Protein kinase A regulates caspase-1 via Ets-1 in
bone stromal cell-derived lesions: a link between
cyclic AMP and pro-inflammatory pathways in
Madson Q. Almeida1, Kit Man Tsang1, Chris Cheadle5, Tonya Watkins5, Jean-Charles Grivel3,
Maria Nesterova1, Raphaela Goldbach-Mansky4and Constantine A. Stratakis1,2,∗
1Section on Endocrinology and Genetics, Program on Developmental Endocrinology and Genetics (PDEGEN),
2Pediatric Endocrinology Inter-Institute Training Program, Eunice Kennedy Shriver National Institute of Child Health
and Human Development (NICHD),3Program on Physical Biology, NICHD and4Translational Autoinflammatory
Disease Section, NIAMS, National Institutes of Health (NIH), Bethesda, MD 20892, USA and5Genomics Core,
Division of Allergy and Clinical Immunology, School of Medicine, Johns Hopkins University, Mason Lord Building,
Center Tower, Room 664, 5200 Eastern Avenue, Baltimore, MD 21224, USA
Received September 10, 2010; Revised September 10, 2010; Accepted October 6, 2010
Patients with genetic defects of the cyclic (c) adenosine-monophosphate (AMP)-signaling pathway and those
with neonatal-onset multisystem inflammatory disease (NOMID) develop tumor-like lesions of the long bones.
The molecular basis of this similarity is unknown. NOMID is caused by inappropriate caspase-1 activity,
which in turn activates the inflammasome. The present study demonstrates that NOMID bone lesions are
derived from the same osteoblast progenitor cells that form fibroblastoid tumors in mice and humans with
defects that lead to increased cAMP-dependent protein kinase A (PKA) signaling. NOMID tumor cells
showed high PKA activity, and an increase in their cAMP signaling led to PKA-specific activation of cas-
pase-1. Increased PKA led to inflammation-independent activation of caspase-1 via over-expression of the
proto-oncogene (and early osteoblast factor) Ets-1. In NOMID tumor cells, as in cells with defective PKA regu-
lation, increased prostaglandin E2 (PGE2) led to increased cAMP levels and activation of Wnt signaling, like
in other states of inappropriate PKA activity. Caspase-1 and PGE2 inhibition led to a decrease in cell prolifer-
ation of both NOMID and cells with abnormal PKA. These data reveal a previously unsuspected link between
abnormal cAMP signaling and defective regulation of the inflammasome and suggest that caspase-1 and
PGE2 inhibition may be therapeutic targets in bone lesions associated with defects of these two pathways.
In humans and mouse models, inappropriate activation of the
pathway leads to the development of bone lesions that vary
(OCM) and chondro- and osteo-sarcomas (C/OS) (1–5). We
and others have demonstrated that these lesions are derived
from early progenitors of the osteoblastic lineage, bone
stromal (also known as ‘stem’) cells (BSCs) (4,5).
Recently, similar lesions were described in children with
neonatal-onset multisystem inflammatory disease (NOMID)
(6). NOMID is caused by an abnormally increased activation
of the potent pro-inflammatory cytokine interleukin-1b
(IL1B); most NOMID patients carry mutations in NLRP3,
the gene encoding cryopyrin (7,8). The latter is an essential
∗To whom correspondence should be addressed at: Section on Endocrinology and Genetics, Program on Developmental Endocrinology and Genetics
(PDEGEN), Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), National Institutes of Health (NIH), 10
Center Drive, Building 10, NIH-Clinical Research Center, Room 1-3216, MSC1103, Bethesda, MD 20892, USA. Tel: +1 3014964686; Fax: +1
3014020574; Email: firstname.lastname@example.org
# The Author 2010. Published by Oxford University Press. All rights reserved.
For Permissions, please email: email@example.com
Human Molecular Genetics, 2011, Vol. 20, No. 1
Advance Access published on October 11, 2010
component of the multimeric inflammasome complex that
activates caspase-1, an enzyme that cleaves the inactive
IL1B precursor (pro-IL1B) to its active form, IL1B (9).
Caspase-1 plays a prominent role in inflammatory responses
and the regulation of apoptosis of various tumor cells (10,11).
Approximately 60% of the NOMID patients have severe
arthropathy that most commonly involves the knees (7).
These patients occasionally develop bulky masses usually in
the physis of the distal femur and the proximal tibia (6).
These are typically the skeletal locations of lesions associated
with abnormal cAMP signaling in both mice (1,5) and humans
(2). The histology of NOMID bone lesions also shows simi-
larities to FD and OCM: cartilage formation is disorganized
at an area proximal to the growth plate; the nearby trabecular
bone is undermineralized and appears to be less dense than
normal, whereas proliferative fibrous tissue fills bone
marrow space with several foci of irregular calcification (6).
The molecular mechanisms underlying the development of
bone lesions in NOMID patients are hitherto unknown.
Prompted by these histological similarities, we investigated
the possibility that NOMID bone lesions could be associated
with increased cAMP signaling. We also explored the hypoth-
esis that inappropriate caspase-1 activation may be involved in
the bone defects of patients with OCM and mice with exten-
sive FD-like lesions and increased cAMP-dependent protein
kinase [protein kinase A (PKA)] signaling, such as those
that are haploinsufficient for the regulatory subunit type 1A
(PRKACA), the Prkar1a+/2Prkaca+/2mice (5). Indeed,
NOMID tumor cells showed high cAMP levels and increased
PKA activity. Like in bone tumor cells from the Prkar1a+/2
Prkaca+/2mice, NOMID tumor cells expressed human BSC
markers and showed an enrichment of the Wnt signaling
pathway in their gene signature. Our functional studies
showed that PKA regulates caspase-1 expression through
Ets-1. Both mouse and human bone tumor cells showed high
prostaglandin E2 (PGE2) levels, a well-known stimulator of
the cAMP/PKA pathway (12,13) and inhibitor of chondrocyte
These data support the hypothesis that an increase in cAMP
signaling through PKA leads to caspase-1 activation by Ets-1,
at least in bone. This is the first time that cAMP and/or PKA is
shown to have an effect on caspase-1, a finding that may have
wider implications for the effects of cAMP on protein
secretion that is regulated by caspase-1, as well as the potential
regulation of the inflammasome by cAMP and/or PKA.
the catalytic subunit
Ets-1 and caspase-1 are up-regulated in bone tumors due
to PKA defects
The Ets-1 gene, which plays an important role in cartilage and
bone formation (15), was significantly over-expressed in
(Prkar1a+/2) and wild-type
(Fig. 1A). Ets-1 was also highly expressed in Prkar1a+/2
Prkaca+/2bone tumors at the protein level by western blot
and immunohistochemistry (IHC) (Fig. 1B and C). The
Ets-2 transcription factor, in contrast, was significantly less
expressed in Prkar1a+/2Prkaca+/2bone tumors (Fig. 1B;
Supplementary Material, Fig. S1).
