IGF-1R signaling in chondrocytes modulates growth plate development by interacting with the PTHrP/Ihh pathway

Abstract

Systemic derangements and perinatal death of generalized insulin-like growth factor 1 (IGF-1) and IGF-1 receptor (IGF-1R) knockout mice preclude definitive assessment of IGF-1R actions in growth-plate (GP) chondrocytes. We generated cartilage-specific Igf1r knockout ((Cart) Igf1r(-/-)) mice to investigate local control of chondrocyte differentiation in the GP by this receptor. These mice died shortly after birth and showed disorganized chondrocyte columns, delayed ossification and vascular invasion, decreased cell proliferation, increased apoptosis, and increased expression of parathyroid hormone-related protein (Pthrp) RNA and protein in their GPs. The increased Pthrp expression in the knockout GPs likely was due to an increase in gene transcription, as determined by the increased activity of a LacZ reporter that was inserted downstream of the endogenous PTHrP promoter and bred into the knockout mice. To circumvent the early death of (Cart) Igf1r(-/-) mice and investigate the role of IGF-1R during postnatal growth, we made tamoxifen (Tam)-inducible, cartilage-specific Igf1r knockout ((TamCart) Igf1r(-/-)) mice. At 2 weeks of age and 7 to 8 days after Tam injection, the (TamCart) Igf1r(-/-) mice showed growth retardation with a disorganized GP, reduced chondrocyte proliferation, decreased type 2 collagen and Indian Hedgehog (Ihh) expression, but increased expression of PTHrP. Consistent with in vivo observations, in vitro knockout of the Igf1r gene by adenoviral expression of Cre recombinase suppressed cell proliferation, promoted apoptosis, and increased Pthrp expression. Our data indicate that the IGF-1R in chondrocytes controls cell growth, survival, and differentiation in embryonic and postnatal GPs in part by suppression of Pthrp expression.

Fig. 1

Ablation of the Igf1r gene in ^CartIgf1r^−/− mice. (A) PCR analyses of genomic DNA extracted from different tissues as specified from the ^CartIgf1r^−/− (KO) and ^CartIgf1r^+/+ (WT) littermates with primer sets for the Cre transgene, floxed-Igf1r allele, and sequences after gene excision (Δ-IGF-1R) as described in “Materials and Methods.” (B) Immunohistochem istry for the expression of IGF-1R in tibial GPs of knockout and wild-type neonates (P₀, same day of birth). Signals were detected by brown DAB stain, and bone sections were counterstained with hematoxylin. RZ=resting zone; PZ=proliferation zone; HZ=hypertrophic zone. Bar=100 μm.

Fig. 2

Skeletal development in ^CartIgf1r^−/− (KO) and ^CartIgf1r^−/− (WT) mice. (A) Whole-mount alizarin red and alcian blue staining of skeletons from 14.5- and 18.5-dpc wild-type and knockout embryos and P₀ neonates. Bar=1 cm. (B) Hematoxylin and eosin staining of tibias and spinal columns from P₀ wild-type and knockout mice. Arrowheads indicate capillaries in the bone marrow of wild-type spine. RZ=resting zone; PZ=proliferation zone; HZ=hypertrophic zone. Bar=200 μm. (C) Immunohistochemistry for the expression of CD31, an endothelial cell marker, in the spine of P₀ wild-type and knockout mice. Signals are indicated by brown DAB stain, and sections were counterstained with hematoxylin. Arrowheads indicate CD31⁺ capillaries. Bar=50 μm.

