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ARTICLE
Notch signaling controls chondrocyte hypertrophy via
indirect regulation of Sox9
Anat Kohn
1,2,
*
, Timothy P Rutkowski
1,2,
*
, Zhaoyang Liu
1,3
, Anthony J Mirando
4
, Michael J Zuscik
1
, Regis J O’Keefe
1,5
and Matthew J Hilton
1,4
RBPjk-dependent Notch signaling regulates both the onset of chondrocyte hypertrophy and the progression to
terminal chondrocyte maturation during endochondral ossification. It has been sugge sted that Notch signaling
can regulate Sox9 transcription, although how this occurs at the molecular level in chondrocytes and whether
this transcriptional regulation mediates Notch control of chondrocyte hypertrophy and cartilage development
is unknown or controversial. Here we have provided conclusive genetic evidence linking RBPjk-dependent
Notch signaling to the regulation of Sox9 expression and chondrocyte hypertrop hy by examining
tissue-specific Rbpjk mutant (Prx1Cre;Rbpjk
f/f
), Rbpjk mutant/Sox9 haploinsufficient
(Prx1Cre;Rbpjk
f/f
;Sox9
f/1
), and control embryos for alterations in SOX9 expression and chondrocyte
hypertrophy duri ng cartilage development. These studies demonstrate that Notch signaling regulates the onset
of chondrocyte maturation in a SOX9-dependent manner, while Notch-mediated regulation of terminal
chondrocyte maturation likely functions independently of SOX9. Furthermore, our in vitro molecular analyses
of the Sox9 promoter and Notch-mediated regulation of Sox9 gene expression in chondrogenic cells identified
the ability of Notch to induce Sox9 expression directly in the acute setting, but suppresses Sox9 transcription
with prolonged Notch signaling that requires protein synthesis of secondary effectors.
Bone Research (2015) 3, 15021; doi:10.1038/boneres.2015.21; Published online: 11 August 2015
INTRODUCTION
The limb skeleton is derived via the process of endochondral
ossification, which begins with the condensation of
mesenchymal progenitor cells within the developing limb-
buds. Cells within condensations undergo chondrogenesis,
creating a cartilage template of the skeletal elements, while
cells at the periphery known as perichondrial cells ultimately
differentiate into osteoblasts that form bone. Chondrocytes
of the developing skeletal elements proliferate with round
disorganized chondroctyes near the epiphyses giving rise
to flattened chondrocytes organized in columns near the
metaphyses. This organization provides directionality to the
longitudinal expansion of cartilage rudiments. As chondro-
cytes approach the center of the elements, they exit the cell
cycle and begin the process of hypertrophic differentiation.
Chondrocyte hypertrophy is a key step during endochodral
bone development where chondrocytes dramatically alter
their morphology and size to generate a mineralizing cartil-
age matrix. Hypertrophic chondrocytes also secrete mole-
cules important in inducing osteoblastogenesis, recruiting
vascular tissue to the primary ossification center, and aiding
in the process of replacing the mineralized cartilage with
bone.
1
When this process is perturbed, chondrodysplasias
and known cartilage and skeletal disorders arise.
Specific transcription factors are responsible for direct-
ing the differentiation pattern of chondrocytes and osteo-
blasts during endochondral ossification. SOX9 is a
transcription factor known to be a master regulator of
chondrogenesis and the differentiated chondrocyte
phenotype.
2
In humans, heterozygous mutations of Sox9
results in campomelic dysplasia, a lethal developmental
disorder characterized by generalized hypoplasia of
1
Department of Orthopaedics and Rehabilitation, The Center for Musculoskeletal Research, University of Rochester Medical Center, Rochester,
NY 14642, USA;
2
Department of Biomedical Genetics, University of Rochester Medical Center, Rochester, NY 14642, USA;
3
Department of Biology,
University of Rochester, Rochester, NY 14642, USA;
4
Department of Orthopaedic Surgery, Duke Orthopaedic Cellular, Developmental, and
Genome Laboratories, Duke University School of Medicine, Durham, NC 27710, USA and
5
Department of Orthopaedic Surgery, Washington
University School of Medicine, St. Louis, MO 63110, USA
Correspondence: Matthew J Hilton (matthew.hilton@dm.duke.edu)
*These Authors contributed equally to this work.
Received: 15 May 2015; Revised: 27 May 2015; Accepted: 28 May 2015
OPEN
Citation: Bone Research (2015) 3, 15021; doi:10.1038/boneres.2015.21
ß 2015 Sichuan University All rights reserved 2095-4700/15
www.nature.com/boneres
endochondral bones and XY sex reversal.
3–4
In mice and
humans, haploinsufficiency of Sox9 is lethal shortly after
birth and phenocopies many of the skeletal anomalies
present in campomelic dysplasia.
5
A study of Sox9
2/2
chi-
meric mice, as well as a study using Prx1Cre to selectively
inactivate Sox9 floxed alleles within the skeletogenic
mesenchyme has identified Sox9 as necessary for the
formation of mesenchymal condensations.
6–7
SOX9
activation of chondrocyte-specific genes is partially
mediated through its activation of two other distantly
related members of the SOX family, SOX5 and SOX6.
8
The
three proteins, together, have been shown to activate
numerous cartilage-specific extracellular matrix genes,
including Col2a1, Col11a2, Matrilin-1, Col27a1, and
Agc1
2
that maintain the differentiated chondrocyte envir-
onment. Recent studies have also shown that over-
expression of Sox9 in hypertrophic chondrocytes results in
delayed terminal chondrocyte maturation and cartilage
matrix turnover, suggesting a specific role in regulating
chondrocyte hypertrophy.
