Retinoic acid-dependent activation of the polycystic kidney disease-1 (PKD1) promoter

The retinoic acids all-trans retinoic acid (AT-RA) and 9-cis retinoic acid (9C-RA) and the retinoic acid receptors RAR and RXR significantly induce transcriptional activity from a 200-bp PKD1 proximal promoter in transfected mammalian cells. This PKD1 promoter region contains Ets, p53, and GC box motifs, but lacks a canonical RAR/RXR motif. Mutagenesis of the Ets sites did not affect RA induction. In contrast, GC box mutations completely blocked stimulation by AT-RA and by RXRβ or RARβ. Mithramycin A, which prevents Sp1 binding, significantly reduced basal promoter activity and suppressed upregulation by AT-RA and RXR. The 200-bp proximal promoter could not be induced by AT-RA in Drosophila SL2 cells, which lack Sp1, but could be activated in these cells transfected with exogenous Sp1. Small interfering RNA knockdown of Sp1 in mammalian cells completely blocked RXRβ upregulation of the promoter. These data indicate that induction of the PKD1 promoter by retinoic acid is mediated through Sp1 elements. RT-PCR showed that AT-RA treatment of HEK293T cells increased the levels of endogenous PKD1 RNA, and chromatin immunoprecipitation showed the presence of both RXR and Sp1 at the PKD1 proximal promoter. These results suggest that retinoids and their receptors may play a role in PKD1 gene regulation

A mutation affecting polycystin-1 mediated heterotrimeric G-protein signaling causes PKD

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the growth of renal cysts that ultimately destroy kidney function. Mutations in the PKD1 and PKD2 genes cause ADPKD. Their protein products, polycystin-1 (PC1) and polycystin-2 (PC2) have been proposed to form a calcium-permeable receptor-channel complex; however the mechanisms by which they function are almost completely unknown. Most mutations in PKD1 are truncating loss-of-function mutations or affect protein biogenesis, trafficking or stability and reveal very little about the intrinsic biochemical properties or cellular functions of PC1. An ADPKD patient mutation (L4132Δ or ΔL), resulting in a single amino acid deletion in a putative G-protein binding region of the PC1 C-terminal cytosolic tail, was found to significantly decrease PC1-stimulated, G-protein-dependent signaling in transient transfection assays. Pkd1ΔL/ΔL mice were embryo-lethal suggesting that ΔL is a functionally null mutation. Kidney-specific Pkd1ΔL/cond mice were born but developed severe, postnatal cystic disease. PC1ΔL protein expression levels and maturation were comparable to those of wild type PC1, and PC1ΔL protein showed cell surface localization. Expression of PC1ΔL and PC2 complexes in transfected CHO cells failed to support PC2 channel activity, suggesting that the role of PC1 is to activate G-protein signaling to regulate the PC1/PC2 calcium channel

Ciclopirox olamine induces ferritinophagy and reduces cyst burden in polycystic kidney disease

Despite the recent launch of tolvaptan, the search for safer polycystic kidney disease (PKD) drugs continues. Ciclopirox (CPX) or its olamine salt (CPX-O) is contained in a number of commercially available antifungal agents. CPX is also reported to possess anticancer activity. Several mechanisms of action have been proposed, including chelation of iron and inhibition of iron-dependent enzymes. Here, we show that CPX-O inhibited in vitro cystogenesis of primary human PKD cyst-lining epithelial cells cultured in a 3D collagen matrix. To assess the in vivo role of CPX-O, we treated PKD mice with CPX-O. CPX-O reduced the kidney-to-body weight ratios of PKD mice. The CPX-O treatment was also associated with decreased cell proliferation, decreased cystic area, and improved renal function. Ferritin levels were markedly elevated in cystic kidneys of PKD mice, and CPX-O treatment reduced renal ferritin levels. The reduction in ferritin was associated with increased ferritinophagy marker nuclear receptor coactivator 4, which reversed upon CPX-O treatment in PKD mice. Interestingly, these effects on ferritin appeared independent of iron. These data suggest that CPX-O can induce ferritin degradation via ferritinophagy, which is associated with decreased cyst growth progression in PKD mice. Most importantly these data indicate that CPX-O has the potential to treat autosomal dominant PKD

Polycystin-1 Regulates Skeletogenesis through Stimulation of the Osteoblast-specific Transcription Factor RUNX2-II*

Polycystin-1 (PC1) may play an important role in skeletogenesis through regulation of the bone-specific transcription factor Runx2-II. In the current study we found that PC1 co-localizes with the calcium channel polycystin-2 (PC2) in primary cilia of MC3T3-E1 osteoblasts. To establish the role of Runx2-II in mediating PC1 effects on bone, we crossed heterozygous Pkd1m1Bei and Runx2-II mice to create double heterozygous mice (Pkd1+/m1Bei/Runx2-II+/-) deficient in both PC1 and Runx2-II. Pkd1+/m1Bei/Runx2-II+/- mice exhibited additive reductions in Runx2-II expression that was associated with impaired endochondral bone development, defective osteoblast-mediated bone formation, and osteopenia. In addition, we found that basal intracellular calcium levels were reduced in homozygous Pkd1m1Bei osteoblasts. In contrast, overexpression of a PC1 C-tail construct increased intracellular calcium and selectively stimulated Runx2-II P1 promoter activity in osteoblasts through a calcium-dependent mechanism. Site-directed mutagenesis of critical amino acids in the coiled-coil domain of PC1 required for coupling to PC2 abolished PC1-mediated Runx2-II P1 promoter activity. Additional promoter analysis mapped the PC1-responsive region to the “osteoblast-specific” enhancer element between -420 and -350 bp that contains NFI and AP-1 binding sites. Chromatin immunoprecipitation assays confirmed the calcium-dependent binding of NFI to this region. These findings indicate that PC1 regulates osteoblast function through intracellular calcium-dependent control of Runx2-II expression. The overall function of the primary cilium-polycystin complex may be to sense and transduce environmental clues into signals regulating osteoblast differentiation and bone development