Systems analysis identifies an essential role for SHANK-associated RH domain-interacting protein (SHARPIN) in macrophage Toll-like receptor 2 (TLR2) responses.
ABSTRACT Precise control of the innate immune response is essential to ensure host defense against infection while avoiding inflammatory disease. Systems-level analyses of Toll-like receptor (TLR)-stimulated macrophages suggested that SHANK-associated RH domain-interacting protein (SHARPIN) might play a role in the TLR pathway. This hypothesis was supported by the observation that macrophages derived from chronic proliferative dermatitis mutation (cpdm) mice, which harbor a spontaneous null mutation in the Sharpin gene, exhibited impaired IL-12 production in response to TLR activation. Systems biology approaches were used to define the SHARPIN-regulated networks. Promoter analysis identified NF-κB and AP-1 as candidate transcription factors downstream of SHARPIN, and network analysis suggested selective attenuation of these pathways. We found that the effects of SHARPIN deficiency on the TLR2-induced transcriptome were strikingly correlated with the effects of the recently described hypomorphic L153P/panr2 point mutation in Ikbkg [NF-κB Essential Modulator (NEMO)], suggesting that SHARPIN and NEMO interact. We confirmed this interaction by co-immunoprecipitation analysis and furthermore found it to be abrogated by panr2. NEMO-dependent signaling was affected by SHARPIN deficiency in a manner similar to the panr2 mutation, including impaired p105 and ERK phosphorylation and p65 nuclear localization. Interestingly, SHARPIN deficiency had no effect on IκBα degradation and on p38 and JNK phosphorylation. Taken together, these results demonstrate that SHARPIN is an essential adaptor downstream of the branch point defined by the panr2 mutation in NEMO.
- SourceAvailable from: Berthe Katrine Fiil[Show abstract] [Hide abstract]
ABSTRACT: Methionine 1-linked ubiquitin chains (Met1-Ub), or linear ubiquitin, has emerged as a central post-translational modification in innate immune signalling. Molecular machinery that assembles, senses and, more recently, disassembles Met1-Ub has been identified, and technical advances have enabled identification of physiological substrates for Met1-Ub in response to activation of innate immune receptors. These discoveries have significantly advanced our understanding of how non-degradative ubiquitin modifications control pro-inflammatory responses mediated by nuclear factor κB and mitogen-activated protein kinases. In this review, we will discuss the current data on Met1-Ub function and regulation, and will point to some of the questions that still remain unanswered.This article is protected by copyright. All rights reserved.FEBS Journal 07/2014; · 3.99 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: The ubiquitin system plays a pivotal role in the regulation of immune responses. This system includes a large family of E3 ubiquitin ligases of over 700 proteins and about 100 deubiquitinating enzymes, with the majority of their biological functions remaining unknown. Over the last decade, through a combination of genetic, biochemical, and molecular approaches, tremendous progress has been made in our understanding of how the process of protein ubiquitination and its reversal deubiquitination controls the basic aspect of the immune system including lymphocyte development, differentiation, activation, and tolerance induction and regulates the pathophysiological abnormalities such as autoimmunity, allergy, and malignant formation. In this review, we selected some of the published literature to discuss the roles of protein-ubiquitin conjugation and deubiquitination in T-cell activation and anergy, regulatory T-cell and T-helper cell differentiation, regulation of NF-κB signaling, and hematopoiesis in both normal and dysregulated conditions. A comprehensive understanding of the relationship between the ubiquitin system and immunity will provide insight into the molecular mechanisms of immune regulation and at the same time will advance new therapeutic intervention for human immunological diseases.Advances in Immunology 01/2014; 124C:17-66. · 5.53 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: SHARPIN regulates immune signaling and contributes to full transcriptional activity and prevention of cell death in response to TNF in vitro. The inactivating mouse Sharpin cpdm mutation causes TNF-dependent multi-organ inflammation, characterized by dermatitis, liver inflammation, splenomegaly, and loss of Peyer's patches. TNF-dependent cell death has been proposed to cause the inflammatory phenotype and consistent with this we show Tnfr1, but not Tnfr2, deficiency suppresses the phenotype (and it does so more efficiently than Il1r1 loss). TNFR1-induced apoptosis can proceed through caspase-8 and BID, but reduction in or loss of these players generally did not suppress inflammation, although Casp8 heterozygosity significantly delayed dermatitis. Ripk3 or Mlkl deficiency partially ameliorated the multi-organ phenotype, and combined Ripk3 deletion and Casp8 heterozygosity almost completely suppressed it, even restoring Peyer's patches. Unexpectedly, Sharpin, Ripk3 and Casp8 triple deficiency caused perinatal lethality. These results provide unexpected insights into the developmental importance of SHARPIN.eLife Sciences 12/2014; 3. · 8.52 Impact Factor
Systems analysis identifies an essential role for SHANK-
associated RH domain-interacting protein (SHARPIN) in
macrophage Toll-like receptor 2 (TLR2) responses
Daniel E. Zaka,1, Frank Schmitza,1, Elizabeth S. Golda, Alan H. Diercksa, Jacques J. Peschona, Joe S. Valvoa,
