Removing or truncating connexin 43 in murine osteocytes alters cortical geometry, nanoscale morphology, and tissue mechanics in the tibia

Hammond, Max A.; Berman, Alycia G.; Pacheco‐Costa, Rafael; Davis, Hannah M.; Plotkin, Lilian I.; Wallace, Joseph M.

doi:10.1016/j.bone.2016.04.021

Cited by 27 publications

(18 citation statements)

References 23 publications

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“…Decreased expression of this important collagen chaperone, which is implicated in osteogenesis imperfecta Drögemuller et al, 2009;Kojima et al, 1998), may also contribute to the defective extracellular matrix and inferior material properties reported in other models of Cx43 deficiency (Bivi et al, 2012b;Lecanda et al, 2000;Watkins et al, 2011). These data confirm a role for Cx43 and the Cx43 CT in the organization and maturation of the collagenous extracellular matrix of bone (Bivi et al, 2012b;Hammond et al, 2016;Pacheco-Costa et al, 2016;Watkins et al, 2011).…”

Section: Discussionmentioning

confidence: 80%

Defective signaling, osteoblastogenesis and bone remodeling in a mouse model of connexin 43 C-terminal truncation

Moorer

Hebert

Tomlinson

et al. 2017

Journal of Cell Science

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In skeletal tissue, loss or mutation of the gap junction protein connexin 43 (Cx43, also known as GJA1) in cells of the osteoblast lineage leads to a profound cortical bone phenotype and defective tissue remodeling. There is mounting evidence in bone cells that the C-terminus (CT) of Cx43 is a docking platform for signaling effectors and is required for efficient downstream signaling. Here, we examined this function, using a mouse model of Cx43 CT-truncation (Gja1 K258Stop). Relative to Gja1 +/− controls, male Gja1 −/K258Stop mice have a cortical bone phenotype that is remarkably similar to those reported for deletion of the entire Cx43 gene in osteoblasts. Furthermore, we show that the Cx43 CT binds several signaling proteins that are required for optimal osteoblast function, including PKCδ, ERK1 and ERK2 (ERK1/2, also known as MAPK3 and MAPK1, respectively) and β-catenin. Deletion of the Cx43 CT domain affects these signaling cascades, impacting osteoblast proliferation, differentiation, and collagen processing and organization. These data imply that, at least in bone, Cx43 gap junctions not only exchange signals, but also recruit the appropriate effector molecules to the Cx43 CT in order to efficiently activate signaling cascades that affect cell function and bone acquisition.

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Section: Discussionmentioning

confidence: 80%

Defective signaling, osteoblastogenesis and bone remodeling in a mouse model of connexin 43 C-terminal truncation

Moorer

Hebert

Tomlinson

et al. 2017

Journal of Cell Science

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“…To explore differences in the distribution of D-spacing values, the histogram and cumulative distribution function (CDF) of each group was generated. The distributions of the control (n = 217) and treated groups (n = 251) were compared using a k-sample Anderson-Darling (A-D) test as previously described [43]. …”

Section: Methodsmentioning

confidence: 99%

β-Aminopropionitrile-Induced Reduction in Enzymatic Crosslinking Causes In Vitro Changes in Collagen Morphology and Molecular Composition

Canelón

Wallace

2016

PLoS ONE

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Type I collagen morphology can be characterized using fibril D-spacing, a metric which describes the periodicity of repeating bands of gap and overlap regions of collagen molecules arranged into collagen fibrils. This fibrillar structure is stabilized by enzymatic crosslinks initiated by lysyl oxidase (LOX), a step which can be disrupted using β-aminopropionitrile (BAPN). Murine in vivo studies have confirmed effects of BAPN on collagen nanostructure and the objective of this study was to evaluate the mechanism of these effects in vitro by measuring D-spacing, evaluating the ratio of mature to immature crosslinks, and quantifying gene expression of type I collagen and LOX. Osteoblasts were cultured in complete media, and differentiated using ascorbic acid, in the presence or absence of 0.25mM BAPN-fumarate. The matrix produced was imaged using atomic force microscopy (AFM) and 2D Fast Fourier transforms were performed to extract D-spacing from individual fibrils. The experiment was repeated for quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Fourier Transform infrared spectroscopy (FTIR) analyses. The D-spacing distribution of collagen produced in the presence of BAPN was shifted toward higher D-spacing values, indicating BAPN affects the morphology of collagen produced in vitro, supporting aforementioned in vivo experiments. In contrast, no difference in gene expression was found for any target gene, suggesting LOX inhibition does not upregulate the LOX gene to compensate for the reduction in aldehyde formation, or regulate expression of genes encoding type I collagen. Finally, the mature to immature crosslink ratio decreased with BAPN treatment and was linked to a reduction in peak percent area of mature crosslink hydroxylysylpyridinoline (HP). In conclusion, in vitro treatment of osteoblasts with low levels of BAPN did not induce changes in genes encoding LOX or type I collagen, but led to an increase in collagen D-spacing as well as a decrease in mature crosslinks.

