Similarly, both TNFa and IL-1b strongly induced mRNA and protein expression of inter-cellular adhesion molecule ICAM-1 and in each case this was prevented by adenovirus expressing the do
Trang 1ICAM-1 expression is highly NF-jB-dependent in A549 cells
No role for ERK and p38 MAPK
Neil S Holden1, Matthew C Catley2, Lisa M Cambridge2, Peter J Barnes2and Robert Newton1
1
Department of Biological Sciences, University of Warwick, Coventry, UK;2Thoracic Medicine, National Heart & Lung Institute, Imperial College Faculty of Medicine, London, UK
The transcription factor nuclear factor jB (NF-jB) is an
activator of multiple cytokines, chemokines and adhesion
molecules, which are important in inflammatory diseases
such as asthma, and is consequently considered as an
attractive therapeutic target In the present study, a
con-stitutively active dominant version of IjBa, IjBaDN, was
introduced into A549 pulmonary cells by
adenovirus-mediated delivery The dominant IjB, but not a null viral
vector, prevented the induction of NF-jB-dependent
transcription by both tumor necrosis factor a (TNFa) and
interleukin-1b (IL-1b) Similarly, both TNFa and IL-1b
strongly induced mRNA and protein expression of
inter-cellular adhesion molecule (ICAM)-1 and in each case this
was prevented by adenovirus expressing the dominant IjB,
but not by the null virus, thereby establishing ICAM-1 as
an NF-jB-dependent gene Numerous studies have
sug-gested key roles for the p38 and extracellular regulated
kinase (ERK) mitogen-activated protein kinase (MAPK) cascades in the activation and transactivation of NF-jB
We show here that SB203580, a selective inhibitor of the p38 MAPK, and PD098059 and UO126, both selective inhibitors of the ERK MAPK cascade, have no effect on TNFa or IL-1b-induced translocation and DNA binding
of NF-jB Furthermore, these inhibitors showed no pharmacologically relevant effect on NF-jB-dependent transcription nor was there any effect on expression of ICAM-1 Taken together these data highlight the potential use of inhibition of the NF-jB signalling pathway in pulmonary inflammatory diseases and suggest that inhi-bitors of the p38 and ERK MAPK pathways may be of lesser effect
Keywords: NF-jB; transactivation; ICAM-1; ERK; p38
The transcription factor nuclear factor-jB (NF-jB) is a
central regulator of the immune system and promotes the
transcription of over 150 genes [1] As many of these genes
are inflammatory, and include cytokines, chemokines,
adhesion molecules as well as other enzymes, such as
inducible nitric oxide synthase and cyclooxengenase-2,
NF-jB may provide an attractive target for therapeutic
intervention in inflammatory diseases [2] NF-jB exists as
a homo or heterodimer made up from subunits from the rel
family of proteins, which in vertebrates, comprises of p65
(RelA), p50/p105, p52/p100, c-Rel and RelB [3] Within
resting cells, NF-jB is retained in the cytoplasm complexed
to an inhibitor protein from the IjB family [3] Stimulation with pro-inflammatory cytokines, such as tumour necrosis factor a (TNFa) or interleukin 1b (IL-1b), activates the IjB kinase (IKK) complex, which then phosphorylates the IjB [3] This leads to ubiquitination of the IjB, targeting it for rapid degradation by the 26S proteosome [3] Degradation
of IjB reveals a nuclear localization signal (NLS) allowing NF-jB to interact with the nuclear import protein karyo-pherin a2 [4] This allows NF-jB to translocate into the nucleus where it can bind to jB sequences in the promoters
of NF-jB-dependent genes to up-regulate transcription [3] There are still many aspects of the NF-jB activation pathway that have yet to be elucidated For example, the mechanisms that enhance the transactivation of NF-jB once it is bound to its consensus sequence remain poorly defined and numerous studies have suggested the involve-ment of the p38 mitogen activated protein kinase (MAPK) and the extracellular signal-regulated kinases (ERK) in the activation or potentiation of NF-jB-dependent transcrip-tion [5] Thus, the selective p38 inhibitor, SB203580, prevents NF-jB/p65-dependent, transactivation without affecting NF-jB DNA binding [6,7] Possible downstream effects of the p38 MAPK include; phosphorylation of histone H3 to enhance recruitment of NF-jB [8], phos-phorylation of the TATA binding protein (TBP) to increase transactivation of the DNA bound NF-jB [9], or phos-phorylation of serine 276 of p65 or cAMP response element binding protein (CREB) binding protein (CBP), events
Correspondence to R Newton, Department of Biological Sciences,
University of Warwick, Coventry CV4 7AL, UK.
