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.
Bicarbonate concentration in saliva is controlled by the action of acid‐base transporters in salivary duct cells. We have previously shown expression of ATP6V1E1 and ATP6V1A in human and rodent salivary ducts and adaptive redistribution to the apical plasma membrane following acute and chronic metabolic acidosis. However, whether such redistribution has any physiological significance has not been elucidated. Using the human submandibular gland cell line HSG, we show for the first time functional expression of V‐ATPase in submandibular duct cells 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, and intracellular H(+) recording with H(+) ‐sensitive dye 2′,7′‐bis‐(carboxyethyl)‐5‐(and‐6)‐carboxyfluorescein, we show that activation of TGF‐β signalling upregulates ATP6V1E1 and ATP6V1B2, and has no effect on ATP6V1A. Following acidosis, neutralization of TGF‐βs or blocking of Smad2/3 phosphorylation 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, V‐ATPase is active in salivary duct cells and involved in intracellular pH regulation following acidosis.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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