OPEN
Loss of menin in osteoblast lineage affects osteocyte–
osteoclast crosstalk causing osteoporosis
Peng Liu
1,2
, Sooyeon Lee
1,2
, Jeanette Knoll
2
, Alexander Rauch
2,9
, Susanne Ostermay
2
, Julia Luther
3
, Nicole Malkusch
1
, Ulf H Lerner
4
,
Mario M Zaiss
5
, Mona Neven
3
, Rainer Wittig
6
, Martina Rauner
7
, Jean-Pierre David
3,5
, Philippe Bertolino
8
, Chang X Zhang
8
and
Jan P Tuckermann*
,1,2
During osteoporosis bone formation by osteoblasts is reduced and/or bone resorption by osteoclasts is enhanced. Currently, only
a few factors have been identified in the regulation of bone integrity by osteoblast-deri ved osteocytes. In this study, we show that
specific disruption of menin, encoded by multiple endocrine neoplasia type 1 (Men1), in osteoblasts and osteocytes caused
osteoporosis despite the preservation of osteoblast differentiation and the bone formation rate. Instead, an increase in osteoclast
numbers and bone resorption was detected that persisted even when the deletion of Men1 was restricted to osteocytes. We
demonstrate that isolated Men1-deficient osteocytes expressed numerous soluble mediators, such as C-X-C motif chemokine 10
(CXCL10), and that CXCL10-mediated osteoclastogenesis was reduced by CXCL10-neutralizing antibodies. Collectively, our data
reveal a novel role for Men1 in osteocyte–osteoclast crosstalk by controlling osteoclastogenesis through the action of soluble
factors. A role for Men1 in maintaining bone integrity and thereby preventing osteoporosis is proposed.
Cell Death and Differentiation (2017) 24, 672–682; doi:10.1038/cdd.2016.165; published online 20 January 2017
The maintenance of bone mass is severely affected in
osteoporotic patients and other bone disorders and can have
multiple etiologies. The complex communication of bone-
forming osteoblasts, bone-residing osteocytes, and bone-
resorbing osteoclasts is still not fully understood. Two hallmark
studies
1,2
have demonstrated that osteocytes are the major
source of the osteoclastogenesis-stimulating factor RANKL
and thus key components in the control of bone mass. This
property goes beyond their previously suggested function of
sensing the mechanical loading of bone.
3
PTH
4
and
sclerostin
5
have been reported to stimulate osteocytes to
support osteoclastogenesis via a RANKL-dependent path-
way. High-mobility group box 1 (HMGB1), which is chemotac-
tic to osteoclasts, is enhanced in apoptotic MLO-Y4 cells;
6
however, there is little information on other factors controlling
osteoclast activity by osteoblast-derived osteocytes.
The nuclear protein menin encoded by the gene Men1
(multiple endocrine neoplasia type 1) has recently been
suggested to control bone mass at higher age through its
influence on the differentiation and function of osteoblasts.
7
In vitro studies in cell lines and primary osteoblasts derived
from 6-month-old mice showed that menin promotes early
osteoblast differentiation in committed cells. Knockdown of
Men1 with antisense oligonucleotides lead to enhanced BMP2
signaling
8–10
and to facilitated inhibition of the late stage of
osteoblast differentiation by potentiating TGFβ-dependent
Smad3 signaling.
11,12
Accordingly, reduced bone mass and
reduced bone formation due to decreased osteoblast pro-
liferation and differentiation and enhanced apoptosis have
been reported using aged osteocalcin-cre mice crossed to
Men1 floxed mice that leads to a conditional deletion of menin
in the late stage of differentiated osteoblasts.
7
On the other
hand, aged osteoblast-specific menin transgenic mice were
found to display increased bone mass.
7
However, whether
Men1 plays a role in bone cells to control bone integrity in
young or middle-aged mice is completely unknown.
Using multiple cre-lines, we comprehensively analyzed the
effects of loss of function of menin throughout the osteoblast
lineage, in osteoclasts, and in osteocytes on bone integrity.
Furthermore, we identified genes under the control of Men1
responsible for the osteocyte-dependent regulation of osteo-
clast formation.