Fax: + 44 247 652 3701, Tel.: + 44 247 657 4187,
E-mail: RNewton@bio.warwick.ac.uk
Abbreviations: CBP, CREB binding protein; ERK, extracellular
signal-regulated kinase; GAPDH, glyceraldehyde-3-phosphate
dehydrogenase; ICAM-1, intercellular adhesion molecule 1; IKK, IjB
kinase; IL-1b, interleukin 1b; MAPK, MAPK/ERK kinase; MEK,
mitogen activated protein kinase; MOI, multiplicity of infection;
NF-jB, nuclear factor jB; TBP, TATA binding protein; MTT,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide;
TNFa, tumour necrosis factor a.
Note: A web site is available at: http://www.bio.warwick.ac.uk/
molphys.asp
(Received 12 September 2003, revised 4 December 2003,
accepted 7 January 2004)
Trang 2which have both been shown to increase the ability of p65
to interact with CBP/p300 [10,11] Similarly, inhibition of
the ERK pathway, with the MEK1 inhibitor, PD098059,
abrogated IKK activity and dominant negative constructs
of ERK prevented NF-jB DNA binding activity [12,13] In
addition, the ERK pathway has been suggested to both
enhance DNA binding activity as well as to phosphorylate
downstream cofactors including CBP/p300 and positive
cofactor 1 to increase the transactivational potential of
NF-jB [10,14]
The pulmonary epithelium is the primary site of contact
with airborne allergens, irritants, pathogens and other
proinflammatory agents that trigger exacerbation in airway
diseases [15] This epithelium is biosynthetically active and
acts as a source of multiple inflammatory cytokines,
chemokines, prostanoids and other mediators as well as
adhesion molecules such as intercellular adhesion molecule
1 (ICAM-1) [15] In this context, ICAM-1 is particularly
important in inflammatory diseases such as asthma, where it
is not only responsible for the recruitment of inflammatory
cells from the blood to the airways, but also acts as a
receptor for many viruses that may exacerbate asthma [16]
Numerous studies suggest a role for NF-jB in ICAM-1
expression, but an unequivocal demonstration in
pulmon-ary epithelial cells is currently lacking As NF-jB is
considered to be a candidate for therapeutic intervention
in airway inflammation, we have investigated the role of
NF-jB in the induction of ICAM-1 expression by
pro-inflammatory cytokines In addition, we have used
NF-jB-dependent transcriptional reporters and ICAM-1 expression
to address the role of p38 and ERK pathways in the
activation of NF-jB dependent transcription in pulmonary
epithelial cells
Materials and methods
Cell culture, cytokines and drugs
A549 cells were grown to confluency in six-well plates as
described previously [17] Cells were cultured overnight in
serum free media before changing to fresh serum free media
containing TNFa or IL-1b (both from R & D systems,
Abingdon, UK) SB203580, PD098059, and U0126 (all
from Calbiochem, Nottingham UK) were dissolved in
dimethylsulfoxide Final concentrations of
dimethylsulfox-ide added to cells were < 0.1% and this had no effect on
any of the responses (data not shown)
NF-jBluciferase reporter cell lines
The NF-jB-dependent reporter cells, A549 6jBtkluc, were
grown as described previously [17] These contain a stably
integrated plasmid with three tandem repeats of the
sequence 5¢-AGC TTA CAA GGG ACT TTC CGC
TGG GGA CTT TCC AGG GA-3¢, which has two copies
of the decameric NF-jB binding site (bold text) upstream of
a minimal thymidine kinase promoter driving a luciferase
gene Confluent cells in 24-well plates were changed to
serum-free medium and treated as indicated before
harvest-ing at 6 h in 1· reporter lysis buffer (Promega,
South-ampton, UK) Luminescence was measured using the
luciferaseassay system (Promega)
