Herstatin is an autoinhibitor of the epidermal growth factor receptor 2 (ErbB2/HER2) that blocks receptor interactions and signaling. This study investigated how herstatin expression affects early epidermal growth factor (EGF) and transforming growth factor beta (TGF-β) signaling pathways and the downstream effects on cell proliferation in mouse fibroblasts. The results showed that herstatin decreased EGF-induced EGFR phosphorylation and delayed receptor downregulation. It also blocked EGF and TGF-β stimulation of the Akt pathway but not the MAPK pathway. While MAPK was fully activated, herstatin prevented TGF-β-induced DNA synthesis and EGF-induced proliferation. These
Introduction, limitations and applications of Microbial consortia engineering. Case study of 'Distributed biological computation with multicellular engineered networks'. Project presentation,
The IRATA safety report for 2013 is out.
Stats are impressive and all segments of construction and industries got something to learn from the IRATA practices.
Introduction, limitations and applications of Microbial consortia engineering. Case study of 'Distributed biological computation with multicellular engineered networks'. Project presentation,
The IRATA safety report for 2013 is out.
Stats are impressive and all segments of construction and industries got something to learn from the IRATA practices.
Channelkit team is now working on a PRO version, with a goal to provide more advanced features that should facilitate and simplify your web research process.
OSU-03012 sensitizes breast cancers to lapatinib-induced cell killing: a role...Enrique Moreno Gonzalez
Lapatinib is characterized as an ErbB1/ErbB2 dual inhibitor and has recently been approved for the treatment of metastatic breast cancer. In this study, we examined mechanisms
associated with enhancing the activity of lapatinib via combination with other therapies.
Evaluating the ability of anti-cancer drugs Etoposide and Staurosporine to in...Davient Bala
Cervical cancer is considered one of the most prevalent cancers affecting Singaporean women.
Although many novel chemotherapeutics have been developed recently, little has been done to
determine the efficiency of current anti-cancer agents working in combination. Here, we aimed to
evaluate the apoptosis induction efficiency of Etoposide and Staurosporine in HeLa cells. Cell cultures
were subjected to either 50 μM Etoposide, 10 nM Staurosporine or both for 24 hours prior visualization
under a fluorescence microscope. We found that Etoposide alone had an efficiency of 16.1% while
Staurosporine alone had 18.3%. However, the polytherapy achieved an efficiency of up to 33.6%,
which indicates an additive effect of both drugs to induce apoptosis. Our results demonstrate that
Etoposide and Staurosporine are both capable of inducing apoptosis in HeLa cells. Furthermore, it
reveals the potential of Etoposide-Staurosporine polytherapy to be a potent combinative treatment
option for cervical cancer patients resistant or sensitive to conventional anti-cancer agents.
2. Herstatin Blocks EGF Activation of Akt and Growth 20619
contrast to dominant negative mutants, herstatin does not
require a membrane anchor to achieve complex formation and
trans inhibition, suggesting that its novel C-terminal domain
may confer high affinity binding to the receptors. Indeed, the
intron-encoded domain, expressed as a recombinant peptide,
binds to HER-2 and the EGFR (29, 32). Although herstatin
inhibits the initial steps of receptor activation, its impact on
ligand binding and intracellular signaling events has not been
examined. In light of the novel structure and receptor binding
properties of herstatin, determination of its effects on signaling
is required to understand its mechanism of action and impact
on the biology of the ErbB receptors.
In this study, we demonstrate that herstatin selectively mod-ulates
signaling cascades triggered by EGF and TGF-!. These
results suggest that this naturally occurring, alternative
HER-2 product provides a novel mechanism for generating
signaling diversity by EGFs.
EXPERIMENTAL PROCEDURES
Cell Culture and Generation of Stable Herstatin EGFR3T3 Clones—
EGFR3T3 cells were derived from NIH3T3 cells by transfection with
human EGFR in mammalian expression vector pCDNA3.1 (Invitrogen).
Stably transfected clones were selected in DMEM " 10% FBS supple-mented
with 0.4 mg/ml G418. A clonal line expressing high levels of
EGFR was transfected with human herstatin in pCDNA3.1/Hygro (In-vitrogen)
using Superfect reagent (Qiagen) as per the manufacturer’s
instructions. Control 3T3 cell lines were generated by transfection with
herstatin alone or with the corresponding empty vector. Stable cell lines
were selected with 0.1 mg/ml hygromycin B and maintained in DMEM
" 10% FBS containing 0.4 mg/ml G418 and 0.1 mg/ml hygromycin B.
Chinese hamster ovary (CHO) cells were grown in DMEM supple-mented
with 10% fetal bovine serum.
Antibodies—Herstatin polyclonal antibody was generated as de-scribed
previously (32) and used at a dilution of 1:10,000. All antibodies
were diluted into TBST (Tris-buffered saline plus 0.005% (v/v) Tween
20). Herstatin monoclonal antibody was a generous gift from Upstate
Biotechnology (Lake Placid, NY) and was used at a 1:1000 dilution.
Antibodies to MAPK and Akt were obtained from Cell Signaling and
used at a 1:1000 dilution. Phospho-specific polyclonal antibodies to
MAPK (phosphorylated at T202 and Y204) and Akt (phosphorylated at
S473) were also obtained from Cell Signaling and used at a 1:1000
dilution. Rabbit polyclonal anti-EGFR antibody was obtained from
Santa Cruz Biotechnology, Inc. and used at a 1:1000 dilution. Mono-clonal
anti-Shc antibody was obtained from Santa Cruz Biotechnology,
Inc. and used at a 1:1000 dilution. Rabbit polyclonal anti-Grb2 antibody
was obtained from Santa Cruz Biotechnology, Inc. and used at a 1:1000
dilution. Rabbit polyclonal anti-p185HER-2/neu antibody was charac-terized
previously (33) and used at a 1:10,000 dilution. Anti-phospho-tyrosine
monoclonal antibody was obtained from Sigma and used at a
1:10,000 dilution.
