Differences in intracellular localisation of ANKH mutants that relate to mechanisms of calcium pyrophosphate deposition disease and craniometaphyseal dysplasia

Abstract

ANKH mutations are associated with calcium pyrophosphate deposition disease and craniometaphyseal dysplasia. This study investigated the effects of these ANKH mutants on cellular localisation and associated biochemistry. We generated four ANKH overexpression-plasmids containing either calcium pyrophosphate deposition disease or craniometaphyseal dysplasia linked mutations: P5L, E490del and S375del, G389R. They were transfected into CH-8 articular chondrocytes and HEK293 cells. The ANKH mutants dynamic differential localisations were imaged and we investigated the interactions with the autophagy marker LC3. Extracellular inorganic pyrophosphate, mineralization, ENPP1 activity expression of ENPP1, TNAP and PIT-1 were measured. P5L delayed cell membrane localisation but once recruited into the membrane it increased extracellular inorganic pyrophosphate, mineralization, and ENPP1 activity. E490del remained mostly cytoplasmic, forming punctate co-localisations with LC3, increased mineralization, ENPP1 and ENPP1 activity with an initial but unsustained increase in TNAP and PIT-1. S375del trended to decrease extracellular inorganic pyrophosphate, increase mineralization. G389R delayed cell membrane localisation, trended to decrease extracellular inorganic pyrophosphate, increased mineralization and co-localised with LC3. Our results demonstrate a link between pathological localisation of ANKH mutants with different degrees in mineralization. Furthermore, mutant ANKH functions are related to synthesis of defective proteins, inorganic pyrophosphate transport, ENPP1 activity and expression of ENPP1, TNAP and PIT-1.

Figure 1

Wt.ANKH overexpression intracellular localisation and biochemical effects of overexpression compared to null vector control in CH-8 and HEK293 cells. Wt.ANKH-GFP (green) in HEK293 cell membrane (A); increased ePPi in HEK293 culture media (B); Alizarin red S staining of CH-8 cells (left) and concentration of ARS/µg protein (right) normalized to ActB confirming reduction in mineralization (C); qPCR in transfected HEK293 cells trended to decrease ENPP1 at 24 H and increase at 48 and 72 H (D), did not significantly change TNAP (E), increased PIT-1 72 H (F); ENPP1 activity in CH-8 cells decreased at 24 H and increased at 48 H (G). H = hours, scale bars = 20 μm; *** indicates p value < 0.01; * indicates p value < 0.05. # indicates p value < 0.1; neg. = Negligible/Raw value below threshold.

Figure 2

P5L overexpression intracellular localisation and biochemical effects compared to wt.ANKH control in CH-8 and HEK293 cells. P5L-GFP (green) mainly localised to HEK293 cytoplasm at 24 H and cell membrane at 72 H (A); increased ePPi in HEK293 culture media (B); ARS staining of CH-8 cells (left) and concentration of ARS/µg protein (right) normalized to ActB confirming increased mineralization (C); no significant change in ENPP1 (D), significant increased TNAP at 24 H and trended increase at 72 H (E), no significant change in PIT-1 (F); ENPP1 activity in CH-8 cells increased at 24, 48 and 72 H (G). H = hours, scale bars = 20 μm; *** indicates p value < 0.01; * indicates p value < 0.05. # indicates p value < 0.1; neg. = Negligible/Raw value below threshold.

Figure 3

S375del overexpression intracellular localisation and biochemical effects compared to wt.ANKH control in CH-8 and HEK293 cells. S375del-GFP (green) localised to HEK293 cytoplasm at all time points (A); little change in ePPi concentration in culture media of HEK293 (B); ARS staining of CH-8 cells (left) and concentration of ARS/µg protein (right) normalized to ActB confirming increased mineralization (C); S375del in HEK293 cells trended to increase ENPP1 and TNAP at 24 H (D,E), significantly increased PIT-1 at 48 H (F); ENPP1 activity in CH-8 cells showed slightly increased at 24 H and decreased at 48 H (G). H = hours, scale bars = 20 μm; *** indicates p value < 0.01; * indicates p value < 0.05. # indicates p value < 0.1; neg. = Negligible/Raw value below threshold.

Figure 4

G389R overexpression intracellular localisation and biochemical effects compared to wt.ANKH control in CH-8 and HEK293 cells with no direct LC3 interaction. G389R-GFP (green) mainly localised to HEK293 cell membrane with many puncta (A); increased ePPi in HEK293 culture media (B); ARS staining of CH-8 cells (left) and concentration of ARS/µg protein (right) normalized to ActB confirming increased mineralization (C); G389R in HEK293 cells increased ENPP1 at 24 H (D), did not significantly change of TNAP and PIT-1 at any time (E,F), ENPP1 activity in CH-8 cells increased at 24 H, then decreased at 48–72 H (G); Live confocal G389R ANKH-GFP (green) and RFP-LC3 (red) showing co-localisation during autophagy induction and rescue (circled) confirmed with intensity over distance (dotted line) (H); HEK293 Lysates of G389R ANKH-GFP and RFP-LC3 (rep of three) at 24 H, with autophagic media for 3 h (A), with normal growth media for 24 H (R); top: anti-GFP Western blot of IP GFP pull down, shows G389R not physically interact with LC3 (I). H = hours, scale bars = 20 μm; *** indicates p value < 0.01; * indicates p value < 0.05. # indicates p value < 0.1; neg. = Negligible/Raw value below threshold.

Figure 5

E490del overexpression intracellular localisation and biochemical effects of compared to wt.ANKH control in CH-8 and HEK293 cells with no direct LC3 interaction. E490del ANKH-GFP (green) localised mainly to cytoplasm and formed puncta at 24, at 72 H majority in membrane (A); increased ePPi in HEK293 culture media (B); ARS staining of CH-8 cells (left) and concentration of ARS/µg protein (right) normalized to ActB confirming increased mineralization (C); E490del in HEK293 cells increased ENPP1 and TNAP at 24 H (D,E); increased PIT-1 at 24–48 H (F); increased ENPP1 activity in CH-8 cells at 24 h (G); Live confocal E490del ANKH-GFP (green) and RFP-LC3 (red) showing co-localisation during autophagy induction and rescue (circled) confirmed with intensity over distance (dotted line) (H); HEK293 Lysates of E490del ANKH-GFP and RFP-LC3 (rep of three) at 24 H, with autophagic media for 3 h (A), with normal growth media for 24 H (R); top: anti-GFP Western blot of IP GFP pull down, shows E490del not physically interacting with LC3 (I). H = hours, scale bars = 20 μm; *** indicates p value < 0.01; * indicates p value < 0.05. # indicates p value < 0.1; neg. = Negligible/Raw value below threshold.