel, the cell population making up the sample was primarily injured
smooth muscle cells (Figure 1G)
, without the contribution of
regenerating endothelium. We also analyzed expression of the receptors
Notch1 through 4 in a comparable manner. In
uninjured endothelium, Notch1 was expressed constitutively,
whereas Notch2, Notch3, and Notch4
exhibited low levels of expression, strikingly similar to background
(Figure 2
and Table 1
). Although the
levels of these Notch genes, particularly Notch2
through 4 were increased in regenerating endothelial cells
(Table 1)
, endothelial expression was modest compared to the induction
in injured intimal smooth muscle cells (Figure 3)
. One interesting observation was that
smooth muscle cell expression of both Notch3 and
Notch4 seemed to be regulated by the presence of endothelial
cells. Although intimal smooth muscle cells in denuded areas expressed
increased levels of the Notch3 and 4 transcripts
(Figure 3, C and D)
, their expression was significantly up-regulated in
areas abutting the regenerating endothelial wound edge (Figure 3; E to
H
). This suggests that interaction of endothelial cells with smooth
muscle cells during vascular repair may contribute to the regulation of
the levels of Notch receptor transcripts.
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Because remodeling vascular cells have the characteristic of being
highly motile, we were interested in evaluating these features in cells
overexpressing a soluble form of the Jagged1 protein. We have
previously established and characterized an in vitro model
of NIH3T3 cells expressing a nontransmembrane form of the extracellular
region of Jagged1.17
This soluble Jagged1 protein is
predicted to be a secreted molecule, and we tested both cell lysates
and conditioned medium for the presence of the soluble Jagged1 protein
(Figure 4A)
. The protein was detectable
both in the cell lysates, and secreted into the conditioned medium of
the cell cultures. We analyzed modifications in cell migration and
phenotypic characteristics caused by the expression and secretion of
the soluble Jagged1 protein. Cells expressing the soluble
Jagged1 construct demonstrated a marked decrease in the rate
of cell migration on plastic as compared to vector control
transfectants (Figure 4)
. The soluble Jagged1 transfectants seemed to
maintain a highly defined wound edge with little invasion of individual
cells into the denuded area (Figure 4B)
. This effect was not seen with
the vector controls, which rapidly demonstrated rogue infiltration to
the denuded area followed by a quick (
24 hours) disintegration of a
defined wound edge. Soluble Jagged1 transfectants were able to migrate
to close the denuded area, albeit at a much slower rate, 24 to 48
hours after the repopulation of the vector control cells (Figure 4C)
.
Because the soluble Jagged1 transfectants were previously reported to
display a spindle shape with decreased pseudopodia-like
processes,17
we evaluated the effects of different
extracellular matrices on cell migration. When cells were seeded on a
fibronectin substrate, cell spreading and attachment was restored, the
soluble Jagged1 cells had a partial rescue of the migration defect, and
individual cell migration into the denuded area was increased (Figure 4D)
. The fibronectin substrate had no significant effect on the vector
control transfectants (data not shown). We also inhibited cell
interaction with the matrix using a peptide integrin inhibitor with
selective, but not total specificity to the vß3 integrin,
SM256,22
and observed that the peptide inhibited the
migration of both the soluble Jagged1 transfectants as well as the
vector controls, and their migration in the presence of the peptide was
indistinguishable (Figure 4E)
.
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Because our evaluation of cell migration included the observations
that 1) the soluble Jagged1 cells maintained a greater degree of cell
contact and migrated more as a sheet rather than the release of
individual cells, and 2) the soluble Jagged1 cell migration defect
could be minimized by increasing cell interaction with the matrix, we
hypothesized that cell-cell and/or cell-matrix interactions were
regulated by soluble Jagged1 production. We also observed that when
plated during routine cell culture, the soluble Jagged1 transfectants
had delayed cell spreading when compared to vector-transfected controls
(Figure 5A)
. Because activation and
phosphorylation of the FAK occurs during attachment and spreading of
cells in vitro,28
we used anti-phosphotyrosine
blotting of immunoprecipitated FAK to evaluate this premise. Compared
to vector-transfected cells, the activation of FAK in soluble Jagged1
transfectants was delayed compared to control vector transfectants,
with equal levels of phosphorylation only seen at later times after
cell plating (Figure 5B)
.