Transient Transfections—Cells were grown to !80% confluence in
6-well plates and then the plasmid DNAs indicated in the figure legends
were introduced using LipofectAMINE reagent (Invitrogen) as per the
manufacturer’s instructions. Transfection efficiencies between samples
were compared by co-transfection with fluorescent green protein ex-pression
plasmid (Invitrogen) and inspection by fluorescence micros-copy.
Proteins were analyzed at 40 h after DNA introduction.
Receptor Internalization Assays—Cells were grown to 70% conflu-ence,
serum-starved for 20 h in 0.5% FBS, washed twice in ice-cold PBS,
and incubated with EGF (Upstate Biotechnology) at 100 ng/ml in cold
DMEM for 2 h at 4 °C. Cells were then rinsed twice with PBS, placed in
pre-warmed DMEM, and returned to 37 °C to allow receptor internal-ization.
At various time points, cells were placed on ice and washed
twice with ice-cold PBS, and then cell surface proteins were labeled
with freshly dissolved EZ-link Sulfo-NHS-LC-Biotin (Pierce) at 0.5
mg/ml in PBS for 30 min at room temperature. To quench the biotiny-lation
reaction, cells were placed on ice and washed twice with cold PBS
containing 0.2 mg/ml bovine serum albumin and twice with PBS. EGFR
from lysed cells was immunoprecipitated (see below). Samples were
resolved by SDS-PAGE in a 6% polyacrylamide gel, electrotransferred
to nitrocellulose membrane, and overlaid with streptavidin-horseradish
peroxidase at 1 "g/ml in TBST (Pierce). Biotinylated proteins were
visualized by exposing blots to x-ray film (X-Omat; Eastman Kodak Co.)
after treatment with Supersignal West Pico reagent (Pierce).
Immunoprecipitations—Cells were washed in PBS and then lysed on
ice in MTG (50 mM Tris, pH 8.0, 100 mM NaCl, 10% (v/v) glycerol, 1 (v/v)
Nonidet P-40, and 2 mM sodium orthovanadate) containing protease
and phosphatase inhibitor mixtures I and II (Sigma; used as per the
manufacturer’s recommendations). Cell lysate was cleared by centrifu-gation,
and protein concentrations were quantified by Bradford assay
(Bio-Rad). EGFR from 150 "g of cell lysate was precipitated by over-night
incubation with 1 "g of anti-EGFR at 4 °C. Signaling complexes
from 500 "g of cell extract were precipitated by overnight incubation
with 2 "g of anti-Grb2 at 4 °C. Immune complexes were bound to 25 "l
of protein G-Sepharose (Amersham Biosciences) by co-incubation for 40
min at 4 °C, centrifuged, and washed three times with 1 ml of ice-cold
MTG (EGFR) or PBS (Grb2). Immune complexes were boiled in SDS-PAGE
sample buffer for 5 min and analyzed as a Western blot.
Western Blot Analysis—Western blotting was conducted as described
previously (29). Briefly, cells were lysed, and protein concentrations
were quantified as described for immunoprecipitations. Lysates were
boiled in SDS-PAGE sample buffer and loaded onto polyacrylamide gels
at 30 "g/lane. After electrophoresis, proteins were transferred onto
nitrocellulose, stained with Ponceau S, incubated with antibody as
described above, and visualized by exposure to x-ray film (X-Omat;
Kodak) after treatment with SuperSignal West Pico reagent or Super-
Signal West Dura reagent (Pierce). Blots were stripped with the Re-Blot
Western blot recycling kit (Chemicon International, Inc.) as per the
manufacturer’s instructions.
[3H]Thymidine Incorporation Assay—Cells were grown to 70% con-fluence,
starved for 24 h in DMEM with 0.5% FBS, and then treated
with various concentrations of TGF-! for 18 h at 37 °C. Ligand was
removed, and cells were incubated in the presence of [3H]thymidine (1
"Ci/ml in DMEM) for 4 h at 37 °C. Cells were washed with cold PBS,
incubated in 10% trichloroacetic acid at room temperature for 10 min,
and washed twice with 5% trichloroacetic acid. DNA was precipitated
with 100% ethanol and then solubilized by incubation in 0.2 N NaOH for
10 min at room temperature. Samples were neutralized with 0.4 N HCl
and counted in a scintillation counter.
EGF Proliferation Assay—Cells were plated in quadruplicate, grown
to confluence, serum-starved for 18 h in DMEM containing 0.5% FBS,
and transferred to growth medium (DMEM containing 0.5% FBS and
10 nM EGF). Three days later, growth medium was removed, and live
cells were quantified with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltet-razolium
bromide (Sigma) as described previously (34).
125I-EGF Binding—The 125I-EGF binding analysis was conducted as
described previously (35). Briefly, cells were grown to 70% confluence,
serum-starved for 24 h, and incubated at 4 °C for 2 h with 175 pM
125I-EGF (NEN) and different amounts of unlabeled EGF (total EGF
concentrations ranged from 175 pM to 10 nM) in binding buffer (DMEM
plus 50 mM Hepes, pH 7.4, and 5% (w/v) bovine serum albumin). Cells
were washed and then extracted in 0.1 N NaOH plus 0.1% (w/v) SDS,
and bound 125I-EGF was quantified by gamma counting.
RESULTS
Herstatin Reduces EGF-stimulated Tyrosine Phosphoryla-tion
and Down-regulation of the EGFR—Previous studies have
shown that transient coexpression of herstatin with the EGFR
in CHO cells results in diminished dimerization and tyrosine
phosphorylation of the receptor in response to EGF (29). To
further characterize the effects of herstatin on EGFR and to
investigate its impact on EGF-induced intracellular signaling,
stable cell lines that express different levels of herstatin were
derived from NIH3T3 cells expressing human EGFR (termed
EGFR3T3 cells). Efforts to stably express herstatin in several
tumor cell lines that overexpress EGFR as well as HER-2 were
thwarted, presumably because of an inhibitory effect on cell
survival. EGFR3T3 cells were transfected with control or her-statin
expression plasmid, and clonal populations were isolated
by selection with hygromycin B. Varied levels of herstatin were
expressed in herstatin-transfected cell lines but not in the
control-transfected cells (Fig. 1A).
