Berberine slows cell growth in autosomal dominant polycystic kidney disease cells.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary monogenic disorder characterized by development and enlargement of kidney cysts that lead to loss of renal function. It is caused by mutations in two genes (PKD1 and PKD2) encoding for polycystin-1 and polycystin-2 proteins which regulate different signals including cAMP, mTOR and EGFR pathways. Abnormal activation of these signals following PC1 or PC2 loss of function causes an increased cell proliferation which is a typical hallmark of this disease. Despite the promising findings obtained in animal models with targeted inhibitors able to reduce cystic cell growth, currently, no specific approved therapy for ADPKD is available. Therefore, the research of new more effective molecules could be crucial for the treatment of this severe pathology. In this regard, we have studied the effect of berberine, an isoquinoline quaternary alkaloid, on cell proliferation and apoptosis in human and mouse ADPKD cystic cell lines. Berberine treatment slows cell proliferation of ADPKD cystic cells in a dose-dependent manner and at high doses (100 µg/mL) it induces cell death in cystic cells as well as in normal kidney tubule cells. However, at 10 µg/mL, berberine reduces cell growth in ADPKD cystic cells only enhancing G0/G1 phase of cell cycle and inhibiting ERK and p70-S6 kinases. Our results indicate that berberine shows a selected antiproliferative activity in cellular models for ADPKD, suggesting that this molecule and similar natural compounds could open new opportunities for the therapy of ADPKD patients.

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 Ca(2+) 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 A(3) 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.

NF-kappaB activation is required for apoptosis in fibrocystin/polyductin-depleted kidney epithelial cells.

Autosomal recessive polycystic kidney disease (ARPKD) is caused by mutations in PKHD1, a gene encoding fibrocystin/polyductin (FC1), a membrane-associated receptor-like protein involved in the regulation of tubular cell adhesion, proliferation and apoptosis. Although it is generally accepted that apoptosis is implicated in ARPKD, the question of whether increased apoptosis is a normal response to abnormal cell proliferation or, instead, it is a primary event, is still subject to debate. In support of the latter hypothesis, we hereby provide evidence that apoptosis occurs in the absence of hyper-proliferation of FC1-depleted kidney cells. In fact, a decrease in cell proliferation, with a concomitant increase in apoptotic index and caspase-3 activity was observed in response to FC1-depletion by PKHD1 siRNA silencing in HEK293 and 4/5 tubular cells. FC1-depletion also induced reduction in ERK1/2 kinase activation, upregulation of the pro-apoptotic protein p53 and activation of NF-kappaB, a transcription factor which reduces apoptosis in many organs and tissues. Interestingly, selective inactivation of NF-kappaB using either an NF-kappaB decoy or parthenolide, a blocker of IKK-dependent NF-kappaB activation, reduced, rather then increased, apoptosis and p53 levels in FC1-depleted cells. Therefore, the proapoptotic function of NF-kappaB during cell death by FC1-depletion in kidney cells is evident.

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.

Methylation analysis of the promoter F of estrogen receptor alpha gene: effects on the level of transcription on human osteoblastic cells.

In this study, the methylation status of the distal promoter F of estrogen receptor alfa (ERalpha) gene in human osteoblastic cells was investigated. The activity of this promoter is responsible for the ERalpha gene transcription in bone tissue. The methylation status of promoter F was here evaluated, for the first time, by direct sequencing of bisulfite-treated genomic DNA, at 10 CpG specific sites localized in a region of about 800 bp. An heterogeneous methylation pattern was observed. The most notable difference was found at four particular CpGs, distant from the exon F transcription start site, showing a methylation status that correlates with the expression level, being ERalpha mRNA transcription reduced in a partially methylated cells but preserved in demethylated cells. The other CpG sites, localized around the transcription start site, were always demethylated except for MG-63 cells showing the lowest level of ERalpha expression. By quantitative RT-PCR analysis we demonstrated that ERalpha gene expression was higher in primary osteoblasts than in bone-derived cells (MG-63 and SaOS-2) and in all cases the ERalpha mRNA is represented by the isoform F. The same 10 CpG sites were investigated in non-osseous cell lines and were found fully methylated in ERalpha-negative breast cancer cells (MDA-MB-231) and completely demethylated in ERalpha-positive breast cancer cells (MCF7). The overall results suggest that methylation of the CpG sites inside ERalpha gene promoter F here analyzed may contribute to ERalpha transcriptional control, directly or indirectly, influencing the tissue specific expression of the gene.

