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Deficiency of polycystin-2 reduces Ca²⁺ channel activity and cell proliferation in ADPKD lymphoblastoid cells¹

GIANLUCA AGUIARI, MANUELA BANZI, STEFANIA GESSI^*, YIQIANG CAI, EMANUELA ZEGGIO, ELISA MANZATI, ROBERTA PIVA, ELISABETTA LAMBERTINI, LUISA FERRARI^||, DORIEN J. PETERS, FRANCESCO LANZA^||, PETER C. HARRIS^**, PIER ANDREA BOREA^*, STEFAN SOMLO and LAURA DEL SENNO²

Department of Biochemistry and Molecular Biology, Departments of
^* Clinical and Experimental Medicine, Pharmacology Unit,
^|| Biomedical Sciences and Advanced Therapies, Haematology Unit, and
Biotechnology Center, University of Ferrara, Ferrara, Italy;
Department of Human and Clinical Genetics, Leiden University Medical Center, Leiden, The Netherlands;
^** Department of Pathology, Mayo Clinic, Rochester, Minnesota, USA; and
Departments of Internal Medicine and Genetics, Yale University School of Medicine, New Haven, Connecticut, USA

²Correspondence: Department of Biochemistry and Molecular Biology, Via L. Borsari 46, 44100 Ferrara, Italy. E-mail: sen{at}unife.it

SPECIFIC AIMS

Polycystin-2 (PC2), the product of the PKD2 gene mutated in type 2 autosomal-dominant polycystic kidney disease (ADPKD), is a Ca²⁺-permeable cation channel found in kidney epithelia and other tissues. The precise expression and subcellular location of PC2 are controversial and there is little information on the role of the endogenous protein in non epithelial cells. Since PC2 is also expressed in B-lymphoblastoid cell lines (LCLs), where Ca²⁺ signaling pathways are important regulators of functional activities, we investigated whether PC2 might play some function in B-LCLs, taking advantage of the reduced levels of PC2 in LCLs from PKD2 subjects.

PRINCIPAL FINDINGS

1. PKD2 and PKD1 RNAs maintain the same reciprocal proportion in LCLs and HEK 293 cells
PKD2 RNA levels in LCLs from non-PKD subjects and in the epithelial kidney HEK293 cell line were studied by real-time RT-PCR analysis (Fig. 1 A). Results demonstrated that the PKD2 RNA was expressed in LCLs in a quantity 8.736-fold lower than in HEK293 cells. The same result was found for the PKD1 RNA (8.719-fold more in HEK293 than in LCLs cells). These data indicated that PKD2 and PKD1 gene expression maintained in the two cell types the same reciprocal proportion.

View larger version (37K): [in this window] [in a new window]   Figure 1. Expression of PKD2 RNA and PC2 in LCLs. A) Real-time RT-PCR analysis of PKD2 and PKD1 RNA in non-PKD-LCLs (n=3) and HEK 293 cells. RNA values are expressed as relative RNA content (means±SE of 4 experiments, each in triplicate). B) Western blot analysis of PC2 in LCLs and HEK 293 cells. Levels of PC2 bands were related to those of either actin or Ponceau red stained bands. PC2 expression in non-PKD LCLs (n=6) was compared with that in HEK293 cells (means±SE of at least 4 experiments). C) LCL cell extracts treated with (+) or without (–) PNGase F and Endo H. D) PC2 subcellular localization. LCL total lysate (Tot), 100,000 x g centrifugation supernatant (S100) and pellet fraction (P100). Fractions from a sucrose gradient centrifugation of LCL lysate analyzed with anti N-ter-PC2, ß1 integrin, calnexin, PDI, and actin antibodies. Electrophoretic results are representative of 3 experiments.

2. PC-2 is expressed in LCLs as a glycosylated ER membrane protein
We found that in LCLs cells (Fig. 1B ) relative expression of PC2 was eightfold lower than in HEK 293 cells (1±0.139 in six non-PKD LCLs vs. 8.05±1.93 in HEK 293). These values were consistent with those found for the PKD2 RNA in LCLs and HEK 293 cell lines.