To investigate the effects of herstatin on ligand-induced ac-tivation
of the receptor, herstatin- and control-transfected
EGFR3T3 cells were serum-starved for 20 h and treated with
saturating concentrations (10 nM) of EGF for 20 min, and
phosphotyrosine levels of the EGFR were assessed. Although
clone 1 produced the greatest amount of herstatin, each of the
three herstatin-expressing clones exhibited a similar reduction
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3. 20620 Herstatin Blocks EGF Activation of Akt and Growth
in tyrosine phosphorylation of the EGFR (Fig. 1B; see also Figs.
4B and 6A), suggesting that maximal inhibition was achieved.
The EGFR expression levels were comparable between paren-tal
cells and the three different clones, showing that expression
of herstatin did not down-regulate the EGFR (Fig. 1B).
Because receptor tyrosine phosphorylation is required for
endocytosis (36), we hypothesized that reduced EGFR auto-phosphorylation
levels in the presence of herstatin alter EGF-mediated
receptor down-regulation from the cell surface. Pa-rental
and herstatin-expressing EGFR3T3 cells were saturated
with EGF at 4 °C and then returned to 37 °C, a temperature
permissive for internalization. After various incubation times,
cell surface proteins were biotinylated, and EGFR was immu-noprecipitated
and visualized by streptavidin-horseradish per-oxidase
overlay. In the parental cell line, cell surface EGFR
was reduced by 80% at 10 min and was undetectable by 20 min
after the removal of the temperature block (Fig. 1C), in agree-ment
with other studies (37, 38). In herstatin-expressing cells,
however, cell surface EGFR was reduced only about 35% at 10
min, and about one-third of the EGFR remained at the cell
surface after 20 min of incubation at 37 °C. Delayed down-regulation
is consistent with previous findings showing that
EGFR degradation, a process contingent upon endosomal loca-tion
of the receptor, is blocked in CHO cells that transiently
express herstatin (29).
Herstatin Inhibits EGF-stimulated Akt Phosphorylation, but
MAPK Is Fully Activated—Signal transduction through the
ErbB family includes both the MAPK and the PI3K/Akt signal-ing
pathways, which are generally stimulated simultaneously
by growth factors (11–13). To examine the role of herstatin in
EGF signaling downstream from the receptor, we treated
EGFR3T3 parental and herstatin-expressing cells with satu-rating
amounts of EGF (16 nM) and observed the kinetics of
MAPK and Akt phosphorylation over a 1-h time course. In the
parental cell line, the highest level of phospho-Akt was de-tected
at 20 min after EGF addition (Fig. 2A). In the herstatin-expressing
cells, however, little phospho-Akt was observed in
EGF-treated cells, with maximal levels reaching only 2% of the
parental controls. Phosphorylation of Akt was almost com-pletely
abolished in the presence of herstatin; interestingly,
there was no reduction of phospho-MAPK levels. In parental
and herstatin-expressing cells, the time course and extent of
MAPK activation were identical; maximal activation was
achieved by 5 min and declined at 20 min after the addition of
EGF (Fig. 2A). To determine whether induction of MAPK in
herstatin-expressing cells was due to ectopic overexpression of
EGFR (39–41), we investigated EGF signaling through endog-enous
receptors in 3T3 cells that express herstatin. As in the
EGFR3T3 cells, we observed preferential EGF activation of
MAPK and abrogation of Akt phosphorylation in the presence
of herstatin (Fig. 2B). These data demonstrate that the signal-ing
profile caused by herstatin expression is not affected by
ectopic overexpression of EGFR.
EGF Induces the Formation of a Signaling Complex Contain-ing
EGFR, phospho-Shc, and Grb2 in Herstatin-expressing
Cells—After EGF treatment, the adaptor protein Shc binds the
autophosphorylation domain of EGFR, is tyrosine-phospho-rylated,
and recruits the Grb2-Sos complex from the cytoplasm
to the plasma membrane (42). To investigate whether EGF
stimulation of MAPK in the presence of herstatin was induced
by EGFR through Shc and Grb2, we immunoprecipitated Grb2
and examined the immune complex by Western blotting. In
both parental and herstatin-expressing cells, EGF-dependent
association of EGFR and Shc with Grb2 was detected (Fig. 3).
Furthermore, Shc was tyrosine-phosphorylated to a similar
extent (Fig. 3), providing evidence for an active signaling com-plex.
Because HER-2 is the preferred heterodimer partner of
EGFR (4–6), endogenous HER-2 may be present in the EGFR-Shc-
Grb2 signaling complex, contributing to EGF stimulation
of the MAPK signaling cascade. However, HER-2 could not be
detected in the signaling complex immunoprecipitated from
500 "g of either parental or herstatin-expressing cells (data not
shown), even though the high titer antibody used (33) detects
p185HER-2 in 20 "g of 3T3 cell extract. These data suggest
that Shc and Grb2 transduce the herstatin-mediated EGF sig-nal
from EGFR to components of the MAPK cascade, with no
evidence of HER-2 involvement.
