13 research outputs found
Mechanically stimulated osteocytes promote the proliferation and migration of breast cancer cells via a potential CXCL1/2 mechanism
Bone represents the most common site for breast cancer metastasis. Bone is a highly dynamic organ that is constantly adapting to its biophysical environment, orchestrated largely by the resident osteocyte network. Osteocytes subjected to physiologically relevant biophysical conditions may therefore represent a source of key factors mediating breast cancer cell metastasis to bone. Therefore, we investigated the potential proliferative and migratory capacity of soluble factors released by mechanically stimulated osteocytes on breast cancer cell behaviour. Interestingly the secretome of mechanically stimulated osteocytes enhanced both the proliferation and migration of cancer cells when compared to the secretome of statically cultured osteocytes, demonstrating that mechanical stimuli is an important physiological stimulus that should be considered when identifying potential targets. Using a cytokine array, we further identified a group of mechanically activated cytokines in the osteocyte secretome, which potentially drive breast cancer metastasis. In particular, CXCL1 and CXCL2 cytokines are highly expressed, mechanically regulated, and are known to interact with one another. Lastly, we demonstrate that these specific factors enhance breast cancer cell migration independently and in a synergistic manner, identifying potential osteocyte derived factors mediating breast cancer metastasis to bone
Primary cilium-mediated MSC mechanotransduction is dependent on Gpr161 regulation of hedgehog signalling
The benefits of physical loading to skeletal mass are well known. The primary cilium has emerged as an
important organelle in bone mechanobiology/mechanotransduction, particularly in mesenchymal stem/stromal
cells, yet the molecular mechanisms of cilium mechanotransduction are poorly understood. In this study, we
demonstrate that Gpr161 is a mechanoresponsive GPCR, that localises to the cilium, and is involved in fluid
shear-induced cAMP signalling and downstream osteogenesis. This Gpr161-mediated mechanotransduction is
dependent on IFT88/cilium and may act through adenylyl cyclase 6 (AC6) to regulate cAMP and MSC osteogenesis. Moreover, we demonstrate that Hh signalling is positively associated with osteogenesis and that Hh gene
expression is mechanically regulated and required for loading-induced osteogenic differentiation through a
mechanism that involves IFT88, Gpr161, AC6, and cAMP. Therefore, we have delineated a molecular mechanism
of MSC mechanotransduction which likely occurs at the cilium, leading to MSC osteogenesis, highlighting novel
mechanotherapeutic targets to enhance osteogenesis
The primary cilium as a novel extracellular sensor in bone
The primary cilium as a novel extracellular sensor in bon
Additional file 1 of 2P-FLIM unveils time-dependent metabolic shifts during osteogenic differentiation with a key role of lactate to fuel osteogenesis via glutaminolysis identified
Additional file 1: Table S1. Gene symbol, name, accession number, unique assay ID and amplicon length of genes used for qPCR. Fig. S1. Alizarin red staining of hMSCs after 21 days of incubation in either Xpan or Osteo+ cell culture media. Table S2. Mahalanobis distance, Hotelling’s T2 stats, F-value, critical F-Values, P-value and significance results of PCA statistical significance analysis. Fig. S2. Supplementation of Osteo+ cell culture medium with lactate (7.5 mM) and metabolic inhibitors. A Alizarin red staining of hMSCs after 14 days of cell culture in several cell culture medium formulations. B Alizarin red quantification per DNA of hMSCs after 14 days of cell culture
TGF beta 1-induced recruitment of human bone mesenchymal stem cells is mediated by the primary cilium in a SMAD3-dependent manner
The recruitment of mesenchymal stem cells (MSCs) is a crucial process in the development,
maintenance and repair of tissues throughout the body. Transforming growth factor-β1 (TGFβ1) is a
potent chemokine essential for the recruitment of MSCs in bone, coupling the remodelling cycle. The
primary cilium is a sensory organelle with important roles in bone and has been associated with cell
migration and more recently TGFβ signalling. Dysregulation of TGFβ signalling or cilia has been linked
to a number of skeletal pathologies. Therefore, this study aimed to determine the role of the primary
cilium in TGFβ1 signalling and associated migration in human MSCs. In this study we demonstrate that
low levels of TGFβ1 induce the recruitment of MSCs, which relies on proper formation of the cilium.