Results
Men1 disruption in the osteoblast lineage severely
affects bone mass but not the bone formation rate. In
order to address the role of Men1 in the entire osteoblast
lineage including osteocytes in vivo, we generated mice
lacking Men1 specifically in the osteoblast lineage by
1
Institute of Comparative Molecular Endocrinology, University of Ulm, Ulm D-89081, Germany;
2
Tissue-specific Hormone Action, Leibniz Institute on Aging–Fritz Lipmann
Institute (FLI), Jena D-07745, Germany;
3
Department of Osteology and Biomechanics, University Medical Center Hamburg-Eppendorf, Hamburg D-20246, Germany;
4
Centre for Bone and Arthritis Research, Department of Internal Medicine and Clinical Nutrition at Institute for Medicine, Sahlgrenska Academy at University of Gothenburg,
Gothenburg SE-41345, Sweden;
5
Department of Internal Medicine 3-Rheumatology and Immunology, Friedrich-Alexander-University Erlangen-Nürnberg (FAU) and
Universitätsklinikum Erlangen, Erlangen D-91054, Germany;
6
Institute for Laser Technologies in Medicine and Metrology at Ulm University, Ulm D-89081, Germany;
7
Division of Endocrinology and Bone Diseases, Department of Medicine III, TU Dresden, Dresden D-01307, Germany and
8
Centre de Recherche en Cancérologie de Lyon,
Inserm U1052, CNRS UMR5286, Université Lyon 1, Lyon F-69000, France
*Corresponding author: JP Tuckermann, Institute of Comparative Molecular Endocrinology, University of Ulm, Ulm D-89081, Germany. Tel: +49 731 50 32600; Fax: +49 731
50 32609; E-mail: jan.tucker mann@uni-ulm.de
9
Current address: Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense, Denmark.
Received 30.6.16; revised 08.12.16; accepted 20.12.16; Edited by E Wagner; published online 20.1.2017
Cell Death and Differentiation (2017) 24, 672–682
Official journal of the Cell Death Differentiation Association
www.nature.com/cdd
Figure 1 Disrupting Men1 in the osteoblast lineage causes bone loss without altering the bone formation rate. (a) Micro CTreconstruction of femurs from 12-week-old female
Men1
flox
and Men1
Runx2Cre
mice. (b–e) Cancellous parameters such as bone volume per tissue volume (BV/TV) (b), trabecular thickness (Tb.Th.) (c), trabecular numbers (Tb.N.)
(d), and trabecular separation (Tb.Sp.) (e) were measured from distal femurs of female Men1
flox
and Men1
Runx2Cre
mice at indicated age by micro CT (n = 4 or 5). *Po0.05,
**Po0.01, ***Po0.001. (f and g) Primary calvarial osteoblasts were isolated from Men1
flox
and Men1
gtRosaCreERT2
mice. After pretreatment with 4-OHT to allow recombination of
the Men1flox allele, cells were cultured with or without osteogenic induction (OI) medium in the absence or presence of BMP2 for 7 and 12 days and stained for alkaline
phosphatase (f) and alizarin red (g), respectively. (h–j) mRNA expression levels of Alpl (h) and Sp7 (i) from 7-day-treated cells as in (f), and Bglap (j) from 12-day-treated cells
as in (g) were analyzed by QRT-PCR (n = 5). *Po0.05, **Po0.01, ***Po0.001 versus untreated Men1
flox
.(k and l) Fluorescent micrographs of dual calcein labeling (k) and
its quantitative analysis of bone formation rate per bone surface (BFR/BS) (l) by dynamic histomorphometry in femurs of 12-week-old female Men1
flox
and Men1
Runx2Cre
mice
(n = 4 or 5). (m) Determination of the serum PINP level (a bone formation marker) in 12-week-old female Men1
flox
and Men1
Runx2Cre
mice (n = 5). Data are represented as
mean ± S.E.M.
Men1 regulates osteocyte–osteoclast crosstalk
P Liu et al
673
Cell Death and Differentiation
crossing Men1
tm1.1Zqw
(hereafter designated Men1
flox
) mice
13
to Tg(Runx2-icre)1Jtuc (hereafter designated Runx2Cre)
mice
14
to obtain Men1
Runx2Cre
mice. Immunohistochemistry
of femurs indicated a decreased menin expression in
osteoblasts and osteocytes of Men1
Runx2Cre
mice
(Supplementary Figure S1a). Successful recombination was
additionally confirmed by crossing a Rosa
mT/mG
(Tomato/
EGFP) reporter mouse
15
to Runx2Cre mice and demonstrat-
ing cre-loxP recombination by GFP-positive osteoblasts and
osteocytes (Supplementary Figure S1b). Men1
Runx2Cre
mice
were viable without gross alterations of the skeleton
(Supplementary Figure S1c) and were born slightly below
the Mendelian ratio (Supplementary Table S1).