9
While previous studies have
shown negative regulation of Sox9 by the b-catenin signal-
ing pathway results in accelerated chondrocyte hyper-
trophy
10
and positive regulation by the TGFb/BMP
signaling pathways induces chondrogenesis,
11–12
the pre-
cise genetic mechanisms regulating Sox9 gene expression
during cartilage development remain ill-defined.
Recent studies have identified the Notch signaling path-
way as a potential regulator of Sox9 in chondrocytes. Briefly,
Notch signaling is initiated when a ligand of the Delta/
Jagged families bind to a Notch receptor (NOTCH1-4).
Binding initiates a series of cleavage events, culminating in
gamma-secretase mediated release of the intracellular por-
tion of the Notch receptor, known as the Notch Intracellular
Domain (or NICD). Once released, NICD translocates to the
nucleus where it binds the transcriptional repressor RBPjk,
converting it to a transcriptional activator. A transcriptionally
active complex composed, in part, of NICD, RBPjk and
MAML drives expression of target genes, such as those of
the Hes/Hey gene family.
13–14
In the absence of NICD,
RBPjk recruits the co-repressors SMRT and SHARP resulting in
a suppression of transcription.
15–16
Recently, two studies
have shown that over-expression of NICD, using either the
Prx1Cre to target the skeletogenic mesenchyme or the
Col2Cre to target chondro-osteoprogenitor cells, results in
inhibition of condensation formation, significantly reduced
levels of Sox9, and, ultimately, an inability to form skeletal
elements.
17–18
These mutan ts are markedly similar to those
observed in mutant embryos lacking Sox9 expression in the
skeletogenic mesenchyme.
6
Furthermore, mutant mice with
over-expression of Sox9 in hypertrophic chondrocytes using
the Col10a1 promoter
9
are histologically very similar to prev-
iously reported mice lacking various components of the
Notch signaling pathway, including the Notch1 and
Notch2 receptors and Rbpjk.
19–20
Interestingly, loss of Rbpjk
in chondro-osteoprogenitor cells leads to increased levels of
Sox9 gene expression in growth plate chondrocytes, specif-
ically in the hypertrophic cells, while over-expression of NICD
leads to decreased Sox9 expression throughout the growth
plate.
18
These data suggest a possible mechanism, by which
RBPjk-dependent Notch signaling is required to modulate
Sox9 expression during cartilage development and chon-
drocyte hypertrophy.
To examine the genetic interaction between RBPjk-
dependent Notch signaling and Sox9 we analyzed tissue-
specific Rbpjk mutant (Prx1Cre;Rbpjk
f/f
), Rbpjk mutant/Sox9
haploinsufficient (Prx1Cre;Rbpjk
f/f
;Sox9
f/1
), and control
embryosforalterationsinchondrocyte hypertrophy and car-
tilage growth. Additionally, chondrogenic cell lines were
used to assess the specific mechanisms by which Notch sig-
naling regulates the Sox9 promoter. The cartilage and
molecular phenotypes observed in our genetic studies com-
bined with our in vitro molecular data demonstrate that
RBPjk-dependent Notch signaling coordinates the onset of
chondrocyte maturation and cartilage growth largely via
indirect regulation of Sox9.
MATERIALS AND METHODS
Mouse strains
All mouse strains including Prx1Cre, Rbpjk
f/f
,andSox9
f/f
are as
previously described.
5,21–22
Prx1Cre and Sox9
f/f
mice were
obtained from the Jackson Laboratory; Rbpjk
f/f
mice were
a generous gift from Dr. Tasuku Honjo (Kyoto Graduate
School of Medicine, Japan). Embryos of the genotypes
Prx1Cre;Rbpjk
f/1
, Prx1Cre;Rbpjk
f/1
;Sox9
f/1
, Prx1Cre;Rbpjk
f/f
,
Prx1Cre;Rbpjk
f/f
;Sox9
f/1
, and Cre-negative littermate con-
trols were produced in Mendelian ratios to be analyzed from
E14.5 to E18.5. All animal studies were performed in accord-
ance with the guidelines set forth by the Institutional Animal
Care and Use Committee.
Analyses of mouse embryos
Embryonic tissues were harvested at E14.5–E18.5, fixed in
10% neutral buf fered formalin , decalcifie d in 1 4% EDTA,
processed, and embedded in paraffin prior to section-
ing at 6 mm. Alcian Blue/Hem atoxylin/Orange G (ABH/
OG) s taining was performed according to standard
methodologies. In situ hybridization was performed
as described previously,
17,19–20, 23–24
using
35
S-labeled
riboprobes. Immunohistochemistry for SOX9 was per-
formed using the Vectastain Elite Rabbit IgG Kit
(Vector) and Santa Cruz Biotechnology SOX9 antibody
(sc20095). SOX9 antibody was prepared in 4% normal
goat serum using a 1:200 dilution without antigen
retrieval. Color reaction was performed using Vector
ImmPACT DAB (Vector); sections were counterstained
with hematoxylin (Zymed).
Notch regulates Sox9 via secondary effectors
A Kohn et al
2
Bone Research (2015) 15021 ß 2015 Sichuan University
Whole-mount skeletal staining of embryos was per-
formed as previously described.
17,25–26
Sox9 luciferase assays
Forty-eight hours before transfection, ATDC5 cells
(RIKEN) were plated in 24-well plates. ATDC5 cel ls were
maintained at 37
6
Cwith5%CO
2
in DMEM/F12 (1:1) sup-
plemented with 5% fetal bovine serum, an d 1% pen icillin/
streptomycin. Transfections were completed u sing
FuGENE HD (Promega) with 600 ng of Flag or NICD1,
200 ng Sox9-pGL3-promoter DNA constructs, and 8 ng
Renilla (Promega). Flag and NICD1 constructs were
transfected first for 24 hours before adding the respect-
ive Sox9-pGL3 constructs and Renilla for 24 additional
hours. After the 48 hours of transfections, ce llular extracts
were collected using the lysis buffer in the dual-lucifer-
ase assay (Promega). Firefly luciferase activi ty was
assessed using 20 mL or microliter of cellular extracts fol-
lowed by immediate analysis of Renilla luciferase activ-
ity. Analysis of the data was performed by normalizing
Sox9-pGL3 luciferase activity t o Renilla luciferase ac tiv-
ity; normalized luc iferase activity was then normalized to
the Flag control of the respective Sox9-pGL3 constructs.
