Rat aquaporin-5 4.3-kb 5′-flanking region differentially regulates expression in salivary gland and lung in vivo

Abstract

We previously cloned a 4.3-kb genomic fragment encompassing 5'-flanking regulatory elements of rat aquaporin-5 (Aqp5) that demonstrated preferential transcriptional activity in lung and salivary cells in vitro. To investigate the ability of Aqp5 regulatory elements to direct transgene expression in vivo, transgenic (TG) mice and rats were generated in which the 4.3-kb Aqp5 fragment directed the expression of enhanced green fluorescent protein (EGFP). RT-PCR revealed relative promoter specificity for the lung and salivary glands in TG mice. Immunofluorescence microscopy showed strong EGFP expression in salivary acinar cells but not in lung type I (AT1) cells, both known sites of endogenous AQP5 expression. Similar results were obtained in TG rats generated by lentiviral transgenesis. EGFP mRNA was detected in both salivary glands and lung. Robust EGFP fluorescence was observed in frozen sections of the rat salivary gland but not in the lung or other tested tissues. The percentage of EGFP-positive acinar cells was increased in parotid and submandibular glands of TG rats receiving a chronic injection of the beta-adrenergic receptor agonist isoproterenol. EGFP-positive cells in the lung that were also reactive with the AT1-cell specific monoclonal antibody VIIIB2 were identified by flow cytometry. These findings demonstrate that the 4.3-kb Aqp5 promoter/enhancer directs strong cell-specific transgene expression in salivary gland and low-level AT1 cell-specific expression in the lung. While these Aqp5 regulatory elements should be useful for functional studies in salivary glands, additional upstream or intronic cis-active elements are likely required for robust expression in the lung.

p300/β-Catenin Interactions Regulate Adult Progenitor Cell Differentiation Downstream of WNT5a/Protein Kinase C (PKC)

“…p300 Regulates Promoter of AT1 Cell-specific Gene Aqp5-Increased p300/␤-catenin interaction during transdifferentiation led us to examine effects of p300 on transcriptional activation of Aqp5, a prototypic marker of AT1 cell differentiation (23,28). We utilized a construct containing a 4.3-kb segment of the rat Aqp5 promoter previously cloned 5Ј to the luciferase (Luc) reporter gene (26) that contains several TCF elements potentially responsive to Wnt signaling (Fig.…”

Integrated Transcriptomic and Epigenomic Analysis of Primary Human Lung Epithelial Cell Differentiation

“…Western blots were performed as previously described [16]. Primary antibodies (all rabbit) were anti-AQP5 (Alomone Labs AQP-005), anti-CAV1 (Abcam ab2910), anti-pro-SFTPC (Millipore AB3786) and anti-LAMIN A/C (sc-20681, Santa Cruz Biotechnology).…”

“…In contrast, examining epigenetic differences between two closely related yet phenotypically distinct cell types might offer more straightforward insights into the relationship between epigenetic changes and the establishment of new expression patterns. Isolated distal lung epithelial cells offer a compelling model system; primary human alveolar epithelial type 2 (AT2) cells can be purified in large numbers from remnant transplant lung, and can be differentiated in vitro in a manner closely mimicking both normal maintenance and regeneration following lung injury [15], [16].…”

Alveolar Epithelium: Beyond the Barrier

“…To elucidate mechanisms regulating AEC phenotype and further characterize interrelationships between AT2 and AT1 cells, we characterized the promoter of the water channel, aquaporin-5, which, within distal lung epithelium, is selectively expressed in AT1 cells (25,26). We used Aqp5 as a prototype AT1 cell gene with which to elucidate molecular mechanisms that regulate phenotype transitions between AT2 and AT1 cells by transcriptional activators (e.g., GATA-binding factor 6 (27) and repressors (e.g., Forkhead box p2 and A1 Foxp2 and FoxA1 (28,29) that are also implicated in lung development through their interactions with other lung-enriched transcription factors (TFs).…”