Caspase-1 gene, which is known to be transcriptionally
regulated by the Ets-1 proto-oncogene (10), was found to be
over-expressed in Prkar1a+/2Prkaca+/2bone tumors both
at the message and protein levels (Figs 1A and 2A and B).
Cryopyrin, a protein highly expressed in polymorphonuclear
cells and chondrocytes, forms a complex with pro-caspase-1
and the adaptor protein pycard (also known as ASC or
apoptosis-associated speck-like protein containing a CARD
domain) that leads to the activation of caspase-1. The inflam-
masome complex leads to the release of active caspase-1,
which in turn activates IL1B through the cleavage of
pro-IL1B (9,16). Nlrp3 (the product of the cryopyrin gene)
(Fig. 1A). IL1B expression was also significantly higher in
Prkar1a+/2Prkaca+/2mice at both the message (Fig. 1A)
and protein levels (Fig. 2C).
We then studied human tumors (OCM) from patients with
PRKAR1A defects. Unfortunately, the only material available
was paraffin-embedded: ICH showed strong staining for
ETS-1, IL1B and cryopyrin and less intense for caspase-1
(Supplementary Material, Fig. S2).
Characterization of NOMID non-lesional and tumor cells
Human bone cells were isolated by enzymatic digestion from
a tumor-like bone lesion (NOMID tumor cells) and normal
cartilage shavings (NOMID non-lesional cells) from the
same patient with NOMID arthropathy (Fig. 3A). Two non-
rs34298354) in the NLRP3 were found in leukocyte DNA
from this patient. The BSC marker CD146 was increased in
NOMID tumor cells compared with non-lesional cells (56.8
versus 40%; median fluorescent intensity, 976 versus 631;
x2¼ 19, P , 0.001) (Fig. 3B).
Microarray analysis was performed in NOMID non-lesional
and tumor cells using the Illumina Beadarrayswsystem. All
genes were displayed in the heat maps constructed by proces-
sing the data using unsupervised hierarchical clustering
(Fig. 3C), as described previously (17). The functional analy-
sis of the whole-genome transcriptome profiling was per-
formed using the DAVID Bioinformatic Resources 2008
(NIAID, NIH) (18) (Fig. 3D and Supplementary Material,
Table S1). Gene set enrichment analysis was used to identify
pathways associated with the NOMID-tumor signature; this
revealed high statistical association with Wnt signaling in
tumor cells compared with non-lesional cells isolated from
the same patient (P ¼ 0.002). In addition, apoptosis and gluta-
mate metabolism pathways were over-expressed in NOMID
tumor cells (P ¼ 0.003 and P ¼ 0.004, respectively).
PKA activity, cAMP levels and PKA subunit expression in
An increase in cAMP-stimulated kinase activity was detected
in NOMID tumor cells when compared with NOMID non-
lesional cells (163 991 + 6222 versus 122 307 + 2114; P ¼
0.02). The former also had high cAMP levels (2.5 + 0.2
166Human Molecular Genetics, 2011, Vol. 20, No. 1
versus 0.14 + 0.05; P , 0.001) (Fig. 4A). To determine
which type of PKA accounted for the higher PKA activity in
(DEAE) cellulose ion-exchange column chromatography fol-
lowed by elution with a linear sodium chloride gradient on
total proteins extracted from NOMID non-lesional and bone
tumor cells. The fractions were then measured for PKA type
I (PKA-I) versus type II (PKA-II) activity: PKA-I complex
was eluted between 40 and 80 mM of NaCl, whereas PKA-II
complex was eluted between 180 and 270 mM of NaCl under
these conditions. There were no free forms of regulatory or
catalytic subunits under these ranges of NaCl concentration.
Only PKA-I activity, and not PKA-II, was significantly
higher in NOMID tumor cells (P ¼ 0.01) (Fig. 4B).
We then assessed PKA regulatory and catalytic subunit
expression. The regulatory subunits RIa and RIIa had
similar protein levels in NOMID non-lesional and tumor
cells, whereas RIIb was undetectable (Fig. 4C). NOMID
tumor cells showed expression of all PKA catalytic subunits
(Ca, Cb, Cg and Prkx), with a relative increase in the Cb
subunit (Fig. 4D). In addition, NOMID tumor cells showed
higher expression levels for both Ets-1 and, as expected,
caspase-1 (Fig. 4E).
In summary, the higher PKA-I activity in NOMID tumor
cells may be primarily explained by an increase in Cb
expression, the catalytic subunit that is primarily responsible
for FD-like lesions and associated tumors in mice with PKA
PKA regulates caspase-1 via Ets-1 proto-oncogene
To examine the role of PKA in the regulation of Ets-1 tran-
scription factor, we analyzed the effects of specific siRNAs
against the PKA regulatory and catalytic subunits in transi-
ently transfected MC3T3 cells. MC3T3 cells are mouse pre-
osteoblasts that express genes encoding BSC and other osteo-
blast markers (19,20). The down-regulation of the target genes
by siRNAs was confirmed by western blot analysis.
Down-regulation of PKA regulatory subunits (RIa, RIIa
and RIIb) did not modify Ets-1 and caspase-1 expression
(Supplementary Material, Fig. S3). The main PKA-I catalytic
subunit is Ca coded by the PRKACA gene. We observed a dra-
matic reduction in Ets-1 and caspase-1 expression after transi-
ent transfection of the MC3T3 cells with catalytic subunit Ca
siRNA (Fig. 5A). Cells transfected with Ets-1 siRNA also
showed a reduction in caspase-1 expression, whereas Ets-2
down-regulation had no effect on caspase-1 protein levels
(Fig. 5B). When cells were transfected with the PRKACA
Figure 1. Ets-1 and caspase-1 inflammasome up-regulation in bone tumors from Prkar1a1/2Prkaca1/2mice. (A) Quantitative RT–PCR analysis showing
Nlrp3, ASC, Casp1, IL1b and Ets-1 over-expression in Prkar1a+/2Prkaca+/2bone tumors (n ¼ 3) compared with Prkar1a+/2bone tumors (n ¼ 3) and
WT bone (n ¼ 3). (B) Western blot analysis showing high Ets-1 protein levels and Ets-2 down-regulation in Prkar1a+/2Prkaca+/2bone tumors. Western
blot analysis was performed in triplicate. (C) Strong positive staining for Ets-1 in bone tumor from Prkar1a+/2Prkaca+/2mice. Prkar1a+/2bone tumor
and bone tissue from Prkaca+/2and WT mice displayed a negative staining (magnification, ×10 and ×40). Gene expression analysis is presented as
Human Molecular Genetics, 2011, Vol. 20, No. 1167
expressing vector, a significant increase in the Ets-1 and
caspase-1 protein expression was seen (Fig. 5C).
