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

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

Common variants at 12p11, 12q24, 9p21, 9q31.2 and in ZNF365 are associated with breast cancer risk for BRCA1 and/or BRCA2 mutation carriers

Abstract Introduction Several common alleles have been shown to be associated with breast and/or ovarian cancer risk for BRCA1 and BRCA2 mutation carriers. Recent genome-wide association studies of breast cancer have identified eight additional breast cancer susceptibility loci: rs1011970 (9p21, CDKN2A/B), rs10995190 (ZNF365), rs704010 (ZMIZ1), rs2380205 (10p15), rs614367 (11q13), rs1292011 (12q24), rs10771399 (12p11 near PTHLH) and rs865686 (9q31.2). Methods To evaluate whether these single nucleotide polymorphisms (SNPs) are associated with breast cancer risk for BRCA1 and BRCA2 carriers, we genotyped these SNPs in 12,599 BRCA1 and 7,132 BRCA2 mutation carriers and analysed the associations with breast cancer risk within a retrospective likelihood framework. Results Only SNP rs10771399 near PTHLH was associated with breast cancer risk for BRCA1 mutation carriers (per-allele hazard ratio (HR) = 0.87, 95% CI: 0.81 to 0.94, P-trend = 3 × 10-4). The association was restricted to mutations proven or predicted to lead to absence of protein expression (HR = 0.82, 95% CI: 0.74 to 0.90, P-trend = 3.1 × 10-5, P-difference = 0.03). Four SNPs were associated with the risk of breast cancer for BRCA2 mutation carriers: rs10995190, P-trend = 0.015; rs1011970, P-trend = 0.048; rs865686, 2df-P = 0.007; rs1292011 2df-P = 0.03. rs10771399 (PTHLH) was predominantly associated with estrogen receptor (ER)-negative breast cancer for BRCA1 mutation carriers (HR = 0.81, 95% CI: 0.74 to 0.90, P-trend = 4 × 10-5) and there was marginal evidence of association with ER-negative breast cancer for BRCA2 mutation carriers (HR = 0.78, 95% CI: 0.62 to 1.00, P-trend = 0.049). Conclusions The present findings, in combination with previously identified modifiers of risk, will ultimately lead to more accurate risk prediction and an improved understanding of the disease etiology in BRCA1 and BRCA2 mutation carriers

Analysis of upstream sequences of the human estrogen receptor gene.

Recently, modulation of some eukaryotic promoters by the estrogen receptor (ER) has been clarified at the molecular level; less is known about the control of expres- sion of the ER gene. In order to understand the mechanisms involved in its transcrip- tional regulation, we subcloned and sequenced a 2.8-kb genomic region upstream of the previously described initiation transcription site of the human ER gene.' A computer-assisted suggests that this region contains some new upstream ORFs and, according to other findings? a putative intronic sequence of 2 kb. RT-PCR experiments performed with specific oligonucleotide primers (FIG.1) demonstrate that (1)the ATG located at -2317 belongs to an upstream ER transcript and (2) this is transcribed in different breast cancer cell lines and tissues, but not in endometrial carcinomas (FIG.2A and B). Accordingly, an mRNA form hybridizing with the probe which includes the ORFs was detected by Northern blot (FIG.2C). The events leading to the production of this additional ER RNA are presently still unknown. However, these data demonstrate that the sequence upstream of -2317 might be a good candidate for an additional ER promoter region that might function differently depending on either tissue type or differentiation status. Moreover, at -1025 of the sequenced region there is an AlT-rich region including a (TA)26 dinucleotide repeat that is polymorphic in the p~pulation.W~e have investigated whether this polymorphism was related to ER gene expression. No differences in the allele number are found between normal and cancer breast samples of the same subject, nor does a relationship exist between the size of polymorphic alleles and the extent of ER content in the breast, endometrial, and kidney cancer cell lines analyzed. However, in these last DNA samples a low heterozygosity is present, suggesting loss of somatic alleles. Therefore, this polymorphism may be a useful marker to study the loss of constitutional heterozygosity on chromosome 6q

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

Acanthosis nigricans-insulin resistance Type A syndrome: analysis of restriction fragments length polymorphisms at the insulin receptor locus

We have identified two sisters (12 and 17 years old) affected by acanthosis nigricans-insulin resistance (AN-IR) type A syndrome and Type 2 (non-insulin-dependent) diabetes mellitus. They presented with acanthosis nigricans, marked hyperinsulinaemia and severe insulin resistance, Type 2 diabetes, no antibodies to the insulin receptor, obesity and virilisation without other endocrine diseases. Both parents and paternal grandmother had Type 2 diabetes. In AN-IR type A syndrome a primary defect of insulin receptor is supposed. The availability of cloned DNA (cDNA) for the human insulin receptor allows examination of the possible role of this gene in this syndrome. Therefore we analysed restriction fragments length polymorphisms (RFLP) for the insulin receptor gene in different members of this family, including diabetic and non diabetic subjects. DNA extracted from white blood cells was digested by seven restriction enzymes, analysed by Southern blotting technique, using a eDNA probe for the insulin receptor of 4.2 kilobases. Insulin receptor DNA fragments appeared the same in all the examined subjects. No association of any RFLP was noted with the syndrome. Therefore, in this family, RFLPs for the insulin receptor gene were uninformative in evaluating the role of this gene in AN-IR type A syndrome, nevertheless the obtained results exclude its marked alterations in the investigated patients