IFT88 deficiency in proximal tubular cells exaggerates cisplatin-induced injury by suppressing autophagy

Primary cilia are widely regarded as specialized sensors in differentiated cells that have been implicated in the regulation of cell proliferation, differentiation, and viability. We previously showed that shortening of primary cilia sensitizes cultured kidney tubular cells to cisplatin-induced apoptosis. IFT88 is an essential component for ciliogenesis and maintenance. Here, we have further examined the effect of proximal tubule-specific IFT88 ablation on cisplatin-induced acute kidney injury (AKI). In the study, more severe AKI occurred in IFT88 knockout mouse than age- and sex-matched wild type mice. Mechanistically, cisplatin stimulated autophagy in kidney tubular cells as an intrinsic protective mechanism. However, renal autophagy was severely impaired in IFT88 knockout mouse. In cultured HK-2 cells, cisplatin induced more apoptosis when IFT88 was knocked down. Tat-beclin 1 peptide, a specific autophagy activator, could partially prevent IFT88-associated cell death during cisplatin treatment, although cilium length was not improved significantly. Re-expression of IFT88 partially restored autophagy in IFT88-knockdown cells and suppressed apoptosis during cisplatin treatment. Taken together, these results indicate that defective autophagy in IFT88-deficient kidney cells and tissues contributes to the exaggerated AKI following cisplatin exposure.

Cell-based assay for ciliopathy patients to improve accurate diagnosis using ALPACA

Skeletal ciliopathies are a group of disorders caused by dysfunction of the cilium, a small signaling organelle present on nearly every vertebrate cell. This group of disorders is marked by genetic and clinical heterogeneity, which complicates accurate diagnosis. In this study, we developed a robust, standardized immunofluorescence approach to accurately diagnose a subset of these disorders. Hereto we determined and compared the cilium phenotype of healthy individuals to patients from three different ciliopathy subgroups, using skin-derived fibroblasts. The cilium phenotype assay consists of three parameters; (1) ciliogenesis, based on the presence or absence of cilium markers, (2) cilium length, measured by the combined signal of an axonemal and a cilium membrane marker, and (3) retrograde intraflagellar transport (IFT), quantified by the area of the ciliary tip. Analysis of the cilium phenotypic data yielded comparable and reproducible results and in addition, displayed identifiable clusters for healthy individuals and two ciliopathy subgroups, i.e. ATD and CED. Our results illustrate that standardized analysis of the cilium phenotype can be used to discriminate between ciliopathy subgroups. Therefore, we believe that standardization of functional assays analyzing cilium phenotypic data can provide additional proof for conclusive diagnosis of ciliopathies, which is essential for routine diagnostic care.

Primary Cilia Formation Does Not Rely on WNT/β-Catenin Signaling

Primary cilia act as crucial regulators of embryo development and tissue homeostasis. They are instrumental for modulation of several signaling pathways, including Hedgehog, WNT, and TGF-β. However, gaps exist in our understanding of how cilia formation and function is regulated. Recent work has implicated WNT/β-catenin signaling pathway in the regulation of ciliogenesis, yet the results are conflicting. One model suggests that WNT/β-catenin signaling negatively regulates cilia formation, possibly via effects on cell cycle. In contrast, second model proposes a positive role of WNT/β-catenin signaling on cilia formation, mediated by the re-arrangement of centriolar satellites in response to phosphorylation of the key component of WNT/β-catenin pathway, β-catenin. To clarify these discrepancies, we investigated possible regulation of primary cilia by the WNT/β-catenin pathway in cell lines (RPE-1, NIH3T3, and HEK293) commonly used to study ciliogenesis. We used WNT3a to activate or LGK974 to block the pathway, and examined initiation of ciliogenesis, cilium length, and percentage of ciliated cells. We show that the treatment by WNT3a has no- or lesser inhibitory effect on cilia formation. Importantly, the inhibition of secretion of endogenous WNT ligands using LGK974 blocks WNT signaling but does not affect ciliogenesis. Finally, using knock-out cells for key WNT pathway components, namely DVL1/2/3, LRP5/6, or AXIN1/2 we show that neither activation nor deactivation of the WNT/β-catenin pathway affects the process of ciliogenesis. These results suggest that WNT/β-catenin-mediated signaling is not generally required for efficient cilia formation. In fact, activation of the WNT/β-catenin pathway in some systems seems to moderately suppress ciliogenesis.

