Dataset Information

Artificial Induction of Native Aquaporin-1 Expression in Human Salivary Cells.

ABSTRACT: Gene therapy for dry mouth disorders has transitioned in recent years from theoretical to clinical proof of principle with the publication of a first-in-man phase I/II dose escalation clinical trial in patients with radiation-induced xerostomia. This trial used a prototype adenoviral vector to express aquaporin-1 (AQP1), presumably in the ductal cell layer and/or in surviving acinar cells, to drive transcellular flux of interstitial fluid into the labyrinth of the salivary duct. As the development of this promising gene therapy continues, safety considerations are a high priority, particularly those that remove nonhuman agents (i.e., viral vectors and genetic sequences of bacterial origin). In this study, we applied 2 emerging technologies, artificial transcriptional complexes and epigenetic editing, to explore whether AQP1 expression could be achieved by activating the native gene locus in a human salivary ductal cell line and primary salivary human stem/progenitor cells (hS/PCs), as opposed to the conventional approach of cytomegalovirus promoter-driven expression from an episomal vector. In our first study, we used a cotransfection strategy to express the components of the dCas9-SAM system to create an artificial transcriptional complex at the AQP1 locus in A253 and hS/PCs. We found that AQP1 expression was induced at a magnitude comparable to adenoviral infection, suggesting that AQP1 is primarily silenced through pretranscriptional mechanisms. Because earlier literature suggested that pretranscriptional silencing of AQP1 in salivary glands is mediated by methylation of the promoter, in our second study, we performed global, chemical demethylation of A253 cells and found that demethylation alone induced robust AQP1 expression. These results suggest the potential for success by inducing AQP1 expression in human salivary ductal cells through epigenetic editing of the native promoter.

SUBMITTER: Wang Z

PROVIDER: S-EPMC5384490 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

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Artificial Induction of Native Aquaporin-1 Expression in Human Salivary Cells.

Wang Z Z Pradhan-Bhatt S S Farach-Carson M C MC Passineau M J MJ

Journal of dental research 20170110 4

Gene therapy for dry mouth disorders has transitioned in recent years from theoretical to clinical proof of principle with the publication of a first-in-man phase I/II dose escalation clinical trial in patients with radiation-induced xerostomia. This trial used a prototype adenoviral vector to express aquaporin-1 (AQP1), presumably in the ductal cell layer and/or in surviving acinar cells, to drive transcellular flux of interstitial fluid into the labyrinth of the salivary duct. As the developme ...[more]

PMID: 28072927

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Project description:The choroid plexus (CP) consists of specialized ependymal cells and underlying blood vessels and stroma producing the bulk of the cerebrospinal fluid (CSF). CP epithelial cells are considered the site of the internal blood-cerebrospinal fluid barrier, show epithelial characteristics (basal lamina, tight junctions), and express aquaporin-1 (AQP1) apically. In this study, we analyzed the expression of aquaporins in the human CP using immunofluorescence and qPCR. As previously reported, AQP1 was expressed apically in CP epithelial cells. Surprisingly, and previously unknown, many cells in the CP epithelium were also positive for aquaporin-4 (AQP4), normally restricted to ventricle-lining ependymal cells and astrocytes in the brain. Expression of AQP1 and AQP4 was found in the CP of all eight body donors investigated (3 males, 5 females; age 74-91). These results were confirmed by qPCR, and by electron microscopy detecting orthogonal arrays of particles. To find out whether AQP4 expression correlated with the expression pattern of relevant transport-related proteins we also investigated expression of NKCC1, and Na/K-ATPase. Immunostaining with NKCC1 was similar to AQP1 and revealed no particular pattern related to AQP4. Co-staining of AQP4 and Na/K-ATPase indicated a trend for an inverse correlation of their expression. We hypothesized that AQP4 expression in the CP was caused by age-related changes. To address this, we investigated mouse brains from young (2 months), adult (12 months) and old (30 months) mice. We found a significant increase of AQP4 on the mRNA level in old mice compared to young and adult animals. Taken together, we provide evidence for AQP4 expression in the CP of the aging brain which likely contributes to the water flow through the CP epithelium and CSF production. In two alternative hypotheses, we discuss this as a beneficial compensatory, or a detrimental mechanism influencing the previously observed CSF changes during aging.

Dataset Information

Artificial Induction of Native Aquaporin-1 Expression in Human Salivary Cells.

Publications

Artificial Induction of Native Aquaporin-1 Expression in Human Salivary Cells.

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