AQP1 expression alterations affect morphology and water transport in Schwann cells and hypoxia-induced up-regulation of AQP1 occurs in a HIF-1α-dependent manner

Abstract

Aquaporin-1 (AQP1) is the principle water channel in the peripheral nervous system (PNS) and is specifically localized to Schwann cells in the PNS. However, the pathophysiological role of AQP1 in peripheral nerves is poorly understood. Here, we utilized RNA interference by lentiviral transduction to specifically down-regulate AQP1 expression and a lentiviral overexpression protocol to up-regulate AQP1 expression, in primary Schwann cell cultures. AQP1 gene silencing resulted in a cell shrinkage phenotype, while AQP1 gene overexpression caused a cell swelling phenotype, as validated by cell volume determinations. Secondly, we utilized an in vitro hypoxia model in Schwann cells to mimic in vivo facial nerve injury. We demonstrated that AQP1 expression was induced within 8h following hypoxia injury in vitro, and that AQP1 knockdown (KD) caused the cells to resist edema following hypoxia. Finally, we investigated the hypoxic regulation of the AQP1 gene, as well as the involvement of Hypoxia-inducible factor-1α (HIF-1α) in AQP1 modulation and we found that KD of HIF-1α decreased hypoxia-dependent induction of endogenous AQP1 expression at both the mRNA and protein levels. Taken together, these results indicate that (1) AQP1 is an important factor responsible for the fast water transport of cultured Schwann cells and is involved in cell plasticity; (2) AQP1 alterations may be a primary factor in hypoxia-induced peripheral nerve edema; (3) HIF-1α participates in the hypoxic induction of the AQP1 gene; (4) AQP1 inhibition might provide a new therapeutic alternative for the treatment of some forms of peripheral nerve edema.

Keywords: AQP1; AQP1-shRNA; AQP1-specific shRNA vectors; AQPs; CoCl(2); GFAP; HIF-1α; HIFs; KD; OMG; PBS; PCR; PNS; Schwann cells; VEGF; [3H]-O-methylglucose; aquaporins; cobalt(II) chloride; glial fibrillary acidic protein; hypoxia-inducible factor-1α; hypoxia-inducible factors; knockdown; lenti-AQP1; lentivirus-AQP1; nerve edema; peripheral nerve system; peripheral nervous system; phosphate-buffered saline; polymerase chain reaction; scr-shRNA; scramble-shRNA vector; shRNA; short hairpin RNA; vascular endothelial growth factor; water transport.