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Osteogenic transcription regulated by exaggerated stretch ......antibody/2ndary antibody 594 –Red)...
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Osteogenic transcription regulated by exaggerated stretch loading via convergent wnt signaling Cassandra M. Juran 1,2 , Elizabeth A. Blaber 1 , Eduardo A.C. Almeida 1 1 Space Biosciences Division, NASA Ames Research Center, Moffett Field, CA 2 Universities Space Research Association, Mountain View, CA Results Methods We hypothesize stretch loading induces gap junction and wnt11 choreographed convergent wnt signaling which regulates a cascade of molecular events terminating in osteogenic gene transcription. Hypothesis Cell and animal studies conducted onboard the International Space Station and formerly the Shuttle flights have provided data illuminating the deleterious biological response of bone to mechanical unloading (Figure 1). Loss of bone mass and inherit microarchitecture is a feature similar to osteoarthritis, the causal mechanism of which has been highly researched. In Vivo down regulation of molecular intra- and inter-cellular signaling cascades has been demonstrated in osteoarthritis and unloading studies. Specific to osteocyte cells the canonical wnt and Connexin43 induced cAMP signaling cascades have been shown as critical regulators. However the intercellular communicative cues and mechanotranductive cascades responsible for osteogenic transcription and stem cell recruitment are still largely unknown. Figure 1. Proximal femur reconstruction from mice flown on 15-day STS-131 mission (B,D,F,) compared to ground controls (A,C,E). BV loss of 17% in flight samples. Background Results Conclusion Acknowledgements: This work is supported by NASA Space Biology Grant NNH14ZTT001N-0062 to E. Blaber and NNH14ZTT001N-003 to E. Almeida. Cellular Connectivity and Intercellular Regulation due to Exaggerated Mechanical Loading Proliferation and Cellular Metabolic Activity with Exaggerated Loading Osteogenic Signaling Expression and Localization after Exaggerated Loading Figure 5. Biostimulator CAD representation. MLOY4 osteocyte-like cells MC3T3-E1 osteoblast-like cells Figure 4. Experimental cell type morphology. MLOY4 represent critical dendritic cell processes inherent to functional osteocytes, while MC3T3-E1 pre- osteoblast cells present no such morphology. Figure 8. Calcein –AM live cell membrane stain and MatLAB automated dendritic process length measurement program and processing. Figure X. Hypothetic Feedback regulation of osteogenic transcription in MLOY4 osteocyte cells due to mechanical loading. This theoretic feedback loop implies the critical importance of coupled wnt signaling control of CX43 intercellular communication channel. Figure 7. Overview of convergent wnt signaling regulation of osteogenic gene transcription pathways in response to stretch loading. CX43 signaling is increased by stretch loading. Co-localization of CX43 and β-catenin is coupled with wnt11/PCP pathway release of Rac1. Nuclear translocation Nuclear factor activation Mechanotrans -ductive signaling Mechanotransductive signaling Nuclear factor activation Table 1: Quantitative Assessment of MLOY4 Dendritic Process Length and Termination Junctions Length (μm) Integer Representation of Shared Terminating Junctions per cell Control (No Stretch) 3.19 ± 2.13 3 48 hours (0.1% strain, 0.1 Hz frequency) 6.50 ± 1.58 7 CX43 w n t 1 w nt 3a w nt 5a w nt 11 -1 0 1 2 5 10 15 20 m RNA expression levels (Experimental/no stretch control fold change) -catenin Rac1 Jun MAPK1 MAPK3 0 2 4 10 15 20 m RNA expression levels (Experimental/no stretch control fold change) RANKL OPG SOST OSX ALPL -4 -2 0 5 10 15 20 m RNA expression levels (Experimental/no stretch control fold change) +9.23 -1.50 -4.32 +1.06 +5.00 -1.16 +11.13 +2.29 -0.01 +0.12 -3.83 +2.55 -2.99 -10.27 -16.58 +/- fold change compared to MC3T3-E1 stretch cultures • Canonical and non- canonical wnt pathways are upregulated during mechanical stimulation of osteocyte cells. • Up regulation of wnt11 is causally associated with Rac1 up regulation. • Rac1 association with β-catenin is in part responsible for nuclear translocation and osteogenic gene transcription. • CX43 and β-catenin co-localization is modulated by Rac1 presence • CX43 sequesters the Rac1/β-catenin complex outside the nuclear membrane. Figure 2. Bone mineral homeostasis is a balance between bone formation by osteoblasts and bone resorption by osteoclasts. Figure 3. Cellular response to mechanical stimulation is dependent on