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. 2010 Jul 28;168(4):971-81.
doi: 10.1016/j.neuroscience.2009.09.020. Epub 2009 Sep 15.

Water permeability through aquaporin-4 is regulated by protein kinase C and becomes rate-limiting for glioma invasion

E S McCoy  1 , B R Haas, H Sontheimer
Affiliations

Water permeability through aquaporin-4 is regulated by protein kinase C and becomes rate-limiting for glioma invasion

E S McCoy et al. Neuroscience. .

Abstract

Glial-derived tumors, gliomas, are highly invasive cancers that invade normal brain through the extracellular space. To navigate the tortuous extracellular spaces, cells undergo dynamic changes in cell volume, which entails water flux across the membrane through aquaporins (AQPs). Two members of this family, AQP1 and AQP4 are highly expressed in primary brain tumor biopsies and both have a consensus phosphorylation site for protein kinase C (PKC), which is a known regulator of glioma cell invasion. AQP4 colocalizes with PKC to the leading edge of invading processes and clustered with chloride channel (ClC2) and K(+)-Cl(-) cotransporter 1 (KCC1), believed to provide the pathways for Cl(-) and K(+) secretion to accomplish volume changes. Using D54MG glioma cells stably transfected with either AQP1 or AQP4, we show that PKC activity regulates water permeability through phosphorylation of AQP4. Activation of PKC with either phorbol 12-myristate 13-acetate or thrombin enhanced AQP4 phosphorylation, reduced water permeability and significantly decreased cell invasion. Conversely, inhibition of PKC activity with chelerythrine reduced AQP4 phosphorylation, enhanced water permeability and significantly enhanced tumor invasion. PKC regulation of AQP4 was lost after mutational inactivation of the consensus PKC phosphorylation site S180A. Interestingly, AQP1 expressing glioma cells, by contrast, were completely unaffected by changes in PKC activity. To demonstrate a role for AQPs in glioma invasion in vivo, cells selectively expressing AQP1, AQP4 or the mutated S180A-AQP4 were implanted intracranially into SCID mice. AQP4 expressing glioma cells showed significantly reduced invasion compared to AQP1 and S180 expressing tumors as determined by quantitative stereology, consistent with a differential role for AQP1 and AQP4 in this process.

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Figures

Figure 1
Figure 1
PKC expression and phosphorylation of AQP4. Cell lysates were separated using 10% SDS-PAGE gels and transferred onto PVDF paper and probed with antibodies Western blot analysis was used to examine PKC expression levels in AQP4-D54 cells as compared to primary astrocyte cultures (A) and localization in migrating tumor cells (B) using PKC antibodies as described in Materials and Methods. (C) Immunoprecipitation showed that pretreatment with either chelerythrine or PMA modulated AQP4 phosphorylation levels but did not alter basal AQP4 expression levels (D).
Figure 2
Figure 2
Functional regulation of AQP4 by PKC. Mean cell volume was measured for 3 min in AQP4-D54 cells (A; untreated n=7, Chel n=6, PMA n=4) and S180 mutant AQP4 (B; n=6). Cells were treated with either chelerythrine or PMA for 45 min before being given a 50% hyposmotic challenge. Representative graphs show water permeability over the first few seconds following hyposmotic challenge. Reciprocal exponential time constant (τ−1) is proportional to osmotic water permeability (C). AQP4+Chel are gliomas expressing WT AQP4 that were treated with chelrythrine. (Significance was assessed using an ANOVA *p<0.05 as compared to control.)
Figure 3
Figure 3
Role of PKC in modulating migration and adhesion in AQP4-expressing tumor cells. (A) Transwell migration assay comparing AQP4-D54 cells treated with inhibitors of various signaling molecules. 40,000 cells were allowed to migrate for 5 hrs through 8 μm FluorBlok filters. Cells were treated with PKC modulators, chelerythrine and PMA, U73122, a PLC inhibitor and U0126 as a MAPK inhibitor. To measure adherence, 100,000 cells were plated onto various matrices [1% BSA (Control), 10 μg/ml collagen I (CN), 10 μg/ml fibronectin III (FN), 20 μg/ml laminin (LN), 20 μg/ml vitronectin (VN)] for 1 hr. Cells were gently washed away and fixed. Five random images were taken and counted. There was no difference in cell adhesion between D54 cells lacking AQP4 expression (B) nor AQP4-expressing cells (C) when treated with chelerythrine.
Figure 4
Figure 4
PKC does not regulate AQP1. Using immunoprecipitation as described in Fig. 1, chelerythrine and PMA treatment did not alter AQP1 phosphorylation (A). Neither water permeability (B) nor cell migration (C) was altered by PKC activity.
Figure 5
Figure 5
PKC modulates phosphorylation of ClC2 and KCC1 but not migration. Using immunfluorescence (A) and immunoprecipitation as described in Fig. 1, chelerythrine reduced PKC mediated ClC2 and KCC1 phosphorylation without altering the basal expression levels of either (B). PKC does not modulate migration in D54 control cells lacking AQP4 (C). Migrating cells were treated acutely with 40 μM DIOA, 12.5 μM CdCl2, or both to block ClC2 and/or KCC1. Inhibition was examined in D54 control cells (D), AQP4-expressing cells (E), and in AQP4 cells treated with chelerythrine (F). (Significance was determined using ANOVA; *p<0.05)
Figure 6
Figure 6
AQP1 expression enhances tumor invasion. AQP1 (n=9), AQP4 (n=8) and S180A-AQP4 (n=8) expressing D54MG were injected intracranially into 6 weeks old mice. Representative images of tumor growth (A). Arrows indicate location of main tumor mass. Fluorescent images demonstrate tumor cell invasion away from the major tumor mass for D54 glioma cells expressing AQP1-GFP, AQP4-DsRed, or AQP4-S180A-DsRed (B). Arrows indicate invading cells. D54 cells expressing AQP1-GFP invaded ∼2 fold over D54-GFP and AQP4-DsRed expressing D54 cells (C). We measured invasion over 500 μm increments and discovered that AQP1 tumors were able to invade greater distances than either D54 or AQP4 cells (D). There were significantly more cells that had migrated distances greater than 1500 μm. Most D54 and AQP4 tumors invaded short distances, while S180A-AQP4 mutants invaded intermediate distances.
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