Luminal flow induces NADPH oxidase 4 translocation to the nuclei of thick ascending limbs

Abstract

Superoxide (O2 −) exerts its physiological actions in part by causing changes in gene transcription. In thick ascending limbs flow‐induced O2 − production is mediated by NADPH oxidase 4 (Nox4) and is dependent on protein kinase C (PKC). Polymerase delta interacting protein 2 (Poldip2) increases Nox4 activity, but it is not known whether Nox4 translocates to the nucleus and whether Poldip2 participates in this process. We hypothesized that luminal flow causes Nox4 translocation to the nuclei of thick ascending limbs in a PKC‐dependent process facilitated by Poldip2. To test our hypothesis, we studied the subcellular localization of Nox4 and Poldip2 using confocal microscopy and O2 − production in the absence and presence of luminal flow. Luminal flow increased the ratio of nuclear to cytoplasmic intensity of Nox4 (N/C) from 0.3 ± 0.1 to 0.7 ± 0.1 (P < 0.01) and O2 − production from 89 ± 15 to 231 ± 16 AU/s (P < 0.001). In the presence of flow PKC inhibition reduced N/C from 0.5 ± 0.1 to 0.2 ± 0.1 (P < 0.01). Flow‐induced O2 − production was also blocked (flow: 142 ± 20 AU/s; flow plus PKC inhibition 26 ± 12 AU/s; P < 0.01). The cytoskeleton disruptor cytochalasin D (1 μmol/L) decreased flow‐induced Nox4 translocation by 0.3 ± 0.01 (P < 0.01); however, it did not reduce flow‐induced O2 −. Flow did not alter Poldip2 localization. We conclude that: (1) luminal flow elicits Nox4 translocation to the nucleus in a PKC‐ and cytoskeleton‐dependent process; (2) Nox4 activation occurs before translocation; and (3) Poldip2 is not involved in Nox4 nuclear translocation. Flow‐induced Nox4 translocation to the nucleus may play a role in O2 −‐dependent changes in thick ascending limbs.

The Role of ROS Homeostasis in ABA-Induced Guard Cell Signaling

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Polymerase δ-interacting Protein 2: A Multifunctional Protein

“…Poldip2 is fairly ubiquitously expressed, having been reported in aorta, 3, 4 lung, 3 kidney, 3, 5, 6 heart 7 and thyroid, 8 as well as many cell types including mouse aortic smooth muscle cells, 9 astrocytes, 10 vascular smooth muscle cells (VSMC), 3, 11 brain endothelial cells, 12 epithelial cells, 12 HeLa, 1, 12-15 fibroblasts, 2, 13, 16, 17 kidney fibroblasts, 18 myoblasts, 19 human umbilical vein endothelial cells, 20 human embryonic kidney cells 293 (HEK 293), 13 MRC5 cells, 21, 22 and cortical neurons. 23 There is also evidence of Poldip2 expression in human breast cancer, 24 human breast adenocarcinoma cells 13 and human lung adenocarcinoma cells.…”

NADPH oxidase 4-derived superoxide mediates flow-stimulated NKCC2 activity in thick ascending limbs

“…Ϫ stimulated by increases in luminal flow (34), and this NADPH oxidase isoform translocates in response to this stimulus (60). Although these studies suggest the importance of NOX4 as a source of O 2…”