Hypoxia and hypoxia mimetics decrease aquaporin 5 (AQP5) expression through both hypoxia inducible factor-1α and proteasome-mediated pathways.

The alveolar epithelium plays a central role in gas exchange and fluid transport, and is therefore critical for normal lung function. Since the bulk of water flux across this epithelium depends on the membrane water channel Aquaporin 5 (AQP5), we asked whether hypoxia had any effect on AQP5 expression. We show that hypoxia causes a significant (70%) decrease in AQP5 expression in the lungs of mice exposed to hypoxia. Hypoxia and the hypoxia mimetic, cobalt, also caused similar decreases in AQP5 mRNA and protein expression in the mouse lung epithelial cell line MLE-12. The action of hypoxia and cobalt on AQP5 transcription was demonstrated by directly quantifying heternonuclear RNA by real-time PCR. Dominant negative mutants of Hypoxia Inducible Factor (HIF-1α) and HIF-1α siRNA blocked the action of cobalt, showing that HIF-1α is a key component in this mechanism. The proteasome inhibitors, lactacystin or proteasome inhibitor-III completely abolished the effect of hypoxia and cobalt both at the protein and mRNA level indicating that the proteasome pathway is probably involved not only for the stability of HIF-1α protein, but for the stability of unidentified transcription factors that regulate AQP5 transcription. These studies reveal a potentially important physiological mechanism linking hypoxic stress and membrane water channels.

Aquaporin 5 regulates cigarette smoke induced emphysema by modulating barrier and immune properties of the epithelium.

Chronic obstructive pulmonary disease (COPD) causes significant morbidity and mortality. Cigarette smoke, the most common risk factor for COPD, induces airway and alveolar epithelial barrier permeability and initiates an innate immune response. Changes in abundance of aquaporin 5 (AQP5), a water channel, can affect epithelial permeability and immune response after cigarette smoke exposure. To determine how AQP5-derived epithelial barrier modulation affects epithelial immune response to cigarette smoke and development of emphysema, WT and AQP5(-/-) mice were exposed to cigarette smoke (CS). We measured alveolar cell counts and differentials, and assessed histology, mean-linear intercept (MLI), and surface-to-volume ratio (S/V) to determine severity of emphysema. We quantified epithelial-derived signaling proteins for neutrophil trafficking, and manipulated AQP5 levels in an alveolar epithelial cell line to determine specific effects on neutrophil transmigration after CS exposure. We assessed paracellular permeability and epithelial turnover in response to CS. In contrast to WT mice, AQP5(-/-) mice exposed to 6 months of CS did not demonstrate a significant increase in MLI or a significant decrease in S/V compared with air-exposed mice, conferring protection against emphysema. After sub-acute (4 weeks) and chronic (6 mo) CS exposure, AQP5(-/-) mice had fewer alveolar neutrophil but similar lung neutrophil numbers as WT mice. The presence of AQP5 in A549 cells, an alveolar epithelial cell line, was associated with increase neutrophil migration after CS exposure. Compared with CS-exposed WT mice, neutrophil ligand (CD11b) and epithelial receptor (ICAM-1) expression were reduced in CS-exposed AQP5(-/-) mice, as was secreted LPS-induced chemokine (LIX), an epithelial-derived neutrophil chemoattractant. CS-exposed AQP5(-/-) mice demonstrated decreased type I pneumocytes and increased type II pneumocytes compared with CS-exposed WT mice suggestive of enhanced epithelial repair. Absence of AQP5 protected against CS-induced emphysema with reduced epithelial permeability, neutrophil migration, and altered epithelial cell turnover which may enhance repair.

Spatial expression of aquaporin 5 in mammalian cornea and lens, and regulation of its localization by phosphokinase A.

PURPOSE: Aquaporins (AQPs) play a significant role in the movement of water across the plasma membrane. In the eye, the cornea and lens are avascular with unique microcirculatory mechanisms to meet the metabolic demands. We have previously shown that AQP0 and AQP1 water channels participate in maintaining lens transparency and homeostasis. In the present investigation, we explored the expression and spatial distribution of AQP5 in the cornea and lens, and its regulation during membrane localization. METHODS: AQP5 expression and cellular localization were investigated by reverse transcription polymerase chain reaction (RT-PCR) using gene-specific primers, and by western blot and immunocytochemistry analyses using specific antibodies. AQP5 phosphorylation was studied using calf intestinal alkaline phosphatase for dephosphorylation. Effects of phosphokinase A (PKA) agonist cyclic AMP (cAMP), and antagonist H-89 on AQP5 expression and localization were studied in vitro using MDCK (Madin-Darby Canine Kidney) cells, and ex vivo using isolated corneas from wild type mice. RESULTS: RT-PCR revealed the presence of AQP5 transcripts in the cornea, lens epithelial cells and fiber cells. Western blotting identified the presence of both non-phosphorylated and phosphorylated forms of AQP5 protein. Immunostaining showed the distribution of AQP5 in the epithelial layer and stromal keratocytes of the cornea, and epithelial and fiber cells of the lens. In vitro and ex-vivo experiments revealed PKA-induced AQP5 internalization; PKA inhibition prevented such internalization. CONCLUSIONS: This is the first report on the spatial expression of AQP5 in the corneal keratocytes and lens epithelial cells, as well as on the regulation of AQP5 localization by PKA in the corneal epithelial cells. PKA-mediated regulation of AQP5 holds promise for therapeutic intervention to control corneal and lens diseases.

The protein kinase A pathway contributes to Hg2+-induced alterations in phosphorylation and subcellular distribution of occludin associated with increased tight junction permeability of salivary epithelial cell monolayers.