To investigate whether the Ca subunit increased Ets-1 and
caspase-1 expression by inhibition of proteosome degradation,
Ca siRNA-transfected cells
lactacystin b-lactone, a proteosome proteolytic activity inhibi-
tor (21). Clasto-lactacystin b-lactone treatment did not rescue
Ets-1 and caspase-1 down-regulation after Ca siRNA transfec-
tion (Fig. 5A).
Given the fact that ETS-1 trans-activation is mediated by
cAMP-responsive element-binding (CREB) protein (22), we
hypothesized that caspase-1 activation by PKA is regulated
through Ets-1. To address this question, we performed simul-
taneous transfections with the PRKACA-expressing vector and
Ets-1 siRNA (Fig. 5C). Ets-1 down-regulation in cells trans-
fected with the PRKACA vector blocked significantly the
increase in caspase-1 expression that was induced by
Ets-1 and caspase-1 expression are regulated by cAMP via
Next, we investigated the effect of cAMP levels on the
expression of Ets-1 and inflammasome molecular partners in
MC3T3 cells. A significant increase in Nlrp3, Caspase-1 and
Ets-1 expression was observed after treatment with forskolin
10 mM (Supplementary Material, Fig. S4A). To evaluate
whether cAMP effects on Nlrp3, Caspase-1 and Ets-1
expression were mediated primarily by PKA, cells were
treated with low concentrations of the PKA inhibitor H89.
The inhibition of PKA activity blocked the increase in the
Nlrp3, Caspase-1 and Ets-1 expression stimulated by cAMP
(Supplementary Material, Fig. S4A). After 6 h of forskolin
treatment, an increase in Ets-1 and caspase-1 expression was
demonstrated by western blot (Supplementary Material,
Fig. S4B). Similarly, the PKA inhibitor H89 also blunted the
cAMP effects on Ets-1 and caspase-1 expression at protein
High PGE2 levels in mouse and NOMID tumor cells
We have recently shown that cAMP levels are increased in the
Prkar1a+/2and Prkar1a+/2Prkaca+/2mouse bone tumor
cells (5), and here (see above) we also showed that NOMID
tumor cells have high cAMP levels. PGE2 is a well-known
activator of cAMP/PKA signaling (12,13) and a relevant
inhibitor of chondrocyte differentiation (14). In addition, the
main enzymes leading to PGE2 elevation, cyclooxygenase-2
(COX-2) and microsomal
(mPGES-1) are induced by IL1B (23). As caspase-1 is a key
Figure 2. Caspase-1 and IL1B over-expression at the protein level in Prkar1a1/2Prkaca1/2bone tumors. (A) Caspase-1 protein levels were higher in bone
tumors from Prkar1a+/2Prkaca+/2mice than from Prkar1a+/2-and WT mice (western blot analysis was performed in triplicate). (B) Stronger caspase-1
immunoreactivity in Prkar1a+/2Prkaca+/2bone tumor when compared with Prkar1a+/2bone lesion (magnification, ×10 and ×20). (C) Prkar1a+/2
Prkaca+/2bone tumor cells inside the bone marrow cavity displayed a high staining for IL1B (magnification, ×10 and ×40).
168 Human Molecular Genetics, 2011, Vol. 20, No. 1
regulator of IL1B synthesis, and in this study both NOMID
and cells from mice with PKA defects showed activated
caspase-1, we measured PGE2 levels in cell culture medium
from human NOMID and mouse Prkar1a+/2and Prkar1a+/2
Prkaca+/2bone tumor cells. Indeed, PGE2 levels were sig-
nificantly higher in Prkar1a+/2and Prkar1a+/2Prkaca+/2
bone tumor cells (691.5 + 130 and 698.7 + 23 pg/ml,
respectively) when compared with MC3T3 cells (46.3 +
P , 0.05) (Fig.
also showed higher PGE2 levels than NOMID non-lesional
cells (337.6 + 13.7 versus 32.3 + 1.8 pg/ml, respectively;
P , 0.05) (Fig. 6B).
6A). NOMID tumorcells
Caspase-1 and PGE2 inhibition decrease proliferation of
cells with PKA and NOMID defects
We then investigated the role of caspase-1 activation in the
proliferation of bone tumor cells in vitro. We used
Ac-YVAD-CMK, a selective and irreversible caspase-1
inhibitor (24). Caspase-1 inhibition significantly reduced
proliferation at 43 + 6.8% of Prkar1a+/2Prkaca+/2bone
tumor cells at 48 h of treatment (Fig. 6C). The caspase inhibi-
tor had no significant effect on MC3T3 and Prkar1a+/2cells.
NOMID non-lesional and tumor cells also showed a dramatic
reduction in proliferation at 60 + 6.1 and 83 + 2.7%, respect-
ively, after 48 h of treatment with the caspase-1 inhibitor
(Fig. 6D). In addition, the inhibition of PGE2 production
through the selective modulation of mPGES-1 expression sig-
nificantlyreduced the viability
Prkar1a+/2Prkaca+/2bone tumor cells after 48 h of treat-
ment (92 + 1.6 and 93 + 1.4%, respectively) (Fig. 6E).
Finally, the blockage of PGE2 synthesis led to a progressive
reduction on proliferation of NOMID non-lesional and tumor
cells (Fig. 6F).
