A novel N-ethyl-N-nitrosourea-induced mutation in phospholipase Cγ2 causes inflammatory arthritis, metabolic defects, and male infertility in vitro in a murine model.

Abstract

It is difficult to identify a single causative factor for inflammatory arthritis because of the multifactorial nature of the disease. This study was undertaken to dissect the molecular complexity of systemic inflammatory disease, utilizing a combined approach of mutagenesis and systematic phenotype screening in a murine model.
In a large-scale N-ethyl-N-nitrosourea mutagenesis project, the Ali14 mutant mouse strain was established because of dominant inheritance of spontaneous swelling and inflammation of the hind paws. Genetic mapping and subsequent candidate gene sequencing were conducted to find the causative gene, and systematic phenotyping of Ali14/+ mice was performed in the German Mouse Clinic.
A novel missense mutation in the phospholipase Cγ2 gene (Plcg2) was identified in Ali14/+ mice. Because of the hyperreactive external entry of calcium observed in cultured B cells and other in vitro experiments, the Ali14 mutation is thought to be a novel gain-of-function allele of Plcg2. Findings from systematic screening of Ali14/+ mice demonstrated various phenotypic changes: an abnormally high T cell:B cell ratio, up-regulation of Ig, alterations in body composition, and a reduction in cholesterol and triglyceride levels in peripheral blood. In addition, spermatozoa from Ali14/+ mice failed to fertilize eggs in vitro, despite the normal fertility of the Ali14/+ male mice in vivo.
These results suggest that the Plcg2-mediated pathways play a crucial role in various metabolic and sperm functions, in addition to initiating and maintaining the immune system. These findings may indicate the importance of the Ali14/+ mouse strain as a model for systemic inflammatory diseases and inflammation-related metabolic changes in humans.

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ARTHRITIS & RHEUMATISM
Vol. 63, No. 5, May 2011, pp 1301–1311
DOI 10.1002/art.30280
© 2011, American College of Rheumatology
A Novel N-ethyl-N-nitrosourea–Induced Mutation in
Phospholipase C?2 Causes Inflammatory Arthritis, Metabolic
Defects, and Male Infertility In Vitro in a Murine Model
Koichiro Abe,1Helmut Fuchs,2Auke Boersma,2Wolfgang Hans,2Philipp Yu,3
Svetoslav Kalaydjiev,4Matthias Klaften,2Thure Adler,4Julia Calzada-Wack,2
Ilona Mossbrugger,2Birgit Rathkolb,5Jan Rozman,6Cornelia Prehn,2
Miriam Maraslioglu,3Yoshie Kametani,7Shin Shimada,7Jerzy Adamski,2
Dirk H. Busch,4Irene Esposito,2Martin Klingenspor,6Eckhard Wolf,5
Wolfgang Wurst,8Valerie Gailus-Durner,2Matilda Katan,9Susan Marschall,2
Dian Soewarto,2Sibylle Wagner,2and Martin Hrabe ˇ de Angelis10
Objective. It is difficult to identify a single caus-
ative factor for inflammatory arthritis because of the
multifactorial nature of the disease. This study was
undertaken to dissect the molecular complexity of sys-
temic inflammatory disease, utilizing a combined ap-
proach of mutagenesis and systematic phenotype
screening in a murine model.
Methods. In a large-scale N-ethyl-N-nitrosourea
mutagenesis project, the Ali14 mutant mouse strain was
established because of dominant inheritance of sponta-
neous swelling and inflammation of the hind paws.
Genetic mapping and subsequent candidate gene se-
quencing were conducted to find the causative gene, and
systematic phenotyping of Ali14/? mice was performed
in the German Mouse Clinic.
Results. A novel missense mutation in the phos-
pholipase C?2 gene (Plcg2) was identified in Ali14/?
mice. Because of the hyperreactive external entry of
calcium observed in cultured B cells and other in vitro
experiments, the Ali14 mutation is thought to be a novel
gain-of-function allele of Plcg2. Findings from system-
atic screening of Ali14/? mice demonstrated various
phenotypic changes: an abnormally high T cell:B cell
Supported in part by the Japan Society for the Promotion of
Science (JSPS grants 19500370 and 22500392 to Dr. Abe) and by the
Research Foundation Itsuu Laboratory (Dr. Abe), by the Nationales
Genomforschungsnetz Deutschland (NGFN-Plus grants 01GS0850,
01GS0851, and 01GS0852 to Drs. Busch, Wolf, and Hrabe ˇ de Angelis),
by the Deutsches Human Genome Projekt (grant-in-aid to Dr. Hrabe ˇ
de Angelis), and by the European Commission (grant LSHG-2006-
037188 to Dr. Hrabe ˇ de Angelis).
1Koichiro Abe, PhD: Tokai University School of Medicine,
Kanagawa, Japan and Helmholtz Zentrum Mu ¨nchen–German Re-
search Center for Environmental Health, Neuherberg, Germany;
2Helmut Fuchs, PhD, Auke Boersma, DVM (current address: Univer-
sity of Veterinary Medicine Vienna, Vienna, Austria), Wolfgang Hans,
PhD, Matthias Klaften, DVM, Julia Calzada-Wack, MD, Ilona Moss-
brugger, DVM, Cornelia Prehn, PhD, Jerzy Adamski, PhD, Irene
Esposito, MD, Valerie Gailus-Durner, PhD, Susan Marschall, PhD,
Dian Soewarto, PhD, Sibylle Wagner, DVM: Helmholtz Zentrum
Mu ¨nchen–German Research Center for Environmental Health,
Neuherberg, Germany;
Philipps-Universita ¨t Marburg, Marburg, Germany;4Svetoslav Kalayd-
3Philipp Yu, MD, Miriam Maraslioglu:
jiev, MD, Thure Adler, DVM, Dirk H. Busch, MD: Helmholtz
Zentrum Mu ¨nchen–German Research Center for Environmental
Health, Neuherberg and Technische Universita ¨t Mu ¨nchen, Munich,
Germany;5Birgit Rathkolb, DVM, Eckhard Wolf, DVM: Helmholtz
Zentrum Mu ¨nchen–German Research Center for Environmental
Health, Neuherberg, and Ludwig-Maximilians-Universita ¨t Mu ¨nchen,
Munich, Germany;
Technische Universita ¨t Mu ¨nchen, Weihenstephan Campus, Freising-
Weihenstephan, Germany;
PhD: Tokai University School of Medicine, Kanagawa, Japan;8Wolf-
gang Wurst, PhD: Helmholtz Zentrum Mu ¨nchen–German Research
Center for Environmental Health, Neuherberg, Technische Univer-
sita ¨t Mu ¨nchen, Munich, Max Planck Institute of Psychiatry, Munich,
and Deutsches Zentrum fu ¨r Neurodegenerative Erkankungen, Mu-
nich, Germany;9Matilda Katan, PhD: Royal Cancer Hospital, Lon-
don, UK;
Mu ¨nchen–German Research Center for Environmental Health,
Neuherberg, and Technische Universita ¨t Mu ¨nchen, Weihenstephan
Campus, Freising-Weihenstephan, Germany.
Address correspondence to Koichiro Abe, PhD, Division of
Basic Medical Science and Molecular Medicine, Tokai University
School of Medicine, Shimokasuya 143, Isehara, Kanagawa 259-1193,
Japan (e-mail: abeko@is.icc.u-tokai.ac.jp); or to Martin Hrabe ˇ de
Angelis, PhD, Institute of Experimental Genetics, Helmholtz Zentrum
Mu ¨nchen, German Research Center for Environmental Health, Ingol-
staedter Landstrasse 1, 85764 Neuherberg, Germany (e-mail:
hrabe@helmholtz-muenchen.de).
Submitted for publication August 20, 2010; accepted in
revised form January 25, 2011.
6Jan Rozman, PhD, Martin Klingenspor, PhD:
7Yoshie Kametani, PhD, Shin Shimada,
10Martin Hrabe ˇ de Angelis, PhD: Helmholtz Zentrum
1301

