Mesenchymal Regulation of Osteogenic Differentiation During Development
Yu, J; Fish, J L; Ealba, E L; Jheon, A H; Butcher, K D; Eames B F; +Schneider, R A
+University of California, San Francisco, CA
[email protected]
INTRODUCTION
With the goal of devising new therapies for disease, birth defects, and
injuries to the skeleton, there is keen interest in understanding how
mesenchymal cells differentiate into osteocytes and ultimately make
bone. A wide range of in vitro and in vivo studies have revealed that
osteogenesis is a complex process involving numerous gene regulatory
networks, reciprocal signaling interactions, and multiple hierarchical
levels of control. Yet what remains unclear are precise mechanisms that
initiate and synchronize the progression of mesenchyme through each
discrete step of osteogenesis including induction, proliferation,
differentiation, osteoid deposition, and mineralization.
This is
significant since a major clinical objective is to engineer mesenchyme
for in vivo applications. To address this issue, we take a systems-level
strategy and experimentally manipulate neural crest mesenchyme
(NCM) destined to form all the bone in the jaw skeleton.
We have established a unique avian chimeric transplant system that
exploits the divergent maturation rates of quail and duck. By
transplanting faster-maturing quail NCM into slower-developing duck
(“quck”) or conversely, duck NCM into quail (“duail”), we can assess
the extent to which each step of osteogenesis is directed by donor NCM,
and more importantly, we can uncover and dissect apart potential
mechanisms. To do so, we assay for donor-induced changes to the
timing of histological events and screen for candidate genes that become
differentially expressed in quck. We then use gain- and loss-of-function
approaches to test if these genes account for an observed phenotype.
Previously we have shown that osteogenesis is significantly
accelerated in chimeric quck and delayed in duail. In both cases, NCM
shifts entire programs for the induction, differentiation, and
mineralization of bone. How NCM accomplishes such a complex task,
and what factors are sufficient to replicate this phenomenon is unknown.
We hypothesized that one likely candidate would be Runx2, since its
expression was altered in chimeras. In vitro, Runx2 can direct
mesenchymal cells down the osteoblast lineage, and trigger expression
of genes encoding major protein components of bone matrix, including
Col1. Moreover, binding of Runx2 to ribosomal DNA leads to direct
repression of ribosomal RNA synthesis to help modulate the switch from
proliferation to differentiation. Similarly, our experiments show that
Runx2 over-expression strongly enhances differentiation and
mineralization in both primary chick mesenchymal cell cultures and a
chick fibroblast cell line. In contrast to such in vitro data, we find that in
vivo over-expression of Runx2 is not sufficient to alter the timing of
either differentiation or mineralization. Thus, NCM must employ
additional molecular mechanisms to control osteogenesis.
METHODS
Eggs of Japanese quail (Coturnix coturnix japonica) and white Pekin
duck (Anas platyrhynchos) were incubated at 37°C until reaching
appropriate embryonic stages (HH). All embryos were handled in
accordance with University and NIH guidelines. Unilateral transplants of
NCM from the rostral mid- and hindbrain was performed in quail and
duck to generate chimeric quck, as described previously.
For over-expression experiments, RCAS-Runx2 virus was injected
into chick NCM at HH8. RCAS-GFP was used for controls. To
visualize mineralization, embryos were fixed in 10% formalin, stained
with alizarin red, and cleared in glycerol. To detect osteoid, samples
were fixed in 4% PFA, embedded, sectioned, and stained with
Milligan’s Trichrome. To measure proliferation, 1ul of BrdU was
injected into the vitelline vein of live embryos 20 minutes before
collection, and sections were stained using a BrdU Kit (Zymed). In situ
hybridization was performed for Runx2 and gag in the RCAS envelope.
In primary cell culture experiments, mesenchymal cells were
harvested from embryos infected with RCAS-Runx2, RCAS-GFP, or no
virus. Epithelium was removed after a brief incubation with 0.05%
trypsin in DMEM, and cells were dissociated. Cells were cultured in
parallel with DF-1 chick fibroblasts in DMEM with 10% FHS. To
initiate osteogenesis, cells we cultured in media supplemented with 15%
embryo juice for up to 72 hours. Osteogenesis was assessed via alizarin
red and qPCR, and proliferation was measured colorimetrically (Wst-1).
