chemistry & biology articleirds have been reported to act as multiple kinase inhibitors (meijer...
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Chemistry & Biology
Article
Indirubin Derivatives Modulate TGFb/BMP Signalingat Different Levels and Trigger Ubiquitin-MediatedDepletion of Nonactivated R-SmadsXinlai Cheng,1,2 Hamed Alborzinia,1 Karl-Heinz Merz,2 Herbert Steinbeisser,3 Ralf Mrowka,4 Catharina Scholl,1
Igor Kitanovic,1 Gerhard Eisenbrand,2 and Stefan Wolfl1,*1Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Im Neuenheimer Feld 364, D-69120 Heidelberg, Germany2Department of Chemistry, University of Kaiserslautern, Erwin-Schrodinger-Str. 52, D-67663 Kaiserslautern, Germany3Institute of Human Genetics, Heidelberg University, Im Neuenheimer Feld 366, D-69120 Heidelberg, Germany4Experimentelle Nephrologie. KIM III, Universitatsklinikum Jena, Am Nonnenplan 2-4, D-07734 Jena, Germany
*Correspondence: [email protected]
http://dx.doi.org/10.1016/j.chembiol.2012.09.008
SUMMARY
Regulatory Smads (R-Smads), Smad1/5/8 andSmad2/3, are the central mediators of TGFb andBMP signaling pathways. Here, we screened indiru-bin derivatives, known kinase inhibitors, and ob-served strong interference with BMP signaling. Wefound that indirubin derivative E738 inhibited bothTGFb and BMP pathways through ubiquitin-protea-some-mediated depletion of total R-Smad pools,although phospho-R-Smad levels were initiallystabilized by GSK3b and cyclin-dependent kinaseinhibition. E738 also enhanced p38 and JNK phos-phorylation, involved in Smad-independent TGFb/BMP signaling. Additionally, using a small siRNAscreen, we showed that depletion of ubiquitin prote-ases USP9x and USP34 significantly reduced totalR-Smad levels, mimicking E738 treatment. In fact,both USP9x and USP34 levels were significantlyreduced in E738-treated cells. Our findings not onlydescribe the complex activity profile of the indirubinderivative E738, but also reveal a mechanism forcontrolling TGFb/BMP signaling, the control ofR-Smad protein levels through deubiquitination.
INTRODUCTION
Transforming growth factor b (TGFb) and bone morphogenetic
protein (BMP) belong to the cytokine growth factor family and
play key roles in embryonic development and organ homeo-
stasis, by regulating cellular differentiation, extracellular matrix
remodeling and proliferation (Hong and Yu, 2009; De Robertis
and Kuroda, 2004; Eivers et al., 2009). Dysregulation of TGFb
and BMP signaling is associated with several diseases, including
cancer (Walsh et al., 2010; Wharton and Derynck, 2009; Pardali
et al., 2010). TGFb/BMP signaling is initiated by ligand binding
to receptor kinases on the cell surface. This induces formation
of heteromeric receptor complexes, which phosphorylate
regulatory Smads (R-Smads), Smad1/5/8 for BMP, or Smad2/3
Chemistry & Biology 19, 1423–143
for TGFb signaling at the C-terminal Mad homology 2 (MH2)
domain. Upon binding, the common partner Smad4 (Co-Smad)
R-Smads are translocated to the nucleus, where activated
Smad complexes bind DNA and regulate gene expression,
including IDs (Massague et al., 2005), CTGF (Abreu et al.,
2002), and SnoN (Pot and Bonni, 2008). Receptor-specific
inhibitors, the BMP-receptor inhibitor dorsomorphin (DM) (Yu
et al., 2008), and the TGFb-receptor inhibitor RepSox (Gellibert
et al., 2004; Ichida et al., 2009) efficiently suppress expression
of target genes by preventing phosphorylation of respective
R-Smads. R-Smads also comprise an N-terminal MH1 domain,
which is connected with the MH2 domain via an interdomain
linker region (Hariharan and Pillai, 2008). Phosphorylation of
this region enhances signaling and mediates turnover (Pera
et al., 2003). TGFb and BMP also activate MAP-kinases,
including ERK, p38, and JNK, independent of R-Smads (Derynck
and Zhang, 2003).
Indirubin is a major active ingredient of a Chinese herbal
prescription named Dangui Luhui Wan, used in traditional
Chinese medicine (TCM) for treatment of various diseases
and has been used clinically to treat chronic myelogenous
leukemia (CML) (Eisenbrand et al., 2004). Indirubin and indiru-
bin derivatives (IRDs) are potent protein kinase inhibitors.
Target kinases include serine/threonine kinases, such as
cyclin-dependent kinases (CDKs) (Hoessel et al., 1999) and
glycogen synthase kinase 3b (GSK3b) (Meijer et al., 2003),
and receptor and nonreceptor tyrosine kinases, like c-Src
(Nam et al., 2005) and FGFR1 (Zhen et al., 2007). The IRD
6-bromoindirubin-30-oxime (BIO) has been used to maintain
pluripotency of mouse and human embryonic stem cells
(ESCs) (Sato et al., 2004) and for generation of epiblast stem
cells (Chou et al., 2008). Because TGFb/BMP signaling is impli-
cated in these processes, we investigated whether IRDs target
this pathway.
In the present work, we establish IRDs as potent regulators
of TGFb/BMP signaling acting at different levels and demon-
strate that total R-Smad levels are regulated by specific ubiqui-
tination signals. Blocking deubiquitination, R-Smads were
rapidly depleted and TGFb/BMP/Smad signaling was sup-
pressed. With these observations, we add an important aspect
to the understanding of the intricate network of TGFb/BMP
signaling.
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Chemistry & Biology
Modulation of Nonactivated R-Smad Levels
RESULTS
IRDs Interfere with BMP Signaling in HeLa Cellsand Human FibroblastsIn an exploratory structure-activity investigation, several IRDs
synthesized in our lab were tested at two concentrations (1 and
5 mM; Figure 1A) in HeLa cells treated with BMP2 (100 ng/ml)
for 24 hr. Although treatment with indirubin-30-oxime (E231) did
not significantly affect phospho- and total Smad1/5/8 levels, ana-
logs carrying either a propanediol group on 30-position (E804), or
a methoxy group at 5-position (E728) or both (E738) led to sig-
nificant depletion of phospho- and total Smad1/5/8. In contrast
5-methoxyindirubin (E723) and the 5-carboxamide (E748a)
showed only a decrease of phospho-Smad1/5/8 at 5 mM. For
E860a, an oxime ether with a large basic substituent in 30-position, no effect was observed at 1 mM, whereas 5 mM almost
completely depleted total and phospho-Smad1/5/8 levels.
The observed differences suggest that separate mechanisms
are required for the regulation of BMP/Smad signaling by IRDs.
Whereas E723 and E748a specifically blocked phosphorylation,
other IRDs also reduced total Smad1/5/8 levels.
