Glutamine metabolism modulates chondrocyte inflammatory response

Osteoarthritis is the most common joint disease in the world with significant societal consequences but lacks effective disease-modifying interventions. The pathophysiology consists of a prominent inflammatory component that can be targeted to prevent cartilage degradation and structural defects. Intracellular metabolism has emerged as a culprit of the inflammatory response in chondrocytes, with both processes co-regulating each other. The role of glutamine metabolism in chondrocytes, especially in the context of inflammation, lacks a thorough understanding and is the focus of this work. We display that mouse chondrocytes utilize glutamine for energy production and anabolic processes. Furthermore, we show that glutamine deprivation itself causes metabolic reprogramming and decreases the inflammatory response of chondrocytes through inhibition of NF-κB activity. Finally, we display that glutamine deprivation promotes autophagy and that ammonia is an inhibitor of autophagy. Overall, we identify a relationship between glutamine metabolism and inflammatory signaling and display the need for increased study of chondrocyte metabolic systems

Abrogation of Cbl–PI3K Interaction Increases Bone Formation and Osteoblast Proliferation

Cbl is an adaptor protein and E3 ligase that plays both positive and negative roles in several signaling pathways that affect various cellular functions. Tyrosine 737 is unique to Cbl and phosphorylated by Src family kinases. Phosphorylated CblY737 creates a binding site for the p85 regulatory subunit of phosphatidylinositol 3 kinase (PI3K) that also plays an important role in the regulation of bone homeostasis. To investigate the role of Cbl–PI3K interaction in bone homeostasis, we examined knock-in mice in which the PI3K binding site on Cbl was ablated due to the substitution of tyrosine 737 to phenylalanine (CblYF/YF, YF mice). We previously reported that bone volume in these mice is increased due to decreased osteoclast function (Adapala et al., J Biol Chem 285:36745–36758, 19). Here, we report that YF mice also have increased bone formation and osteoblast numbers. In ex vivo cultures bone marrow-derived YF osteoblasts showed increased Col1A expression and their proliferation was also significantly augmented. Moreover, proliferation of MC3T3-E1 cells was increased after treatment with conditioned medium generated by culturing YF bone marrow stromal cells. Expression of stromal derived factor-1 (SDF-1) was increased in YF bone marrow stromal cells compared to wild type. Increased immunostaining of SDF-1 and CXCR4 was observed in YF bone marrow stromal cells compared to wild type. Treatment of YF condition medium with neutralizing anti-SDF-1 and anti-CXCR4 antibodies attenuated MC3T3-E1 cell proliferation. Cumulatively, these results show that abrogation of Cbl–PI3K interaction perturbs bone homeostasis, affecting both osteoclast function and osteoblast proliferation

Comprehensive Genome-Wide Transcriptomic Analysis of Immature Articular Cartilage following Ischemic Osteonecrosis of the Femoral Head in Piglets

<div><p>Objective</p><p>Ischemic osteonecrosis of the femoral head (ONFH) in piglets results in an ischemic injury to the immature articular cartilage. The molecular changes in the articular cartilage in response to ONFH have not been investigated using a transcriptomic approach. The purpose of this study was to perform a genome-wide transcriptomic analysis to identify genes that are upregulated in the immature articular cartilage following ONFH.</p><p>Methods</p><p>ONFH was induced in the right femoral head of 6-week old piglets. The unoperated femoral head was used as the normal control. At 24 hours (acute ischemic-hypoxic injury), 2 weeks (avascular necrosis in the femoral head) and 4 weeks (early repair) after surgery (n = 4 piglets/time point), RNA was isolated from the articular cartilage of the femoral head. A microarray analysis was performed using Affymetrix Porcine GeneChip Array. An enrichment analysis and functional clustering of the genes upregulated due to ONFH were performed using DAVID and STRING software, respectively. The increased expression of selected genes was confirmed by a real-time qRTPCR analysis.</p><p>Results</p><p>Induction of ONFH resulted in the upregulation of 383 genes at 24 hours, 122 genes at 2 weeks and 124 genes at 4 weeks compared to the normal controls. At 24 hours, the genes involved in oxidoreductive, cell-survival, and angiogenic responses were significantly enriched among the upregulated genes. These genes were involved in HIF-1, PI3K-Akt, and MAPK signaling pathways. At 2 weeks, secretory and signaling proteins involved in angiogenic and inflammatory responses, PI3K-Akt and matrix-remodeling pathways were significantly enriched. At 4 weeks, genes that represent inflammatory cytokines and chemokine signaling pathways were significantly enriched. Several index genes (genes that are upregulated at more than one time point following ONFH and are known to be important in various biological processes) including <i>HIF-1A</i>, <i>VEGFA</i>, <i>IL-6</i>, <i>IL6R</i>, <i>IL-8</i>, <i>CCL2</i>, <i>FGF2</i>, <i>TGFB2</i>, <i>MMP1</i>, <i>MMP3</i>, <i>ITGA5</i>, <i>FN and Col6A1</i> were upregulated in the immature articular cartilage following ONFH. A qRTPCR analysis of selected genes confirmed the upregulated expression observed in the microarray analysis.</p><p>Conclusion</p><p>Immature articular cartilage responds to ONFH by the upregulation of genes involved in hypoxic stress response, angiogenesis, matrix remodeling and inflammation. This study provides novel insights into the multi-faceted role of immature articular cartilage, with inflammation as a key component, following ONFH in piglets.</p></div

Genes involved in inflammatory response are significantly enriched in the immature articular cartilage at 4 weeks following the induction of ONFH.

