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Design Logic of a CannabinoidReceptor Signaling NetworkThat Triggers Neurite Outgrowth
Kenneth D. Bromberg, Avi Ma
’
ayan, Susana R. Neves, Ravi Iyengar
*Cannabinoid receptor 1 (CB1R) regulates neuronal differentiation. To understand the logic underlyingdecision-making in the signaling network controlling CB1R-induced neurite outgrowth, we profiled theactivation of several hundred transcription factors after cell stimulation. We assembled an in silicosignaling network by connecting CB1R to 23 activated transcription factors. Statistical analyses of thisnetwork predicted a role for the breast cancer 1 protein BRCA1 in neuronal differentiation and a newpathway from CB1R through phosphoinositol 3-kinase to the transcription factor paired box 6 (PAX6).Both predictions were experimentally confirmed. Results of transcription factor activation experimentsthat used pharmacological inhibitors of kinases revealed a network organization of partial OR gatesregulating kinases stacked above AND gates that control transcription factors, which together allow fordistributed decision-making in CB1R-induced neurite outgrowth.
S
ignaling through the cannabinoid receptor 1 (CB1R), which couples to the heterotri-mericguaninenucleotide
–
bindingproteins(G proteins) G
i
and G
o,
regulates many physio-logical processes. In cultured mouse Neuro2Acells,CB1Rstimulationinducesneuriteoutgrowththrough a signaling pathway from G
a
o
that ac-tivates the protein kinase c-Src and the transcrip-tion factor signal transducer and activator of transcription3(Stat3)(
1
,
2
).CB1Rsignalingalsohas a key role during central nervous systemdevelopment and in the adult brain (
3
,
4
).Furthermore,CB1R has beenshown to modulateseveral neurological disorders (
5
). However, theorganization of the CB1R signaling network in-volved in cellular state-change decisions has not been well defined. Delineation of the organiza-tion of signaling networks is useful in identifyingemergent decision-making capabilities (
6
). Todo so, we started with delineating individual pathways (
1
,
2
). However, simply verifying the presenceandfunctionofindividualpathwayswillnot advance our knowledge of the design of complex cellular regulatory networks and their decision-making capabilities. A key challenge insystems biology is to identify, experimentallyverify, and understand the organizations of com- plex regulatory systems.To broadly define the cellular network reg-ulating CB1R-induced neurite outgrowth, we in-tegrated transcription factor activity profiling,networkbiology,andcellbiology.First,theCB1R-triggered activation of multiple transcription fac-tors was profiled during neurite outgrowth. Wethen developed an in silico network in which theactivated transcription factors were linked toknown interactors and pathways that regulatethem to identify new components and pathwaysinvolved in neuronal differentiation. Predictionswereexperimentallytestedinculturedandprimaryneurons.Wethenusedselectivepharmacologicalinhibitors in transcription factor activation ex- periments to determine the hierarchy betweenthree key kinases and transcription factors.Theseexperiments allowed for construction of a mapwhere partial OR gates at the level of G proteinsregulating kinases are stacked on top of ANDgates at the level of kinases regulating transcrip-tion factors, allowing for a distributed decision-making capability within the network.
CB1R-regulated transcription factors.
Weassayed transcription factor activation in re-sponsetotheCB1RagonistHU-210inNeuro2Acells by using a commercial array (
7
). Spotted onthis array are 345 oligonucleotide transcriptionfactor
–
binding sites (table S1), enabling the ac-tivation of a large number of transcription factorsto be assayed simultaneously [see (
7
) for arraydetails]. Studies have indicated that CB1R ac-tivation of G
a
o
can stimulate Stat3 (
2
), so we
B
******
N o r m a l i z e d n e u r i t e o u t g r o w t h
****
SustainedEarlyLate
*****
00.250.50.7511.251.5
c - M y b T x n D N C R E B S t a t 3 D B D S t a t 3 Y - > F p c D N A 3 U S F 1 s i R N A S P I 1 s i R N A N F Y A s i R N A c - M y b s i R N A P A X 6 s i R N A S m a d 3 s i R N A N R 3 C 1 s i R N A S C s i R N A L u c s i R N A
SustainedEarly
A
DMSOHU-21020 min
Activated TFs
AP-2
α
CEBP
α
NFYAc-MYBCREBNR3C1RAR
α
STAT3SMAD3GATA2PAX6RUNX2TCF1USF1SPI1
Fig. 1.
