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Center for Endocrinology, Metabolism, and Molecular Medicine (W.L., W.L.L), and Robert H. Lurie Cancer Center (Y.L.), Department of Medicine, Northwestern University Medical School and Veterans Affairs Chicago Healthcare System, Lakeside Division, Chicago, Illinois 60611
Address all correspondence and requests for reprints to: William L. Lowe, Jr., M.D., Center for Endocrinology, Metabolism, and Molecular Medicine, Tarry 15–703, Northwestern University Medical School, 303 East Chicago Avenue, Chicago, Illinois 60611. E-mail: wlowe{at}nwu.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B-dependent
transcription. Treatment of bovine pulmonary artery endothelial cells
(PAEC) with IGF-I activated the mitogen-activated protein
kinases extracellular signal-regulated kinase (ERK)1/2 and ERK5.
In contrast, IGF-I had no effect on either c-Jun
amino-terminal kinase or p38 kinase activity. IGF-I also activated
phosphatidylinositol (PI) 3-kinase, as reflected by increased
phosphorylation of Akt. There was no evidence of cross-talk between the
ERK and PI 3-kinase pathways in PAEC. In PAEC transiently transfected
with pTK81-NFB-Luc, which contained four copies of the NF-B DNA
binding site 5' to a minimal promoter and the luciferase gene,
treatment with 50 ng/ml IGF-I increased luciferase activity 1.8-fold.
Inhibition of ERK activity using PD98059 and PI 3-kinase activity with
LY 294002 abrogated the induction of NF-B-dependent transcription by
IGF-I, suggesting that both pathways contribute to the effect of IGF-I
on NF-Bdependent transcription. In contrast to the effect of
tumor necrosis factor-
on NF-B activation, Western blot analyses
demonstrated that IGF-I had no effect on IB phosphorylation and
degradation or nuclear translocation and DNA binding of NF-B. These
data suggest a direct of effect of IGF-I on nuclear NF-B.
IGF-I also increased endothelial cell migration approximately 2-fold,
as demonstrated using a Boyden chamber apparatus. IGF-I-induced
endothelial cell migration was inhibited, in part, by LY 294002 but not
PD98059. Together, these studies demonstrate that IGF-I activates
multiple signaling pathways in endothelial cells with little evidence
for cross-talk between the pathways. Moreover, these pathways appear to
mediate both overlapping and distinct effects in that activation of
both PI 3-kinase and the ERKs contributed to the stimulation of
NF-B-dependent transcription by IGF-I, whereas only PI 3-kinase
mediated IGF-I-stimulated endothelial cell migration. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The cellular events that follow IGF-I binding and account for its biological effects are still being elucidated, but signaling pathways that are activated upon IGF-I binding have been defined. Ligand binding to the receptor activates the intrinsic tyrosine kinase activity of the receptor and results in phosphorylation of members of a family of insulin receptor substrates and other molecules, including Shc, Crk, and Grb2 (9, 10). Subsequent to these initial events, two signaling pathways that mediate many of the effects of IGF-I are activated, the phosphatidylinositol 3-kinase (PI 3-kinase) and mitogen-activated protein kinase (MAPK) pathways (9, 10). In vascular smooth muscle cells, these pathways contribute to the regulation of IGF-I-induced DNA synthesis and cell migration (11, 12).
The purpose of the present study was to begin to define the signaling
pathways important for IGF-I-mediated effects in endothelial cells. To
that end, we have defined signaling pathways activated by IGF-I in
endothelial cells and their contribution to two important IGF-I
mediated effects in these cells: cell migration and activation of the
transcription factor nuclear factor (NF)-B. Endothelial cell
migration is important for repair of the endothelium at sites of
vascular injury, whereas NF-B demonstrates increased expression at
sites of high probability for atherosclerosis and, upon activation,
both protects cells from apoptosis and participates in inflammatory
processes (13, 14, 15, 16, 17).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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MAPK immune complex assays
For the kinase assays, cells were plated at a density of 5
x 105 cells per 75-cm2
plate. The cells were preincubated in serum-free medium with 0.25% BSA
(DMEM + 0.25% BSA) for 14–16 h before being treated with growth
factors. Antibodies directed against extracellular signal-regulated
kinase (ERK)-2, c-Jun amino-terminal kinase-1 (JNK1), p38
kinase, and ERK5 were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The substrate protein for the JNK immune
complex assay was a glutathione-S-transferase
(GST)-c-Jun fusion protein, which was generated as described
previously (19). The substrate for the p38 kinase assay
was GST-ATF2, which was prepared using the plasmid pGST-ATF2 (kindly
provided by Dr. Roger Davis, Howard Hughes Medical Institute,
University of Massachusetts Medical School, Wooster, MA). The
substrate for the ERK2 and ERK5 assays was myelin basic protein
(Sigma, St. Louis MO). The activity of ERK2, JNK1, p38
kinase, and ERK5 was determined using immune complex assays as
described previously (19, 20, 21). The final reaction products
were separated by SDS-PAGE followed by autoradiography.
