The Ca2+ channel TRPV4 is dispensable for Ca2+ influx and cell volume regulation during hypotonic stress response in human keratinocyte cell lines

Ritzmann, Dirk; Jahn, Magdalena; Heck, Susanne; Jung, Cristina; Cesetti, Tiziana; Couturier, Nathalie; Rudolf, Rüdiger; Reuscher, Naemi; Buerger, Claudia; Rauh, Oliver; Fauth, Torsten

doi:10.1016/j.ceca.2023.102715

Cited by 4 publications

(7 citation statements)

References 60 publications

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“…Keratinocytes immortalized by the expression of SV40 large T antigen and hTERT (NHK‐SV/TERT3‐5) were purchased from Evercyte GmbH. The proprietary cell line NHK‐E6/E7 was generated by lentiviral transduction of juvenile NHKs (purchased from PromoCell) using HPV16‐E6/E7 human papillomavirus oncogenes (BRAIN Biotech AG) 32 . The latter three cell types were cultured in KGM‐2 (PromoCell).…”

Section: Methodsmentioning

confidence: 99%

“…The proprietary cell line NHK-E6/E7 was generated by lentiviral transduction of juvenile NHKs (purchased from PromoCell) using HPV16-E6/E7 human papillomavirus oncogenes (BRAIN Biotech AG). 32 The latter three cell types were cultured in KGM-2 (PromoCell).…”

Section: Cells and Cell Culturementioning

confidence: 99%

“…However, the degree of similarity with primary keratinocytes is divergent especially with regard to their ability to differentiate and reconstitute a stratified epidermis and a comprehensive analysis comparing cell lines immortalized by different approaches is lacking. Thus, we aimed at comparing the commercially available cell line NHK‐SV/TERT (NHEK/SV‐TERT3‐5, Evercyte GmbH), generated from adult abdominal keratinocytes by overexpression of SV40 large T antigen and hTERT, 30,31 and our own cell line NHK‐E6/E7 32 generated from juvenile keratinocytes after retroviral transfection with HPV oncogenes E6/E7. These cell lines are evaluated regarding their growth and differentiation capacities and suitability as in vitro models in comparison to primary NHK and HaCaT cells.…”

Section: Introductionmentioning

confidence: 99%

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Different immortalized keratinocyte cell lines display distinct capabilities to differentiate and reconstitute an epidermis in vitro

Jahn,

Lang,

Diehl

et al. 2023

Experimental Dermatology

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Dermatological research relies on the availability of suitable models that most accurately reflect the in vivo situation. Primary keratinocytes obtained from skin reduction surgeries are not only limited by availability but have a short lifespan and show donor‐specific variations, which hamper the understanding of general mechanisms. The spontaneously immortalized keratinocyte cell line HaCaT displays chromosomal aberrations and is known to differentiate in an abnormal manner. To overcome these issues, we validated different engineered immortalized cell lines created from primary human keratinocytes (NHK) as model systems to study epidermal function. Cell lines either immortalized by the expression of SV40 large T antigen and hTERT (NHK‐SV/TERT) or by transduction with HPV E6/E7 (NHK‐E6/E7) were analysed for their growth and differentiation behaviour using 2D and 3D culture systems and compared to primary keratinocytes. Both cell lines displayed a robust proliferative behaviour but were still sensitive to contact inhibition. NHK‐E6/E7 could be driven into differentiation by Ca2+ switch, while NHK‐SV/TERT needed withdrawal from any proliferative signal to initiate a delayed onset of differentiation. In 3D epidermal models both cell lines were able to reconstitute a stratified epidermis and functional epidermal barrier. However, only NHK‐E6/E7 showed a degree of epidermal maturation and stratification that was comparable to primary keratinocytes.

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Section: Methodsmentioning

confidence: 99%

Section: Cells and Cell Culturementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Different immortalized keratinocyte cell lines display distinct capabilities to differentiate and reconstitute an epidermis in vitro

Jahn,

Lang,

Diehl

et al. 2023

Experimental Dermatology

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“…However, volume regulation is not exclusive of AQP4 and TRPV4 functional complex being TRPV4 a volume-sensor (Ritzmann et al, 2023;Toft-Bertelsen et al, 2017, 2018 reacting to cell volume increases irrespective of the specific molecular mechanisms triggering cell swelling, which can be prompted by osmotic changes via AQPs or other factors leading to an influx of water into the cell (Toft-Bertelsen et al, 2017). The coupling between TRPV4-mediated calcium influx and RVD is still unclear but likely coupled to downstream unidentified effectors, promoting the loss of electrolytes and water (Mola et al, 2016;Toft-Bertelsen et al, 2018).…”

