RNA sequencing uncovers alterations in corneal endothelial metabolism, pump and barrier functions of Slc4a11 KO mice

Ogando, Diego G.; Bonanno, Joseph A.

doi:10.1016/j.exer.2021.108884

Cited by 6 publications

(11 citation statements)

References 27 publications

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“…SLC4A11 KO mice show corneal edema, stromal lactate accumulation, mitochondrial ROS, and alterations in extracellular matrix and junctional morphology at 7–12 weeks of age [ 35 , 45 , 46 , 49 , 50 ]. RNA-Seq analysis of freshly isolated corneal endothelium from 12-week-old WT and KO mice indicate several gene pathway expression alterations that shed light on a cause for the corneal edema [ 51 ]. Not surprisingly, given the increase in oxidative stress in the SLC4A11 KO, genes for glutathione metabolism were upregulated.…”

Section: Gene Expression Changes In Slc4a11 Knock-outmentioning

confidence: 99%

“…Atp1b2, a component of the Na/K-ATPase, was also downregulated. Using these findings, Ogando et al [ 51 ] found reductions in lactate production, oxygen consumption, glutaminolysis, and Na/K-ATPase activity. There was also upregulation of several junctional proteins (e.g., occludins and claudins) but downregulation of Actin2, which can link cadherins to the actin cytoskeleton.…”

Section: Gene Expression Changes In Slc4a11 Knock-outmentioning

confidence: 99%

“…Endothelium permeability to fluorescein diffusion was more than 50% greater in KO tissue, indicating a loss of the barrier function. Ogando et al [ 51 ] concluded that the metabolic alterations, reduced lactate:H + and Na/K-ATPase transport capacity, and alteration of the osmotic barrier function together can explain the significant edema seen in the SLC4A11 KO.…”

Section: Gene Expression Changes In Slc4a11 Knock-outmentioning

confidence: 99%

“…Using comparative transcriptome analysis of primary cultures of human corneal endothelial cells and SLC4A11 KO MCEC (mouse corneal endothelial cells), Zhang et al [ 44 ] also found inhibition of cell metabolism, ion transport (e.g., SLC4A4-NBCe1), and mitochondrial function, along with the activation of AMPK-p53/ULK1, consistent with increased mitophagy and mitochondrial dysfunction. Downregulation of the sodium bicarbonate transporter (NBCe1) was a key finding with the cultured cells, but this was not seen in KO tissue [ 51 ]. Another key difference was that MCT4 (SLC16A4) was upregulated in cultured cells.…”

Section: Gene Expression Changes In Slc4a11 Knock-outmentioning

confidence: 99%

“…In this regard, Parker [ 54 ] and Nehrke [ 58 ] have put forth a hypothesis, yet to be tested, that SLC4A11 facilitates lactate:H + flux and the corneal endothelium pump by actively balancing pH i . SLC4A11 KO shows reduced lactate production and MCT4 expression in corneal endothelium, suggesting that there is some functional association [ 51 , 52 ] and that SLC4A11 activity is linked to lactate transport and specifically to MCT4, which is also on the basolateral membrane. H + flux via SLC4A11 can proceed in either direction to balance pH i , according to Nehrke; however the electrochemical gradient favors H + influx.…”

Section: Slc4a11 Plasma Membrane Functionmentioning

confidence: 99%

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The H+ Transporter SLC4A11: Roles in Metabolism, Oxidative Stress and Mitochondrial Uncoupling

Bonanno¹,

Shyam²,

Choi³

et al. 2022

Cells

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Solute-linked cotransporter, SLC4A11, a member of the bicarbonate transporter family, is an electrogenic H+ transporter activated by NH3 and alkaline pH. Although SLC4A11 does not transport bicarbonate, it shares many properties with other members of the SLC4 family. SLC4A11 mutations can lead to corneal endothelial dystrophy and hearing deficits that are recapitulated in SLC4A11 knock-out mice. SLC4A11, at the inner mitochondrial membrane, facilitates glutamine catabolism and suppresses the production of mitochondrial superoxide by providing ammonia-sensitive H+ uncoupling that reduces glutamine-driven mitochondrial membrane potential hyperpolarization. Mitochondrial oxidative stress in SLC4A11 KO also triggers dysfunctional autophagy and lysosomes, as well as ER stress. SLC4A11 expression is induced by oxidative stress through the transcription factor NRF2, the master regulator of antioxidant genes. Outside of the corneal endothelium, SLC4A11’s function has been demonstrated in cochlear fibrocytes, salivary glands, and kidneys, but is largely unexplored overall. Increased SLC4A11 expression is a component of some “glutamine-addicted” cancers, and is possibly linked to cells and tissues that rely on glutamine catabolism.

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Section: Gene Expression Changes In Slc4a11 Knock-outmentioning

confidence: 99%

Section: Gene Expression Changes In Slc4a11 Knock-outmentioning

confidence: 99%

Section: Gene Expression Changes In Slc4a11 Knock-outmentioning

confidence: 99%

Section: Gene Expression Changes In Slc4a11 Knock-outmentioning

confidence: 99%

Section: Slc4a11 Plasma Membrane Functionmentioning

confidence: 99%

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The H+ Transporter SLC4A11: Roles in Metabolism, Oxidative Stress and Mitochondrial Uncoupling

Bonanno¹,

Shyam²,

Choi³

et al. 2022

Cells

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Effect of Physiological Oxygen on Primary Human Corneal Endothelial Cell Cultures

Patel

Gonzalez

Paone

et al. 2022

Trans. Vis. Sci. Tech.

