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Mechanical forces regulate cell functions through multiple pathways. G protein-coupled estrogen receptor (GPER) is a seven-transmembrane receptor that is ubiquitously expressed across tissues and mediates the acute cellular response to estrogens. Here, we demonstrate an unidentified role of GPER as a cellular mechanoregulator. G protein-coupled estrogen receptor signaling controls the assembly of stress fibers, the dynamics of the associated focal adhesions, and cell polarization via RhoA GTPase (RhoA). G protein-coupled estrogen receptor activation inhibits F-actin polymerization and subsequently triggers a negative feedback that transcriptionally suppresses the expression of monomeric G-actin. Given the broad expression of GPER and the range of cytoskeletal changes modulated by this receptor, our findings position GPER as a key player in mechanotransduction.

The invasive properties of cancer cells are intimately linked to their mechanical phenotype, which can be regulated by intracellular biochemical signalling. Cell contractility, induced by mechanotransduction of a stiff fibrotic matrix, and the epithelial&ndash, mesenchymal transition (EMT) promote invasion. Metastasis involves cells pushing through the basement membrane into the stroma&mdash, both of which are altered in composition with cancer progression. Agonists of the G protein-coupled oestrogen receptor (GPER), such as tamoxifen, have been largely used in the clinic, and interest in GPER, which is abundantly expressed in tissues, has greatly increased despite a lack of understanding regarding the mechanisms which promote its multiple effects. Here, we show that specific activation of GPER inhibits EMT, mechanotransduction and cell contractility in cancer cells via the GTPase Ras homolog family member A (RhoA). We further show that GPER activation inhibits invasion through an in vitro basement membrane mimic, similar in structure to the pancreatic basement membrane that we reveal as an asymmetric bilayer, which differs in composition between healthy and cancer patients.

Tamoxifen has been used for many years to target estrogen receptor signalling in breast cancer cells. Tamoxifen is also an agonist of the G protein-coupled estrogen receptor (GPER), a GPCR ubiquitously expressed in tissues that mediates the acute response to estrogens. Here we report that tamoxifen promotes mechanical quiescence in hepatic stellate cells (HSCs), stromal fibroblast-like cells whose activation triggers and perpetuates liver fibrosis in hepatocellular carcinomas. This mechanical deactivation is mediated by the GPER/RhoA/myosin axis and induces YAP deactivation. We report that tamoxifen decreases the levels of hypoxia-inducible factor-1 alpha (HIF-1α) and the synthesis of extracellular matrix proteins through a mechanical mechanism that involves actomyosin-dependent contractility and mechanosensing of tissue stiffness. Our results implicate GPER-mediated estrogen signalling in the mechanosensory-driven activation of HSCs and put forward estrogenic signalling as an option for mechanical reprogramming of myofibroblast-like cells in the tumour microenvironment. Tamoxifen, with half a century of safe clinical use, might lead this strategy of drug repositioning.

Alkaptonuria (AKU) is a rare inherited disease resulting from a deficiency of the enzyme homogentisate 1,2-dioxygenase which leads to the accumulation of homogentisic acid (HGA). AKU is characterized by severe cartilage degeneration, similar to that observed in osteoarthritis. Previous studies suggest that AKU is associated with alterations in cytoskeletal organization which could modulate primary cilia structure/function. This study investigated whether AKU is associated with changes in chondrocyte primary cilia and associated Hedgehog signaling which mediates cartilage degradation in osteoarthritis. Human articular chondrocytes were obtained from healthy and AKU donors. Additionally, healthy chondrocytes were treated with HGA to replicate AKU pathology (+HGA). Diseased cells exhibited shorter cilia with length reductions of 36% and 16% in AKU and +HGA chondrocytes respectively, when compared to healthy controls. Both AKU and +HGA chondrocytes demonstrated disruption of the usual cilia length regulation by actin contractility. Furthermore, the proportion of cilia with axoneme breaks and bulbous tips was increased in AKU chondrocytes consistent with defective regulation of ciliary trafficking. Distribution of the Hedgehog-related protein Arl13b along the ciliary axoneme was altered such that its localization was increased at the distal tip in AKU and +HGA chondrocytes. These changes in cilia structure/trafficking in AKU and +HGA chondrocytes were associated with a complete inability to activate Hedgehog signaling in response to exogenous ligand. Thus, we suggest that altered responsiveness to Hedgehog, as a consequence of cilia dysfunction, may be a contributing factor in the development of arthropathy highlighting the cilium as a novel target in AKU.

