Effects Of AQP1on Morphology And Water Transport In Schwann Cells And Mechanism Research Of Hypoxia-induced Up-regulation Of AQP1

【Background】The facial nerve as a kind of peripheral nerve, which cause nerve edema and even formvicious cycle of ‘edema-ischemic-edema’in the tortuous and narrow osseous facial canalfollowing injury. The special anatomy of facial canal can aggravate facial nerve injury.Therefore, how to prevent or alleviate facial nerve edema in the clinical treatment of facialparalysis is essential.Aquaporins (AQPs), as a family of membrane water channel proteins, have beendemonstrated multiple physiological functions in diverse fluid transporting tissuesincluding nervous system. Numerous evidences have proven that AQP1was the principlewater channel in the peripheral nervous system (PNS). In our previous works, we havedemonstrated that facial nerve tissues expressed AQP1and successfully established amouse facial nerve lacerations model. Western Blot detection revealed that the expressionof AQP1was obviously increased after facial nerve injury. The change of AQP1expressionin the level of protein and mRNA highly coincided with the degree of the nerve edema inthe facial canal, which indicated that AQP1was closely related to facial nerve edema dueto injury (unpublished data). In addition, immunocytochemical studies showed that AQP1was localized to Schwann cells. But, the causal relationship of AQP1expression and facialnerve edema is still unclear. It is unknown if the increased level of AQP1expressionaggravates facial edema or if the alterations of AQP1levels are an accompanyingphenomenon of facial nerve edema. As such, further elucidation of the pathophysiologicalroles of AQP1in Schwann cells is critical for our understanding of its role in this intricateprocess.Therefore, in this study, we established RNA interference and transgenic protocol bylentivirus transduction that specifically down-regulated and up-regulated the expression ofAQP1respectively in order to analyze its functional role in Schwann cells. Moreover, wemade Schwann cells hypoxia model to mimick facial nerve injury in vivo and studied thepathological role of AQP1in vitro hypoxia condition that mimicked by cobalt(II) chloride(CoCl2) by PCR, western blot analysis and immunofluorescence. Finally, we studied thehypoxic regulation of the AQP1gene and analyze the role of the AQP1promoter in thisprocess. We have also investigated the involvement of HIF-1α in AQP1modulation. Thesefindings will provide theoretical basis for the treatment of facial nerve edema injury.【Objective】 1. To investigate the causal relationship of AQP1expression and Schwann cells swelling.2. To determine the AQP1gene function in facial nerve edema following injury and toprovide a new therapeutic alternative for the treatment of facial nerve edema.3. To study the effect of HIF-1α on AQP1expression in pathological condition of hypoxiaand molecular mechanism of hypoxic induction of endogenous AQP1expression.【Methods】1. Validation of AQP1expression on mouse facial nerve Schwann cells byimmunofluorescence staining(1) Schwann cells were prepared from primary cell cultures of facial nerve tissues from3day old C57BL/6mice by trypsin digestion and purified with a difference-speedadherence method.(2) AQP1expression was analyzed together with the Schwann cell marker S-100bydouble immunostaining experiments.2. Effects of lentivirus-mediated AQP1-shRNA on morphology and water transport inSchwann cells(1) Construction and identification of AQP1-shRNA lentiviral expression vector. Thedesigned and synthesised Single-Stranded primer were annealed to Double-Stranded oligosequences and subcloned into linear pLKO.1-TRC lentiviral plasmid vector digested byenzyme Age I and EcoR I．Screening positive clone after transformed into DH5a competentcells and identified by PCR amplification and DNA sequencing．After sequencingconfirmation，each of the recombinant vectors and packaging vectors werecotransducted into293T cells，and the recombinant lentivirus were packaged．ThenSchwann cells were infected by purified lentivirus and screening effective shRNAsequence by RT-PCR and western blot analysis.(2) Primary Schwann cells in culture were transfected with AQP1-shRNA and CTRL(src-shRNA) and photographed during gene silencing every24h for6days using adigital photocamera connected to a phase-contrast microscope.