In LCLs, PC2 was heavily N-glycosylated and PNGase and EndoH-sensitive, as found in kidney epithelial cells (Fig. 1C ). Subcellular localization studies showed that PC2 was present in the P100 membrane fraction and comigrated, in density gradient centrifugation, with the ER membrane resident protein calnexin. Thus, our findings indicate that PC2 retains an ER membrane localization in LCLs as it does in kidney epithelial cells.

3. PAF-evoked Ca²⁺ levels and cell proliferation are reduced in LCLs from PKD subjects
Results obtained in seven PKD2- (5 truncating and 2 unknown mutations), nine PKD1-, and eight control LCLs showed a marked PC2 reduction in PKD2- vs. PKD1-LCLs, and no difference between PKD1- and control LCLs (0.386±0.062 in PKD2-, 0.97±0.129 in controls, and 0.97±0.1 in PKD1-LCLs, P<0.001 in PKD2 vs. PKD1). Real-time RT-PCR analysis of PKD2 RNA in three controls and three PKD2-LCLs showed that the PC2 reduction in PKD2-LCLs was not associated with reduced PKD2 RNA content.

Since PC2 is structurally and functionally related to a Ca²⁺ channel, we measured in Fura2/M-loaded LCLs the changes in cytoplasm Ca²⁺ concentrations ([Ca²⁺]_I) induced by the platelet-activating factor (PAF), a potent phospholipid molecule inducing a rapid release of Ca²⁺ from internal stores in B-lymphoblastoid cells. The 1 µM PAF treatment of a non-PKD-LCL (Nor) produced a sustained increase in [Ca²⁺]_I, which was smaller in a PKD2-LCLs (PK2) (Fig. 2 A). The average [Ca²⁺]_i elevation in four PKD2-LCLs was significantly lower than in four control LCLs (104±8.2 nM vs. 271±7. 9 nM, P<0.001). The loss of PC2 was therefore associated with a reduction in PAF-stimulated [Ca²⁺]_i, supporting a PC2 role in LCLs Ca²⁺ homeostasis. In the four PKD1-LCLs, the PAF-induced [Ca²⁺]_i was significantly smaller than in control LCLs (149±16.9 in PKD1 vs. 271±7.9 nM in control, P=0.005). No statistically significant difference was found between PKD2- and PKD1-LCLs values. Therefore, also an allelic mutation of PKD1 gene resulted in a reduction of PAF-stimulated [Ca²⁺]_i in the presence of PC2 normal levels.

View larger version (35K): [in this window] [in a new window]   Figure 2. PAF-induced Ca2+ mobilization and cell proliferation are reduced in PKD-LCLs. A) Example of the PAF-evoked Ca2+ response in a non-PKD- (Nor) and a PKD2- (PK2) LCL. Cells were loaded with Fura2/AM and stimulated with 1 µM PAF at the time indicated by the arrow. PAF-evoked [Ca2+]i were estimated as differences between peak and basal [Ca2+]I. Means were from 4 non-PKD- (Nor), 4 PKD2- (PK2), and 4 PKD1-LCLs (PK1) (**P<0.001 and *P<0.005). B) Examples of cell growth in a non-PKD-, PKD1-, and PKD2-LCL cultured in the presence of 10% or 2.5% FCS. Cell duplication time was calculated in 4 non-PKD- and 8 PKD-LCLs (PKD, 4 PKD1, and 4 PKD2) (means±SE of at least 3 experiments in triplicate). Number of cells positive for the Ki67 antigen in 4 non-PKD- and 4 PKD2-LCLs expressed as % of total number of cells. Values represent means ± SE of 4 experiments.

In B-LCLs, the increase in [Ca²⁺]_i controls a diverse range of cell functions, including gene expression and cell proliferation. In PKD-LCLs, cell proliferation was lower than in control cells (Fig. 2B ). In the presence of low serum (2.5%), the cell duplication time was significantly higher in PKD- than in control LCLs (55.69±2.02 h vs. 42.41±2.32 h, P<0.001). Accordingly, in control LCLs, the number of cells that were positive to the Ki67 antigen, a marker of cell proliferation, was higher than in PKD2-LCLs (38.03±5.79% vs. 17.34±1.47%, respectively).