32P incorporation into substrate proteins was
quantified using a STORM 860 Phosphor-Imager (Molecular Dynamics, Inc., Sunnyvale, CA).
Western blot analysis
Western blots were probed with polyclonal antibodies directed
against phospho-ERK1/2, phospho-Akt, Akt, phospho-IB, and IB
(New England Biolabs, Inc., Beverly, MA) at a dilution of
1:1,000 or with a polyclonal antibody directed against ERK1 and ERK2
(Santa Cruz Biotechnology, Inc.) at a dilution of
1:7,500.
Western blot analyses were performed as described previously (19, 20). Briefly, cell lysates were prepared in cell lysis buffer [150 mM NaCl, 1% NP-40, 0.5% deoxycholate, 0.1% SDS, 50 mM Tris (pH 7.5), 1 µM Na orthovanadate, 2 mM sodium pyrophosphate, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 0.7 µg/ml leupeptin], and protein content of the lysate was determined using the Coomassie blue protein assay (Bio-Rad Laboratories, Inc. Richmond, CA). Forty micrograms of protein were size separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes in a semidry apparatus. The membranes were blocked in 20 mM Tris (pH 7.6), 137 mM NaCl, 0.1% Tween-20 (TBST) and 4% nonfat dry milk for 90 min at room temperature. Membranes were incubated for 3 h at 22 C in TBST containing nonfat milk and primary antibody, washed three times for 15 min at 22 C in TBST, and incubated for 90 min at room temperature in TBST containing nonfat dry milk and second antibody (1:7,500 dilution). Following three washes in TBST, immunoreactive bands were detected using the enhanced chemiluminescence (ECL) detection system from Amersham Pharmacia Biotech (Arlington Heights, IL), according to the manufacturer’s instructions.
Electrophoretic mobility shift assay
For the preparation of nuclear extracts, cells were harvested
after the indicated treatment in 5 ml PBS containing 1 µM
EDTA, 1 mM dithiothreitol (DTT), and 1 mM PMSF.
The cells were resuspended in 400 µl cold buffer A [10
mM HEPES (pH 7.9), 10 mM NaCl, 0.1
mM EDTA, 0.1 mM EGTA, 1 mM DTT, 1
mM PMSF, 1 µg/ml pepstatin A, 10 µg/ml leupeptin, 10
µg/ml aprotinin, 0.1 mM p-aminobenzamindine)
and incubated on ice for 15 min. Following addition of 25 µl of 10%
NP-40 and vigorous vortexing for 10 sec, cell pellets were resuspended
in 50 µl of cold buffer C [20 mM HEPES (pH
7.9), 0.4 M KCl, 1 mM EDTA,
1 mM EGTA, 1 mM DTT, 1
mM PMSF, 20% glycerol, 1 µg/ml pepstatin A, 10
µg/ml leupeptin, 10 µg/ml aprotinin, and 0.1
mM p-aminobenzamindine] and mixed
at 4 C for 15 min. The mixture was clarified, and the protein content
of the supernatant was determined using the Coomassie blue protein
assay. Nuclear extracts were stored at -70 C.
For the electrophoretic mobility shift assays, annealed
oligonucleotides containing the consensus DNA binding sequence for
NF-B (5'-AGTTGAGGGGACTTTCCCAGGC-3'; 5'-GCCTGGGAAAGTCCCCTCAACT-3') or
the E-box of the adenovirus major late transcription factor promoter
(5'-ATAGGTGTAGGCCACGTGACCGGGTGT-3'; 5'-ACACCCGGTCACGTGGCCTACACCTAT-3')
were radiolabeled with 
-32P-ATP using T4
polynucleotide kinase as described previously (22).