Characterization of the Effects of EGF Concentration on In-tracellular
Signaling in the Presence and Absence of Hersta-tin—
Previous studies have shown that at very low EGF con-centrations,
MAPK activation occurs in the absence of Akt
activation and EGFR tyrosine phosphorylation (43). We there-fore
examined whether signaling in EGFR3T3 cells exhibits a
similar sensitivity to EGF concentration. Maximal stimulation
of MAPK occurred independently of Akt activation in
EGFR3T3 cells treated with 0.01 nM EGF (Fig. 4A), suggesting
that ectopic overexpression of EGFR did not eliminate sensi-tivity
to very low concentrations of EGF. At 0.1 nM EGF, a
subsaturating concentration, maximal stimulation of Akt was
observed in parental cells, but stimulation of Akt was inhibited
in the herstatin-expressing cells. Therefore, preferential inhi-bition
of the Akt pathway by herstatin could reflect a quanti-tative
reduction in effective EGF concentration by competitive
FIG. 1. Herstatin expression, EGFR tyrosine phosphorylation,
and receptor down-regulation in parental and herstatin-trans-fected
EGFR3T3 cells. A, 30 "g of lysate from parental and herstatin-expressing
EGFR3T3 cells was separated by 7.5% SDS-PAGE and
subjected to Western blot analysis for herstatin as described under
“Experimental Procedures.” B, herstatin- and mock-transfected
EGFR3T3 cells were serum-starved for 20 h and then incubated with 10
nM EGF for 20 min at 37 °C. Cell extracts were resolved by 6% SDS-PAGE
and analyzed as a Western blot using anti-phosphotyrosine
antibody. The blot was stripped as described under “Experimental
Procedures” and reprobed with anti-EGFR. Results are representative
of three independent experiments. C, herstatin (clone 1)- and mock-transfected
EGFR3T3 cells were serum-starved as described above,
incubated with 16 nM EGF at 4 °C for 2 h, and incubated at 37 °C for the
durations indicated. Cell surface proteins were biotinylated as de-scribed
under “Experimental Procedures.” EGFR was immunoprecipi-tated
from 150 "g of lysate, and immune complexes were separated by
6% SDS-PAGE, transferred to nitrocellulose, and then overlaid with
streptavidin-horseradish peroxidase. Films were scanned by imaging
densitometry (BioRad, model GS700) to quantitate streptavidin-horse-radish
peroxidase signal. Similar results were observed in two inde-pendent
experiments.
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4. Herstatin Blocks EGF Activation of Akt and Growth 20621
FIG. 2. EGF-induced signaling in parental and herstatin-expressing EGFR3T3 cells. A, herstatin (clone 1)- and mock-transfected
EGFR3T3 cells were serum-starved and incubated with 16 nM EGF at 37 °C for the durations indicated. Cell lysates were separated by 7.5% and
10% SDS-PAGE and then subjected to Western blot analysis using antibodies specific for phospho-Akt (phospho-Ser473) and phospho-p42/p44
MAPK (phospho-T202 and Y204). Exposed films were scanned by imaging densitometry to quantitate phospho-Akt signal. Blots were stripped and
probed with Akt antibody. Results are representative of six independent experiments. B, herstatin- and mock-transfected 3T3 cells were
serum-starved and incubated with 10 nM EGF at 37 °C for the durations indicated. Cell lysates were separated by 7.5% and 10% SDS-PAGE and
then subjected to Western blot analysis as described above.
inhibition. However, after treatment with either 1 or 100 nM
EGF (#100 times KD), EGFR phosphotyrosine levels were di-minished
!10-fold, and Akt activation was reduced to the same
extent (Fig. 4B). These data suggest that herstatin has a qual-itative
impact on intracellular signaling that is independent of
EGF concentration.
Herstatin Expression Does Not Alter the Binding Affinity of
EGF for EGR3T3 Cells—Previous studies demonstrated that
herstatin binds to the extracellular domain of the HER-2 re-ceptor
(32) and forms a stable complex with EGFR (29). Inhi-bition
of EGFR by herstatin could be caused by interference
with EGF binding. To examine this possibility, we character-ized
the binding affinity of EGF to parental and herstatin-expressing
clones of EGFR3T3 cells. The cells were incubated
with subsaturating amounts (175 pM) of 125I-EGF, and its dis-placement
by unlabeled EGF was measured (Fig. 5). The dis-placement
curve exhibited by the parental cells was indistin-guishable
from that of cell lines that expressed either high
(clone 1) or low (clones 2 and 3) levels of herstatin (see Fig. 1A).
Moreover, Scatchard analysis of the binding data, as described
in the legend to Fig. 6, revealed the same apparent dissociation
constant of !500 pM in the absence and presence of different
levels of herstatin. These studies show that herstatin expres-sion
does not prevent EGF binding or alter the EGF binding
affinity, suggesting that herstatin is not a competitive inhibitor
of EGF-mediated EGFR activation. Herstatin therefore modu-lates
signaling of receptors that are complexed with EGF.
Herstatin Inhibits TGF-!-induced Receptor Phosphorylation
and Akt Phosphorylation, Whereas MAPK Activation Is Unaf-fected—
TGF-! is an EGFR ligand that has increased mitogenic
and transforming potency compared with EGF (44). Although
these ligands compete for receptor binding, they exhibit subtly
different binding properties to the EGFR (45) and show distinct
co-receptor-dependent signal potentiation (37). Despite these
differences, parental and herstatin-expressing EGFR3T3 cells
treated with TGF-! displayed signaling profiles similar to
those observed in response to EGF stimulation. Depression of
EGFR tyrosine phosphorylation occurred in the herstatin-ex-pressing
cells, particularly at high (100 nM) concentrations of
TGF-! (Fig. 6A). Moreover, phospho-Akt levels were markedly
decreased, whereas MAPK activation was unaffected at both
low (1 nM) and high (100 nM) concentrations of TGF-! (Fig. 6A).
Herstatin Expression Does Not Alter FGF-2 Stimulation of Akt—
The strong suppression of EGF- and TGF-!-induced Akt phospho-rylation
in the EGFR3T3 cells that stably express herstatin could
be an indirect effect of herstatin expression or chronic ErbB recep-tor
inhibition. To examine the integrity of the PI3K/Akt pathway,
we monitored phospho-Akt levels after treatment with FGF-2, a
growth factor that activates a heterologous receptor tyrosine ki-nase.
Parental and herstatin-expressing EGFR3T3 cells were se-rum-
starved, exactly as done before EGF treatment, and then cells
were incubated with saturating amounts of FGF-2 (10 nM) for 20
min. FGF-2 treatment stimulated the tyrosine phosphorylation of
an 119-kDa protein, the approximate size of the FGF receptor (Fig.
6B). Furthermore, FGF-2 increased phospho-Akt levels to an equiv-alent
extent in the presence and absence of herstatin (Fig. 6B).
