A Phe377del mutation in ANK leads to impaired osteoblastogenesis and osteoclastogenesis in a mouse model for craniometaphyseal dysplasia (CMD)

Abstract

Craniometaphyseal dysplasia (CMD) is a rare genetic disorder with hyperostosis of craniofacial bones and widened metaphyses in long bones. Patients often suffer from neurological symptoms due to obstruction of cranial foramina. No proven treatment is available and the pathophysiology is largely unknown. A Phe377 (TTC(1130-1132)) deletion in exon 9 of the pyrophosphate (PPi) transporter ANK leads to CMD-like features in an Ank(KI/KI) mouse model. Here, we investigated the effects of CMD-mutant ANK on mineralization and bone mass at a cellular level. Ank(KI/KI) osteoblast cultures showed decreased mineral deposition. Expression of bone mineralization regulating genes Mmp13, Ocn, Osx and Phex was reduced in Ank(KI/KI) osteoblasts, while the Fgf23 mRNA level was highly elevated in Ank(KI/KI) calvarial and femoral bones. Since ANK is a known PPi transporter, we examined other regulators of Pi/PPi homeostasis Enpp1 and Tnap. Significantly increased ENPP1 activity may compensate for dysfunctional mutant ANK leading to comparable extracellular PPi levels in Ank(+/+) osteoblasts. Similar to Ank(KI/KI) bone marrow-derived macrophage cultures, peripheral blood cultures from CMD patients exhibited reduced osteoclastogenesis. Cell-autonomous effects in Ank(KI/KI) osteoclasts resulted in disrupted actin ring formation and cell fusion. In addition, Ank(KI/KI) osteoblasts failed to adequately support osteoclastogenesis. Increased bone mass could partially be rescued by bone marrow transplants supporting our hypothesis that reduced osteoclastogenesis contributes at least in part to hyperostosis. We conclude that the Phe377del mutation in ANK causes impaired osteoblastogenesis and osteoclastogenesis resulting in hypomineralization and a high bone mass phenotype.

Figures

Figure 1.

Ank expression, matrix and mineral formation in osteoblast cultures. qPCR of Ank expression (A) in calvarial and femoral bone and (B) during in vitro calvarial osteoblast differentiation. Data were normalized to 18S RNA. Data presented are average with SD from three independent experiments. bP < 0.01 indicates significant difference in fold-change compared with Ank+/+ calvarial osteoblasts at day 0. Expression in Ank+/+ calvaria and Ank+/+ calvarial osteoblasts at day 0 were used as calibrators. No difference between Ank+/+ and AnkKI/KI groups at any time point. (C) Ank+/+ and AnkKI/KI mCOBs stained for ALP and von Kossa at culture days 7, 14 and 21. AnkKI/KI cultures show reduced mineral nodule formation. (D) Ank+/+ and AnkKI/KI BMSCs stained for ALP at culture days 7, 14 and 21. (E) Ank+/+ and AnkKI/KI BMSCs stained for ALP followed by von Kossa staining (left panel) and crystal violet (right panel) at day 14. (F) Ank+/+ and AnkKI/KI BMSCs stained for alizarin red S at culture days 7, 14 and 21. Histogram represents corresponding calcium-binding levels. aP < 0.05 indicates significant difference.

Figure 2.

Effects of Phe377del Ank on ePPi and on regulators for ePPi, Tnap and Enpp1 in mCOBs. (A) ePPi assays in Ank+/+ and AnkKI/KI mCOBs (left panel) as well as in wild-type and Anknull/null mCOBs (right panel). Statistical analysis was performed by Student's t-test. bP < 0.01. (B) Gene expression analysis by qPCR of Tnap and Enpp1 in Ank+/+ and AnkKI/KI mCOBs in differentiation medium for 0, 7, 14 and 21 days. No significant difference was found between Ank+/+ and AnkKI/KI cultures during osteoblast differentiation. (C) Enzymatic activities of TNAP and PC-1 in cultured Ank+/+ and AnkKI/KI mCOBs at days 0, 7, 14 and 21. bP < 0.01 indicates statistical significance by one-way ANOVA.