Differential expression of microRNA501-5p affects the aggressiveness of clear cell renal carcinoma.

Renal cell carcinoma is a common neoplasia of the adult kidney that accounts for about 3% of adult malignancies. Clear cell renal carcinoma is the most frequent subtype of kidney cancer and 20-40% of patients develop metastases. The absence of appropriate biomarkers complicates diagnosis and prognosis of this disease. In this regard, small noncoding RNAs (microRNAs), which are mutated in several neoplastic diseases including kidney carcinoma, may be optimal candidates as biomarkers for diagnosis and prognosis of this kind of cancer. Here we show that patients with clear cell kidney carcinoma that express low levels of miR501-5p exhibited a good prognosis compared with patients with unchanged or high levels of this microRNA. Consistently, in kidney carcinoma cells the downregulation of miR501-5p induced an increased caspase-3 activity, p53 expression as well as decreased mTOR activation, leading to stimulation of the apoptotic pathway. Conversely, miR501-5p upregulation enhanced the activity of mTOR and promoted both cell proliferation and survival. These biological processes occurred through p53 inactivation by proteasome degradation in a mechanism involving MDM2-mediated p53 ubiquitination. Our results support a role for miR501-5p in balancing apoptosis and cell survival in clear cell renal carcinoma. In particular, the downregulation of microRNA501-5p promotes a good prognosis, while its upregulation contributes to a poor prognosis, in particular, if associated with p53 and MDM2 overexpression and mTOR activation. Thus, the expression of miR501-5p is a possible biomarker for the prognosis of clear cell renal carcinoma.

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.

Polycystin-1 promotes PKCalpha-mediated NF-kappaB activation in kidney cells.

Polycystin-1 (PC1), the PKD1 gene product, is a membrane receptor which regulates many cell functions, including cell proliferation and apoptosis, both typically increased in cyst lining cells in autosomal dominant polycystic kidney disease. Here we show that PC1 upregulates the NF-kappaB signalling pathway in kidney cells to prevent cell death. Human embryonic kidney cell lines (HEK293(CTT)), stably expressing a PC1 cytoplasmic terminal tail (CTT), presented increased NF-kappaB nuclear levels and NF-kappaB-mediated luciferase promoter activity. This, consistently, was reduced in HEK293 cells in which the endogenous PC1 was depleted by RNA interference. CTT-dependent NF-kappaB promoter activation was mediated by PKCalpha because it was blocked by its specific inhibitor Ro-320432. Furthermore, it was observed that apoptosis, which was increased in PC1-depleted cells, was reduced in HEK293(CTT) cells and in porcine kidney LtTA cells expressing a doxycycline-regulated CTT. Staurosporine, a PKC inhibitor, and parthenolide, a NF-kappaB inhibitor, significantly reduced the CTT-dependent antiapoptotic effect. These data reveal, therefore, a novel pathway by which polycystin-1 activates a PKCalpha-mediated NF-kappaB signalling and cell survival.

The cytoplasmic C-terminus of polycystin-1 increases cell proliferation in kidney epithelial cells through serum-activated and Ca(2+)-dependent pathway(s).