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Purpose Primary human corneal endothelial cells (HCEnCs) cultured in room air are exposed to significantly higher O 2 concentrations [O 2 ] than what is normally present in the eye. We evaluated the growth and metabolism of HCEnCs cultured under physiological [O 2 ] (2.5%; [O 2 ] 2.5 ) and room air ([O 2 ] A ). Methods Primary cultures of HCEnCs from normal donors and donors with Fuchs dystrophy were grown at [O 2 ] 2.5 and [O 2 ] A . Growth and morphology were compared using phase-contrast microscopy, zonula occludens (ZO-1) localization, cell density measurements, and senescence marker staining. CD44 (cell quality) and HIF-1α (hypoxia-inducible factor-1α) levels were evaluated by Western blotting. Cell adaptability to a reversal of [O 2 ] growth conditions was measured with cell viability assays, and cell metabolism was assessed via oxygen consumption and extracellular acidification rates. Results HCEnCs grown at [O 2 ] A and [O 2 ] 2.5 displayed similar morphologies, ZO-1 localization, CD44 expression, and senescence. Cells from donors with Fuchs dystrophy grew better at [O 2 ] 2.5 than at [O 2 ] A . HIF-1α was undetectable. Cells displayed greater viability at [O 2 ] 2.5 than at [O 2 ] A . HCEnCs showed significantly greater proton leak ( P < 0.01), nonmitochondrial oxygen consumption ( P < 0.01), and spare capacity ( P < 0.05) for oxygen consumption rates, and greater basal glycolysis ( P < 0.05) with a decreased glycolytic reserve capacity ( P < 0.05) for extracellular acidification rates. Conclusions Primary HCEnCs show unique metabolic characteristics at physiologic [O 2 ]. The effect of [O 2 ] for optimization of HCEnC culture conditions should be considered. Translational Relevance With the advance of cell-based therapeutics for corneal endothelial diseases, [O 2 ] should be considered an important variable in the optimization of HCEnC culture conditions.

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Investigation of the functional impact of CHED- and FECD4-associated SLC4A11 mutations in human corneal endothelial cells

Chung,

Chen,

Choo

et al. 2024

PLoS ONE

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Mutations in the solute linked carrier family 4 member 11 (SLC4A11) gene are associated with congenital hereditary endothelial dystrophy (CHED) and Fuchs corneal endothelial dystrophy type 4 (FECD4), both characterized by corneal endothelial cell (CEnC) dysfunction and/or cell loss leading to corneal edema and visual impairment. In this study, we characterize the impact of CHED-/FECD4-associated SLC4A11 mutations on CEnC function and SLC4A11 protein localization by generating and comparing human CEnC (hCEnC) lines expressing wild type SLC4A11 (SLC4A11WT) or mutant SLC4A11 harboring CHED-/FECD4-associated SLC4A11 mutations (SLC4A11MU). SLC4A11WT and SLC4A11MU hCEnC lines were generated to express either SLC4A11 variant 2 (V2WT and V2MU) or variant 3 (V3WT and V3MU), the two major variants expressed in ex vivo hCEnC. Functional assays were performed to assess cell barrier, proliferation, viability, migration, and NH3-induced membrane conductance. We demonstrate SLC4A11-/- and SLC4A11MU hCEnC lines exhibited increased migration rates, altered proliferation and decreased cell viability compared to SLC4A11WT hCEnC. Additionally, SLC4A11-/- hCEnC demonstrated decreased cell-substrate adhesion and membrane capacitances compared to SLC4A11WT hCEnC. Induction with 10mM NH4Cl led SLC4A11WT hCEnC to depolarize; conversely, SLC4A11-/- hCEnC hyperpolarized and the majority of SLC4A11MU hCEnC either hyperpolarized or had minimal membrane potential changes following NH4Cl induction. Immunostaining of primary hCEnC and SLC4A11WT hCEnC lines for SLC4A11 demonstrated predominately plasma membrane staining with poor or partial colocalization with mitochondrial marker COX4 within a subset of punctate subcellular structures. Overall, our findings suggest CHED-associated SLC4A11 mutations likely lead to hCEnC dysfunction, and ultimately CHED, by interfering with cell migration, proliferation, viability, membrane conductance, barrier function, and/or cell surface localization of the SLC4A11 protein in hCEnC. Additionally, based on their similar subcellular localization and exhibiting similar cell functional profiles, protein isoforms encoded by SLC4A11 variant 2 and variant 3 likely have highly overlapping functional roles in hCEnC.

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RNA sequencing uncovers alterations in corneal endothelial metabolism, pump and barrier functions of Slc4a11 KO mice

Cited by 6 publications

References 27 publications

The H+ Transporter SLC4A11: Roles in Metabolism, Oxidative Stress and Mitochondrial Uncoupling

The H+ Transporter SLC4A11: Roles in Metabolism, Oxidative Stress and Mitochondrial Uncoupling

Effect of Physiological Oxygen on Primary Human Corneal Endothelial Cell Cultures

Investigation of the functional impact of CHED- and FECD4-associated SLC4A11 mutations in human corneal endothelial cells

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