Strikingly, micro computed tomography (micro CT) revealed
a strong bone loss in the distal femur of female Men1
Runx2Cre
mice (Figures 1a–e). This bone loss was observed in 4-week-
old mice and persisted upon aging (Figures 1b–e). Similarly,
bone loss was observed in male Men1
Runx2Cre
mice
(Supplementary Table S2), resembling osteoporosis.
Although trabecular thickness remained unaltered, the strong
decrease of trabecular bone mass in Men1
Runx2Cre
mice was
found to be caused by a low number of trabeculae and
an increased trabecular separation (Figures 1b–e and
Supplementary Table S2). Notably, cortical bone thickness in
the femur was unchanged (Supplementary Table S3).
Histomorphometry and micro CT analysis of tibiae and
vertebrae confirmed the trabecular bone loss (Supple-
mentary Figures S1d–h and Supplementary Table S2). Next,
we analyzed whether the osteoporotic phenotype was
specifically dependent on the elimination of Men1 in the
osteoblast lineage.
First, we corroborated that the observed bone loss strictly
depended on elimination of Men1 in the osteoblast lineage.
Mice with a disruption of Men1 in the myeloid lineage
(Lyz2
tm1(cre)Ifo
,
16
hereafter designated Men1
LysMCre
mice)
including osteoclasts (Supplementar y Figures S2a and b)
did not display any alterations in trabecular bone volume,
trabecular thickness, numbers, and separation
(Supplementary Figures S2c–f). In contrast, eliminating
Men1 in the early differentiated osteoblast lineage using
another osteoblast-specific cre-line, Tg(Sp7-tTA,tetO-EGFP/
cre)1Amc (hereafter designated OsxCre) mice,
17
reproduced
the severe osteoporosis as observed in Men1
Runx2Cre
mice.
The resulting Men1
OsxCre
mice also displayed a strong
decrease of trabecular bone volume and trabecular numbers
and an increased trabecular separation, whereas trabecular
thickness was only slightly affected (Supplementary Table S2).
Second, we asked whether loss of Men1 influenced the
expression of osteoblast marker genes and osteoblast
numbers in vivo. The mRNA expression of osteoblast marker
genes such as Alpl, Col1a1, Runx2, and Bglap was similar in
the calvarial bone of Men1
Runx2Cre
mice compared with control
(Men1
flox
) (Supplementary Figures S3a–d). In line with this,
dynamic histomorphometry of vertebrae did not detect
significant changes of osteoblast numbers and osteoblast
surface (Supplementary Figures S3e and f). Osteocyte
number was significantly higher in Men1
Runx2Cre
mice
(Supplementary Figure S3g).
Furthermore, we asked whether Men1 deficiency could
influence osteoblast differentiation in vitro as suggested in
previous studies.
7,12
To eliminate Men1 efficiently in osteo-
blast progenitor cells, we cultivated mesenchymal progenitor
cells and primary calvarial osteoblasts derived from Men1
flox
mice crossed to a mouse line with ubiquitous expression of a
Cre-ERT2 fusion protein (Gt(ROSA)26Sor
tm9(Cre/ESR1)Arte
,
hereafter designated gtRosaCreERT2), allowing a dramatic
decrease in Men1 expression upon tamoxifen treatment
(Supplementary Figures S3o, p, and s). Disruption of Men1
in vitro did not affect the growth of calvarial osteoblasts
(Supplementary Figure S3h). Intriguingly, the differentiation of
Men1-deficient calvarial osteoblasts and mesenchymal pro-
genitor cells was unaffected and the cells responded normally
to BMP2-induced differentiation as indicated by histochemical
staining (Figures 1f, g and Supplementary Figures S3i–l) and
marker gene expression (Figures 1h–j, Supplementary
Figures S3m and n). In accordance with these results, no
major differences were observed in the BMP2-induced
phosphorylation of Smad1/5/8 (Supplementary Figure S3p)
and induction of the BMP2–Smad target genes Id1 and Dlx5
(Supplementary Figures S3q and r) between control and
Men1-deficient cells.
As it was previously reported that inhibiting Men1 expres-
sion with antisense oligonucleotides could affect TGFβ
signaling,
11
we also analyzed the TGFβ-induced phosphor-
ylation of Smad3 as well as the expression of TGFβ-induced
genes in the absence of Men1 in osteoblasts. No significant
difference was found at the level of Smad3 phosphorylation,
nor at expression levels of the TGFβ target genes Serpine and
Skil in the absence of Men1 (Supplementary Figures S3s–u).
In accordance with the unaltered osteoblast differentiation,
there was no difference in the bone formation rate in femurs
(Figures 1k and l) and vertebrae (Supplementary Figures S3v
and w) of 12-week-old Men1
flox
and Men1
Runx2Cre
mice.