(3) Cell volume of CTRL and AQP1shRNA treated cells was determined daily duringAQP1-KD by [3H]-O-methylglucose (OMG) equilibration method.(4) Cell viability was measured by MTT assay.(5) Apoptosis effects of AQP1-shRNA on Schwann cells were detected by flow cytometry.3. Effects of lentivirus-mediated AQP1overexpression on morphology and water transportin Schwann cells (1) Construction and identification of lentiviral expression vector containing mouse AQP1gene.Mouse AQP1gene was inserted into lentiviral vector (pCDH-CMV-MCS-EF1-copGFP),which was confirmed by PCR and sequencing analysis.The virus packagingplasmids (pCDH-CMV-MCS-EF1-copGFP-AQP1, psPAX2and pMD) werecontransfected into293T cells, and the Lentivirus-AQP1was harvested from293T cells.The Lentivirus-AQP1was used to infect C57BL/6mouse Schwann cells in vitro and theexpression of AQP1mRNA and protein in infected mouse Schwann cells was detected byquantitative real-time PCR and western blot analysis.(2) Primary Schwann cells in culture were transfected with Lentivirus-AQP1and CTRL(empty vector)and photographed during gene overexpression every24h for6days using adigital photocamera connected to a phase-contrast microscope.(3) Cell volume of CTRL and Lentivirus-AQP1treated cells was determined daily duringAQP1-KD by [3H]-O-methylglucose (OMG) equilibration method.4. Effects of AQP1expression on morphology and water transport in Schwann cellsfollowing hypoxia(1) Made Schwann cells hypoxia model to mimick facial nerve injury in vivo andidentified the alteration of AQP1expression in vitro hypoxia condition that mimicked bycobalt(II) chloride (CoCl2) by PCR, western blot analysis and immunofluorescence.(2) CTRL and AQP1-shRNA treated cells were induced hypoxia with CoCl2respectively.Phase contrast microscopy analysis the morphology alteration of CTRL and AQP1-shRNAtreated cells following hypoxia. Cell volume was also determined.5. Molecular mechanism of hypoxic induction of endogenous AQP1expression(1) HIF-1α expression in Schwann cells was identified after hypoxia injury by PCR andwestern blot analysis.(2) Knocked down HIF-1α with specific siRNA and analyzed the effect thatdown-regulation of HIF-1α had on the hypoxia-induced overexpression of endogenousAQP1.【Results】1. Primary Schwann cells demonstrated AQP1protein expression.2. RT-PCR analysis and Western blot assay confirmed that AQP1expression inAQP1-shRNA cells was much lower than that in scr-RNA cells. AQP1-shRNA caused cellshrinkage in cultured Schwann cells by38.9%at day4(p <0.001vs. control) and43.6%at day6(p <0.001vs. control). MTT assay showed that the cell viability in AQP1-shRNA cells was lower compared to cells transduced with scr-shRNA(P<0.05). Flow cytometryassay showed that AQP1gene silencing could not affect the apoptosis and necrosis rates ofSchwann cells.3. PCR and sequencing revealed that the pCDH-CMV-MCS-EF1-copGFP-AQP1plasmid was correctly constructed. The expression of AQP1mRNA and protein inSchwann cells infected with the Lentivirus-AQP1was increased significantly comparedwith negative control lentivirus. Lenti-AQP1caused cell swelling in cultured Schwanncells by68.3%at day4(p <0.001vs. control) and78.5%at day6(p <0.001vs. control).4. Hypoxia induced overexpression of AQP1mRNA and protein. AQP1was stronglyexpressed on membrane of Schwann cells following CoCl2induced hypoxia. AQP1-KDtreated cells can resist edema following hypoxia injury. The morphology of AQP1-KDtreated cells had no obvious alteration following treated with CoCl2. However, the CTRLcells presented obvious swelling that were mostly characterized by round shape. Cellvolume determinations also validated the results(p <0.001).5. The expression of HIF-1α mRNAand protein was increased following CoCl2inducedhypoxia. HIF-1αsiRNAreduced hypoxic induction of endogenousAQP1expression.【Conclusions】1. AQP1seems to be the major factor responsible for the fast water transport of culturedSchwann cells.2. AQP1is a protein involved in cell plasticity.3. AQP1alteration may be a primary factor in ischemia-induced and hypoxia-inducedperipheral nerve edema.4. Our results support the view that HIF-1α participates in the hypoxic induction of theAQP1gene.5. AQP1inhibition might provide a new therapeutic alternative for the treatment of someforms of peripheral nerve edema.