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. 2009 S. Karger AG, Basel.
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. 2009 S. Karger AG, Basel.
Authors: Michael Pujari-Palmer; Shiuli Pujari-Palmer; Xi Lu; Thomas Lind; Håkan Melhus; Thomas Engstrand; Marjam Karlsson-Ott; Hakan Engqvist Journal: PLoS One Date: 2016-10-04 Impact factor: 3.240