In presence of 10% fetal bovine serum (FBS), the influence of
IRDs on BMP/Smad signaling was much weaker (Figure 1B) and
depletion of phospho- and total Smad1/5/8 was observed only
with 5 mM E738. The lack of activity of other IRDs in presence
of FBS indicates that FBS activates pathways, which attenuate
the effect of IRDs.
Experiments were repeated with human foreskin fibroblasts
(HFF) and comparable results were obtained (Figure 1C; Fig-
ure S1A available online). The influence of IRDs on phospho-
and total Smad1/5/8 levels correlated with their structures in all
experiments. E738, being the most active IRD in both cell lines,
was chosen for further experiments.
E738 Decreases Total Smad1/5/8 Levels
in Different Cell Lines
We next studied the effects of E738 on BMP/Smad signaling in
C2C12, HFF, NIH 3T3, HEK293, HaCat, and Jurkat cells. Probing
cell lysates with phospho- and total-Smad1/5/8-specific anti-
bodies a dose-dependent reduction of phospho- and total
Smad1/5/8 was observed in all six cell lines (Figures 1D and
S1B–S1D). Consistent with these results, immunostaining of
C2C12 cells treated 12 hr with 1 mM E738 showed nearly
complete depletion of the total Smad1/5/8 reservoir (Figure 1E).
Comparison of E738 with Other Inhibitors
and Modulators of BMP/TGFb Signaling
The IRD BIO is a well-known GSK3b (IC50, 5 nM) and CDK
(CDK1/CycB IC50, 320 nM and CDK5/p25 IC50, 83 nM) inhibitor
(Meijer et al., 2003) and has been used in mouse somatic cell
reprogramming and maintaining of mouse iPSCs (induced
pluripotent stem cells), which involves modulation of BMP and
TGFb signaling (Ichida et al., 2009; Li et al., 2009; Yuan et al.,
2011; Xu et al., 2008). We therefore compared the effect of
E738 on BMP2 signaling in HeLa cells with BIO and BMP and
TGFb inhibitors, dorsomorphin (DM) and RepSox, (Zhu et al.,
2010; Ichida et al., 2009; Yuan et al., 2011). As shown in Fig-
ure 2A, treatment with E738 (1 mM) reduced the concentration
of total and phospho-Smad1/5/8, whereas BIO (5 mM) lowered
Smad1/5/8 phosphorylation, but only marginally decreased
total Smad1/5/8 levels. DM (1 mM) blocked Smad1/5/8 phos-
1424 Chemistry & Biology 19, 1423–1436, November 21, 2012 ª2012
phorylation, but did not alter total Smad1/5/8 levels, consistent
with inhibition of BMP-receptor signaling (Yu et al., 2008). Treat-
ment with 10 mM RepSox neither inhibited Smad1/5/8 phos-
phorylation nor changed total Smad1/5/8 levels. Interestingly,
Repsox augmented phosphorylation of Smad1/5/8.
Further analysis of TGFb/BMP/Smad signaling in HeLa cells
stimulated with TGFb or FBS (Figure 2B) confirmed that only
E738 strongly reduced both, phosphorylated and total Smad
levels. Using quantitative western blot analysis of total R-Smad
levels, we determined concentration/activity profiles for E738,
BIO, and GSK3b inhibitors, CHIR99021 (GSKi) and LiCl (Li
et al., 2011; Klein and Melton, 1996), in HeLa cells in presence
of BMP2, TGFb, or 10% FBS (Figure S2A). Whereas GSKi and
LiCl did not influence total R-Smad levels at all concentrations,
higher concentrations of BIO (5 and 10 mM) also mediated
depletion of R-Smads in presence of BMP2 and TGFb. In pres-
ence of FBS (10%) BIO up to 10 mM did not influence R-Smad
levels. IC50 values are summarized in Figure 2C. Depletion of
R-Smads by E738 occurred also in medium without FBS or
growth factors (Figure S2A).
To investigate the specificity of R-Smad depletion by E738, we
analyzed Smad4 and Smad7 protein levels. These inhibitory
Smads were not changed by E738 in HeLa cells in presence of
BMP2 or TGFb (Figure 2D). Thus, only R-Smad levels are sensi-
tive to E738 treatment.
The effect of E738 on BMP/TGFb target gene transcription
was evaluated by qRT-PCR (Figure 2E). Expression of ID1, a
well-established BMP target (Clement et al., 2000) regulating
cell differentiation by interaction with MyoD (Benezra et al.,
1990), was significantly repressed. Expression of ID2, another
BMP and TGFb signaling target (Clement et al., 2000), was
reduced to 50%. Expression of CTGF, a BMP antagonist and
TGFb agonist (Derynck and Zhang, 2003), and of SnoN, a
TGFb downstream gene (Deheuninck and Luo, 2009), was
decreased to �30% and 10%, respectively.
BMP2 target gene expression was also analyzed in compar-
ison with DM, RepSox, and BIO. The results obtained clearly
show that E738 inhibits both, BMP and TGFb-specific target
genes. DM only blocks expression of BMP target genes (Fig-
ure 2F). Treatment with RepSox enhanced expression of ID1
and ID2 and slightly decreased CTGF mRNA. The comple-
mentary activity of DM and RepSox fits with specific receptor
inhibition and opposing effects of BMP2 and TGFb signals.
Distinct from other inhibitors, BIO suppressed ID1 expression,
whereas ID2 and CTGF mRNA levels were clearly increased.
Taken together, the IRD E738 blocks TGFb/BMP/Smad
signaling through specific depletion of R-Smad pools without
affecting other Smad levels and thus uniquely modulates
TGFb/BMP/Smad signaling.
IRD E738 Is a Multiple Kinase InhibitorKinase Inhibitor Activity Is Indispensable
for R-Smad Depletion
IRDs have been reported to act as multiple kinase inhibitors
(Meijer et al., 2006). Based on the cocrystallization structure of
indirubin E231 in the ATP-binding pocket of CDK2, replacement
of C-atom at 70-position with N-atom should deprive IRDs of
protein kinase inhibitory activity (Hoessel et al., 1999; Eisenbrand
et al., 2008). The resulting 70-aza-indirubin showed no inhibitory
Elsevier Ltd All rights reserved
Figure 1. Impact of IRDs on the Activation of BMP-Signaling through Smad1/5/8
(A–C) Phospho-Smad1/5/8 ([(1(Ser463/465)/5(Ser463/465)/8(Ser426/428)) and total Smad1/5/8 levels (pan-Smad1/5/8) were detected by immunoblot
after 24 hr treatment with IRDs as indicated in HeLa cells with the stimuli of BMP2 (100 ng/ml) (A), with 10% FBS (B), or in HFF cells with 15% FBS (C).
(D) C2C12, HFF, and NIH 3T3 cells were treated with increasing concentrations of E738 in presence of BMP2 (100 ng/ml). DMSO was used as mock and b-Actin
as loading control. Forty micrograms of protein were loaded per lane.
