Gremlin1 is required for skeletal development and postnatal skeletal homeostasis

Abstract

Gremlin is an antagonist of bone morphogenetic proteins, and its overexpression causes suppressed osteoblastogenesis and osteopenia. Inactivation of Grem1 results in severe developmental defects, but the consequences of the global inactivation of Grem1 on the postnatal skeleton are not known. To study the function of gremlin, Grem1 was inactivated by homologous recombination, and mice were maintained in a C57BL/6/FVB mixed genetic background due to embryonic and neonatal lethality in the uniform C57BL/6 background. Grem1 null mice exhibited developmental skeletal abnormalities, leading to incomplete formation of metatarsal bones and of fore limbs and hind limbs. Grem1 null mice exhibited decreased weight and body fat and shortened femoral length. Bone histomorphometric and microarchitectural analyses of distal femurs revealed decreased bone volume and increased bone formation in 1-month-old Grem1 null mice. Trabecular femoral bone volume was restored in older Grem1 null female mice, and to a lesser extent in male mice. Vertebral microarchitecture confirmed the osteopenia observed in 1-month-old Grem1 null mice and demonstrated recovery of trabecular bone in older female, but not in older male Grem1 null mice, which exhibited persistent vertebral osteopenia. In conclusion, Grem1 is not only necessary for skeletal development, but also for postnatal skeletal homeostasis; its inactivation causes osteopenia, which is partially reversed in a spatial, temporal, and sex-dependent manner due to an increase in bone formation.

Figure 1

Weight, body composition (% fat), femoral length and real time RT-PCR of calvarial RNA extracts from 1, 3 and 6 month old male (upper panels) and female (lower panels) Grem1 null mice (filled circles and black bars) and wild type littermate controls (open circles and white bars). The weight in g, also obtained in 10 day old mice, (A); % body fat (B); femoral length in mm (C); and Grem1 copy number corrected for Rpl38 (D) are shown. Please note Grem1 was undetectable in Grem1 null mice so that bars are not depicted. Values are means ± SEM, n = 4 to 14. *Significantly different from control mice, p < 0.05.

Figure 2

Representative photographs and skeletal X-rays of 3 month old Grem1 null mice and wild type (WT) littermate controls. Arrows point to absent fibula and metatarsal bones.

Figure 3

Representative histological sections and calcein/demeclocycline labeling of femoral sections from 10 day and 1, 3 and 6 month old male and female Grem1 null mice and wild type littermate controls. Sections were stained with von Kossa without counter stain (final magnification 40×) at all ages or unstained and examined under fluorescence microscopy at 1, 3 and 6 months of age (final magnification 100×).

Figure 4

Representative vertebral microarchitecture assessed by μCT scanning of 1, 3 and 6 month old male and female Grem1 null mice and wild type littermate controls.