Luciferase assays were performed in triplicate and
repeated three times. Statistical analysis was performed
using a two-tailed, unpaired t-test.
In vitro Notch activation and protein synthesis inhibition
Six-well tissue culture plates were coated with
RetroNectin (Takara) solution (20 mg?mL
21
in 1X PBS, 1
mL per well) at 4
6
C overnight while gently rocking. The
following day, each well was blocked with 1 mL 2%
BSA (Sigma) for 30 minutes at room temperature and
briefly washed with 1X PBS. The plate was then coated
with anti-IgG (Sigma) (10 mg?mL
21
in 1X PBS, 1 mL per
well) at 4
6
C overnight while gently rocking. The following
day, each well was blocked with 1 mL 2% BSA for
30 minutes at room temperature and briefly washed with
1X PBS. The plate was next coated with either IgG
(Sigma) (10 mg?mL
21
in 1X PBS, 1 mL per well) or
Recombinant JAG1 protein (R&D Systems) (10 mg?mL
21
in 1X PBS, 1 mL per well) at 4
6
C overnight while gently
rocking. The following day, each well was blocked with 1
mL 2% BSA for 30 minutes at room temperature and briefly
washed with 1X PBS. Coated plates were air dried for 1 hour
in the tissue culture hood, sealed with parafilm and stored
at 4
6
C until use. ATDC5 cells were seeded onto the IgG-
or JAG1-coated plates at a density of 500 000 cells per
well in ITS media 6 the protein synthesis inhibitor, cyclohex-
imide (10 mg?mL
21
) (Sigma). RNA was harvested at 0, 2, 4,
and 8 hours after seeding and quantitative reverse tran-
scription polymerase chain reaction (RT-PCR) was per-
formed to measure levels of Hes1, Hey1,andSox9 gene
expression.
RESULTS
Loss of RBPjk leads to inappropriate expression of SOX9 in
hypertrophic chondrocytes
In previous studies, we have demonstrated that RBPjk-
dependent Notch signaling is necessary for normal onset
of chondrocyte maturation as well as terminal hyper-
trophy.
20
In these studies, the Prx1Cre transgene was used
to selectively target removal of Rbpjk from the skeleto-
genic mesenchyme of the developing limb. While a clear
role for RBPjk-dependent Notch signaling during chondro-
cyte maturation was established, the downstream
mechanisms have not been determined. Previous studies
have shown that Sox9 is expressed in the epiphyseal resting
and proliferating chondrocytes but not in the hypertrophic
chondrocytes.
27
Interestingly, immunohistochemistry for
SOX9 reveals persistent, inappropriate expression of SOX9
protein within deeper zones of the hypertrophic region in
Prx1Cre;Rbpjk
f/f
mutants, compared to littermate controls
(Figure 1a and b). This is consistent with data showing that
over-expression of NICD in chondro-osteoprogenitor and
mesenchymal progenitor cells leads to reduced Sox9
gene expression in cartilage and limb-bud mesenchyme,
respectively.
18
Conversely, when RBPjk is removed, Sox9
gene expression is elevated in all chondrocytes.
18
Collectively, these data suggest a possible mechanism
for RBPjk-dependent Notch signaling regulation of chon-
drocyte hypertrophy via Sox9 regulation.
SOX9 IHC, E14.5 Humerus
Control
Prx1 Cre;Rbpjk
f/f
a
b
Figure 1. Loss of RBPjk leads to inappropriate expression of SOX9 in
hypertrophic chondrocytes. Immunohistochemistry for SOX9 in control
(a) and Prx1Cre;Rbpjk
f/f
(b) E14.5 humerus sections.
Notch regulates Sox9 via secondary effectors
A Kohn et al
3
ß 2015 Sichuan University Bone Research (2015) 15021
Sox9 haploinsufficiency rescues delays in onset of
chondrocyte hypertrophy due to loss of Rbpjk
To begin to understand the genetic interactions between
RBPjk-dependent Notch signaling and Sox9, we removed
asinglecopyofSox9 in the background of Rbpjk mutants.
For completeness, the breeding strategy allowed for the
generation of the following genotypes: Cre-negative con-
trols, Rbpjk heterozygous (Prx1Cre;Rbpjk
f/1
), double hetero-
zygous (Prx1Cre;Rbpjk
f/1
;Sox9
f/1
), Rbpjk mutants (Prx1Cre;
Rbpjk
f/f
), and Rbpjk mutant/Sox9 haploinsufficient (Prx1Cre;
Rbpjk
f/f
;Sox9
f/1
)embryos.
