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Institute of Endocrinology, Sheba Medical Center (R.B., M.H., N.W., R.H., A.K., A.G.), Tel Hashomer 52621; and Faculty of Life Sciences, Bar-Ilan University (R.B., U.N.), Ramat Gan 51905, Israel
Address all correspondence and request for reprints to: Dr. Avraham Geier, Institute of Endocrinology, Sheba Medical Center, 52621 Tel Hashomer, Israel. E-mail: geiera{at}bezeqint.net
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IGF-I exerts its biological effects (cell proliferation, differentiation, and survival) by binding to a tyrosine kinase receptor, the IGF-I receptor (IGF-IR) (19, 20). This binding results in a conformational change and cross-phosphorylation between the ß-subunits of the receptor complex, leading to further activation of the protein-tyrosine kinase activity (21). This activation induces a rapid phosphorylation of insulin receptor substrate-1 (IRS-1), IRS-2, and Shc, which are the major substrates for IGF-IR. The tyrosyl-phosphorylated IRS proteins serve as docking sites for numerous SH2 domain-containing proteins, including the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3K) and Grb2. PI3K then initiates phospholipid turnover, and Grb2/SOS activation results in initiation of the mitogen-activated protein kinase (MAPK) signal transduction cascade. It has been suggested that both PI3K, through its downstream target the serine-threonine kinase Akt, as well as the MAPK pathways are involved in IGF-I inhibition of apoptosis (4, 22). Recent results show that IGF-I, through Akt or MAPKs, can promote cell survival by phosphorylating, and thereby inhibiting, BAD, the proapoptotic member of the Bcl-2 family (23, 24, 25). Furthermore, it has been demonstrated that MAPKs induce phosphorylation of Bcl-2, which may stabilize the Bcl-2-Bax heterodimerization and support survival (26).
The antiapoptotic effects of ATA were usually attributed to its ability
to inhibit the endogenous endonuclease activity involved in
fragmentation of DNA into 180-bp oligonucleosome integer fragments.
This assumption was derived from cell-free studies that demonstrated
that ATA inhibits many nucleic acid-binding proteins, including RNA
polymerase, replicase, exonuclease III, deoxyribonuclease I,
ribonuclease A, S1 nuclease, and various restriction nucleases
(27, 28). However, because of its charge, ATA seems to be
membrane impermeant (29). Indeed, ATA appears to block the
apoptotic death of trophic factor-deprived PC12 cells by acting at
points upstream of c-Jun kinase activation (30), which
contrasts the assumption that ATA acts as an endonuclease inhibitor.
Moreover, it was shown that ATA antagonizes NMDA binding to the NMDA
receptor (7), blocks the binding of gp120, the human
immunodeficiency virus coat protein to CD4 molecule (31),
and prevents the binding of interferon-
to its receptor
(32). These findings support the idea that the site of
action of ATA is at the surface of cells rather than on endonucleases
in the nucleus.
A similar mechanism was proposed for ATA’s action in PC12 cells
(33). It has been suggested that activating a certain
growth factor receptor tyrosine kinase underlies the protective effect
of ATA, as MAPKs, Shc proteins, and phospholipase C-
were tyrosine
phosphorylated in ATA-treated PC12 cells.
An additional endonuclease inhibitor, the sulfonated aromatic polyanion Evans blue (EB), was found to be a survival agent, and similar to ATA, it was shown to protect renal tubular cells against DNA strand break and death induced by H2O2 or hypoxia/reoxygenation injuries (6). To understand the molecular mechanism by which ATA and EB mediate cell survival, we investigated the possibility that activation of IGF-I signaling underlies the marked cytoprotective effect of these drugs. Therefore, we examined the effects of the polyanions on the activation of several key proteins in the IGF-I signal transduction pathway.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Antibodies
Monoclonal antiphosphotyrosine antibody was purchased from
Transduction Laboratories (Lexington, KY). Polyclonal anti-IRS-1 and
IRS-2 antibodies and the polyclonal antibody directed against the
regulatory subunit of PI3K were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Polyclonal
antiphospho-specific Akt (Ser473) antibody was
obtained from New England Biolabs, Inc. (Beverly, MA).