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ratio, up-regulation of Ig, alterations in body composi-
tion, and a reduction in cholesterol and triglyceride
levels in peripheral blood. In addition, spermatozoa
from Ali14/? mice failed to fertilize eggs in vitro, despite
the normal fertility of the Ali14/? male mice in vivo.
Conclusion. These results suggest that the Plcg2-
mediated pathways play a crucial role in various meta-
bolic and sperm functions, in addition to initiating and
maintaining the immune system. These findings may
indicate the importance of the Ali14/? mouse strain as
a model for systemic inflammatory diseases and
inflammation-related metabolic changes in humans.
Inflammatory arthritis, including rheumatoid ar-
thritis, often leads to significant destruction of articular
tissue, resulting in physical disability (1). The disease is
associated not only with immobility, but also with ca-
chexia and depression (2). These impairments obviously
cause social isolation and lead to diminishing health-
related quality of life. Despite the accumulated evidence
regarding systemic inflammatory diseases, only treat-
ments targeting symptoms are available. Inflammatory
arthritis is a multifactorial disease that is induced by
complex combinations of genetic and environmental
influences. Thus, characterization of a single factor
triggering spontaneous inflammation is a major chal-
lenge in the field.
Phosphoinositide-specific phospholipase C
(PLC) is a signal transduction effector involving various
cell functions (3). PLC hydrolyzes phosphatidylinositol
4,5-triphosphate, a component of the plasma membrane,
and generates second messenger molecules, inositol
1,4,5-triphosphate (IP3) and diacylglycerol (DAG) (4).
DAG remains in the plasma membrane and activates
protein kinase C, while IP3induces the release of
calcium ions from the endoplasmic reticulum. DAG and
IP3mediate transduction of the signals from highly
specific receptors of hormones, neurotransmitters, anti-
gens, and growth factors to downstream, intracellular
targets. Therefore, they contribute to the regulation of
various biologic functions, such as cell motility, fertiliza-
tion, and immunity.
PLC enzymes consist of 13 isozymes belonging to
6 different subtypes (?, ?, ?, ?, ?, and ?). Phospholipases
C?1 and C?2 (Plcg1 and Plcg2, respectively) have been
identified as the PLC? subtypes (3). Plcg1-null embryos
appear normal at embryonic day (E) 8.5 but fail to
develop beyond E9 (5). This highlights the widespread
importance of Plcg1. In contrast, Plcg2 is most highly
expressed in hematopoietic organs such as the spleen
and lymph node, and it plays a key role in constructing
the immune system (6,7). In human B cells, loss of Plcg2
signaling results in an immunodeficiency syndrome
called X-linked agammaglobulinemia (8). Consistent
with this, Plcg2-knockout mice show defects in the
functioning of B cells, platelets, mast cells, and natural
killer (NK) cells (9). Furthermore, analysis of a sponta-
neous null allele of Plcg2, abnormal lymphatics, revealed
that Plcg2 plays an important role in separation of blood
and lymphatic vessels (10). These findings indicate a
crucial role of Plcg2 signaling in initiating and maintain-
ing the immune system.
To increase the variety of disease models, large-
scale mutagenesis programs have been carried out
worldwide using N-ethyl-N-nitrosourea (ENU) in mice
(11) and other vertebrates. In the ENU mutagenesis
project in Munich, we use the inbred C3HeB/FeJ (C3H)
mouse strain for harboring mutations (12–14). This
enables the analysis of complex phenotypes without the
bias of polymorphic genetic interferences, such as mod-
ifier effects from genetic background (15). Recently, this
forward genetics approach in mice was used for intensive
study of inflammatory arthritis (16–20). In the present
study, we describe a novel dominant-mutant mouse
strain, Ali14 (Ali for abnormal limb), representing a
novel gain-of-function allele of Plcg2. In addition to the
described abnormalities in the immune system, Ali14/?
mice displayed various metabolic changes and in vitro
infertility. Our findings may reveal how a single muta-
tion leads to systemic inflammatory diseases and related
symptoms in humans.
MATERIALS AND METHODS
Mice. The Ali14 mutation was generated in the Munich
mouse mutagenesis project, as described previously (12,14).
Briefly, we injected male C3H mice (The Jackson Laboratory)
intraperitoneally with ENU (Serva Electrophoresis). The Ali14
strain is maintained by backcrossing to wild-type (WT) female
C3H mice. For genetic mapping analysis, C57BL/6J (BL/6)
mice (The Jackson Laboratory) were used. For systematic
phenotyping at the German Mouse Clinic, 74 mice were
obtained from Ali14/? and C3H WT mating pairs. The mice
were genotyped by direct sequencing of polymerase chain
reaction (PCR) products using specific primers (Plcg2-ex16L,
5?-GTGAATGCTGGGGTGATGTC-3?; Plcg2-ex16R, 5?-
GAGCTAAGGATGCTCAAGCC-3?).
Genetic mapping and candidate sequencing. Genomic
DNA was extracted automatically from the tail tips of arthritic
N2mice (Agowa) and genotyped using 75 single-nucleotide
polymorphism (SNP) markers and matrix-assisted laser de-
sorption ionization–time-of-flight (MALDI-TOF) mass spec-
trometry (Sequenom), as previously described (19). For can-
didate sequencing analysis, we selected PCR primers beside
the coding exons of Plcg2, using ExonPrimer (available at
1302ABE ET AL