Polycystin-1 (PC1) is a large transmembrane protein important in renal differentiation and defective in most cases of autosomal dominant polycystic kidney disease (ADPKD), a common cause of renal failure in adults. Although the genetic basis of ADPKD has been elucidated, molecular and cellular mechanisms responsible for the dysregulation of epithelial cell growth in ADPKD cysts are still not well defined. We approached this issue by investigating the role of the carboxyl cytoplasmic domain of PC1 involved in signal transduction on the control of kidney cell proliferation. Therefore, we generated human HEK293 cells stably expressing the PC1 cytoplasmic tail as a membrane targeted TrkA-PC1 chimeric receptor protein (TrkPC1). We found that TrkPC1 increased cell proliferation through an increase in cytoplasmic Ca2+ levels and activation of PKC alpha, thereby upregulating D1 and D3 cyclin, downregulating p21waf1 and p27kip1 cyclin inhibitors, and thus inducing cell cycle progression from G0/G1 to the S phase. Interestingly, TrkPC1-dependent Ca2+ increase and PKC alpha activation are not constitutive, but require serum factor(s) as parallel component. In agreement with this observation, a significant increase in ERK1/2 phosphorylation was observed. Consistently, inhibitors specifically blocking either PKC alpha or ERK1/2 prevented the TrkPC1-dependent proliferation increase. NGF, the TrkA ligand, blocked this increase. We propose that in kidney epithelial cells the overexpression of PC1 C-terminus upregulates serum-evoked intracellular Ca2+ by counteracting the growth-suppression activity of endogenous PC1 and leading to an increase in cell proliferation.

Expression of the human oestrogen receptor-alpha gene is regulated by promoter F in MG-63 osteoblastic cells.

(O)estrogen receptor-alpha (ERalpha), a hormone-dependent transcription factor belonging to the steroid/thyroid-hormone-receptor superfamily, plays an essential role in the development and maintenance of the skeleton. Here we report the analysis of an unexplored sequence inside the bone-specific distal promoter F (PF) with respect to the regulation of ERalpha gene expression in bone. This sequence, 785 bp in size, is localized upstream of the assigned transcription start site of exon F, at -117140 bp from the originally described transcription start site +1. It contains a TA reach box, a conventional CAAT box and potential regulatory elements for many transcription factors, including Cbfa1 [OSE2 (osteoblast-specific element) core binding factor], GATA-1 [(A/T)GATA(A/G) binding protein], Sox5 [sex-determining region Y (SRY)-type HMG bOX protein, belonging to a subfamily of DNA-binding proteins with an HMG domain], Sry, AP1 (activator protein 1) and CP2 (activator of gamma-globin). It is able to strongly activate the luciferase reporter gene in MG-63 osteoblastic-like cells, but not in MCF7 breast-cancer cells. This is in agreement with different transcripts that we found in the two cell types. The footprinting and electrophoretic mobility-shift assays (EMSAs) showed that, inside the region analysed, there were some sequences that specifically reacted to nuclear proteins isolated from MG-63 cells. In particular, we identified two regions, named PF a and PF b, that do not present binding sites for known transcription factors and that are involved in a strong DNA-protein interaction in MG-63, but not in MCF7, cells. The analysis of three transcription factors (GATA-1, Sry and Sox) that might bind the identified footprinted areas suggested a possible indirect role of these proteins in the regulation of ERalpha gene expression in bone. These data provide evidence for different promoter usage of the ERalpha gene through the recruitment of tissue-specific transcription activators and co-regulators.

Osteoblastic differentiation induced by transcription factor decoy against estrogen receptor alpha gene.

Osteoblasts have been shown to express both isoforms of estrogen receptor (ER alpha and ER beta). As a tool for the study of endogenous regulation of these genes the decoy strategy was employed. Human MG-63 osteoblast-like cells were transfected with a DNA decoy molecule containing a putative negative cis-element (DNA-102) located in the C distal promoter of ER alpha gene. Using real-time quantitative RT-PCR, we found that the DNA-102, but not scrambled DNA, produced a 36-fold increase in the level of total ER alpha mRNA and a 12-fold increase in the level of mRNA for the F isoform that is transcribed from the upstream F promoter, which is predominantly used in osteoblasts. This effect appears to be controlled by estrogen since 17-beta-estradiol downregulated the mRNA increase. Notably, the same decoy was able to induce a 6-fold increase in ER beta mRNA transcription, indicating the coregulation of the ER alpha and ER beta expression. An increase in OPN but not in BMP4 expression was also observed. In addition, in decoy-treated cells, the cell growth decreased together with an increase in alkaline phosphatase activity. These findings indicated that DNA-102 decoy was able to induce a more differentiated osteoblastic phenotype. The augmentation of ER alpha and ER beta expression by the decoy approach may offer a further possibility for patient response to estrogenic therapy in the treatment of diseases related to estrogen deficiency.

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.