Mineral formation in joints caused by complete or joint-specific loss of ANK function

To reveal the ANK complete loss of function phenotype in mice, we generated conditional and null alleles. Mice homozygous for the null allele exhibited widespread joint mineralization, similar in severity to animals harboring the original ank allele. A delayed yet similar phenotype was observed in mice with joint-specific loss of ANK function.

The ANK pyrophosphate regulator was originally identified and proposed to play a key role in articular cartilage maintenance based on a single spontaneous mouse mutation (ank) that causes severe generalized arthritis. A number of human mutations have subsequently been reported in the human ortholog (ANKH), some of which produce skull and long bone defects with no apparent defects in joints or articular cartilage. None of the currently known mouse or human mutations clearly eliminate the function of the endogenous gene.

Two new Ank alleles were generated using homologous recombination in mouse embryonic stem (ES) cells. Joint range of motion assays and muCT studies were used to quantitatively assess phenotypic severity in wildtype, heterozygous, and homozygous mice carrying either the null (Anknull) or original (Ankank) allele. A Gdf5-Cre expressing line was crossed to mice harboring the conditional (Ankfloxp) allele to eliminate ANK function specifically in the joints. Histological stains and beta-galactosidase (LACZ) activity were used to determine the correlation between local loss of ANK function and defective joint phenotypes.

Anknull/Anknull mice develop severe ectopic postnatal crystal deposition in almost every joint of the body, leading to eventual joint fusion and loss of mobility. The severity of phenotype in these mice is indistinguishable from that of Ankank/Ankank mice. In addition, despite the widespread expression of Ank in many tissues, the specific deletion of Ank in joints also produces joint mineralization and ankylosis.

These studies show that ANK function is required locally in joints to inhibit mineral formation and that the Ank gene plays a key role in postnatal maintenance of joint mobility and function.