Figure 3.

Gene expression in mCOBs and bone tissues from Ank+/+ and AnkKI/KI mice. (A) qPCR of decreased Mmp13, Ocn, Runx2, Sp7 (Osx) and Phex in day 21 AnkKI/KI calvarial osteoblast cultures normalized to expression levels of Ank+/+ cells. Data from three independent experiments were pooled. aP < 0.05, bP < 0.01. (B) qPCR analysis of Fgf23, DMP1, MEPE and Phex in calvariae and (C) in femurs. RNA samples isolated from 8- to10-week-old Ank+/+ and AnkKI/KI mice.

Figure 4.

Bone explantation in Ank+/+ (+/+) and AnkKI/KI (KI/KI) mice. (A) X-ray and μCT images of femurs prior to and 3 weeks after intramuscular explantation. Ank+/+ (+) and AnkKI/KI (KI) femurs explanted into (→) Ank+/+ and AnkKI/KI mice. The Table shows trabecular bone volume in femoral diaphyses (n = 6 per group). Bone volume of KI/KI bones transplanted into KI/KI recipient mice is significantly larger than bone volume of +/+ bones transplanted into +/+ mice, aP < 0.05, and +/+ bones transplanted into KI/KI recipient mice, bP < 0.05. Statistics performed by ANOVA.

Figure 5.

In vitro osteoclast assays in human peripheral blood cultures derived from CMD patients and sex- and age-matched healthy controls. (A) Representative images of TRAP staining (at day 15) of cultures from CMD patients and controls (top). Resorption assays on calcium-phosphate-coated slides after fixation and von Kossa staining (below). White areas represent the resorbed area, while black areas show the remaining mineralized surface. (B) Histograms show number of mature osteoclasts per well and percentile of the resorbed area.

Figure 6.

In vitro murine osteoclast assays. (A) Increased Ank mRNA in mature Ank+/+ and AnkKI/KI BMM cultures by qPCR. (B) Rhodamine-phalloidin staining of actin and DAPI nuclear staining of mature osteoclasts. Note the disrupted actin belt (*) in AnkKI/KI BMMs, which are generally smaller in area. (C) Decreased fusion efficiency (percentage of nuclei number in mature osteoclasts to total number of nuclei in culture) of AnkKI/KI BMMs. bP < 0.01. (D) qPCR of DC-Stamp expression in BMMs during osteoclastogenesis. Decreased DC-Stamp mRNA level in day 2 AnkKI/KI BMM cultures containing pre-fusion osteoclasts. bP < 0.01. (E) Cocultures of primary mCOBs and BMMs. Resorption assay evaluated after 12 days of plating on calcium-phosphate coated slides. Replacement with Ank+/+ mCOBs or Ank+/+ BMMs partially rescues resorption activity of AnkKI/KI mCOB or AnkKI/KI BMM cultures, respectively (one-way ANOVA with Tukey's multiple-comparison to the Ank+/+ mCOB-Ank+/+ BMM group, aP < 0.05; bP < 0.01). There is no significant difference between replacement with AnkKI/KI mCOB or AnkKI/KI BMM cultures.

Figure 7.

μCT analysis of femurs and mandibles after BMT. Images show longitudinal sections of femurs highlighting the bone shape, trabeculation in diaphyseal region and cross-section through mandibles. Ank+/+ (+/+) and AnkKI/KI (KI/KI) bone marrow transplanted (→) into Ank+/+ and AnkKI/KI recipient mice (Ank+/+ → Ank+/+ mice, n = 8; AnkKI/KI → AnkKI/KI mice, n = 6; Ank+/+ → AnkKI/KI mice, n = 8; AnkKI/KI → Ank+/+ mice, n = 6). Ank+/+ BMT partially rescued the high mandibular bone mass of AnkKI/KI mandibles. The trabeculation in diaphyses of AnkKI/KI femurs was reduced in the group of AnkKI/KI mice receiving Ank+/+ bone marrow and slightly increased in Ank+/+ mice that received AnkKI/KI bone marrow.