The insulin-like growth factor-1 binding protein acid-labile subunit alters mesenchymal stromal cell fate

Abstract

Age-related osteoporosis is accompanied by an increase in marrow adiposity and a reduction in serum insulin-like growth factor-1 (IGF-1) and the binding proteins that stabilize IGF-1. To determine the relationship between these proteins and bone marrow adiposity, we evaluated the adipogenic potential of marrow-derived mesenchymal stromal cells (MSCs) from mice with decreased serum IGF-1 due to knockdown of IGF-1 production by the liver or knock-out of the binding proteins. We employed 10-16-week-old, liver-specific IGF-1-deficient, IGFBP-3 knock-out (BP3KO) and acid-labile subunit knock-out (ALSKO) mice. We found that expression of the late adipocyte differentiation marker peroxisome proliferator-activated receptor gamma was increased in marrow isolated from ALSKO mice. When induced with adipogenic media, MSC cultures from ALSKO mice revealed a significantly greater number of differentiated adipocytes compared with controls. MSCs from ALSKO mice also exhibited decreased alkaline-phosphatase positive colony size in cultures that were stimulated with osteoblast differentiation media. These osteoblast-like cells from ALSKO mice failed to induce osteoclastogenesis of control cells in co-culture assays, indicating that impairment of IGF-1 complex formation with ALS in bone marrow alters cell fate, leading to increased adipogenesis.

FIGURE 1.

Effects of als and igfbp-3 gene inactivation on primary osteoblast cultures. Osteoblast cultures derived from control, LID, ALSKO, and BP3KO 16-week-old male mice were maintained for 14 days and stained for alkaline phosphatase activity. Stained area was normalized to the control value. Data are presented as mean ± S.E. of n > 5 mice per genotype. *, p < 0.05 versus control.

FIGURE 2.

Effects of als and igfbp-3 gene inactivation on bone marrow-derived adipocyte differentiation. Bone marrow cells were isolated from 8–10-week-old control (n = 5), LID (n = 5), ALSKO (n = 4), and BP3KO (n = 4) mice and cultured in adipogenic media, as described under “Experimental Procedures.” Adipocytes were detected by Oil-Red-O staining 14 days following adipogenesis induction and are presented as number of adipocytes per well. Data are expressed as mean ± S.E. *, p < 0.05 versus control.

FIGURE 3.

Recombinant ALS inhibits adipogenesis of MSC cells. MSC cultures derived from 8–10-week-old control mice (n = 5) were induced with adipogenic media in the presence of insulin (10 μg/ml) and rhIGF-1 (10 nm), rhIGFBP-3 (1 μg/ml), or rhALS (0.01 μg/ml). Data are expressed as mean ± S.E. *, p < 0.05 versus control.

FIGURE 4.

Expression of adipogenic markers in bone marrow of IGF mutant mice. Gene expression of c/ebp-α, ap2, and pparγ was assessed by real-time PCR in marrow taken from 8–16-week-old control (n = 4), LID (n = 3), ALSKO (n = 5), and BP3KO (n = 4) mice. Data are expressed as mean ± S.E. *, p < 0.05 versus control mice.

FIGURE 5.

Co-culture of osteoblasts derived from ALSKO mice with BM-nonadherent cells derived from control mice failed to support osteoclastogenesis. A, schematic of the co-culture protocol. B, osteoblast-like cells derived from 8–10-week-old control (n = 5), LID (n = 5), ALSKO (n = 4), and BP3KO (n = 4) mice were co-cultured with BM-nonadherent cells derived from control mice and stained for TRAP after 7 days in culture. TRAP+ cells were counted in 3 wells per mouse per genotype. Data are expressed as mean ± S.E. *, p < 0.05 versus control.