Rapid degradation of progressive ankylosis protein (ANKH) in craniometaphyseal dysplasia

Abstract

Mutations in the progressive ankylosis protein (NP_473368, human ANKH) cause craniometaphyseal dysplasia (CMD), characterized by progressive thickening of craniofacial bones and widened metaphyses in long bones. The pathogenesis of CMD remains largely unknown, and treatment for CMD is limited to surgical intervention. We have reported that knock-in mice (AnkKI/KI) carrying a F377del mutation in ANK (NM_020332, mouse ANK) replicate many features of CMD. Interestingly, ablation of the Ank gene in AnkKO/KO mice also leads to several CMD-like phenotypes. Mutations causing CMD led to decreased steady-state levels of ANK/ANKH protein due to rapid degradation. While wild type (wt) ANK was mostly associated with plasma membranes, endoplasmic reticulum (ER), Golgi apparatus and lysosomes, CMD-linked mutant ANK was aberrantly localized in cytoplasm. Inhibitors of proteasomal degradation significantly restored levels of overexpressed mutant ANK, whereas endogenous CMD-mutant ANK/ANKH levels were more strongly increased by inhibitors of lysosomal degradation. However, these inhibitors do not correct the mislocalization of mutant ANK. Co-expressing wt and CMD-mutant ANK in cells showed that CMD-mutant ANK does not negatively affect wt ANK expression and localization, and vice versa. In conclusion, our finding that CMD mutant ANK/ANKH protein is short-lived and mislocalized in cells may be part of the CMD pathogenesis.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1

Reduced expression of CMD-mutant ANK/ANKH: Representative images of immunoblotting with FLAG or ANK antibodies of (A) ROS cells overexpressing 3xFLAG-tagged wt, -F377del and –S375del ANK; white asterisk indicates 3xFLAG-ANK band immunoblotted with FLAG antibody (B) mature osteoclasts from Ank+/+, Ank+/KI, AnkKI/KI and AnkKO/KO BMM cultures (C) stem cells from human exfoliated deciduous teeth (SHEDs) differentiated for 0, 7, 14, and 21 days in α-MEM medium supplemented with 50 μg/ml ascorbic acid and 8 mM β-glycerophosphate. Actin and GAPDH are loading controls. Experiments were repeated at least three times.

Figure 2

CMD-mutant ANK mislocalizes in multinucleated osteoclasts compared to wt ANK: Representative confocal images of mature osteoclasts with immunostaining showed (A) ANK_wt localized to plasma membrane, ER, Golgi and lysosomes in Ank+/+ OCs, shown by co-staining of ANK (red) with the ER, Golgi and lysosome markers Calnexin, TGN38 and LAMP1 (green), respectively. AnkKO/KO OCs served as a negative control; Scale bar = 50 μm (B) ANK immunostaining in BMM-derived mature OCs from Ank+/+ and AnkKI/KI mice. Intense expression of ANK_wt associated with membranous organelles (white arrows) was not observed for CMD mutant ANK in AnkKI/KI OCs. ANK (red), nuclei staining with Hoechst (blue). Scale bar = 50 μm. Experiments were repeated at least three times.

Figure 3

CMD mutant-ANK protein is rapidly degraded compared to wt ANK: Pulse-chase assays of MEFs overexpressing 3xFLAG-wt or -S375del mutant ANK. Cells were labeled with [35S]-methionine and chased for 0, 1, 2, 4 and 8 hours. Total cell lysates were subjected to immunoprecipitation with a FLAG antibody and further analyzed by SDS-PAGE and autoradiography. Data averaged from three independent experiments shown in the histogram. Data presented: mean ± S.D.

Figure 4

Exogenous CMD mutant-ANK protein is mainly degraded via the ubiquitin-proteasome system (UPS): Immunoblotting with FLAG antibody showed (A) inhibitors of proteasomal degradation MG132, bortezomib and epoxomicin rescue 3xFLAG-F377del ANK expression (blue arrows) in transfected ROS cells in a dose-dependent manner (B) exogenous CMD mutant ANK (3xFLAG-tagged F377del or -S375del) was restored more by MG132 (5 μM for 9 hours) than by BFA (100 ng/ml for 6 hours) in transfected MEFs. Actin and GAPDH are loading controls. (C) Immunoblotting with FLAG and ubiquitin antibodies showed increased steady-state levels of 3xFLAG-wt and mutant ANK in transfected MEFs in the presence of K0 or K48R ubiquitin constructs. GAPDH as loading control. Experiments were repeated at least three times. No: no ubiquitin transfected; Ratio between 3xFLAG-ANK and K0 or K48R ubiquitin was shown as 1:1, 1:2 and 1:3.

Figure 5

Levels of 3xFLAG-tagged wt, -S375del, and -F377del ANK proteins are increased in yeast strains lacking specific ER-localized E3 ligases. Immunoblot with FLAG antibody showed increased steady-state levels of 3xFLAG-ANK in yeast strains lacking the Hrd1 E3 ligase (hrd1Δ and doa10Δ hrd1Δ yeast deletion backgrounds), but not in a doa10Δ single deletion strain. Ponceau staining served as loading control.

Figure 6

Inhibitors of lysosomal degradation rescue the expression of endogenous CMD mutant ANK: Representative immunoblotting with ANK/ANKH antibody showed that BFA, an inhibitor of lysosomal degradation, rescues ANK_F377del expression better than inhibitors of proteasomal degradation (A) in mouse BMM-derived osteoclasts treated with proteasomal inhibitors: MG: 3 μM MG132, Kif: 1 μM (3rd lane) and 3 μM (4th lane) kifunensine, and lysosomal inhibitors: CQ: chloroquine 50 μM, BFA: bafilomycin a1 100 ng/ml (B) in osteoblast explant cultures (top panel) of one CMD patient and from SHEDs from one control and two CMD patients (bottom panel) differentiated in ascorbic acid and β-glycerophosphate for 14 days (MG: 5 μM MG132 for 9 hours, Epo: 1 μM epoxomicin for 9 hours, Kif: 1 μM kifunensine for 9 hours, BFA: 100 ng/ml bafilomycin a1 for 6 hours). GAPDH as loading control. Experiments were repeated at least three times, Untx: unreated.

Figure 7

Localization of wt and mutant ANK in the presence of proteasomal and lysosomal inhibitors. (A) Immunocytochemistry of ROS cells transfected with 3xFLAG-wt or –F377del ANK in the presence or absence of MG132 using FLAG antibody (green) and Hoechst 33342 stain for nuclei (blue). Scale bar = 50 μm. (B) ANK immunocytochemistry with ANK antibodies of BMM-derived osteoclasts untreated or treated with 0.001% DMSO (control), 10 μM MG132 for 9 hrs or 100 ng/ml BFA for 6 hrs. ANK (red), nuclei (blue). Identical exposure time was used for each image. Scale bar = 50 μm.

Figure 8

Expression and localization of overexpressed wt ANK in the presence of mutant ANK. (A) FLAG immunoblots of MEF cells transfected with SNAP 3xFLAG-tagged wt or/and 3xFLAG–tagged F377del ANK in with various doses. (B) Representative confocal images of MEF cells untransfected, transfected with SNAP 3xFLAG-wt alone, CLIP 3xFLAG-F377del ANK alone, SNAP 3xFLAG-wt plus CLIP 3xFLAG-F377del ANK (1:1 ratio) or SNAP 3xFLAG-wt plus CLIP 3xFLAG-F377del ANK (2:1 ratio). Scale bar = 50 μm. All experiments were repeated at least three times. Enlargement of boxed areas from merged images are shown in the right column.