Histological examination of E14.5 embryos with ABH/OG
staining (Figure 2) revealed the expected delay in onset of
hypertrophy in Rbpjk mutant embryos, as indicated by the
shorter hypertrophic zone (Figure 2a2) compared to litter-
mate controls (Figure 2a1). Interestingly, Rbpjk mutant/
Sox9 haploinsufficient embryos (Figure 2a4) revealed a
longer hypertrophic zone compared to controls, indi-
cating acceleration in chondrocyte hypertrophy. The
longer hypertrophic zone was also seen in double hetero-
zygous embryos (Figure 2a3), suggesting that Sox9 hap-
loinsufficiency is sufficient to accelerate chondrocyte
hypertrophic differentiation. To ensure that the acceler-
ated hypertrophic phenotype was due to Sox9 haploinsuf-
ficiency and not a result of double heterozygosity of Rbpjk
and Sox9, we examined Sox9 heterozygous embryos
(Prx1Cre;Sox9
f/1
). At E14.5, loss of a single allele of Sox9
results in a longer hypertrophic zone compared to litter-
mate controls (Figure 2a5 and a6). Finally, quantitative
analysis of the hypertrophic zone revealed a significantly
Length of hypertrophic zone Length of hypertrophic zone
b1
b2
Prx1Cre Prx1Cre
Control Rbpjk
f/f
Rbpjk
f/+
; Sox9
f/+
Rbpjk
f/f
; Sox9
f/+
Control
Sox9
f/+
E 14.5 Humerus
a1
a2
a3
a6a5
a4
Percent of total length
40
30
20
10
0
WT Rbpjk
Dbl Het Rbpjk; Sox9
*
*
*
*
*
Sox9 Het
Percent of total length
40
30
20
10
0
WT
*
Figure 2. Notch regulates the onset of chondrocyte hypertrophy via Sox9.(a) Histological analyses of control (a1), Prx1Cre;Rbpjk
f/f
(a2 ),
Prx1Cre;Rbpjk
f/1
;Sox9
f/1
(a3), Prx1Cre;Rbpjk
f/f
;Sox9
f/1
(a4), as well as, control (a5)andPrx1Cre;Sox9
f/1
embryonic tibia sections at E14.5. (b)
Quantification of the lengths of the hypertrophic zone, expressed as a percentage of the total length of the element. (b1)WT– wild-type, Rbpjk
(Prx1Cre;Rbpjk
f/f
), Dbl Het (Prx1Cre;Rbpjk
f/1
;Sox9
f/1
), RBPJk; Sox9 (Prx1Cre;Rbpjk
f/f
;Sox9
f/1
). (b2)WT– wild-type, Sox9 Het (Prx1Cre;Sox9
f/1
).
Notch regulates Sox9 via secondary effectors
A Kohn et al
4
Bone Research (2015) 15021 ß 2015 Sichuan University
shorter hypertrophic zone in Rbpjk mutants compared to
controls, while the Rbpjk mutant/Sox9 haploinsufficient
hypertrophic zones were significantly longer than both
controls and Rbpjk mutants and were similar to Rbpjk/
Sox9 double heterozygous mice (Figure 2b1). Removal of
a single Rbpjk allele in the Sox9 haploinsufficient back-
ground had no significant effect on the Sox9 heterozygous
change in hypertrophy (Figure 2b1 and b2). Similar histo-
logical changes in the onset of chondrocyte hypertrophy
were observed in all elements examined from both fore-
limb and hindlimb (data not shown).
Cartilage elements of the limb skeleton were further
analyzed for common markers of chondrocyte hyper-
trophy using in situ hybridization (Figure 3). As observed in
Figure 2, histological analysis by ABH/OG staining
revealed a smaller hypertrophic zone in Rbpjk mutant
embryos, but a larger hypertrophic zone in Rbpjk
mutant/Sox9 haploinsufficient embryos (Figure 3a–c). As
we previously reported,
20
molecular analysis using in situ
hybridization revealed significantly smaller Ihh and
Col10a1 domains in Rbpjk mutant embryos compared to
controls (Figure 3d, e, g, h). Furthermore, molecular ana-
lysis for Mmp13 showed a small number of cells expressing
Mmp13 in the control sections (Figure 3j, orange circle),
while no Mmp13 expressing cells are detected in Rbp jk
mutant sections (Figure 3k). Conversely, in situ hybridiza-
tion for Ihh and Col10a1 reveals larger expression domains
in Rbpjk mutant/Sox9 haploinsufficient embryos, and a
wide domain of Mmp13 expressing cells (Figure 3f, i, l).
These data indicate that during chondrocyte hyper-
trophy, Sox9 is likely downstream of RBPjk-dependent
Notch signaling such that a reduction in Sox9 expression
in Rbpjk mutants largely corrects or over-corrects the
delay in chondrocyte hypertrophy.
Control Rbpjk
f/f
Rbpjk
f/f
; Sox9
f/+
Prx1Cre
E 14.5 Humerus
Mmp13
Col10a1
Ihh ABH/OG
abc
de f
gh i
jkl
Figure 3. Notch regulates the onset of chondrocyte hypertrophy via Sox9. Histological and molecular analysis of control, Prx1Cre;Rbpjk
f/f
and
Prx1Cre;Rbpjk
f/f
;Sox9
f/1
embryonic tibia sections at E14.5. ABH/OG staining (a, b, c). In situ hybridization for markers of chondrocyte maturation –
Indian Hedgehog (Ihh)(d, e, f), Collagen 10a1 (Col10a1)(g, h, i)andMatrix Metaloproteinase 13 (Mmp13)(j, k, l). Yellow arrowheads indicate primary Ihh
expressing domains. Yellow double-headed arrows indicate Col10a1 expression domain. Orange circle highlights Mmp13 expression.
