The Pyrophosphate Transporter ANKH is Expressed in Kidney and Bone Cells and Colocalises to the Primary Cilium/Basal Body Complex

Abstract

Background/Aims: ANKH encodes a putative pyrophosphate transporter named ANKH, which regulates tissue calcification. ANKH is a transmembrane protein with at least 8 predicted transmembrane domains. Sequence analysis reveals a possible cilial localisation motif immediately after the last transmembrane segment. Here we aim to determine the subcellular localisation of ANKH in ciliated epithelial cells and murine tissue and identify colocalisation using ciliary/basal body markers. Methods: Using murine kidney, renal epithelial cells and osteoblast cells we investigated the expression and localisation of ANKH using RT-PCR, Western blotting and immunocytochemistry. Results: Here we confirm endogenous expression of ANKH mRNA and protein in whole mouse kidney as well as mouse renal epithelial cell lines M1 and mpkCCDcl4 and the osteoblast cell line MC3T3-E1. Using antibodies directed towards ANKH, we confirm cilial and basal body localisation in renal tissues and renal epithelial cells, in addition to a centrosomal localisation in dividing mpkCCDcl4 cells. We also establish that the osteoblast cell line MC3T3-E1 forms an epithelioid cell layer, with junctional complex formation and primary cilia expression. ANKH is also seen within cilial and basal body structures of MC3T3-E1 cells. An ANKH-3XFLAG construct expressed in mpkCCDcl4 cells also localises to the primary cilium/basal body complex confirming this localisation. Conclusion: We conclude that the transmembrane protein ANKH is expressed in cilia and basal body structures, and postulate a sensory role at this location.

Pulsed electromagnetic fields stimulate osteogenic differentiation and maturation of osteoblasts by upregulating the expression of BMPRII localized at the base of primary cilium

“…Kwon et al found that primary cilia mediate the osteogenic and anti-resorptive responses of dynamic fluid flow depending on the decreases of intracellular cAMP levels and adenylyl cyclase 6 located at primary cilium [48]. Carr et al reported the human homolog of the mutated gene in the ank/ank mouse (ANKH), a widely expressed protein limiting and regulating calcification within bones, is expressed in basal body of primary cilium in the osteoblast cell line MC3T3-E1 [49]. Xiao et al shown that polycystin-1 (PC1) plays an important role in skeletogenesis through regulation of the bone-specific transcription factor Runx2-II, which co-localizes with the calcium channel polycystin-2 (PC2) in primary cilia of MC3T3-E1 osteoblasts [50].…”

Extracellular Pyrophosphate in the Kidney: How Does It Get There and What Does It Do

“…Localisation studies may suggest new ways in which to investigate the functions of ANKH. We have shown that ANKH localises to the primary cilium and basal body, as well as the apical membrane, of renal epithelial cell lines and murine renal tissue [14]. This places ANKH in a very suitable position from which to sense luminal PPi in the urine (fig.…”

iTRAQ-Based Proteomic Analysis of Neonatal Kidney from Offspring of Protein Restricted Rats Reveals Abnormalities in Intraflagellar Transport Proteins

“…Experimental models also provided data supporting a link between adverse intrauterine environment and adult disease. Both uteroplacental insufficiency and calorie restriction in pregnant rats led to high blood pressure in their adult offspring [33]. We have previously shown that a low maternal protein diet during pregnancy results in lower kidney index, glomeruli number and decreased glomerular filtration rate in IUGR offspring, with an increased incidence of hypertension [8].…”

“…These proteins, including IFT80, IFT88and IFT144, belong to the primary cilium complex A (144) and B(80,88) module [34]. The primary cilium, a slim microtubule-based organelle that projects from the surface of vertebrate cells, has been located on almost every mammalian cell type [33]. Cilia are essential for certain developmental signaling pathways and mechanosensory functions, like detecting flow or mechanical movement and inducing an influx of Ca 2+ [24].…”