Our recent studies showed that Prkar1a+/2Prkaca+/2mice
developed bone tumors that bore histological resemblance to
those that develop in humans with NOMID (5). Prkar1a+/2
Figure 3. Molecular characterization of NOMID cells . (A) NOMID cells have a fibroblastoid and spindle-shaped appearance at phase contrast microscopy (mag-
nification, ×10). (B) The BSC marker CD146 was increased in NOMID tumor cells compared with NOMID non-lesional cells. (C) Heatmap visualization of
gene expression data from NOMID non-lesional and tumor cells. (D) Functional analysis of whole-genome transcriptome profiling showing up-regulation of Wnt
signaling and apoptosis pathways in NOMID tumor cells. BSC, bone stem cell; MFI, median fluorescent intensity.
Human Molecular Genetics, 2011, Vol. 20, No. 1 169
Prkaca+/2mouse lesions involved a particular subpopulation
of cells that could be identified as belonging to the osteoblastic
lineage, derived from stromal cells (BSCs), and were localized
in specific locations of the skeleton always proximal to the
growth plate (5). The present study suggests that NOMID
bone tumors are also derived from the homologous, if not
identical, cells that are cAMP/PKA-sensitive and proliferate
in the same skeletal areas that are affected in humans and
mice with PKA defects.
The molecular mechanism shared by the two very distinct
disorders was elucidated in this report and was hitherto unsus-
pected: cAMP/PKA activates caspase-1 via the transcription
factor Ets-1; this leads to increased IL1B and consequently
PGE2, which in turn increases cAMP levels further, feeding
into a vicious cycle of PKA and caspase-1 activation within
the affected bone tissues. This could explain the histological
features of a PKA defect-like condition in NOMID; it also
led to the identification of PGE2 as the main culprit behind
the increased cAMP levels in human and mouse bone
lesions with PKA defects.
What is remarkable in the identified cross-talk between the
two pathways is that each can activate the other outside of
their usual context: caspase-1 activation in cells with PKA
defects was associated with an increase in cAMP levels and
further activation of the PKA pathway (employing PGE2),
without the involvement of any other exogenous signals.
Vice versa, PKA activation of caspase-1 in NOMID cells led
to IL1B and PGE2 increases without any need for activation
of pro-inflammatory markers. It should be noted that in both
human (2,6) and mouse (1,5) bone tumors caused by
NOMID or PKA defects, there was never any histological or
other evidence of inflammation. Arthropathy in NOMID is
not caused by synovitis or joint effusion, but by abnormal
endochondral bone formation and defective chondrocyte apop-
tosis (25,26). Lack of inflammation in NOMID bone tumors
was somewhat of a puzzle in this pro-inflammatory condition,
but it can now easily be explained by the findings of this study.
Another novel finding of this investigation is the regulation
of Ets-1 by PKA. The Ets family of proteins consists of a large
number of evolutionarily conserved transcription factors, and
inappropriate expression of ETS-1 has been linked to a
variety of human cancers (27). ETS-1 is known to transform
cell lines, promote autonomous growth in culture and lead to
tumor formation in vivo (28–30). Ets-1 mRNA is first detected
Figure 4. High PKA activity, cAMP levels and b catalytic subunit (Cb) expression in NOMID tumor cells. (A) NOMID tumor cells presented high cAMP levels
when compared with NOMID non-lesional cells. (B) PKA-I activity after cAMP stimulus was significantly higher in NOMID tumor cells than in non-lesional
cells (P ¼ 0.01). (C) RIa and RIIa protein levels were similar in NOMID non-lesional and tumor cells. (D) NOMID tumor cells showed a remarkable induction
in the expression of Cb subunit. Expression levels of the other catalytic subunits (Ca, Cg and Prkx) were similar in NOMID non-lesional and tumor cells. (E)
NOMID tumor cells presented higher expression levels of Ets-1 and caspase-1. All the experiments (cAMP and PKA assays and western blot analysis) were
performed in triplicate. cAMP levels are presented as mean + SD.
170 Human Molecular Genetics, 2011, Vol. 20, No. 1
at day 14 of mouse development in mesenchymal cells in the
developing limbs, tail, vertebrate and other sites (31), where
bone development occurs and where tumors in NOMID and
PKA defects occur. During bone formation, Ets-1 and Ets-2
peak at different stages: Ets-1 is expressed in mesenchymal
cells (31) regulating genes that are involved in proliferation,
whereas Ets-2 expression peaks during osteoblast differen-
tiation and matrix maturation (32). The signal transduction
pathways that regulate ETS-1 expression in bone were
unknown. In this study, we showed that PKA regulates
caspase-1 expression through Ets-1 activation. Caspase-1
up-regulation induced by PKA was blocked by Ets-1 down-
regulation, suggesting a direct effect of PKA on Ets-1 tran-
scription. Ets-1 transactivation requires the recruitment of
p300 and CREB protein (22,33). It was known that Ets-1 acti-
vates the transcription of caspase-1 by direct binding to a func-
tional activation site in the caspase-1 promoter (10).
The findings of this study have important implications for
therapeutics, since the two pathways can now be targeted
interchangeably (or, maybe, simultaneously) in bone lesions
associated with either PKA defects or NOMID. Almost all
NOMID manifestations (fever, urticarial rash, aseptic menin-
gitis and arthropathy) are driven by increased IL1B and
respond to the systemic administration of anakinra, an IL-1
antagonist (34). In this study, we showed that inhibition of
caspase-1, the enzyme that cleaves pro-IL1B to its active
form IL1B, significantly decreased proliferation of both
NOMID cells and Prkar1a+/2Prkaca+/2bone tumor cells,
suggesting that this drug may be helpful in controlling mani-
festations of patients with PKA defects as well.
Increased IL1B expression in both NOMID and Prkar1a+/2
bone tumor cells was also associated with
increased PGE2through induction of COX-2 and mPGES-1
pathway plays a key role in the development of many
tumors (35), and it has been demonstrated that human
mesenchymal stem cell proliferation is regulated by PGE2
through activation of cAMP-dependent PKA-I (12), consistent
with the findings in this study. PGE2suppresses the maturation
of growth plate chondrocytes and inhibits the expression of
colX, VEGF, MMP-13 and alkaline phosphatase in a dose-
dependent manner (14). PGE2 rapidly activates CREB
through PKA signaling and results in the subsequent transcrip-
tional activation of PKA/CREB-dependent genes (14). Inter-
actions between PGE2and Wnt signaling pathway regulate
proliferation and regeneration of stem cells. PGE2modifies
the Wnt signaling cascade at the level of b-catenin degra-
dation through cAMP/PKA (13). Indeed, the gene signature
Figure 5. Protein kinase A regulates caspase-1 via Ets-1 proto-oncogene activation . (A) Ets-1 and caspase-1 expression had a significant down-regulation after
transient transfection with Ca siRNA. (B) Western blot analysis showed a reduction in caspase-1 expression in MC3T3 cells transfected with Ets-1 siRNA. Ets-2
down-regulation had no effect on caspase-1 protein expression. (C) PRKACA-expressing vector promoted a significant increase in the Ets-1 and caspase-1
protein expression. Simultaneous transfection with Ets-1 siRNA and PRKACA-expressing vector blocked caspase-1 up-regulation induced by Ca subunit.