the mechanism of cell deformation. Of import in this study is strain applied through the cell attachments, in contrast to fluid flow studies which are regulated by membrane strains. A B C Figure 6: Florescence imaging demonstrating cell morphologies (actin – Green), connexin localization (CX43 primary antibody/2 nd ary antibody 594 – Red) and counterstained with DAPI (nuclear –Blue). Figure A) and insert are MLOY4 osteocyte cells after 48 hours of stretch culture, B) and insert are MC3T3-E1 osteoblast cells, and C) and insert are co- cultured MLOY4 and MC3T3-E1. A) illustrates typical MLOY4 morphology with extended cell processes along the direction of stretch (indicated by white arrow) and localized CX43 presence at the process peripheries and nuclear envelope. While in A) and C) CX43 is highly expressed at membrane interfaces in B) the extent of localization is non- specific in osteoblast cells. Osteoblast-Osteocyte co-cultured cells C) demonstrate both increased CX43 presence within the cell and membranous localization inherit to mechanistic communication both intercellular and intracellular. Nuclear translocation MLOY4 osteocyte-like and MC3T3-E1 osteoblast-like cells were cultured in a custom designed biostimulator (Figure 5) and allowed to acclimate for 48 hours before 48 hours of stretch loading. Stretch loading was imparted by 2 Arduino controlled linear drive motors set to 0.1% tensile strain and 0.1Hz cyclic application. Measure of CX43 localization, cell number, metabolism, and phenotypic expression were taken at 10 minutes, 2, 12, 24 and 48 hours. Culture conditions were maintained at 5% CO 2 37°C and 90% humidity. Culture media was supplemented by 1% anti-anti, 10% FBS and changed every 48 hours such as to not interrupt the stimulation regime. The self organization of osteocyte cells is a critical metric of the cell population function. MLOY4 cells cultured in unloaded conditions will form dense overlapping networks with short dendritic process lengths and little CX43 membrane localization. In vivo examination of osteocyte networks have been shown to be highly interconnected (Figure 3) and these connections are made between dendritic cell processes with lengths averaging 20-30μm. Within the osteon the cell processes are organized in the canaliculus network, the microarchitecture of which amplifies mechanical stress sensed by the processes which in turn lengthens the process. This supposition is supported by our measurement of MLOY4 dendritic cell process lengthening under stretch loading stimulation. Additionally, the shared terminating junctions were quantified and demonstrated greater MLOY4 population interconnectivity when cells were exposed to stretch loading. Figure 8. Analysis of MLOY4 viability during stretch stimulation. *p <0.05 Cell number and metabolic activity of cultured MLOY4 cells demonstrate cellular viability. Stretch stimulation has a negative correlation with proliferation compared to unloaded controls. Cellular metabolism initially increases due to new stress applied to the cells however after acclimation metabolism reaches steady state after 24 hours. Bone is a dynamic tissue undergoing constant remodeling and repair from stem cell precursors in the bone marrow (Figure 2). Osteocytes are believed to be responsible for the controlled regulation of cell activity in living bone. Thus how mechanical stimulation modulates biochemical activity of the osteocyte is a definitive factor in the study of bone biology and homeostasis maintenance. In this investigation, MLO-Y4 osteocyte-like and MC3T3-E1 osteoblast-like cells (control cell) were culture under dynamic tensile conditions and evaluated for expression of CX43 and wnt-signaling proteins influential in driving cell-cell communication as well as stem cell recruitment and differentiation. A significant feature of interest in mechanical regulation of bone biology is the mechanism of loading experienced by the cell modulates the cells reaction. Many of the previous osteocyte studies investigating response to loading have evaluated how the cells respond to fluid flow induced shear adjacent to cells in monolayer. This model however, is not representative of the critical osteocyte dendritic process activation within the canaliculus (Fig 3A). Thus our experiential design will utilize stretch loading, such that the cell process directly experience load to better represent the physiologic response of cells in vivo (Fig 3B). *
Transcript of Osteogenic transcription regulated by exaggerated stretch ......antibody/2ndary antibody 594 –Red)...