<p>The genes and the pathways that were significantly enriched (p<0.05, based on t-tests, Bonferroni, FDR) belong to inflammation. The STRING10 network diagram indicates that the different genes are closely related to the inflammatory responses. The clusters of genes indicating chemokines/cytokines/receptors and the relationship to growth factors and matrix related genes show the functional link between matrix and recruitment of inflammatory cells. The nodes indicate individual genes and the connecting lines indicate the relationship. Shorter lines indicate closer relationship. The evidence plot utilizes multiple lines to indicate stronger evidence for the proximity in a functional relationship.</p

Response to hypoxic stress (HIF-1 pathway) and cell survival signaling pathways (PI3K-Akt, MAPK pathways) are the most significantly enriched functions in the immature articular cartilage at 24 hours following the induction of ONFH.

<p>The genes and the specific pathways that were significantly enriched (p<0.05, based on t-tests, Bonferroni, FDR) are listed. The network diagram (STRING10 software) indicates a close relationship of different genes based on the functional roles in biological pathways. The clusters of genes indicating specific biological functions (cell survival and signaling, matrix and growth factors, hypoxic stress response) are indicated using boxes with dashed lines. The nodes indicate individual genes and the connecting lines indicate the relationship. Shorter lines indicate closer relationship. This evidence plot utilizes multiple lines to indicate stronger evidence for the proximity in a functional relationship.</p

Angiogenesis, matrix remodeling and inflammation are the most significantly enriched functions in the immature articular cartilage at 2 weeks following the induction of ONFH.

<p>The genes and the specific pathways that were significantly enriched (p<0.05, based on t-tests, Bonferroni, FDR) are listed. The STRING10 network diagram was used to indicate the closeness of relationship of different genes based on the functional roles in biological pathways. The clusters of genes indicating matrix-related functions (e.g. <i>FN</i>, <i>TNC</i>, <i>MMP1</i>, <i>MMP3</i>), angiogenesis (e.g. <i>VEGF</i>) and inflammation (e.g. <i>IL8</i>, <i>OSMR</i>) are shown. The nodes indicate individual genes and the connecting lines indicate the relationship. Shorter lines indicate closer relationship. The evidence plot utilizes multiple lines to indicate stronger evidence for the proximity in a functional relationship.</p

Index genes representing the major roles of the immature articular cartilage.

<p>The STRING10 network diagram indicates the functional clustering of the index genes. The nodes indicate individual genes and the connecting lines indicate the relationship. Shorter lines connect the closely related genes. Multiple connectors indicate stronger evidence for the functional relationship.</p

Biological processes, molecular functions and biological pathways upregulated in the immature articular cartilage at 24 hours following ONFH.

<p>Biological processes, molecular functions and biological pathways upregulated in the immature articular cartilage at 24 hours following ONFH.</p

Biological processes, molecular functions and biological pathways upregulated in the immature articular cartilage at 2 weeks following ONFH.

<p>Biological processes, molecular functions and biological pathways upregulated in the immature articular cartilage at 2 weeks following ONFH.</p

Selected index genes showed increased expression in the immature articular cartilage by qRTPCR analysis.

<p>The microarray results were validated by real-time qRTPCR analysis of the expression of selected index genes at 24 hours, 2 weeks and 4 weeks following the induction of ONFH. <b>(A)</b> The hypoxic response (<i>HIFA</i>, hypoxia inducible factor 1 alpha), angiogenesis (<i>VEGFA</i>, vascular endothelial growth factor alpha), <b>(B)</b> cytokines/cytokine receptors (<i>IL6/IL6R</i>, interleukin 6/interleukin 6 receptor), <b>(C)</b> chemokines (<i>IL8</i>, interleukin 8, CCL2, chemokine (C-C) motif ligand-2 <b>(D)</b> matrix related factors (<i>FN</i>, fibronectin; <i>ITGA5</i>, integrin alpha v subunit), <b>(E)</b> growth factors (<i>FGF2</i>, fibroblast growth factor-2) and <b>(F)</b> transcription factor (<i>NFkB p65</i>, nuclear factor kappa b, subunit p65) were confirmed by qRTPCR. The mRNA levels were normalized to 18sRNA and the data is shown as fold difference relative to normal articular cartilage. A total of n = 4 pigs were used per time point. Statistical analysis was performed using ANOVA and Tukey’s multiple comparison tests. P value * <0.05, **<0.01, ***<0.001, ****<0.0001</p