Western blot analysis was confirmed in three independent experiments.
Human Molecular Genetics, 2011, Vol. 20, No. 1 171
of NOMID tumor-like bone lesion cells showed a significant
enrichment with expression of genes of the Wnt signaling
pathway, similar to what we have shown elsewhere for PKA
subunit defects (36–38). Interestingly, Wnt signaling acti-
vation is shared by all human lesions and mouse tissues
affected by increased cAMP signaling that we have studied
so far (37,39–41). Thus, COX2-, PGE2- and/or Wnt inhibitors
can potentially be useful in the treatment of patients with the
In conclusion, we identified a key link between cAMP sig-
naling and the inflammasome in bone cells (Fig. 7). This
finding has potentially more general implications, such as,
for example, on the possible effect of cAMP on caspase-1-
mediated regulation of protein secretion. It may also lead to
new therapies in patients with NOMID or those with PKA
MATERIALS AND METHODS
Prkar1a heterozygous mice (Prkar1a+/2), which contain one
null allele of Prkar1aD2, were generated previously in our lab-
oratory (1). Prkaca heterozygous mice (Prkaca+/2), which
have a neomycin resistance cassette to replace exons 6–8 of
the Prkaca gene (42), were purchased from Mutant Mouse
Figure 6. Mouse and NOMID tumor cells secret PGE2 and show high sensibility to caspase-1 and mPGES-1 inhibition. (A) PGE2 levels were significantly
higher in Prkar1a+/2and Prkar1a+/2Prkaca+/2bone tumor cells when compared with MC3T3 cells. (B) NOMID tumor cells also showed higher PGE2
levels than NOMID non-lesional cells. (C) Caspase-1 inhibition significantly reduced proliferation of Prkar1a+/2Prkaca+/2bone tumor cells. The caspase
inhibitor had no effect on MC3T3 cells and Prkar1a+/2bone tumor cells. (D) NOMID cells showed a significant decrease in proliferation after caspase-1 inhi-
bition. (E) The inhibition of PGE2 production by a selective inhibitor of mPGES-1 significantly reduced viability of Prkar1a+/2and Prkar1a+/2Prkaca+/2
bone tumor cells. (F) NOMID non-lesional and tumor cells also had a progressive reduction on proliferation after mPGES-1 blockage. PGE2 levels and cell
proliferation are shown as mean + SD. All the experiments were performed in triplicate. PGE2, prostaglandin E2; mPGES1, microsomal prostaglandin E
172 Human Molecular Genetics, 2011, Vol. 20, No. 1
129X1-Prkacatm1Gsm/Mmnc). Prkar1a+/2and Prkaca+/2
mice were crossed to generate Prkar1a+/2Prkaca+/2mice
on a mixed C57BL/6 129Sv/B6 hybrid background (5). All
animals were genotyped as described previously. Animal
work in this study was carried out in accordance with Insti-
tutional Laboratory Animal Care and Use Committee guide-
lines under animal protocol 06-033 (at the NIH, Bethesda,
Primary cell cultures
Primary culture of Prkar1a+/2and Prkar1a+/2Prkaca+/2
bone tumors was established as described previously (5).
Tissues from the bone lesion and non-affected cartilage of a
same patient with NOMID were isolated from cartilage shav-
ings by enzymatic digestion (43).
Total RNA was extracted from NOMID non-lesional and
tumor cells using the TRIZOL reagent method. Three biologi-
cal replicates for each of the two cell lines were used for
microarray analysis. Preparation of cRNA from total RNA,
hybridization in Expression BeadChips, scanning and image
analysis were done as described previously (44). Normaliza-
tion and analysis of microarray data were performed as
described previously (37). The raw and normalized microarray
data reported in this paper have been deposited in the Gene
Expression Omnibus database (accession no. GSE21835).
Real-time quantitative RT–PCR
Quantitative real-time PCR was performed in the ABI Prism
7700 Sequence Detector using TaqMan Gene Expression
Assays according to the manufacturer’s instructions (Applied
Biosystems, Foster City, CA, USA). The assay IDs were
Nlrp3, Mm00840904_m1; ASC, Mm00445747_g1; Casp1
Mm00438023_m1; Il1b Mm00434228_m1
Mm00468970_m1, GAPDH 4308313. The relative quantifi-
cation was performed using the 22DDCTmethod (45).
siRNA and construct transfections
MC3T3 cells were transfected with 100 nM MISSIONw
siRNA (Sigma-Aldrich, St Louis, MO, USA) specific to
mouse RIa (SASI_Mm01_00116787), RIIa (SASI_Mm01_
Mm01_00217223), Ets-1 (SASI_Mm01_00173246), Ets-2
(SASI_Mm01_00130872) or non-targeting pool siRNA using
N-TER peptide transfection reagent (Sigma-Aldrich), as per
manufacturer’s instruction. Ca siRNA-transfected cells were
treated with clasto-lactacystin b-lactone (5 mM), a proteosome
proteolytic activity inhibitor (21).
MC3T3 cells were also transfected with the retroviral
vectors OT1521 (mock) or OT1529 (PRKACA-expressing
metallothionine-1 promoter driving the expression of construct
for PKA subunits Ca, as described previously (46). All exper-
iments were performed in triplicate.
Western blot analysis
Western blot analysis was performed following standard pro-
cedures (5,47). The following primary antibodies were used:
RIa (610610, BD transduction), RIIa (C-20, Santa Cruz Bio-
technology, Santa Cruz, CA, USA), RIIb (H-90, Santa Cruz
Biotechnology), Ca (C-20, Santa Cruz Biotechnology), Cb
(C-20), PRKX (a gift from Dr Robert M. Kotin, NHLBI,
NIH, Bethesda, MD, USA), caspase-1 (M-20, Santa Cruz
Biotechnology), Ets-1 (ab26096, Abcam, Cambridge, MA,
USA), Ets-2 (C-20, Santa Cruz Biotechnology), GAPDH
(ab9485, Abcam) and histone H1 (FL-219, Santa Cruz
All IHC was performed in collaboration with Histoserv, Inc.