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Osteogenic transcription regulated by exaggerated stretch loading via convergent wnt signaling
Cassandra M. Juran1,2, Elizabeth A. Blaber1, Eduardo A.C. Almeida11Space Biosciences Division, NASA Ames Research Center, Moffett Field, CA
2Universities Space Research Association, Mountain View, CA
Results Methods
We hypothesize stretch loading induces gap junction and wnt11 choreographed convergent wnt signaling which regulates a cascade of molecular events terminating in osteogenic gene transcription.
Hypothesis
Cell and animal studies conducted onboard the International Space Station andformerly the Shuttle flights have provided data illuminating the deleterious biologicalresponse of bone to mechanical unloading (Figure 1). Loss of bone mass and inheritmicroarchitecture is a feature similar to osteoarthritis, the causal mechanism ofwhich has been highly researched. In Vivo down regulation of molecular intra- andinter-cellular signaling cascades has been demonstrated in osteoarthritis andunloading studies. Specific to osteocyte cells the canonical wnt and Connexin43induced cAMP signaling cascades have been shown as critical regulators. Howeverthe intercellular communicative cues and mechanotranductive cascades responsiblefor osteogenic transcription and stem cell recruitment are still largely unknown.
Figure 1. Proximal femurreconstruction from miceflown on 15-day STS-131mission (B,D,F,) compared toground controls (A,C,E). BVloss of 17% in flight samples.
Background
Results
Conclusion
Acknowledgements: This work is supported by NASA Space Biology Grant NNH14ZTT001N-0062 to E. Blaber and NNH14ZTT001N-003 to E. Almeida.
Cellular Connectivity and Intercellular Regulation due to Exaggerated Mechanical Loading
Proliferation and Cellular Metabolic Activity with Exaggerated Loading
Osteogenic Signaling Expression and Localization after Exaggerated Loading
Figure 5. BiostimulatorCAD representation.
MLOY4 osteocyte-like cells
MC3T3-E1 osteoblast-like cells
Figure 4. Experimental cell type morphology. MLOY4represent critical dendritic cell processes inherent tofunctional osteocytes, while MC3T3-E1 pre-osteoblast cells present no such morphology.
Figure 8. Calcein –AMlive cell membrane stainand MatLAB automateddendritic process lengthmeasurement programand processing.
Figure X. HypotheticFeedback regulation ofosteogenic transcriptionin MLOY4 osteocytecells due to mechanicalloading. This theoreticfeedback loop impliesthe critical importanceof coupled wnt signalingcontrol of CX43intercellularcommunicationchannel.
Figure 7. Overview of convergent wnt signaling regulationof osteogenic gene transcription pathways in response tostretch loading. CX43 signaling is increased by stretchloading. Co-localization of CX43 and β-catenin is coupledwith wnt11/PCP pathway release of Rac1.