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http://ihg2.helmholtz-muenchen.de/ihg/ExonPrimer.html), and
pools of C3H and Ali14/? genomic DNA samples were
sequenced using a standard Sanger sequencing procedure
(ABI3100 Genetic Analyzer, Big Dye terminator chemistry;
Applied Biosystems).
Histologic and immunohistochemical analyses. Or-
gans were fixed in 4% buffered formalin and embedded in
paraffin for histologic examinations, and formic acid was used
for decalcification of paw samples (17). Myeloperoxidase
(MPO) staining and immunohistochemical analyses were per-
formed as previously described (21).
Clinical chemical analyses. Plasma levels of alkaline
phosphatase, aspartate amylase, creatine kinase, aspartate
aminotransferase, alanine aminotransferase, ferritin, transfer-
rin, lipase, glucose, cholesterol, triglycerides, uric acid, urea,
potassium, sodium chloride, calcium, inorganic phosphate, and
iron were measured using an AU 400 autoanalyzer and its
adapted reagents (Olympus).
Flow cytometry and calcium fluorimetry. Flow cytom-
etry was performed as previously described in detail (18,22).
Briefly, cells were stained with combinations of anti-IgD,
anti-B220, anti-CD44, anti-CD45, anti-CD3, anti-CD4, anti-
CD62L, and anti-Ly6C and analyzed using a FACSCalibur
(BD Biosciences), with results assessed using either FlowJo
(Tree Star) or CellQuest Pro (BD Biosciences) software. In
vitro calcium monitoring was carried out as described previ-
ously (16). Briefly, WT and mutant Plcg2 proteins were
expressed in cultured WEHI-231 B cells by retroviral transfec-
tion of Plcg2 expression constructs, based on the MIGR1
vector. For measurements of intracellular calcium flux, cells
were loaded with Fura Red (Molecular Probes) and stimulated
with 5 ?g/ml of goat anti-murine IgM (Star 86; Serotech).
Fluorescence of the cells was detected by flow cytometry.
Adoptive transfer of splenocytes. Splenocytes obtained
from Ali14/? mice or WT littermates were labeled with
carboxyfluorescein succinimidyl ester (CFSE) dye using the
CellTrace CFSE Cell Proliferation Kit (Invitrogen) according
to the manufacturer’s guidelines. WT recipients were injected
intravenously with the CFSE-labeled splenocytes (1 ? 107).
Clinical scores were determined in the recipient mice up to 18
days after injection, and the mice were then killed for flow
cytometric and histologic analyses.
Dual x-ray absorptiometry (DXA) and peripheral
quantitative computed tomography (QCT) analysis. We used a
pDEXA Sabre X-ray Bone Densitometer (Norland Medical
Systems) for capturing various bone and fat-related param-
eters, as previously described (17,23,24). Briefly, the entire
mouse body region was assayed with a 0.02 gm/cm2histogram
averaging-width setting. Peripheral QCT analysis was carried
out using Stratec XCT Research SA? (Stratec Medizintech-
nik).
Measurement of body composition by nuclear mag-
netic resonance (NMR) imaging. Body composition was deter-
mined using a Burker’s Whole Animal Body Composition
Analyzer (Minispec Bruker), based on time-domain NMR
imaging. Calibration was done using dissected lean muscle and
fat tissue.
In vitro fertilization (IVF) and sperm motility mea-
surement. IVF experiments were performed as described
previously (25). Sperm quality was measured using an IVOS
Sperm Analyzer (version 12.1c; Hamilton Research) as previ-
ously described (26). The motility (percentage of all motile
spermatozoa) and progressivity (percentage of spermatozoa
with a minimum velocity of ?60 ?m/second and straightness of
?50%) were evaluated.
RESULTS
Identification of the Ali14/? strain in the Munich
ENU mutagenesis project. The Ali14/? mouse line was
established in the large-scale Munich ENU mutagenesis
project on the basis of dominant inheritance of swollen
footpads and rubor on the ears of adult mice (Figures
1A and B and data not shown). Only male offspring were
found to exhibit the phenotypes. We kept the Ali14
mutation by backcrossing with WT C3H mice more than
10 times to reduce unrelated mutations induced by
ENU. Radiographs of the hind paws of Ali14/? mice
showed destruction of the distal phalanges, lower bone
density, and sealed joints of the phalanges (Figures 1C
and D). Histologic analysis of the distal phalanges of
Ali14/? mice indicated the presence of inflammatory
infiltrates into soft tissue and increased hematopoiesis in
Figure 1. Radiographic and immunohistologic analyses of swelling
and inflammation in the hind paws of Ali14/? mice. A and B, Assessment
of the gross morphologic appearance of the hind paws of wild-type
(WT) (A) and Ali14/? (B) mice indicated swollen digits and redness of
the footpads in Ali14/? mice. C and D, Radiographs of the hind paws
of WT (C) and Ali14/? (D) mice revealed destruction of the nails and
distal phalanxes as well as sealed phalangeal joints in Ali14/? mice.
E and F, Hematoxylin and eosin staining of sections of the phalanges
of WT (E) and Ali14/? (F) mice demonstrated abnormally thin
compact bones and increased hematopoiesis in bone marrow in the
phalanges of Ali14/? mice. Infiltrated inflammatory cells were also
observed in the dermis. Original magnification ? 5.
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the bone marrow (Figures 1E and F). In addition, severe
dermatitis and ulceration were observed in the ear
auricles of Ali14/? mice, while in the spleen, increased
hematopoiesis was observed in red pulps (results avail-
able at http://abe.med.u-tokai.ac.jp/index.html or from
the corresponding author upon request).
Immunohistochemical analysis using a granulo-
cyte marker, MPO, showed that numerous positively
staining cells were found in the regions of dermatitis in
the ear auricles (results available from the correspond-
ing author upon request). In contrast to the results with
MPO, markers for B cells (B220), T cells (CD3), and
macrophages (Mac3) stained only a minor population of
the infiltrates (results not shown).
Association of the Ali14 mutation with an amino
acid substitution in Plcg2. We started genetic mapping
of the Ali14 mutation using the BL/6 mouse as a
partner strain. However, none of the (C3H-Ali14/? ?
BL/6-?/?)F1mice exhibited the inflammatory arthritis
phenotype. We randomly selected the F1mice for
crossing to the original WT strain, C3H-?/?, to reduce
suppressive effects in the next generation. As antici-
pated, some of the mating pairs produced offspring with
swollen paws. We subjected genomic DNA from 56
phenotype-positive mice to MALDI-TOF mass spec-
trometry with 75 genome-wide SNP markers. Among
the 75 SNP markers, one marker, rs4227428, exhibited
the highest logarithm of odds (LOD) score (LOD 2.72),
and the LOD scores for all other markers were below
1.28 (Figure 2A).
The rs4227428 SNP locates on the distal region of
chromosome 8. We identified Plcg2 as a possible candi-
date gene in this region, because a previously identified
gain-of-function mutation in the Plcg2 gene, the Plcg2Ali5
mutation, causes a similar arthritis phenotype (16).
Subsequently, we identified an AT-to-GC transition in
exon 16 of Plcg2 that distinguishes C3H-?/? and C3H-
Ali14/? mice (Figure 2B). DNA sequencing of the same
region in WT BL/6, BALB/cByJ, CAST/EiJ, and 129SvJ
mouse genomes revealed no polymorphisms (Figure 2B
and results not shown).
The Ali14 mutation causes an amino acid substi-
tution at tyrosine-495, to a cysteine residue. Interest-
ingly, the tyrosine residue is conserved among various
vertebrates (Figure 2C). It is located not within the
catalytic domain but within a split pleckstrin homology
(spPH) domain that is specific to the PLC? family
(Figure 2D). Importantly, the Ali14 mutation leads to
greater responsiveness to a variety of upstream signals,
such as epidermal growth factor (EGF) in vitro (27).
Therefore, these observations indicate that Ali14 is a
gain-of-function allele of Plcg2, and this mutation is
hereafter referred to as Plcg2Ali14.
Immune system characteristics of Plcg2Ali14/?
mice. Because the functions of Plcg2 in lymphocytes are
well characterized, we analyzed the expression patterns
of lymphocytes and related parameters in Plcg2Ali14/?
mice. In B cell populations in the peripheral blood of
Figure 2. Identification of the Ali14 mutation by positional candidate
cloning.A,Standardgenome-widegeneticmappingusingsingle-nucleo-
tide polymorphism markers identified the distal region of chromosome
8 as the Ali14 candidate region, according to logarithm of odds (LOD)
scores. B, An AT-to-GC transition on the Ali14 genome was identified
in exon 16 of the phospholipase C?2 gene (Plcg2). C, Among various
classes of vertebrates, the Ali14 mutation causes an amino acid
substitution (Tyr495–Cys) (arrow) of Plcg2 in conserved tyrosine. D,
The domain structure of Plcg2 is shown. Arrow indicates the position
of the Ali14 mutation. spPH ? split pleckstrin homology (domain).
1304 ABE ET AL