Fig. 3

Effects of Igf1r knockout on proliferation and apoptosis of GPCs in ^CartIgf1r^−/− (KO) and ^CartIgf1r^+/+ (WT) mice. (A) Cell proliferation in tibial GPs of P₀ wild-type and knockout mice was assessed by immunocytochemistry with antiserum against PCNA and BrdU. Sections were counterstained with hematoxylin. Bar graphs show numbers of PCNA⁺ or BrdU⁺ cells as a percent of total cell number in the PZ. Bar=100 μm. (B) Apoptotic cells in the P₀ wild-type and knockout GPs were detected by TUNEL staining, as described in “Materials and Methods.” Bar graphs show numbers of TUNEL⁺ cells as a percent of total cell number in each zone specified. Results are expressed as means±SD. (C) Cell apoptosis in tibial GPs of P₀ wild-type and knockout mice was further assessed by immunocytochemistry with antiserum against active caspase-3 (brown). RZ=resting zone; PZ=proliferating zone; HZ=hypertrophic zone in both B and C. Bar=50 μm.

Fig. 4

Effects of Igf1r knockout on the expression of critical components of the PTHrP/Ihh feedback mechanism in E18.5 ^CartIgf1r^−/− (KO) and ^CartIgf1r^+/+ (Cont) embryos. (A) mRNA levels from the tibia-femur joints from control (solid bars) and knockout (open bars) mice were quantified by qPCR for the expression of Ihh, Patched, Pthrp, and Opn genes. RNA expression was presented as percent expression of L19, a housekeeping gene, and expressed as means±SD. ^ap<.05 KO versus Cont. (B) β-Gal staining of ^CartPthrpIgf1r^−/− (KO) and control (Cont) embryos. (Panel 1) Whole-mount staining of ^CartPthrpIgf1r^−/− embryos. (Panel 2) Staining of β-Gal activity in femurs from knockout and control embryos. (Panels 3 and 4) Sections of tibia from control and knockout embryos. (Panels 5 and 6) High magnification for panels 3 and 4. Scale bar for panels 3 to 6 =100 μm.

Fig. 5

Postnatal growth and GP development in ^TamCartIgf1r^−/− (KO) and ^TamIgf1r^flox/flox (Cont) mice. Postnatal deletion of IGF-1R in chondrocytes was induced by injecting ^TamCartIgf1r^flox/flox mice with four doses of Tam (0.2 mg/mouse) at 2-day intervals starting at 5 days. For controls (^TamIgf1r-^flox/flox), Igf1r^flox/flox mice, which did not express the Cre transgene, were injected with Tam as described earlier. Mice were studied on day 14 (3 days after the last injection). (A) Photographs of the 14-day-old control and knockout littermates. (B) Immunohistochemistry of tibial GPs of control and knockout mice for the expression of IGF-1R was performed as described earlier. (C) H&E staining of tibial GPs of control and knockout mice. PZ= proliferating zone; HZ=hypertrophic zone. (D–G) Immuno histochemistry of tibial GPs of control and knockout mice for the expression of PCNA (D), α₁(II) (E), Ihh (F), and PTHrP (G). Signals were indicated by brown DAB stain. Sections for immunohistochemistry were counterstained with hematoxylin. Bar=100 μm.

Fig. 6

Effect of Igf1r KO on proliferation, apoptosis, and gene expression in cultured GPCs. GPCs from 2-to4-day-old Igf1r^flox/flox mice infected with Ad-Cre (1 to 8 pfu/cell) or Ad-DNR (8 pfu/cell) viruses or mock solution. (A) Protein and RNA expression of IGF-1R in cultured cells was determined by Western blotting and qPCR, respectively, 72 hours after infection. mRNA levels were normalized to the level of L19 as a loading control and presented as percent of the levels in mock-infected cultures (control). (B) Proliferation of GPCs was determined 72 hours after infection. The cells were labeled with 5-bromo-2-deoxyuridine (BrdU) for 4 hours, and the BrdU signals were detected by DAB stain (arrows). Bar graphs show numbers of BrdU⁺ cells as PERCENT of total cell number in the cultures. (C) Apoptosis was assessed by TUNEL staining, and signals were detected by DAB-stain (arrows). Bar graphs show numbers of TUNEL⁺ cells as percent of total cell number in the cultures. (D) RNA expression of Opn and Pthrp in cultured cells was determined 72 hours after infection by qPCR. All results are expressed as mean±SD. ^ap<.05 versus control cultures.