(E) C2C12 cells were treated with 0.5 mMor 1 mME738 for 12 hr in the presence of BMP2 and analyzed by immunostaining with antibody against total Smad1/5/8
including DAPI staining of nuclei.
See also Figure S1.
Chemistry & Biology
Modulation of Nonactivated R-Smad Levels
Chemistry & Biology 19, 1423–1436, November 21, 2012 ª2012 Elsevier Ltd All rights reserved 1425
Figure 2. E738-Mediated Inactivation of BMP- and TGFb-Associated R-Smads
(A and B) Interference of E738 (1 mM) on TGFb/BMP/Smad signaling in comparison to BIO (5 mM), DM (1 mM), and RepSox (10 mM)with stimuli of BMP2 (100 ng/ml)
(A) or TGFb (20 ng/ml) (B), or FBS (10%) in HeLa cells for 24 hr treatment detected by immunoblot.
(C) IC50 values of compound concentrations depleting total R-Smad levels quantitatively analyzed by immunoblot from at least three independent experiments.
HeLa cells were treated with various concentration of E738, BIO, LiCl, or GSKi for 6 hr in presence of BMP (100 ng/ml), TGFb (20 ng/ml), 10% FBS, or without
growth factor.
(D) Smad4 and Smad7 protein levels were not influenced by E738 after 24 hr treatment.
Chemistry & Biology
Modulation of Nonactivated R-Smad Levels
1426 Chemistry & Biology 19, 1423–1436, November 21, 2012 ª2012 Elsevier Ltd All rights reserved
Chemistry & Biology
Modulation of Nonactivated R-Smad Levels
activity when screened against �200 protein kinases, although
70-aza-indirubin was clearly cytotoxic in tumor cells (Eisenbrand
et al., 2008). We used two 70-aza-indirubins E846 and E847
(structures in Figure S2B). These aza-IRDs neither inhibited
phosphorylation of Stat3, nor mediated depletion of R-Smads,
whereas counterpart IRDs (E728 and E804) did (Figure 2G).
Thus, inhibition of protein kinase activity could be indispensable
for R-Smad depletion.
E738-specific inhibition of protein kinases was analyzed
by in vitro inhibition of selected recombinant protein kinases
measuring residual activity after treatment with 1 mM E738 in
presence of 1 mM ATP (Cheng et al., 2010). E738 reduced the
residual activity of GSK3b to only 6% and significantly
decreased activity of CDKs (CDK1/CycB1, 16%; CDK2/CycA,
10%; CDK2/CycE, 8%; CDK4/CycD1, 23%; CDK5/p35, 16%).
E738 also inhibited kinases associated with Stat3 phosphory-
lation (JAK2, 44%; KIT, 44%; Src, 25%) and activity of b-Raf
was reduced to 45% (Figure S3A).
We next analyzed the effect of E738 on GSk3b/Wnt and
Stat3 signaling in the cellular context. Treatment of HeLa cells
with increasing concentrations of E738 for 1 hr (Figures 3A and
S3B) clearly showed a concentration-dependent decrease of
Stat3 phosphorylation and stabilization of nonphosphorylated
b-catenin (NP-b-catenin, Ser33/37/Thr41), which accumulates
upon GSK3b inhibition. E804 (5 mM) a known inhibitor of phos-
pho-Stat3 signaling was included as reference (Nam et al.,
2005).
Inhibition of GSK3b was also analyzed in HEK293 cells con-
taining a TopFlash-luciferase reporter for recording of Wnt/
b-catenin signaling (Xu et al., 2004). Treatment with E738 at
1 mM induced luciferase quite similar to 20 mM LiCl (Figure 3B).
E738 Shows a Distinct Gene Activation Profile
in Xenopus Embryos
We also analyzed expression of Wnt and TGFb/nodal target
genes in Xenopus embryos treated with E738 in comparison
with the GSK3b inhibitor LiCl and SB505124 (SB5), a selective
ALK4, 5, and 7 inhibitor (Klein and Melton, 1996; Skirkanich
et al., 2011). Treatment with E738 resulted in a very distinct
expression profile of Wnt and TGFb/nodal target genes (Fig-
ure 3C). Expression of the Wnt target genes nodal related 3
(nr3) and siamois (sia) (McKendry et al., 1997; Carnac et al.,
1996) was strongly induced by LiCl and also increased after
treatment with E738. In contrast to the LiCl-specific response,
the TGFb target genes brachyury (bra) and goosecoid (gsc)
(Skirkanich et al., 2011) were strongly repressed by E738. In
addition, expression of fox1e, which is repressed by nodal-
signaling (Luxardi et al., 2010), was upregulated by E738. These
findings are in good agreement with previous expression pro-
files from embryos treated with SB5 (Skirkanich et al., 2011)
and prove that E738 inhibits both GSK3b and TGFb/nodal
signaling in early Xenopus embryos.
(E) Expression of ID1, ID2,CTGF, and SnoN in HeLa cells treated with 1 mME738
value of nontreated cells and show the mean ± SD of quadruplicates of three ind
(F) Expression of ID1, ID2, andCTGF in HeLa cells treatedwith 1 mME738, 1 mMDM
before and show the mean ± SD.
(G) HeLa Cells were treated with IRDs or Aza-IRDs (1 mM for each) for 24 hr with
(Smad1/5/8 or Smad2/3).
See also Figure S2.
Chemistry & Biology 19, 1423–143
The above results characterize E738 as a potent kinase inhib-
itor, affecting GSK3b as well as Stat3 signaling, and indicate
that kinase inhibition might be indispensable for depletion of
R-Smads. Nevertheless, inhibition of BMP/TGFb and nodal
signaling is clearly distinct from effects observed with other
GSK3b inhibitors.
E738-Mediated R-Smad Depletion Is Independentof R-Smad ActivationIt had been shown that inhibition of GSK3b and CDKs is involved
in controlling turnover of phosphorylated R-Smads and termina-
tion of signaling (Fuentealba et al., 2007; Alarcon et al., 2009;
Sapkota et al., 2007). Because E738 inhibited GSK3b and
CDKs in vitro and showed Wnt-like signaling in reporter cells,
we investigated the influence of E738 on the duration of
Smad1/5/8 phosphorylation and on degradation of activated
p-Smad1/5/8. In a first experimental design HeLa cells were
stimulated with a pulse of BMP2 for 1 hr prior to E738 treatment
(Figure 3D, upper). Without E738, the BMP2 induced p-Smad1/
5/8 signal peaked 0.5 hr after removal of BMP2 (1.5 hr after
adding BMP2) and decreased in the next 1 hr; no change of
total Smad1/5/8 was observed (Figure 3D). When E738 was
added at the time point of BMP2 removal, p-Smad1/5/8 levels
did not decrease for �1.5 hr after removal of BMP2 (2.5 hr after
adding BMP2) (Figure 3D). In presence of E738, a slight reduc-
tion of total Smad1/5/8 was already detectable after 2 hr,
whereas the level of p-Smad1/5/8 was still higher than in control
without E738.
