Acidosis-induced V-ATPase trafficking in salivary ducts is initiated by cAMP/PKA/CREB pathway via regulation of Rab11b expression

Journal Cellular Physiology

et al. 2019

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Bicarbonate concentration in saliva is controlled by the action of acid–base transporters in salivary duct cells. We show for the first time expression of ATP6V1B1 in submandibular gland and introduce transforming growth factor‐beta (TGF‐β) as a novel regulator of V‐ATPase subunits. Using QRT‐PCR, immunoblotting, biotinylation of surface proteins, immunofluorescence, chromatin immunoprecipitation, and intracellular H(+) recording with H(+)‐sensitive dye 2′,7′‐bis‐(carboxyethyl)‐5‐(and‐6)‐carboxyfluorescein we show that in the human submandibular gland (HSG) cell line, activation of TGF‐β signaling upregulates ATP6V1E1 and ATP6V1B2, downregulates ATP6V1B1, and has no effect on ATP6V1A. TGF‐β1 effects on ATP6V1B1 are mediated through the canonical, the soluble adenylate cyclase, and ERK signaling. A CREB binding sequence was identified in the ATP6V1B1 promoter and CREB binding decreased after TGF‐β1 treatment. Following acidosis, a bafilomycin‐sensitive and Na+‐independent cell pH recovery was observed in HSG cells, an effect that was not influenced after disruption of acidic lysosomes. Moreover, neutralization of TGF‐βs, inhibition of TGF‐β receptor, or inhibition of the canonical pathway decreased membrane expression of ATP6V1A and prevented the acidosis‐induced increased V‐ATPase activity. The results suggest multiple modes of action of TGF‐β1 on V‐ATPase subunits in HSG cells: TGF‐β1 may regulate transcription or protein synthesis of certain subunits and trafficking of other subunits in a context‐dependent manner. Moreover, surface V‐ATPase is active in salivary duct cells and involved in intracellular pH regulation following acidosis.

“…1.21 ± 0.03 fold) as previously reported (Oehlke et al, 2012). ATP6V1A1 redistribution towards the cell membrane was also prevented in the presence of SIS3.…”

Section: Tgf-β1 Regulates Atp6v1b1 Expression Via Multiple Signalinsupporting

confidence: 87%

Differential regulation of vacuolar H⁺‐ATPase subunits by transforming growth factor‐β1 in salivary ducts

Kluge

Namkoong

Journal Cellular Physiology

et al. 2019

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“…Since exposure of cells to extracellular acid–base changes for 6 hr has been shown to induce incorporation or retrieval of acid–base transporters, including NBCe1, to/from the cell membrane in other cells paradigms (Oehlke, Martin, Osterberg, & Roussa, ; Oehlke, Schlosshardt, Feuerstein, & Roussa, ; Oehlke, Speer, & Roussa, ), we have performed biotinylation of surface proteins and enrichment. Subsequent immunoblotting (Figure d) revealed that NBCe1 membrane expression was comparable between control and alkalotic astrocytes (1.00 ± 0.07 fold, not significant, using the two‐tailed unpaired Student's t test, n = 6).…”

Section: Resultsmentioning

confidence: 99%

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

Giannaki

Theparambil

et al. 2019

Glia

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The electrogenic sodium bicarbonate cotransporter 1, NBCe1 (SLC4A4), is the major bicarbonate transporter expressed in astrocytes. It is highly sensitive for bicarbonate and the main regulator of intracellular, extracellular, and synaptic pH, thereby modulating neuronal excitability. However, despite these essential functions, the molecular mechanisms underlying NBCe1‐mediated astrocytic response to extracellular pH changes are mostly unknown. Using primary mouse cortical astrocyte cultures, we investigated the effect of long‐term extracellular metabolic alkalosis on regulation of NBCe1 and elucidated the underlying molecular mechanisms by immunoblotting, biotinylation of surface proteins, intracellular H+ recording using the H+‐sensitive dye 2′,7′‐bis‐(carboxyethyl)‐5‐(and‐6)‐carboxyfluorescein, and phosphoproteomic analysis. The results showed significant downregulation of NBCe1 activity following metabolic alkalosis without influencing protein abundance or surface expression of NBCe1. During alkalosis, the rate of intracellular H+ changes upon challenging NBCe1 was decreased in wild‐type astrocytes, but not in cortical astrocytes from NBCe1‐deficient mice. Alkalosis‐induced decrease of NBCe1 activity was rescued after activation of mTOR signaling. Moreover, mass spectrometry revealed constitutively phosphorylated S255‐257 and mutational analysis uncovered these residues being crucial for NBCe1 transport activity. Our results demonstrate a novel mTOR‐regulated mechanism by which NBCe1 functional expression is regulated. Such mechanism likely applies not only for NBCe1 in astrocytes, but in epithelial cells as well.

“…Total RNA was isolated from cortical astrocytes as described earlier (Oehlke, Schlosshardt, Feuerstein, & Roussa, 2012) and subsequently 1.0 mg was reverse-transcribed. Primers for NBCe1 were (corresponding to nucleotides 62-114; Genbank accession number AF210250; Giffard et al, 2000): 5 0 -TGGAGGATGAAGCTGTCC -3 0 as forward primer and 5 0 -ACACACATGTTTAAGGAAGGAA -3 0 as reverse primer.…”

Section: Quantitative Real-time Pcrmentioning

confidence: 99%

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

Ophoven

Schrödl-Häußel

et al. 2017

Glia

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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.