Incubation of radiolabeled DNA with cellular nuclear extracts,
separation of DNA-protein complexes using low ionic strength native
PAGE, and detection of signals were performed as described previously
(22). To verify the specificity of the binding reaction, a
100-fold excess of unlabeled oligonucleotide was added to the reaction
before adding the labeled probe.
Transient transfection and luciferase assays
pTK81-NFB-Luc was constructed by cloning two copies of
annealed oligonucleotide containing the sequence
5'-CGCGGGGACTTTCCCgtacGGGGACTTTCCCG-CGgtac-3' in the antisense
orientation 5' to the luciferase gene in the KpnI site in
the vector pTK81-Luc. This sequence contained two copies (indicated in
capital letters) of a NF-B response element. For
transfection assays, cells were plated onto 12-well plates at a density
of 1 x 105 cells per well. Plasmid DNA was
transfected into cells using the cationic lipid Lipofectin (Life Technologies, Inc., Gaithersburg, MD) according to the
manufacturer’s instruction. Cells were incubated in Opti-mem with
lipofectin and plasmid DNA for 4 h and then transferred to
serum-free DMEM + 0.25% BSA. For experiments using the indicated
inhibitors, inhibitors were added 30 min before treating the cells with
either IGF-I or tumor necrosis factor (TNF)-. After treatment for
6 h, cells were harvested, and luciferase assays were performed as
described previously (22). Light emission was measured
with an AutoLumatLB953 luminometer (E.G.& G. Berthold, Bad Wildbad,
Germany). The luciferase activity present in each sample was normalized
using the protein content of the sample. All luciferase assays were
performed in triplicate.
Cell migration assay
The migration assay was performed using a modified 48-well
Boyden chamber apparatus (Neuroprobe Inc., Cabin John, MD). IGF-I was
diluted to 50 ng/ml in serum-free DMEM + 0.25% BSA and loaded into the
lower wells of the Boyden chamber in triplicate. The wells were
subsequently covered with a polyvinylpyrrolidone-free filter with
8-µm pores (Nucleopore Corp., Pleasanton, CA) coated with type I
collagen (Vitrogen, Collagen Corp., Palo Alto, CA). The cells were
serum starved with serum-free DMEM + 0.1% BSA overnight and then
trypsinized and resuspended at a density of 1 x
106 cells per ml. Cells (50,000 in 50 µl) were
loaded into the upper wells of the Boyden chamber. For assays with
inhibitors, the cells were treated with DMEM + 0.1% BSA containing the
indicated inhibitor for 30 min and then trypsinized and washed. The
cells were then resuspended in DMEM + 0.1% BSA containing the
inhibitor and loaded into the upper well of the Boyden chamber in
triplicate. The chambers were incubated for 6 h at 37 C in a 5%
CO2 and 95% air atmosphere. At the end of the
incubation time, the cells attached to the filter were fixed and
stained in DifQuick stain ( Allegiance Healthcare Corp., Waukegan,
IL). The number of cells that had migrated to the lower chamber
was determined by counting the number of cells in a 6x magnification
field of each well. All studies were done in triplicate.
Statistical analyses
Values are reported as the mean ± SEM.
P values were calculated using the one-way repeated measures
ANOVA with Tukey’s pairwise multiple comparison procedure or
Kruskal-Wallis one-way ANOVA on Ranks with the Dunnett’s pairwise
multiple comparison procedure, as appropriate, using SigmaStat
2.0 software (Jandel Corp., San Rafael, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). IGF-I-stimulated
ERK phosphorylation was evident and maximal 15 min after treatment with
IGF-I with a return to basal levels 120 min after IGF-I treatment (Fig. 1A). The ability of IGF-I to stimulate ERK phosphorylation was then
compared with the effect of two well described growth factors for
endothelial cells: vascular endothelial cell growth factor (VEGF) and
basic fibroblast growth factor (bFGF) (Fig. 1B). As can be seen, the
effect of IGF-I was similar to that of VEGF but slightly less than that
of bFGF. Commensurate with the ability of IGF-I to increase ERK
phosphorylation, treatment with IGF-I also increased ERK2 activity, as
demonstrated using an immune complex assay (Fig. 1C). The IGF-I-induced
increase in ERK2 activity was similar to that stimulated by bFGF and
VEGF.