These data demonstrate the functional integrity of the PI3K/Akt
pathway and suggest that the reduction of phospho-Akt levels
caused by herstatin expression is specific to EGF- and TGF-!-
induced signaling.
FIG. 3. EGF induces EGFR-Shc-Grb2 complex formation in
herstatin-expressing EGFR3T3 cells. Herstatin (clone 1)- and
mock-transfected EGFR3T3 cells were serum-starved and incubated
with 10 nM EGF at 37 °C for 20 min. Proteins complexed to anti-Grb2
were immunoprecipitated from 500 "g of cell lysate, separated by 8%
and 12% SDS-PAGE, and then subjected to Western blot analysis using
the antibodies indicated. The anti-phosphotyrosine blot was stripped
and reprobed with anti-Shc antibody. Results are representative of two
independent experiments.
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5. 20622 Herstatin Blocks EGF Activation of Akt and Growth
Herstatin Uncouples MAPK from Akt Activation in Tran-siently
Transfected CHO Cells—To evaluate whether the un-coupling
of phospho-Akt from MAPK activation by herstatin
was a feature confined to the 3T3 cell background, we exam-ined
the EGF signaling profile in CHO cells, which do not
express endogenous EGFR (25). The cells were transiently
transfected with EGFR and different levels of herstatin expres-sion
plasmids and then treated with EGF. Fig. 6C illustrates
that EGF induction of MAPK phosphorylation were unaffected
by expression of different amounts of herstatin. In contrast,
Akt activation was inhibited in proportion to herstatin expres-sion
levels.
Herstatin Depresses Mitogenic Stimulation by EGF and
TGF-!—To test whether herstatin affected the proliferative func-tions
of the EGFR growth factors, TGF-!-induced mitogenesis
was assessed by measuring DNA synthesis. Cells were first
forced into quiescence by serum starvation for 40 h and then
treated with different concentrations of TGF-!. The ligand was
removed, and cells were incubated with [3H]thymidine to quan-tify
DNA synthesis. In the presence of herstatin, we observed a
striking decrease in TGF-!-induced [3H]thymidine incorporation
that was not overcome at high ligand concentrations (Fig. 7A).
The impact of herstatin on EGF-mediated cell proliferation was
examined by quantitation of live cells by the 3-(4,5-dimethylthia-zol-
2-yl)-2,5-diphenyltetrazolium bromide assay (34). Equal
numbers of EGFR3T3 control and herstatin-transfected cells
were plated, serum-starved, and then treated for 72 h with ve-hicle
or EGF. Herstatin expression resulted in a significant re-duction
in viable cells in the absence of EGF (p $ 0.007; Fig. 7B),
which may reflect diminished survival under conditions of serum
deprivation. Whereas EGF treatment significantly increased the
control EGFR3T3 cells (p $ 0.006; Fig. 7B), cells expressing
herstatin displayed no significant proliferation in response to
EGF treatment. These results demonstrate that herstatin inter-rupts
TGF-!- and EGF-mediated mitogenic signal transduction,
resulting in inhibition of proliferation.
DISCUSSION
Investigating the mechanisms employed by ErbB inhibitors
and their impact on signaling is critical to understanding the
biology of these receptors and to developing anti-receptor tyro-sine
kinase therapeutics. Whereas several inhibitors of the
EGFR have been investigated, herstatin is distinguished by its
novel structure, consisting of subdomains I and II of the HER-2
ECD and an intron encoded-C-terminal domain (32). Further-more,
herstatin is the only naturally occurring inhibitor of the
EGFR in mammalian cells that exerts its action on the initial
events in receptor activation: dimerization and autophospho-rylation
(29, 32). In this study, we show that herstatin selec-tively
modulates the intracellular signaling pathways stimu-lated
by EGFR ligands. EGF binds to its receptor with normal
affinity in the presence of herstatin, yet receptor tyrosine phos-phorylation
and down-regulation are suppressed. Whereas her-statin
allows full ligand stimulation of the MAPK pathway and
its upstream effector, Shc, Akt phosphorylation is selectively
blocked, resulting in suppression of cell growth.
Herstatin is a secreted protein that binds to and inhibits the
EGFR (29). Although the binding site has not been mapped,
previous observations suggest that herstatin associates with
the ECD of ErbB receptors (32). We therefore examined
whether herstatin may interfere with binding of EGF. Our
results demonstrate that neither the binding affinity nor the
overall number of EGF binding sites was significantly altered
in cells that expressed either low or very high levels of hersta-tin.
Therefore, herstatin appears to inhibit EGFR that is occu-pied
by growth factor. Two other classes of EGFR inhibitors
compete with ligand binding, including the Drosophila ligand
FIG. 4. Effects of EGF concentra-tion
on signaling in parental and her-statin-
expressing EGFR3T3 cells. A,
herstatin (clone 1)- and mock-transfected
EGFR3T3 cells were serum-starved,
treated with 0, 0.01, and 10 nM EGF at
37 °C for 20 min, and analyzed by West-ern
blot as described in the Fig. 2 legend.
Staining with Ponceau S confirms equal
loading. B, herstatin (clone 1)- and mock-transfected
EGFR3T3 cells were treated
with l or 100 nM EGF as described in A.
Exposed films were scanned by imaging
densitometry to quantitate phosphoty-rosine
signal. Results are representative
of two independent experiments.
FIG. 5. Displacement of 125I-EGF by unlabeled EGF in parental
and herstatin-expressing EGFR3T3 cells. Herstatin and mock-transfected
EGFR3T3 cells were plated at 5 % 104 cells/well in 24-well
plates and serum-starved. Cells were incubated for 2 h at 4 °C with 175
pM 125I-EGF and unlabeled EGF at total concentrations ranging from
175 pM to 10 nM. Displacement of bound 125I-EGF by unlabeled EGF
was plotted as the maximum percentage bound 125I-EGF. The dissoci-ation
constants (KD) of EGF for herstatin- and mock-transfected
EGFR3T3 clones were estimated by Scatchard analysis: bound/free
versus bound was plotted using KaleidaGraph software (Synergy Anal-ysis,
1997); the slopes of regression lines as calculated by the program
provided the estimate of &1/KD. All regression line R2 values exceeded
0.99.