Adenovirus infection A549 cells were either infected with empty Ad5 expression vector or a vector expressing an IjBa protein with an N-terminal deletion (Ad5IjBaDN) [18] This deletion renders IjBa immune to signal-induced proteolysis and generates a constitutive inhibitor of NF-jB As previously described, cells were infected at a multiplicity of infection (MOI) of 10, a level which we have shown previously to infect > 95% of cells, and then cultured for 24 h prior to treatment with drugs or cytokines [19]
Cell viability assay Cell viability was assessed by using the 3-(4,5-dimethylthi-azol-2-yl)-2,5-diphenyl-tetrazolium bromide [thiazolyl blue tetrazolium bromide (MTT)] assay (Sigma, Poole, UK) according to the manufacturer’s instructions
Semi-quantitative reverse transcription PCR RNA isolation, reverse transcription, primers, PCR conditions, and cycling parameters for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were as described previously [17] Primer pairs for ICAM-1 (Accession No: BC015969) (5¢fi3¢) were (forward) CCG TGT ACT GGA CTC CAG AA, (reverse) AGG TGT AGC TGC ATG GCA TA Cycling parameters for ICAM-1 were: 94C,
30 s; 58C, 30 s; 72 C, 30 s The number of amplification cycles used was that necessary to achieve exponential amplification where product formation was proportional
to starting cDNA and was established empirically [17] Following amplification, PCR products (10 lL) were run
on 2.0% agarose gels stained with ethidium bromide After densitometric analysis usingTOTALLABsoftware, version 1 (Nonlinear Dynamics), data were expressed as the ratio of ICAM-1/GAPDH
Electrophoretic mobility shift assay (EMSA) Nuclear proteins were isolated 1 h after stimulation and the consensus NF-jB (5¢-AGT TGA GGG GAC TTT CCC AGG-3¢) probe (Promega) radioactively labelled as des-cribed previously [20] Specificity of binding was determined
by the prior addition of 100-fold excess unlabeled consensus oligonucleotide Reactions were separated on 7% native acrylamide gels before vacuum drying and autoradiography Western blot analysis
Cells were harvested in 100 lL of 10 mMTris/HCl (pH 7.5), 0.15MNaCl, 1.5 mMMgCl2, 0.65% (v/v) NP-40, 0.5 mM phenylmethylsulfonyl fluoride, 0.5 mMdithiothreitol Sam-ples were run on 10% SDS polyacrylamide gels and transferred to Hybond-ECL nitro-cellulose paper (Amer-sham Pharmacia, Bucks, UK) using standard techniques Membranes were probed with mouse monoclonal anti-human GAPDH Ig (#4699–9555) (Biogenesis Ltd, Poole, UK) or mouse polyclonal anti-ICAM-1 (sc-8439) Ig (Santa Cruz Biotechnology, CA, USA) at dilutions of 1 : 20 000 and 1 : 1000, respectively Alternatively, membranes were probed for phospho-hsp27 (#2401), or ERK 1/2 (#9100)
Trang 3(both phospho and pan ERK 1/2) (New England BioLabs,
Hertfordshire, UK) at a dilution of 1 : 1000 and incubated
overnight at 4C In all cases, proteins were visualized using
ECL (Amersham Pharmacia Biotech, Bucks, UK)
accord-ing to the manufacturer’s instructions
Results
The establishment ofICAM-1 as an NF-jB-dependent
gene
Western blot analysis revealed extremely low levels of
ICAM-1 protein in untreated cells, but following treatment
with either TNFa or IL-1b, ICAM-1 protein was rapidly
induced (Fig 1) This was first apparent by 2 h
poststim-ulation and levels of ICAM-1 protein continued to rise over
the 18 h of the experiment To examine the role of NF-jB
in the induction of ICAM-1, cells were infected with an
adenoviral vector that over-expresses IjBaDN, a dominant
inhibitor of NF-jB [18] We have described previously the
effectiveness of this construct in repressing both NF-jB
DNA binding and transcriptional activity in A549 cells [19]
Both TNFa and IL-1b produced a robust increase in
reporter activity, measured 6 h poststimulation as
des-cribed previously [21] In each case, this activity was reduced
to basal levels by the IjBaDN expressing virus (Fig 2) The
null, or empty, viral vector showed no effect on induction
of reporter activity (Fig 2) Likewise, there was no effect of