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“…Each standard site ROI was a set of 7 slices, perpendicular to the proximal-distal axis. As previously described [3, 10], a custom MATLAB (MathWorks, Natick, MA) program was used to calculate the following parameters: total bone area (B.Ar), marrow area (Ma.Ar), cortical area (Ct.Ar), average cortical width (Ct.Wi), periosteal bone perimeter (Ps.Pm), endocortical bone perimeter (Ec.Pm), maximum and minimum second moment of inertia ( I max and I min , respectively), width of the anteroposterior axis (AP.Wi), width of the mediolateral axis (ML.Wi), and AP.Wi to ML.Wi ratio (AP.Wi/ML.Wi) according to standard guidelines.…”

Section: Methodsmentioning

confidence: 99%

“…The second moment of inertia about the anteroposterior axis and the extreme fiber in tension were obtained from µCT images using a 7 slice region centered on the fracture site and were used to map load-displacement to stress-strain curves. As previously described [3, 10], pre- and postyield mechanical properties were calculated using a custom MATLAB program.…”

Section: Methodsmentioning

confidence: 99%

Even with rehydration, preservation in ethanol influences the mechanical properties of bone and how bone responds to experimental manipulation

Vesper

Hammond

Allen

et al. 2017

Bone

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Typically, bones are harvested at the time of animal euthanasia and stored until mechanical testing. However, storage methods are not standardized, and differential effects on mechanical properties are possible between methods. The goal of this study was to investigate the effects that two common preservation methods (freezing wrapped in saline-soaked gauze and refrigerating ethanol fixed samples) have on bone mechanical properties in the context of an in vitro ribosylation treatment designed to modify mechanical integrity. It was hypothesized that there would be an interactive effect between ribose treatment and preservation method. Tibiae from twenty five 11 week old female C57BL/6 mice were separated into 2 preservation groups. Micro-CT scans of contralateral pairs assessed differences in geometry prior to storage. After 7 weeks of storage, bones in each pair of tibiae were soaked in a solution containing either 0 M or 0.6 M ribose for 1 week prior to 4 point bending tests. There were no differences in any cortical geometric parameters between contralateral tibiae. There was a significant main effect of ethanol fixation on displacement to yield (−16.3%), stiffness (+24.5%), strain to yield (−13.9%), and elastic modulus (+18.5%) relative to frozen specimens. There was a significant main effect of ribose treatment for yield force (+13.9%), ultimate force (+9.2%), work to yield (+22.2%), yield stress (+14.1%), and resilience (+21.9%) relative to control-soaked bones. Postyield displacement, total displacement, postyield work, total work, total strain, and toughness were analyzed separately within each preservation method due to significant interactions. For samples stored frozen, all six properties were lower in the ribose-soaked group (49%–68%) while no significant effects of ribose were observed in ethanol fixed bones. Storage in ethanol likely caused changes to the collagen matrix which prevented or masked the embrittling effects of ribosylation that were seen in samples stored frozen wrapped in saline-soaked gauze. These data illustrate the clear importance of maintaining hydration if the eventual goal is to use bones for mechanical assessments and further show that storage in ethanol can alter potential to detect effects of experimental manipulation (in this case ribosylation).

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Removing or truncating connexin 43 in murine osteocytes alters cortical geometry, nanoscale morphology, and tissue mechanics in the tibia

Cited by 27 publications

References 23 publications

Defective signaling, osteoblastogenesis and bone remodeling in a mouse model of connexin 43 C-terminal truncation

Defective signaling, osteoblastogenesis and bone remodeling in a mouse model of connexin 43 C-terminal truncation

β-Aminopropionitrile-Induced Reduction in Enzymatic Crosslinking Causes In Vitro Changes in Collagen Morphology and Molecular Composition

Even with rehydration, preservation in ethanol influences the mechanical properties of bone and how bone responds to experimental manipulation

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