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Plcg2Ali14/?mice, the frequency of IgD?B220? double-
positive cells was drastically reduced (45.5% in
Plcg2Ali14/?mice versus 80.5% in WT mice) (Figure 3A).
In T cell populations, the proportion of activated/
memory CD44?Ly6C? cells within the CD8 compart-
ment was increased in Plcg2Ali14/?mouse peripheral
blood (37.5%) as compared with that in WT mouse
peripheral blood (16.2%) (Figure 3A). These results
indicate that Plcg2Ali14/?mice display increased expres-
sion of T cells and decreased expression of B cells, as has
also been detected in Plcg2Ali5/?mutant mice (16).
Populations of granulocytes and NK cells were not
changed (results not shown).
We also measured levels of different Ig isotypes
in the plasma of Plcg2Ali14/?mice, since increased Ig
levels are often observed in patients with inflammatory
arthritis. Results of bioplex bead array assays demon-
strated that IgG2a, IgG3, and IgA were expressed at
normal levels in Plcg2Ali14/?mouse plasma (Figure 3B).
However, plasma levels of IgG1, IgG2b (only in male
mutants), and IgM in Plcg2Ali14/?mice were significantly
increased (for IgG1 [in both sexes], P ? 0.01 versus WT
mice; for IgG2b [in males] and IgM [in both sexes], P ?
0.05 versus WT mice) (Figure 3B). Although we mea-
sured plasma levels of autoantibodies (anti-DNA and
rheumatoid factor), no significant differences were de-
tected (results not shown).
In previous studies of Plcg2Ali5/?mice, increased
and sustained external calcium entry were observed in B
cells (16). Therefore, we analyzed the levels of external
calcium entry using an in vitro system. We used 4
different Plcg2 constructs, generated with either no
mutation (WT-Plcg2), the Ali5 mutation (Ali5-Plcg2),
the Ali14 mutation (Ali14-Plcg2), or both the Ali5 and
Ali14 mutations (Ali5/Ali14-Plcg2). These constructs
were introduced into cultured WEHI-231 B cells by
retroviral transfection, and calcium mobilization was
assayed by calcium fluorimetry. Interestingly, the cells
transfected with Ali14-Plcg2 showed the highest initial
peak of calcium entry and longest duration of sustained
calcium levels when compared with cells transfected with
any of the other constructs (Figure 3C).
To verify the immunologic origin of the inflam-
matory arthritis phenotype, we performed adoptive
transfer experiments in which CFSE-labeled Ali14/?
splenocytes were injected intravenously into WT recipi-
ents. However, no arthritis phenotype was observed to
develop within 18 days after injection (n ? 4), although
certain numbers of CFSE-positive donor cells were
detected in the lymphoid organs of the recipients, as
revealed by flow cytometry (results not shown).
Figure 3. A, Lymphocyte phenotypes were assessed by flow cytometry
in wild-type (WT) (?/?) and Plcg2Ali14/?mice, for populations of
IgD?B220? B cells (upper panels) and CD44?Ly6C? T cells (lower
panels). Values in each segment are the percentage of positive cells. B,
Plasma Ig levels were determined by enzyme-linked immunosorbent
assay. Bars show the mean ? SEM levels of Ig subclasses in male (M)
and female (F) WT and Plcg2Ali14/?mice. ? ? P ? 0.05; ?? ? P ? 0.01.
C, External calcium entry (assessed as fluorescence units over time) in
a cultured B cell line was assayed by calcium fluorimetry. The MIGR1
retrovirus vector was used to introduce various Plcg2 constructs.
FITC ? fluorescein isothiocyanate; APC ? allophycocyanin.
Ali14, A NOVEL GAIN-OF-FUNCTION MUTATION IN MURINE PHOSPHOLIPASE C1305