IdentificationofpositiveandnegativeregulatorsofCB1R-inducedneuriteoutgrowth. (
A
) Arrays of transcription factor activation in Neuro2A cells treatedwith DMSO as a control or 2
m
M HU-210 (CB1R agonist) for 20 min. The rightpanel highlights several of the activated transcription factors. The colors in thepanelcorrespondtothecircledspotsinthearrays.TCF1,Tcellfactor1.(
B
)Effectsof transcription factor inhibition on neurite outgrowth. Neuro2A cells weretransfectedwiththeindicatedsiRNAsortransfectedwithDNStat3constructs[Y
→
FandDNA-bindingdomain(DBD)],DNCREB,wild-typec-Myb,orpcDNA3(seefig.S11 for construct expression) and then stimulated with HU-210 to induce neuriteoutgrowth.Errorbars,mean±SEM(
n
=3independentexperiments);*,
P
<0.05(statistically significant difference by Student
’
s
t
test) versus the control LucsiRNA; **,
P
< 0.05 (Student
’
s
t
test) versus control pcDNA3. The figure is acompositeofmultipleexperiments.ThesiRNAtransfectionswereperformedastwo experimental sets. Set 1: Luc, AP-2, PAX6, c-Myb, and USF1. Set 2: Luc,NR3C1, Smad3, RAR
a
, CEBP
a
, NFYA, and SPI1. Transfections of each DN con-structwerefirstdoneindependentlyandthenrepeatedasoneexperimentalset.Depletionoftranscriptionfactorexpressionwasconfirmedbyquantitativereal-timereverse transcription polymerase chain reaction (RT-PCR) or immunoblot (fig. S12).
Department of Pharmacology and Systems Therapeutics,Mount Sinai School of Medicine, New York, NY 10029,USA.*To whom correspondence should be addressed. E-mail:ravi.iyengar@mssm.edu
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expected to observe activation of Stat3 on thearray. Mouse Neuro2A cells were treated withHU-210 (2
m
M) for 20, 60, 120, and 360 min toassess transcription factor activation. Ongoingtranscription was required for at least 360 min toinduce neurite outgrowth in response to CB1R signaling (fig. S1). Nuclear extracts were ob-tained and processed for hybridization to thearray. The Entrez Gene names of all the tran-scription factors activated over the 360-min timecourse are displayed in table S2. All of the tran-scription factor
–
activation arrays described intable S2 are shown in fig. S2A, and severaltranscriptionfactorsthatwere activatedat20minarehighlightedinFig.1A.Activatedtranscriptionfactors fell into three main categories: those that were activated early and transiently, such asStat3, Smad3, and Smad4; those that displayedsustained activation, including c-Myb and paired box 6 (PAX6); and those that were activated at later times, such as forkhead box I1 (FOXI1) andupstream transcription factor 1 (USF1). In all, 33transcription factors were activated over the 6-hour time course of CB1R stimulation. Becausethe activations of homeobox D8 (HOXD8),HOXD9, and HOXD10 and Smad3 and Smad4wereeachrepresentedassinglespotsonthearray,they were grouped together in table S2. For thecomputational analysis (see below), they wereused individually. Stat3 was activated at 20 and60 min, and this activation was confirmed by gelshiftanalysis(fig.S3A).cAMPresponseelement
–
binding protein (CREB), a transcription factor known to be involved in neurite outgrowth (
8
),was also activated, and this result was verifiedwhen CREB was phosphorylated on Ser
133
in
Fig. 2.