Antti Niemistöb, Irina Podolskya, Shannon G. Fallena, Rosa Suena, Tetyana Stolyara, Carrie D. Johnsona,
Kathleen A. Kennedya, M. Kristina Hamiltonc, Owen M. Siggsd, Bruce Beutlerd,2, and Alan Aderema,2
aSeattle Biomedical Research Institute, Seattle, WA 98109;bDepartment of Signal Processing, Tampere University of Technology, 33101, Tampere, Finland;
cDepartment of Anatomy, Physiology and Cell Biology, University of California, Davis, CA 95616; anddThe Scripps Research Institute, La Jolla, CA 92037
Contributed by Bruce Beutler, May 18, 2011 (sent for review April 11, 2011)
Precise control of the innate immune response is essential to ensure
Systems-level analyses of Toll-like receptor (TLR)-stimulated macro-
phages suggested that SHANK-associated RH domain-interacting
esis was supported by the observation that macrophages derived
from chronic proliferative dermatitis mutation (cpdm) mice, which
harbor a spontaneous null mutation in the Sharpin gene, exhibited
impaired IL-12 production in response to TLR activation. Systems
biology approaches were used to define the SHARPIN-regulated
networks. Promoter analysis identified NF-κB and AP-1 as candidate
transcriptionfactors downstreamofSHARPIN,and network analysis
suggested selective attenuation of these pathways. We found that
the effects of SHARPIN deficiency on the TLR2-induced transcrip-
tome were strikingly correlated with the effects of the recently de-
scribed hypomorphic L153P/panr2 point mutation in Ikbkg [NF-κB
Essential Modulator (NEMO)], suggesting that SHARPIN and NEMO
interact. We confirmed this interaction by co-immunoprecipitation
analysis and furthermore found it to be abrogated by panr2. NEMO-
dependent signaling was affected by SHARPIN deficiency in a man-
ner similar to the panr2 mutation, including impaired p105 and
ERK phosphorylation and p65 nuclear localization. Interestingly,
SHARPIN deficiency had no effect on IκBα degradation and on p38
and JNK phosphorylation. Taken together, these results demon-
strate that SHARPIN is an essential adaptor downstream of the
branch point defined by the panr2 mutation in NEMO.
innate immunity|signal transduction|pattern-recognition|ubiquitylation
flammatory sequelae are mitigated at a number of levels. Prin-
cipal among these is the precise identification of the threat and
the appropriate tailoring of the response. Infectious agents are
precisely identified by a variety of pattern recognition receptors,
including Toll-like receptors (TLRs), which recognize molecular
motifs that are specific to the pathogen (6). Although much is
known about the mechanisms through which TLRs mediate im-
mune responses, a number of important questions remain un-
answered (7). Central to these is a complete knowledge of all of
the critical components within the TLR-signaling pathways and
how dynamic interactions between them lead to the appropriate
coordination of host defense. The precise titration of the re-
sponse requires multiple levels of regulation that include cross-
talk and feedback between various signaling pathways and gene
regulatory networks operating on very different spatial and tem-
poral scales. Systems biology provides a framework in which this
complexity can be addressed. Systems approaches combine prior
knowledge and biological insight with global measurement tech-
nologies and computational methods both to reveal regulatory
system. We have used these approaches to identify transcription
he innate immune system is critical for host defense but,
unchecked, can cause severe inflammatory disease (1–5). In-
factors that function within regulatory circuits to coordinately
amplify and attenuate TLR-mediated responses (8–10). Systems-
level analysis can also be used to contextualize and elucidate the
function of naturally occurring or induced mutations that impact
immune phenotypes. The present work has used this approach to
functionally link two mutations, chronic proliferative dermatitis
mutation (cpdm) and panr2, in the TLR pathway. cpdm is a spon-
taneous null mutation in the Sharpin gene (SHANK-associated
RH domain-interacting protein) (11), and panr2 is a chemically
induced hypomorphic mutation in the Ikbkg gene encoding
NEMO (NF-κB Essential Modulator) (12).
By computationally examining transcriptional and epigenomic
profiles of macrophages activated with a variety of pathogen-
derived agonists, we identified SHARPIN as a potential regulator
of TLR responses. SHARPIN was initially described to interact
with the Shank family of proteins in the postsynaptic density of
excitatory synapses (13) and has subsequently been shown to in-
teract with several other proteins including EYA1 (14) andPTEN
(15); however, the functional significance of these interactions
remains unknown. A role for SHARPIN in immune regulation
was first revealed by the identification of mutations within the
Sharpin gene in two lines of mice displaying a Th2-dominated
cpdm phenotype (11).
Our systems analysis reported here demonstrates that TLR
responses in macrophages are markedly impaired by SHARPIN
deficiency and that SHARPIN controls expression of a subset of
TLR2-induced and NF-κB– and AP-1–dependent genes that over-
laps with those affected by the hypomorphic panr2 mutation in
NEMO. It has recently been reported that SHARPIN is a com-
ponent of the linear ubiquitin chain assembly complex (LUBAC)
that modifies NEMO, thereby promoting the activation of NF-κB
by multiple receptors (16–18). These data complement our results
SHARPIN and NEMO, as well as the other LUBAC component
RBCK1. Our data demonstrate that SHARPIN controls a branch
point in the TLR2/NF-κB/AP-1 pathways that is necessary for the
production of proinflammatory cytokines, including the Th1-
skewing factor IL-12.
