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 M), 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.