Research ArticleMicrocytosis in ank/ank mice and the role of ANKH in promoting erythroid differentiation
Introduction
The mutated gene, ank, of the spontaneous mutant mice progressive ankylosis, was first identified in 2000, and is a nonsense mutation in the exon 11 of the Ank gene [1]. Although the gene product is quite ubiquitously expressed, the only significant phenotype found in the homozygous mutant mice is characterized by pathologic calcium apatite crystal deposition in the synovial and subsynovial spaces followed by chondro-osteophyte formation and eventual bony ankylosis of the affected joint. There was also a report on defective splenic T cell mitogenic response to PHA and ConA in these mutant mice [2]. Heterozygous ank mice have a normal phenotype, but loss of the Ank function in homozygous ank mice causes increased intracellular [PPi] (i[PPi]), and decreased extracellular [PPi] (e[PPi]) levels [1]. Recently, an Ank null mouse was generated and the phenotype of the homozygous Ank null mice was indistinguishable from that of ank/ank mice [3]. Furthermore, mice with joint-specific deletion of Ank alleles also showed joint mineralization and ankylosis, indicating that Ank function is required locally in joints to prevent mineral formation and ensure joint mobility and function [3].
The Ank protein is a multipass transmembrane protein which regulates PPi export from the intracellular to the extracellular compartments. The human ANKH protein is very similar to the mouse Ank protein, differing by nine of the entire 492 amino acids. Both Ank and NPP-1 were required to elevate e[PPi] constitutively and following TGFβ induction [4], suggesting that Ank may facilitate export of NTP, a substrate for NPP-1 on the outer cell membrane. A decrease in intracellular NTP would reduce the available intracellular NTP for intracellular hydrolysis to [PPi], lowering i[PPi] and e[PPi] levels. In humans, autosomal dominant craniometaphyseal dysplasia (CMD) is caused by ANKH mutations [5], [6]. Heterozygous ANKH mutations were detected in at least five multiplex families with chondrocalcinosis (calcium pyrophosphate dihydrate deposition disease) [7], [8], [9], [10], [11]. It remains unclear how these mutations lead to pathogenesis of the disease, though they appear to be dominant negative mutations in CMD and gain-of-function mutations in chondrocalcinosis [12].
The observation that Ank is a polypeptide growth factor-, serum- and tumor promoter-inducible gene in fibroblasts led to the proposal that in cells with high metabolic activity, Ank is required for efficient removal of excess i[PPi] generated by consumption of ATP [13]. The notion that Ank/ANKH might have function(s) additional to that of exporting PPi is not new. Upregulation of MMP13 expression was induced by over-expression of Ank in primary bovine chondrocytes [4]. In the course of studying the ank/ank mice, we found that they have microcytosis. In this study, we specifically asked whether Ank/ANKH play a role in erythropoiesis. We examined Ank expression in various differentiation stages of erythropoiesis. As redundancy of proteins with similar functions might complicate the identification of some tissue-specific function(s) of Ank in the ank/ank mutant mice, instead of performing in vitro knock down of Ank/ANKH in culture cells, we used the approach of over-expressing ANKH in specific cell types which might enable the detection of novel ANKH function(s). K562 cells were derived from a patient with chronic myeloid leukemia (CML) in terminal blast crisis [14], and were commonly used as a model for erythroid differentiation as these leukemic cells can be induced to undergo erythroid differentiation by various agents such as hemin, butyrate, cisplatin and ara-C [15], [16]. Here we report the generation of stable ANKH transfectants in K562 cells, and show that ANKH promotes early erythroid differentiation.
Section snippets
Breeding and maintenance of ank/ank mice
A breeding pair of ank/+ mice (C3FeB6-A/Aw-j –ank+/−) was obtained from the Jackson Laboratory (Bar Harbor, ME) and a breeding colony was maintained in the animal facility, Toronto Western Research Institute. ank/ank mice were initially generated by crossbreeding heterozygous offspring of the breeding pairs. Later, heterozyotes from larger litters were used as breeders, as in general, they give more mutant mice. Mice were genotyped using tail DNA as described by Ho et al. [1]. We arbitrarily
ank/ank mice have microcytosis
Blood was obtained from ank/ank and wild-type littermates (12 for each group) and erythrocyte indices are summarized in Table 2. There were no age and gender differences in these parameters with each group (mutant vs. wild-type mice). Compared to normal littermates, the mutant mice had significantly lower mean corpuscular volume (MCV; 50.6 ± 0.59 [normal] vs. 43.9 ± 0.39 [mutant]; p-value < 0.001) and mean corpuscular hemoglobin (MCH [pg/RBC]; 16.0 ± 0.64 [normal] vs. 13.6 ± 0.15 [mutant]; p-value = 0.002)
Discussion
In this study, we showed that ank/ank mice which have a loss of Ank function develop red cell microcytosis. Inflammation is a potential factor which might influence red cell size and number [31]. In the literature, both the presence of joint inflammation [32], [33] and the lack of evidence of inflammation in the joints of ank/ank mice [34] were reported. In our colony of ank/ank mice, we observed synovial proliferation, increase of calcified matrix, joint narrowing and small calcific deposits (
Acknowledgments
Supported by grants from the Canadian Institutes of Health Research, and the Arthritis Center of Excellence.
We wish to thank Dr. JA Winkles (American Red Cross) for his gift of anti-Ank antibodies, and Dr. R. Reithmeier (University of Toronto) for helpful discussions on the RBC proteome.
We also thank Gordana Kuruzar for the technical help in the collection of blood samples from the mice.
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