Dataset Information

Cell Confluence Modulates TRPV4 Channel Activity in Response to Hypoxia.

ABSTRACT: Transient receptor potential vanilloid 4 (TRPV4) is a polymodal Ca²⁺-permeable channel involved in various hypoxia-sensitive pathophysiological phenomena. Different tools are available to study channel activity, requiring cells to be cultured at specific optimal densities. In the present study, we examined if cell density may influence the effect of hypoxia on TRPV4 activity. Transiently TRPV4-transfected HEK293T cells were seeded at low or high densities corresponding to non-confluent or confluent cells, respectively, on the day of experiments, and cultured under in vitro normoxia or hypoxia. TRPV4-mediated cytosolic Ca²⁺ responses, single-channel currents, and Ca²⁺ influx through the channel were measured using Ca²⁺ imaging/microspectrofluorimetric assay, patch-clamp, and Bioluminescence Resonance Energy Transfer (BRET), respectively. TRPV4 plasma membrane translocation was studied using confocal microscopy, biotinylation of cell surface proteins, and BRET. Our results show that hypoxia exposure has a differential effect on TRPV4 activation depending on cell confluence. At low confluence levels, TRPV4 response is increased in hypoxia, whereas at high confluence levels, TRPV4 response is strongly inhibited, due to channel internalization. Thus, cell density appears to be a crucial parameter for TRPV4 channel activity.

SUBMITTER: Barbeau S

PROVIDER: S-EPMC9313184 | biostudies-literature | 2022 Jul

REPOSITORIES: biostudies-literature

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Cell Confluence Modulates TRPV4 Channel Activity in Response to Hypoxia.

Barbeau Solène S Joushomme Alexandre A Chappe Yann Y Cardouat Guillaume G Baudrimont Isabelle I Freund-Michel Véronique V Guibert Christelle C Marthan Roger R Berger Patrick P Vacher Pierre P Percherancier Yann Y Quignard Jean-François JF Ducret Thomas T

Biomolecules 20220707 7

Transient receptor potential vanilloid 4 (TRPV4) is a polymodal Ca<sup>2+</sup>-permeable channel involved in various hypoxia-sensitive pathophysiological phenomena. Different tools are available to study channel activity, requiring cells to be cultured at specific optimal densities. In the present study, we examined if cell density may influence the effect of hypoxia on TRPV4 activity. Transiently TRPV4-transfected HEK293T cells were seeded at low or high densities corresponding to non-confluen ...[more]

PMID: 35883510

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Project description:The polymodal transient receptor potential vanilloid 4 (TRPV4) channel, a member of the TRP channel family, is a calcium-permeable cationic channel that is gated by various stimuli such as cell swelling, low pH and high temperature. Therefore, TRPV4-mediated calcium entry may be involved in neuronal and glia pathophysiology associated with various disorders of the central nervous system, such as ischemia. The TRPV4 channel has been recently found in adult rat cortical and hippocampal astrocytes; however, its role in astrocyte pathophysiology is still not defined. In the present study, we examined the impact of cerebral hypoxia/ischemia (H/I) on the functional expression of astrocytic TRPV4 channels in the adult rat hippocampal CA1 region employing immunohistochemical analyses, the patch-clamp technique and microfluorimetric intracellular calcium imaging on astrocytes in slices as well as on those isolated from sham-operated or ischemic hippocampi. Hypoxia/ischemia was induced by a bilateral 15-minute occlusion of the common carotids combined with hypoxic conditions. Our immunohistochemical analyses revealed that 7 days after H/I, the expression of TRPV4 is markedly enhanced in hippocampal astrocytes of the CA1 region and that the increasing TRPV4 expression coincides with the development of astrogliosis. Additionally, adult hippocampal astrocytes in slices or cultured hippocampal astrocytes respond to the TRPV4 activator 4-alpha-phorbol-12,-13-didecanoate (4?PDD) by an increase in intracellular calcium and the activation of a cationic current, both of which are abolished by the removal of extracellular calcium or exposure to TRP antagonists, such as Ruthenium Red or RN1734. Following hypoxic/ischemic injury, the responses of astrocytes to 4?PDD are significantly augmented. Collectively, we show that TRPV4 channels are involved in ischemia-induced calcium entry in reactive astrocytes and thus, might participate in the pathogenic mechanisms of astroglial reactivity following ischemic insult.

Project description:Investigating macrophage plasticity emerges as a promising strategy for promoting tissue regeneration and can be exploited by regulating the transient receptor potential vanilloid 4 (TRPV4) channel. The TRPV4 channel responds to various stimuli including mechanical, chemical, and selective pharmacological compounds. It is well documented that treating cells such as epithelial cells and fibroblasts with a TRPV4 agonist enhances the Ca2+ influx to the cells, which leads to secretion of pro-inflammatory cytokines, while a TRPV4 antagonist reduces both Ca2+ influx and pro-inflammatory cytokine secretion. In this work, we investigated the effect of selective TRPV4 modulator compounds on U937-differentiated macrophages encapsulated within three-dimensional (3D) matrices. Despite offering a more physiologically relevant model than 2D cultures, pharmacological treatment of macrophages within 3D collagen matrices is largely overlooked in the literature. In this study, pro-inflammatory macrophages were treated with an agonist, 500 nM of GSK1016790A (TRPV4(+)), and an antagonist, 10 mM of RN-1734 (TRPV4(-)), to elucidate the modulation of the TRPV4 channel at both cellular and extracellular levels. To evaluate macrophage phenotypic alterations within 3D collagen matrices following TRPV4 modulator treatment, we employed structural techniques (SEM, Masson's trichrome, and collagen hybridizing peptide (CHP) staining), quantitative morphological measures for phenotypic assessment, and genotypic methods such as quantitative real-time PCR (qRT-PCR) and immunohistochemistry (IHC). Our data reveal that pharmacological modulation of the macrophage TRPV4 channel alters the cytoskeletal structure of macrophages and influences the 3D structure encapsulating them. Moreover, we proved that treating macrophages with a TRPV4 agonist and antagonist enhances the expression of pro- and anti-inflammatory genes, respectively, leading to the upregulation of surface markers CD80 and CD206. In the TRPV4(-) group, the CD206 gene and CD206 surface marker were significantly upregulated by 9- and 2.5-fold, respectively, compared to the control group. These findings demonstrate that TRPV4 modulation can be utilized to shift macrophage phenotype within the 3D matrix toward a desired state. This is an innovative approach to addressing inflammation in musculoskeletal tissues.

Dataset Information

Cell Confluence Modulates TRPV4 Channel Activity in Response to Hypoxia.

Publications

Cell Confluence Modulates TRPV4 Channel Activity in Response to Hypoxia.

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