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.

The temporal dynamic expression of Sonic Hedgehog (SHH) and signaling during early midbrain dopaminergic (mDA) neuron development is one of the key players in establishing mDA progenitor diversity. However, whether SHH signaling is also required during later developmental stages and in mature mDA neurons is less understood. We study the expression of SHH receptors Ptch1 and Gas1 (growth arrest-specific 1) and of the transcription factors Gli1, Gli2 and Gli3 in mouse midbrain during embryonic development [embryonic day (E) 12.5 onwards)], in newborn and adult mice using in situ hybridization and immunohistochemistry. Moreover, we examine the expression and regulation of dopaminergic neuronal progenitor markers, midbrain dopaminergic neuronal markers and markers of the SHH signaling pathway in undifferentiated and butyric acid-treated (differentiated) MN9D cells in the presence or absence of exogenous SHH in vitro by RT-PCR, immunoblotting and immunocytochemistry. Gli1 was expressed in the lateral mesencephalic domains, whereas Gli2 and Gli3 were expressed dorsolaterally and complemented by ventrolateral expression of Ptch1. Co-localization with tyrosine hydroxylase could not be observed. GAS1 was exclusively expressed in the dorsal mesencephalon at E11.5 and co-localized with Ki67. In contrast, MN9D cells expressed all the genes investigated and treatment of the cells with butyric acid significantly upregulated their expression. The results suggest that SHH is only indirectly involved in the differentiation and survival of mDA neurons and that the MN9D cell line is a valuable model for investigating early development but not the differentiation and survival of mDA neurons.

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