Osx-Cre directed ablation of site-1 protease in mice results in kyphosis and scoliosis

Abstract

Purpose: Site-1 protease (S1P) is a proprotein convertase that is primarily known for processing of latent, endoplasmic reticulum (ER) membrane-bound transcription factors SREBPs and ATF6 to their free and active form. SREBPs are involved in cholesterol and fatty acid homeostasis; ATF6 is involved in ER stress response. Cartilage-specific ablation of S1P in mice (S1Pcko) using Col2-Cre results in poor cartilage with a drastic decrease of collagen type IIB (Col IIB) in the matrix and a complete lack of endochondral bone formation, even though the molecular program for chondrocyte maturation is not disrupted. In a postnatal model using Col2-CreERT mice, ablation of S1P in postnatal chondrocytes gradually eliminates the primary growth plate with a gradual abolition of Ihh and type X collagen expression in the growth plate indicating a cessation of hypertrophic chondrocyte differentiation. The goal of this study is to understand the importance of S1P in chondrocyte maturation in the growth plate. Methods: The osterix protein is expressed in prehypertrophic chondrocytes (as well as in osteoblasts). S1Pf/f mice (homozygous for the S1Pflox allele in which exon 2 of S1P is floxed) were crossed with Osx-Cre mice (in which the Cre recombinase is under the influence of the Osterix (Osx) promoter) to obtain mice with S1P ablation in prehypertrophic chondrocytes (S1POsx mice). S1POsx mice and wild type littermates were studied by histology, immunofluorescence (IF), and micro-computed tomography (μCT) techniques. Results: Mice with a homozygous deletion for S1P (S1Pf/f;Osx-Cre mice or S1Pcko-Osx) suffer from severe chondrodysplasia that is visible in their gross morphology beginning from postnatal day 7. Mice which are heterozygous for S1P deletion (S1P+/f-Osx) have a size intermediate to the S1Pcko-Osx mice and their wild type littermates; additionally, these mice are also hunchbacks (kyphosis) which gets more pronounced with age. Interestingly, μCT analysis not only confirmed the kyphosis in S1P+/f-Osx mice, but also revealed scoliosis in the S1Pcko-Osx mice. Micro-CT analysis also showed decreased bone mineralization in both mutant mice with the S1Pcko-Osx mice showing a more drastic decrease than the heterozygote resulting in fragile bones. In embryonic studies, S1Pcko-Osx mice exhibited a delay in the onset for endochondral bone formation, while the S1P+/f-Osx showed normal onset of endochondral bone formation as the wild type. In immunofluorescence studies for type II collagen (Col II) distribution, while the Col2-Cre-directed S1Pcko mice exhibited Col II entrapment in all zones of the growth plate including the resting and proliferative zone, S1Pcko-Osx mice showed Col II entrapment only in the hypertrophic zone, but not the resting and proliferative zones; negligible Col II entrapment was observed in the growth plate of S1P+/f-Osx mice. Conclusions: These results indicate the indispensable nature of S1P for cartilage and overall skeletal development. S1P is essential for normal growth plate activity and cartilage formation. The current study also endorses the importance of S1P in normal skeletal development and is important for both axial and appendicular skeletal development. For the first time, we demonstrate an important functional link between S1P and diseases of the vertebral column. These studies also suggest an indispensable role for S1P in osteoblasts. Thus understanding the contributions of S1P in cartilage, bone and vertebrae development will contribute to the understanding of overall skeletal homeostasis.