In the second experimental design, we pretreated HeLa cells
with E738 for 1 hr before adding BMP2, removed E738 after
3 hr, and continued BMP2 treatment for 2 hr. In a parallel time
course experiment cells were treated with DM instead of E738
(Figure S3C). E738 pretreatment enhanced Smad1/5/8 phos-
phorylation even before BMP2 was added, whereas BMP2-
induced phosphorylation was blocked, as with DM. After
removal of compounds, Smad1/5/8 phosphorylation recovered
in DM treated cells but not after E738 treatment.
Influence of E738 on Phosphorylation of Linker
Region of Activated Smad1/5/8
Phosphorylation of Smad1/5/8 at the C termini and the linker
region are a prerequisite for ubiquitination by Smurf1 E3 ligase.
Linker phosphorylation at S206 is crucial for this specific recog-
nition (Fuentealba et al., 2007; Alarcon et al., 2009, Zhu et al.,
1999). We therefore performed time course studies of Smad1/
5/8 phosphorylation at S206 with E738 (1 mM). In absence of
E738 (Figure 3E, left) maximum intensity of BMP-induced phos-
phorylation at C termini and S206 simultaneously appeared
after incubation for 1 hr and decreased afterward. In presence
of E738 (Figure 3E, right) concurrent C-tail and linker phos-
phorylation of Smad1/5/8 occurred as well but with 1 hr delay.
Similar stabilization of linker phosphorylation was observed in
for 24 hr in presence of BMP2 quantified by qRT-PCR. Data were normalized to
ependent experiments.
, 5 mMBIO, or 10 mMRepSox relative to BMP2 alone. Data were normalized as
BMP2 and analyzed by immunoblot on the level of p-Stat3 and total R-Smads
6, November 21, 2012 ª2012 Elsevier Ltd All rights reserved 1427
Figure 3. Regulation of Protein Kinases Involved in Smad-Signaling by E738 and Modulation of Target Gene Expression
(A) HeLa cells were treated with or without IRDs as indicated for 1 hr in the presence of BMP2 and protein levels were analyzed by immunoblot as labeled on
the panel.
(B) Induction of Wnt-luciferase reporter assay (TopFlash) in HEK293 cells measured online in living cells after 1, 2, and 3 days with 1 mM E738 or 20 mM LiCl
in presence of 10%, 0.5%, or without FBS. Luminescence counts are presented as mean ± SD of 24 measurements.
Chemistry & Biology
Modulation of Nonactivated R-Smad Levels
1428 Chemistry & Biology 19, 1423–1436, November 21, 2012 ª2012 Elsevier Ltd All rights reserved
Chemistry & Biology
Modulation of Nonactivated R-Smad Levels
the experiment adding E738 after a 1 hr BMP2 pulse (Figure 3D).
This delay in degradation of phosphorylated R-Smads is in good
agreement with the effect of other GSK3b inhibitors, which
prevent degradation of activated R-Smads (Fuentealba et al.,
2007). In contrast, looking at total Smad1/5/8 levels, we found
a decrease as early as 30 min upon addition of E738. After 6 hr
the amount was markedly reduced, and after 24 hr total
Smad1/5/8 was no longer detectable (Figure 3E).
E738 Treatment Leads to MAPK Phosphorylation
BMPs can activate MAPKs including ERK, p38, and JNK in
a Smad-independent fashion to regulate various biological
processes, like osteoblast apoptosis (Hay et al., 2001) and
vascular development (Yang et al., 2007). We analyzed regula-
tion of MAPKs in E738-treated HeLa cells. Treatment with
E738 enhanced phosphorylation of p38 and JNK, but not
ERK1/2 in the presence of BMP2 after 1 hr (Figure 3A). E738-
mediated JNK and p38 phosphorylation significantly increased
after 6 hr (Figure 3E). The activation of p38 by E738 was further
confirmed in a dose-dependent analysis after 6 hr treatment
(Figure 3F).
Taken together, the results presented so far demonstrate
complex E738-orchestrated TGFb/BMP signaling: (1) inhibition
of GSK3b/CDKs stabilizing phosphorylated R-Smads in the
short-term range, (2) degradation of total R-Smads decreasing
signaling activity in the long-term range, and (3) phosphoryla-
tion of p38/JNK MAPKs to activate Smad-independent TGFb/
BMP signaling.
Role of Ubiquitination in E738-Mediated Depletionof R-Smad PoolsR-Smad Depletion by E738 Is Independent
of MAP-Kinase Signaling
As already mentioned, turnover of activated R-Smads is medi-
ated by phosphorylation of the linker region through MAP-
kinases p38 and JNK, which facilitates binding of the ubiquitin
ligase Smurf1 (Sapkota et al., 2007). Because E738 induced
phosphorylation of p38 and JNK (Figures 3A, 3E, and 3F), we
analyzed whether p38 or JNK are required for R-Smad degrada-
tion by E738. Coincubation of HeLa cells with E738 and MAP-
kinase inhibitors SP600125 (JNKi), SB203580 (P38i), and
U0126 (ERKi) alone or in multiple combinations did not attenuate
the level of phospho-Smad1/5/8 and did not block the reduction
of total Smad1/5/8 levels after 6 and 24 hr of BMP2 treatment
(Figures 4A and 4B) or when stimulated with TGFb (Figure S4A).
The specific phosphorylation of MAPKs was clearly reduced by
the corresponding inhibitors. Thus, MAPKs should not play a role
in E738-mediated degradation of R-Smads.
We also applied several other inhibitors in combination with
E738 (Figures 4C, 4D, S4B, and S4C). Cells treated with the
translation inhibitor cycloheximide (CHX) showed a weaker
signal for total Smad1/5/8, most likely due to reduced Smad
synthesis (Figures 4C and S4B), whereas DM even enhanced
E738-mediated total R-Smad reduction (Figures 4D and S4C).
(C) Expression of Wnt and nodal/TGFb target genes during gastrula in Xenopus la
normalized to ODC, in comparison to untreated DMSO control and show the me
(D–F) HeLa cells were treated with 1 mM E738 after a 1 hr pulse of BMP2 (D), or in
lysates were analyzed by immunoblot using specific antibodies as indicated.
See also Figure S3.
Chemistry & Biology 19, 1423–143
Effect of E738 Treatment on R-Smad Expression
The effect of CHX promoted us to investigate if E738 inter-
feres with Smad expression on the mRNA level. Quantitative
RT-PCR revealed that 24 hr E738 treatment repressed
Smad1 and Smad2 expression to 60% and 30%, respectively,
whereas Smad4 mRNA levels remained unchanged (Figure 4E).
Expression of Smad-associated E3 ligases, Smurf1 and
Smurf2, was also reduced to 50% (Smurf1) and 30% (Smurf2).
This effect of E738 on R-Smad transcription is not sufficient
to explain the strong reduction observed on the protein level.
Thus, major degradation must occur directly on the protein
level.