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Bone- and fat-related parameters in Plcg2Ali14/?
mice. To ascertain whether the phenotypes observed in
Plcg2Ali14/?mice were limited to the peripheral paws or
were a systemic feature in the body, we used DXA
analysis. Although results from DXA are of semiquan-
titative quality, DXA enables a fast scan of whole-body
composition. As shown in Figures 4A and B, Plcg2Ali14/?
mice displayed significantly reduced bone mineral con-
tent and bone mineral density as compared to that in
WT mice of both sexes (both P ? 0.0001 in males and
P ? 0.01 in females).
We next used peripheral QCT to characterize
the bone phenotype of Plcg2Ali14/?mice in more detail.
In the distal femoral metaphysis, total bone density
and total bone content were significantly reduced in
Plcg2Ali14/?mice of both sexes (results available at
http://abe.med.u-tokai.ac.jp/index.html or from the cor-
responding author upon request). This was mainly due
to a significant reduction in trabecular bone density and
cortical bone content. Concurrently, the trabecular bone
area was increased, whereas the cortical bone area was
reduced.
In addition to the bone defects, male Plcg2Ali14/?
mice showed a significantly reduced fat mass, as revealed
by DXA (Figure 4C). Although DXA analysis of fat
mass is, unlike other methods, a semiquantitative ap-
proach, the significant reduction in body weight ob-
served in male Plcg2Ali14/?mice (Figure 4D) strongly
supports the notion that body fat is reduced in
Plcg2Ali14/?mice. NMR analysis of the body composition
of Plcg2Ali14/?mice confirmed this notion; male
Plcg2Ali14/?mice exhibited significantly reduced fat mass
(mean ? SEM 6.5 ? 0.8 units in Plcg2Ali14/?mice versus
8.4 ? 1.4 units in WT mice; P ? 0.005) and reduced lean
mass (19.6 ? 1.3 units in Plcg2Ali14/?mice versus 21.6 ?
0.7 units in WT mice; P ? 0.01). In male Plcg2Ali14/?
mice, we detected a significant genotype effect on the
relationship between body mass and fat mass content.
Interestingly, female Plcg2Ali14/?mice also showed ten-
dencies toward having reduced fat mass and reduced
body weight (Figures 4C and D), but this was not
observed in the batch of mice used for NMR analysis.
Novel phenotypes identified by plasma biochem-
ical screening. In the German Mouse Clinic (28), we
screened various biochemical plasma parameters to
identify novel phenotypes in Plcg2Ali14/?mice. Of the 20
parameters measured in the clinical chemical screening,
the values for cholesterol, triglycerides, and alkaline
phosphatase were found to be abnormal in Plcg2Ali14/?
mouse plasma (Table 1). In male Plcg2Ali14/?mice, the
mean value for cholesterol was 2.8 mM, which was 1.5
mM lower than that in WT mouse plasma. Female
Plcg2Ali14/?mice also had a slightly lower concentration
of cholesterol (mean 2.4 mM in female Plcg2Ali14/?mice
versus 2.9 mM in female WT mice). The between-group
differences in this parameter were significant in both
male Plcg2Ali14/?mice and female Plcg2Ali14/?mice, as
determined by Welch’s t-test (P ? 0.001 in males and
P ? 0.01 in females, versus WT mice).
With regard to peripheral triglyceride levels,
Plcg2Ali14/?mice showed significantly lower concentra-
tions than were observed in WT mice (mean 2.7 mM and
1.9 mM in male and female Plcg2Ali14/?mice, respec-
tively versus 4.3 mM and 2.8 mM in male and female WT
mice, respectively). Alkaline phosphatase activity was
found to be significantly reduced in both male and
female Plcg2Ali14/?mice (mean 66.3 units/liter and 122.9
units/liter in male and female Plcg2Ali14/?mice, respec-
tively versus 96.9 units/liter and 142.4 units/liter in male
and female WT mice, respectively). Blood glucose values
were significantly reduced only in male mice of the
Plcg2Ali14/?strain (97.9 mg/dl in male Plcg2Ali14/?mice
versus 131.3 mg/dl in male WT mice). In the steroid
metabolism screening, significantly reduced levels of
Figure 4. Bone- and fat-related phenotypes in male (M) and female
(F) Plcg2Ali14/?mice. A–C, Dual x-ray absorptiometry analysis revealed
reduced bone mass, measured as bone mineral content (BMC) (A) and
bone mineral density (BMD) (B), as well as reduced fat mass (C) in
Plcg2Ali14/?mice (solid bars) compared with wild-type mice (open
bars). D, Body weight was also reduced in Plcg2Ali14/?mice. Bars show
the mean ? SEM. ? ? P ? 0.01; § ? P ? 0.0001.
1306ABE ET AL