Author contributions: D.E.Z., F.S., E.S.G., A.H.D., J.J.P., B.B., and A.A. designed research;
D.E.Z., F.S., A.H.D., J.S.V., I.P., S.G.F., R.S., T.S., C.D.J., K.A.K., M.K.H., and O.M.S. performed
research; D.E.Z., F.S., A.H.D., and A.N. analyzed data; and D.E.Z., F.S., E.S.G., A.H.D., and
A.A. wrote the paper.
The authors declare no conflict of interest.
Data deposition: Microarray data from this study have been deposited in the Gene Ex-
pression Omnibus (GEO) database (accession no. GSE29947).
1D.E.Z. and F.S. contributed equally to this article.
2To whom correspondence may be addressed. E-mail: firstname.lastname@example.org or
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| July 12, 2011
| vol. 108
| no. 28www.pnas.org/cgi/doi/10.1073/pnas.1107577108
SHARPIN Deficiency Impairs TLR Responses in Macrophages. We
identified SHARPIN as a potential regulator of macrophage
responses over the course of our systems-level transcriptional and
epigenomic analyses of combinatorial TLR pathway activation.
To evaluate the role of SHARPIN in innate immunity, we ana-
lyzed TLR responses in macrophages derived from cpdm mice,
which bear a null mutation in the Sharpin gene (11). IL-12p40
production was markedly impaired in response to nearly all TLR
ligands evaluated, including Pam3CSK4(TLR2), LPS (TLR4),
CpG-B (TLR9), and R848 (TLR7) (Fig. 1A). The cpdm mutation
also strongly attenuated macrophage production of IL-12p40 in
response to infection with Listeria monocytogenes, which signals
through TLR2, TLR5, and various Nod-like receptor family
members (19–21), (Fig. 1B). Because IL-12p40 production in
effects of SHARPIN deficiency on the response to this ligand in
greater detail. Quantitative real-time PCR (qRT-PCR) analysis
revealed marked attenuation of Il12b and Tnf mRNA expression
as early as 1–2 h poststimulation (Fig. 1C). In addition to Il12b,
induction of another Th1-promoting cytokine, Il18, was also ab-
rogated (Fig. 1C). These results demonstrate that SHARPIN
plays a major role in proinflammatory cytokine induction in re-
sponse to TLR activation in macrophages.
Systems Analysis Predicts That SHARPIN Regulates NF-κB and AP-1.
We applied the tools of systems biology to identify the pathways
controlled by SHARPIN. Transcriptome analysis of wild-type
macrophages identified 400 genes induced threefold or more by
a 12-h stimulation with Pam3CSK4in two independent experi-
ments (Fig. 2A and Dataset S1). SHARPIN deficiency arising
from the cpdm mutation resulted in threefold impaired induction
of 87 of these genes, including many proinflammatory cytokines
(Fig. 2A and Dataset S1). To identify the transcription factors
(TFs) that mediate the effect of SHARPIN on macrophage
to scan the proximal promoter sequences of all 400 Pam3CSK4-
regulated genes, and we then applied the Gene Set Enrichment
Analysis (GSEA) algorithm (23) to determine which TFs were
associated with impaired Pam3CSK4responses. The only TF-
binding sites that were over-represented in the promoters of
SHARPIN-dependent genes relative to the overall set of 400
Pam3CSK4-induced genes were NF-κB and AP-1 (Fig. 2). This
result suggests that SHARPIN may be required for maximal NF-
κB and AP-1 activation in response to TLR2 stimulation in
To further explore the link between SHARPIN, NF-κB, and
AP-1, we performed network analysis using Cytoscape (24) to
visualize direct protein–protein and protein–DNA interactions
obtained from InnateDB (25) (Fig. 2C). This analysis provides
a literature-based context for our TF-binding predictions. Many
of the genes with impaired induction in SHARPIN-deficient
macrophages are established targets of the NF-κB TFs RELA/
p65, NFKB1/p50, and c-REL/REL, including Il12b, Il1a, Il1b, Il6,
and Nos2. Notably, several of the genes not affected by SHAR-
PIN deficiency are also known direct targets of NF-κB TFs, in-
cluding Icam1, Itgal, Mmp9, and Cxcl10. This result suggests that
SHARPIN deficiency results in selective inhibition of TLR2-
induced NF-κB activation in macrophages. The link between
SHARPIN and AP-1 was similarly evaluated (Fig. 2D). Once
again, SHARPIN deficiency resulted in the inhibition of a subset
ofTLR2-induced genesthat areknown directtargets ofAP-1 TFs.
Therefore, although the GSEA suggested that loss of SHARPIN
significantly impairs activation of both NF-κB and AP-1, it likely
does not result in complete ablation of these pathways.
We analyzed the link between SHARPIN, NF-κB, and AP-1 in
greater depth by integrating the SHARPIN-dependent gene set
defined above with our database of transcriptome responses in
mutant macrophages. These included null mutations in the Nfkb1,
Tnf, Atf3, and Il10 genes and ENU-induced hypomorphic point
mutations in Map3k8 (sluggish) encoding TPL2 (26) and Ikbkg
(panr2) encoding NEMO (12). The effects of SHARPIN de-
ficiency on TLR2-activated macrophage transcriptomes did not
resemble the effects of Nfkb1, Map3k8, Atf3, and Il10 mutations
(Fig. 3A and Dataset S2), indicating that the dominant role of
SHARPIN is not specifically associated with these genes. The
effects of TNF deficiency were significantly correlated with the
effects of SHARPIN deficiency (P < 1 × 10−15), although the
effects were generally twofold less than the effects of SHARPIN
(Fig. 3A and Dataset S2). The impaired TNF induction that we
observed in cpdm macrophages (Fig. 1C) thus only partially
and littermate controls [+/()] were stimulated with the indicated TLR ligands for 12 h. Secreted IL-12p40 protein was measured in the supernatant by ELISA.Ligand
(B) cpdm and control BMM were infected with L. monocytogenes at multiplicity of infection (MOI) of 2 and 10 for 8 h. Secreted IL-12p40 protein was measured
Error bars indicate mean and SEM from two independent experiments. Significance levels: *P < 0.05, **P < 0.01, ***P < 0.001, and ns (not significant).
