Gianluca Aguiari

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

Polycystin‐2 (PC2), encoded by the PKD2 gene, mutated in 10‐15% of autosomal‐dominant polycystic kidney disease (ADPKD) patients, is a Ca2+‐permeable cation channel present in kidney epithelia and other tissues. As PC2 was found expressed in B‐lymphoblastoid cells (LCLs) and Ca2+ signaling pathways are important regulators of B cell function activities, we investigated whether PC2 plays some role in B‐LCLs. In LCLs, PC2 was found mainly in ER membranes but ~8 times less than in kidney HEK293 cells. The same reductions were found in PKD2 and PKD1 RNA; thus, PKD genes maintained, in LCLs, the same reciprocal proportion as they do in kidney cells. In LCLs obtained from subjects carrying PKD2 mutations (PKD2‐LCLs) and showing reduced PC2 levels, intracellular Ca2+ concentrations evoked by platelet‐activating factor (PAF), were significantly lower than in non‐PKD‐LCLs. This reduction was also found in PKD1‐LCLs but without PC2 reductions. Likewise, cell proliferation, which is controlled by Ca2+, was reduced in PKD2‐ and PKD1‐LCLs. Moreover, in LCLs with PKD2 nonsense mutations, aminoglycoside antibiotics reduced the PC2 defect by promoting readthrough of stop codons. Therefore, PC2 and PC1 are functionally expressed in LCLs, which provide a model, easily obtainable from ADPKD patients, to study PKD gene expression and function.

Effects of a Hydroxyapatite-based Biomaterial on Gene Expression in Osteoblast-like Cells

Biostite® is a hydroxyapatite-derived biomaterial that is used in periodontal and bone reconstructive procedures due to its osteoconductive properties. Since the molecular effects of this biomaterial on osteoblasts are still unknown, we decided to assess whether it may specifically modulate osteoblast functions in vitro. We found that a brief exposure to Biostite® significantly reduced the proliferation of MG-63 and SaOS-2 osteoblast-like cells to ~ 50% of the plateau value. Furthermore, gene array analysis of MG-63 cells showed that Biostite® caused a differential expression of 37 genes which are involved in cell proliferation and interaction, and related to osteoblast differentiation and tissue regeneration. Results were confirmed by RT-PCR, Western blot, and by an increase in alkaline phosphatase (ALP) specific activity. Biostite® also increased levels of polycystin-2, a mechano-sensitive Ca2+ channel, a promising new marker of bone cell differentiation. Biostite®, therefore, may directly affect osteoblasts by enhancing chondro/osteogenic gene expression and cytoskeleton-related signaling pathways, which may contribute to its clinical efficacy.

Expression of polycystin-1 C-terminal fragment enhances the ATP-induced Ca2+ release in human kidney cells

Polycystin-1 (PC1) is a membrane protein expressed in tubular epithelia of developing kidneys and in other ductal structures. Recent studies indicate this protein to be putatively important in regulating intracellular Ca(2+) levels in various cell types, but little evidence exists for kidney epithelial cells. Here we examined the role of the PC1 cytoplasmic tail on the activity of store operated Ca(2+) channels in human kidney epithelial HEK-293 cell line. Cells were transiently transfected with chimeric proteins containing 1-226 or 26-226 aa of the PC1 cytoplasmic tail fused to the transmembrane domain of the human Trk-A receptor: TrkPC1 wild-type and control Trk truncated peptides were expressed at comparable levels and localized at the plasma membrane. Ca(2+) measurements were performed in cells co-transfected with PC1 chimeras and the cytoplasmic Ca(2+)-sensitive photoprotein aequorin, upon activation of the phosphoinositide pathway by ATP, that, via purinoceptors, is coupled to the release of Ca(2+) from intracellular stores. The expression of TrkPC1 peptide, but not of its truncated form, enhanced the ATP-evoked cytosolic Ca(2+) concentrations. When Ca(2+) assays were performed in HeLa cells characterized by Ca(2+) stores greater than those of HEK-293 cells, the histamine-evoked cytosolic Ca(2+) increase was enhanced by TrkPC1 expression, even in absence of external Ca(2+). These observations indicate that the C-terminal tail of PC1 in kidney and other epithelial cells upregulates a Ca(2+) channel activity also involved in the release of intracellular stores.