this virus on basal activation of the reporter (data not
shown) [19]
Having established that IjBDN over-expression is
effect-ive at preventing induction of NF-jB transcriptional
activity, this virus was used to test the role of NF-jB in
the induction of ICAM-1 expression As in previous
experiments, unstimulated cells expressed very little
ICAM-1 protein and this was also true of ICAM-1 mRNA
(Fig 3) Upon stimulation with either TNFa or IL-1b,
ICAM-1 mRNA and protein was dramatically increased
Prior infection with the IjBaDN expressing adenovirus, but not the null virus, totally prevented the expression of ICAM-1 in response to stimulation by both TNFa, and IL-1b These data therefore establish that the induction of ICAM-1 by both TNFa and IL-1b is highly NF-jB-dependent in A549 cells
Effect of SB203580, PD098059 and U0126 on NF-jB DNA binding and NF-jB-dependent transcription
To examine the role of the p38 and the ERK MAPK pathways, we examined the effects of the p38 MAPK inhibitor, SB203580, the MEK1 inhibitor, PD098059 and the MEK 1/2 inhibitor, U0126, on TNFa- and IL-1b-induced NF-jB translocation and DNA binding As induction of NF-jB DNA binding was shown previously
to be maximal at 1 h poststimulation [21], the cells were stimulated for 1 h with TNFa or IL-1b prior to analysis by EMSA Following each treatment, two NF-jB DNA binding complexes were observed and these were both removed by competition with the addition of 100-fold excess
of cold oligonucleotide (Fig 4A) Densitometric analysis of these complexes indicated a 10-fold increase in NF-jB DNA binding when compared to unstimulated cells (Fig 4A, lower panels) In each case the prior addition of SB203580, PD098059, or U0126 revealed no effect on the induction of NF-jB DNA binding
To investigate the potential role these MAPK cas-cades on NF-jB-dependent transcription, 6jBtk cells
Fig 1 Time course of ICAM-1 expression in TNFa and IL-1b
stimu-lated cells Cells were either not stimustimu-lated (NS) or stimustimu-lated with
TNFa (10 ngÆmL)1) or IL-1b (1 ngÆmL)1) and harvested at 0, 2, 6 and
18 h post stimulation, prior to Western blot analysis of ICAM-1 A
representative blot is shown and optical densities (n ¼ 3) are plotted as
arbitrary units as means ± SEM.
Fig 2 Validation of an adenovirus expressing a constitutively active IjB A549 6jBtk cells were either infected or not at a MOI of 10 with either Ad5IjBDN or null virus as indicated After changing to serum free media, cells were either unstimulated or treated with TNFa (10 ngÆmL)1) or IL-1b (1 ngÆmL)1) as indicated Cells were harvested after 6 h for luciferase activity determination Data (n ¼ 4) are expressed as percentage of stimulated cells and are plotted as means ± SEM.
Trang 4were stimulated with either TNFa or IL-1b in the presence
of absence of various concentrations of SB203580,
PD098059 or UO126 At concentrations from 0.01 and
1 lMthese inhibitors showed little or no effect on luciferase
levels (Fig 4B) However decreased luciferase activity was
observed with 10 lMSB203850 and this appeared to reach a
plateau by 100 lM With PD098059 and UO126 a variable
effect was observed at 10 lM and by 100 lM a marked
repression was apparent (Fig 4B) However, in each case,
these concentrations were 10 to 100-fold greater than the
reported Kivalues and so probably result from nonspecific
effects of these kinase inhibitors ([22] and refs therein) To
assess the effect of these compounds on cell viability, MTT
assays were performed on cells preincubated with a range
of concentrations of the inhibitors With the exception of
U0126, which showed a 30% loss of cell viability at 100 lM,
MTT analysis revealed little or no effect of PD098059,
SB203580 or U0126 on cell viability at any of the
concentrations tested (0.01–100 lM; data not shown)
Effect of MEK and p38 MAPK inhibitors on ICAM-1
expression in A549 cells