Zak et al.PNAS
| July 12, 2011
| vol. 108
| no. 28
accounts for the overall defect. Contrary to all other mutants ex-
amined,theeffects ofthehypomorphicNEMO mutationpanr2on
TLR2 responses were very highly correlated with the effects of
the mutations tracking each other qualitatively and quantitatively
(Fig. 3B). Detailed qRT-PCR temporal analysis in independent
experiments confirmed that SHARPIN deficiency and the NEMO
panr2 mutation similarly impair Pam3CSK4-induced expression of
Il1a and Il1b (Fig. 3C), with the effect of the panr2 mutation being
somewhat stronger than that of cpdm. These effects were specific,
as Pam3CSK4-induced Nfkbia expression was only marginally
affected by either mutation (Fig. 3D). Such remarkable overlap
between the effects of these mutations, identified through our
systems biology analysis, led us to predict that SHARPIN and
NEMO interact functionally in a manner abrogated by the
SHARPIN Interacts with NEMO. HA-tagged SHARPIN co-immuno-
precipated with Flag-tagged wild-type NEMO, confirming our hy-
pothesis that SHARPIN and NEMO interact in cells (Fig. 4A).
Expression of NEMO harboring the panr2 mutation, L153P,
resulted in abrogation of this interaction (Fig. 4A). Thus, the over-
whelming similarity between the effects of SHARPIN deficiency
and the NEMO panr2 mutation is likely to result from the specific
loss of this interaction.
Given that TLR2 responses in panr2 macrophages were slightly
more attenuated than those in SHARPIN-deficient cpdm mac-
rophages(Fig.3),wetestedwhetherinteractions between NEMO
and the SHARPIN paralog RBCK1 (RBCC protein interacting
with PKC 1; also known as HOIL-1L) (11), were similarly abro-
gated. RBCK1 has recently been shown to interact with and
polyubiquitinate NEMO as part of the NF-κB-activating LUBAC
(27). V5-tagged RBCK1 readily co-immunoprecipitated with
Flag-tagged wild-type NEMO. As in the case of SHARPIN, this
interaction was abrogated by the panr2 mutation (Fig. 4B).
SHARPIN Controls a Branch of NEMO-Dependent Signaling. TLR2-
induced signaling was affected by SHARPIN deficiency in a
manner mirroring, but generally weaker than, the reported effects
of panr2 mutation in NEMO. These include impaired phosphor-
ylation of p105 and ERK (Fig. 5A). Phosphorylation of p105 is
dependent on I-kappa-B kinase (IKK) complex activation and
leads to p105 degradation, TPL2 activation, and ERK phosphor-
ylation (28). Thus, simultaneous impairment in p105 and ERK
phosphorylation mutually reinforce each other and suggest a spe-
cific abrogation of the p105 kinase activity of the IKK complex in
the absence of SHARPIN–NEMO interactions. Neither SHAR-
PIN deficiency nor the NEMO panr2 mutation results in complete
ablation of IKK complex activity because IκBα degradation
was not impaired by either (Fig. 5B). Thus, SHARPIN–NEMO
interactions potentially control a branch point in IKK activity—
ablating IKK p105 kinase activity while having no effect on IKK-
induced IκBα degradation. In addition to stabilizing TPL2, p105
functions as an IκB itself, sequestering p50 homodimers (29) as
well as p65- and c-Rel–containing heterodimers in the cytoplasm
(30). This function for p105 in macrophages is supported by our
observation that SHARPIN deficiency results in moderately im-
paired TLR2-induced nuclear localization of p65 (Fig. 5C).
Systems biology approaches have the capacity to unravel the bi-
ological complexity that underlies the exquisite precision of in-
12 h, and RNA was extracted and analyzed by microarray (Agilent). A total of 400 genes (rows) induced at least threefold by Pam3CSK4in control BMM in two
independent experiments are shown. Genes are sorted according to impairment (top) or enhancement (bottom) of responses in cpdm BMM in two in-
dependent experiments. (Left) Pink intensity indicates increasing expression relative to unstimulated wild-type BMM. Values for each gene are scaled relative
to the overall maximum value observed for that gene. (Right) Orange intensity indicates increasing GSEA enrichment scores for NF-κB– and AP-1–binding sites.