4. Aminoglycoside antibiotics increase the reduced PC-2 levels in PKD2-LCLs with premature stop mutations
We found that aminoglycoside antibiotics, known to promote read-through of eukaryotic stop codons, increased the PC2 expression level in PKD2-LCLs heterozygous for an in-frame UAA stop codon (Y386X or R186X). In PKD2-LCLs with the R186X mutation, the 48 h treatment with 50 µg/mL gentamicin (G) and 200 µg/mL isepamicin (I) produced a 1.86 ± 0.475 and a 1.75 ± 0.48 nonsignificant increase in PC2 levels, respectively, not observed in non-PKD-LCL (0.88±0.14 and 1.00±0.16 in G and I, respectively). Consistently, 3 day treatment with G (50 µg/mL) produced a 16% increase in PAF-evoked Ca²⁺ release in R186X-PKD2 LCLs, demonstrating that aminoglycoside treatment caused an increase in functional PC2.

CONCLUSIONS AND SIGNIFICANCE

This report characterizes for the first time functional aspects of PC2 in LCLs, taking advantage of the reduced levels of PC2 in LCLs obtained from PKD2 subjects. Our observations provide evidence that B-LCLs are a useful model to study PKD2 gene expression and modulation. Since PKD2 mutations are phenotypically manifested at heterozygous level in PKD2-LCLs as a constant reduction in PC2, one could propose LCL PC2 analysis as a preliminary assay before DNA and RNA analysis when linkage analysis is impossible, as in ADPKD sporadic cases. Moreover, since two PKD2-LCLs heterozygous for nonsense mutations showed an increase in PC2 levels when cultured in the presence of aminoglycosidic antibiotics, the LCLs could be a model by which to analyze therapeutic strategies aimed at suppressing PKD gene mutations.

Detection of PC2 as a glycosylated protein localized mainly in ER membranes in LCLs strongly suggests a role for PC2 as an intracellular Ca²⁺ release channel, as proposed in kidney cells. Consistent with this hypothesis, we found that in PKD2-LCLs heterozygous for truncating PKD2 mutations, the marked reduction in PC2 resulted in a decrease in PAF-mediated Ca²⁺ mobilization. This observation revealed that PC2 is critical for the regulation of intracellular Ca²⁺ levels in LCLs, as shown in epithelial cells.

That both PKD2 and PKD1 RNA expression maintains in LCLs the same quantity ratio as in HEK293 kidney cells suggests that polycystins may play similar roles in the two different cell lineages by interacting in a functional PC complex, like that identified in kidney cells. Consistently, the observation that the decrease in PAF-mediated Ca²⁺ mobilization was found not only in PKD2 but also in PKD1-LCLs strongly supports the involvement of PC1 in the control of LCL intracellular Ca²⁺ homeostasis. Thus, in lymphoblastoid cells the two proteins function in the same pathway, so that the loss of activity of either protein results in the same manifestation as the reduction in cell proliferation, a Ca²⁺-regulated pathway in B-LCLs shown in Fig. 3 .

View larger version (36K): [in this window] [in a new window]   Figure 3. Proposed model for the role of PC2 in LCLs. The scheme suggests that PC2, interacting with PC1 either at the plasma or the endoplasmic reticulum (ER) membranes mediates LCL Ca2+ influx or Ca2+ release from the ER, taking part in a variety of cellular events, including cell proliferation. In ADPKD LCLs, where there is an impaired function of the PC complex because of a PC2 reduction (*) in PKD2-LCLs or a PC1 functional loss (*) in PKD1-LCLs, the reduction in cytoplasmic Ca2+ affects the Ca2+-mediated signaling, and then cell proliferation.

Contributing in complex with PC1 to the control of intracellular calcium homeostasis in LCLs, PC2 proves to be a molecular component of the still elusive Ca²⁺ influx mechanism in lymphoblasts.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0687fje

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