Notch regulates Sox9 via secondary effectors
A Kohn et al
5
ß 2015 Sichuan University Bone Research (2015) 15021
Cartilage element shortening due to Sox9
haploinsufficiency is rescued by loss of Rbpjk
Examination of ABH/OG stained full-length tibia sections
(Figure 4a) revealed that skeletal elements of the
Rbpjk mutants (Figure 4a2), the double heterozygous
(Figure 4a3) and the Rbpjk mutant/Sox9 haploinsufficient
embryos (Figure 4a4) were all significantly smaller than lit-
termate controls and displayed bowed skeletal elements
(Figure 4a1). Double heterozygous mutants displayed the
shortest skeletal elements, which were comparable to
Sox9 heterozygous mutant embryos (Prx1Cre;Sox9
f/1
)in
both length and curvature of the elements (Figure 4a3
and 4b2). Quantification revealed that the tibias of all three
mutants were significantly shorter than controls, and that the
double heterozygous were significantly shorter than Rbpjk
mutants or the Rbpjk mutant/Sox9 haploinsufficient embryos
(Figure 4c). Interestingly, removal of both Rbpjk floxed alleles
in the Sox9 haploinsufficient background restores tibia length
equivalent to the Rbpjk mutant size, although does not cor-
rect the curvature or bowing of the Sox9 heterozygous or
double heterozygous mutants. Furthermore, BrdU analyses
showed no significant difference in chondrocyte prolifera-
tion between all mutant genotypes (data not shown), sug-
gesting the rescued length in Rbpjk mutant/Sox9 haploin-
sufficient embryos as compared to double heterozygous or
Sox9 heterozygous mutants is likely due to alterations in
hypertrophic differentiation. Collectively, these data sug-
gest that complete removal of Rbpjk-dependent Notch
signaling elevates Sox9 expression to a level that can
counteract some of the chondrogenic effects oberserved
in Sox9 happloinsuficient mutant mice.
Sox9 haploinsufficiency does not rescue delays in
chondrocyte terminal hypertrophy due to loss of Rbpjk
We further analyzed limbs from E18.5 embryos in a similar
manner to those from E14.5. ABH/OG staining of proximal
tibia growth plates revealed the long hypertrophic zone
characteristic of the delayed terminal chondrocyte
hypertrophy observed in Rbpjk mutants, compared to
controls (Figure 5a1 and a2). Interestingly, the double het-
erozygous embryos appeared to have relatively normal
growth plates without any significant change in length of
the hypertrophic zone, however, the overall size of the
cartilage growth plate was slightly smaller than controls
(Figure 5a3). Interestingly, the growth plate and hyper-
trophic zone of the Rbpjk mutant/Sox9 haploinsufficient
embryos phenocopied that of Rbpjk mutants, specifically
in regard to the expanded hypertrophic zone (Figure 5a2
and a4). In situ hybridization for chondrocyte hypertrophy
markers, Ihh, Col10a1, and Mmp13, was performed on
E18.5 tibia sections. Analysis of the proximal growth plate
revealed expanded domains of Ihh, Col10a1, and Mmp13
in both the Rbpjk mutants and Rbpjk mutant/Sox9 haploin-
sufficient embryos compared to controls, while the
double heterozygous embryos reveal domains similar in
size to controls with a mild enhancement in Mmp13
(Figure 5a4–a12). Consistent with the results observed in
Figure 1, IHC results demonstrate that SOX9 protein persists
deeper into the hypertrophic zone of Rbpjk mutants due to
the enhanced Sox9 expression as compared to controls
(Figure 5b1 and b2). Interestingly, while SOX9 persistence
within the hypertrophic chondrocytes was reduced in
Rbpjk mutant/
Sox9 haploinsufficient embryos compared
to Rbpjk mutants, the progression through terminal chon-
drocyte hypertrophy was still delayed (identified by the
expanded hypertrophic zones) (Figure 5b2 and b3).
These data indicate that RBPjk-dependent Notch signal-
ing regulation of terminal chondrocyte hypertrophy and
cartilage matrix turnover may function independent of
Sox9 transcriptional control.
Notch mediated suppression of Sox9 requires
secondary effectors
The Notch signaling pathway has been shown to be an
important regulator of Sox9, although the mechanism of this
regulation is controversial. Here, we performed a rigorous
analysis of the known Sox9 promoter and Sox9 gene express-
ion in the context of Notch signaling activation. To achieve
this, we first utilized Sox9 luciferase constructs containing vari-
ous sizes of the Sox9 promoter. As shown in Figure 6a, the only
construct containing RBPjk consensus sequences is the 6.8 kb
known promoter fragment. Using ATDC5 cells, we co-trans-
fected the 6.8 kb (6.8 kb WT) construct with either Flag (con-
trol) or NICD1 over-expression plasmids. As expected, the
over-expression of NICD1 suppressed Sox9 luciferase activity
(Figure 6b). According to Chen et al., the RBPjk consensus
sequence, located 3 kb upstream of the transcriptional start
site, is the site significantly enriched of the NICD/RBPjk
complex.
28
To determine if this site is responsible for the sup-
pression of Sox9, we made point mutations designed to
prevent the binding of RBPjk to its consensus sequence
(TGGGAA to TCCGAA).
29
After co-transfecting the 6.8 kb
mutant (6.8 kb MT) construct with Flag or NICD1, we
observed suppression of Sox9 luciferase activity despite the
RBPjk mutation (Figure 6b). To rule out the possibility that
NICD may be binding to another RBPjk site in the 6.8 kb pro-
moter region, we utilized a Sox9 luciferase construct contain-
ing only 1 kb of promoter sequence upstream of the
transcriptional start site, which does not contain any RBPjk
binding sites. We co-transfected the 1 kb Sox9 construct with
Flag or NICD1, and observed a similar level of suppression as
seen with the 6.8 kb construct (Figure 6c). Interestingly, most
or all of the Notch-mediated suppression of Sox9 luciferase
activity was lost when the promoter fragments were
reduced to contain only 0.5 kb to 0.32 kb of the Sox9
promoter (Figure 6c). These data provide evidence that
Notch regulates Sox9 via secondary effectors
A Kohn et al
6
Bone Research (2015) 15021 ß 2015 Sichuan University
Percent change
Compared to Control
c
Control
Rbpjk
f/f
Rbpjk
f/+
;Sox9
f/+
Rbpjk
f/f
;Sox9
f/+
Pex1Cre
E18.5 Tibias
a1 a2 a3 a4
E18.5 Tibias
Control Prx1Cre;Sox9
f/+
b1 b2
100
90
80
70
60
Control
Rbpjk
Double het
Rbpjk; Sox9
*
*
*
*
*
Figure 4. Cartilage element length reduction due to Sox9 haploinsufficiency is rescued by loss of RBPjk-dependent Notch signaling. (a) ABH/OG
staining of control (a1), Prx1Cre;Rbpjk
f/f
(a2), Prx1Cre;Rbpjk
f/1
;Sox9
f/1
(a3) and Prx1Cre;Rbpjk
f/f
;Sox9
f/1
(a4) E18.5 full length tibia sections. (b) ABH/OG
staining of control (b1) and Prx1Cre;Sox9
f/1
(b2) E18.5 tibia sections. (c) Quantification of the size difference of E18.5 tibia sections.