Polyclonal anti-active MAPK antibody was purchased from Promega Corp. (Madison, WI), and monoclonal anti-MAPK (extracellular
signal-regulated kinases 1 and 2) antibody was obtained from
Zymed Laboratories, Inc. (San Francisco, CA). Polyclonal
anti-Grb2 and anti-IGF-IR ß-subunit antibodies, and polyclonal
antibodies to erbB1 and erbB4 were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
Cell culture
Breast cancer cells (MCF-7) were grown in DMEM supplemented with
6% FBS (Biological Industries, Bet Haemek, Israel), as previously
described (1). Parental NIH-3T3 cells, NIH-3T3 cells
overexpressing the wild-type human IGF-IR (NWTc43), or the
kinase-defective receptor (NKR-1), a gift from D. LeRoith (NIH,
Bethesda, MD), were grown in DMEM supplemented with 10% FBS as
previously described (34). Breast cancer cells (MDA-231)
and rat hepatoma cells (FaO) were grown in RPMI 1640 medium
supplemented with 6% or 10% FBS, respectively, as previously
described (10, 35).
Phosphorylation/activation experiments
Subconfluent monolayers cells, grown in 60-mm dishes, were
deprived of serum for 20 h before each experiment. The medium was
aspirated, and cells were washed twice and incubated with the indicated
reagents in serum-free medium at 37 C under the experimental
conditions. The reaction was terminated by removing the medium and
freezing cell monolayers with liquid nitrogen. In some experiments
cells were preincubated with 50 µM sodium orthovanadate
for 3 h.
Survival and proliferation analysis
Subconfluent cells grown in 35-mm dishes were deprived of serum
for 20 h and then exposed to fresh serum-deprived medium in the
absence or presence of the indicated additives. After treatment, the
medium was recovered, and the cells were detached with trypsin. The
medium and the detached cells were pooled, and viability was estimated
by trypan blue assay, as previously described (1). In some
experiments cells deprived of serum for 20 h were exposed to 30
µg/ml CHX in the absence or presence of increasing concentrations of
fractionated ATA, crude ATA, EB, or IGF-I. Cell death was estimated as
described above.
Protein analysis
Cells were solubilized at 4 C with buffer A [50 mM
HEPES, 2 mM sodium orthovanadate, 80 mM
ß-glycerophosphate, 50 mM NaF, 10 mM sodium
pyrophosphate, 2 mM sodium EGTA, 2 mM sodium
EDTA, 1% Triton x-100, 10% glycerol, and protease inhibitor mixture
(Sigma), 1:1000, pH 7.4]. The solubilized cells were
centrifuged at 12,000 x g for 15 min at 4 C. Aliquots
of the supernatants were normalized for protein, mixed with
concentrated (5x) Laemmli sample buffer, and resolved on 7.5%, 10%,
or 12% SDS-PAGE.
For immunoprecipitation, cells were solubilized in buffer B (50 mM Tris-HCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM sodium EGTA, 1 mM sodium orthovanadate, and 1 mM NaF, protease inhibitor mixture, 1:1000, pH 7.4). Aliquots (0.5–1.0 mg) were immunoprecipitated at 4 C with antibodies to IRS-1, IRS-2, IGF-IR, and Grb2, coupled to protein A or A/G Sepharose beads. The immunocomplexes were washed three times with buffer B and resolved on SDS-PAGE.
Electrophoretic transfer of proteins to nitrocellulose papers was carried out as previously described (35). Blots were incubated with the indicated antibodies, and proteins were detected by enhanced chemiluminescence.