Nuclear architecture, a function of both chromatin and nucleoskeleton structure, is known to change with stem cell differentiation and differs between various somatic cell types. These changes in nuclear architecture are associated with the regulation of gene expression and genome function in a cell-type specific manner. Biophysical stimuli are known effectors of differentiation and also elicit stimuli-specific changes in nuclear architecture. This occurs via the process of mechanotransduction whereby extracellular mechanical forces activate several well characterized signaling cascades of cytoplasmic origin, and potentially some recently elucidated signaling cascades originating in the nucleus. Recent work has demonstrated changes in nuclear mechanics both with pluripotency state in embryonic stem cells, and with differentiation progression in adult mesenchymal stem cells. This review explores the interplay between cytoplasmic and nuclear mechanosensitivity, highlighting a role for the nucleus as a rheostat in tuning the cellular mechano-response.

Engineering tissues with a structure and spatial composition mimicking those of native articular cartilage (AC) remains a challenge. This study examined if infrapatellar fat pad-derived stem cells (FPSCs) can be used to engineer cartilage grafts with a bulk composition and a spatial distribution of matrix similar to the native tissue. In an attempt to mimic the oxygen gradients and mechanical environment within AC, FPSC-laden hydrogels (either 2 mm or 4 mm in height) were confined to half of their thickness and/or subjected to dynamic compression (DC). Confining FPSC-laden hydrogels was predicted to accentuate the gradient in oxygen tension through the depth of the constructs (higher in the top and lower in the bottom), leading to enhanced glycosaminoglycan (GAG) and collagen synthesis in 2 mm high tissues. When subjected to DC alone, both GAG and collagen accumulation increased within 2 mm high unconfined constructs. Furthermore, the dynamic modulus of constructs increased from 0.96 MPa to 1.45 MPa following the application of DC. There was no synergistic benefit of coupling confinement and DC on overall levels of matrix accumulation; however in all constructs, irrespective of their height, the combination of these boundary conditions led to the development of engineered tissues that spatially best resembled native AC. The superficial region of these constructs mimicked that of native tissue, staining weakly for GAG, strongly for type II collagen, and in 4 mm high tissues more intensely for proteoglycan 4 (lubricin). This study demonstrated that FPSCs respond to joint-like environmental conditions by producing cartilage tissues mimicking native AC. Copyright © 2016 John Wiley & Sons, Ltd.

Primary cilia are single non-motile organelles that provide a highly regulated compartment into which specific proteins are trafficked as a critical part of various signaling pathways. The absence of primary cilia has been shown to prevent differentiation of human mesenchymal stem cells (hMSCs). Changes in primary cilia length are crucial for regulating signaling events; however it is not known how alterations in cilia structure relate to differentiation. This study tested the hypothesis that changes in primary cilia structure are required for stem cell differentiation. hMSCs expressed primary cilia that were labeled with acetylated alpha tubulin and visualized by confocal microscopy. Chemically induced differentiation resulted in lineage specific changes in cilia length and prevalence which were independent of cell cycle. In particular, adipogenic differentiation resulted in cilia elongation associated with the presence of dexamethasone, while insulin had an inhibitory effect on cilia length. Over a 7-day time course, adipogenic differentiation media resulted in cilia elongation within 2 days followed by increased nuclear PPARγ levels; an early marker of adipogenesis. Cilia elongation was associated with increased trafficking of insulin-like growth factor-1 receptor β (IGF-1Rβ) into the cilium. This was reversed on inhibition of elongation by IFT-88 siRNA transfection, which also decreased nuclear PPARγ. This is the first study to show that adipogenic differentiation requires primary cilia elongation associated with the recruitment of IGF-1Rβ onto the cilium. This study may lead to the development of cilia-targeted therapies for controlling adipogenic differentiation and associated conditions such as obesity. Stem Cells 2015;33:1952–1961