Novel role for polycystin-1 in modulating cell proliferation through calcium oscillations in kidney cells

Abstract.  Objectives: Polycystin‐1 (PC1), a signalling receptor regulating Ca2+‐permeable cation channels, is mutated in autosomal dominant polycystic kidney disease, which is typically characterized by increased cell proliferation. However, the precise mechanisms by which PC1 functions on Ca2+ homeostasis, signalling and cell proliferation remain unclear. Here, we investigated the possible role of PC1 as a modulator of non‐capacitative Ca2+ entry (NCCE) and Ca2+ oscillations, with downstream effects on cell proliferation. Results and discussion: By employing RNA interference, we show that depletion of endogenous PC1 in HEK293 cells leads to an increase in serum‐induced Ca2+ oscillations, triggering nuclear factor of activated T cell activation and leading to cell cycle progression. Consistently, Ca2+ oscillations and cell proliferation are increased in PC1‐mutated kidney cystic cell lines, but both abnormal features are reduced in cells that exogenously express PC1. Notably, blockers of the NCCE pathway, but not of the CCE, blunt abnormal oscillation and cell proliferation. Our study therefore provides the first demonstration that PC1 modulates Ca2+ oscillations and a molecular mechanism to explain the association between abnormal Ca2+ homeostasis and cell proliferation in autosomal dominant polycystic kidney disease.

Polycystin-1 regulates amphiregulin expression through CREB and AP1 signalling: implications in ADPKD cell proliferation.

In autosomal dominant polycystic kidney disease (ADPKD), renal cyst development and enlargement, as well as cell growth, are associated with alterations in several pathways, including cAMP and activator protein 1 (AP1) signalling. However, the precise mechanism by which these molecules stimulate cell proliferation is not yet fully understood. We now show by microarray analysis, luciferase assay, mutagenesis, and chromatin immunoprecipitation that CREB and AP1 contribute to increased expression of the amphiregulin gene, which codifies for an epidermal growth factor-like peptide, in ADPKD cystic cells, thereby promoting their cell growth. Increased amphiregulin (AR) expression was associated with abnormal cell proliferation in both PKD1-depleted and -mutated epithelial cells, as well as primary cystic cell lines isolated from ADPKD kidney tissues. Consistently, normal AR expression and proliferation were re-established in cystic cells by the expression of a mouse full-length PC1. Finally, we show that anti-AR antibodies and inhibitors of AP1 are able to reduce cell proliferation in cystic cells by reducing AR expression and EGFR activity. AR can therefore be considered as one of the key activators of the growth of human ADPKD cystic cells and thus a new potential therapeutic target.

Nonspecific Cation Current Associated with Native Polycystin-2 in HEK-293 Cells

Mutations in either PKD1 or PKD2 gene are associated with autosomal dominant polycystic kidney disease, the most common inherited kidney disorder. Polycystin-2 (PC2), the PKD2 gene product, and the related protein polycystin-L, function as Ca(2+)-permeable, nonselective cation channels in different expression systems. This work describes a nonspecific cation current (I(CC)) that is present in native HEK-293 cells and highly associated with a PC2-channel activity. The current is voltage dependent, activating for potentials that are positive to -50 mV and inactivating in a few milliseconds. It is sensitive to Cd(2+), Gd(3+), La(3+), SKF96365, and amiloride. After silencing of PC2 by RNA interfering, cells show a reduced current that is restored by transfection with normal but not truncated PC2. Consistently, I(CC) is abolished by perfusion with an anti-PC2 antibody. Furthermore, heterologous expression of the PC1 cytoplasmic tail significantly increases I(CC) peak amplitude compared with native cells. This is the first characterization of such a current in HEK-293 cells, a widely used expression system for ion channels. These cells, therefore, could be regarded as a suitable and readily accessible tool to study interactions between native PC2/PC1 complex and other membrane proteins, thus contributing to the understanding of autosomal dominant polycystic kidney disease pathogenesis.

Multidrug therapy for polycystic kidney disease: a review and perspective.

Autosomal dominant polycystic kidney disease (ADPKD) is a renal disorder characterized by the development of cysts in both kidneys leading to end-stage renal disease (ESRD) by the fifth decade of life. Cysts also occur in other organs, and phenotypic alterations also involve the cardiovascular system. Mutations in the PKD1 and PKD2 genes codifying for polycystin-1 (PC1) and polycystin-2 (PC2) are responsible for the 85 and 15% of ADPKD cases, respectively. PC1 and PC2 defects cause similar symptoms; however, lesions of PKD1 gene are associated with earlier disease onset and faster ESRD progression. The development of kidney cysts requires a somatic ‘second hit’ to promote focal cyst formation, but also acute renal injury may affect cyst expansion, constituting a ‘third hit’. PC1 and PC2 interact forming a complex that regulates calcium homeostasis. Mutations of polycystins induce alteration of Ca2+ levels likely through the elevation of cAMP. Furthermore, PC1 loss of function also induces activation of mTOR and EGFR signaling. Impaired cAMP, mTOR and EGFR signals lead to activation of a number of processes stimulating both cell proliferation and fluid secretion, contributing to cyst formation and enlargement. Consistently, the inhibition of mTOR, EGFR activity and cAMP accumulation ameliorates renal function in ADPKD animal models, but in ADPKD patients mild results have been shown. Here we briefly review major ADPKD-related pathways, their inhibition and effects on disease progression. Finally, we suggest to reduce abnormal cell proliferation with possible clinical amelioration of ADPKD patients by combined inhibition of cAMP-, EGFR- and mTOR-related pathways.