(Germantown, MD, USA). The following primary antibodies
were used: Ets-1 (ab26096, Abcam), Ets-2 (C-20, Santa
Cruz Biotechnology), caspase-1 (M-20, Santa Cruz Biotech-
nology) and IL1B (H-153, Santa Cruz Biotechnology).
NOMID normal and tumor cells (1 × 105) were collected from
cultures and fixed using BD Cytofix/CytopermTMFixation/
Permeabilization Solution Kit (BD Biosciences, San Jose,
CA, USA). Subsequently, NOMID cells were stained with
PE or fluorescein isothiocyanate (FITC)-tagged primary anti-
bodies (CD44-FITC and CD146-PE, BD Biosciences) at 48C
for 30 min.
Figure 7. Schematic representation of pro-inflammatory pathway induction by
cAMP/PKA signaling in BSC-derived lesions. PGE2, prostaglandin E2;
mPGES1, microsomal prostaglandin E synthase-1; COX2, cyclooxygenase-2;
CREB, cAMP-responsive element-binding protein.
Human Molecular Genetics, 2011, Vol. 20, No. 1173
PKA activity, cAMP assay and DEAE cellulose
PKA enzymatic activity was measured as described previously
(48). cAMP levels were determined by
System (Amersham Biosciences, Piscataway, NJ, USA).
DEAE cellulose chromatography was performed as described
in Cheadle et al. (44).
3H Biotrak Assay
Cell treatment, proliferation assay and PGE2 levels
MC3T3 cells were stimulated by forskolin (10 mM) associated
or not with the PKA activity inhibitor H89 (50 nM). MC3T3,
Ac-YVAD-CMK (10, 30 and 50 mM; Cayman Chemical,
Ann Arbor, MI, USA), a selective and irreversible caspase-1
inhibitor (24), or with CAY1052 (1,3 and 10 mM; Cayman
Chemical), a selective mPGES-1 inhibitor (49). After 48 h
of treatment, cell proliferation was evaluated using the CellTi-
ter 96wAQueous One Solution Cell Proliferation Assay
(Promega, Madison, WI, USA). PGE2 concentration was
measured in the cell culture medium using the Prostaglandin
E2 ELISA Kit-Monoclonal (Cayman Chemical).
All statistical analyses were performed with SPSS 16.0 (SPSS
Inc., Chicago, IL, USA). Continuous data are expressed as
mean + SD. All experiments were performed in triplicate. A
two-sample t-test was used for paired samples. The Kolmo-
gorov–Smirnov (K–S) test was employed to analyze the
differences between NOMID tumor and normal cells at the
flow cytometry. A P-value less than 0.05 was considered
Supplementary Material is available at HMG online.
We would like to thank Dr Xibin Wang and Dr Hang Pham for
their help with the NOMID cell line experiments. We also
thank Dr J. Aidan Carney (Mayo Clinic, Rochester, MN,
USA) for providing samples of human tumors associated
Conflict of Interest statement. The authors have nothing to
This work was supported by US National Institutes of Health,
Eunice Kennedy Shriver National Institute of Child Health and
Human Development intramural project Z01-HD-000642-04
(to C.A.S.) and the NIAMS Intramural Research.
data (50) add support to PKA and/or involvement of
Prkar1a in early osteoblast proliferation, and this pathway’s
significant involvement in human osteosarcoma. It would be
interesting to study caspase-1 and/or the inflammasome in
this human tumor.
1. Kirschner, L.S., Kusewitt, D.F., Matyakhina, L., Towns, W.H. II, Carney,
J.A., Westphal, H. and Stratakis, C.A. (2005) A mouse model for the
Carney complex tumor syndrome develops neoplasia in cyclic
AMP-responsive tissues. Cancer Res., 65, 4506–4514.
2. Carney, J.A., Boccon-Gibod, L., Jarka, D.E., Tanaka, Y., Swee, R.G.,
Unni, K.K. and Stratakis, C.A. (2001) Osteochondromyxoma of bone: a
congenital tumor associated with lentigines and other unusual disorders.
Am. J. Surg. Pathol., 25, 164–176.
3. Stratakis, C.A., Kirschner, L.S. and Carney, J.A. (2001) Clinical and
molecular features of the Carney complex: diagnostic criteria and
recommendations for patient evaluation. J. Clin. Endocrinol. Metab., 86,
4. Pavel, E., Nadella, K., Towns, W.H. II and Kirschner, L.S. (2008)
Mutation of Prkar1a causes osteoblast neoplasia driven by dysregulation
of protein kinase A. Mol. Endocrinol., 22, 430–440.
5. Tsang, K.M., Starost, M.F., Nesterova, M., Boikos, S.A., Watkins, T.,
Almeida, M.Q., Harran, M., Li, A., Collins, M.T., Cheadle, C. et al.
(2010) Alternate protein kinase A activity identifies a unique population
of stromal cells in adult bone. Proc. Natl Acad. Sci. USA, 107, 8683–
6. Hill, S.C., Namde, M., Dwyer, A., Poznanski, A., Canna, S. and
Goldbach-Mansky, R. (2007) Arthropathy of neonatal onset multisystem
inflammatory disease (NOMID/CINCA). Pediatr. Radiol., 37, 145–152.
7. Aksentijevich, I., Nowak, M., Mallah, M., Chae, J.J., Watford, W.T.,
Hofmann, S.R., Stein, L., Russo, R., Goldsmith, D., Dent, P. et al. (2002)
De novo CIAS1 mutations, cytokine activation, and evidence for genetic
heterogeneity in patients with neonatal-onset multisystem inflammatory
disease (NOMID): a new member of the expanding family of
pyrin-associated autoinflammatory diseases. Arthritis Rheum., 46, 3340–
8. Feldmann, J., Prieur, A.M., Quartier, P., Berquin, P., Certain, S., Cortis,
E., Teillac-Hamel, D., Fischer, A. and de Saint Basile, G. (2002) Chronic
infantile neurological cutaneous and articular syndrome is caused by
mutations in CIAS1, a gene highly expressed in polymorphonuclear cells
and chondrocytes. Am. J. Hum. Genet., 71, 198–203.
9. Petrilli, V., Dostert, C., Muruve, D.A. and Tschopp, J. (2007) The
inflammasome: a danger sensing complex triggering innate immunity.