Proteasome Inhibition Prevents E738-Mediated
R-Smad Degradation
The ubiquitin-proteasome pathway is the major route of pro-
tein degradation. Treatment with the proteasome inhibitor
MG132 efficiently rescued E738-mediated R-Smad degrada-
tion in HeLa cells stimulated by BMP2 (Figures 4F, 4G, and
S4D), or TGFb (Figure S4E), and in HFF cells (Figure S4F).
Because MG132 counteracted E738 activity on R-Smad degra-
dation, we postulated that MG132 might also promote cell
survival in presence of E738. Cotreatment with MG132 improved
cell survival in presence of FBS, BMP2, or TGFb (Figure 5A).
Cleavage of PARP, a marker of apoptosis, was significantly
prevented by cotreatment compared to either E738 or MG132
alone (Figure 5B).
Looking at ubiquitination, we found that E738 changed the
size distribution of ubiquitinated proteins and ubiquitination
precursors also in the presence of MG132, showing a significant
reduction of lower molecular weight fragments (Figure 5C).
DM and LiCl did not influence the ubiquitination pattern (Fig-
ure 5C). Moreover, accumulation of p-Smad1/5/8 was observed
in presence of MG132 alone or in combination with E738, but
not with DM (Figures 4G and 5C).
Smad1/5/8-specific ubiquitination was analyzed by immuno-
precipitation of Smad1/5/8 following incubation with MG132
and E738 in HeLa cells. Cotreatment with E738 clearly enhanced
Smad1/5/8 ubiquitination, but did not change Smurf1 coimmu-
noprecipitation (Figure 5D).
These results illustrate that E738 does not inhibit BMP
receptor-mediated phosphorylation of Smad1/5/8 and that
ubiquitin-mediated degradation is required to remove both,
nonactivated and activated Smad1/5/8.
USP9x and USP34 Are Involved in Maintenance
of R-Smad Pools
Our results demonstrated that E738 is able to promote ubiquiti-
nation of R-Smads (Figure 5D) and thus interferes with TGFb/
BMP signaling. We next performed a small siRNA screen target-
ing 16 proteins, which are crucial in TGFb/BMP-, WNT-, Stat3-
signaling, or play a role in ubiquitination or apoptosis. For each
gene, two published siRNAs were used (Table S1).
Smurf1 is the ubiquitin ligase required for degradation of
activated R-Smads (Fuentealba et al., 2007). Because E738
evis analyzed by qRT-PCR. Fold changes were calculated from mRNA levels,
an ± SD of triplicate measurements.
a time course study (E), or with various concentrations of E738 for 6 hr (F). Cell
6, November 21, 2012 ª2012 Elsevier Ltd All rights reserved 1429
Figure 4. Cotreatment with the Proteasome Inhibitor MG132 Blocks E738-Induced R-Smad Degradation
(A and B) HeLa cells were incubated with E738 (5 mM) or in combination with SP600125 (JNKi, 10 mM), SB203580 (P38i, 10 mM), and U0126 (ERKi, 10 mM), or with
inhibitor combinations (JNKi+P38i, 2i) or (JNKi+P38i+ERKi, 3i) for 6 hr (A) or 24 hr (B).
(C) HeLa cells were stimulated with BMP2 for 24 hr in the presence or absence of E738 (1 mM), cycloheximide (CHX, 10 mM), or both.
(D) E738 at increasing concentrations as indicated in combination with DM (1 mM).
(E) The expression levels of Smad1, Smad2, Smad4, Smurf1, and Smurf2 were analyzed by qRT-PCR. Data were normalized to the value of nontreated cells,
show the mean ± SD of quadruplicates, and are representative of three independent experiments.
(F and G) BMP2-stimulated HeLa cells were treated with DM (1 mM), E738, MG132 (MG), or in combination as indicated for 24 hr (F) or 6 hr (G).
See also Figure S4.
Chemistry & Biology
Modulation of Nonactivated R-Smad Levels
clearly reduced total Smad1/5/8 levels in presence of DM (Fig-
ure 4D) and in absence of growth factors (Figures 2C and
S2A), and reduced expression of Smurf1 and Smurf2 (Figure 4E),
we assumed that Smurfs might not be involved in degradation
of nonactivated Smad1/5/8. Indeed, in Smurf1-depleted HeLa
cells the reduction of R-Smad degradation was insignificant
(Figure 5E).
1430 Chemistry & Biology 19, 1423–1436, November 21, 2012 ª2012
TAB1 and TAB2 are key elements of Smad-independent
TGFb/BMP-signaling (Derynck and Zhang, 2003). Depletion of
TAB1 and TAB2 by siRNA significantly reduced phosphorylation
of p38 in HeLa cells, but did not influence total R-Smad levels
(Figure 5F). Thus, E738-mediated activation of MAPKs is
TAB1/2-dependent, whereas degradation through ubiquitination
of R-Smads is TAB1/2-independent.
Elsevier Ltd All rights reserved
Figure 5. Effects of E738 and MG132 on Cell Proliferation and Ubiquitination
(A) SRB assays were performed to analyze the inhibitory effect of E738 (1 mM) with or without MG (5 mM) on the proliferation of HeLa cells after 24 hr treatment in
the presence of FBS (10%), BMP2, or TGFb for 24 hr. Data were normalized to value of nontreated cells. Mean ± SD of quadruplicates representing three
independent experiments.
(B) Effect of E738 and MG132 on PARP cleavage. Cells were treated with BMP2 or TGFb in presence of E738 (1 mM) with or without MG (5 mM) for 24 hr.
(C) BMP2-stimulated HeLa cells were treated with DM (1 mM), E738 (1 mM), BIO (5 mM), MG (5 mM), LiCl (30 mM), or E738 (1 mM) in combination with MG as
indicated for 24 hr.
(D) Immunoprecipitation of 4 hr compound-treated HeLa cells with Smad1/5/8 antibody, analyzed by immunoblot for ubiquitin and Smurf1.
(E and F) HeLa cells were transfected with Smurf1 siRNA or random siRNA control and treated with E738 (1 mM) in presence of BMP2 for 24 hr (E), or with TAB1/2
siRNAs and then treated with E738 (5 mM) for 6 hr (F).
Chemistry & Biology
Modulation of Nonactivated R-Smad Levels
Ubiquitin proteases USP9x and USP34 were reported to con-
tribute to accumulation of b-catenin (Murray et al., 2004; Lui et al.,
2011). Moreover, USP9x had been shown to influence ubiq-
Chemistry & Biology 19, 1423–143
uitination of Smad4 to enhance TGFb/Smad signaling (Dupont
et al., 2009). RNA interference of USP34 in HeLa cells reduced
the level of total Smad1/5/8, but not Smad2/3 (Figures 6A and
6, November 21, 2012 ª2012 Elsevier Ltd All rights reserved 1431
Figure 6. Role of USP9x and USP34 in the Control of R-Smad Levels
(A and B) HeLa cells were transfected with increasing concentrations of USP34 siRNA (A) or USP9x siRNA (B). Protein levels were analyzed after 6 hr
BMP2-stimulation in presence or without with E738 (5 mM).