Page 7

dehydroepiandrosterone (DHEA) were detected only in
male Plcg2Ali14/?mice (mean 35.4 pg/ml versus 127.0
pg/ml in male WT mice; P ? 0.01).
Normal sperm motility, but impaired IVF ability,
in male Plcg2Ali14/?mice. Although the mutation was
transmitted to the next generation at a normal rate by
natural mating (results not shown), we found that sper-
matozoa from male Plcg2Ali14/?mice failed to achieve
fertilization in vitro. Table 2 shows a summary of the
findings from IVF experiments using spermatozoa from
Plcg2Ali14/?mice, performed using a standard procedure
as described in Materials and Methods. Notably, the
2-cell cleavage rate of Plcg2Ali14/?mouse spermatozoa
was less than 5% (Table 2). Overall, we used 1,959
oocytes, but only 16 embryos were developed using
Plcg2Ali14/?mouse spermatozoa (0.8%). In contrast, in
experiments using WT mouse spermatozoa, 82.1% of
262 oocytes developed to 2-cell stage embryos. However,
the motility and progressivity of sperm from Plcg2Ali14/?
mice were comparable with those of WT mouse sperm
(results available at http://abe.med.u-tokai.ac.jp/index.
html or from the corresponding author upon request).
DISCUSSION
In this study, we identified a novel dominant
mutation, Ali14, causing spontaneous inflammation of
the peripheral paws of mice. From genetic mapping
analysis and candidate sequencing, we identified an
AT-to-GC transition in the coding region of Plcg2.
Furthermore, additional phenotypes of Plcg2Ali14/?mice
were analyzed intensively in a systematic phenotyping
center, the German Mouse Clinic (28,29). After analyz-
ing more than 240 parameters in this study, we found
novel functions of Plcg2 in vivo. This detailed phenotype
description gives insights into the hidden roles of PLC
signaling in vivo.
A line of experimental evidence indicates that the
Ali14 mutation is a gain-of-function mutation of Plcg2.
Plcg2 catalyzes formation of the second messengers, IP3
Table 1.
Results of laboratory chemical analyses of blood plasma from Plcg2Ali14/?mice*
Parameter
Male Female
Wild-type
(n ? 7)
Plcg2Ali14/?
(n ? 8)P
Wild-type
(n ? 10)
Plcg2Ali14/?
(n ? 11)P
Albumin, gm/liter
Urea, mM
Cholesterol, mM
Triglycerides, mM
AP, units/liter
Glucose, mg/dl
DHEA, pg/ml
26.3 ? 0.52
10.3 ? 0.30
4.3 ? 0.14
4.3 ? 0.29
96.9 ? 1.99
131.3 ? 8.2
127.0
24.3 ? 0.25
9.0 ? 0.17
2.8 ? 0.13
2.7 ? 0.30
66.3 ? 4.83
97.9 ? 8.7
35.4
?0.01
?0.01
?0.001
?0.01
?0.001
?0.05
?0.01
27.2 ? 0.43
9.4 ? 0.41
2.9 ? 0.09
2.8 ? 0.14
142.4 ? 6.41
118.8 ? 7.5
94.0
25.8 ? 0.18
8.3 ? 0.26
2.4 ? 0.05
1.9 ? 0.20
122.9 ? 4.05
128.5 ? 5.2
91.8
?0.05
?0.05
?0.01
?0.01
?0.05
NS
NS
* Values are the mean ? SEM. The parameters selected were those in which a significant difference between groups was found on duplicate tests
performed 3 weeks apart. AP ? alkaline phosphatase; NS ? not significant; DHEA ? dehydroepiandrosterone.
Table 2.
Results of in vitro fertilization (IVF) experiments using sperm from male Plcg2Ali14/?mice*
IVF experiment
Parental strain
No. of oocytesNo. of 2-cell embryosCleavage rate, %MaleFemale
1
2
3
4
5
6
7
8
Total
Plcg2Ali14/?sperm
Wild-type sperm
Plcg2Ali14/?
Plcg2Ali14/?
Plcg2Ali14/?
Plcg2Ali14/?
Plcg2Ali14/?
Wild-type
Wild-type
Wild-type
Wild-type
Wild-type
Wild-type
Wild-type
Wild-type
Wild-type
Wild-type
Wild-type
450
322
468
455
264
67
150
45
00.0
4.3
0.0
0.4
0.0
77.6
86.7
73.3
14
0
2
0
52
130
33
1,959
262
160.8
82.1 215
* Plcg2Ali14/?mice (C3HeB/FeJ [C3H] background) and C3H wild-type mice were used for IVF experiments. In all experiments, fresh spermatozoa
from different mice were used.
Ali14, A NOVEL GAIN-OF-FUNCTION MUTATION IN MURINE PHOSPHOLIPASE C1307