Curr. Opin. Immunol., 19, 615–622.
10. Pei, H., Li, C., Adereth, Y., Hsu, T., Watson, D.K. and Li, R. (2005)
Caspase-1 is a direct target gene of ETS1 and plays a role in
ETS1-induced apoptosis. Cancer Res., 65, 7205–7213.
11. Mouawad, R., Antoine, E.C., Gil-Delgado, M., Khayat, D. and Soubrane,
C. (2002) Serum caspase-1 levels in metastatic melanoma patients:
relationship with tumour burden and non-response to biochemotherapy.
Melanoma Res., 12, 343–348.
12. Kleiveland, C.R., Kassem, M. and Lea, T. (2008) Human mesenchymal
stem cell proliferation is regulated by PGE2 through differential activation
of cAMP-dependent protein kinase isoforms. Exp. Cell Res., 314,
13. Goessling, W., North, T.E., Loewer, S., Lord, A.M., Lee, S.,
Stoick-Cooper, C.L., Weidinger, G., Puder, M., Daley, G.Q., Moon, R.T.
et al. (2009) Genetic interaction of PGE2 and Wnt signaling regulates
developmental specification of stem cells and regeneration. Cell, 136,
14. Li, T.F., Zuscik, M.J., Ionescu, A.M., Zhang, X., Rosier, R.N., Schwarz,
E.M., Drissi, H. and O’Keefe, R.J. (2004) PGE2 inhibits chondrocyte
differentiation through PKA and PKC signaling. Exp. Cell Res., 300,
15. Raouf, A. and Seth, A. (2000) Ets transcription factors and targets in
osteogenesis. Oncogene, 19, 6455–6463.
16. Ting, J.P., Willingham, S.B. and Bergstralh, D.T. (2008) NLRs at the
intersection of cell death and immunity. Nat. Rev. Immunol., 8, 372–379.
17. Eisen, M.B., Spellman, P.T., Brown, P.O. and Botstein, D. (1998) Cluster
analysis and display of genome-wide expression patterns. Proc. Natl
Acad. Sci. USA, 95, 14863–14868.
174Human Molecular Genetics, 2011, Vol. 20, No. 1
18. Huang da, W., Sherman, B.T. and Lempicki, R.A. (2009) Systematic and Download full-text
integrative analysis of large gene lists using DAVID bioinformatics
resources. Nat. Protoc., 4, 44–57.
19. Quarles, L.D., Yohay, D.A., Lever, L.W., Caton, R. and Wenstrup, R.J.
(1992) Distinct proliferative and differentiated stages of murine
MC3T3-E1 cells in culture: an in vitro model of osteoblast development.
J. Bone Miner. Res., 7, 683–692.
20. Sudo, H., Kodama, H.A., Amagai, Y., Yamamoto, S. and Kasai, S. (1983)
In vitro differentiation and calcification in a new clonal osteogenic cell
line derived from newborn mouse calvaria. J. Cell Biol., 96, 191–198.
21. Dick, L.R., Cruikshank, A.A., Destree, A.T., Grenier, L., McCormack,
T.A., Melandri, F.D., Nunes, S.L., Palombella, V.J., Parent, L.A.,
Plamondon, L. et al. (1997) Mechanistic studies on the inactivation of the
proteasome by lactacystin in cultured cells. J. Biol. Chem., 272, 182–188.
22. Yang, C., Shapiro, L.H., Rivera, M., Kumar, A. and Brindle, P.K. (1998)
A role for CREB binding protein and p300 transcriptional coactivators in
Ets-1 transactivation functions. Mol. Cell Biol., 18, 2218–2229.
23. Park, J.Y., Pillinger, M.H. and Abramson, S.B. (2006) Prostaglandin E2
synthesis and secretion: the role of PGE2 synthases. Clin. Immunol., 119,
24. Rabuffetti, M., Sciorati, C., Tarozzo, G., Clementi, E., Manfredi, A.A. and
Beltramo, M. (2000) Inhibition of caspase-1-like activity by
Ac-Tyr-Val-Ala-Asp-chloromethyl ketone induces long-lasting
neuroprotection in cerebral ischemia through apoptosis reduction and
decrease of proinflammatory cytokines. J. Neurosci., 20, 4398–4404.
25. Masumoto, J., Dowds, T.A., Schaner, P., Chen, F.F., Ogura, Y., Li, M.,
Zhu, L., Katsuyama, T., Sagara, J., Taniguchi, S. et al. (2003) ASC is an
activating adaptor for NF-kappa B and caspase-8-dependent apoptosis.
Biochem. Biophys. Res. Commun., 303, 69–73.
26. Dowds, T.A., Masumoto, J., Zhu, L., Inohara, N. and Nunez, G. (2004)
Cryopyrin-induced interleukin 1beta secretion in monocytic cells:
enhanced activity of disease-associated mutants and requirement for ASC.
J. Biol. Chem., 279, 21924–21928.
27. Hsu, T., Trojanowska, M. and Watson, D.K. (2004) Ets proteins in
biological control and cancer. J. Cell Biochem., 91, 896–903.
28. Seth, A. and Papas, T.S. (1990) The c-ets-1 proto-oncogene has oncogenic
activity and is positively autoregulated. Oncogene, 5, 1761–1767.
29. Topol, L.Z., Tatosyan, A.G., Ascione, R., Thompson, D.M., Blair, D.G.,
Kola, I. and Seth, A. (1992) C-ets-1 protooncogene expression alters the
growth properties of immortalized rat fibroblasts. Cancer Lett., 67, 71–78.
30. Seth, A. and Watson, D.K. (2005) ETS transcription factors and their
emerging roles in human cancer. Eur. J. Cancer, 41, 2462–2478.
31. Kola, I., Brookes, S., Green, A.R., Garber, R., Tymms, M., Papas, T.S.
and Seth, A. (1993) The Ets1 transcription factor is widely expressed
during murine embryo development and is associated with mesodermal
cells involved in morphogenetic processes such as organ formation. Proc.
Natl Acad. Sci. USA, 90, 7588–7592.
32. Vary, C.P., Li, V., Raouf, A., Kitching, R., Kola, I., Franceschi, C.,
Venanzoni, M. and Seth, A. (2000) Involvement of Ets transcription
factors and targets in osteoblast differentiation and matrix mineralization.
Exp. Cell Res., 257, 213–222.