In vitro autophosphorylation of IGF-IR
IGF-IR from untreated MCF-7 cells was purified by WGA affinity
chromatography as previously described (36). Samples of
the resultant WGA-purified receptors were incubated without or with ATA
or IGF-I for 30 min at 22 C. Autophosphorylation was initiated by the
addition of 1 mM ATP in buffer C (50 mM HEPES,
10 mM MgAc, 4 mM MnAc, and 0.05% Triton X-100,
pH 7.4). The reaction was continued for 1 min at 22 C, then stopped
using Laemmli sample buffer. Samples were analyzed by SDS-PAGE,
transferred to a nitrocellulose membrane, and immunoblotted with
antiphosphotyrosine antibody as described above.
PI3K activation
PI3K activity associated with IRS-1 was measured as previously
described (4). Briefly, cells were stimulated with the
indicated additives, lysed, and equal amounts of protein from cell
lysates were immunoprecipitated with anti-IRS-1 antibody. The
precipitates were incubated in vitro in the presence of
phosphatidylinositol and [-32P]ATP (3000
Ci/mmol; Amersham Pharmacia Biotech UK Ltd.,
Buckinghamshire, UK) for 10 min. The products of the kinase
reaction were analyzed by TLC. Detection of phosphorylated lipids was
performed by autoradiography.
IGF-I binding assay
Subconfluent cells, grown in 12-well plates, were deprived of
serum for 20 h. Cultures were rinsed twice with ice-cold PBS.
[125I]IGF-I (2 ng/ml) was then added in
combination with various concentration of ATA, suramin, or fuchsin
(0–1000 µg/ml) for 3 h at 4 C. Thereafter, the cells were
washed twice with PBS to remove unbound ligand and then lysed in 0.5
N NaOH. Released radioactivity was measured in a
-counter. Nonspecific binding, determined in the presence of unbound
IGF-I (1000 ng/ml), did not exceed 15% of the total binding.
Fractionation of ATA
ATA fractions were prepared as previously described
(37). Five hundred milligrams of commercial ATA were
dissolved in 2 ml buffer D (2 M KSCN and 10 mM
sodium phosphate, pH 7.2). The solution was applied to a 2 x 68
cm Bio- P-4 column (Bio-Rad Laboratories, Inc., Richmond,
CA) in buffer D. The void volume fractions from three preparations were
pooled and separated on a 2 x 68-cm gel column of P-10
(Bio-Rad Laboratories, Inc.) in buffer D. ATA polymers in
the elution fractions were precipitated by adjusting the pH to 3.0–3.5
and concentrated by centrifugation at 6000 x g for 15
min. The precipitated polymers were washed twice in 150
mM NaCl/3 mM HCl and
lyophilized to dryness.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, MCF-7 cells die in the absence of
serum, leaving about 20% viable cells after 4 days in culture. ATA and
EB as well as TPA protected cell viability similar to IGF-I. Moreover,
in the presence of ATA and EB, but not TPA, MCF-7 cells continued to
proliferate, similar to cells in the presence of IGF-I (Fig. 1B). Our
findings that ATA and EB mimic these effects of IGF-I and the
increasing evidence that the site of ATA’s action is at the surface of
cells suggest that the aromatic polyanions and IGF-I may share a common
signaling pathway.
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, the agents examined stimulated the
phosphorylation of several proteins, but only ATA and EB, similar to
IGF-I, enhanced tyrosine phosphorylation of 95- and 185-kDa proteins,
which may correspond to IGF-IR and IRS-1/2, respectively.
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, ATA and EB, similar to
IGF-I, induced phosphorylation of the IGF-IR ß-subunit. Stripping and
reblotting with an anti-IGF-IR antibody confirmed that similar amounts
of the receptor were present in the immunoprecipitates.
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, phosphorylation reached a
maximum after exposure to 100–200 µg/ml of either ATA or EB compared
with 100 ng/ml IGF-I. At these concentrations maximal phosphorylation
was achieved after 20–60 min of treatment with ATA, EB, and IGF-I
(Fig. 3C). Interestingly, the phosphorylation induced by ATA and EB did
not decline up to 120 min, whereas the phosphorylation induced by IGF-I
decreased gradually, indicating a sustained activation of the IGF-IR by
the aromatic polyanions.