RESULTS
In chimeric quck, mineralization proceeds on the timetable of quail
donor NCM (3 stages earlier than in control duck). This premature
initiation of mineralization is readily apparent via alizarin red staining in
the dermal bones of the jaw. We also observe acceleration in osteoid
deposition, alkaline phosphatase activity (a marker for osteoblast
differentiation), and a premature decrease in proliferation (BrdU).
Furthermore, these changes correspond to early up-regulation of genes
encoding transcription factors essential for mineralization, such as
Runx2, Dlx5, Msx1, and Twist1.
Similarly, in vitro retroviral over-expression of Runx2, both in DF-1
cells (a chick fibroblast cell line), and in primary mesenchymal cells
isolated from chick mandibles, decreases proliferation (Wst-1), while
promoting differentiation and mineralization (as measured by Alizarin
Red staining). In contrast, high levels of Runx2 over-expression in vivo
(as visualized by in situ hybridization) were not sufficient to reproduce
the equivalent phenotype of early proliferation, osteoid deposition, or
mineralization as seen in quck. We did however, observe ectopic
mineralization in embryos treated with RCAS-Runx2.
DISCUSSION
Consistent with previous reports, we find using two approaches
(primary mesenchymal cells, and a chick fibroblast line) that Runx2
over-expression in vitro is able to decrease proliferation and increase
osteogenic differentiation. However, Runx2 over-expression in vivo
does not have the same effect and thus is not sufficient to account for the
ability of NCM to induce the changes to proliferation and mineralization
that we observe in chimeras. There are several interpretations as to why
our in vitro results differ from our in vivo results, particularly as they
relate to the complex local signaling environment. Moreover, NCM
may act through a number of these mechanisms to drive osteogenesis.
TGFβ and Erk MAPK Modulation of Runx2 activity
Many signaling pathways modulate the activity of Runx2 and may
attenuate the effects of its over-expression at the mRNA level. Posttranslationally, Runx2 proteins can be degraded by Smurfs (E3 ubiquitin
ligases), if they do not undergo p300-dependent acetylation as a
downstream action of TGFβ. In addition, Runx2 may need to be
phosphorylated by Erk and p38 MAPK and transactivated before
inducing osteoblast expression of genes involved in bone formation,
such as MMP13. TGFβ may also need to act in concert with Erk MAPK
signaling to activate AP-1 transcription complexes, which can then bind
and function as co-activators of Runx2. Additionally, high
concentrations of Runx2 can increase expression of TβRI, incurring a
regulatory feedback loop whereby TβRI recruits histone deacetylases to
act as transcriptional co-repressors at Runx2 target promoters, ultimately
limiting the progression of osteoblast differentiation. We are currently
exploring many of these possibilities in ongoing studies.
Signals promoting osteoblast differentiation after commitment
Runx2 is the first transcription factor required for determination of
the osteoblast lineage, while Sp7 (Osterix) and canonical Wnt-signaling
further direct the fate of mesenchymal cells to osteoblasts. Without coover-expression of these factors in vivo, mesenchymal cells may never
progress past the stage of immature osteoblasts. In future experiments,
we will test whether manipulating these molecules and/or TGFβ
signaling pathway members in conjunction with Runx2 over-expression
is sufficient alter timing of differentiation and mineralization in vivo.
Results from our studies will help elucidate molecular mechanisms
through which NCM controls osteogenesis and define developmental
periods when tissues are competent to respond to inductive signals,
which is a critical first step for designing novel therapeutic strategies.
ACKNOWLEDGEMENTS:
Supported by R01 DE016402 from the NIDCR and R21 AR052513
from the NIAMS to R.A.S; and CIHR postdoctoral fellowship to A.H.J.
Paper No. 236 • ORS 2011 Annual Meeting