(B) Details of NF-κB and AP-1 GSEA. Genes are ordered according to impairment (left) or enhancement (right) of responses in cpdm BMM. Red line: differences
between Pam3CSK4responses in cpdm and wild-type BMM for ordered genes. Blue bars: presence of NF-κB– or AP-1–binding sites in promoters of ordered
genes. Orange lines: GSEA enrichment scores for NF-κB or AP-1, calculated using the effect of cpdm mutation on Pam3CSK4responses (red line) and binding
site information (blue bars). Dashed lines: median and 95% quantile maximum enrichment scores in permuted datasets. (C and D) Cytoscape interaction
networks for NF-κB (C) and AP-1 (D). White, blue, and pink nodes (genes) are induced by Pam3CSK4in a manner not affected, strongly impaired, or enhanced,
respectively, by SHARPIN deficiency. Gray nodes: genes not induced by Pam3CSK4. Orange borders: genes predicted to regulate the networks as part of NF-κB
(C) or AP-1 (D). Green and black edges (connecting genes and gene products): known protein–DNA and protein–protein interactions, respectively.
SHARPIN is predicted to regulate TLR2-induced NF-κB and AP-1 activation. (A) cpdm and control BMM were stimulated with Pam3CSK4(300 ng/mL) for
| www.pnas.org/cgi/doi/10.1073/pnas.1107577108Zak et al.
nate immune responses. This precision is achieved by a large
number of signaling networks that influence each other by subtle
feed-forward and feedback mechanisms. Systems approaches
usually begin with large-scale measurements of transcriptomes or
proteomes, and the data are then computationally analyzed to
provide testable hypotheses that are evaluated by more traditional
approaches. Measurement technologies are now robust and sen-
sitive; however, biological inference technologies are still being
developed. We have developed a number of computational tools
that have enabled us to identify transcriptional control mecha-
nisms governing innate immune responses. For example, we used
to confirm that the transcription factor ATF3 functions as a neg-
ative regulator of a subset of NF-κB–dependent genes that are
induced by TLR4 (8). A follow-up systems analysis further refined
our understanding of this process by demonstrating the subtle
interplay between ATF3 and C/EBPδ in fine-tuning the response:
NF-κB acting as an initiator, ATF3 acting as an attenuator, and
C/EBPδ actingasan amplifier(9). Further studiesshowedthat the
interactions within this regulatory circuit occur at the epigenetic
level. The in vivo relevance of this network was confirmed in a
mouse sepsis model (9, 31).
Perhaps the most powerful tool in unraveling the immune re-
sponse has been genetic analysis of the mouse. This analysis
has been enabled by targeted gene deletion studies, chemical- or
radiation-induced mutations as well as mutations that arose spon-
taneously. Whereas gene-targeting experiments are often initiated
on the basis of a priori assumptions about predicted gene function
within established pathways, phenotypic screens of mutagenized
mice can reveal unique and unpredicted components of such
pathways. However, it is difficult and labor intensive to establish
mechanisms linking mutations to their respective phenotypes.
Systems biology may be of assistance in this process. Massively
genes affected by mutations causing phenotypes of interest. It will
mutations in desired genes. As illustrated in the present paper,
mice harboring mutations that cause interesting immune pheno-
for NF-κB, ATF3, and C/EBPδ, which are generated in a highly
standardized manner, could be used as a comparator to identify
signaling pathways that are functionally associated with mutated
genes of interest. For example, a gene associated with an innate
immune phenotype could be compared with a compendium of
TLR-induced signaling and gene regulatory networks; if the net-
work generated overlaps with the network triggered by a known
in an associated pathway. The fact that these networks are gener-
ated using thousands of data points (e.g., entire transcriptomes)
makes it far less likely that such an overlap occurs by chance.
Such a comparative transcriptomic approach was applied in the
present study to link the cpdm mutation in SHARPIN to pathways
known to regulate TLR responses. The SHARPIN-dependent
genes specifically overlapped with genes regulated by the panr2
hypomorphic mutation in NEMO (12), and the extraordinarily
strong association between the effects of these mutants suggested
that SHARPIN might interact with NEMO. This prediction was
confirmed; SHARPIN and NEMO interact, and interestingly,
we demonstrated that the panr2 mutation in NEMO impairs this
mutants and respective wild-type (WT) controls were stimulated with Pam3CSK4(300 ng/mL) for 12 h, and RNA was extracted and analyzed by microarray
(Agilent: cpdm, panr2, Nfkb1, Atf3, Il10; Affymetrix: sluggish and Tnf). A total of 251 genes (rows) induced at least twofold in two independent wild-type
replicates for each mutant are shown. Genes are colored according to impairment (blue) or enhancement (red) of Pam3CSK4responses in indicated mutants
compared with respective wild type. (B) Correlation between the effects of cpdm mutation (black dots) and panr2 mutation (red dots) on macrophage
responses to Pam3CSK4, plotted against the effects of cpdm mutation (R[panr2 vs. cpdm] = 0.82, P < 1 × 10−15). Impairments or enhancements consistently
observed in two independent experiments are plotted. (C and D) BMM derived from homozygous cpdm mice, cpdm littermate controls [+/()], hemizygous
panr2 mice, and wild-type panr2 littermate controls (+/Y) were stimulated with Pam3CSK4(300 ng/mL) for 0–12 h. RNA was harvested and reverse-transcribed
into cDNA. (C) Il1a and Il1b transcript levels were measured by SYBR Green qRT-PCR. Error bars indicate mean and SEM from two independent experiments.
(D) Nfkbia transcript levels were measured by Taqman qRT-PCR.
