In this study, we generated chondrocyte-specific Igf1r
knockout mice to investigate the direct actions of IGF-1R in chondrocytes during embryonic and postnatal skeletal development. Our data confirm a critical role for this receptor in sustaining proliferation, survival, and differentiation of the chondrocyte and its interaction with the PTHrP/Ihh signaling pathway. Deleting Igf1r
in chondrocytes in CartIgf1r−/−
mice led to their hypoproliferation and increased apoptosis. The actions of IGF-1R on chondrocytes appear to be direct because acute knockout of Igf1r
in cultured chondrocytes produced similar growth and apoptotic effects. The IGF-1R in chondrocytes is also critically involved in cell differentiation because its deletion delays cell maturation and hypertrophy, decreases the expres sion of α1
(II), and blocks vascular invasion into the cartilage. These dysregulated activities together caused disorganized growth plates and a shortened skeleton.(27,28)
The morphologic changes—expanded PZ and shortened HZ—in the GP of knockout mice further indicate unsynchronized cell proliferation and differentiation. These morphologic changes are more evident in the tibial GP but are less profound in the femoral GP, suggesting a site-dependent effect of Igf1r
The hypoproliferation phenotype of Igf1r
knockout mice was clearly demonstrated by the reduced PCNA expression and decreased BrdU incorporation in the tibial GP. The latter observation indicates a disproportionally prolonged S phase of cell cycle in knockout GPCs. Interestingly, another chondro-cyte-specific Igf1r
KO mouse model made by Long and colleagues did not show a reduction in BrdU labeling in the femoral GP in comparison with Wt controls.(29)
The authors suggested that the unchanged BrdU+
cell fraction might be due to the proportional lengthening of all phases of the cell cycle in chondrocytes in the PZ, resulting in an overall decrease in proliferation without a significant change in the ratio of BrdU+
cells over the total number of cells.(29)
This is different from our observations with the tibial GP in our model. It is possible that these discordant results of BrdU labeling are due in part to a site-specific effect of the gene knockout because we assessed the BrdU labeling in the tibial GP in this study, whereas Long and colleagues examined the femoral GP. This site-dependent effect is further supported by less change in the morphology of the femoral GP than of the tibial GP in our knockout mice and in the model of Long and colleagues, which showed relatively normal cell organization in the femoral GP of the knockout mice.(29)
In addition to hypoproliferation and increased apoptosis of chondrocytes, CartIgf1r−/−
mice had a significant delay in chondrocyte terminal differentiation. The latter change likely caused the accumulation of cells in the PZ, resulting in its disorganization and expansion in the knockout mice, similar to that seen in the global Igf1
The delayed terminal differentiation also was reflected by a shortened HZ and a reduced bone length.
The similarity in the skeletal phenotypes between the CartIgf1r−/−
and global Igf1
knockout mice indicates that IGF-1 is likely the main ligand acting on the receptor in the chondrocytes, although a role for IGF-2 is not excluded by our data. The degree of skeletal defect in the CartIgf1r−/−
mice, however, is less profound than that in the generalized Igf1r
knockout mice, suggesting that the function of IGF-1R in other relevant cells, such as osteoblasts,(23)
also may contribute to the GP development. Alternatively, the IGF1-1R may be involved in earlier chondro genic events that take place before the activation of the α1
(II) promoter that we used to drive the expression of the Cre
transgene in our knockout mice.
Ihh and PTHrP constitute a feedback loop to control the pace of chondrocyte proliferation and maturation. Interestingly, ablation of Igf1r
increased the expression of PTHrP protein and RNA, and this was likely due to an increase in gene transcription based on the study using a β-Gal reporter system. The increased PTHrP expression might have contributed to the delayed cell differentiation and mineralization and the reduced α1
(II) expression in the GPs in our knockout mouse models(32)
because their phenotypes are similar to those of transgenic mice overexpressing PTHrP.(33)
However, the latter mice have increased proliferation and decreased apoptosis. It appears that Igf1r
knockout prevents the increased growth and survival of chondrocytes otherwise expected with increased PTHrP expression, suggesting that the increased expression of PTHrP is an effort to compensate for the reduced proliferation in the Igf1r
knockout. It remains unclear how the Igf1r
knockout affects PTHrP expression in GPCs. It has been proposed that Ihh is a critical modulator that promotes PTHrP expression in the GP. We, however, observed a decrease in the expression of Ihh protein in the prehypertrophyic chondrocytes that are immediately adjacent to the PTHrP-expressing cells in the GP of 2-week-old TamCartIgf1r−/−
mice, supporting an Ihh-independent regulation of PTHrP expression by Igf1r
This notion is further supported by the observation that the expression of Pthrp
was upregulated even in cell layers of the GP that do not express this gene normally.
It is worth pointing out that we did not dissect out subregions of GPs in preparing RNA for the qPCR analyses. Because we normalized gene expression to the expression of ribosomal protein L19 gene, inclusion of RNA from the disproportionally expanded PZ, which does not express Pthrp, might have underestimated the impact of Igf1r knockout on the Pthrp expression assessed by qPCR. This diluting effect may explain in part the discrepancy between the modest change (approximately 35%) in Pthrp RNA levels assessed by qPCR, as shown in , compared with the more robust Pthrp reporter activity determined by the β-Gal staining in . Furthermore, the potentially longer half-life of the LacZ transcript compared with that of Pthrp mRNA also may contribute to the enhanced sensitivity in detecting the transcription activity of the Pthrp gene. Nevertheless, these observations, together with study of cultured chondrocytes, support the impact of Igf1r knockout on PTHrP expression in GPCs.
Our studies also demonstrate an important role for the IGF-1R in the induction of angiogenesis and vascular invasion in the GPs. In our mouse models, Igf1r
knockout delays development of the vasculature and overall formation of the ossification center in the spinal column. These data are consistent with previous studies demonstrating that IGF-1R signaling is required to mediate the hypoxia-inducible factor 1(HIF-1)-induced angiogenesis and expression vascular endothelial growth factor (VEGF).(34,35)
Our TamCarIgf1r−/− mice are the first to allow assessment of IGF-1R actions in cartilage development during postnatal growth studies. Our initial characterization of the mice demonstrates that the IGF-1R modulates functions in postnatal GPs, as it does in embryonic GPs. This inducible knockout model will be invaluable in future studies on the role of IGF-1R signaling in the development of cartilage diseases such as osteoarthritis and endochondral repair of bone fractures.