Deficiency of polycystic kidney disease-1 gene (PKD1) expression increases A3 adenosine receptors in human renal cells: Implications for cAMP-dependent signalling and proliferation of PKD1-mutated cystic cells

Cyst growth and expansion in autosomal dominant polycystic kidney disease (ADPKD) has been attributed to numerous factors, including ATP, cAMP and adenosine signalling. Although the role of ATP and cAMP has been widely investigated in PKD1-deficient cells, no information is currently available on adenosine-mediated signalling. Here we investigate for the first time the impact of abnormalities of polycystin-1 (PC1) on the expression and functional activity of adenosine receptors, members of the G-protein-coupled receptor superfamily. Pharmacological, molecular and biochemical findings show that a siRNA-dependent PC1-depletion in HEK293 cells and a PKD1-nonsense mutation in cyst-derived cell lines result in increased expression of the A(3) adenosine receptor via an NFkB-dependent mechanism. Interestingly, A(3) adenosine receptor levels result higher in ADPKD than in normal renal tissues. Furthermore, the stimulation of this receptor subtype with the selective agonist Cl-IB-MECA causes a reduction in both cytosolic cAMP and cell proliferation in both PC1-deficient HEK293 cells and cystic cells. This reduction is associated with increased expression of p21(waf) and reduced activation not only of ERK1/2, but also of S6 kinase, the main target of mTOR signalling. In the light of these findings, the ability of Cl-IB-MECA to reduce disease progression in ADPKD should be further investigated. Moreover, our results suggest that NFkB, which is markedly activated in PC1-deficient and cystic cells, plays an important role in modulating A(3)AR expression in cystic cells.

Polymerase-chain reaction as a tool for investigations on sequence-selectivity of DNA-drugs interactions

Sequence-selectivity of DNA-binding drugs was recently reported in a number of studies employing footprinting and gel retardation approaches. In this paper we performed polymerase-chain reaction (PCR) experiments to study the in vitro effects of distamycin, daunomycin, chromomycin and mithramycin. As model systems we employed the human estrogen receptor (ER) gene and the Harvey-ras (Ha-ras) oncogene, in order to obtain PCR products significantly differing for the A + T/G + C frequency ratio. Distamycin, daunomycin, chromomycin and mithramycin are indeed known to differentially bind to different DNA regions depending upon the DNA sequences recognized. The main conclusion of our experiments is that distamycin, daunomycin, chromomycin and mithramycin inhibit polymerase-chain reaction in a sequence-dependent manner. Distamycin inhibits indeed PCR mediated amplification of AT-rich regions of the human estrogen receptor gene, displaying no inhibitory effects on PCR-mediated amplification of GC-rich sequences of Ha-ras oncogene. By contrast daunomycin, chromomycin and mithramycin were found to inhibit PCR-mediated amplification of the Ha-ras GC-rich oncogene sequences. We propose that polymerase-chain reaction technique could be applied to study the in vivo interactions of DNA-binding drugs to specific genes in intact cells.

Mutations in autosomal dominant polycystic kidney disease 2 gene: Reduced expression of PKD2 protein in lymphoblastoid cells

The polycystic kidney disease 2 (PKD2) gene, encoding a 968-amino acid integral membrane protein with six predicted membrane-spanning domains and intracellular NH2 and COOH termini, is mutated in approximately 15% of the cases of autosomal dominant polycystic kidney disease (ADPKD), a common genetic disease frequently resulting in renal failure. For a better understanding of the cause of this disorder, we searched for mutations in the PKD2 gene in two PKD2-linked families characterized by different clinical phenotypes. A common polymorphism, a nonsense mutation, and a frameshift mutation were found. Both mutations are predicted to produce truncated proteins of 314 and 386 amino acids, arrested at the first extracellular loop of the protein. Restriction enzyme analysis of polymerase chain reaction (PCR) and reverse transcriptase (RT)-PCR products, respectively, showed that mutations cosegregated with the disease and mutated alleles were expressed at the messenger RNA level in lymphoblastoid cell lines. However, in these cells, Western blot analysis showed only PKD2 normal protein, and it was expressed at a lower level than that found in cells without the PKD2 mutation. These findings suggest that in lymphoblastoid cells, the truncated protein product of the mutant allele may not be stable.