To determine whether ATA is capable of directly activating the IGF-IR,
WGA partially purified IGF-IR was employed in a cell-free
phosphorylation system. Figure 3D demonstrates that ATA and IGF-I
stimulated phosphorylation of the WGA-purified receptor preparation.
The band observed at 95 kDa is consistent with the reported
autophosphorylation of the ß-subunit of the IGF-IR.
Tyrosine phosphorylation of IRS-1/IRS-2 and activation of
PI3K
The 185-kDa tyrosine-phosphorylated protein(s) depicted in Fig. 2
may correspond to (or contain) IRS-1 and IRS-2. To examine this
possibility, cells were stimulated with ATA, EB, or IGF-I. Solubilized
proteins were immunoprecipitated with anti-IRS-1 and IRS-2 antibodies,
separated by SDS-PAGE, and immunoblotted with antiphosphotyrosine
antibody. As shown in Fig. 4A
(upper panel), ATA and EB, similar to IGF-I, stimulated the
tyrosine phosphorylation of IRS-1. Similar phosphorylation of IRS-2 is
shown in Fig. 4B (upper panel). IRS-1 and IRS-2 act as
docking proteins, which in their tyrosine-phosphorylated state
associate with and activate several protein molecules that transmit the
downstream signal of IGF-I. Thus, IRS-1 and IRS-2 immunoprecipitates
were tested for the presence of PI3K and Grb2. Lysates from treated
cells were immunoprecipitated with IRS-1 and IRS-2 antibodies, and the
immunoprecipitates were immunoblotted with an antibody directed to the
p85 subunit of PI3K. As shown in Fig. 4A (lower panel), p85
precipitated with IRS-1 in either ATA- or EB-treated as well as in
IGF-I-treated cells. Similar results were achieved with IRS-2, as
demonstrated in Fig. 4B (lower panel). Moreover, ATA and EB,
similar to IGF-I, increased the IRS-1-associated PI3K activity, whereas
suramin and heparin did not (Fig. 4C). The association of IRS-1 with
Grb2 was evaluated in lysates from treated cells that were
immunoprecipitated with anti-Grb2 antibody and immunoblotted with
antibodies to either phosphotyrosine or IRS-1. As shown in Fig. 4D, IRS-1 coprecipitated with Grb2 in ATA- and EB-treated as well as in
IGF-I-treated cells.
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, ATA and EB, similar to
IGF-I, but not suramin, stimulated the phosphorylation of Akt.
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, the two phosphorylated isoforms, Erk-1 (p44) and Erk-2 (p42),
were clearly detected in blots prepared from cells treated with ATA,
EB, TPA, and IGF-I, but not in suramin- and heparin-treated cells.
ATA and EB compete for IGF-I binding
As ATA and EB activate the IGF-IR, we assessed their ability to
compete for IGF-I binding to the IGF-IR. Two additional polyanions,
fuchsin and suramin, structurally related to ATA and EB, respectively,
that did not activate the IGF-IR were also examined. A typical
concentration- response plot showing inhibition of
[125I]IGF-I binding to MCF-7 cells is
illustrated in Fig. 6. ATA and EB
inhibited [125I]IGF-I binding in a
dose-dependent manner with IC50 of 45 and 130
µg/ml, respectively. The IC50 value for suramin
was 80 µg/ml, whereas fuchsin up to 200 µg/ml had only a minor
inhibitory effect. These data provide further evidence that ATA and EB
interact directly with the IGF-IR. Suramin interference with IGF-I
binding, could be explained by its interacting with IGF-I directly
rather than with its binding sites, as suggested previously (38, 39).
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), in contrast to the
increase in phosphorylation induced by the potent phosphatases
inhibitor vanadate (Fig. 7B). Similar results indicated that EB is not
a phosphatase inhibitor (data not shown).