Construction of networks and identificationof BRCA1 and a PI3K-AKT-PAX6 pathway as regula-tors of CB1R-induced neurite outgrowth. (
A
) Eightmammalian protein-protein interaction databasesand one signaling network were consolidated intoa single network made of 67,379 human protein-protein and protein-ligand interactions (I). This net-work was filtered by removing interactions fromresearch articles that reported more than three in-teractions. The lists of activated and nonactivatedtranscription factors (TFs) at 20 min were used asinput nodes to find direct and neighboring inter-actionsandtoidentifypathsfromtheCB1Rreceptorto the transcription factors (II), enabling us to iden-tify and rank regulators within the network (III). (
B
)A subnetwork created by finding the shortest pathsofamaximumofsevensteps fromthe HU-210 node(HU)tothe23activatedtranscriptionfactors(orangenodes) at 20 min. (I) Paths were found for 17 out ofthe23factors.BRCA1,PI3K,andAKT1arehighlighted(green nodes). HU and CB1R nodes are highlightedinblue.(II)PathwayconnectingCB1RtoPAX6throughPI3K and AKT1 (edited manually after literature re-view). (III)Table showing the ranking ofcomponentsinpathwaysdetectedin acontrolsubnetwork(fig.S4and table S3) versus the activated subnetwork usingthe ranking method described in the supporting on-linematerial(SOM)(
7
).I,II,andIIIin(B)correspondto I, II, and III in (A), respectively. CREBBP, CREB-binding protein; PIK3CA, PIK3 catalytic, alpha; PIP2,phosphatidylinositol 4,5-bisphosphate; PIP3, phos-phatidylinositol3,4,5-trisphosphate.(
C
)Subnetworkcreated by finding the shortest paths of a maximumof two steps between the 23 activated transcriptionfactors.Nineteenofthefactorswereconnectedusingthis method (orange nodes). A binomial proportionstest wasusedtoprune out most of thelessimportantintermediates. BRCA1 is highlighted in green.
B
Gene name Score
BRCA11.165CREBBP0.985FLT10.837PIK3CA0.609YBX10.559GRB20.522PIP20.514EP3000.474PIP30.421KCNIP30.418PLCB10.376AKT10.342FYN0.326
IIIIII
A
FilterI. Create asubnetwork thatconnects theactivated TFsList ofactivatedTFsList ofnon-activatedTFsExperimentalresults fromTF activationarraysIntergrated PPIand SignalingNetworksIII. Rank listof regulatorswithinnetworkII. Find pathwaysfrom HU-210 toactivated TFs andnon-activated TFs
C
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response to HU-210 (fig. S3B). It is likely that CREB is activated through
bg
subunit of G
o
(G
bg
)
–
mediated stimulation of p42 and p44mitogen-activated protein kinase (MAPK) (
9
).MAPK was also activated in response to CB1R stimulation, and the treatment of cells with theupstream MAPK kinase (MEK)
–
1,2 kinase in-hibitor PD 98059 (PD) attenuated phosphoryl-ation of both MAPK and CREB (fig. S3E).Transfectionofadominant-negative(DN)CREBconstruct inhibited cannabinoid-induced neuriteoutgrowth, albeit to a lesser extent than did DNStat3 (Fig. 1B). Retinoic acid receptor (RAR),another well-known regulator of neurite out-growth (
10
), was also activated on the array, andthis finding was confirmed by gel shift analysis(fig. S3C). We also examined several tran-scription factors, including c-Myb, activating protein 2
a
(AP-2
a
), and PAX6, that have not been shown to have a role in neurite outgrowth.Gel shift analysis confirmed the activation c-Myb(fig. S3D). These results validate several of thetranscription factors that were activated on thearrays.To test whether the activated transcriptionfactors might regulate the induction of neuriteoutgrowth, we assessed 10 of the activatedfactors, representing all three categories (early,sustained, and late activation) in addition toCREB and Stat3. Depletion of AP-2
a
, PAX6,and spleen focus forming virus proviral inte-gration oncogene 1 (SPI1) with RNA interfer-ence (RNAi) inhibited cannabinoid-inducedneurite outgrowth by ~60%, and RNAi of RAR
a
was also slightly inhibitory (Fig. 1B). Incontrast, RNAi of Smad3, c-Myb, and nuclear transcription factor
–
Y
a
(NFYA) led to an en-hancement of HU-210
–
stimulated neurite out-growth. Ectopic expression of c-Myb reducedneurite outgrowth by ~30%. Off-target gene-silencing effects of RNAi seemed unlikely becauseCB1R-induced neurite outgrowth was similar incells treated with luciferase (Luc) small interferingRNA (siRNA) and a separate scrambled (SC)siRNA. These results suggest that transcriptionfactor profiling is able to detect both positive andnegative regulators of neurite outgrowth.
In silico network construction and predictingnew components and pathways.