The effect of SHARPIN deficiency on macrophage TLR2 responses specifically mirrors the NEMOpanr2mutation. (A) BMM derived from the indicated
Zak et al. PNAS
| July 12, 2011
| vol. 108
| no. 28
interaction (Fig. 4). Although qualitatively and quantitatively
associated, the effects of the panr2 mutation on macrophage
responses appeared stronger than the effects of SHARPIN de-
ficiency; there is a residual level of proinflammatory cytokine in-
duction in SHARPIN-deficient macrophages that is not observed
in panr2 macrophages (Fig. 3). This suggested that the panr2 mu-
tation was also able to impair a SHARPIN-independent pathway.
The SHARPIN paralog, RBCK1/HOIL-1L (13), which recently
was shown to interact with NEMO as part of the LUBAC complex
(27), was an attractive candidate to mediate the SHARPIN-
independent pathway. This hypothesis was reinforced by our ob-
servation that the panr2 mutation ablates the RBCK1–NEMO
interaction as well (Fig. 4B). This contention is strengthened by
a recent observation that SHARPIN and RBCK1 are present in
distinct LUBAC complexes that are both capable of polyub-
iquitinating NEMO (16–18).
Given the interaction between SHARPIN and NEMO, we ex-
amined whether the known signaling pathways that are impaired
by the panr2 mutation (12) were similarly affected by SHARPIN
deficiency. To facilitate the interpretation of the results, we have
constructed a model that is presented in Fig. S1. Like panr2, TLR-
induced phosphorylation of p105 was ablated in SHARPIN-
deficient macrophages with the consequential interruption of NF-
κB p65 translocation to the nucleus (Fig. 5). Similarly, ERK
phosphorylation was significantly decreased, although to a lesser
phosphorylation is mediated exclusively through SHARPIN and
that SHARPIN-dependent ERK phosphorylation may occur via
the p105-dependent TPL2 pathway (Fig. S1A). It is possible that
the ERK phosphorylation that occurs in the absence of SHAR-
PIN, but is nevertheless impaired by the panr2 mutation, involves
the SHARPIN homolog RBCK1 (Fig. S1A). These signaling
defects were observed in the absence of any effects on IκBα deg-
radation or p38 and JNK phosphorylation.
The expanded model suggests a bifurcation in the MyD88
pathway that occurs at NEMO. On the one hand, there are the
signals that are ablated by the L153P/panr2 mutation (Fig. S1A).
We have shown that SHARPIN controls part of this branch. This
branch is essential for maximal induction of many proinflam-
matory cytokines, including IL-12, IL-1α, IL-1β, IL-18, and TNF.
On the other hand, there are the signals that are unaffected by the
L153P/panr2 mutation. This branch controls the induction of
a different set of genes (Fig. S1B). Interestingly, NF-κB and AP-1
are effector TFs for both branches of the pathway. This suggests
a previously unappreciated specificity in NF-κB and AP-1 activi-
ties. It is possible that different members of these TF families
mediate the differential responses. This specificity may also arise
at the level of the IKK complex itself, given that IKK-dependent
phosphorylation of p105 (32) is SHARPIN-dependent and IKK-
dependent phosphorylation of IκBα (32) is not.
The etiology of hyper-eosinophilic skin inflammation in
SHARPIN-deficient mice remains unclear. Reciprocal engraft-
ment and hematopoietic reconstitution experiments indicate that
disease initiation is dependent upon SHARPIN deficiency within
the skin (33). However, disease progression is associated with an
imbalanced Th2-dominated T-cell response (34, 35). Thus, both
skin-intrinsic and -extrinsic mechanisms contribute to disease.
Exogenous IL-12 attenuates the severity of disease in SHARPIN-
deficient mice, suggesting that IL-12 insufficiency contributes to
disease pathogenesis (34). Notably, we have shown that SHAR-
PIN is essential for the induction of two myeloid-derived cyto-
kines important for Th1-polarized immune responses, IL-12 and
IL-18, in response to TLR2 activation. The panr2 mouse does not
display the skin phenotypes associated with SHARPIN-deficient
mice. Although differences in genetic backgrounds may contrib-
ute to these phenotypic distinctions, it is also possible that the
cpdm and control BMM were stimulated with Pam3CSK4(300 ng/mL) for the
indicated times. Equal amounts of cell lysates were analyzed by immuno-
blotting for p105, ERK, JNK, and p38 MAPK phosphorylation (A) and IκBα
protein levels (B). (C) BMM were stimulated with Pam3CSK4(300 ng/mL) for
30 min. Cells were fixed, stained with anti-p65, and imaged. Relative nuclear-
to-cytoplasmic localization of p65 (p65Nuc./p65Cyto.) for individual BMM was
quantified using automated image analysis. Each point represents an in-
dividual control macrophage (filled circles) or cpdm BMM (open circles).
Representative results from one of two independent experiments are shown;
bars indicate median values. Significant differences were observed between
stimulated and unstimulated control BMM and between stimulated cpdm
and stimulated control BMM (P < 0.001 and P < 0.05, respectively).
SHARPIN controls a specific branch of NEMO-dependent signaling.
mutation. Lysates of HEK293T cells expressing tagged SHARPIN, RBCK1,
NEMO (wild type), and NemoL153P (panr2) were subjected to immunopre-
cipitation using anti-FLAG beads and analyzed by Western blot. (A) HA-
tagged SHARPIN readily co-immunoprecipitates with FLAG-tagged wild-type
(WT) NEMO but not with panr2 NEMO. (B) HA-tagged SHARPIN and V5-
tagged RBCK1 readily co-immunoprecipitate with wild-type NEMO but not
with panr2 NEMO. Results representative from one of at least two in-
dependent experiments are shown.