Mesenchymal stem cells (MSCs) are an attractive cell source for tissue engineering applications due to their multipotentiality and increased expansion potential compared to mature cells. However, the full potential of MSCs for cellular therapies is not realised, due, in part, to premature proliferative senescence and impaired differentiation capacity following expansion under 20% oxygen. Bone marrow MSCs reside under reduced oxygen levels (4%-7% oxygen), thus this study investigates the effects of uninterrupted physiological oxygen tensions (2%, 5%) on MSC expansion and subsequent differentiation. Expansion potential was evaluated from colony formation efficiency, population-doubling rates, and cellular senescence. Colony formation was significantly reduced under 5% oxygen compared to 2% and 20% oxygen. Population-doubling time was initially shorter with 20% oxygen, but subsequently no significant differences in doubling time were detected between the oxygen conditions. MSCs expanded with 20% oxygen contained a greater proportion of senescent cells than those under physiological oxygen levels, indicated by a three to fourfold increase in β-galactosidase staining. This may be related to the approximately twofold enhanced mitochondrial oxygen consumption under this culture condition. Chondrogenic differentiation was achieved following expansion at each oxygen condition. However, osteogenesis was only achieved for cells expanded and differentiated at 20% oxygen, indicated by alkaline phosphatase activity and alizarin red staining. These studies demonstrate that uninterrupted hypoxia may enhance long-term MSC expansion, but results in a population with impaired osteogenic differentiation potential. Thus, novel differentiation conditions are required to enable differentiation to nonchondrogenic lineages using hypoxia-cultured MSCs.

Alkaptonuria (AKU) is an ultra-rare genetic disease, in which the accumulation of a toxic metabolite, homogentisic acid (HGA) leads to the systemic development of ochronotic aggregates. These aggregates cause severe complications mainly at the level of joints with extensive degradation of the articular cartilage. Primary cilia have been demonstrated to play an essential role in development and the maintenance of articular cartilage homeostasis, through their involvement in mechanosignalling and Hedgehog signalling pathways. Hedgehog signalling has been demonstrated to be activated in osteoarthritis (OA) and to drive cartilage degeneration in vivo. The numerous similarities between OA and AKU suggest that primary cilia hedgehog signalling may also be altered in AKU. Thus, we characterised an AKU cellular model in which healthy chondrocytes were treated with HGA (66 µM) to replicate AKU cartilage pathology. We investigated the degree of activation of the hedgehog signalling pathway and how treatment with inhibitors of the receptor Smoothened (Smo) influenced hedgehog activation and primary cilia structure. The results obtained in this work provide a further step in the comprehension of the pathophysiological features of AKU, suggesting a potential therapeutic approach to modulate AKU cartilage degradation processes through manipulation of the hedgehog pathway. This article is protected by copyright. All rights reserved

Mesenchymal stem cell (MSC) differentiation is mediated by soluble and physical cues. In this study, we investigated differentiation-induced transformations in MSC cellular and nuclear biophysical properties and queried their role in mechanosensation. Our data show that nuclei in differentiated bovine and human MSCs stiffen and become resistant to deformation. This attenuated nuclear deformation was governed by restructuring of Lamin A/C and increased heterochromatin content. This change in nuclear stiffness sensitized MSCs to mechanical-loading-induced calcium signaling and differentiated marker expression. This sensitization was reversed when the ‘stiff’ differentiated nucleus was softened and was enhanced when the ‘soft’ undifferentiated nucleus was stiffened through pharmacologic treatment. Interestingly, dynamic loading of undifferentiated MSCs, in the absence of soluble differentiation factors, stiffened and condensed the nucleus, and increased mechanosensitivity more rapidly than soluble factors. These data suggest that the nucleus acts as a mechanostat to modulate cellular mechanosensation during differentiation. DOI: http://dx.doi.org/10.7554/eLife.18207.001

Mechanical cues direct the lineage commitment of mesenchymal stem cells (MSCs). In this study, we identified the operative molecular mechanisms through which dynamic tensile loading (DL) regulates changes in chromatin organization and nuclear mechanics in MSCs. Our data show that, in the absence of exogenous differentiation factors, short term DL elicits a rapid increase in chromatin condensation, mediated by acto-myosin based cellular contractility and the activity of the histone-lysine N-methyltransferase EZH2. The resulting change in chromatin condensation stiffened the MSC nucleus, making it less deformable when stretch was applied to the cell. We also identified stretch induced ATP release and purinergic calcium signaling as a central mediator of this chromatin condensation process. Further, we showed that DL, through differential stabilization of the condensed chromatin state, established a ‘mechanical memory’ in these cells. That is, increasing strain levels and number of loading events led to a greater degree of chromatin condensation that persisted for longer periods of time after the cessation of loading. These data indicate that, with mechanical perturbation, MSCs develop a mechanical memory encoded in structural changes in the nucleus which may sensitize them to future mechanical loading events and define the trajectory and persistence of their lineage specification.