As previous reports implicating MAPK pathways in
NF-jB-dependent transcription have suggested the involvement
of mechanisms that impact on the promoter architecture,
and this may not always be faithfully reproduced in a reporter system, we examined the effect of SB203580 and PD098059 on ICAM-1 expression as an endogenous indicator of NF-jB transcriptional activity As in previous experiments, stimulation of A549 cells with TNFa or IL-1b caused a considerable up regulation of ICAM-1 expression
at 6 h poststimulation compared to unstimulated cells (Fig 5) Pre-incubation of cells with a range of concentra-tions of PD098059 or SB203580 showed little or no effect on ICAM-1 expression in either TNFa or IL-1b stimulated cells suggesting that neither the p38 MAPK nor the MEK1-ERK pathways are involved in the expression of ICAM-1
in this system (Fig 5)
Validation of the SB203580, PD098059 and U0126
in A549 cells
To validate the inhibitory action of SB203580, the phos-phorylation of heat shock protein 27 (hsp27), a downstream target of p38 MAPK was analysed [22] Both TNFa and IL-1b markedly induced hsp27 phosphorylation and this was prevented by preincubation with SB203580 (Fig 6) Analysis of GAPDH expression confirmed equality of loading Following densitometric analysis, EC50values of 0.21 and 0.23 lMwere calculated for inhibition of TNFa-and IL-1b-stimulated hsp27 phosphorylation, respectively These values are consistent with the published IC50 for SB203580 (0.6 lM) indicating the inhibition of p38 at pharmacologically relevant concentrations [23]
Similarly, the MEK inhibitors, PD098059 and U0126, were validated by analysis of phosphorylation of ERKs 1 and 2, which lie downstream of MEK1 [24] Both TNFa and IL-1b upregulated phosphorylation of p42/44 ERKs and preincubation with PD098059 abrogated this response Following densitometry, EC50values of 0.26 and 0.58 lM were calculated for TNFa- and IL-1b-stimulated cells, respectively These are consistent with published values for MEK 1 inhibition (5 lM) [25] As a loading and expression control, the expression of total p42/44 was also examined Likewise, U0126 completely prevented the phosphorylation
of p42/44 with EC50values of 0.23 and 0.1 lMfor TNFa and IL-1b stimulated cells, respectively This is again consistent with the published IC50 value for MEK1/2 inhibition (0.065 lM) suggesting that both PD098059 and U0126 are functionally active in A549 cells [26]
Discussion
The adhesion molecule ICAM-1 has been shown by numerous studies to be an important factor in many allergic diseases such as asthma, where it not only plays a critical role in airway inflammation and the development of hyper-responsiveness [27], but also acts as a receptor for infection
by rhinoviral and respiratory syncytial viruses, which both increase exacerbations in asthma [28,29] The regulation of ICAM-1 production is therefore a potential target for the development of new therapeutics in asthma Various studies have implicated NF-jB in the transcriptional regulation of ICAM-1 in a variety of cells [30–33], however, these studies often rely on reporter systems, which may not faithfully mimic the architecture of the endogenous promoter In the current study, we have over-expressed a dominant form of
Fig 3 Effect of an adenovirus expressing a constitutively active IjB on
ICAM-1 expression Cells were either infected or not at a MOI of 10
with either Ad5IjBDN or null virus as indicated After changing to
serum free media, cells were either not stimulated (NS) or treated with
TNFa (10 ngÆmL)1) or IL-1b (1 ngÆmL)1) as indicated Cells were
either harvested after 6 h for semiquantitative RT-PCR analysis or
after 24 h for Western blot analysis of both ICAM-1 and GAPDH
expression Representative blots are shown, and data (n ¼ 6) were
normalized to GAPDH expression and are plotted as means ± SEM.