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v, 5,
vß3, vß5, and 5ß1 on the surface of vector control and
soluble Jagged1-transfected cells (data not shown). However,
further analysis of the focal adhesion complexes by immunofluorescence
confirmed the biochemical differences in the soluble Jagged1
transfectants (Figure 6; A to D
). When
cells were plated on plastic, vector-transfected control cells
displayed abundant vinculin-positive focal adhesion sites. However, the
soluble Jagged1 transfectants had significantly fewer focal adhesion
sites and of smaller size. This difference was particularly exaggerated
when the cells were plated on a collagen substrate (Figure 6, C and D)
,
a condition previously shown to support the branching chord-like
morphology of the soluble Jagged1 transfectants.17
Immunostaining with antibodies against phosphotyrosine, a well-known
histochemical marker of focal adhesion sites28
yielded
results similar to those described for vinculin staining (data not
shown). The defects in the focal adhesion sites are consistent with the
delayed cell spreading and slower phosphorylation in the soluble
Jagged1 transfectants described above. The attenuation of focal
adhesion sites may at least partially explain the decreased migratory
activity of the soluble Jagged1 transfectants. However, the decrease in
motility could be also attributed to the strengthening of intercellular
contacts, and the highly defined wound edge in the soluble
Jagged1 transfectants supports this explanation. To explore the effect
of soluble Jagged1 transfection on cell-cell contacts, we performed
immunofluorescence staining of confluent cultures of vector and soluble
Jagged1 transfectants using a pan-cadherin antibody that detects all
cadherins, or an antibody against ß-catenin. The immunofluorescence
preparations were studied using confocal microscopy under standard
conditions of illumination and registration, which permitted the
objective comparison of the cell clones. We found a significant
increase of both cadherins and ß-catenin expression in the cell-cell
contacts of soluble Jagged1-expressing cells compared to vector
controls (Figure 6; E to H
). These findings provide evidence for the
regulation of both cell-matrix and cell-cell adhesion molecules by
endogenous Notch signaling through soluble Jagged1.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our in vitro studies have focused on a system using a secreted form of the extracellular portion of the Jagged1 ligand in NIH3T3 cells in an effort to understand how perturbation of the Notch signaling system affects cell phenotype. Recent data have suggested that the active form of a Delta/Serrate/Jagged ligand may be more highly regulated than previously expected. In addition to the transmembrane-bound ligand, Delta has been shown to be cleaved from the cell surface, generating a soluble agonist for Notch activity.36 Conversely, secreted forms of Delta and Serrate have been shown to act as dominant-negative forms of the ligands in Drosophila embryos,37 and we have recently reported similar data for the soluble form of Jagged1 in mammalian cells.47 Although immobilization of the extracellular domain of Delta was shown to be required for Notch-mediated inhibition of myoblast differentiation and HES1 transactivation,38 similar studies with the soluble form of Jagged1 have not been performed. The initial rationale for producing the secreted form of the Jagged1 ligand was the discovery of this form expressed by human endothelial cells undergoing in vitro angiogenesis.18 Indeed, previous studies in this system have verified that the production of the soluble Jagged1 form changes cell morphology, decreases contact inhibition of cell growth, and stimulates angiogenesis in a chick chorioallantoic membrane.17 As soluble Delta ligand has been shown to be generated in vivo, proteolytic cleavage of the Jagged1 ligand may also be a mechanism for the regulation of function, and it will be critical to establish the active forms of these ligands during normal embryonic development as well as disease processes such as Alagille syndrome.
Our findings that the presence of the soluble Jagged1 protein decreases cell adhesion and migration, probably as a result of inhibiting the formation or stability of focal adhesion complexes, have implications for understanding downstream events involved in vascular repair. In addition, the increases in ß-catenin and cadherins in the intercellular junctions of soluble Jagged1 transfectants provide a consistent explanation for the decreased rates of migration in the soluble Jagged1 population. Increased expression of cadherin has been implicated in contact-mediated inhibition of cell migration39 as well as acting as a tumor suppressor for growth and invasion of tumors in vitro and in vivo.40-42 Interestingly, cellular migration and invasion are key features of remodeling vascular cells. The observations that soluble Jagged1 protein inhibits cell-matrix interaction, focal adhesion formation, and cellular migration while increasing cell-cell contacts suggests that endogenous Jagged/Notch signaling may act to maintain cell interaction with the matrix and to activate the migratory ability of cells, possibly by decreasing cell-cell contacts. This interpretation is consistent with the in vivo expression of Jagged/Notch genes particularly at the leading wound edge in regenerating endothelium, where cells are actively migrating to cover the denuded surface. Also particularly in the smooth muscle cell population, one would expect that invasion of cells through the internal elastic lamina would require the attenuation of cell-cell contacts and an increase in cell-matrix interaction to allow singly migrating cells to enter the intimal compartment. Although cadherins and focal adhesions have not been established as direct downstream targets of Notch signaling, recent data have shown that 1) perturbation of Notch signaling in Xenopus embryos leads to changes in the segmental expression pattern of the paraxial protocadherin, which is expressed during convergence extension cell movements in gastrulating embryos;43,44 and 2) expression of a constitutively activated Notch4 receptor disrupts contact inhibition of proliferation in mammary epithelial cells in vitro, and stimulates invasion and migration into a collagen gel.45 These studies are consistent with our observations that both cell-matrix and cell-cell interactions can be influenced by the Jagged/Notch pathway. Our results in combination with the earlier report of soluble Jagged1 cells regulating angiogenesis in the chick chorioallantoic membrane assay17 suggest that both microvessel and large vessel phenotype may be controlled through Notch signaling. Furthermore, a recent report showing that either Jagged1 or Notch4/int3 induced microvessel-like structures in a rat brain-derived endothelial cell line in vitro46 supports earlier studies suggesting that Notch signaling regulates cellular differentiation and phenotype throughout the vascular tree.18 We would predict that in large vessels in vivo, the expression of the Jagged/Notch genes reflect a functional role in modulating these processes in cellular migration and invasion.
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Acknowledgements |
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Footnotes |
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Supported by the American Heart Association (SDG9630017N to L. L.) and the National Institutes of Health (grants RR15555 to V. L., L. L., and T. M., and HL35627 to T. M.).
Accepted for publication May 3, 2001.
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