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, A and B). Similarly, neither
VEGF nor bFGF was able to activate these MAPKs. In contrast, JNK 1
activity was markedly increased by treating cells for 15 min with 5
ng/ml TNF-, whereas activation of p38 kinase in the PAEC was
demonstrated by inducing hyperosmolarity with increasing concentrations
of raffinose, a trisaccharide that is unable to cross the plasma
membrane (Fig. 2, A and B). ERK5 activity, which is activated by
oxidative stress, shear stress, and some growth factors (24, 25), was also increased by treatment of PAEC with IGF-I. In
cells treated with 50 ng/ml IGF-I, maximal ERK5 activity was present
within 15–30 min (Fig. 3A), and IGF-I
increased ERK5 activity 2.5-fold (Fig. B). Epidermal growth factor
(EGF), which has been shown previously to increase ERK5 activity
(24), stimulated a similar increase in ERK5 activity,
whereas TNF- had no effect.
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). Treatment of cells for 15 min
with 50 ng/ml IGF-I stimulated a marked increase in Akt
phosphorylation. VEGF and bFGF also increased Akt phosphorylation,
although their effect was not as marked as that of IGF-I. In contrast,
TNF- had no effect on Akt phosphorylation. Time course studies
demonstrated that Akt phosphorylation was evident within 5 min of IGF-I
treatment with maximal stimulation at 15 min (Fig. 4B). Interestingly,
the effect of IGF-I on Akt phosphorylation was sustained for up to
2 h.
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).
Similarly, 20 µM LY 294002 abrogated IGF-I-induced Akt
phosphorylation, but 20 µM PD98059 had no effect on
IGF-I-stimulated phosphorylation of Akt (Fig. 5, A and B). Finally,
activation of ERK5 by IGF-I has not been previously documented, and the
signaling pathway that mediates this effect of IGF-I is unknown.
Treatment of cells with 20 µM LY 294002 had no effect on
IGF-I-induced ERK5 activity, whereas 20 µM PD98059
partially inhibited ERK5 activation by IGF-I (Fig. 5C). Similarly, 100
nM Calphostin C, an inhibitor of protein kinase C,
partially abrogated IGF-I-induced ERK5 activity, suggesting that
protein kinase C participates in activation of ERK5 by IGF-I.
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B activityB-dependent transcriptional activity. To address this, PAEC were
transiently transfected with pTK81-NFB-Luc, a fusion gene that
contained four copies of the NF-B DNA binding site 5' to a minimal
thymidine kinase promoter and the luciferase gene in the vector pTK81.
Treatment of cells for 6 h with 50 ng/ml IGF-I increased
luciferase activity 1.8-fold (Fig. 6, top panel), whereas in cells transfected with pTK81, IGF-I
increased luciferase activity only 1.1 ± 0.0-fold (mean ±
SEM, n = 3). Treatment of cells with either
20 µM PD98059 or LY 294002 inhibited the
IGF-I-induced increase in luciferase activity (Fig. 6, top
panel), suggesting that both the ERK and PI 3-kinase pathways
contribute to IGF-I-induced activation of NF-B. For comparison, the
effect of TNF-, a cytokine that has been shown previously to
increase NF-B-mediated transcription, was examined. TNF-
stimulated a more robust 3.2-fold increase in NF-B activity (Fig. 6, bottom panel), and, interestingly, despite the apparent lack
of effect of TNF- on ERK1/2 or PI 3-kinase activity, both LY 294002
and PD98059 partially abrogated TNF--induced luciferase activity
(Fig. 6, bottom panel), suggesting that both pathways, in
some manner, facilitate activation of NF-B by TNF-.
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B activity. A key event in the activation of NF-B is release
of functional NF-B dimers from the inhibitory protein, IB, by
phosphorylation and subsequent degradation of IB (15).
Release of the NF-B dimers results in their translocation to the
nucleus. To determine whether IGF-I stimulates IB phosphorylation
and degradation, PAEC were treated with 50 ng/ml IGF-I for varying
periods of time, and Western blot analyses were performed using
antibodies directed against IB and phospho-IB. As a control,
cells were also treated with 5 ng/ml TNF-. Interestingly, IGF-I had
no effect on either IB phosphorylation or total IB levels (Fig. 7A). In contrast, TNF- stimulated the
phosphorylation of IB, with increased phospho-IB levels present
within 5 min of TNF- treatment, and increased IB degradation,
with a decrease in IB levels also present within 5 min of treatment
with TNF- (Fig. 7B). Consistent with previous studies, recovery of
IB levels was evident within 60 min of TNF- treatment
(15).
