Direct measurement of TRPV4 and PIEZO1 activity reveals multiple mechanotransduction pathways in chondrocytes

Servin‐Vences, M. Rocio; Moroni, Mirko; Lewin, Gary R.; Poole, Kate

doi:10.7554/elife.21074

Cited by 210 publications

(285 citation statements)

References 81 publications

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“…The inactivation kinetics of the currents observed in vector-transfected cells were slow, and do not resemble those observed when Piezo1 is overexpressed in HEK cells (Fig.1c left)(Bae et al, 2011; Coste et al, 2010). However, slower inactivation kinetics of Piezo1-dependent currents in other cell types has been observed in cell-attached recordings (Bae et al, 2015; Lee et al, 2014; Pathak et al, 2014; Peyronnet et al, 2013; Servin-Vences et al, 2017). We previously created a more reliable mechanically silent cell line for expression studies by inactivating endogenous Piezo1 in HEK cells (HEK-P1KO) using CRISPR/Cas9 to truncate all Piezo1 alleles (Lukacs et al, 2015).…”

Section: Resultsmentioning

confidence: 95%

Endogenous Piezo1 Can Confound Mechanically Activated Channel Identification and Characterization

Dubin¹,

Murthy²,

Lewis³

et al. 2017

Neuron

127

116

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Summary A gold standard for characterizing mechanically-activated (MA) currents is via heterologous expression of candidate channels in naïve cells. Two recent studies described MA channels using this paradigm. TMEM150c was proposed to be a component of a MA channel partly based on a heterologous expression approach. In another study, Piezo1’s N-terminal “propeller” domain was proposed to constitute an intrinsic mechanosensitive module based on expression of a chimera between a pore-forming domain of the mechano-insensitive ASIC1 channel and Piezo1. When we attempted to replicate these results we found each construct conferred modest MA currents in a small fraction of naïve HEK cells similar to the published work. Strikingly, these MA currents were not detected in cells in which endogenous Piezo1 was CRISPR/Cas9-inactivated. These results highlight the importance of choosing cells lacking endogenous MA channels to assay the mechanotransduction properties of various proteins.

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Section: Resultsmentioning

confidence: 95%

Endogenous Piezo1 Can Confound Mechanically Activated Channel Identification and Characterization

Dubin¹,

Murthy²,

Lewis³

et al. 2017

Neuron

127

116

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“…TRPV4 ion channel can be activated by various stimuli, including osmotic stress, mechanical loading, and heat. TRPV4 plays a key role in regulating calcium homeostasis and matrix metabolism in cartilage, while TRPV4‐dysfunction is associated with the development of osteoarthritis . In the compressed cartilage, both mechanical loading and osmotic stress can activate the TRPV4 channel and initiate the [Ca 2+ ] i transients.…”

Section: Resultsmentioning

confidence: 99%

Calcium signaling of in situ chondrocytes in articular cartilage under compressive loading: Roles of calcium sources and cell membrane ion channels

Zhou

Chen

et al. 2017

Journal Orthopaedic Research

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Mechanical loading on articular cartilage can induce many physical and chemical stimuli on chondrocytes residing in the extracellular matrix (ECM). Intracellular calcium ([Ca ] ) signaling is among the earliest responses of chondrocytes to physical stimuli, but the [Ca ] signaling of in situ chondrocytes in loaded cartilage is not fully understood due to the technical challenges in [Ca ] imaging of chondrocytes in a deforming ECM. This study developed a novel bi-directional microscopy loading device that enables the record of transient [Ca ] responses of in situ chondrocytes in loaded cartilage. It was found that compressive loading significantly promoted [Ca ] signaling in chondrocytes with faster [Ca ] oscillations in comparison to the non-loaded cartilage. Seven [Ca ] signaling pathways were further investigated by treating the cartilage with antagonists prior to and/or during the loading. Removal of extracellular Ca ions completely abolished the [Ca ] responses of in situ chondrocytes, suggesting the indispensable role of extracellular Ca sources in initiating the [Ca ] signaling in chondrocytes. Depletion of intracellular Ca stores, inhibition of PLC-IP pathway, and block of purinergic receptors on plasma membrane led to significant reduction in the responsive rate of cells. Three types of ion channels that are regulated by different physical signals, TRPV4 (osmotic and mechanical stress), T-type VGCCs (electrical potential), and mechanical sensitive ion channels (mechanical loading) all demonstrated critical roles in controlling the [Ca ] responses of in situ chondrocyte in the loaded cartilage. This study provided new knowledge about the [Ca ] signaling and mechanobiology of chondrocytes in its natural residing environment. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:730-738, 2018.