Furthermore, we demonstrate that receptors and downstream signalling components in canonical TGFβ
signalling localize to the cilium and that TGFβ1 signalling is associated with activation of SMAD3 at the
ciliary base. These findings demonstrate a novel role for the primary cilium in the regulation of TGFβ
signalling and subsequent migration of MSCs, and highlight the cilium as a target to manipulate this key
pathway and enhance MSC recruitment for the treatment of skeletal diseases
Physiological cyclic hydrostatic pressure induces osteogenic lineage commitment of human bone marrow stem cells: a systematic study
Background: Physical loading is necessary to maintain bone tissue integrity. Loading-induced fluid shear is
recognised as one of the most potent bone micromechanical cues and has been shown to direct stem cell
osteogenesis. However, the effect of pressure transients, which drive fluid flow, on human bone marrow stem cell
(hBMSC) osteogenesis is undetermined. Therefore, the objective of the study is to employ a systematic analysis of
cyclic hydrostatic pressure (CHP) parameters predicted to occur in vivo on early hBMSC osteogenic responses and
late-stage osteogenic lineage commitment.
Methods: hBMSC were exposed to CHP of 10 kPa, 100 kPa and 300 kPa magnitudes at frequencies of 0.5 Hz, 1 Hz
and 2 Hz for 1 h, 2 h and 4 h of stimulation, and the effect on early osteogenic gene expression of COX2, RUNX2
and OPN was determined. Moreover, to decipher whether CHP can induce stem cell lineage commitment, hBMSCs
were stimulated for 4 days for 2 h/day using 10 kPa, 100 kPa and 300 kPa pressures at 2 Hz frequency and cultured
statically for an additional 1–2 weeks. Pressure-induced osteogenesis was quantified based on ATP release, collagen
synthesis and mineral deposition.
Results: CHP elicited a positive, but variable, early osteogenic response in hBMSCs in a magnitude- and frequencydependent
manner, that is gene specific. COX2 expression elicited magnitude-dependent effects which were not
present for RUNX2 or OPN mRNA expression. However, the most robust pro-osteogenic response was found at the
highest magnitude (300 kPa) and frequency regimes (2 Hz). Interestingly, long-term mechanical stimulation utilising
2 Hz frequency elicited a magnitude-dependent release of ATP; however, all magnitudes promoted similar levels of
collagen synthesis and significant mineral deposition, demonstrating that lineage commitment is magnitude
independent. This therefore demonstrates that physiological levels of pressures, as low as 10 kPa, within the bone
can drive hBMSC osteogenic lineage commitment.
Conclusion: Overall, these findings demonstrate an important role for cyclic hydrostatic pressure in hBMSCs and
bone mechanobiology, which should be considered when studying pressure-driven fluid shear effects in hBMSCs
mechanobiology. Moreover, these findings may have clinical implication in terms of bioreactor-based bone tissue
engineering strategies
Wanted dead or alive: skeletal structure alteration of cold-water coral desmophyllum pertusum (lophelia pertusa) from anthropogenic stressors
Ocean acidification (OA) has provoked changes in the carbonate saturation state that may alter the formation and structural biomineralisation of calcium carbonate exoskeletons for marine organisms. Biomineral production in organisms such as cold-water corals (CWC) rely on available carbonate in the water column and the ability of the organism to sequester ions from seawater or nutrients for the formation and growth of a skeletal structure. As an important habitat structuring species, it is essential to examine the impact that anthropogenic stressors (i.e., OA and rising seawater temperatures) have on living corals and the structural properties of dead coral skeletons; these are important contributors to the entire reef structure and the stability of CWC mounds. In this study, dead coral skeletons in seawater were exposed to various levels of pCO2 and different temperatures over a 12-month period. Nanoindentation was subsequently conducted to assess the structural properties of coral samples’ elasticity (E) and hardness (H), whereas the amount of dissolution was assessed through scanning electron microscopy. Overall, CWC samples exposed to elevated pCO2 and temperature show changes in properties which leave them more susceptible to breakage and may in turn negatively impact the formation and stability of CWC mound development. </p
Mesenchymal stem cell mechanotransduction is cAMP dependent and regulated by adenylyl cyclase 6 and the primary cilium
Mechanical loading is a potent stimulus of bone adaptation, requiring
