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

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

Effects of serum and compressive loading on the cartilage matrix synthesis and spatiotemporal deposition around chondrocytes in 3D culture

The aim of this study was to investigate the effects of serum and compressive dynamic loading on the cartilaginous matrix spatiotemporal distribution around chondrocytes in vitro. Murine chondrocytes suspended in agarose were cultured in serum-free media or in varying concentrations of serum with or without compressive dynamic loading. Gene expression was assayed by quantitative polymerase chain reaction. Immunohistochemistry was performed for type II collagen and type VI collagen, aggrecan, or cartilage oligomeric matrix protein (COMP) to study the effect of serum and dynamic loading on the spatiotemporal distribution of cartilage matrix components. Chondrocytes in serum-free culture exhibited negligible differences in type II collagen, aggrecan, and COMP mRNA expression levels over 15 days of cultivation. However, higher serum concentrations decreased matrix gene expression. Expression of the matrix metalloproteinases (MMP)-3 and MMP-13 mRNA increased over time in serum-free or reduced serum levels, but was significantly suppressed in 10% fetal bovine serum (FBS). Compressive loading significantly stimulated MMP-3 expression on days 7 and 15. Immunohistochemical analysis demonstrated that maximum pericellular matrix deposition was achieved in 10% FBS culture in the absence of compressive loading. The pericellular distribution of type II and VI collagens, aggrecan, and COMP proteins tended to be more co-localized in the pericellular region from day 9 to day 21; compressive loading helped promote this co-localization of matrix proteins. The results of this study suggest that the quantity, quality, and spatial distribution of cartilaginous matrix can be altered by serum concentrations and compressive loading

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

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. This article has an associated First Person interview with the first author of the paper

The ER retention of Col II in S1P<i>^cko</i> chondrocytes.

<p>(A–F) Double-labeled immunofluorescence analyses for Col II THD (IIF antibody) and calnexin in E16.5 femurs in WT (<b>A–C</b>) and S1P<i>^cko</i> (<b>D–F</b>). Signals from calnexin (<b>A, D</b>) are shown in green (white arrows) and those from Col II THD (<b>B, E</b>) are shown in red (white arrowheads) with composite signals shown in <b>C</b> and <b>F</b>. (<b>G–L</b>) Double-labeled immunofluorescence analyses for Col II THD and XBP-1 in E16.5 femurs in WT (<b>G–I</b>) and S1P<i>^cko</i> (<b>J-L</b>). Individual signals from XBP-1 (<b>G, J</b>) are shown in green (white arrows) and those from Col II THD (<b>H, K</b>) are shown in red (white arrowheads), with composite signals shown in <b>I</b> and <b>L</b>. All images shown are for mature columnar proliferative chondrocytes in the WT and a corresponding region in S1P<i>^cko</i> (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105674#pone.0105674.s001" target="_blank">Fig. S1A</a>). Bar (all panels): 10 µm.</p

COMP and aggrecan are not intracellularly retained in S1P<i>^cko</i> chondrocytes.

<p>Double-labeled immunofluorescence analyses for COMP and calnexin in E16.5 femurs in WT (A–C) and S1P<i>^cko</i> (<b>D–F</b>). Individual signals from calnexin (<b>A, D</b>) are shown in green (white arrows) and those from COMP (<b>B, E</b>) are shown in red (white arrowheads), with composite signals in <b>C</b>, <b>F</b>. The relative distribution of Col II THD (red) and COMP (green) proteins for WT (<b>G</b>) and S1P<i>^cko</i> (<b>H</b>) are shown as composites from double-labeled immunolabeling analyses in E16.5 femurs. Occasional entrapment of COMP is indicated by arrows marked with an asterisk. Composite signals showing the localization of aggrecan (Agc) (red) relative to calnexin (green) in E16.5 WT (<b>I</b>) or S1P<i>^cko</i> (<b>J</b>) femurs. All images shown are for mature columnar proliferative chondrocytes in the WT and a corresponding region in S1P<i>^cko</i>. Bar (all panels): 10 µm.</p

A schematic summarizing the results from this study and possible additional roles for S1P in cartilage development.

<p>S1P is historically known for processing the transcription factors ATF6 and SREBPs. Two relatively recently discovered activities include the processing of BBF2H7 and the α/β subunit precursor of N-acetylglucosamine-1-phosphotransferase complex (GNPTAB). Of these four known activities, only SREBPs activities relating to cholesterol and fatty acid homeostasis are down-regulated in S1P<i>^cko</i> chondrocytes, with maximal down-regulation of Scd1. However this does not appear to be responsible for the S1P<i>^cko</i> mutant phenotypes as pro-Col IIB retention in chondrocytes or lack of endochondral bone development is not seen in <i>Scd1−/−</i> mice. The retention of pro-Col IIB in the ER could be due to changes in ER membrane lipid composition, though unlikely as pro-Col IIB retention is not seen in <i>Scd1−/−</i> mice. Processing of additional, yet unidentified, S1P substrates in chondrocytes likely modulate pro-Col IIB trafficking from the ER. Lack of endochondral bone formation is probably due to the abnormal cartilage matrix devoid of Col II, or due to unidentified S1P-regulated processes. In the figure, blue arrows indicate activities that are normal in S1P<i>^cko</i> chondrocytes; red arrows indicate activities that are down-regulated or abnormal in S1P<i>^cko</i>. Dashed arrows with a question mark indicate the possibility of these mechanisms directed by novel S1P substrates, but nothing more is known as yet.</p

Intact ER stress response in S1P<i>^cko</i> chondrocytes.