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“…VGCCs play critical roles in the differentiation, proliferation, and metabolic activities of chondrocytes (55). We previously showed that blockage of T‐type VGCC reduced load‐induced osteoarthritis in mice (51). The functions of VGCCs in the spontaneous calcium signaling of chondrocytes may further reveal their roles in osteoarthritis development and be worth future research efforts.…”

Section: Discussionmentioning

confidence: 99%

“…8 ) Mechanosensitive ion channels: Sample was treated with 20 µM GdCl 3 . 9 ) TRPV4 channel: 10 µM GSK205 was used to block the TRPV4 channel (16, 51). 10 ) Voltage‐gated calcium channel (VGCC): 17.7 µM NNC 55‐0396 was used to block the T‐type VGCC (52, 53); 100 µM verapamil was used to block the L‐type VGCC (54–56).…”

Section: Methodsmentioning

confidence: 99%

Spontaneous calcium signaling of cartilage cells: from spatiotemporal features to biophysical modeling

Zhou

et al. 2019

FASEB j.

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Intracellular calcium ([Ca2+]i) oscillation is a fundamental signaling response of cartilage cells under mechanical loading or osmotic stress. Chondrocytes are usually considered as nonexcitable cells with no spontaneous [Ca2+]i signaling. This study proved that chondrocytes can exhibit robust spontaneous [Ca2+]i signaling without explicit external stimuli. The intensity of [Ca2+]i peaks from individual chondrocytes maintain a consistent spatiotemporal pattern, acting as a unique “fingerprint” for each cell. Statistical analysis revealed lognormal distributions of the temporal parameters of [Ca2+]i peaks, as well as strong linear correlations between their means and sds. Based on these statistical findings, we hypothesized that the spontaneous [Ca2+]i peaks may result from an autocatalytic process and that [Ca2+]i oscillation is controlled by a threshold‐regulating mechanism. To test these 2 mechanisms, we established a multistage biophysical model by assuming the spontaneous [Ca2+]i signaling of chondrocytes as a combination of deterministic and stochastic processes. The theoretical model successfully explained the lognormal distribution of the temporal parameters and the fingerprint feature of [Ca2+]i peaks. In addition, by using antagonists for 10 pathways, we revealed that the initiation of spontaneous [Ca2+]i peaks in chondrocytes requires the presence of extracellular Ca2+, and that the PLC‐inositol 1,4,5‐trisphosphate pathway, which controls the release of calcium from the endoplasmic reticulum, can affect the initiation of spontaneous [Ca2+]i peaks in chondrocytes. The purinoceptors and transient receptor potential vanilloid 4 channels on the plasma membrane also play key roles in the spontaneous [Ca2+]i signaling of chondrocytes. In contrast, blocking the T‐type or L‐type voltage‐gated calcium channel promoted the spontaneous calcium signaling. This study represents a systematic effort to understand the features and initiation mechanisms of spontaneous [Ca2+]i signaling in chondrocytes, which are critical for chondrocyte mechanobiology.—Zhou, Y., Lv, M., Li, T., Zhang, T., Duncan, R., Wang, L., Lu, X. L. Spontaneous calcium signaling of cartilage cells: from spatiotemporal features to biophysical modeling. FASEB J. 33, 4675–4687 (2019). http://www.fasebj.org

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Direct measurement of TRPV4 and PIEZO1 activity reveals multiple mechanotransduction pathways in chondrocytes

Cited by 210 publications

References 81 publications

Endogenous Piezo1 Can Confound Mechanically Activated Channel Identification and Characterization

Endogenous Piezo1 Can Confound Mechanically Activated Channel Identification and Characterization

Calcium signaling of in situ chondrocytes in articular cartilage under compressive loading: Roles of calcium sources and cell membrane ion channels

Spontaneous calcium signaling of cartilage cells: from spatiotemporal features to biophysical modeling

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