CYP27B1 null mice with LacZreporter gene display no 25-hydroxyvitamin D3-1alpha-hydroxylase promoter activity in the skin

Abstract

The hormonally active form of vitamin D(3),1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], is synthesized in the kidney through a tightly regulated reaction catalyzed by 25-hydroxyvitamin D(3)-1alpha-hydroxylase (1alpha-hydroxylase), the product of the CYP27B1 gene. Through gene targeting in embryonic stem cells, we engineered a mouse strain in which the coding region of the 1alpha-hydroxylase gene is replaced by the genes for beta-galactosidase (lacZ) and neomycin resistance. Null mice produced no detectable 1alpha-hydroxylase transcript. The mice grew normally when maintained on a balanced diet containing 1,25(OH)(2)D(3) but rapidly developed rickets when phosphorus and 1,25(OH)(2)D(3) were restricted. Rickets was curable through administration of 1,25(OH)(2)D(3) but not its biological precursor, 25-hydroxyvitamin D(3). Upon administration of a diet low in calcium and devoid of any form of vitamin D(3), beta-galactosidase activity was detected in the kidneys of the -/- and +/- mice and in placentas harvested from -/- females bred with -/- males. No beta-galactosidase activity was detected in skin sections or in primary keratinocyte cultures from -/- animals. Our results demonstrate we have generated 1alpha-hydroxylase null mice that display phenotypes characteristic of vitamin D-dependency rickets type I. From the histochemical analysis of reporter gene expression in these mice, we conclude that acute 1,25(OH)(2)D(3) deficiency in otherwise healthy animals does not stimulate local production of 1,25(OH)(2)D(3) in the skin. These findings stand in contrast to previously published reports of 1,25(OH)(2)D(3) production in keratinocytes.

Fig. 1.

Agarose electrophoresis of products generated from RT-PCR of total kidney RNA from +/+, +/–, and –/– mice by using 1α-hydroxylase- and β-actin-specific primers. pGEM DNA markers (Promega) are shown in the first lane. PCR product sizes are 345 bp for 1α-hydroxylase, and 350 bp for β-actin.

Fig. 2.

Silver nitrate staining of wrist long bones for analysis of rickets induction and rescue. (A) Wrists of +/+, +/–, and –/– animals fed a high-calcium phosphorus-free diet. E, EP, and D indicate the epiphysis, epiphyseal plate, and diaphysis of the growing bone, respectively. (B) Wrists of rachitic –/– mice after 6-day treatment with vehicle, 25-OH-D₃ (250 ng/day) or 1,25(OH)₂D₃ (25 ng/day). The zone of newly calcified cartilage in the 1,25(OH)₂D₃-treated mouse is indicated by the brackets.

Fig. 3.

Reporter gene activity in kidneys of 1α-hydroxylase mutant mice. (A–C) Gross stains of kidneys (longitudinal bisection) from –/–, +/–, and +/+ mice after 8-day administration of diet lacking calcium and any form of vitamin D₃. (D–F) Stains of thin kidney sections after 8 days of experimental diet (×40). (G) Stains of kidney from a –/– mouse after 1 day on the experimental diet. (H and I) Kidney stains of a –/– mouse that received the maintenance diet only.

Fig. 4.

Extrarenal 1α-hydroxylase promoter activity in placental tissue. X-Gal staining of fetuses and placentas harvested after mating of –/– (A) and +/+ (B) mice. Promoter activity was stimulated during midgestation by depriving dams of calcium and any form of vitamin D₃, as described in Materials and Methods.

Fig. 5.

X-Gal stains of skin sections and cultured keratinocytes. (A and B) X-Gal stains (×100) of frozen skin sections from –/– and +/+ mice after 8-day administration of diet lacking calcium and any form of vitamin D₃. The epidermis, dermis, and hypodermis are indicated as E, D, and HD, respectively. Hair follicles (HF) extend into the dermis, and the fat (F) and connective tissue (CT) layers of the hypodermis are clearly visible. (C and D) X-Gal stains of keratinocytes (×400) from –/– and +/+ newborns grown in nondifferentiating conditions.