Download Paper No. 156 • 54th Annual Meeting of the Orthopaedic Research

Beta-Catenin’s Role in the Mechanosensitivity of Osteochondroprogenitor Cells
Thomas D. Falls1, Radhika Atit3, Eric J. Anderson2, Melissa L. Knothe Tate1,2
of Biomedical Engineering, Case Western Reserve University, Cleveland, OH; 2Department of Mechanical & Aerospace Engineering,
Case Western Reserve University, Cleveland, OH; 3Department of Biology, Case Western Reserve University, Cleveland, OH
[email protected]
1Department
Introduction: The role of biochemical signals in determining cell fate during
embryonic development has been documented, but neither the mechanical signals
present in utero nor the specific signals needed to guide progenitor cells toward a
specific lineage are currently known [1]. This study examined the effects of fluid
flow shear stress on embryonic osteochondroprogenitor cells lacking β-catenin,
which has recently been found to play a key role in cell-cell adhesion and mechanotransduction. We hypothesized that fluid flow induced shear stress modulates the
fate of osteochondroprogenitor cells in the presence and absence of β-catenin.
Our approach was to investigate transcription activity associated with
osteogenic, chondrogenic, and adipogenic fate in response to application of controlled shear stress via fluid flow in the absence of biochemical signals and to determine whether transcription activity changes in equivalent subsets of primary mesenchymal stem cells derived from conditional β-catenin knockouts. Thereafter, we
developed an idealized model to investigate the effect of cell spacing on the effective stress imparted to individual cells to determine the feasibility of our working
hypothesis. Specifically, we aimed to:
i. identify the population of osteochondroprogenitor cells in the mesodermal
core of E11.5 mouse embryo forelimbs using histology as a first step toward our
goal to use the identified cells for fluid flow shear stress studies,
ii. determine the effects of β-catenin on the mechanosensitivity of osteochondroprogenitor cells exposed to fluid flow shear stress in a parallel plate flow chamber,
iii. model the effects of cells acting as stress concentrators, in the absence of
adherens junctions, using computational fluid dynamics.
Materials and Methods: Forelimbs of E11.5 Dermo1-Cre;R26R-GFP
embryos were removed, fixed in 4% cold PFA, cryosectioned, stained with antibodies against GFP and either Sox9 or Runx2, and counterstained with DAPI.
The same procedure was used for cells cultured from the mesoderm of the forelimbs. The mesodermal core was dissected from forelimbs of E11.5 Dermo1Cre;β-catenin^c/c embryos [3], placed in 0.25% trypsin EDTA overnight, and
then incubated at 37 C. BGJb growth media (+10% FBS, 2% antibiotic) was used
to culture the cells. Cells were seeded onto treated glass coverslips and media was
flowed over the cells at 1 dyn/cm^2 for 30 minutes. After flow, total RNA was
immediately isolated from the cells, and real-time PCR was performed for markers including types I & 2 collagen, Runx2, Sox9, Osx, Msx2, Aggrecan, and PParγ. TBP served as the endogenous control. A previously described Computational
Fluid Dynamics (CFD) model [2] was adapted to evaluate stress concentration on
individual cells protruding from the lower surface of the flow chamber. The fluid
flow induced shear stress around and over the surface of the cells was calculated
statically as a measure of shear stress distributions over the cells at a given point in
time (no temporal component, no cell remodeling). Using the computational
model, the cells were deformed by 5, 10, 20 and 50% to mimic deformation under
shear, which flattened the cell surface and caused the edges to bulge outwards. In
addition, the relationship between cell spacing and imparted shear stress to the cell
surface was investigated by comparing stresses between the leading and the trailing
cell for both the full- and half-cell length spacings.
Results: The tissue specific gene targeting eliminated β-catenin expression,
rendering that tissue unable to transduce wnt signaling, and resulting in osteochondroprogenitor cells lacking β-catenin to stabilize n-cadherin based cell-cell
junctions. Based on immunohistochemistry of the forelimbs of Dermo1-Cre;
R26R-GFP mice, ~59% of the Dermo1 positive cells also expressed Sox9 throughout the mesodermal core. In addition, circa 56% of the Dermo1 expressing cells
also expressed Runx2, but in a much smaller, proximally located domain.
Immunocytochemistry of primary cells isolated and cultured (P1) from the mesodermal core corroborated immunohistochemistry-based data on entire isolated
limbs; the majority of the cells co-expressed Sox9, but a minority of cells coexpressed Runx2. By the second subculture (P2), however, the majority of Dermo1
positive cells were co-expressing Runx2 as well. Hence, we inferred that the correct
population of β-catenin^c/c cells could be identified and isolated for the
mechanosensitivity studies.
Exposure of primary mesodermal cells dissociated from the murine limbbud at
E11.5 to 1 dyn/cm^2 shear stress via fluid flow significantly upregulated Col1a1
transcription in the cells lacking β-catenin and downregulated transcription in cells
not lacking β-catenin. Transcription of Sox9, Runx2, Osx, AGC, and Ppar-γ was
not significantly affected by exposure to shear stress. Previous studies show that
cells lacking β-catenin do not reassociate in culture to the same degree as normal
cells after dissociation from the mesoderm. Using computer models, we demonstrated that isolated cells (lacking β-catenin) would be exposed to higher levels of
shear stress than reassociated normal cells.
Discussion: The experimental and predictive computational data show, for the
first time to our knowledge, that gene transcription activity of primary embryonic
mesenchymal cells can be modulated by mechanical cues even in the absence of βcatenin, a protein that links cadherins to the cytoskeleton. As the cells were derived
from the mesoderm of E11.5 mice prior to lineage commitment and the cells were
exposed to shear stress for 30 minutes, it is premature to refer to the change in transcription activity as indicative of fate modulation. Furthermore, dissociation of the
cells and subsequent culture changed the developmental context of the cells.
Nonetheless, taken together with computational predictions, conditional β-catenin
mutant cells are exposed to higher effective fluid flow induced shear stress due to
loss of connection with adjacent cells. β-catenin mutant cells significantly upregulate the transcription of Type I collagen in response to flow induced shear, effectively increasing the resistance to flow in their immediate pericellular space and
thereby adapting their environment to dampen stress and reduce cell deformation.
In contrast, cells from the littermates downregulate the transcription of Type I collagen, which may act to increase the transduction of mechanical signals to the
newly formed semiconfluent sheet of cells originally dissociated from a three
dimensional loose mesenchymal structure. These studies provide a glimpse into the
role of mechanical cues in modulating gene transcription activity by pluripotent
cells at a time in de novo development before which cell fate has been determined.
In the future, they may provide a new basis to generate target tissue types by guiding cell lineage commitment through delivery of appropriate mechanical signals.
References: [1] Anderson, E.J. and M.L. Knothe Tate. Tissue Eng, 2007. [2]
Anderson, E.J. et al. Biomed Eng Online, 2006. [3] Brault, V. et al., Development,
2001.
Paper No. 156 • 54th Annual Meeting of the Orthopaedic Research Society