Nuclear translocation
Nuclear factor
activation
Mechanotrans-ductive signaling
Mechanotransductive signaling
Nuclear factor activation
Table 1: Quantitative Assessment of MLOY4 Dendritic Process Length and Termination Junctions
Length (µm) Integer Representation ofShared Terminating Junctions
per cell
Control (No Stretch) 3.19 ± 2.13 3
48 hours (0.1% strain, 0.1 Hz frequency)
6.50 ± 1.58 7
C X 4 3 w n t 1 w n t 3 a w n t 5 a w n t 1 1
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+9.23
-1.50
-4.32
+1.06
+5.00
-1.16
+11.13
+2.29
-0.01+0.12
-3.83
+2.55
-2.99
-10.27
-16.58
+/- fold change compared to MC3T3-E1 stretch cultures
• Canonical and non-canonical wnt pathways are upregulated during mechanical stimulation of osteocyte cells.
• Up regulation of wnt11 is causally associated with Rac1 up regulation.
• Rac1 association with β-catenin is in part responsible for nuclear translocation and osteogenic gene transcription.
• CX43 and β-catenin co-localization is modulated by Rac1 presence
• CX43 sequesters the Rac1/β-catenin complex outside the nuclear membrane.
Figure 2. Bonemineralhomeostasis is abalance betweenbone formationby osteoblastsand boneresorption byosteoclasts.
Figure 3. Cellular response tomechanical stimulation isdependent on the mechanismof cell deformation. Of importin this study is strain appliedthrough the cell attachments, incontrast to fluid flow studieswhich are regulated bymembrane strains.
A B C
Figure 6: Florescence imaging demonstrating cell morphologies (actin – Green), connexin localization (CX43 primaryantibody/2ndary antibody 594 – Red) and counterstained with DAPI (nuclear –Blue). Figure A) and insert are MLOY4osteocyte cells after 48 hours of stretch culture, B) and insert are MC3T3-E1 osteoblast cells, and C) and insert are co-cultured MLOY4 and MC3T3-E1. A) illustrates typical MLOY4 morphology with extended cell processes along thedirection of stretch (indicated by white arrow) and localized CX43 presence at the process peripheries and nuclearenvelope. While in A) and C) CX43 is highly expressed at membrane interfaces in B) the extent of localization is non-specific in osteoblast cells. Osteoblast-Osteocyte co-cultured cells C) demonstrate both increased CX43 presence withinthe cell and membranous localization inherit to mechanistic communication both intercellular and intracellular.
Nuclear translocation
MLOY4 osteocyte-like and MC3T3-E1osteoblast-like cells were cultured in a customdesigned biostimulator (Figure 5) and allowedto acclimate for 48 hours before 48 hours ofstretch loading. Stretch loading was impartedby 2 Arduino controlled linear drive motors setto 0.1% tensile strain and 0.1Hz cyclicapplication. Measure of CX43 localization, cellnumber, metabolism, and phenotypicexpression were taken at 10 minutes, 2, 12, 24and 48 hours.
Culture conditions were maintained at 5%CO2 37°C and 90% humidity. Culturemedia was supplemented by 1% anti-anti,10% FBS and changed every 48 hourssuch as to not interrupt the stimulationregime.
The self organization of osteocyte cells is acritical metric of the cell populationfunction. MLOY4 cells cultured in unloadedconditions will form dense overlapping
networks with short dendritic process lengths and little CX43 membranelocalization. In vivo examination of osteocyte networks have been shown to behighly interconnected (Figure 3) and these connections are made between dendriticcell processes with lengths averaging 20-30µm. Within the osteon the cell processesare organized in the canaliculus network, the microarchitecture of which amplifiesmechanical stress sensed by the processes which in turn lengthens the process. Thissupposition is supported by our measurement of MLOY4 dendritic cell processlengthening under stretch loading stimulation. Additionally, the shared terminatingjunctions were quantified and demonstrated greater MLOY4 populationinterconnectivity when cells were exposed to stretch loading.
Figure 8. Analysis of MLOY4 viabilityduring stretch stimulation. *p
