TGF‐β signaling directly regulates transcription and functional expression of the electrogenic sodium bicarbonate cotransporter 1, NBCe1 (SLC4A4), via Smad4 in mouse astrocytes

Abstract

The electrogenic sodium bicarbonate cotransporter NBCe1 (SLC4A4) expressed in astrocytes regulates intracellular and extracellular pH. Here, we introduce transforming growth factor beta (TGF‐β) as a novel regulator of NBCe1 transcription and functional expression. Using hippocampal slices and primary hippocampal and cortical astrocyte cultures, we investigated regulation of NBCe1 and elucidated the underlying signaling pathways by RT‐PCR, immunoblotting, immunofluorescence, intracellular H(+) recording using the H(+) ‐sensitive dye 2′,7′‐bis‐(carboxyethyl)‐5‐(and‐6)‐carboxyfluorescein, mink lung epithelial cell (MLEC) assay, and chromatin immunoprecipitation. Activation of TGF‐β signaling significantly upregulated transcript, protein, and surface expression of NBCe1. These effects were TGF‐β receptor‐mediated and suppressed following inhibition of JNK and Smad signaling. Moreover, 4‐aminopyridine (4AP)‐dependent NBCe1 regulation requires TGF‐β. TGF‐β increased the rate and amplitude of intracellular H+ changes upon challenging NBCe1 in wild‐type astrocytes but not in cortical astrocytes from Slc4a4‐deficient mice. A Smad4 binding sequence was identified in the NBCe1 promoter and Smad4 binding increased after activation of TGF‐β signaling. The data show for the first time that NBCe1 is a direct target of TGF‐β/Smad4 signaling. Through activation of the canonical pathway TGF‐β acts directly on NBCe1 by binding of Smad4 to the NBCe1 promoter and regulating its transcription, followed by increased protein expression and transport activity.

Functional expression of electrogenic sodium bicarbonate cotransporter 1 (NBCe1) in mouse cortical astrocytes is dependent on S255‐257 and regulated by mTOR

“…We have previously elucidated two regulatory mechanisms for astrocytic NBCe1 transport activity. NBCe1 is regulated by 4AP through increased protein abundance and surface expression (Schrödl-Häußel et al, 2015), and is a direct target of TGF-β, a crucial molecular player in the context of epilepsy (Khakipoor et al, 2017 We showed that extracellular alkalosis downregulates NBCe1 activity in mouse cortical astrocytes with unchanged surface protein abundance and cell survival. We also demonstrated that activation of mTOR is sufficient to rescue alkalosis-induced decreased NBCe1 activity.…”

“…We have previously elucidated two regulatory mechanisms for astrocytic NBCe1 transport activity. NBCe1 is regulated by 4AP through increased protein abundance and surface expression (Schrödl‐Häußel et al, ), and is a direct target of TGF‐β, a crucial molecular player in the context of epilepsy (Khakipoor et al, ). Here we have extended our previous work by first addressing the questions if astrocytic NBCe1 is regulated after long‐term extracellular alkalosis and, if yes, what are the underlying molecular mechanisms.…”

“…The changes in [H + ] i were monitored using the ratio F (440)/ F (488). The ratio was converted into pH and absolute intracellular proton concentrations ([H + ] i ) by using the nigericin‐based (4 μM) calibration technique (Khakipoor et al, ; Theparambil et al, ). Cells were perfused with calibration solution containing (in mM): KCl 145, NaH 2 PO 4 0.4, Na 2 HPO 4 1.6, Glucose 5, MgCl 2 6H 2 O 1, Ca‐DiGluconat H 2 O 1.3, adjusted at pH 6.5, 7.0, 7.5, and 8.0.…”

“…All protocols were carried out in accordance with German ethical guidelines for laboratory animals and approved by the Institutional (Khakipoor et al, 2017). Cortices were dissected and immediately collected in cold HBSS on ice.…”

TGF-β Signaling Regulates SLC8A3 Expression and Prevents Oxidative Stress in Developing Midbrain Dopaminergic and Dorsal Raphe Serotonergic Neurons

“…In neurons lacking TGF-β signaling, the number of MDA-and 4-hydroxynonenal (4-HNE)-positive cells was significantly increased, accompanied with increased cellular 4-HNE abundance. These results suggest that TGF-β contributes to the regulation of SLC8A3 expression in developing dopaminergic and dorsal raphe serotonergic neurons, thereby preventing oxidative stress.Cellular ionic homeostasis is a prerequisite for proper cellular function and survival, whereby TGF-βs are known to regulate several channels and transporters within or outside the central nervous system [6][7][8][9]. Particularly, the link between TGF-βs and Ca 2+ homeostasis has been documented in several cellular paradigms: In cortical neurons, TGF-β regulates L-type Ca 2+ channels through MEK, JNK1/2, and p38 MAPK signaling [10]; it increases store-operated Ca 2+ entry into megakaryocytes [11]; and it enhances Ca 2+ influx pathways and the expression of transient receptor potential canonical channels (TRPCs) in human cardiac fibroblasts.…”