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6. Herstatin Blocks EGF Activation of Akt and Growth 20623
FIG. 6. Herstatin effects on signaling induced by EGF, TGF-!, and FGF-2. A, herstatin (clone 1)- and mock-transfected EGFR3T3 cells
were serum-starved and treated with l or 100 nM TGF-! at 37 °C for 20 min and analyzed as described in Fig. 4B. Results are representative of
two independent experiments. B, herstatin (clone 1)- and mock-transfected EGFR3T3 cells were serum-starved and treated with 100 nM FGF-2 at
37 °C for 20 min, and cell lysate was subjected to Western blot analysis using antibodies against phosphotyrosine and phospho-Akt. The blot was
stripped and incubated with Akt antibody. C, CHO cells were grown to 80% confluence and transfected with 1.5 "g of EGFR and 0.5 or 1.5 "g of
herstatin expression plasmids. 20 h after transfection, cells were serum-starved, treated with 10 nM EGF for 20 min at 37 °C, and analyzed for
phospho-Akt and phospho-p42/p44 MAPK by Western blot. Equal loading was confirmed by Ponceau S staining.
Argos (46, 47) and the EGFR monoclonal antibody C225 (48).
Because herstatin inhibits ligand-occupied receptor, it may be
effective even when growth factors are overproduced by tumors
(49).
In this study, we demonstrate that herstatin uncouples in-tracellular
signaling pathways triggered by EGF and TGF-!.
Whereas EGFR tyrosine phosphorylation and Akt activation
are inhibited, Shc and MAPK are fully activated. This is in
contrast to the intracellular signaling effects of several other
inhibitors of EGFR including the quinazoline inhibitors (12),
the p185neu dominant negative mutant (15), and the C225
monoclonal antibody inhibitor (14), which suppress EGF-in-duced
phosphorylation of both MAPK and Akt. Similar to her-statin,
these inhibitors reduce EGFR tyrosine phosphorylation
and cause prolonged retention of the EGFR at the cell surface.
In agreement with previous studies (43), we also observed
uncoupling of MAPK and Akt signaling cascades at very low
concentrations of EGF (0.01 nM), suggesting that either limit-ing
amounts of ligand or the presence of herstatin preferen-tially
stimulated the MAPK cascade. The signaling effects of
herstatin can not be explained by a reduction in the effective
concentration of growth factor; the EGF binding affinity was
unaffected by herstatin, and selective suppression of Akt sig-naling
was observed at both subsaturating (0.1 nM) and very
high ligand concentrations (100 nM).
Because phosphorylation of specific tyrosine residues on ac-tivated
receptors is responsible for the recruitment and activa-tion
of distinct intracellular signaling molecules (51), inhibition
of some, but not all, EGFR phosphorylation sites by herstatin
may cause preferential recruitment of effector molecules (52).
Evaluation of this possibility will require phosphopeptide map-ping
of the EGFR from the herstatin-expressing cells. However,
even in the absence of EGFR tyrosine phosphorylation, either
by kinase-impaired EGFR (39–41) or EGFR missing its tyro-sine
phosphorylation sites (53), MAPK is activated, whereas
Akt is not stimulated by EGF (16, 54). In these cases (53, 55),
as well as with herstatin, EGFR appears to be involved in
activation of the MAPK cascade, as shown by its presence in a
signaling complex containing tyrosine phosphorylated Shc and
Grb2. An endogenous receptor tyrosine kinase or a cytoplasmic
kinase such as src (56) may also participate in MAPK activa-tion
by EGF. Endogenous HER-2, the preferred EGFR hetero-meric
partner, is unlikely to be responsible for MAPK activa-tion
because herstatin effectively disrupts HER-2 homomeric
FIG. 7. TGF-!-induced DNA synthesis and EGF-induced prolif-eration
of parental and herstatin-expressing EGFR3T3 cells. A,
herstatin (clone 1)- and mock-transfected EGFR3T3 cells were plated at
5 % 104 in triplicate, serum-starved, and treated with TGF-! at the
concentrations indicated. At 18 h, cells were incubated with [3H]thymi-dine
as described under “Experimental Procedures.” Levels of [3H]thy-midine
incorporation are expressed as a percentage of the untreated
control. Error bars indicate sample mean ' S.D. Results are represent-ative
of three independent experiments. B, herstatin (clone 1)- and
mock-transfected EGFR3T3 clones were grown to 90% confluence, se-rum-
starved, and treated with 10 nM EGF. At 72 h, live cells were
quantified by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-mide
assay as described under “Experimental Procedures.” Error bars
indicate sample mean ' S.D. Results are representative of two inde-pendent
experiments.
Downloaded from http://www.jbc.org/ at Harvard Libraries on December 1, 2014
7. 20624 Herstatin Blocks EGF Activation of Akt and Growth
and heteromeric interactions (29). Although we were unable to
detect HER-2 in the EGF-induced signaling complex, other
endogenous tyrosine kinases may be involved in MAPK activa-tion
in herstatin-expressing cells.
A major question in signaling through receptor tyrosine ki-nases
concerns the mechanism by which growth factors can
stimulate diverse cellular responses (52, 57, 58). For the ErbB
receptors, one level of diversity is achieved through generation
of different ErbB dimer pairs that have been found to differen-tially
stimulate intracellular signaling pathways (10, 51, 52,
57). Alternatively, signaling by the same dimer pair may be
altered in response to activation by different growth factors (8,
22, 52). Additionally, EGF ligands have been shown to promote
diverse cellular responses, depending on the cell type (59). The
results presented here point to a novel mechanism of generat-ing
diversity by which a single ligand, EGF, can achieve altered
signal output within a given cell context. Our studies suggest
that herstatin, a HER-2 receptor variant expressed in fetal
kidney and liver cells, can selectively alter EGF signal output,
resulting in growth arrest.