<p>(<b>A</b>) RNA from the chondroepiphyseal cartilage of E16.5 WT (lane 1) and S1P<i>^cko</i> (lane 2) used in microarray analysis were converted to cDNA and amplified by XBP-1 PCR primers to identify spliced (s) XBP-1 mRNA in a 4.8% polyacrylamide gel. (<b>B</b>) XBP-1 was amplified by PCR using XBP-1 PCR primers 3S and 12AS with cDNA derived from E16.5 WT (lanes 1, 3, 5) and S1P<i>^cko</i> (lanes 2, 4, 6) epiphyseal cartilage RNA and the PCR product restriction digested with <i>Pst</i>I which selectively cuts the un-spliced (u) XBP-1 mRNA, and the resulting products visualized in a 2% agarose gel. In lanes 1 and 2, the cDNA used are from the same embryos used in (<b>A</b>) and for genome-wide expression profiling. Each lane in lanes 3–6 show analyses from RNA pooled from the chondroepiphysis of two different embryos. Thus a total of five WT and five S1P<i>^cko</i> embryos were analyzed. The inverse of the gels are shown in both (<b>A</b>) and (<b>B</b>) to enhance visualization of spliced XBP-1 mRNA. (<b>C–F</b>) Expression signaling for two ATF6-driven genes, BiP and Sdf2l1, as seen by in situ hybridization analyses in WT and S1P<i>^cko</i> cartilage. BiP expression is seen in the ulna, carpal, and metacarpal regions in E16.5 WT (<b>C</b>) and S1P<i>^cko</i> (<b>D</b>) forelimbs. Sdf2l1 expression is seen in the femur of E15.5 WT (<b>E</b>) and S1P<i>^cko</i> (<b>F</b>). Bar: 10 µm. (<b>G</b>) A scatter plot generated from quantitative real-time PCR analysis in the murine UPR RT² Profiler PCR Array system comparing the relative expression of 84 genes between WT and S1P<i>^cko</i> chondrocytes. A log transformation plot is shown in which the relative gene expression level of each gene (2^-ΔCt) in WT is plotted against the corresponding value in S1P<i>^cko</i> to indicate fold changes (2^-ΔΔCt). The black line indicates no fold change (fold change of 1). The pink lines indicate a fold change of 2 (gene expression threshold). All genes within these two lines are considered to be similar in expression to WT. Only Ddit3 or Insig-1 were significantly differentially expressed among the 84 genes profiled. A total of four WT and four S1P<i>^cko</i> embryos were profiled.</p

Quantitative real-time PCR analyses of genes down-regulated in S1P<i>^cko</i> chondrocytes when compared to WT littermates.

<p>RNA was harvested and pooled from the chondroepiphyseal cartilage of five E16.5 S1P<i>^cko</i> or five E16.5 WT embryos.</p

Normal endochondral bone development and Col II deposition in <i>Scd1</i>−/− mice.

<p>(<b>A, B</b>) Sections of E15.5 humerus were stained with Safranin O, Fast green, and hematoxylin showing normal onset of endochondral bone development in <i>Scd1</i>−/− mice. (<b>C–F</b>) Double-labeled immunofluorescence analyzing Col II deposition in <i>Scd1</i>−/− mice using IIF and IIA antibodies. Colors represent antibody localizations as follows: green, Col IIA (IIA antibody); red, Col II THD (IIF antibody); yellow, colocalization of both antibodies; blue, DAPI-stained nuclei. Panels <b>C</b> and <b>D</b> show the matrix around early immature chondrocytes in the resting zone; panels <b>E</b> and <b>F</b> show the matrix around mature columnar chondrocytes in the proliferative zone. Bar: (A, B): 500 µm; (C–F): 50 µm.</p

Specific retention of pro-Col IIB in S1P<i>^cko</i> chondrocytes.

<p>Double-labeled immunofluorescence analyses were done for pro-Col IIB (red, IIBN antibody) and calnexin (green) in E16.5 femurs in WT (A) and S1P<i>^cko</i> (B) and only the composite for this analysis is shown. In a separate analysis (symbolized by the dashed line between panels A/B and C/D), double-labeled immunofluorescence was done for Col II THD (red) (<b>C–F</b>) and pro-Col IIB (green) (<b>E, F</b>) in E16.5 femurs in WT (<b>C, E</b>) and S1P<i>^cko</i> (<b>D, F</b>) with composite signals shown in <b>E</b> and <b>F</b>. Panels <b>G-L</b> show double-labeled immunofluorescence analyses for Col II THD (red) (<b>G, J</b>) and Col IIA (green; IIA antibody) (<b>H, K</b>) in E16.5 femurs in WT (<b>G–I</b>) and S1P<i>^cko</i> (<b>J–L</b>). Composite signals are shown in panels <b>I</b> and <b>L</b>. All images shown are for mature columnar proliferative chondrocytes in the WT and a corresponding region in S1P<i>^cko</i>. Bar (all panels): 10 µm.</p