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, ATA and EB, similar to IGF-I,
stimulated phosphorylation of the IGF-IR in NIH-3T3 cells that
overexpressed the wild-type human IGF-IR (NWTc43), but not in NIH-3T3
cells that overexpressed the kinase-defective receptor (NKR-1). These
findings support the idea that the aromatic polyanions interact with
and activate directly the IGF-IR.
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), ErbB4
(Fig. 9B), or ErbB2 (not shown) in MDA-231 cells. ErbB3 was not
detected in these cells. In contrast, the highly homologous insulin
receptor was heavily phosphorylated by EB and only weakly by ATA in FaO
cells (Fig. 9C), suggesting that ATA is a selective activator for the
IGF-IR.
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). The ability of the
various ATA fractions to induce the phosphorylation of IRS proteins
(p185) and to protect CHX-treated MCF-7 cells from death was
investigated. As shown in Fig. 10, B and C, both the intensity of
IRS-1/IRS-2 tyrosine phosphorylation and the survival of MCF-7 cells
were increased by the high Mr ATA polymers. Low
Mr fractions (<1300) had neither the potency to
stimulate IRS protein phosphorylation nor the ability to inhibit cell
death when tested at a final concentration of 200 µg/ml (data not
shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study we supply evidence to suggest that the nonpeptide molecules ATA and EB promote cell survival and proliferation by activation of the IGF-IR signaling pathway. We have demonstrated in MCF-7 cells that ATA and EB, similar to IGF-I, stimulated tyrosine phosphorylation of the IGF-IR and its major substrates, IRS-1 and IRS-2, and activated central components of the IGF-I signal transduction pathway. Both compounds increased phosphorylation of the IGF-IR in a time- and dose-dependent manner, yet at higher concentrations than the natural ligand. The generality of our observation in MCF-7 cells is supported by the findings that IGF-IR was activated by the two aromatic polyanions in NIH-3T3 cells overexpressing the human IGF-IR as well as in MDA-231 and PC12 cells (data not shown).
The mechanism by which ATA and EB activate the IGF-IR is still unclear. ATA and EB are negatively charged molecules at physiological pH (42). Because of their charge, they seem to be membrane impermeant (29). Therefore, we assume that the negatively charged groups in these compounds interact with positively charged groups on the extracellular domains of the IGF-IR. This binding may lead to a conformational change in the receptor, resulting in activation of its protein-tyrosine kinase. Binding data, which demonstrated a dose-dependent displacement of IGF-I by ATA and EB, support the idea that the two polyanions act as competitive agonists. However, these compounds bind with a lower affinity than the natural ligand to the same binding region on the IGF-IR. Alternatively, the polyanions may interact at a receptor site distinct from the IGF-I-binding site, which, in turn, could induce a conformational change such that IGF-I affinity to the receptor was reduced. The possibility that ATA and EB inhibit IGF-I binding by interacting with IGF-I directly, thus effectively lowering the available IGF-I concentration, cannot be excluded. Suramin, which also inhibited IGF-I binding (but did not activate the IGF-IR), has been shown previously to inhibit the binding of various growth factors, including IGF-I, to cell surface receptors, leading to a decrease in their biological activities. It has been indicated that this inhibition occurs through a direct action on the ligands, rather than on the receptors (38, 39).
Direct binding and activation of the IGF-IR by ATA are further supported by our results in a cell-free system, which indicate that ATA, similar to IGF-I, stimulated the phosphorylation of WGA-purified IGF-IR. Activation of a putative, unknown tyrosine kinase capable of phosphorylating the IGF-IR can be excluded, because neither drug induced phosphorylation of an IGF-IR with a defective ATP-binding domain. In support of this view are findings that the transforming nonreceptor tyrosine kinase Src can directly phosphorylate the kinase-defective IGF-IR in vitro (43). The possibility that ATA or EB enhances tyrosine phosphorylation by inhibition of tyrosine phosphatases is unlikely, because ATA did not augment the maximal phosphorylation induced by IGF-I, in contrast to the increase in phosphorylation induced by the phosphatase inhibitor vanadate. Taken together these results suggest a common mechanism of action shared by the aromatic polyanions and IGF-I.