Page 8

and DAG. IP3mediates release of intracellular Ca2?
from the endoplasmic reticulum, and the increased
concentration of Ca2?then returns to the basal level for
a certain time. The cultured cells expressing Ali14-Plcg2
proteins exhibited a high initial peak of Ca2?concentra-
tion and a prolonged return-to-basal time (Figure 3C),
suggesting that Plcg2-mediated signals are enhanced
more in Plcg2Ali14/?mice. The Ali14 mutation causes an
amino acid substitution in an spPH domain of Plcg2. PH
domains function as structural modules for membrane
association and protein–protein interaction involving
inosito-lipid–mediated intracellular signaling (30). Re-
cently, it was reported that Rac GTPases cause marked
stimulation of Plcg2 (31). Furthermore, this stimulation
is regulated by interaction between Rac and the spPH
domains of Plcg2 (32). Our previous results indicated
that the Ali14-Plcg2 protein enhances Rac activation of
Plcg2 without increasing Rac binding, and also enhances
the response to EGF stimulation (27). Therefore, Ali14-
mutated proteins affect basal enzymatic activity slightly
but amplify signals more strongly than WT proteins.
Taken together, these findings strongly indicate that the
Ali14 mutation is a gain-of-function allele of Plcg2.
Abnormalities in the adoptive immune system of
Plcg2Ali14/?mice are similar to the phenotypes of
Plcg2Ali5/?mice. In the mouse peripheral blood, in
addition to an abnormal T cell:B cell ratio, up-regulation
of IgM and IgG1 is identical between Plcg2Ali14/?mice
and Plcg2Ali5/?mice (16). In contrast, Plcg2-knockout
mice show decreased IgM levels and do not show an
induction of an increase in intracellular Ca2?in B cells
(9,33). The increased populations of T cells in
Plcg2Ali14/?mice must be secondary effects, because
Plcg1 predominates in T cells (7). Results of analysis of
T cells in Plcg2Ali5/?mice support this notion, in that Ali5
T cells do not exhibit an enhanced Ca2?response by T
cell receptor stimulation (16).
Transfer of bone marrow cells from Plcg2Ali5/?
mice results in reconstitution of inflammatory arthritis
in irradiated WT mice. However, Plcg2Ali5/?mice with a
double mutation of RAG2 (RAG2-knockout allele),
which lack mature lymphocytes, show the arthritis phe-
notype (16). Overall, bone marrow–derived cells are
responsible for the phenotype, but lymphocytes are not
essential to trigger inflammation in Plcg2Ali5/?mice.
Similarly, the adoptive transfer experiments in this study
revealed that a certain number of Ali14/? splenocytes,
most of which consist of lymphocytes, contribute very
little to arthritis induction. Therefore, it is most likely
that the functions of lymphocytes, including autoreactive
T cell responses and autoantibodies, are not involved in
the initial phase of arthritis in Ali14/? mice.
In Plcg2-deficient mice, a loss of collagen-
induced platelet aggregation, impaired degranulation of
mast cells, and dysfunction of NK cells were observed
(9). Furthermore, Plcg2 is involved in neutrophil activa-
tion and neutrophil-mediated arthritis induction in the
K/BxN serum-transfer model (34,35). Mast cells and
neutrophils are mostly derived from bone marrow and
circulate in the peripheral blood. However, they seldom
stay in lymphoid organs, such as the spleen. This life
cycle is suitable for explaining the pathogenesis of
arthritis in Ali14/? mice. Thus, it is interesting to
analyze the myeloid-lineage cells as candidates for a
primary trigger of spontaneous inflammation in
Plcg2Ali5/?and Plcg2Ali14/?mice.
Using DXA and peripheral QCT analyses, we
found a significant decrease in the bone mineral content
and bone mineral density in Plcg2Ali14/?mice. Plcg2-
knockout mice have been found to have an osteopetrotic
phenotype because of the decrease in the number of
osteoclasts in these mice (36), suggesting that Plcg2-
mediated signals are necessary for osteoclast formation.
Therefore, it is important to analyze whether factors
downstream of Plcg2 signaling, such as nuclear factor of
activated T cells and NF-?B, are active in osteoclasts in
Plcg2Ali14/?mice. It is likely that both constitutive oste-
oclast activation and decreased bone formation lead to
the osteoporotic phenotype in Plcg2Ali14/?mice. Further
analyses using biomarkers of bone formation and re-
sorption are necessary to depict molecular mechanisms
of bone defects in Plcg2Ali14/?mice.
In female Plcg2Ali14/?mice, swollen paws are
rarely detected in our rearing system. This difference
between sexes is also obvious in Ali5/? mice, despite the
fact there is no between-sex difference in lymphocyte
abnormality (16). We hypothesize that regulation of sex
hormones may be related to the modification of the
arthritis phenotype in Ali14/? mice. The results from
the steroid hormone screen support this notion, in that
DHEA levels were reduced only in male Ali14/?-
heterozygous mice (Table 1). The testosterone concen-
tration was also reduced in male mice, although a
statistically significant difference was not detected (re-
sults not shown). Androgens are able to inhibit cutane-
ous wound healing, possibly by modulating inflamma-
tory responses (37,38). This unknown mechanism may
be involved in inflammatory responses via the Plcg2-
mediated signaling cascade.
The rate of IVF using spermatozoa from
Plcg2Ali14/?mice was extremely low, despite normal
1308ABE ET AL