SHARPIN interacts with NEMO in a manner abrogated by the panr2
| www.pnas.org/cgi/doi/10.1073/pnas.1107577108Zak et al.
panr2 mutation is permissive for those pathways critical for der-
mal/epidermal homeostasis. Additionally, impaired TNF-induced
activation of NF-κB within keratinocytes is responsible for skin
inflammation associated with epidermallesions in NF-κB pathway
of these findings, it is likely that TNF receptor signaling within the
epidermis is less affected by panr2 than by SHARPIN deficiency.
Future efforts will be directed toward a comprehensive analysis of
receptor-specific and cell type-specific requirements for SHAR-
PIN in NF-κB– and AP-1–dependent gene regulation.
Materials and Methods
Mice. Sharpincpdm, C57BL/KaLawRij controls for Sharpincpdm, Il10−/−, Nfkb1−/−,
and Tnf−/−mice were obtained from the Jackson Laboratory. Atf3−/−,
Ikbkgpanr2, and Map3k8sluggishmice have been described (8, 12, 26). For
microarrays, macrophages derived from female homozygous Sharpincpdm
mice were compared with age- and sex-matched wild-type controls. For all
other experiments involving Sharpincpdm, macrophages derived from homo-
zygous mutants were compared with littermate controls. For all experiments
involving Ikbkgpanr2, macrophages derived from male hemizygous mutants
were compared with wild-type littermate control males. For all other strains,
macrophages derived from mutant strains were compared with macrophages
derived from age- and sex-matched C57BL/6controls(JacksonLaboratory). All
work was approved by the Institute for Systems Biology Institutional Animal
Health Guide for Care and Use of Laboratory Animals as its standard.
Bone Marrow-Derived Macrophage Cultures. Bone marrow was collected from
femurs and cultured for 7–10 d in complete RPMI containing 10% heat-
inactivated FCS (HyClone Laboratories), 100 U/mL penicillin, 100 μg/mL strep-
tomycin, 2 mM L-glutamine, and 50 ng/mL recombinant human Macrophage
Colony-Stimulating Factor (rhM-CSF) (Peprotech).
Ligands. TLR ligands were obtained as follows: Pam3CSK4 (EMC micro-
collections GmbH), Salmonella minnesota R595 (Re) ultra-pure LPS (List Bi-
ological), CpG-ODN1826 (CpG-B; Invivogen), R848 (GL Synthesis), PolyI:C
(Amersham Biosciences), and Fugene-6 (Roche).
Macrophage Infections. Bonemarrow-derivedmacrophageswereplatedinsix-
well cell-culture plates at 1 × 106cells/well. The following day, wild-type
Listeria monocytogenes 10403s (a generous gift from Dan Portnoy, University
ofCalifornia,Berkeley, CA)was addedatmultiplicitiesofinfection (MOIs)of2
and10.Macrophageswere then incubated for 1hat 37°Cbeforechangingto
cell culture media containing 15 μg/mL gentamicin to kill all extracellular
bacteria. Cells were then incubated for an additional 7 h before supernatants
ELISA. IL-12p40 protein levels were measured in supernatants using Duoset
DY499 according to the manufacturer’s instructions (R&D). Tests for signif-
icant differences were performed by Bonferonni posttests of repeated
measures two-way ANOVA (Graphpad PRISM).
qRT-PCR, Microarrays and Analysis, Expression Constructs and Cloning,
Immunoprecipitation and Immunoblotting, and Immunofluorescence. De-
tailed methods are provided in the SI Materials and Methods.
ACKNOWLEDGMENTS. This work was supported by the National Institutes
of Health Contract HHSN272200700038C (to A.A.), Grants 5R01AI032972 and
5R01AI025032 (to A.A.), and by Academy of Finland application no. 213462,
Finnish Programme for Centres of Excellence in Research 2006–2011 (A.N.).
1. Janeway CA, Jr., Medzhitov R (2002) Innate immune recognition. Annu Rev Immunol
2. Aderem A, Ulevitch RJ (2000) Toll-like receptors in the induction of the innate
immune response. Nature 406:782–787.
3. Medzhitov R (2001) Toll-like receptors and innate immunity. Nat Rev Immunol 1:
4. Nathan C (2002) Points of control in inflammation. Nature 420:846–852.
5. Kobayashi KS, Flavell RA (2004) Shielding the double-edged sword: Negative
regulation of the innate immune system. J Leukoc Biol 75:428–433.
6. Kawai T, Akira S (2010) The role of pattern-recognition receptors in innate immunity:
Update on Toll-like receptors. Nat Immunol 11:373–384.
7. Zak DE, Aderem A (2009) Systems biology of innate immunity. Immunol Rev 227:
8. Gilchrist M, et al. (2006) Systems biology approaches identify ATF3 as a negative
regulator of Toll-like receptor 4. Nature 441:173–178, and correction (2008) 451:1022.
9. Litvak V, et al. (2009) Function of C/EBPdelta in a regulatory circuit that discriminates
between transient and persistent TLR4-induced signals. Nat Immunol 10:437–443.