Notch regulates Sox9 via secondary effectors
A Kohn et al
7
ß 2015 Sichuan University Bone Research (2015) 15021
the NICD1 suppression of Sox9 likely occurs via secondary
effectors and does not utilize the RBPjk binding sites in a
suppressive manner.
To elucidate the mechanism of the Notch suppression of
Sox9, we used a bioinformatic approach to analyze the 1
kb upstream fragment (TRANSFAC; Biobase). Interestingly,
we located a conserved N-box consensus sequence
(CACCAG) (Figure 6a) at 2681 to 2676. The N-box is one
of two known binding sites of the HES/HEY family of tran-
scription factors. HES/HEY factors are well characterized
as downstream Notch target genes. Upon binding to the
N-box, HES/HEY factors will recruit co-repressors inhibiting
gene transcription.
30
Additionally, it has been shown in
the developing limb that Notch signaling can induce Hes/
Hey gene expression.
17
Interestingly, the 0.5 kb and 0.32 kb
Sox9 luciferase constructs utilized in Figure 6c both lack the
identified N-box. These constructs are deficient in suppres-
sing Sox9 luciferase expression suggesting that Notch sup-
pression of Sox9 is mediated via secondary effectors that
likely include one or more of the HES/HEY factors.
Control
a1
a2 a3 a4
a5
a6
a7
a8
a9
a10
a11
a12
a
Prx1Cre
Rbpjk
f/f
Rbpjk
f/f
;Sox9
f/+
Control
Prx1Cre
Rbpjk
f/f
Rbpjk
f/f
;Sox9
f/+
b
SOX9 IHC,E18.5 Tibias
Control
Prx1Cre
Rbpjk
f/f
Rbpjk
f/f
;Sox9
f/+
b1
b2
b3
Figure 5. Notch regulation of terminal chondrocyte maturation is not likely to be mediated via Sox9.(a) Histological and molecular analysis of control,
Prx1Cre;RBPjk
f/f
, Prx1Cre;RBPjk
f/f
;Sox9
f/1
embryonic tibia sections at E18.5. ABH/OG staining (a1–a3). Markers of chondrocyte maturation – Indian
Hedgehog (Ihh)(a4–a6), Collagen 10a1 (Col10a1)(a7–a9) and Matrix Metaloproteinase 13 (Mmp13)(a10–a12) were analyzed by in situ hybridization. (b)
Immunohistochemistry for SOX9 in control (b1), Prx1Cre;RBPjk
f/f
(b2), and Prx1Cre;RBPjk
f/f
;Sox9
f/1
(b3) embryonic tibia sections at E18.5. Yellow
double-headed arrows indicate the length of the SOX9 expressing hypertrophic chondrocyte domain. Red double-headed arrows indicate the total
length of the hypertrophic zone.
Notch regulates Sox9 via secondary effectors
A Kohn et al
8
Bone Research (2015) 15021 ß 2015 Sichuan University
–1.0 kb –0.5 kb –0.32 kb
1.5
1.0
0.5
0
1.5
1.0
0.5
0
WT
MT
Sox9 promoter luciferase
Sox9 promoter luciferase
a
b
c
**
Flag
NICD
Flag
NICD
*
*
*
–6.8 kb WT
–6.8 kb MT
TGGGAA
TCCGAA
CACCAG
CACCAG
CACCAG
–1.0 kb
–0.5 kb
–0.32 kb
Luc
Luc
Luc
Luc
Luc
IgG
JAG1
Hes1 – cycloheximide d2d1
d3
d5
d4
d6
Hes1 + cycloheximide
150
100
50
20
10
0
2 000
1 500
1 000
100
0
0 h 2 h 4 h 8 h
0 h 2 h 4 h 8 h
0 h 2 h 4 h 8 h 0 h 2 h 4 h 8 h
0 h 2 h 4 h 8 h 0 h 2 h 4 h 8 h
8
6
4
2
0
8
6
4
2
0
8
6
4
2
0
8
6
4
2
0
10
*
*
*
Hes1 – cycloheximide
*
*
*
Sox9 – cycloheximide
*
*
*
Hes1 + cycloheximide
*
*
*
*
*
*
*
Sox9 + cycloheximide
d
Figure 6. Notch signaling inhibits Sox9 gene expression via secondary effectors. ( a) Diagram for the localization of a RBPjk consensus binding site
(yellow box is wild-type sequence and blue box is mutant sequence) and N-box consensus binding site (red box) in the Sox9 promoter and luciferase
constructs. Core consensus sequences are listed in diagram. (b) ATDC5 cells were co-transfected with Flag or NICD1 over-expression vectors and either
a wild-type 6.8 kb Sox9-Luciferase complex (26.8 kb WT) or 6.8 kb construct with a mutated RBPjk binding site (26.8 kb MT). Luciferase levels were
measured 24 hours after transfection. (c) ATDC5 cells were co-transfected with Flag or NICD1 over-expression vectors and either a 1 kb, 0.5 kb, or 0.32
kb Sox9-Luciferase deletion constructs. Luciferase levels were measured 24 hours after transfection. (d) Quantitative RT-PCR assessing Hes1 (d1, d2),
Hey1 (d3, d4), and Sox9 (d5, d6) gene expression in ATDC5 cells at 0-, 2-, 4-, and 8-hour post-culture on IgG versus JAG1 coated plates in the absence
(d1, d3, d5) or presence (d2, d4, d6) of cycloheximide.