Correspondingly, the serum PINP level was not changed in
Men1
Runx2Cre
mice (Figure 1m). However, the bone formation
rate in 12-month-old Men1
Runx2Cre
mice was significantly
reduced (Supplementary Figures S3x and y). This corrobo-
rates the finding of Kanazawa et al.
7
who described impaired
osteoblast function in 9- and 12-month-old mice in which Men1
was eliminated by an osteocalcin-cre.
In summary, despite a possible role for Men1 in bone
formation during aging, no drastic changes of osteoblast
marker gene expression, osteoblast number, and osteoblast
function were observed in young and middle-aged mice
lacking Men1 in the osteoblast lineage.
Men1 deficiency in the osteoblast lineage, specifically in
osteocytes, leads to an enhanced osteoclastogenesis.
We observed a strong increase of osteoclast numbers and
surface in femurs (Figures 2a and b) and vertebrae
(Supplementary Figures S4a and b). These increases were
also seen in calvaria (Supplementary Figures S4c–e) and
were associated with a high porosity (Supplementary
Figure S4f). Bone resorption as determined by serum
C-terminal telopeptide (CTX) level was increased in
12-week-old Men1
Runx2Cre
mice (Figure 2c). To test whether
Men1-deficient osteoblasts could cause greater osteoclasto-
genesis, we performed osteoblast–osteoclast co-culture by
using control or Men1-deficient osteoblasts and wild-type
osteoclast precursors. Surprisingly, the Men1-deficient
Men1 regulates osteocyte–osteoclast crosstalk
P Liu et al
674
Cell Death and Differentiation
osteoblasts could not trigger higher osteoclast numbers
(Figure 2d).
Osteocytes were recently established as major producers of
RANKL in bone and therefore important regulators of
osteoclastogenesis.
1,2
As deletion of Men1 by the cre-loxP
system in osteoblasts also affects Men1 expression in
osteocytes (Supplementary Figures S1a and b), we further
tested the capacity of Men1-deficient osteocytes to stimulate
osteoclast formation ex vivo. To this end, we isolated primary
osteocyte-enriched fractions from Men1
gtRosaCreERT2
mice.
These cells expressed higher Dmp1 and lower Alpl levels
when compared with primary osteoblasts, confirming the
efficacy of the isolation of osteocytes (Supplementary Figures
S5a and b). Following 4-hydroxytamoxifen (4-OHT) treatment
to eliminate the Men1 gene (Supplementary Figure S5c), the
osteocyte-enriched fraction was co-cultivated with wild-type
bone marrow cells (BMCs) containing osteoclast progenitor
cells. As a result, osteoclast number and osteoclast area were
significantly higher in the co-culture containing Men1-deficient
osteocytes (Figures 2e–g).
Men1 deficiency in osteocytes leads to an enhanced
osteoclastogenesis in vivo. The in vitro co-culture
experiments suggested that the increased osteoclast number
in the mice lacking Men1 in the osteoblastic lineage was
conferred by Men1-deficient osteocytes rather than osteo-
blasts. To confirm this hypothesis in vivo, we generated mice
lacking Men1 in osteocytes, but not in early differentiated
osteoblasts, by crossing Men1
flox
mice to Tg(Dmp1-cre)1Jqfe
(hereafter designated Dmp1Cre) mice
18
to obtain
Men1
Dmp1Cre
mice. Loss of menin in osteocytes in
Men1
Dmp1Cre
mice was confirmed by immunohistochemistry
(Supplementary Figure S5d) and by crossing Men1
Dmp1Cre
mice to Rosa
mT/mG
reporter mice that displayed a strongly
diminished Men1 expression in GFP-positive cells (Figure 3a
and Supplementary Figure S5e). As Dmp1 is also expressed
in mature osteoblasts, we stained osteocalcin as a mature
osteoblast marker in Men1
Dmp1Cre
; Rosa
mT/mG
reporter mice
(Supplementar y Figure S5f) to determine the degree of
recombination in these cells. We found that 29 ± 9% of
osteocalcin-stained mature osteoblasts were EGFP positive,
indicating that a minor fraction of the late-stage differentiated
osteoblasts is also mutant in addition to osteocytes in
Men1
Dmp1Cre
; Rosa
mT/mG
mice. Micro CT analysis revealed
a severe osteoporosis in the distal femur of adult
Men1
Dmp1Cre
mice (Figures 3b–f). The decrease of trabecular
bone mass in Men1
Dmp1Cre
mice was because of a low
number of trabeculae and an increased trabecular separa-
tion. Trabecular thickness was again unchanged. Osteoblast
number, osteoblast surface, and osteocyte number were not
altered in Men1
Dmp1Cre
mice (Figures 3g–i). Accordingly, the
bone formation rate and serum PINP level were not different
between Men1
flox
mice and Men1
Dmp1Cre
mice (Figures 3j–l).
