Conditional deletion of insulin-like growth factor-I in collagen type 1alpha2-expressing cells results in postnatal lethality and a dramatic reduction in bone accretion

Abstract

IGF-I acts through endocrine and local, autocrine/paracrine routes. Disruption of both endocrine and local IGF-I action leads to neonatal lethality and impaired growth in various tissues including bone; however, the severity of growth and skeletal phenotype caused by disruption of endocrine IGF-I action is far less than with total IGF-I disruption. Based on these data and the fact that bone cells express IGF-I in high abundance, we and others predicted that locally produced IGF-I is also critical in regulating growth and bone accretion. To determine the role of local IGF-I, type 1alpha2 collagen-Cre mice were crossed with IGF-I loxP mice to generate Cre+ (conditional mutant) and Cre- (control) loxP homozygous mice. Surprisingly, approximately 40-50% of the conditional mutants died at birth, which is similar to total IGF-I disruption, but not observed in mice lacking circulating IGF-I. Expression of IGF-I in bone and muscle but not liver and brain was significantly decreased in the conditional mutant. Accordingly, circulating levels of serum IGF-I were also not affected. Disruption of local IGF-I dramatically reduced body weight 28-37%, femur areal bone mineral density 10-25%, and femur bone size 18-24% in growing mice. In addition, mineralization was reduced as early as during embryonic development. Consistently, histomorphometric analysis determined impaired osteoblast function as demonstrated by reduced mineral apposition rate (14-30%) and bone formation rate (35-57%). In conclusion, both local and endocrine IGF-I actions are involved in regulating growth of various tissues including bone, but they act via different mechanisms.

FIG. 1

Cell type-specific iCre expression in embryos and newborn mice. Immunostaining was performed for iCre in cross-sections from the neck region of embryos at d 12.5 (A and B) and tibia (C and D) and liver (E and F) of newborn mice. Control (A, C, and E) and conditional mutant (B, D, and F) mice are shown. Arrows indicate iCre-specific immunostaining in bone-forming regions. iCre was not detectable in the livers of conditional mutants.

FIG. 2

mRNA expression of IGF-I (A), iCre (B), and Col1 (C) in 16.5-d-old embryos. Data were analyzed for ΔCT values using ANOVA and are presented as mean ± sem relative to control gene expression [peptidylprolyl isomerase A (PPIA)]. Means with different letters (a, b, c, d) are significantly different at P ≤ 0.05. Leg, n = 5–7; liver, n = 4; lung, n = 4.

FIG. 3

Reduced local IGF-I expression. Data from 12-wk-old mice are expressed as fold change from controls and presented as mean ± sem, n = 6–17. *, Significant difference between conditional mutant and control mice (P < 0.05). Control values are set at 1-fold.

FIG. 4

Reduced mineralization in conditional mutant mice. Embryos at d 16.5 were stained for Alizarin Red and Alcian Blue. Control (B, D, F, and H) and conditional mutant (A, C, E, and G) mice.

FIG. 5

Reduced body weight (A) and length (B) in conditional mutants between 2 and 12 wk of age (n = 7–12 for 2 wk of age; n = 17–19 for 12 wk of age). All data are expressed as mean ± sem. At 2 wk of age, a significant effect of genotype was observed at P < 0.001. Solid line, Controls; dashed line, conditional mutants. *, Significant difference between conditional mutant and control mice (P < 0.0001).

FIG. 6

Reduced BMC (A) and BMD (B) in conditional mutant mice between 2 and 12 wk of age (n = 7–12 for 2 wk of age; n = 17–19 for 12 wk of age). All data are expressed as mean ± sem. Solid line with closed square, Control femur; dashed line with closed square, conditional mutant femur; solid line with closed circle, control vertebrae; dashed line with closed circle, conditional mutant vertebrae; solid line with open triangle, control total body; dashed line with open triangle, conditional mutant total body. *, Significant difference between conditional mutant and control mice (P < 0.05).

FIG. 7

Dynamic histomorphometry demonstrates reduced bone formation and bone size in conditional mutant mice at 2 wk of age. BFR, Bone formation rate; BS, bone surface; MAR, mineral apposition rate. Controls, n = 6–8; conditional mutants, n = 8–12. *, P < 0.05 vs. control; #, P ≤ 0.07 vs. control. Data are expressed as a percent of controls and presented as mean ± sem.

FIG. 8

Reduced bone and total volume in conditional mutants as determined by µCT analysis. A and C, Control. B and D, Conditional mutant. A and B, Cortical bone in cross-section of femur mid-shaft. C and D, Trabecular bone in cross-section of femur distal end. E, Controls (n = 5); conditional mutants (n = 5). *, Significant difference from control at P ≤ 0.01. Data are expressed as a percent of controls and presented as mean ± sem.

FIG. 9

Reduced rate of gain between 2 and 4 wk of age in conditional mutants. F, Femur; TB, total body; V, vertebrae. *, P < 0.05 vs. control; #, P ≤ 0.07 vs. control. All data are expressed as a percent of controls and presented as mean ± sem.

FIG. 10

Increased IGF-II and IGFBP-3 expression in the bones of conditional mutants during embryonic and early postnatal development. Gene expression was determined by real-time RT-PCR. Embryos, n = 4; 2 wk of age, n = 8; 12 wk of age, n = 7–8. *, Significant difference from controls at P ≤ 0.05. Data are expressed as fold change from controls and presented as mean ± sem. Control values are set at 1-fold.