Intracellular Calcium Release Modulates Polycystin-2 Trafficking Ayako Miyakawa1,2, Cristián Ibarra1, Seth Malmersjö1, Anita Aperia3, Peter Wiklund2 and Per Uhlén1*
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Miyakawa et al. BMC Nephrology 2013, 14:34 http://www.biomedcentral.com/1471-2369/14/34 RESEARCH ARTICLE Open Access Intracellular calcium release modulates polycystin-2 trafficking Ayako Miyakawa1,2, Cristián Ibarra1, Seth Malmersjö1, Anita Aperia3, Peter Wiklund2 and Per Uhlén1* Abstract Background: Polycystin-2 (PC2), encoded by the gene that is mutated in autosomal dominant polycystic kidney disease (ADPKD), functions as a calcium (Ca2+) permeable ion channel. Considerable controversy remains regarding the subcellular localization and signaling function of PC2 in kidney cells. Methods: We investigated the subcellular PC2 localization by immunocytochemistry and confocal microscopy in primary cultures of human and rat proximal tubule cells after stimulating cytosolic Ca2+ signaling. Plasma membrane (PM) Ca2+ permeability was evaluated by Fura-2 manganese quenching using time-lapse fluorescence microscopy. Results: We demonstrated that PC2 exhibits a dynamic subcellular localization pattern. In unstimulated human or rat proximal tubule cells, PC2 exhibited a cytosolic/reticular distribution. Treatments with agents that in various ways affect the Ca2+ signaling machinery, those being ATP, bradykinin, ionomycin, CPA or thapsigargin, resulted in increased PC2 immunostaining in the PM. Exposing cells to the steroid hormone ouabain, known to trigger Ca2+ oscillations in kidney cells, caused increased PC2 in the PM and increased PM Ca2+ permeability. Intracellular Ca2+ buffering with BAPTA, 2+ inositol 1,4,5-trisphosphate receptor (InsP3R) inhibition with 2-aminoethoxydiphenyl borate (2-APB) or Ca /Calmodulin- dependent kinase inhibition with KN-93 completely abolished ouabain-stimulated PC2 translocation to the PM. Conclusions: These novel findings demonstrate intracellular Ca2+-dependent PC2 trafficking in human and rat kidney cells, which may provide new insight into cyst formations in ADPKD. Keywords: Polycystin-2, Protein trafficking, Calcium signaling, Kidney cells, Autosomal dominant polycystic kidney disease Background this protein is expressed in the endoplasmic reticulum Autosomal dominant polycystic kidney disease (ADPKD) (ER) or in the plasma membrane (PM). The amount of is the most commonly inherited monogenetic disease, PC2 present in the ER, where it functions as a Ca2+-acti- affecting more than 1 in 1000 live births, causing renal vated intracellular Ca2+ release channel [3], is signifi- failure [1]. ADPKD is caused by mutation in two asso- cantly larger than the amount of PC2 expressed in the ciated proteins, polycystin-1 or −2 (PC1 and PC2), which PM, where it functions as a non-selective cation channel are essential for the formation and maintenance of a [5,6]. In addition, PC2 has also been documented in the proper structure of the renal tubule. These mutations in primary cilium of kidney epithelial cells, where it contri- PC1 and PC2 are responsible for approximately 85% and butes to the mechano-sensing machinery by mediating 15% of all ADPKD cases, respectively [2]. It is well estab- Ca2+ entry in response to flow rate changes [7]. Never- lished that PC2 acts as an ion channel permeable to cal- theless, the exact function and mechanism of PC2 cium ions (Ca2+) [3]. Interestingly, loss of PC2 channel activation in the cilium and/or other subcellular orga- function and subsequent impaired Ca2+ signaling may nelles remain largely unknown. contribute to ADPKD pathogenesis [4]. A controversial Ca2+ signaling is a vital mechanism in many cell types, aspect of PC2-mediated signaling in ADPKD is whether controlling diverse cellular processes, such as: secretion, mechano-transduction, cell death, gene expression, or * Correspondence: [email protected] 1 proliferation (for review see [8]). Under certain conditions, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, 2+ Stockholm SE-171 77, Sweden via a sophisticated interplay between Ca channels and Full list of author information is available at the end of the article © 2013 Miyakawa et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Miyakawa et al. BMC Nephrology 2013, 14:34 Page 2 of 8 http://www.biomedcentral.com/1471-2369/14/34 transporters located in the PM and/or on the membrane Reagents of internal organelles, such as the ER, sustained oscillatory Reagents and concentrations were as follows: ouabain Ca2+ signaling can occur [9]. These Ca2+ oscillations en- (1 μM for hPT cells and 100 μM for rPT cells since code important information in their frequency and ampli- rodents are more resistant to ouabain than humans tude, which is decoded by cells using different Ca2+-effectors, [12], Sigma-Aldrich), ATP (25 μM, Sigma-Aldrich), such as protein kinases, phosphatases, proteases or tran- bradykinin (20 nM, Sigma-Aldrich), ionomycin (1 μM, scription factors, which in turn regulate adaptive cellular Sigma-Aldrich), thapsigargin (1 μM, Sigma-Aldrich), bis responses. Intracellular Ca2+ signaling mishandling is (2-aminophenoxy)ethane tetraacetic acid (BAPTA, 10 involved in the pathogenesis or progression of several dis- μM, Molecular Probes), 2-aminoethoxydiphenyl borate ease conditions and particularly in kidney disease, where (2-APB, 100 μM, Sigma-Aldrich), KN-93 (10 μM, Sigma- intracellular Ca2+ signaling appears to be linked to cystic Aldrich), LY294002 (10 μM, Sigma-Aldrich), wortmanin formation during ADPKD [10]. (5 μM, Sigma-Aldrich), FK506 (20 nM, Sigma-Aldrich), In the current study, we investigated the dynamic na- Go6983 (1 μM, Sigma-Aldrich), calphostin (5 μM, ture of PC2 localization in primary human and rat Sigma-Aldrich), and cycloheximide (CHX, 100 μM, kidney proximal tubule cells. We found that PC2 trans- Sigma-Aldrich). Cells were treated with inducers of Ca2+ locates from a basal, reticular-like localization to a PM signaling (ouabain, ATP, bradykinin) 3 h prior immunos- localization when cells were challenged with agents that taining and with inhibitors 15 min prior Ca2+ signaling- raise the basal concentration of intracellular Ca2+.PC2 inducers. trafficking was inhibited either by intracellular Ca2+ re- lease blockers or by inhibitors of key Ca2+-dependent Immunocytochemistry proteins. Taken together, these results demonstrate that Immunocytochemistry of PC2 in hPT and rPT cells was PC trafficking in kidney cells is regulated by intracellular performed according to standard protocol. Cells were Ca2+ and may contribute to the general understanding fixed with 4% paraformaldehyde for 15 min and then of ADPKD. permeabilized for 10 min with 0.3% Triton X-100. After blocking with 1% BSA for 1 h, cells were incubated over- night at 4°C with anti-PC2 polyclonal antibodies (gene- Methods rously provided by Dr. Stefan Somlo, Department of Cells cultures Internal Medicine, Yale University School of Medicine, Primary cultures of rat proximal tubule (rPT) cells New Haven, CT, USA and Dr. Jing Zhou, Renal Division, were prepared as described previously [11]. Briefly, Department of Medicine, Brigham and Women’s Hos- kidneys from 20-day-old female Sprague Dawley rats pital and Harvard Medical School, Boston, MA, USA) were used to prepare rPT cells. Cells were cultured in against amino acids 103 to 203 (YCB9) [13] or amino supplemented DMEM (20 mM Hepes, 24 mM acids 44 to 62 [7] on the N-terminus, respectively. Both NaHCO3, 10 mg/ml penicillin, 10 mg/ml strepto- antibodies showed similar PC2 pattern in proximal tu- mycin and 10% fetal bovine serum (FBS)) on glass bule cells (Additional file 1: Figure S1). All images pre- coverslips for 48–72 h in 5% CO2 at 37°C. Cells were sented here are using the antibody against YCB9. Cells starvedin1%FBSandculturedintheabsenceof were incubated for 1 h at room temperature with antibiotics for 24 h before the experiment. Primary Alexa488 fluorescent secondary antibody (1:500, Mo- cultures of human proximal tubule (hPT) cells were lecular Probes). Staining with only secondary antibody prepared as described. Briefly, approximately 10 g of was used as control (Additional file 2: Figure S2). Slides fresh renal tissue, obtained from human nephrectomy were scanned with similar exposure time using a Leica samples, was dissected, and the obtained cortex was TCS SP inverted confocal laser scanning microscope minced and then enzymatically digested with 1 mg/ml equipped with a 40x/1.4 NA oil-immersion objective. of type 4 collagenase. The resulting suspension was filtered and then centrifuged at 200 x g for 5 min. Calcium imaging The pellet was washed three times with ice-cold For Ca2+ experiments cells were loaded with 5 μM Fura- Hank’s balanced salt solution and finally resuspended 2/AM (Invitrogen) at 37°C for 1 h. Calcium measure- in DMEM/Ham’s Nutrient Mixture F12, supplemen- ments were performed at 37°C in a heated chamber ted with 10% FBS, insulin-transferrin-selenium, hydro- (QE-1, Warner Instruments) with a cooled CCD camera cortisone and antibiotics. The final cell suspension (ORCA-ERG, Hamamatsu) mounted on an upright was then plated onto coverslips coated with type IV microscope (Axioskop 2 FS, Zeiss) equipped with a collagen. All experiments were ethically approved by 40x/0.8 NA water dipping lens. Excitation at 340 and the Swedish Ethical Committee North, numbers 183/03 380 nm was carried out with a monochromator (rat) and 03–143 (human). (Polychrome IV, TILL Photonics). Devices were Miyakawa et al. BMC Nephrology 2013, 14:34 Page 3 of 8 http://www.biomedcentral.com/1471-2369/14/34 controlled and data were recorded and analyzed with the computer software MetaFluor (Molecular Devices). All experiments were performed in physiological buffer (100 mM NaCl, 4 mM KCl, 25 mM NaHCO3, 1.5 mM CaCl2, 1.1 mM MgCl2, 1 mM NaH2PO4,10mM D-glucose, and 20 mM HEPES, pH 7.4). An oscillating cell was defined as a cell that displayed two or more Ca2+ peaks, of which each peak’s amplitude was at least 10% over baseline. PM permeability was measured using the Fura-2 fluorescence quenching technique with Mn2+.