(C and D) Cell extracts of BMP2-stimuated HeLa cells with or without E738 were prepared at indicated time points and used for immunoblot, or for immuno-
precipitation with antibodies specific for USP9x (C) or Smad1/5/8 (D) antibody and subsequent immunoblot analysis.
(E) Schematic model of BMP signaling orchestrated by E738.
See also Figure S5.
Chemistry & Biology
Modulation of Nonactivated R-Smad Levels
1432 Chemistry & Biology 19, 1423–1436, November 21, 2012 ª2012 Elsevier Ltd All rights reserved
Chemistry & Biology
Modulation of Nonactivated R-Smad Levels
S5A). In USP9x-depleted HeLa cells, both R-Smads were
reduced (Figures 6B and S5B). In direct comparison with E738
treatment, the effect of USP9x and USP34 depletion on R-
Smad degradation was less pronounced, which may be ex-
plained by limited efficiency of siRNA-mediated depletion of
USP9x and USP34. Interestingly, E738 treatment led to a sig-
nificant decrease of USP34 and USP9x (Figures 6A and 6B).
Further analysis of the effect of E738 onUSP9x andUSP34 levels
revealed that both were reduced as early as 2 hr after treatment
with E738 in presence of BMP2 (Figure 6C) and TGFb (Fig-
ure S5C). Coimmunoprecipitation with an USP9x-specific anti-
body and subsequent immunoblot for Smad1/5/8 showed a clear
interaction of Smad1/5/8 with USP9x (Figures 6C and S5C). The
reverse experiment, coimmunoprecipitation with a Smad1/5/8
antibody, confirmed USP9x and Smad1/5/8 interaction and re-
vealed interaction of USP34 with Smad1/5/8 (Figure 6D).
These experiments clearly demonstrate a direct control of
nonactivated R-Smad levels through ubiquitination, which
involves ubiquitin proteases, USP9x and USP34, to control
ubiquitination and prevent depletion of nonactivated R-Smads.
This mechanism is efficiently blocked by E738 and can explain
the initially observed inhibition of BMP and TGFb signaling
through this IRD.
DISCUSSION
TGFb signaling, including TGFb- and BMP-mediated pathways,
is a central component in the control of tissue formation, embry-
onic development, and maintenance of stem cells. IRDs have
been shown to prevent migration and invasion of tumor endothe-
lial cells in vitro and in vivo (Williams et al., 2011) and to generate
ESC-like cells (Chou et al., 2008). Because TGFb/BMP signaling
plays an important role in these events (Xu et al., 2008), we postu-
lated that IRDs influence TGFb/BMP signaling. Screening of our
own indirubin derivatives collection resulted in identification of
IRDs, in particular E738, which inhibited R-Smad signaling by
promoting R-Smad degradation in all of seven cell lines tested.
In the TGFb/BMP signaling cascade, Smad2/3 is phosphory-
lated by TGFb-receptor type 1, ALK4/5/7, whereas Smad1/5/8
is phosphorylated by ALK1/2/3/6 after activation by BMP (Mas-
sague et al., 2005). Cross-activation between these pathways is
possible (Valdimarsdottir et al., 2006; Goumans et al., 2002) and
even TGFb/BMP independent phosphorylation of R-Smads was
reported (Zhu et al., 2007). Treatment with receptor inhibitors,
like DM and RepSox, directly inhibited R-Smad phosphorylation.
In comparison, E738 showed a long-term and robust block of R-
Smad activation, including noncanonically activated R-Smads.
Like other IRDs, E738 is a potent protein kinase inhibitor that
clearly inhibits GSK3b, CDKs, and other kinases. The strong
inhibition of GSK3b, an activity shared by other IRDs including
BIO (Eisenbrand et al., 2004), is sufficient to explain stabiliza-
tion of phospho-Smad1/5/8 in the short term and fits well with
results by Fuentealba et al. (2007). Reduced expression of
Smurf1, required for ubiquitination and degradation of activated
Smad1/5/8, should further contribute to stabilization of phos-
phorylated R-Smads by E738. These mechanisms, however,
cannot explain the strong inhibition of TGFb/BMP signaling
observed with E738. Other GSK3b inhibitors, except for BIO in
some conditions, did not directly inhibit TGFb/BMP signaling.
Chemistry & Biology 19, 1423–143
BIO, however, is also an IRD and thus may share inhibitory activ-
ities with E738.
Inhibition of TGFb/BMP signaling through R-Smad depletion
was completely blocked by MG132, an inhibitor of the ubiqui-
tin-proteasome degradation pathway. Ubiquitin-mediated
degradation is the known mechanism for turnover of activated
R-Smads with Smurf1 as main E3 ligase (Fuentealba et al.,
2007; Zhu et al., 1999).
With our experiments, we clearly show that ubiquitination
also controls nonactivated R-Smad levels, which is independent
of Smurf1. Consistent with this, knock-down of Smurf1 only
rescued a trace amount of total Smad1/5/8, because activated
Smad1/5/8 were no longer degraded.
MAPKs p38 and JNK are known to mediate turnover of
R-Smads after activation by TGFb, BMP, and other growth
factors phosphorylating R-Smads in the linker region (Pera
et al., 2003; Sapkota et al., 2007). E738 also activated p38 and
JNK. Using small molecule MAP-kinase inhibitors and siRNA-
mediated knock-down of TAB1/2 we could exclude this pathway
from participation in the observed R-Smad degradation.
The proteasome inhibitor MG132 not only rescued R-Smads
from degradation, but also prevented E738-induced PARP
cleavage and apoptosis, linking E738 activity directly with the
ubiquitin-proteasome pathway. Indeed, two ubiquitin-specific
proteases, USP9x and USP34, were identified to play a role in
stabilization and degradation of R-Smads. Both were dramati-
cally repressed by E738 in HeLa cells as early as 2 hr after
treatment.
USP9x and USP34 had been reported to stabilize b-catenin
and enhance Wnt signaling (Murray et al., 2004; Lui et al.,
2011). Our results add an additional regulatory activity, control
of R-Smad levels, and reveal a direct interaction with R-Smads.
This provides, to our knowledge, a novel interaction between
Wnt and TGFb/BMP signaling. In fact, treatment of Xenopus
embryos with E738 induced Wnt/Gsk3b target genes and simul-
taneously blocked TGFb (activin/nodal) activity.
Taken together, our findings clearly show that E738 regulates
TGFb/BMP signaling at four regulatory levels: (1) stabilization
of receptor-activated R-Smads through inhibition of CDKs
and GSK3b, (2) TAB1/2-dependent activation of p38 and JNK
MAP-kinases, and (3) reduction of R-Smad levels by down-
regulation of R-Smad expression, and (4) increased ubiquitina-
tion of nonphosphorylated R-Smads by blocking USP9x and
USP34 activity (Figure 6E).