Although thetranscription factor arrays indicate that manytranscription factors are activated during neu-rite outgrowth, they do not provide informationabout the cell signaling pathways and compo-nents that lead to their activation. We identifiedthe upstream signaling pathways and compo-nents regulating the activated transcription fac-tors by constructing a network in silico. For thiswe used available protein-protein interactiondatabases, graph-theory analysis, and statisticaltests. We consolidated eight existing mammalian protein-protein interactions networks, the Bio-molecular Interaction Network Database (BIND)(
11
), the Human Protein Reference Database(HPRD) (
12
), the Molecular Interaction data- base (MINT) (
13
), the Database of InteractingProteins (DIP) (
14
), IntAct (
15
), BioGRID (
16
),Reactome (
17
), and the Protein-Protein Inter-action Database (PPID) (
18
), with a neuronalsignaling network we developed (
19
). To remove potentially low-confidence interactions, such asinteractions reported from yeast two-hybridscreens, we filtered the nine consolidated data sets by removing all articles reporting more thanthree interactions. This method reduced the num- ber of interactions in the consolidated databasefrom 67,379 to 15,494 (Fig. 2A). Applying a shortest-path analysis, we first automaticallyfound undirected paths of a limited path length(two steps, all direct- and second-neighbor inter-actions) between all the transcription factors,knowing the consensus-binding sequences onthe transcription factor
–
activation array. Com- bining all the paths from this search resulted in a subnetwork made of 444 nodes and 1873 inter-actions from 1843 unique references. We mergedthis subnetwork with a large-scale curated signal-ing network we developed from the neuroscienceliterature (
19
). Again applying shortest-path anal-ysis, we searched for directed paths with a lim-ited threshold path length (seven steps) from theCB1R agonist HU-210 to the transcription fac-tors associated with the consensus sequences onthe array. We found paths from HU-210 to 104transcription factors, including 17 of the 23 tran-scription factors that were activated within 20 minin table S2 (Fig. 2B) and 87 transcription factorsthat were not activated (fig. S4). Counting andcomparing the number of times that componentsappeared in pathways to activated factors or non-activated factors enabled us to identify intermediatecomponents in pathways predicted to participatein the regulation of the activated transcriptionfactors (Fig. 2B and algorithm S1). BRCA1, the breastcancersusceptibilityprotein(
20
),was rankedhighest as the most specific regulator of the
CB
N o r m a l i z e d n e u r i t e o u t g r o w t h
*
A
BRCA1siRNADMSOLucsiRNADMSOBRCA1siRNAHU-210LucsiRNAHU-210SCsiRNAHU-210
00.30.60.91.21.51.8
N o r m a l i z e d B R C A 1 m R N A
Time (min)
***
60 120 180 240 300 360000.250.500.751
HU-210MergeHU-210Hoescht(nuclearstain)HU-210Stat3DMSOHoescht(nuclearstain)DMSOStat3BRCA1 siRNALuc siRNA
Fig. 3.
Regulation of CB1R-induced neuriteoutgrowthbyBRCA1.(
A
)EffectofBRCA1siRNAoncannabinoid-induced neurite outgrowth. Neuro2Acells were transfected with Luc siRNA or BRCA1siRNA and stimulated with 2
m
M HU-210 to induceneurite outgrowth orwith DMSO as a control.Amounts of neurite outgrowth in cells exposed toLuc siRNA and HU-210 were normalized to 1, andbaseline amounts of neurite outgrowth were nor-malizedto0(
7
).TreatmentofcellswithanSCsiRNA(SCsiRNAHU)resultedinsimilaramountsofcannabinoid-induced neurite outgrowth as Luc siRNA (Luc siRNA HU). Error bars, mean ± SEM (
n
= 3 independentexperiments),*,
P
<0.01(student
’
s
t
test)versusLucsiRNAHU-210control.(
B
)RegulationofStat3localization.Neuro2Acells weretransfectedwithLuc siRNA or BRCA1siRNAandtreatedwith DMSOor HU-210 for 20min.Cells were fixed, permeablized, and stained with Stat3 antibodies. Purple and yellow arrows indicate cytosolicand nuclear Stat3 localization, respectively. Scale bars indicate distance in micrometers. Nuclei were visualizedwith Hoescht stain. (
C
) Decreased BRCA1 expression in response to CB1R stimulation. Neuro2A cells werestimulated with HU-210 and RNA was isolated at the indicated times. Quantitative real-time RT-PCR wasperformed as described in the SOM (
7
). Error bars, mean ± SEM (
n
= 4 independent experiments); *,
P
< 0.01(Student
’
s
t
test) versus 0 min control.
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