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

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.