Osteoclast number, osteoclast surface, and CTX level were
significantly elevated in Men1
Dmp1Cre
mice (Figures 3m–o),
whereas RANKL and OPG levels were not changed
(Supplementar y Figures S5g and h). Thus, a similar bone
phenotype was observed in Men1
Dmp1Cre
mice as shown in
Men1
Runx2Cre
and Men1
OsxCre
mice. Together with the
observed increase of osteoclastogenesis by osteocytes,
these observations strongly suggest that osteoporosis by
loss of Men1 is executed by osteocytes in these three
different conditional knockout mouse strains.
Menin suppresses CXCL10 to regulate osteocyte–osteo-
clast crosstalk. We could not detect differences in Tnfsf11
(RANKL) and Tnfrsf11b (OPG) expression in the bone of
Men1
Dmp1Cre
mice (Supplementary Figures S6a and b). We
Figure 2 Bone resorption is enhanced in Men1
Runx2Cre
mice, caused by Men1 deficiency in osteocytes. (a and b) Osteoclast numbers per bone perimeter (N.Oc/B.Pm) (a)
and osteoclast surface per bone surface (Oc.S/BS) (b) in sections of femoral trabecular bone were measured by histomorphometry (n = 4 or 5). (c) Determination of resorption by
assessment of the serum level of the biomarker CTX in 12-week-old female Men1
flox
and Men1
Runx2Cre
mice (n = 5). (d) Number of multinucleated TRAP-positive osteoclasts was
counted from primary co-cultures of wild-type BMCs with Men1
flox
or Men1
gtRosaCreERT2
primary osteoblasts (n = 3). (e – g) Osteoclastogenesis of pri mary co-cultures of wild-type
BMCs with Men1
flox
or Men1
gtRosaCreERT2
primary osteocytes was visualized by TRAP staining (e). Number of multinucleated TRAP-positive cells (f) and their area (g) were
determined (n = 3). *Po0.05, **Po0.01. Data are represented as mean ± S.E.M. Scale bar: 25 μm
Men1 regulates osteocyte–osteoclast crosstalk
P Liu et al
675
Cell Death and Differentiation
Figure 3 Men1 deficiency in osteocytes leads to an enhanced osteoclastogenesis in vivo. (a) Real-time PCR analysis of Men1 mRNA expression in FACS-sorted GFP-
positive cells from femoral bone of Men1
Dmp1Cre
mice crossed to Rosa
mT/mG
mice in animals heterozygous and homozygous for Men1flox. (b–f) Micro CT reconstruction (c)of
femurs from 12-week-old female Men1
flox
and Men1
Dmp1Cre
mice. Cancellous parameters such as BV/TV (b), Tb.Th. ( d), Tb.N. (e), and Tb.Sp. (f) were measured from distal
femurs of 12-week-old female Men1
flox
and Men1
Dmp1Cre
mice by micro CT (n = 4 or 5). (g–i) Osteoblast number per bone perimeter (N.Ob/B.Pm) (g), osteoblast surface per
bone surface (Ob.S/BS) (h), and osteocyte number per bone area (N.Ot/B.Ar) (i) in trabecular bone of distal femoral sections from 12-week-old female Men1
flox
and Men1
Dmp1Cre
mice were measured by histomorphometry (n = 4 or 5). (j and k) Fluorescent micrographs of dual calcein labeling (j) and its quantitative analysis of BFR/BS (k) in femoral
sections of 12-week-old female Men1
flox
and Men1
Dmp1Cre
mice (n = 4 or 5). (l) Deter mination of the serum PINP level in 12-week-old female Men1
flox
and Men1
Dmp1Cre
mice
(n = 5). (m and n) N.Oc/B.Pm (m) and Oc.S/BS (n) in trabecular bone of distal femoral sections from 12-week-old female Men1
flox
and Men1
Dmp1Cre
mice were measured by
histomorphometry (n = 4 or 5). (o) Determination of resorption from the serum CTX level in 12-week-old female Men1
flox
and Men1
Dmp1Cre
mice (n = 5). *Po0.05, **Po0.01,
***Po0.001. Data are represented as mean ± S.E.M.
Men1 regulates osteocyte–osteoclast crosstalk
P Liu et al
676
Cell Death and Differentiation