Cartilage-Specific Ablation of Site-1 Protease in Mice Results in the Endoplasmic Reticulum Entrapment of Type IIB Procollagen and Down-Regulation of Cholesterol and Lipid Homeostasis

Abstract

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.

Transcriptional control of chondrocyte specification and differentiation

“…It includes SREBP1 and 2, proteins essential for cholesterol metabolism. These factors may be critical in GPCs, as suggested by the observation that chondrocyte-specific inactivation of the gene for S1P, a proprotein convertase required to cleave SREBP precursor proteins into mature forms, results in severe chondrodysplasia in the mouse, with significant downregulation of SREBP-dependent pathways [143, 144]. …”

Emerging targets in osteoarthritis therapy

“…The UPR is important for the normal differentiation program in chondrocytes proceeding to hypertrophy and apoptosis during growth plate development. [88–90]. Inflammatory, mechanical, and oxidative stress can induce ER stress and the UPR in cartilage via C/EBP homologous protein (CHOP) and X-box protein 1 (XBP1) [••47], both of which potentiate IL-1β-induced oxidative stress and pro-catabolic responses [91]; however, XBP1 can promote chondrocyte survival under certain conditions [92].…”

Site-1 protease regulates skeletal stem cell population and osteogenic differentiation in mice

“…S6) at E16.5. Our previous studies had demonstrated that S1P ablation in the chondrocyte lineage resulted in intracellular Col II entrapment with abnormal cartilage that impeded endochondral bone development (Patra et al, 2014a(Patra et al, , 2007. However, S1P-ablation in the Osx lineage results in cartilage matrix very similar to WT.…”

“…S1P cko mice (Col2-Cre driven) suffered from a complete lack of vascular invasion where pro-Col IIB entrapment was absolute (Patra et al, 2014a(Patra et al, , 2007. This suggests that clearing of the entrapped pro-Col IIB in this zone by UPR may be necessary before vascular invasion can begin.…”

“…Antibodies to the type II collagen (Col II) triple helical domain (THD), type IIA procollagen (Col IIA) and the type IIB procollagen ( pro-Col IIB) and their use in double-labeled IF to analyze the cartilage matrix are as reported (Patra et al, 2014a(Patra et al, , 2007. IF for PECAM-1, Runx2, type I collagen (Col I), osteocalcin, Cxcl-12 and Gremlin1 proteins was performed on frozen hind limb sections after fixing tissues overnight with 4% formaldehyde (Duan et al, 2015).…”