Cloning and Targeted Deletion of the Mouse Fetuin Gene

Abstract

We proposed that the ␣ 2 -Heremans Schmid glycoprotein/fetuin family of serum proteins inhibits unwanted mineralization. To test this hypothesis in animals, we cloned the mouse fetuin gene and generated mice lacking fetuin. The gene consists of seven exons and six introns. The cystatin-like domains D1 and D2 of mouse fetuin are encoded by three exons each, whereas a single terminal exon encodes the carboxyl-terminal domain D3. The promoter structure is well conserved between rat and mouse fetuin genes within the regions shown to bind transcription factors in the rat system. Expression studies demonstrated that mice homozygous for the gene deletion lacked fetuin protein and that mice heterozygous for the null mutation produced roughly half the amount of fetuin protein produced by wild-type mice. Fetuin-deficient mice were fertile and showed no gross anatomical abnormalities. However, the serum inhibition of apatite formation was compromised in these mice as well as in heterozygotes. In addition, some homozygous fetuin-deficient female ex-breeders developed ectopic microcalcifications in soft tissues. These results corroborate a role for fetuin in serum calcium homeostasis. The fact that generalized ectopic calcification did not occur in fetuin-deficient mice proves that additional inhibitors of phase separation exist in serum.

Fetuin-B, a second member of the fetuin family in mammals

“…On a structural basis, fetuin-B is clearly homologous to fetuin-A, as shown by a shared arrangement in three domains, including two cystatin-like domains D1 and D2 and a domain D3 that is itself divided into the proline-rich D3a and C-terminal D3b subdomains. All these features suggest that the exon\intron arrangement described previously for the human, rat and mouse FETUA genes, namely three exons each for D1 and D2 and a single large exon for D3 [13,39,40], is likely to be conserved in the FETUB gene. The FETUB gene assignments also argue for this view.…”

“…Consistent with the importance of fetuins in development, AHSG is involved in osteogenesis and bone resorption [11,12]. These functions have been ascribed to the capacities of AHSG to control calcium-salt precipitation in blood [13] and to accumulate in bone matrix [14] as well as to counteract a transforming growth factor-β activity required for bone mineralization [12,15]. AHSG modulates some insulin-driven and kinase-mediated signal-transduction pathways, possibly in a tissue-specific fashion [16].…”

Vascular Calcification

“…In light of this in vitro evidence, early control or prevention of hyperphosphatemia may be key in reducing coronary calcification and the resulting morbidity and mortality as a result of cardiovascular disease for patients on dialysis. Matrix gla-protein (31) Arterial, valve, and cartilage calcification ␤-glucosidase (klotho) (32) Vascular calcification, rapid aging Desmin (33) Neonatal cardiomyopathy with calcification Carbonic anhydrase II (34) Vascular calcification of small arteries Fetuin (35) Decreased serum hyaluronic acid inhibitory activity Osteoprotegrin (36) Osteoporosis, vascular calcification Osteopontin (37) Increased calcification of subcutaneously implanted bioprosthetic valve tissue…”

“…It should be added that definitive data on the presence of local and systemic inhibitors of calcification have been accumulating over the past several years and come largely from studies involving gene knockout mice. These data implicate the involvement of several gene products in ectopic calcification (Table 1) (31)(32)(33)(34)(35)(36)(37) and suggest that the matrix gla-protein gene, osteoprotegrin, and osteopontin may serve as natural inhibitors of cardiovascular calcification that may be either constitutively expressed or induced to prevent ectopic calcification. Indeed, vascular calcification may involve an active and dynamic balance of procalcifying and anticalcifying mechanisms.…”

Low serum vitamin K in PXE results in defective carboxylation of mineralization inhibitors similar to the GGCX mutations in the PXE-like syndrome

“…This serum protein carries calcium phosphate and has been shown to have a major function in preventing pathological calcification. [29][30][31] The extent of the protective mechanism by fetuin-A, however, seems to be less than that exerted by MGP, as fetuin-A-deficient mice only develop minor calcified lesions compared with the extensive calcifications in MgpÀ/À mice. 30 However, fetuin-A is closely related to the extracellular transport of MGP.…”