No abstract

Site-1 protease (S1P) is a proprotein convertase with essential functions in the conversion of precursor proteins to their active form. In earlier studies, we demonstrated that S1P ablation in the chondrocyte lineage results in a drastic reduction in endochondral bone formation. To investigate the mechanistic contribution of S1P to bone development we ablated S1P in the osterix lineage in mice. S1P ablation in this lineage results in osteochondrodysplasia and variable degrees of early postnatal scoliosis. Embryonically, even though Runx2 and osterix expression are normal, S1P ablation results in a delay in vascular invasion and endochondral bone development. Mice appear normal when born, but by day 7 display pronounced dwarfism with fragile bones that exhibit significantly reduced mineral density, mineral apposition rate, bone formation rate and reduced osteoblasts indicating severe osteopenia. Mice suffer from a drastic reduction in bone marrow mesenchymal progenitors as analyzed by colony-forming unit-fibroblast assay. Fluorescence-activated cell sorting analysis of the skeletal mesenchyme harvested from bone marrow and collagenase-digested bone show a drastic reduction in hematopoietic lineage-negative, endothelial-negative, CD105+ skeletal stem cells. Bone marrow mesenchymal progenitors are unable to differentiate into osteoblasts in vitro, with no effect on adipogenic differentiation. Postnatal mice have smaller growth plates with reduced hypertrophic zone. Thus, S1P controls bone development directly by regulating the skeletal progenitor population and their differentiation into osteoblasts.

The proprotein convertase site-1 protease (S1P) converts latent ER-membrane bound transcription factors SREBPs and ATF6 to their active forms. SREBPs are involved in cholesterol and fatty acid homeostasis whereas ATF6 is involved in unfolded protein response pathways (UPR). Cartilage-specific ablation of S1P in mice (S1Pcko) results in abnormal cartilage devoid of type II collagen protein (Col II). S1Pcko mice also lack endochondral bone development. To analyze S1Pcko cartilage we performed double-labeled immunofluorescence studies for matrix proteins that demonstrated that type IIB procollagen is trapped inside the ER in S1Pcko chondrocytes. This retention is specific to type IIB procollagen; other cartilage proteins such as type IIA procollagen, cartilage oligomeric matrix protein (COMP) and aggrecan are not affected. The S1Pcko cartilage thus exhibits COMP-, aggrecan-, and type IIA procollagen-derived matrices but is characterized by the absence of a type IIB procollagen-derived matrix. To understand the molecular reason behind S1Pcko phenotypes we performed genome-wide transcriptional profiling of cartilage isolated from S1Pcko and wild type littermates. While the UPR pathways are unaffected, the SREBPs-directed cholesterol and fatty acid pathways are significantly down-regulated in S1Pcko chondrocytes, with maximal down-regulation of the stearoyl-CoA desaturase-1 (Scd1) gene. However, mouse models that lack Scd1 or exhibit reduction in lipid homeostasis do not suffer from the ER retention of Col II or lack endochondral bone. These studies indicate an indispensable role for S1P in type IIB procollagen trafficking from the ER. This role appears not to be related to lipid pathways or other current known functions of S1P and is likely dependent on additional, yet unknown, S1P substrates in chondrocytes.

Site-1 protease (S1P) is a proprotein convertase with essential functions in lipid homeostasis and unfolded protein response pathways. We previously studied a mouse model of cartilage-specific knock-out of S1P in chondroprogenitor cells. These mice exhibited a defective cartilage matrix devoid of type II collagen protein (Col II) and displayed chondrodysplasia with no endochondral bone formation even though the molecular program for endochondral bone development appeared intact. To gain insights into S1P function, we generated and studied a mouse model in which S1P is ablated in postnatal chondrocytes. Postnatal ablation of S1P results in chondrodysplasia. However, unlike early embryonic ablations, the growth plates of these mice exhibit a lack of Ihh, PTHrP-R, and Col10 expression indicating a loss of chondrocyte hypertrophic differentiation and thus disruption of the molecular program required for endochondral bone development. S1P ablation results in rapid growth plate disruption due to intracellular Col II entrapment concomitant with loss of chondrocyte hypertrophy suggesting that these two processes are related. Entrapment of Col II in the chondrocytes of the prospective secondary ossification center precludes its development. Trabecular bone formation is dramatically diminished in the primary spongiosa and is eventually lost. The primary growth plate is eradicated by apoptosis but is gradually replaced by a fully functional new growth plate from progenitor stem cells capable of supporting new bone growth. Our study thus demonstrates that S1P has fundamental roles in the preservation of postnatal growth plate through chondrocyte differentiation and Col II deposition and functions to couple growth plate maturation to trabecular bone development in growing mice.Site-1 protease (S1P 3 ; also known as the membrane-bound transcription factor protease, site-1) is a proprotein convertase that plays a vital role in maintaining lipid homeostasis and unfolded protein response through regulated intramembrane proteolysis pathways. During lipid homeostasis, S1P is involved in the proteolytic maturation of the latent endoplasmic reticulum membrane-bound sterol regulatory element-binding protein transcription factors that direct the synthesis of proteins involved in cholesterol and fatty acid synthesis and uptake (1, 2). During unfolded protein response, S1P is involved in the maturation of the endoplasmic reticulum membrane-bound transcription factors ATF6 (3), old astrocyte specifically induced substance (4), and cAMP-responsive element-binding protein H (5).Several recent studies have also implicated a role for S1P in skeletal development, although the exact molecular nature of this requirement is not known. Mammalian skeletal development takes place largely through the process of endochondral ossification in which mesenchymal cell condensates differentiate to form chondrocytes. Chondrocytes secrete the type II collagen (Col II)-rich cartilaginous anlagen to form a template for the development of the skeletal structura...