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B and NF-B
DNA binding in response to IGF-I or TNF- treatment were next
examined. To define the effect of the above ligands on nuclear
translocation of NF-B, nuclear extracts were prepared from PAEC
treated for varying periods of time with either IGF-I or TNF-, and
Western blot analyses were performed using an antibody directed against
the p65 subunit of NF-B (Fig. 8A).
Again, differing effects of IGF-I and TNF- were observed. Consistent
with its effect on IB phosphorylation and degradation, TNF-
increased the levels of p65 in the nucleus, whereas IGF-I had no effect
on nuclear p65 levels. Gel shift analyses were used to examine the
effect of IGF-I and TNF- on DNA binding of NF-B. Treatment with
IGF-I had no effect on NF-B DNA binding activity (Fig. 8B). In
contrast, TNF- stimulated a marked increase in NF-B DNA binding
activity (Fig. 8C). As a control, protein binding to an oligonucleotide
containing the E box of the adenovirus major late promoter was
determined. Protein binding to this oligonucleotide was unaffected by
either IGF-I or TNF- (Fig. 8, B and C). Together, the above data
suggest that IGF-I regulates NF-B transcriptional activity
downstream of NF-B nuclear translocation and DNA binding, whereas
TNF- stimulates NF-B activity via a pathway that modulates
nuclear translocation and DNA binding.
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). To examine the effect of 20
µM LY 294002 and 20 µM PD98059 on
IGF-I-induced cell migration, the cells were pretreated for 30 min with
the respective inhibitor before initiation of the migration assay (Fig. 9). Inhibition of IGF-I-induced PI 3-kinase activity using LY 294002
attenuated the effect of IGF-I on endothelial cell migration; in
contrast, PD98059 had no effect on endothelial cell migration. These
data suggest that activation of PI 3-kinase contributes to
IGF-I-stimulated endothelial cell migration but that ERK activation is
not required.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A transcription factor that may be important for atherosclerosis and
has been associated with endothelial cell activation and survival as
well as endothelial cell dysfunction and vascular inflammation is
NF-B (13, 14, 40, 41). NF-B is ubiquitously
expressed and exists as a dimer composed of members of the NF-B/Rel
family (15). In endothelial cells, a heterodimer of
NF-B1 (p50) and p65 (RelA) is the predominant species of NF-B
(42) In unstimulated cells, NF-B is retained in the
cytoplasm by inhibitory proteins of the IB family (15).
Upon cellular activation, IB is phosphorylated by a multisubunit
IB kinase complex, which targets IB for ubiquination and
degradation and liberates NF-B, resulting in nuclear translocation
of NF-B and activation of transcription (15). Previous
studies have demonstrated that IGF-I increases NF-B DNA binding in
vascular smooth muscle cells and NF-B DNA binding, nuclear p65
levels, and NF-B-dependent gene transcription in neural cells
(43, 44). The role of IGF-I in regulating NF-B binding
and activity in endothelial cells has not been previously examined,
except for one study that showed a transient increase in whole-cell p65
levels 30 min following treatment with IGF-I (45). The
present study demonstrated that IGF-I increases NF-B-dependent
transcription in endothelial cells, although, in contrast to the
previous studies in vascular smooth muscle and neural cells, IGF-I had
no effect on either nuclear p65 levels or NF-B DNA binding.
Moreover, IGF-I had no effect on IB phosphorylation or degradation.
The induction of NF-B-dependent transcription by TNF-, on the
other hand, was associated with IB phosphorylation and degradation,
increased nuclear p65 levels, and NF-B DNA binding. As has been
described previously, subsequent to TNF- treatment, IB levels
recovered, with full recovery evident 60 min after TNF- treatment
(15, 46). Although phospho-IB levels also appeared to
increase, IB levels were sustained during the recovery phase.
Nuclear translocation is a key event in NF-B activation, but protein
phosphorylation also appears to contribute to regulation of NF-B
activity (15). A recent study demonstrated that both PI
3-kinase and Akt were able to stimulate the transcriptional activity of
NF-B by targeting the basal levels of nuclear NF-B as opposed to
stimulating nuclear translocation (47). Thus, the effect
of IGF-I in PAEC may be mediated by phosphorylation of basal nuclear
NF-B. Consistent with that possibility, the present study
demonstrated that the effect of IGF-I on NF-B transcriptional
activity in endothelial cells was inhibited by LY 294002, an inhibitor
of PI 3-kinase. The stimulation of NF-B-dependent transcription by
IGF-I in neural cells also was inhibited by PI 3-kinase
(44).