This study examines how differentiation of human mesenchymal stem cells regulates the interaction between the cell membrane and the actin cortex controlling cell behavior. Micropipette aspiration was used to measure the pressure required for membrane-cortex detachment which increased from 0.15 kPa in stem cells to 0.71 kPa following chondrogenic differentiation. This effect was associated with reduced susceptibility to mechanical and osmotic bleb formation, reduced migration and an increase in cell modulus. Theoretical modelling of bleb formation demonstrated that the increased stiffness of differentiated cells was due to the increased membrane-cortex adhesion. Differentiated cells exhibited greater F-actin density and slower actin remodelling. Differentiated cells also expressed greater levels of the membrane-cortex ezrin, radixin, moeisin (ERM) linker proteins which was responsible for the reduced blebability, as confirmed by transfection of stem cells with dominant active ezrin-T567D-GFP. This study demonstrates that stem cells have an inherently weak membrane-cortex adhesion which increases blebability thereby regulating cell migration and stiffness.

The aim of this study was to explore how cell–matrix interactions and extrinsic mechanical signals interact to determine stem cell fate in response to transforming growth factor-b3 (TGF-b3). Bone marrow derived mesenchymal stem cells (MSCs) were seeded in agarose and fibrin hydrogels and subjected to dynamic compression in the presence of different concentrations of TGF-b3. Markers of chondrogenic, myogenic and endochondral differentiation were assessed. MSCs embedded within agarose hydrogels adopted a spherical cell morphology, while cells directly adhered to the fibrin matrix and took on a spread morphology. Free-swelling agarose constructs stained positively for chondrogenic markers, with MSCs appearing to progress towards terminal differentiation as indicated by mineral staining. MSC seeded fibrin constructs progressed along an alternative myogenic pathway in long-term free-swelling culture. Dynamic compression suppressed differentiation towards any investigated lineage in both fibrin and agarose hydrogels in the short-term. Given that fibrin clots have been shown to support a chondrogenic phenotype in vivo within mechanically loaded joint defect environments, we next explored the influence of long term (42 days) dynamic compression on MSC differentiation. Mechanical signals generated by this extrinsic loading ultimately governed MSC fate, directing MSCs along a chondrogenic pathway as opposed to the default myogenic phenotype supported within unloaded fibrin clots. In conclusion, this study demonstrates that external cues such as the mechanical environment can override the influence specific substrates, scaffolds or hydrogels have on determining mesenchymal stem cell fate. The temporal data presented in this study highlights the importance of considering how MSCs respond to extrinsic mechanical signals in the long term.

The inherent biocompatibility of fibrin hydrogels makes them an attractive material for use in a wide range of tissue engineering applications. Despite this, their relatively low stiffness and high compliance limits their potential for certain orthopaedic applications. Enhanced mechanical properties are desirable so as to withstand surgical handling and in vivo loading after implantation and additionally, can provide important cues to cells seeded within the hydrogel. Standard methods used to enhance the mechanical properties of biological scaffolds such as chemical or thermal crosslinking cannot be used with fibrin hydrogels as cell seeding and gel formation occurs simultaneously. The objective of this study was to investigate the use of plastic compression as a means to improve the mechanical properties of chondrocyte-seeded fibrin hydrogels and to determine the influence of such compression on cell viability within these constructs. It was found that the application of 80% strain to fibrin hydrogels for 30 min (which resulted in a permanent strain of 47.4%) produced a 2.1-fold increase in the subsequent compressive modulus. Additionally, chondrocyte viability was maintained in the plastically compressed gels with significant cellular proliferation and extracellular matrix accumulation observed over 28 days of culture. In conclusion, plastic compression can be used to modulate the density and mechanical properties of cell-seeded fibrin hydrogels and represents a useful tool for both in theatre and in vitro tissue engineering applications.