Hg(2+) is commonly used as an inhibitor of many aquaporins during measurements of transcellular water transport. To investigate whether it could also act on the paracellular water transport pathway, we asked whether addition of Hg(2+) affected transport of radiolabeled probes through tight junctions of a salivary epithelial cell monolayer. Inclusion of 1 mM Hg(2+) decreased transepithelial electrical resistance by 8-fold and augmented mannitol and raffinose flux by 13-fold, which translated into an estimated 44% increase in pore radius at the tight junction. These Hg(2+)-induced effects could be partially blocked by the protein kinase A (PKA) inhibitor N-[2-((p-bromocinnamyl) amino) ethyl]-5-isoquinolinesulfonamide, 2HCl (H89), suggesting that both-PKA dependent and PKA-independent mechanisms contribute to tight junction regulation. Western blot analyses showed a 2-fold decrease in tight junction-associated occludin after Hg(2+) treatment and the presence of a novel hyperphosphorylated form of occludin in the cytoplasmic fraction. These findings were corroborated by confocal imaging. The results from this study reveal a novel contribution of the PKA pathway in Hg(2+)-induced regulation of tight junction permeability in the salivary epithelial barrier. Therapeutically, this could be explored for pharmacological intervention in the treatment of dry mouth, Sjögren's syndrome, and possibly other disorders of fluid transport.

Identification and characterization of novel human transcripts embedded within HMGA2 in t(12;14)(q15;q24.1) uterine leiomyoma.

The high mobility group A2 protein (HMGA2) has been implicated in the pathogenesis of mesenchymal tumors such as leiomyoma, lipoma and hamartoma. HMGA2 was pinpointed by mapping the breakpoints in the chromosomal translocations in 12q15, especially the t(12;14) that is commonly seen in uterine leiomyoma. It is generally assumed that altered expression of HMGA2 is an early event in the pathway to tumor formation. Here, we show evidence that three novel transcripts, A15, B6 and D12 are located within the HMGA2 gene itself and are transcribed from the opposite strand. These embedded transcripts are expressed at 6-20-fold higher levels in tumors compared to matched myometrium from the same patients. We estimate that the domain of increased expression extends 500kb on chromosome 12q15, and encompasses the majority of t(12;14) translocation breakpoints. However, a corresponding domain of consistently altered expression is not seen on chromosome 14 or outside of the chromosome 12 multiple aberration region. These data suggest that t(12;14) breakpoints contribute to the pathogenesis of uterine leiomyoma by interrupting a complex regulation of HMGA2 and other genes embedded within and around it. We also discovered a novel laminin receptor gene, transcribed from the opposite strand, within the promoter region of HMGA2. Although the roles for these embedded transcripts are still unknown, preliminary data suggest that they are members of the family of non-coding RNA and that they may play an important role in the pathology of uterine leiomyoma.

Decreased expression of aquaporin-5 in bleomycin-induced lung fibrosis in the mouse.

The expression of aquaporin-5, the major water channel expressed in alveolar, tracheal, and upper bronchial epithelium, is significantly down-regulated during acute lung injury. In the present study, the expression of aquaporin-5 in two different mouse models of lung fibrosis was evaluated. Lung fibrosis was induced by intratracheal and by subcutaneous infusion of bleomycin. The expression of aquaporin-5 was investigated by immunohistochemical studies and by polymerase chain reaction. There were many cells with loss of aquaporin-5 immunoreactivity in type I alveolar epithelial cells in the mouse models of lung fibrosis. Immunohistochemistry of lung tissue in aquaporin-5 knockout mice revealed a fibrotic phenotype with increased deposition of extracellular collagen type I in thickened alveolar walls. Semiquantitative analysis of aquaporin-5 mRNA expression showed more abundant content of aquaporin-5 in the lung of the normal mouse compared to the mouse with lung fibrosis. The results of this study showed, for the first time, that chronic lung injury and lung fibrosis is associated with decreased protein and mRNA expression of aquaporin-5 in the lung.

Cyclic AMP regulates aquaporin 5 expression at both transcriptional and post-transcriptional levels through a protein kinase A pathway.

The membrane water channel aquaporin 5 (AQP5) plays an important role in transporting water across the apical surface of the alveolar epithelium and the epithelia of submucosal glands in the upper airway and nasopharynx. It is thus a potentially important target for modulating the fluid content of upper airway and nasopharyngeal secretions in disorders such as cystic fibrosis. Here, we have used an in vitro system to identify a molecular mechanism through which transcriptional and post-transcriptional regulation of AQP5 is accomplished. In a murine lung epithelial cell line (MLE-12), the addition of chlorophenylthio-cAMP (cpt-cAMP) causes a 4-fold increase in AQP5 mRNA and protein levels and induces translocation of AQP5 to the apical plasma membrane. Treatment with forskolin and isoproternol also caused similar increases in AQP5 expression both in vitro and in mouse lung tissue slices. The addition of actinomycin D abolished the cAMP-mediated increase in AQP5 mRNA and showed that there was no increase in the half-life of AQP5 mRNA, and inhibition of protein kinase A by H-89 blocked the cpt-cAMP-mediated increase of AQP5. Pretreatment of cells with cycloheximide blocked the cpt-cAMP-mediated increase of AQP5 mRNA, indicating that de novo protein synthesis is essential for increased AQP5 transcription. Immunofluorescent micrographs of cells treated with cpt-cAMP showed a significantly stronger AQP5 signal at the plasma membrane as compared with untreated cells. These results show that cAMP regulates AQP5 at multiple levels, by increasing synthesis of AQP5 mRNA and by triggering translocation of AQP5 to the plasma membrane.