The Ras/MAPK pathway was previously suggested to be the
major mitogenic signaling pathway initiated by the EGFR (60).
In the presence of herstatin, full stimulation of MAPK by
TGF-! was insufficient to drive entry into S phase, as sug-gested
by the suppression of DNA synthesis. Moreover, EGF
stimulation of MAPK was insufficient to stimulate cell prolif-eration
in the presence of herstatin. Because Akt activation
was strongly inhibited, components of the PI3K signaling path-way
that are required for growth factor-induced proliferation
may not be activated in the presence of herstatin. This finding
is in agreement with recent observations that interruption of
the MAPK pathway by EGFR blockade with quinazoline inhib-itors
is not the cause of G1 arrest, but rather interruption of
PI3K function is required (12).
Signaling through the EGF receptor is often enhanced in hu-man
cancers through overexpression of the receptor and auto-crine
stimulation by ligands produced by the tumor (49). En-hanced
EGFR signaling in several carcinomas is directly coupled
to inappropriate phospho-Akt survival signals, rendering many
cancers resistant to apoptotic signals, including those activated
by radiation and chemotherapies (12, 19). EGF stimulation of
Akt kinase activity has been proposed as a major mechanism
behind enhanced survival conferred by inappropriate EGF sig-naling.
The activation of Akt appears to be both required and
sufficient for the antiapoptotic function of EGF (19). Results
presented here point to the effectiveness of herstatin in blockage
of Akt activation and inhibition of proliferation stimulated by
EGF. Previous studies also demonstrate the effectiveness of her-statin
in blocking proliferation stimulated by HER-2 overexpres-sion,
which often occurs in the same tumor cells that have en-hanced
EGFR signaling. The results presented here further
support the potential utility of herstatin in the development of
therapeutics against cancers with ErbB receptor involvement.
Acknowledgments—We thank L. S. Shamieh for helpful discussions
and critical reading of the manuscript, M. E. Sommer for generating the
EGFR3T3/herstatin cell lines, and M. C. Denton for constructing the
herstatin and EGFR expression plasmids.
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![Herstatin Blocks EGF Activation of Akt and Growth 20619
contrast to dominant negative mutants, herstatin does not
require a membrane anchor to achieve complex formation and
trans inhibition, suggesting that its novel C-terminal domain
may confer high affinity binding to the receptors. Indeed, the
intron-encoded domain, expressed as a recombinant peptide,
binds to HER-2 and the EGFR (29, 32). Although herstatin
inhibits the initial steps of receptor activation, its impact on
ligand binding and intracellular signaling events has not been
examined. In light of the novel structure and receptor binding
properties of herstatin, determination of its effects on signaling
is required to understand its mechanism of action and impact
on the biology of the ErbB receptors.
In this study, we demonstrate that herstatin selectively mod-ulates
signaling cascades triggered by EGF and TGF-!. These
results suggest that this naturally occurring, alternative
HER-2 product provides a novel mechanism for generating
signaling diversity by EGFs.
EXPERIMENTAL PROCEDURES
Cell Culture and Generation of Stable Herstatin EGFR3T3 Clones—
EGFR3T3 cells were derived from NIH3T3 cells by transfection with
human EGFR in mammalian expression vector pCDNA3.1 (Invitrogen).
Stably transfected clones were selected in DMEM " 10% FBS supple-mented
with 0.4 mg/ml G418. A clonal line expressing high levels of
EGFR was transfected with human herstatin in pCDNA3.1/Hygro (In-vitrogen)
using Superfect reagent (Qiagen) as per the manufacturer’s
instructions. Control 3T3 cell lines were generated by transfection with
herstatin alone or with the corresponding empty vector. Stable cell lines
were selected with 0.1 mg/ml hygromycin B and maintained in DMEM
" 10% FBS containing 0.4 mg/ml G418 and 0.1 mg/ml hygromycin B.
Chinese hamster ovary (CHO) cells were grown in DMEM supple-mented
with 10% fetal bovine serum.
Antibodies—Herstatin polyclonal antibody was generated as de-scribed
previously (32) and used at a dilution of 1:10,000. All antibodies
were diluted into TBST (Tris-buffered saline plus 0.005% (v/v) Tween
20). Herstatin monoclonal antibody was a generous gift from Upstate
Biotechnology (Lake Placid, NY) and was used at a 1:1000 dilution.
Antibodies to MAPK and Akt were obtained from Cell Signaling and
used at a 1:1000 dilution. Phospho-specific polyclonal antibodies to
MAPK (phosphorylated at T202 and Y204) and Akt (phosphorylated at
S473) were also obtained from Cell Signaling and used at a 1:1000
dilution. Rabbit polyclonal anti-EGFR antibody was obtained from
Santa Cruz Biotechnology, Inc. and used at a 1:1000 dilution. Mono-clonal
anti-Shc antibody was obtained from Santa Cruz Biotechnology,
Inc. and used at a 1:1000 dilution. Rabbit polyclonal anti-Grb2 antibody
was obtained from Santa Cruz Biotechnology, Inc. and used at a 1:1000
dilution. Rabbit polyclonal anti-p185HER-2/neu antibody was charac-terized
previously (33) and used at a 1:10,000 dilution. Anti-phospho-tyrosine
monoclonal antibody was obtained from Sigma and used at a
1:10,000 dilution.
Transient Transfections—Cells were grown to !80% confluence in
6-well plates and then the plasmid DNAs indicated in the figure legends
were introduced using LipofectAMINE reagent (Invitrogen) as per the
manufacturer’s instructions. Transfection efficiencies between samples
were compared by co-transfection with fluorescent green protein ex-pression
plasmid (Invitrogen) and inspection by fluorescence micros-copy.
Proteins were analyzed at 40 h after DNA introduction.