Both hydrostatic pressure (HP) and cell-matrix interactions have independently been shown to regulate the chondrogenic differentiation of mesenchymal stem cells (MSCs). The objective of this study was to test the hypothesis that the response of MSCs to hydrostatic pressure will depend on the biomaterial within which the cells are encapsulated. Bone-marrow-derived MSCs were seeded into either agarose or fibrin hydrogels and exposed to 10 MPa of cyclic HP (1 Hz, 4 h per day, 5 days per week for 3 weeks) in the presence of either 1 or 10 ng ml(-1) of TGF-β3. Agarose hydrogels were found to support a spherical cellular morphology, while MSCs seeded into fibrin hydrogels attached and spread, with clear stress fiber formation. Hydrogel contraction was also observed in MSC-fibrin constructs. While agarose hydrogels better supported chondrogenesis of MSCs, HP only enhanced sulfated glycosaminoglycan (sGAG) accumulation in fibrin hydrogels, which correlated with a reduction in fibrin contraction. HP also reduced alkaline phosphatase activity in the media for both agarose and fibrin constructs, suggesting that this stimulus plays a role in the maintenance of the chondrogenic phenotype. This study demonstrates that a complex relationship exists between cell-matrix interactions and hydrostatic pressure, which plays a key role in regulating the chondrogenic differentiation of MSCs.

Various forms of mechanical stimulation have been shown to enhance chondrogenesis of mesenchymal stem cells (MSCs). However, the response of MSCs undergoing chondrogenesis to such signals has been shown to depend on the temporal application of loading. The objective of this study was to determine the effect of dynamic compression on cartilage-matrix-specific gene expression and to relate this response to the local biochemical environment and cell phenotype at the time of loading. At 0, 7, 14, and 21 days extracellular matrix (ECM) deposition within MSC-seeded agarose hydrogels due to transforming growth factor-β3 stimulation was determined biochemically and histologically, and then reverse transcription-polymerase chain reaction was used to examine the effects of dynamic compression on cartilage-matrix-specific gene expression. The results of these experiments show that the local environment in the core of the constructs is more favorable for chondrogenesis in comparison to the annulus, as evident from both ECM synthesis and gene expression. Additionally, we found that the response of the cells to mechanical stimulus varied with both the spatial region within the constructs and the temporal application of loading. Dynamic compression applied at day 21 was found to enhance levels of cartilage matrix gene expression following a peak in expression levels at day 14 in free swelling constructs, suggesting that mechanical signals play a key role in the maintenance of a chondrogenic phenotype. The application of mechanical stimulus to enhance cartilage ECM synthesis may be an important tool in regenerative medicine-based cartilage repair. The results of this study suggest that a chondrogenic phenotype and/or a well-developed pericellular matrix must first be established before dynamic compression can have a positive effect on cartilage-matrix-specific gene expression.

Engineering organs and tissues with the spatial composition and organisation of their native equivalents remains a major challenge. One approach to engineer such spatial complexity is to recapitulate the gradients in regulatory signals that during development and maturation are believed to drive spatial changes in stem cell differentiation. Mesenchymal stem cell (MSC) differentiation is known to be influenced by both soluble factors and mechanical cues present in the local microenvironment. The objective of this study was to engineer a cartilaginous tissue with a native zonal composition by modulating both the oxygen tension and mechanical environment thorough the depth of MSC seeded hydrogels. To this end, constructs were radially confined to half their thickness and subjected to dynamic compression (DC). Confinement reduced oxygen levels in the bottom of the construct and with the application of DC, increased strains across the top of the construct. These spatial changes correlated with increased glycosaminoglycan accumulation in the bottom of constructs, increased collagen accumulation in the top of constructs, and a suppression of hypertrophy and calcification throughout the construct. Matrix accumulation increased for higher hydrogel cell seeding densities; with DC further enhancing both glycosaminoglycan accumulation and construct stiffness. The combination of spatial confinement and DC was also found to increase proteoglycan-4 (lubricin) deposition toward the top surface of these tissues. In conclusion, by modulating the environment through the depth of developing constructs, it is possible to suppress MSC endochondral progression and to engineer tissues with zonal gradients mimicking certain aspects of articular cartilage.