Section: Introductionmentioning

confidence: 99%

Aquaporin‐4 and transient receptor potential vanilloid 4 balance in early postnatal neurodevelopment

Cibelli,

Mola,

Saracino

et al. 2024

Glia

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In the adult brain, the water channel aquaporin‐4 (AQP4) is expressed in astrocyte endfoot, in supramolecular assemblies, called “Orthogonal Arrays of Particles” (OAPs) together with the transient receptor potential vanilloid 4 (TRPV4), finely regulating the cell volume. The present study aimed at investigating the contribution of AQP4 and TRPV4 to CNS early postnatal development using WT and AQP4 KO brain and retina and neuronal stem cells (NSCs), as an in vitro model of astrocyte differentiation. Western blot analysis showed that, differently from AQP4 and the glial cell markers, TRPV4 was downregulated during CNS development and NSC differentiation. Blue native/SDS‐PAGE revealed that AQP4 progressively organized into OAPs throughout the entire differentiation process. Fluorescence quenching assay indicated that the speed of cell volume changes was time‐related to NSC differentiation and functional to their migratory ability. Calcium imaging showed that the amplitude of TRPV4 Ca2+ transient is lower, and the dynamics are changed during differentiation and suppressed in AQP4 KO NSCs. Overall, these findings suggest that early postnatal neurodevelopment is subjected to temporally modulated water and Ca2+ dynamics likely to be those sustaining the biochemical and physiological mechanisms responsible for astrocyte differentiation during brain and retinal development.

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“…Removal of the distal N-terminus results in loss of sensitivity to swelling whereas its replacement with a truncated domain of the cognate TRPV1 converted TRPV4 into a shrinking sensor (Toft-Bertelsen, Yarishkin et al 2019). The channel has been implicated in osmosignaling in neurons, glia, epithelial cells, endothelial cells, heterologously expressing HEK293 cells and oocytes (Liedtke and Friedman 2003, Ryskamp, Jo et al 2014, Phuong, Redmon et al 2017, Toft-Bertelsen, Yarishkin et al 2019, Lapajne, Lakk et al 2020, Sonkusare and Laubach 2022 but not keratinocytes (Ritzmann, Jahn et al 2023).…”

Section: Introductionmentioning

confidence: 99%

Osmosensing in trabecular meshwork cells

Baumann,

Yarishkin,

Lakk

et al. 2024

Preprint

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Aqueous humor drainage from the anterior eye constitutes a key determinant of intraocular pressure (IOP) under homeostatic and pathological conditions. Swelling of the trabecular meshwork (TM) increases its flow resistance but the mechanisms that sense and transduce osmotic gradients remain poorly understood. We used optical molecular analyses, optical imaging and electrophysiology to investigate TM osmotransduction and its role in calcium and chloride homeostasis. Anisosmotic conditions elicited proportional changes in TM cell volume. Swelling, but not shrinking, evoked increases in intracellular calcium concentration [Ca2+]TM. Hypotonicity-evoked calcium signals were sensitive to HC067047, a selective blocker of TRPV4 channels, whereas the agonist GSK1016790A promoted swelling under isotonic conditions. TRPV4 inhibition partially suppressed hypotonicity-induced volume increases and reduced the magnitude of the swelling-induced membrane current, with a substantial fraction of the swelling-evoked current abrogated by Cl-channel antagonists DIDS and niflumic acid. The volume-sensing transcriptome of primary human TM cells showed expression of TRPV4, TRPM4, AQP1, and TMEMC3B genes. Cl-channel expression was dominated by ANO6 transcripts, auxiliary levels of ANO3, ANO7 and ANO10 and modest expression of LTTRC genes that encode volume-activated anion channels. Thus, TRPV4-mediated cation influx works with Cl-efflux to sense and respond to osmotic stress, potentially contributing to pathological swelling, calcium overload and intracellular signaling that could exacerbate functional disturbances in inflammatory disease and glaucoma.

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The Ca2+ channel TRPV4 is dispensable for Ca2+ influx and cell volume regulation during hypotonic stress response in human keratinocyte cell lines

Cited by 4 publications

References 60 publications

Different immortalized keratinocyte cell lines display distinct capabilities to differentiate and reconstitute an epidermis in vitro

Different immortalized keratinocyte cell lines display distinct capabilities to differentiate and reconstitute an epidermis in vitro

Aquaporin‐4 and transient receptor potential vanilloid 4 balance in early postnatal neurodevelopment

Osmosensing in trabecular meshwork cells

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