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Modulation of P-glycoprotein activity by cannabinoid molecules in HK-2 renal cells.

ABSTRACT: 1. Endogenous and synthetic cannabinoid molecules have been investigated as possible MDR-1/P-glycoprotein (P-gp) modulators in HK-2-immortalized renal cells, using calcein acetoxymethylester (calcein-AM) as a P-gp substrate. 2. Among the endocannabinoid molecules tested, anandamide (AEA), but not 2-arachidonoyl-glycerol (2-AG) or palmitoyl-ethanolamide (PEA), increased the intracellular fluorescence emitted by calcein, a metabolic derivative of the P-gp substrate calcein-AM, indicative of a reduction in transport capacity. 3. All the three synthetic cannabimimetics tested, that is, R-(+)-methanandamide (R(+)-MET), AM 251 and CP55,940 significantly increased calcein accumulation in the cytosol. 4. RT-PCR demonstrated that HK-2 cells do not express CB1 or CB2 cannabinoid receptors. 5. R(+)-MET, AM251 and CP55,940 were also evaluated as modulators of P-gp expression, by Western blot analysis. Only AM251 weakly enhanced the protein levels (by 1.2-fold) after a 4-day-long incubation with the noncytotoxic drug concentration 2 microM. 6. The present data provide the first evidence that the endocannabinoid AEA and different synthetic cannabinoids may inhibit the P-gp activity in vitro via a cannabinoid receptor-independent mechanism.

SUBMITTER: Nieri P

PROVIDER: S-EPMC1751877 | biostudies-literature | 2006 Jul

REPOSITORIES: biostudies-literature

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Modulation of P-glycoprotein activity by cannabinoid molecules in HK-2 renal cells.

Nieri Paola P Romiti Nadia N Adinolfi Barbara B Chicca Andrea A Massarelli Ilaria I Chieli Elisabetta E

British journal of pharmacology 20060522 5

1. Endogenous and synthetic cannabinoid molecules have been investigated as possible MDR-1/P-glycoprotein (P-gp) modulators in HK-2-immortalized renal cells, using calcein acetoxymethylester (calcein-AM) as a P-gp substrate. 2. Among the endocannabinoid molecules tested, anandamide (AEA), but not 2-arachidonoyl-glycerol (2-AG) or palmitoyl-ethanolamide (PEA), increased the intracellular fluorescence emitted by calcein, a metabolic derivative of the P-gp substrate calcein-AM, indicative of a redu ...[more]

PMID: 16715117

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Project description:The PI3K/Akt pathway is interconnected to protein kinase CK2, which directly phosphorylates Akt1 at S129. We have previously found that, in HK-2 renal cells, downregulation of the CK2 regulatory subunit β (shCK2β cells) reduces S129 Akt phosphorylation. Here, we investigated in more details how the different CK2 isoforms impact on Akt and other signaling pathways. We found that all CK2 isoforms phosphorylate S129 in vitro, independently of CK2β. However, in HK-2 cells the dependence on CK2β was confirmed by rescue experiments (CK2β re-expression in shCK2β HK-2 cells), suggesting the presence of additional components that drive Akt recognition by CK2 in cells. We also found that CK2β downregulation altered the phosphorylation ratio between the two canonical Akt activation sites (pT308 strongly reduced, pS473 slightly increased) in HK-2 cells. Similar results were found in other cell lines where CK2β was stably knocked out by CRISPR-Cas9 technology. The phosphorylation of rpS6 S235/S236, a downstream effector of Akt, was strongly reduced in shCK2β HK-2 cells, while the phosphorylation of two Akt direct targets, PRAS40 T246 and GSK3β S9, was increased. Differently to what observed in response to CK2β down-regulation, the chemical inhibition of CK2 activity by cell treatment with the specific inhibitor CX-4945 reduced both the Akt canonical sites, pT308 and pS473. In CX-4945-treated cells, the changes in rpS6 pS235/S236 and GSK3β pS9 mirrored those induced by CK2β knock-down (reduction and slight increase, respectively); on the contrary, the effect on PRAS40 pT246 phosphorylation was sharply different, being strongly reduced by CK2 inhibition; this suggests that this Akt target might be dependent on Akt pS473 status in HK-2 cells. Since PI3K/Akt and ERK1/2/p90rsk pathways are known to be interconnected and both modulated by CK2, with GSK3β pS9 representing a convergent point, we investigated if ERK1/2/p90rsk signaling was affected by CK2β knock-down and CX-4945 treatment in HK-2 cells. We found that p90rsk was insensitive to any kind of CK2 targeting; therefore, the observation that, similarly, GSK3β pS9 was not reduced by CK2 blockade suggests that GSK3β phosphorylation is mainly under the control of p90rsk in these cells. However, we found that the PI3K inhibitor LY294002 reduced GSK3β pS9, and concomitantly decreased Snail1 levels (a GSK3β target and Epithelial-to-Mesenchymal transition marker). The effects of LY294002 were observed also in CK2β-downregulated cells, suggesting that reducing GSK3β pS9 could be a strategy to control Snail1 levels in any situation where CK2β is defective, as possibly occurring in cancer cells.

Project description:AimThe aim of the study was to investigate the potential role of BMP6 in TGF-beta1-mediated changes in HK-2 cells.MethodsBMP6 was purified via heparin affinity and reverse phase liquid chromatography. The purity, specificity, and bioactivity of BMP6 were determined by SDS-PAGE, Western blot assays, and the induction of alkaline phosphatase (ALP) activity, respectively. Cell proliferation, morphology, and expression levels of alpha-SMA and E-cadherin were assessed by cell viability, microscopy, and Western blot assays, respectively. In addition, cell adhesion abilities were determined by counting the number of attached cells. The expression of fibronectin, collagen IV, matrix metalloproteinases 2 (MMP-2), and tissue inhibitors of matrix metalloproteinases 2 (TIMP-2) were analyzed using RT-PCR. MMP-2 activity was analyzed by zymography, whereas the activation of the MAPKs and Smad signaling were analyzed using Western blot assays and a reporter gene assay, respectively.ResultsOur results indicated that recombinant BMP6 induced ALP activity in a dose-dependent and time-course-dependent manner. Treatment with TGF-beta1 reduced both the cell proliferation and the expression of E-cadherin, induced a morphological transformation, decreased the expression and activity of MMP-2, and increased the expression levels of alpha-SMA, fibronectin, and TIMP-2 in HK-2 cells. All of these effects were inhibited when cells were treated with TGF-beta1 in combination with rhBMP6, whereas rhBMP6 alone demonstrated no such effect. Treatment with TGF-beta1, rhBMP6, or a combination of both had no effect on the expression of collagen IV. In addition, the administration of rhBMP6 prevented the enhanced adhesion behavior triggered by TGF-beta1. Furthermore, the addition of rhBMP6 abrogated the JNK and Smad2/3 signaling that was activated by TGF-beta1.ConclusionBMP6 ameliorated the TGF-beta1-induced changes in HK-2 cells. The suppression of TGF-beta1-mediated JNK and Smad2/3 signaling activation were implicated in these effects.Acta Pharmacologica Sinica (2009) 30: 994-1000; doi: 10.1038/aps.2009.56; published online 22 June 2009.

Dataset Information

Modulation of P-glycoprotein activity by cannabinoid molecules in HK-2 renal cells.

Publications

Modulation of P-glycoprotein activity by cannabinoid molecules in HK-2 renal cells.

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