The intricate regulation of TGFb/BMP signaling by IRDs may
explain the versatile applications of IRDs, including a potential
benefit of IRDs in stem cell reprogramming, which had been
shown for BIO (Sato et al., 2004; Chou et al., 2008). Furthermore,
our results add an important part to the control of TGFb/BMP
signaling, namely the control of R-Smad abundance by ubiquiti-
nation and deubiquitination. Given that IRDs influence these
pathways in several distinct layers, our results suggest, that
E738 and other IRDs may lead to new therapies in regenerative
medicine, fibrosis, and chronic diseases.
SIGNIFICANCE
TGFb and BMP signaling pathways are crucial in the con-
trol of cell differentiation, embryonic development, and
6, November 21, 2012 ª2012 Elsevier Ltd All rights reserved 1433
Chemistry & Biology
Modulation of Nonactivated R-Smad Levels
proliferation. Overactivation of these signaling pathways is
associated with a number of diseases. It is well known that
TGFb/BMP receptor signaling is regulated at several levels.
In particular, duration of R-Smad activation is controlled by
ubiquitination of activated R-Smads, which leads to degra-
dation of transcriptionally active phosphorylated R-Smads.
So far, total R-Smad levels had not been considered as
an important regulatory element in TGFb/BMP signaling.
Here, we show drastic depletion of total R-Smad levels of
bothmajor TGFb-family signaling pathways. In recent years,
it had been shown that protein stability is not only controlled
at the level of ubiquitination but also by deubiquitination
through ubiquitin-specific proteases. Using the new indiru-
bin derivative, E738, we not only observed impaired TGFb/
BMP signaling but also a rapid decline of total R-Smad
levels. Analyzing the effects of E738 in detail, we found inter-
ference with TGFb/BMP signaling at clearly separated
levels: short term stabilization of R-Smad activation through
GSK3b inhibition, activation of noncanonical signaling
through MAP-kinases p38 and JNK, and strong depletion
of total R-Smads, through protein degradation and reduced
transcription. Using RNA interference and protein interac-
tion studies, we could clearly show that total R-Smad level
control could be linked to the deubiquitinating proteases
USP9x and USP34. Depletion of these enzymes by siRNA
led to a concomitant reduction of total R-Smad levels of
both mayor pathways, Smad1/5/8 for BMP-signaling, as
well as Smad2/3 for TGFb downstream signals. Our work
clearly demonstrates that TGFb/BMP-signaling is efficiently
regulated by the relative abundance of total R-Smad
pools. Furthermore, this mechanism is accessible for small
molecule intervention, which will lead to new options for
intervention with prosurvival and cell differentiation TGFb/
BMP signals.
EXPERIMENTAL PROCEDURES
Materials
IRDs were synthesized. Structures and purities were determined by 13C- and1H-nuclear magnetic resonance (NMR) spectroscopy and elemental analyses
(Eisenbrand et al., 2004; Hoessel et al., 1999; Cheng et al., 2010). Dorsomor-
phin (DM), LiCl, and RepSox were purchased from Calbiochem (Germany).
P38i (SB203580), JNKi (SP600125), ERKi (U0126), SB505124 (SB5), and
MG132 were obtained from Sigma-Aldrich (Germany). The Xenopus experi-
ments were performed according to the German animal protection laws and
approved by the government of the state of Baden-Wurttemberg.
Cell Culture
HeLa, HEK293, HaCat, and C2C12 cells were cultured in DMEM (GIBCO,
Germany) containing 10% FBS (PAA, Germany) and 1% Pen/Strep (GIBCO,
Germany). Jurkat cells were cultured in RPMI with 10% FBS and 1%
Pen/Strep. HFF cells were cultured in Dulbecco’s modified Eagle medium
(DMEM) containing 15% FBS and 1% Pen/strep. NIH 3T3 cells were cultured
in DMEM with 10% NCS (GIBCO, Germany) and 1% Pen/Strep. Cells were
maintained under 5% CO2 at 37�C in humidified atmosphere. After 24 hr pre-
incubation in serum-free medium, cells were treated with compounds and
proteins as detailed in the text. Oligonucleotides for siRNA interference were
synthesized using previously reported sequences (Table S1) and riboxx
siRNA-design (Riboxx, Radebeul, Germany). Transfections were performed
using the riboxxFECT reagent according to themanufacturer’s instructions fol-
lowed by 30 hr incubation in DMEM with 5% FBS. Before adding substances,
transfected cells were maintained in medium without FBS for 18 hr. Then cells
1434 Chemistry & Biology 19, 1423–1436, November 21, 2012 ª2012
were treated with TGFb (20 ng/ml) or BMP2 (100 ng/ml) and compounds as
indicated in the text.
Immunoblot
Cell pellets were homogenized in lysis buffer containing 1 mM EDTA, 0.5%
Triton X-100, 5 mM NaF, 6 M Urea, 1 mM Na3VO4, 10 mg/ml Pepstatin,
100 mMPMSF, and 3 mg/ml Aprotinin in PBS. Forty micrograms of total protein
per sample were resolved on 10% SDS-PAGE and blotted to membrane
(Immobilon, Millipore, Germany) by semi-dry transfer. Antibodies against the
following proteins were used for immunoblotting: p-Smad1/5/8 (C-terminal),
p-Smad1 (S206), p-JNK, JNK, p-p38-MAPK, p38-MAPK, p-Erk1/2, Erk1/2,
p-Smad3, pan-Smad2/3, Smad4, p-Stat3, Stat3, non-p-b-catenin 41/37/33,
ubiquitin, USP9x, TAB1, TAB2 (all from Cell Signaling, NEB, Germany), b-actin
(Sigma Aldrich), Smad1/5/8 (Santa Cruz, Germany), Smad7 (Epitomics,
Biomol, Germany), and USP34 (Bethyl, Biomol, Germany). Corresponding
goat anti-mouse and goat ant-rabbit IgG/HRP conjugates were from
KPL (Germany). All antibodies were applied according to manufacturers’
recommendations. Immunoblots were developed with ECL solution (Pierce,
Germany) and images were recorded with a digital imaging system (LAS
3000, Fuji).
Immunostaining
Serum-deprived C2C12 cells were coincubated with BMP2 (100 ng/ml) and
E738 at 0.5 mM or 1 mM for 12 hr. Cells were fixed in 4% paraformaldehyde,
permeabilized at room temperature for 10 min using PBS containing 0.1%
Triton X-100, and afterward blocked 1 hr at room temperature in PBS con-
taining 5% goat serum and 0.1% Tween 20. Cells were incubated overnight
at 4�C with Smad1/5/8 antibody (Santa Cruz) and corresponding signals
were detected using FITC-conjugated secondary antibody (BD Bioscience,
Germany). Nuclear staining was performed with DAPI (1 mg/ml) for 5 min.