ATA and EB are known to prevent the death of various cell types
(2, 3, 6). We have shown previously that ATA, similar to
IGF-I, can modulate apoptotic signaling and suppress apoptosis
(1, 10). Phosphorylation of IRS proteins and activation of
PI3K, Akt, and MAPK proteins by ATA and EB indicate the ability of both
aromatic polyanions to activate pivotal elements in IGF-I signaling
that are essential for cell survival (24, 25). Our
findings that ATA and EB mimic the effects of IGF-I may be of great
relevance to their antiapoptotic activity, as IGF-I is a major
cellular survival factor (13, 14, 15). The specificity of this
action is underlined by our findings that other aromatic and
nonaromatic polyanions, such as suramin and heparin, did not activate
this cascade. TPA, which was a survival factor in MCF-7 cells,
activated MAPKs probably by a different pathway, the PKC-Raf-1
mechanism (26), as the drug did not stimulate the
phosphorylation of IRS proteins. Thus, the enhanced cell survival and
the increased proliferation of MCF-7 cells induced by ATA and EB could
be mediated via activation of the IGF-I signaling cascade. In support
of this idea are our findings with fractionated ATA. Upon fractionation
by size exclusion chromatography, we found that fractions that enhanced
the intensity of tyrosyl-phosphorylated IRS-1/IRS-2 also increased the
survival of MCF-7 cells in the presence of CHX, whereas fractions
devoid of IRS phosphorylation activity had no survival ability. In
addition, we found that neither IGF-I nor ATA enhanced the survival of
parental NIH-3T3 cells treated with CHX. By contrast, in NIH-3T3
overexpressing a functional human IGF-IR, 15% and 8% of cell death
were found in the presence of IGF-I and ATA, respectively, compared
with 60% in the presence of CHX only (Wertheim, N., and H. Kanety,
unpublished observations). The idea that the protective ability of ATA
was mediated by the tyrosine phosphorylation cascade was previously
suggested by Okada and Koizumi (33). These researchers
found that MAPKs, Shc proteins, and phospholipase C- were tyrosine
phosphorylated in ATA-treated PC12 cells. They speculated that ATA
activated a certain membranal receptor tyrosine kinase that was not the
nerve growth factor or EGF receptor. Our finding that the IGF-IR
signaling cascade is stimulated by ATA in PC12 cells (unpublished
results) offers a candidate for the speculated receptor tyrosine
kinase.
Recently, it was shown that ATA protected PRL-dependent Nb2 lymphocytes against staurosporine-induced apoptosis (44). In these cells ATA was found to activate the Janus kinase (JAK)-STAT (signal transducer and activator of transcription) signaling pathway, but not through the PRL or the interleukin-2 receptor. As IGF-I receptors were detected in these cells (45), and IGF-IR was found to activate the JAK-STAT pathway (46, 47), activation of the IGF-IR may be one mechanism by which ATA affects the JAK-STAT pathway in Nb2 lymphocytes.
In summary, the results of this study indicate that ATA and EB activate the IGF-IR signal transduction pathway. ATA molecules are selective activators of the IGF-IR, whereas EB also activates the insulin receptor. The survival/proliferation-promoting effects of ATA and EB in MCF-7 cells could be transduced via this pathway. Further understanding of the molecular mechanism by which these compounds protect cell viability may be useful in designing a novel class of nonpeptide antiapoptotic drugs for treatment of a number of pathological conditions, ranging from ischemia or drug toxicity in heart and kidney to neurodegenerative disorders such as Alzheimer’s and Parkinson’s diseases.
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Acknowledgments |
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Footnotes |
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Received November 20, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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to its receptor. Biochem Biophys Res Commun 172:1298–1303[CrossRef][Medline]
IR-3). J Biol Chem 268:2655–2661-induced phosphorylation
of insulin receptor substrate-1 (IRS-1). J Biol Chem 270:23780–23784This article has been cited by other articles:
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