Trang 5IjBa to unequivocally demonstrate that the induction of
ICAM-1 expression is NF-jB-dependent in TNFa- and
IL-1b-stimulated pulmonary epithelial A549 cells
As noted above, both the p38 and ERK MAPK
cascades are variously implicated in the activation of
NF-jB However, as many of these studies rely on transfection
analysis of reporter plasmids, which are prone to artefacts,
or on the effects of single high dose inhibitors, which may
lead to pharmacologically unrelated events, to define
functional roles for MAPK, they should be treated with
a degree off caution ([22] and references therein) Thus, in contrast to the observation that dominant negative ERKs completely prevented NF-jB DNA binding [12,13], the present analysis showed no effect of either the MEK 1 inhibitor PD098059 or the MEK 1/2 inhibitor U0126 on NF-jB translocation and DNA binding As a similar result was observed with the p38 MAPK inhibitor, SB203580, these data indicate that neither the ERK nor p38 MAPK pathways play a significant role in the activation of NF-jB DNA binding in A549 cells However, numerous studies
Fig 4 Effect of PD098059, SB203580 and
U0126 on NF-jB DNA binding, and
NF-jB-dependent transcription (A) Cells were
pre-incubated with PD098059, SB203580 or
U0126 (all at 10 l M ) and then stimulated
with either TNFa (10 ngÆmL)1) or IL-1b
(1 ngÆmL)1) After 1 h, nuclear extracts were
prepared and analysed by EMSA
Represen-tative blots are shown (n ¼ 4) XS indicates
the presence of a 100-fold excess of cold
NF-jB probe Specific complexes, defined by
competition (XS), are indicated, and data,
expressed as a percentage of stimulated cells
are plotted as means ± SEM (B) 6jBtk A549
cells were preincubated with various
concen-trations of PD098059, SB203580 or U0126
(0.01–100 l M ) before stimulation with either
TNFa (10 ngÆmL)1) or IL-1b (1 ngÆmL)1).
Cells were harvested after 6 h for luciferase
activity determination Data (n ¼ 5) was
expressed as percentage of stimulated cells and
are plotted as means ± SEM.
Fig 5 Effect of p38 MAPK and MEK
inhi-bitors on ICAM-1 expression Cells were
pre-incubated with various concentrations of
PD098059, SB203580 or U0126 (0.01–30 l M )
before stimulation with either TNFa
(10 ngÆmL)1) or IL-1b (1 ngÆmL)1) Cells were
harvested after 6 h for Western blot analysis
of ICAM-1 expression Representative blots
are shown and data (n ¼ 2), expressed as
percentage of stimulated cells, are plotted as
means ± SEM.
Trang 6have implicated roles for p38 and MEK 1/2 cascades in the
transactivation of NF-jB downstream of DNA binding
[5–7], through modulation of various components of the
basal transcriptional machinery such as TBP [9], or even
NF-jB/p65 itself [10,11] In addition, p38-dependent
phosphorylation may lead to modification of histone
proteins causing changes in the chromatin structure of
NF-jB-dependent genes and allowing increased access of
the basal transcription machinery to the DNA [8] More
recently, the p38 downstream kinase, mitogen- and
stress-activated protein kinase-1 (MSK-1) was shown to
phos-phorylate p65 at Ser276 and this was shown to be essential
for association with CBP/p300 and thus up-regulation of
NF-jB transactivation [10] However, in the present study,
inhibitors of the p38 and ERK MAPK pathways were all
shown to be functionally active at the correct
pharmaco-logical concentrations, yet had no effect on the activation
of NF-jB-dependent transcription as determined by
luci-ferasereporter assay However, as transcriptional reporters
may not mimic the true physiological architecture
exhib-ited by an endogenous promoter within cells, ICAM-1 was
also used as an endogenous NF-jB-dependent reporter
However, this analysis confirmed the reporter data
indica-ting that neither the p38 nor the ERK MAPK pathways
play a major role in NF-jB-dependent transcription in these cells Finally, it should be noted that the use of the p38 MAPK inhibitor, SB203580, only implicates the a and
b isofroms of p38, as SB203580 has little or no effect on p38 c and d and it is possible that this could explain discrepancies between over-expression and inhibitor based studies [34]
In conclusion, the data presented here firmly demon-strates that the adhesion molecule ICAM-1 is a highly NF-jB-dependent gene in A549 pulmonary epithelial cells and is therefore a useful endogenous reporter of NF-jB-dependent transcription Furthermore, despite the extensive evidence documenting roles for the p38 and ERK MAPK pathways in NF-jB-dependent transcription, we found that highly selective inhibitors of these pathways had no effect on activation of NF-jB DNA binding, NF-jB-dependent transcription, or on the endogenous NF-jB-dependent gene ICAM-1 Taken together, these data highlight the potential utility of inhibiting the NF-jB signalling pathway in pulmonary inflammatory diseases and suggest that inhibi-tors of the p38 and ERK MAPK pathways may be of lesser effect in this cell type
Acknowledgements
N S H and M C C are MRC collaborative students supported by Novartis Pharmaceuticals and Aventis Pharmaceuticals, respectively.
L M C was funded by a grant from Novartis Pharmaceuticals. References
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