Page 9

sperm motility and progressivity in these mice (Table 2
and results not shown). This indicates that not only
Ali14, but also WT (?) haploid sperm from Plcg2Ali14/?
mice showed impaired fertility in vitro. The WT haploid
spermatozoa in Plcg2Ali14/?mice might contain mutant
Plcg2Ali14proteins, since all of the spermatozoa are
originated from diploid spermatogonial (Ali14/?) stem
cells. If the Plcg2 proteins were produced in spermato-
genesis and accumulated in the cytoplasm, all mature
spermatozoa in Ali14/? mice share the mutant proteins.
This may also explain the complete sterility of Plcg2Ali5/?
mice (16). Because Plcg2Ali14/?mice show normal fertil-
ity in vivo, once fertilization occurs, the Plcg2 pathways
are not essential for further development. Therefore, we
speculate that the Plcg2-mediated pathway might be
involved in the acrosome reaction, but not in the zygote
formation. PLCs are actually activated during sperm
capacitation (39). Furthermore, Ca2?oscillation and
Plcg are tightly linked in the acrosome reaction (40).
Therefore, it would be worthwhile to visualize the
acrosome reaction and calcium mobilization using fluo-
rescent molecular probes in Ali14 and Ali5 spermatozoa.
Using simplified methods, we detected a reduc-
tion in body weight and fat mass only in male Plcg2Ali14/?
mice. NMR analysis of Plcg2Ali14/?mice confirmed this
male-specific reduction in fat and lean mass in quanti-
tative levels (results available from the corresponding
author upon request). In addition, the concentration of
peripheral triglycerides was also significantly decreased
in male heterozygotes. Since only male heterozygous
mice show the arthritis phenotype in our rearing system,
the reduction in fat mass could be a consequence of
spontaneous inflammation induced by the Ali14 muta-
tion. In humans, cachexia is characterized by weight loss
that involves depletion of host adipose tissue and skel-
etal muscle mass (2). It occurs with a number of diseases,
including cancer, acquired immunodeficiency syndrome,
and major trauma.
Rheumatoid cachexia is characterized by reduced
body cell mass in patients with rheumatoid arthritis (41).
Cachexia-like phenotypes were also found in animal
models of inflammatory arthritis; human tumor necrosis
factor ? (TNF?)–transgenic mice show weight loss, as
well as a reduction in fat and lean mass (42). This line of
evidence strongly suggests that cachexia is related to
increased levels of TNF?. Although there has been no
direct evidence to support the notion of an interaction
between TNF? and Plcg2, a number of reports indicate
a possibility of the interaction. In bone marrow–derived
macrophages, Plcg2 is necessary for full production of
TNF?, because TNF? levels induced by lipopolysaccha-
ride are clearly reduced in Plcg2 conditional-knockout
mice (43). In humans, TNF? induces cyclooxygenase 2
in lung epithelial cells. However, this induction is
strongly attenuated by U73122, a general inhibitor of
PLCs (44). Thus, Plcg2 is a key molecule in TNF?-
mediated signaling in lung epithelial cells. Therefore, it
is likely that a gain-of-function mutation in Plcg2 results
in the cachexia-like phenotype, via overproduction of
TNF? or activation of the TNF?-mediated pathways.
In the present study, we have thus characterized
a novel ENU-induced dominant mutation, Ali14, whose
phenotype presents as swollen peripheral limbs. By
genetic mapping and candidate sequencing, we identi-
fied Ali14 as a missense mutation in Plcg2. In addition to
inflammatory infiltrates in the paws, we found various
metabolic defects in Ali14 heterozygous mice by system-
atic phenotyping. Furthermore, IVF experiments with
spermatozoa from Ali14 heterozygotes resulted in ex-
tremely low IVF rates, despite the normal fertility of the
heterozygous mice in vivo. Recently, we obtained Ali14
homozygous mice, which exhibit much more severe
arthritis and an earlier onset of arthritis, at ?3 weeks of
age, in both sexes. This preliminary study indicated that
Ali14 is a semidominant mutation; it is predicted that the
compound-mutant mice with Ali14 and a knockout allele
(Ali14/?) show phenotypes similar to those of Ali14
homozygous (Ali14/Ali14) mice. Since the homozygous
mice do not show diminished survival rates (they survive
long enough to reach sexual maturity), the metabolic
abnormality and fertility, as well as inflammatory arthri-
tis phenotype, in these mice can be compared with those
of heterozygotes. These findings could extend the un-
derstanding of the complex molecular effects of inflam-
mation to a whole body, such as that observed in
rheumatoid cachexia.
ACKNOWLEDGMENTS
We gratefully acknowledge the excellent technical as-
sistance of Michael Schulz, Reinhard Seeliger, Sabrina Bothur,
Michaela Grandl, Elfie Holupierek, Katrin Laube, Jacqueline
Mueller, Elenore Samson, Florian Schleicher, Daniela
Schmidt, Waldemar Schneider, Ann-Elisabeth Schwarz, Lucie
Thurmann, and Susanne Wittich, as well as all members of the
ENU core facility and the animal caretaker team. We also
thank the members of the German Mouse Clinic for compre-
hensive phenotyping and discussions.
AUTHOR CONTRIBUTIONS
All authors were involved in drafting the article or revising it
critically for important intellectual content, and all authors approved
the final version to be published. Dr. Abe had full access to all of the
Ali14, A NOVEL GAIN-OF-FUNCTION MUTATION IN MURINE PHOSPHOLIPASE C1309

Page 10

data in the study and takes responsibility for the integrity of the data
and the accuracy of the data analysis.
Study conception and design. Abe, Fuchs, Boersma, Hans, Yu, Rath-
kolb, Rozman, Esposito, Klingenspor, Wurst, Gailus-Durner,
Marschall, Soewarto, Wagner, Hrabe ˇ de Angelis.
Acquisition of data. Abe, Boersma, Hans, Yu, Kalaydjiev, Klaften,
Mossbrugger, Rathkolb, Rozman, Prehn, Maraslioglu, Kametani, Shi-
mada, Adamski, Esposito, Klingenspor, Wolf, Gailus-Durner, Soew-
arto, Wagner.
Analysis and interpretation of data. Abe, Fuchs, Boersma, Hans, Yu,
Kalaydjiev, Klaften, Adler, Calzada-Wack, Mossbrugger, Rathkolb,
Rozman, Prehn, Maraslioglu, Kametani, Shimada, Adamski, Busch,
Esposito, Klingenspor, Katan, Wagner, Hrabe ˇ de Angelis.
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