SummaryAquaporin (AQP) is a water-selective channel protein. In the brain, AQPs play critical roles in the production of cerebrospinal fluid and in edema formation. In contrast, the expression and role of AQPs in spinal cord are unclear. We aimed to investigate the localization of AQP1 and AQP4 in normal rat spinal cord compared with the expression of marker proteins for astrocytes, neurons, and endothelial cells. Immunohistochemistry demonstrated that AQP1 and AQP4 are expressed along all levels of the spinal cord from the cervical to lumbar levels. AQP1 immunolabeling was observed in the dorsal horns in the gray matter, whereas the labeling was weak and mainly seen close to glia limitans in the white matter. AQP1 was co-labeled with marker proteins for unmyelinated neuronal fibers (peripherin) and endothelial cells (RECA-1) of blood vessels that had penetrated through the glia limitans. In contrast, AQP1 did not colocalize with GFAP, an astrocyte marker, at any level of the spinal cord. AQP4 was exclusively localized at the astrocytes, but AQP4 expression in spinal cord exhibited a less polarized and more spatial distribution than that of brain astrocytes. The observed characteristic localization and expression patterns of AQP1 and AQP4 could provide insights toward gaining an understanding of the role of AQPs in the spinal cord. (J Histochem Cytochem 62:598-611, 2014)

Background Increased expression of MDR1 gene is one of the major mechanisms responsible for multidrug resistance in cancer cells. Two alternative promoters, upstream and downstream, are responsible for transcription of MDR1 gene in the human. However, the molecular mechanism regarding the transactivation of MDR1 upstream promoter (USP) has not been determined. Methods Dual-luciferase reporter gene assays were used to assess the effect of Nkx-2.5 on MDR1 USP activity using reporter plasmids for human MDR1 USP and its mutants. MDR1 mRNA level was examined by quantitative real-time PCR. The direct binding of Nkx-2.5 to the USP of MDR1 was evaluated by promoter enzyme immunoassays and chromatin immunoprecipitation assays. Results Nkx-2.5 significantly stimulates the transactivation of MDR1 USP and increases MDR1 mRNA expression in MCF7 breast cancer cells. Reporter gene assays with deleted MDR1 USPs showed that the Nkx-2.5-binding site is located between positions -71 and +12. Mutation of the Nkx-2.5-binding site at nucleotide +4 to +10 markedly reduced the Nkx-2.5-mediated activation of MDR1 USP activity. A promoter binding immunoassay and a chromatin immunoprecipitation assay revealed that Nkx-2.5 binds directly to the region +4/+10 of human MDR1 USP. Conclusion The results in the present study show Nkx-2.5 is a positive regulator for the transactivation of MDR1 USP in MCF7 breast cancer cells. Our findings will help elucidate the regulatory mechanism responsible for the multidrug resistant cancer phenotype.

MicroRNA (miRNA), a small RNA synthesized from non‐coding RNA, acts as an important post‐transcriptional regulator. We aimed to identify the vasopressin‐responsive miRNAs from kidney inner medullary collecting duct (IMCD) cells, and particularly to demonstrate aquaporin‐2 (AQP2)‐targeting miRNAs. Microarray chip assay (Affymetrix GeneChip miRNA 3.0 Array) was carried out in IMCD tubule suspension of rat kidney in the absence or the presence of dDAVP stimulation (10‐8 M, 2 h). The results demonstrated 19 miRNAs, including both precursors and mature miRNAs, which showed 1.3‐fold changes in the expression in response to dDAVP stimulation (P<0.05). Additionally, vasopressin‐responsive miRNA networks were predicted by in silico analysis, which exhibited potential target genes. To identify AQP2‐targeting miRNAs, in silico analysis using TargetScan 6.2 was performed. Four miRNAs (miR‐32, miR‐137, miR‐216a, and miR‐216b) targeting 3’UTR of rat AQP2 mRNA were predicted. Target seed regions of miR‐32 and miR‐137 were conserved at the 3’UTR (476 ‐ 483) of rat AQP2 mRNA. RT‐qPCR and immunoblot analysis demonstrated that dDAVP‐induced AQP2 up‐regulation was significantly attenuated in mpkCCDc14 cells, when the cells were transfected by miRNA‐mimic of miR‐32 or miR‐137. This study provides a novel insight on the regulation of AQP2 protein expression via RNA interference, i.e., AQP2‐targeting miRNAs.