the replenishment of the osteoblast from a progenitor population. One
such progenitor is themesenchymal stemcell (MSC), which undergoes
osteogenic differentiation in response to oscillatory fluid shear. Yet, the
mechanism mediating stem cell mechanotransduction, and thus the
potential to target this therapeutically, is poorly understood. In this study,
we demonstrate that MSCs utilise cAMP as a second messenger in
mechanotransduction, which is required for flow-mediated increases in
osteogenic gene expression. Furthermore, we demonstrate that this
mechanosignalling is dependent on the primary cilium and the ciliary
localised adenylyl cyclase 6. Finally, we also demonstrate that this
mechanotransduction mechanism can be targeted therapeutically to
enhance cAMP signalling and early osteogenic signalling, mimicking
the beneficial effect of physical loading. Our findings therefore
demonstrate a novel mechanism of MSC mechanotransduction
that can be targeted therapeutically, demonstrating a potential
mechanotherapeutic for bone-loss diseases such as osteoporosis
TRPV4-mediates oscillatory fluid shear mechanotransduction in mesenchymal stem cells in part via the primary cilium
Skeletal homeostasis requires the continued replenishment of the bone forming osteoblast from a mesenchymal stem cell (MSC) population, a process that has been shown to be mechanically regulated. However, the mechanisms by which a biophysical stimulus can induce a change in biochemical signaling, mechanotransduction, is poorly understood. As a precursor to loading-induced bone formation, deciphering the molecular mechanisms of MSC osteogenesis is a critical step in developing novel anabolic therapies. Therefore, in this study we characterize the expression of the
mechanosensitive calcium channel Transient Receptor Potential subfamily V member 4 (TRPV4) in MSCs and demonstrate that TRPV4 localizes to areas of high strain, specifically the primary cilium. We demonstrate that TRPV4 is required for MSC mechanotransduction, mediating oscillatory fluid shear induced calcium signaling and early osteogenic gene expression. Furthermore, we demonstrate that TRPV4 can be activated pharmacologically eliciting a response that mirrors that seen with mechanical
stimulation. Lastly, we show that TRPV4 localization to the primary cilium is functionally significant, with MSCs with defective primary cilia exhibiting an inhibited osteogenic response to TRPV4 activation. Collectively, this data demonstrates a novel mechanism of stem cell mechanotransduction, which can be targeted therapeutically, and further highlights the critical role of the primary cilium in MSC biology
Loss of adenylyl cyclase 6 in leptin receptor-expressing stromal cells attenuates loading-induced endosteal bone formation
Bone marrow stromal/stem cells represent a quiescent cell population that replenish the osteoblast bone-forming cell pool with age
and in response to injury, maintaining bone mass and repair. A potent mediator of stromal/stem cell differentiation in vitro and bone
formation in vivo is physical loading, yet it still remains unclear whether loading-induced bone formation requires the osteogenic differentiation of these resident stromal/stem cells. Therefore, in this study, we utilized the leptin receptor (LepR) to identify and trace
the contribution of bone marrow stromal cells to mechanoadaptation of bone in vivo. Twelve-week-old Lepr-cre;tdTomato mice were
subjected to compressive tibia loading with an 11 N peak load for 40 cycles, every other day for 2 weeks. Histological analysis
revealed that Lepr-cre;tdTomato+ cells arise perinatally around blood vessels and populate bone surfaces as lining cells or osteoblasts
before a percentage undergo osteocytogenesis. Lepr-cre;tdTomato+ stromal cells within the marrow increase in abundance with age,
but not following the application of tibial compressive loading. Mechanical loading induces an increase in bone mass and bone formation parameters, yet does not evoke an increase in Lepr-cre;tdTomato+ osteoblasts or osteocytes. To investigate whether adenylyl
cyclase-6 (AC6) in LepR cells contributes to this mechanoadaptive response, Lepr-cre;tdTomato mice were further crossed with AC6fl/fl
mice to generate a LepR+ cell-specific knockout of AC6. These Lepr-cre;tdTomato;AC6fl/fl animals have an attenuated response to compressive tibia loading, characterized by a deficient load-induced osteogenic response on the endosteal bone surface. This, therefore,
shows that Lepr-cre;tdTomato+ cells contribute to short-term bone mechanoadaptation. © 2020 The Authors. JBMR Plus published by
Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research