Notch regulates Sox9 via secondary effectors
A Kohn et al
9
ß 2015 Sichuan University Bone Research (2015) 15021
To further determine whether Notch inhibits Sox9
expression directly or via downstream target gene activa-
tion, we initiated Notch signaling in ATDC5 cells using
Jagged1 (JAG1) coated plates and assessed Notch-
induced gene expression (Figure 6d). Cultures were main-
tained in the presence or absence of the protein synthesis
inhibitor, cycloheximide, in order to assess whether trans-
lation of downstream Notch target genes is required for
Sox9 gene regulation. RNA was isolated from cultures at
0, 2, 4, and 8 hours of culture, and quantitative RT-PCR was
performed to measure levels of the established Notch tar-
get genes, Hes1 and Hey1,
31
as well as Sox9. The culture of
ATDC5 cells on JAG1 coated plates versus IgG coated
control plates leads to significant up-regulation of both
Hes1 and Hey1 gene expression across all time points
(Figure 6d1–d4). When we conducted this experiment in
the presence of cycloheximide (Figure 6d2 and d4), similar
levels of Hes1 and Hey1 up-regulation were observed indi-
cating that both Hes1 and Hey1 transcription are directly
activated by RBPjk-dependent Notch signaling, as has
been previously reported.
31
Interestingly, when we exam-
ine Sox9 transcriptional regulation in the absence of cyclo-
heximide, we see an initial mild up-regulation of Sox9 gene
expression at 2 hours, but then see a sustained inhibition of
Sox9 expression at 4 and 8 hours (Figure 6d5). When we
assess Notch signaling effects on Sox9 gene expression in
the presence of cycloheximide, we find the same but sig-
nificant mild up-regulation of Sox9 at 2 hours. However, the
inhibition of Sox9 gene expression seen in the absence of
cycloheximide at 4 and 8 hours (Figure 6d5) is lost when
ATDC5 cells are cultured in the presence of cycloheximide
and JAG1 activation (Figure 6d6). These results indicate
that Notch signaling can initially promote Sox9 gene
expression directly through the NICD/RBPjk transcriptional
activating complex, but the inhibition of Sox9 requires the
translation of Notch-dependent target genes.
DISCUSSION
Our previous work identified RBPjk-dependent Notch signal-
ing as a necessary regulator of both the onset of chondro-
cyte hypertrophy and the progression to terminal
chondrocyte maturation, although the mechanism remains
unknown.
20
Here we have provided conclusive genetic
evidence linking RBPjk-dependent Notch signaling to the
regulation of Sox9 expression and chondrocyte hyper-
trophy. In Rbpjk mutant embryos, haploinsufficiency of
Sox9 was able to rescue the delays in onset of chondrocyte
hypertrophy, but had little if any impact on delayed progres-
sion to terminal chondrocyte maturation and cartilage
matrix turnover. Interestingly, limb length reductions char-
acteristic of Sox9 haploinsufficiency were partially resolved
when RBPjk-dependent Notch signaling was completely
removed, suggesting that a delicate regulation and
balance of Sox9 expression is required to coordinate chon-
drocyte hypertrophy and cartilage growth. Furthermore, our
in vitro results demonstrate the ability of Notch signaling to
acutely enhance Sox9 expression likely through direct RBPjk-
dependent regulation, while continuous Notch signaling
suppresses Sox9 via secondary effectors.
SOX9 is well established as the master regulator of chon-
drogenesis and a factor required for the maintenance of the
immature chondrocyte phenotype,
5–7,32–3 3
and thus a
potential target of RBPjk-dependent Notch function in car-
tilage development. Our work, as well as others, has shown
that Sox9 is normally down-regulated in hypertrophic chon-
drocytes,
27
indicating that Sox9 down-regulation is required
for the onset of hypertrophy. A recent study has specifically
addressed this by using a BAC-Col10a1 promoter to drive
continuous expression of Sox9 in hypertrophic chondro-
cytes.
9
Maintenance of SOX9 within hypertrophic chondro-
cytes results in an elongated zone of hypertrophic
chondrocytes at E18.5, strikingly similar to the elongated
hypertrophic zone caused by the delayed progression to
terminal chondrocyte maturation observed in Notch mutant
embryos.
19–20
While Hattori et al.
9
did not exam ine the onset
of chondrocyte hypertrophy, at E14.5, using their Sox9 over-
expressing mutants; our Prx1Cre transgene induced Sox9
heterozygous mutant mice demonstrate accelerated
hypertrophy via the down-regulation of Sox9. Therefore,
our data are consistent with a specific role for SOX9 in regu-
lating the onset of chondrocyte hypertrophy. Based on
these data and our understanding of Notch signaling, we
explored the genetic relationship between Notch signaling
and Sox9 in the context of chondrocyte hypertrophy. While
the delayed onset of chondrocyte hypertorphy due to loss of
RBPjk could be fully rescued by the removal of a single allele
of Sox9, the same was not true concerning delayed terminal
chondrocyte maturation. As we have suggested, it is there-
fore not likely that Sox9 is involved in RBPjk-dependent Notch
regulation of terminal chondrocy te hypertrophy, however,
we cannot rule out the possibility that removal of a single
allele of Sox9 was not significant enough of a knock-down
to effect the terminal chondrocyte maturation phenotype.
In our current genetic model utilizing the Prx1Cre, removal of
both Sox9 alleles was impossible, as SOX9 is required for con-
densation formation and early chondrogenesis.
