Site-1 protease is essential for endochondral bone formation in mice

Abstract

Site-1 protease (S1P) has an essential function in the conversion of latent, membrane-bound transcription factors to their free, active form. In mammals, abundant expression of S1P in chondrocytes suggests an involvement in chondrocyte function. To determine the requirement of S1P in cartilage and bone development, we have created cartilage-specific S1P knockout mice (S1Pcko). S1Pcko mice exhibit chondrodysplasia and a complete lack of endochondral ossification even though Runx2 expression, Indian hedgehog signaling, and osteoblastogenesis is intact. However, there is a substantial increase in chondrocyte apoptosis in the cartilage of S1Pcko mice. Extraction of type II collagen is substantially lower from S1Pcko cartilage. In S1Pcko mice, the collagen network is disorganized and collagen becomes entrapped in chondrocytes. Ultrastructural analysis reveals that the endoplasmic reticulum (ER) in S1Pcko chondrocytes is engorged and fragmented in a manner characteristic of severe ER stress. These data suggest that S1P activity is necessary for a specialized ER stress response required by chondrocytes for the genesis of normal cartilage and thus endochondral ossification.

Site-1 Protease Is Essential to Growth Plate Maintenance and Is a Critical Regulator of Chondrocyte Hypertrophic Differentiation in Postnatal Mice

“…The femurs and humeri were fixed in 10% formalin for 24 -48 h, dehydrated, and embedded in methyl methacrylate (14), and 50-m thick sections were cut, mounted on glass slides, and visualized for incorporation of calcein (FITC filter) and alizarin (TRITC filter) by fluorescent microscopy (Olympus BX51), and images were digitized and merged (Olympus DP70). T mice do indeed cause loss of S1P activity on tamoxifen administration, we injected tamoxifen into pregnant mothers at 10.5 and 13.5 days post-coitus to test if this results in chondrodysplasia and lack of endochondral bone as seen in the S1P cko (S1P f/f ; Col2Cre) mice (8). Mice were harvested and analyzed at E18.5.…”

“…Apoptosis of chondrocytes was analyzed by a TUNEL assay using the in situ cell death detection kit (Roche Applied Science) according to the manufacturer's instructions with nuclei counterstained by DAPI. Detection of Col II protein by immunohistochemistry was done on hyaluronidase (1% for 30 min at 37°C)-treated paraffin-embedded tissues using the IIF antibody described previously (8,13) with HRP-conjugated goatanti-rat secondary antibody and a DAB substrate (Invitrogen) with tartrazine as counterstain. Double-labeled immunofluorescence with IIF and IIA antibodies was performed as described previously (8, 13) on hyaluronidase or proteinase K (10 g/ml in 10 mM Tris-HCl, pH 7.5, for 20 min at 37°C) treated paraffin-embedded tissues with the exception that both primary (or secondary) antibodies were added simultaneously rather than sequentially.…”

“…Tartrate-resistant acid phosphatase-positive cells were stained using standard procedures with hematoxylin as counterstain. In situ hybridization (ISH) analyses with 35 Slabeled probes were done as described previously (8,12). Apoptosis of chondrocytes was analyzed by a TUNEL assay using the in situ cell death detection kit (Roche Applied Science) according to the manufacturer's instructions with nuclei counterstained by DAPI.…”

“…We have shown that S1P is essential to endochondral bone development as cartilage-specific ablation of S1P activity in mice (S1P cko ) results in severe chondrodysplasia (8). S1P cko mice do not form any endochondral bone.…”

Inhibition of Proprotein Convertase SKI-1 Blocks Transcription of Key Extracellular Matrix Genes Regulating Osteoblastic Mineralization

“…Third, SKI-1 is required for normal cartilage morphogenesis in zebrafish (28). Finally, conditional inactivation of SKI-1 in mice using a type II collagen CRE recombinase transgene leads to abnormal growth plate calcification (29). The purpose of this study was to determine whether SKI-1 protease controls initiation of osteoblastic mineralization.…”