Receptor Internalization Assays—Cells were grown to 70% conflu-ence,
serum-starved for 20 h in 0.5% FBS, washed twice in ice-cold PBS,
and incubated with EGF (Upstate Biotechnology) at 100 ng/ml in cold
DMEM for 2 h at 4 °C. Cells were then rinsed twice with PBS, placed in
pre-warmed DMEM, and returned to 37 °C to allow receptor internal-ization.
At various time points, cells were placed on ice and washed
twice with ice-cold PBS, and then cell surface proteins were labeled
with freshly dissolved EZ-link Sulfo-NHS-LC-Biotin (Pierce) at 0.5
mg/ml in PBS for 30 min at room temperature. To quench the biotiny-lation
reaction, cells were placed on ice and washed twice with cold PBS
containing 0.2 mg/ml bovine serum albumin and twice with PBS. EGFR
from lysed cells was immunoprecipitated (see below). Samples were
resolved by SDS-PAGE in a 6% polyacrylamide gel, electrotransferred
to nitrocellulose membrane, and overlaid with streptavidin-horseradish
peroxidase at 1 "g/ml in TBST (Pierce). Biotinylated proteins were
visualized by exposing blots to x-ray film (X-Omat; Eastman Kodak Co.)
after treatment with Supersignal West Pico reagent (Pierce).
Immunoprecipitations—Cells were washed in PBS and then lysed on
ice in MTG (50 mM Tris, pH 8.0, 100 mM NaCl, 10% (v/v) glycerol, 1 (v/v)
Nonidet P-40, and 2 mM sodium orthovanadate) containing protease
and phosphatase inhibitor mixtures I and II (Sigma; used as per the
manufacturer’s recommendations). Cell lysate was cleared by centrifu-gation,
and protein concentrations were quantified by Bradford assay
(Bio-Rad). EGFR from 150 "g of cell lysate was precipitated by over-night
incubation with 1 "g of anti-EGFR at 4 °C. Signaling complexes
from 500 "g of cell extract were precipitated by overnight incubation
with 2 "g of anti-Grb2 at 4 °C. Immune complexes were bound to 25 "l
of protein G-Sepharose (Amersham Biosciences) by co-incubation for 40
min at 4 °C, centrifuged, and washed three times with 1 ml of ice-cold
MTG (EGFR) or PBS (Grb2). Immune complexes were boiled in SDS-PAGE
sample buffer for 5 min and analyzed as a Western blot.
Western Blot Analysis—Western blotting was conducted as described
previously (29). Briefly, cells were lysed, and protein concentrations
were quantified as described for immunoprecipitations. Lysates were
boiled in SDS-PAGE sample buffer and loaded onto polyacrylamide gels
at 30 "g/lane. After electrophoresis, proteins were transferred onto
nitrocellulose, stained with Ponceau S, incubated with antibody as
described above, and visualized by exposure to x-ray film (X-Omat;
Kodak) after treatment with SuperSignal West Pico reagent or Super-
Signal West Dura reagent (Pierce). Blots were stripped with the Re-Blot
Western blot recycling kit (Chemicon International, Inc.) as per the
manufacturer’s instructions.
[3H]Thymidine Incorporation Assay—Cells were grown to 70% con-fluence,
starved for 24 h in DMEM with 0.5% FBS, and then treated
with various concentrations of TGF-! for 18 h at 37 °C. Ligand was
removed, and cells were incubated in the presence of [3H]thymidine (1
"Ci/ml in DMEM) for 4 h at 37 °C. Cells were washed with cold PBS,
incubated in 10% trichloroacetic acid at room temperature for 10 min,
and washed twice with 5% trichloroacetic acid. DNA was precipitated
with 100% ethanol and then solubilized by incubation in 0.2 N NaOH for
10 min at room temperature. Samples were neutralized with 0.4 N HCl
and counted in a scintillation counter.
EGF Proliferation Assay—Cells were plated in quadruplicate, grown
to confluence, serum-starved for 18 h in DMEM containing 0.5% FBS,
and transferred to growth medium (DMEM containing 0.5% FBS and
10 nM EGF). Three days later, growth medium was removed, and live
cells were quantified with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltet-razolium
bromide (Sigma) as described previously (34).
125I-EGF Binding—The 125I-EGF binding analysis was conducted as
described previously (35). Briefly, cells were grown to 70% confluence,
serum-starved for 24 h, and incubated at 4 °C for 2 h with 175 pM
125I-EGF (NEN) and different amounts of unlabeled EGF (total EGF
concentrations ranged from 175 pM to 10 nM) in binding buffer (DMEM
plus 50 mM Hepes, pH 7.4, and 5% (w/v) bovine serum albumin). Cells
were washed and then extracted in 0.1 N NaOH plus 0.1% (w/v) SDS,
and bound 125I-EGF was quantified by gamma counting.
RESULTS
Herstatin Reduces EGF-stimulated Tyrosine Phosphoryla-tion
and Down-regulation of the EGFR—Previous studies have
shown that transient coexpression of herstatin with the EGFR
in CHO cells results in diminished dimerization and tyrosine
phosphorylation of the receptor in response to EGF (29). To
further characterize the effects of herstatin on EGFR and to
investigate its impact on EGF-induced intracellular signaling,
stable cell lines that express different levels of herstatin were
derived from NIH3T3 cells expressing human EGFR (termed
EGFR3T3 cells). Efforts to stably express herstatin in several
tumor cell lines that overexpress EGFR as well as HER-2 were
thwarted, presumably because of an inhibitory effect on cell
survival. EGFR3T3 cells were transfected with control or her-statin
expression plasmid, and clonal populations were isolated
by selection with hygromycin B. Varied levels of herstatin were
expressed in herstatin-transfected cell lines but not in the
control-transfected cells (Fig. 1A).
To investigate the effects of herstatin on ligand-induced ac-tivation
of the receptor, herstatin- and control-transfected
EGFR3T3 cells were serum-starved for 20 h and treated with
saturating concentrations (10 nM) of EGF for 20 min, and
phosphotyrosine levels of the EGFR were assessed. Although
clone 1 produced the greatest amount of herstatin, each of the
three herstatin-expressing clones exhibited a similar reduction
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