Immunoprecipitation
Immunoprecipitation was performed according to the manufacturers protocol
(Cell Signaling Technologies). Briefly, cells were lysed in lysis buffer (100 mM
TRIS/HCl pH 7.5, 150 mM NaCl, 1 mM MgCl2, 0.25% NP-40 substitution,
5 mM NaF, 1 mM Na3VO4, 10 mg/ml pepstatin, 100 mM PMSF, and 3 mg/ml
aprotinin) and incubated with rabbit serum (DAKO) for 1 hr on ice and
protein-A Sepharose beads (Upstate, Millipore) for 30 min. Supernatant was
incubated with anti-Smad1/5/8 (1:100, Santa Cruz) or anti-USP9x (1:100,
Cell Signaling Technologies,) antibodies overnight at 4�C. Immune complexes
were precipitated with protein-A Sepharose bead for 1 hr at 4�C and analyzed
by immunoblotting. Clean-Blot IP Detection Reagent (Thermo, Germany) was
used for background reduction.
Quantitative RT-PCR
Quantitative real-time (qRT) reverse-transcription-PCR was performed ac-
cording to manufacturer’s instructions (Light Cycler, Roche, Germany). Briefly,
total RNAwas isolated fromcells using TRIzol reagent. cDNAwasgeneratedby
reverse-transcription with AMV reverse transcriptase (Promega, Germany) of
equivalent quantities of RNA, and qRT-PCRwas performed using SYBRGreen
PCR master mix on Light Cycler 480 (Roche, Germany). The following primer
(Eurofins, Germany) pairs were used for the amplification of ID1, ID2, CTGF,
SnoN, Smad1, Smad2, Smad4, Smurf1, and Smurf2, respectively: (ID1) 5 s:
CATGAACGGCTGTTACTCAC; 3as: GTTCCAACTTCGGATTCCGAG; (ID2)
5 s:GACTGCTACTCCAAGCTCAAG; 3as: GTGATGCAGGCTGACAATAGTG;
(CTGF) 5 s: CCAGACCCACTATGATTAGAGC; 3as:GAGGCGTCATTGGTAAC;
(SnoN) 5 s: GCGAACCTGTACTTCTGTTCCTG; 3as:GAAATGCTGCTGGTC
AC; (Smad1) 5 s: GTGCTGGTTCCAAGACACAG; 3as ATTGGGAGAGTGAG
GAAACG; (Smad2) 5 s: AGAAGTCAGCTGGTGGGTCT; 3as: TGAGTGGTGA
GGCTTTCTC; (Smad4) 5 s: TGCCATAGACAAGGTGGAG; 3as: AGCCTCCC
ATCCAATGTTC; (Smurf1) 5 s: GATACCGGATACCAGCGTTTG; 3as:GGTTC
CTATTCTGTCTCGTG and (Smurf2) 5 s: ACAGAGGACAACGCAACAAG; 3as:
ATTACGGATCTCCCATCAG. b-Actin 5 s: CTGACTACCTCATGAAGATCCTC;
3as: CATTGCCAATGGTGATGACCTG was used as control.
Kinase Assay and Cell Proliferation Assay
A radiometric protein kinase assay (33PanQinase activity assay) was used for
measuring activity of protein kinases performed by ProQinase as previously
Elsevier Ltd All rights reserved
Chemistry & Biology
Modulation of Nonactivated R-Smad Levels
described (Cheng et al., 2010). Sulforhodamine B binding (SRB) assay was
used to evaluate cell proliferation (Cheng et al., 2010).
Analysis of Gene Expression Patterns in Xenopus laevis Embryos
Embryos were obtained by in vitro fertilization and cultured with inhibitors
at 14�C from stage 2 to stage 10.5 (early gastrula), RNA was isolated from
embryonic tissue, and cDNA synthesis was performed using random primers.
For each experiment, individual samples were analyzed in triplicate. Quantita-
tive PCR was performed in a q-TOWER (Jena Analytics) under conditions
described in Houston et al. (2002) and Skirkanich et al. (2011). Samples
were normalized to ornithine decarboxylase (ODC) and compared to appro-
priate DMSO/medium control. Primer sequences for qRT-PCR were: ODC:
Forward: 50-TGCACATGTCAAGC-30, Reverse: 50-GCCCATCACACGTT-30;fox1e: Forward: 50-CATGGAGCCCCAGATAAAAG-30, Reverse: 50-TTGGGTC
CAAGGTCCAATAA-30; bra: Forward: 50-TTCTGAAGGTGAGCATGTCG-30,Reverse: 50-GTTTGACTTTGCTAAAAGAGACAGG-30; goosecoid: Forward: 50-TTACCAATGAACAACTGGA-30, Reverse: 50-TTCCACTTTTGGGCATTTTC-30;nr3: Forward: 50-CCAAAGCTTCATCGCTAAAAG-30, Reverse: 50-AAAAGAAG
GGAGGCAAATACG-30; sia: Forward: 50-TCTGGTAGAACTTTACTCTGTTTT
GG-30, Reverse: 50-AACTTCATGGTTTTGCTGACC-30.
TopFlash-Luciferase Reporter Assay
Gsk3b inhibition in living cells was analyzed in Super TOP-Flash (STF) cells
(kindly provided by Jeremy Nathans, Johns Hopkins School of Medicine,
Baltimore, MD). STF cells are HEK293 cells stably transfected with a luciferase
gene containing seven LEF/TCF-binding sites in the promoter (Xu et al., 2004).
For on line measurement, cells were cultured in DMEM without phenol red
containing 10 mM HEPES buffer, 10% FCS, 4.5 g/l glucose and luciferin
(250 mM) in 96-well plates. Measurements were performed in a customized
TopCount plate reader (Perkin-Elmer) modified in a way that plates and
measurement chamber are kept constantly at 35�C ensuring cell viability
during measurement periods of several days. Substances were added at
beginning of measurements as indicated in the text.
Statistical Analysis
The statistical significance of compared measurements was assessed using
Student’s one-tailed or two-tailed t test (Microsoft Excel).
SUPPLEMENTAL INFORMATION
Supplemental Information includes one table and five figures and can be found
with this article online at http://dx.doi.org/10.1016/j.chembiol.2012.09.008.
ACKNOWLEDGMENTS
We thank Elke Lederer, Marie-Sofie Karipidis, and Holger Treiber for technical
assistance; Anna Ahomaa, Kristina Buchner, and Kristina Herold for help per-
forming experiments; Pavlo Holenya for suggestions on experiments and
methods; Peter Hortschansky for recombinant BMP2; Joachim Nickel for
recombinant TGFb; Steven Dooley for providing inhibitors and reagents; and
Jeremy Nathans for making available the Super-TopFlash cells. We thank
Christiane Peuckert for critical reading and corrections. We also thank Aris
Moutsakas and Carl-Hendrik Heldin for critical discussions and comments
on our manuscripts. This work was supported by BMBF MedSys, Drugs-iPS
(FKZ0315298[A,B]) coordinated by Ralf Mrowka.
Received: December 20, 2011
Revised: September 1, 2012
Accepted: September 6, 2012
Published: November 21, 2012
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