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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SCIENCE
VOL 320 16 MAY 2008
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RESEARCH ARTICLES
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