The present studies also demonstrated that the MEK1 inhibitor PD98059
was able to attenuate IGF-I-induced NF-B activity. These data are
consistent with a role for ERK1 and -2, in addition to PI 3-kinase, in
IGF-I-induced NF-B activity, although, given the effect of PD98059
on IGF-I-induced ERK5 activity, a role for ERK5 in the stimulation of
NF-B-induced transcription by IGF-I cannot be excluded. Previous
studies have suggested a role for the ERKs in NF-B activation. In
cobalt chloride-treated endothelial cells, NF-B DNA binding was
inhibited by PD98059 (44), whereas in other cell types,
inhibition of ERK activation with PD98059 was associated with either
decreased, no change, or increased NF-kB activity
(48, 49, 50, 51, 52, 53). Interestingly, the effect of ERK inhibition is
not universally dependent upon changes in NK-B nuclear translocation
or DNA binding. For example, the inhibition by PD98059 of
NF-B-dependent IL-6 production in monocytes treated with okadaic
acid was independent of NF-B DNA binding (53).
Similarly, the regulation of NF-B-dependent transcription but not
nuclear translocation was dependent upon ERK activation in Cos cells
treated with TNF- (54). Together with the present
studies, these data suggest that the ERKs are capable of modulating
NF-B activity via a mechanism that is independent of nuclear
translocation of NF-B.
An additional factor important in atherosclerosis is vascular cell migration. Endothelial cell migration may enhance vascular repair and, thus, attenuate the formation of atherosclerotic plaques by contributing to the repair of desquamated areas in the endothelial cell lining (1, 2). Previous studies have demonstrated the ability of IGF-I to stimulate endothelial cell migration (26, 27), although the mechanism for this effect in endothelial cells has not been examined. In the present study, we confirmed the ability of IGF-I to stimulate endothelial cell migration and have now demonstrated that this effect is dependent, in part, upon activation of PI 3-kinase and independent of ERK activation. Activation of PI 3-kinase also plays an important role in IGF-I-induced motility of Schwann cells and migration of vascular smooth muscle and colonic epithelial cells (11, 12, 55, 56). In contrast to endothelial cells, however, inhibition of ERK activity at least partially attenuates the effect of IGF-I on the migration of vascular smooth muscle and colonic epithelial cells.
In summary, despite the recognition for many years that endothelial
cells express IGF-I receptors, there is little known about the signal
transduction pathways that are important for IGF-I-mediated effects in
these cells. We have now demonstrated that IGF-I activates multiple
signaling pathways in endothelial cells, including ERK1, -2, and -5 and
PI 3-kinase, with little evidence for cross-talk between these
signaling pathways. The different signaling pathways appear to mediate
both overlapping and distinct effects in that activation of both
PI 3-kinase and the ERKs contribute to stimulation of NF-B-dependent
transcription by IGF-I, whereas only PI 3-kinase mediates
IGF-I-stimulated endothelial cell migration.
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Acknowledgments |
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Footnotes |
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Received November 14, 2000.
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 H. B. Koon, G. J. Bubley, L. Pantanowitz, D. Masiello, B. Smith, K. Crosby, J. Proper, W. Weeden, T. E. Miller, P. Chatis, et al. Imatinib-Induced Regression of AIDS-Related Kaposi's Sarcoma J. Clin. Oncol., February 10, 2005; 23(5): 982 - 989. [Abstract] [Full Text] [PDF]
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M. Qiao, P. Shapiro, R. Kumar, and A. Passaniti Insulin-like Growth Factor-1 Regulates Endogenous RUNX2 Activity in Endothelial Cells through a Phosphatidylinositol 3-Kinase/ERK-dependent and Akt-independent Signaling Pathway J. Biol. Chem., October 8, 2004; 279(41): 42709 - 42718. [Abstract] [Full Text] [PDF]
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J. K. G. Crean, D. Finlay, M. Murphy, C. Moss, C. Godson, F. Martin, and H. R. Brady The Role of p42/44 MAPK and Protein Kinase B in Connective Tissue Growth Factor Induced Extracellular Matrix Protein Production, Cell Migration, and Actin Cytoskeletal Rearrangement in Human Mesangial Cells J. Biol. Chem., November 8, 2002; 277(46): 44187 - 44194. [Abstract] [Full Text] [PDF]
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, P <
0.05 vs. the activity in cells treated with 50 ng/ml
IGF-I. B, Effect of TNF-