33. Jayaraman, G., Srinivas, R., Duggan, C., Ferreira, E., Swaminathan, S.,
Somasundaram, K., Williams, J., Hauser, C., Kurkinen, M., Dhar, R. et al.
(1999) p300/cAMP-responsive element-binding protein interactions with
ets-1 and ets-2 in the transcriptional activation of the human stromelysin
promoter. J. Biol. Chem., 274, 17342–17352.
34. Goldbach-Mansky, R., Dailey, N.J., Canna, S.W., Gelabert, A., Jones, J.,
Rubin, B.I., Kim, H.J., Brewer, C., Zalewski, C., Wiggs, E. et al. (2006)
Neonatal-onset multisystem inflammatory disease responsive to
interleukin-1beta inhibition. N. Engl. J. Med., 355, 581–592.
35. Greenhough, A., Smartt, H.J., Moore, A.E., Roberts, H.R., Williams,
A.C., Paraskeva, C. and Kaidi, A. (2009) The COX-2/PGE2 pathway: key
roles in the hallmarks of cancer and adaptation to the tumour
microenvironment. Carcinogenesis, 30, 377–386.
36. Horvath, A., Mathyakina, L., Vong, Q., Baxendale, V., Pang, A.L., Chan,
W.Y. and Stratakis, C.A. (2006) Serial analysis of gene expression in
adrenocortical hyperplasia caused by a germline PRKAR1A mutation.
J. Clin. Endocrinol. Metab., 91, 584–596.
37. Almeida, M.Q., Muchow, M., Boikos, S., Bauer, A.J., Griffin, K.J., Tsang,
K.M., Cheadle, C., Watkins, T., Wen, F., Starost, M.F. et al. (2010)
Mouse Prkar1a haploinsufficiency leads to an increase in tumors in the
Trp53+/2 or Rb1+/2 backgrounds and chemically induced skin
papillomas by dysregulation of the cell cycle and Wnt signaling. Hum.
Mol. Genet., 19, 1387–1398.
38. Iliopoulos, D., Bimpaki, E.I., Nesterova, M. and Stratakis, C.A. (2009)
MicroRNA signature of primary pigmented nodular adrenocortical
disease: clinical correlations and regulation of Wnt signaling. Cancer
Res., 69, 3278–3282.
39. Bourdeau, I., Antonini, S.R., Lacroix, A., Kirschner, L.S., Matyakhina, L.,
Lorang, D., Libutti, S.K. and Stratakis, C.A. (2004) Gene array analysis of
macronodular adrenal hyperplasia confirms clinical heterogeneity and
identifies several candidate genes as molecular mediators. Oncogene, 23,
40. Bimpaki, E.I., Iliopoulos, D., Moraitis, A. and Stratakis, C.A. (2009)
MicroRNA signature in massive macronodular adrenocortical disease and
implications for adrenocortical tumorigenesis. Clin. Endocrinol. (Oxf.),
41. Hsiao, H.P., Kirschner, L.S., Bourdeau, I., Keil, M.F., Boikos, S.A.,
Verma, S., Robinson-White, A.J., Nesterova, M., Lacroix, A. and
Stratakis, C.A. (2009) Clinical and genetic heterogeneity, overlap with
other tumor syndromes, and atypical glucocorticoid hormone secretion in
adrenocorticotropin-independent macronodular adrenal hyperplasia
compared with other adrenocortical tumors. J. Clin. Endocrinol. Metab.,
42. Skalhegg, B.S., Huang, Y., Su, T., Idzerda, R.L., McKnight, G.S. and
Burton, K.A. (2002) Mutation of the Calpha subunit of PKA leads to
growth retardation and sperm dysfunction. Mol. Endocrinol., 16, 630–
43. Wang, X., Manner, P.A., Horner, A., Shum, L., Tuan, R.S. and Nuckolls,
G.H. (2004) Regulation of MMP-13 expression by RUNX2 and FGF2 in
osteoarthritic cartilage. Osteoarthritis Cartil., 12, 963–973.
44. Cheadle, C., Nesterova, M., Watkins, T., Barnes, K.C., Hall, J.C., Rosen,
A., Becker, K.G. and Cho-Chung, Y.S. (2008) Regulatory subunits of
PKA define an axis of cellular proliferation/differentiation in ovarian
cancer cells. BMC Med. Genomics, 1, 43.
45. Livak, K.J. and Schmittgen, T.D. (2001) Analysis of relative gene
expression data using real-time quantitative PCR and the 2(-Delta Delta
C(T)) method. Methods, 25, 402–408.
46. Nesterova, M., Yokozaki, H., McDuffie, E. and Cho-Chung, Y.S. (1996)
Overexpression of RII beta regulatory subunit of protein kinase A in
human colon carcinoma cell induces growth arrest and phenotypic
changes that are abolished by site-directed mutation of RII beta.
Eur. J. Biochem., 235, 486–494.
47. Neary, C.L., Nesterova, M., Cho, Y.S., Cheadle, C., Becker, K.G. and
Cho-Chung, Y.S. (2004) Protein kinase A isozyme switching: eliciting
differential cAMP signaling and tumor reversion. Oncogene, 23, 8847–
48. Rohlff, C., Clair, T. and Cho-Chung, Y.S. (1993) 8-Cl-cAMP induces
truncation and down-regulation of the RI alpha subunit and up-regulation
of the RII beta subunit of cAMP-dependent protein kinase leading to type
II holoenzyme-dependent growth inhibition and differentiation of HL-60
leukemia cells. J. Biol. Chem., 268, 5774–5782.
49. Guerrero, M.D., Aquino, M., Bruno, I., Terencio, M.C., Paya, M., Riccio,
R. and Gomez-Paloma, L. (2007) Synthesis and pharmacological
evaluation of a selected library of new potential anti-inflammatory agents
bearing the gamma-hydroxybutenolide scaffold: a new class of inhibitors
of prostanoid production through the selective modulation of microsomal
prostaglandin E synthase-1 expression. J. Med. Chem., 50, 2176–2184.
50. Molyneux, S.D., Di Grappa, M.A., Beristain, A.G., McKee, T.D., Wai,
D.H., Paderova, J., Kashyap, M., Hu, P., Maiuri, T., Narala, S.R. et al.
(2010) Prkar1a is an osteosarcoma tumor suppressor that defines a
molecular subclass in mice. J. Clin. Invest., 120, 3310–3325.
Human Molecular Genetics, 2011, Vol. 20, No. 1175