10. Ramsey SA, et al. (2008) Uncovering a macrophage transcriptional program by
integrating evidence from motif scanning and expression dynamics. PLOS Comput
11. Seymour RE, et al. (2007) Spontaneous mutations in the mouse Sharpin gene result in
multiorgan inflammation, immune system dysregulation and dermatitis. Genes
12. Siggs OM, et al. (2010) A mutation of Ikbkg causes immune deficiency without
impairing degradation of IkappaB alpha. Proc Natl Acad Sci USA 107:3046–3051.
13. Lim S, et al. (2001) Sharpin, a novel postsynaptic density protein that directly interacts
with the shank family of proteins. Mol Cell Neurosci 17:385–397.
14. Landgraf K, et al. (2010) Sipl1 and Rbck1 are novel Eya1-binding proteins with a role
in craniofacial development. Mol Cell Biol 30:5764–5775.
15. He L, Ingram A, Rybak AP, Tang D (2010) Shank-interacting protein-like 1 promotes
tumorigenesis via PTEN inhibition in human tumor cells. J Clin Invest 120:2094–2108.
16. Ikeda F, et al. (2011) SHARPIN forms a linear ubiquitin ligase complex regulating NF-
κB activity and apoptosis. Nature 471:637–641.
17. Tokunaga F, et al. (2011) SHARPIN is a component of the NF-κB-activating linear
ubiquitin chain assembly complex. Nature 471:633–636.
18. Gerlach B, et al. (2011) Linear ubiquitination prevents inflammation and regulates
immune signalling. Nature 471:591–596.
19. Zenewicz LA, Shen H (2007) Innate and adaptive immune responses to Listeria
monocytogenes: A short overview. Microbes Infect 9:1208–1215.
20. Warren SE, et al. (2010) Cutting edge: Cytosolic bacterial DNA activates the
inflammasome via Aim2. J Immunol 185:818–821.
21. Leber JH, et al. (2008) Distinct TLR- and NLR-mediated transcriptional responses to an
intracellular pathogen. PLoS Pathog 4:e6.
22. Vadigepalli R, Chakravarthula P, Zak DE, Schwaber JS, Gonye GE (2003) PAINT: A
promoter analysis and interaction network generation tool for gene regulatory
network identification. OMICS 7:235–252.
23. Subramanian A, et al. (2005) Gene set enrichment analysis: A knowledge-based
approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci USA
24. Smoot ME, Ono K, Ruscheinski J, Wang PL, Ideker T (2011) Cytoscape 2.8: New
features for data integration and network visualization. Bioinformatics 27:431–432.
25. Lynn DJ, et al. (2008) InnateDB: Facilitating systems-level analyses of the mammalian
innate immune response. Mol Syst Biol 4:218.
26. Xiao N, et al. (2009) The Tpl2 mutation Sluggish impairs type I IFN production and
increases susceptibility to group B streptococcal disease. J Immunol 183:7975–7983.
27. Tokunaga F, et al. (2009) Involvement of linear polyubiquitylation of NEMO in NF-
kappaB activation. Nat Cell Biol 11:123–132.
28. Beinke S, Robinson MJ, Hugunin M, Ley SC (2004) Lipopolysaccharide activation of the
TPL-2/MEK/extracellular signal-regulated kinase mitogen-activated protein kinase
cascade is regulated by IkappaB kinase-induced proteolysis of NF-kappaB1 p105. Mol
Cell Biol 24:9658–9667.
29. Savinova OV, Hoffmann A, Ghosh G (2009) The Nfkb1 and Nfkb2 proteins p105 and
p100 function as the core of high-molecular-weight heterogeneous complexes. Mol
30. Sriskantharajah S, et al. (2009) Proteolysis of NF-kappaB1 p105 is essential for T cell
antigen receptor-induced proliferation. Nat Immunol 10:38–47.
31. Gilchrist M, et al. (2010) A key role for ATF3 in regulating mast cell survival and
mediator release. Blood 115:4734–4741.
32. Heissmeyer V, Krappmann D, Hatada EN, Scheidereit C (2001) Shared pathways of
IkappaB kinase-induced SCF(betaTrCP)-mediated ubiquitination and degradation for
the NF-kappaB precursor p105 and IkappaBalpha. Mol Cell Biol 21:1024–1035.
33. Gijbels MJ, HogenEsch H, Bruijnzeel PL, Elliott GR, Zurcher C (1995) Maintenance of
donor phenotype after full-thickness skin transplantation from mice with chronic
proliferative dermatitis (cpdm/cpdm) to C57BL/Ka and nude mice and vice versa.
J Invest Dermatol 105:769–773.
34. HogenEsch H, et al. (2001) Increased expression of type 2 cytokines in chronic
proliferative dermatitis (cpdm) mutant mice and resolution of inflammation
following treatment with IL-12. Eur J Immunol 31:734–742.
35. HogenEsch H, Dunham A, Seymour R, Renninger M, Sundberg JP (2006) Expression of
chitinase-like proteins in the skin of chronic proliferative dermatitis (cpdm/cpdm)
mice. Exp Dermatol 15:808–814.
36. Wullaert A, Bonnet MC, Pasparakis M (2011) NF-κB in the regulation of epithelial
homeostasis and inflammation. Cell Res 21:146–158.
Zak et al.PNAS
| July 12, 2011
| vol. 108
| no. 28