6
Our data demonstrate that haploinsufficiency of Sox9
results in significant shortening and bowing of the endo-
chondral elements. These data are consistent with prev-
iously reported Sox9 mutants, including Prx1Cre;Sox9
fx/1
embryos analyzed by Akiyama et al., which displayed
hypoplasia and bowing of various endochondral ele-
ments.
6
Global haploinsufficiency of Sox9 results in
more severe limb element hypoplasia and bowing as
compared to the Prx1Cre targeted Sox9 mutants.
5
Interestingly, at E18.5, Sox9 global heterozygous embryos
Notch regulates Sox9 via secondary effectors
A Kohn et al
10
Bone Research (2015) 15021 ß 2015 Sichuan University
display an approximately two-fold increase in the length
of their hypertrophic zone. This expansion is likely due to
early onset of hypertrophy, as opposed to the delays in
terminal chondrocyte hypertrophy observed in Notch
loss-of-function mutants.
5,18–20
An extension of the hyper-
trophic zone due to early onset in chondrocyte hyper-
trophy has been previously reported in b-catenin GOF
embryos,
34
which has also been shown to down-regulate
SOX9 during the transition to chondrocyte hypertrophy by
specifically targeting SOX9 protein for proteosomal degra-
dation.
35
Here, we demonstrated that complete removal
of RBPjk-dependent Notch signaling in the Sox9 haploin-
sufficient background was enough to restore some Sox9
expression and promote cartilage growth similar to that
observed in Rbpjk mutants, but not enough to correct
the bowing observed in all Sox9 haploinsufficient mutants.
In recent work using the Col2Cre transgene to study
RBPjk-dependent and –independent Notch signaling
effects on vertebral formation, Chen et al. proposed a
model whereby RBPjk-dependent Notch signaling sup-
pression of Sox9 is important for normal axial skeletogen-
esis.
36
Controversially, they proposed a mechanism by
which the NICD-RBPjk complex directly inhibits Sox9 gene
expression via direct binding to the Sox9 promoter. While
provocative, there is no evidence to suggest NICD-RBPjk
complexes can function as transcriptional repressors since
NICD recruitment to RBPjk and DNA is known to displace
transcriptional repressors ultimately leading to transcrip-
tional activation. Chen et al. provided chromatin immu-
noprecipitation (ChIP) assays detecting the recruitment of
the NICD-RBPjk complex to RBPjk binding sites in the Sox9
promoter as evidence of their claim. While it is possible and
probable that NICD-RBPjk complexes bind to the Sox9 pro-
moter, it is more likely that NICD-RBPjk may have a physio-
logical role in directly promoting Sox9 expression in specific
contexts. This is supported by data that demonstrate the
ability of Notch to induce Sox9 gene expression in embry-
onic stem cells, pancreatic cells, and Mu¨ller glial cells.
37–39
Furthermore, the work presented here in chondrogenic
cells demonstrates that site specific mutations of the
RBPjk binding site (proposed by Chen et al. as the repress-
ive element) does not yield any change in Notch-
mediated suppression of Sox9. Alternatively, we propose
that Notch signaling leads to an indirect suppression of
Sox9 via the induction of alternative downstream targets.
This is also supported by our own data, which demon-
strates that Notch-induced inhibition of Sox9 in chondro-
genic cells requires active protein synthesis. We therefore
hypothesize that one or more of the HES/HEY family of
repressive bHLH transcription factors, some of the primary
downstream targets of RBPjk-dependent Notch signaling,
act as mediators of Notch signaling to repress Sox9 gene
expression. This is further supported by the loss of Notch-
mediated suppression of Sox9 driven luciferase activity
when the Sox9 promoter was truncated to exclude a
putative HES/HEY binding site. Further studies will be
required to elucidate the exact role HES/HEY factors may
play in specifically regulating Sox9 expression and cartil-
age development.
AUTHORS’ CONTRIBUTION
Authors’ roles: Study design: Matthew J. Hilton. Study con-
duct: Anat Kohn, Timothy P. Rutkowski, Zhaoyang Liu, and
Anthony J. Mirando. Data collection: Anat Kohn, Timothy P.
Rutkowski, and Zhaoyang Liu. Data analysis: Anat Kohn,
Timothy P. Rutkowski, and Zhaoyang Liu. Data interpretation:
AnatKohn,TimothyP.Rutkowski,ZhaoyangLiu,RegisJ.
O’Keefe, Matthew J. Hilton, and Michael J. Zuscik. Drafting
manuscript: Anat Kohn, Timothy P. Rutkowski, Zhaoyang Liu,
Regis J. O’Keefe, Matthew J. Hilton, and Michael J. Zuscik.
Approving final version of manuscript: Anat Kohn, Timothy P.
Rutkowski, Zhaoyang Liu, Regis J. O’Keefe, Matthew J. Hilton,
and Michael J. Zuscik. Anat Kohn, Timothy P. Rutkowski,
Zhaoyang Liu, and Matthew J. Hilton take responsibility for
the integrity of the data analysis.
Competing Interests
The authors declare no conflict of interest.
Acknowledgements
This work was supported in part by the following United States National
Institute of Health grants: R01 grants (AR057022 and AR063071), R21 grant
(AR059733 to MJH), a P30 Core Center grant (AR061307), and a T32 training
grant that supported both AK and TPR (AR053459 to Regis J. O’Keefe and
Michael J. Zuscik). The NICD and FLAG control plasmids were a gift from Dr.
Raphael Kopan (Cincinnati Children’s Hospital ) and the 6.8 kb Sox9-
Luciferase plasmid was a kind gift from Dr. Peter Koopman (University of
Queensland). We would like to gratefully acknowledge the technical
expertise and assistance of Sarah Mack, Kathy Maltby, and Ashish Thomas
within the Histology, Biochemistry, and Molecular Imaging Core in t he
Center for Musculoskeletal Research at the University of Rochester Medical
Center.
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