Primary cilia formation does not rely on WNT/β-catenin signaling

Primary cilia act as crucial regulators of embryo development and tissue homeostasis. They are instrumental for modulation of several signaling pathways, including Hedgehog, WNT, and TGF-β. However gaps exist in our understanding of how cilia formation and function is regulated.Recent work has implicated WNT/β-catenin signaling pathway in the regulation of ciliogenesis, yet the results are conflicting. One model suggests that WNT/β-catenin signaling negatively regulates cilia formation, possibly via effects on cell cycle. In contrast second model proposes a positive role of WNT/β-catenin signaling on cilia formation, mediated by the re-arrangement of centriolar satellites in response to phosphorylation of the key component of WNT/β-catenin pathway, β-catenin.To clarify these discrepancies, we investigated possible regulation of primary cilia by the WNT/β-catenin pathway in cell lines (RPE-1, NIH3T3, HEK293) commonly used to study ciliogenesis. We used WNT3a to activate or LGK974 to block the pathway, and examined initiation of ciliogenesis, cilium length, and percentage of ciliated cells. We show that the treatment by WNT3a has no- or lesser inhibitory effect on cilia formation. Importantly, the inhibition of secretion of endogenous WNT ligands using LGK974 blocks WNT signaling but does not affect ciliogenesis. Finally, using knock-out cells for key WNT pathway components, namely DVL1/2/3, LRP5/6 or AXIN1/2 we show that neither activation nor deactivation of the WNT/β-catenin pathway affects the process of ciliogenesis.These results suggest that WNT/β-catenin-mediated signaling is not generally required for efficient cilia formation. In fact, activation of the WNT/β-catenin pathway in some systems seems to moderately suppress ciliogenesis.

Long-Pentraxin 3 Affects Primary Cilium in Zebrafish Embryo and Cancer Cells via the FGF System

Primary cilium drives the left-right asymmetry process during embryonic development. Moreover, its dysregulation contributes to cancer progression by affecting various signaling pathways. The fibroblast growth factor (FGF)/FGF receptor (FGFR) system modulates primary cilium length and plays a pivotal role in embryogenesis and tumor growth. Here, we investigated the impact of the natural FGF trap long-pentraxin 3 (PTX3) on the determination of primary cilium extension in zebrafish embryo and cancer cells. The results demonstrate that down modulation of the PTX3 orthologue ptx3b causes the shortening of primary cilium in zebrafish embryo in a FGF-dependent manner, leading to defects in the left-right asymmetry determination. Conversely, PTX3 upregulation causes the elongation of primary cilium in FGF-dependent cancer cells. Previous observations have identified the PTX3-derived small molecule NSC12 as an orally available FGF trap with anticancer effects on FGF-dependent tumors. In keeping with the non-redundant role of the FGF/FGR system in primary cilium length determination, NSC12 induces the elongation of primary cilium in FGF-dependent tumor cells, thus acting as a ciliogenic anticancer molecule in vitro and in vivo. Together, these findings demonstrate the ability of the natural FGF trap PTX3 to exert a modulatory effect on primary cilium in embryonic development and cancer. Moreover, they set the basis for the design of novel ciliogenic drugs with potential implications for the therapy of FGF-dependent tumors.

Ca2+-Dependent Regulation by the Cyclic AMP Pathway of Primary Cilium Length in LLC-PK1 Renal Epithelial Cells

The primary cilium is a sensory organelle projecting from the apical surface of renal epithelial cells. Dysfunctional cilia have been linked to a number of genetic diseases known as ciliopathies, which include autosomal dominant polycystic kidney disease (ADPKD). Previous studies have determined that renal epithelial primary cilia express both the polycystin-2 (PC2, TRPP2) channel and the type-2 vasopressin receptor (V2R), coupled to local cAMP production. However, little is known as to how Ca2+ and cAMP signals lead to changes in the length of the primary cilium. Here, we explored how cAMP signals regulate the length of the primary cilium in wild type LLC-PK1 renal epithelial cells. Primary cilia length was determined by immunocytochemical labeling of the ciliary axoneme. Treatment of cells with the cAMP analog 8-Br-cAMP (1 mM) in normal external Ca2+ (1.2 mM) produced a 25.3% increase (p < 0.0001) in the length of the primary cilium, a phenomenon also observed in cells exposed to high external Ca2+ (6.2 mM). However, exposure of cells to vasopressin (AVP, 10 μM), which also increases cAMP in primary cilia of LLC-PK1 cells, mimicked the effect of 8-Br-cAMP in normal, but not in high Ca2+. Further, specific gene silencing of PC2 expression further increased primary cilium length after 8-Br-cAMP treatment, in normal, but not high Ca2+. The encompassed data indicate a crosstalk between the cAMP and Ca2+ signals to modulate the length of the primary cilium, in a phenomenon that implicates the expression of PC2.Significance StatementMorphological changes in primary cilia have been linked to genetic disorders, including autosomal dominant polycystic kidney disease (ADPKD), a major cause of kidney disease. Both cAMP and Ca2+ are universal second messengers that regulate polycystin-2 (PC2, TRPP2), a Ca2+ permeable non-selective cation channel implicated in ADPKD, and expressed in the primary cilium of renal epithelial cells. Despite current interest, little is known as to how second messenger systems and how aberrant regulation of PC2 may link primary cilium structure with cyst formation in ADPKD. Here we determined that both the cAMP analog 8-Br-cAMP and vasopressin increase the length of the primary cilium in renal epithelial cells. However, this phenomenon depends of external Ca2+ and PKD2 gene silencing. Proper cAMP signaling may be essential in the control of the primary cilium of renal epithelial cells, and the onset of cyst formation in ADPKD.