Notch signaling in postnatal joint chondrocytes, but not subchondral osteoblasts, is required for articular cartilage and joint maintenance

Abstract

Objective Notch signaling has been identified as a critical regulator in cartilage development and joint maintenance, and loss of Notch signaling in all joint tissues results in an early and progressive osteoarthritis (OA)-like pathology. This study investigated the targeted cell population within the knee joint in which Notch signaling is required for normal cartilage and joint integrity. Methods Two loss-of-function mouse models were generated with tissue-specific knockout of the core Notch signaling component, RBPjκ. The AcanCreERT2 transgene specifically removed Rbpjκ floxed alleles in postnatal joint chondrocytes, while the Col1Cre(2.3kb) transgene deleted Rbpjκ in osteoblast populations, including subchondral osteoblasts. Mutant and control mice were analyzed via histology, immunohistochemistry, real-time qPCR, X-ray, and microCT imaging at multiple time-points. Results Loss of Notch signaling in postnatal joint chondrocytes results in a progressive OA-like pathology, and triggered the recruitment of non-targeted fibrotic cells into the articular cartilage potentially due to mis-regulated chemokine expression from within the cartilage. Upon recruitment, these fibrotic cells produced degenerative enzymes that may lead to the observed cartilage degradation and contribute to a significant portion of the age-related OA-like pathology. On the contrary, loss of Notch signaling in subchondral osteoblasts did not affect normal cartilage development or joint maintenance. Conclusions RBPjκ-dependent Notch signaling in postnatal joint chondrocytes, but not subchondral osteoblasts, is required for articular cartilage and joint maintenance.

Molecular mechanisms underlying osteoarthritis development: Notch and NF-κB

“…Similar results have been reported by the same group using Acan-Cre ERT2 and Rbpj-flox mice [ 27 ]. Interestingly, fibrotic cells and high levels of Mmp13 are observed in the superficial zone of Rbpj-knockout cartilage [ 27 ].…”

“…In one study, Rbpj knockout in limb mesenchymal progenitor cells using Prx1-Cre, or in adult chondrocytes using Col2a1-Cre ERT2 , promoted OA development within 8 months without surgical induction [26]. Similar results have been reported by the same group using Acan-Cre ERT2 and Rbpj-flox mice [27]. Interestingly, fibrotic cells and high levels of Mmp13 are observed in the superficial zone of Rbpj-knockout cartilage [27].…”

Molecular mechanisms underlying osteoarthritis development: Notch and NF-κB

“…Similar results have been reported by the same group using Acan-Cre ERT2 and Rbpj-flox mice [ 27 ]. Interestingly, fibrotic cells and high levels of Mmp13 are observed in the superficial zone of Rbpj-knockout cartilage [ 27 ].…”

“…In one study, Rbpj knockout in limb mesenchymal progenitor cells using Prx1-Cre, or in adult chondrocytes using Col2a1-Cre ERT2 , promoted OA development within 8 months without surgical induction [26]. Similar results have been reported by the same group using Acan-Cre ERT2 and Rbpj-flox mice [27]. Interestingly, fibrotic cells and high levels of Mmp13 are observed in the superficial zone of Rbpj-knockout cartilage [27].…”

NOTCH2 sensitizes the chondrocyte to the inflammatory response of tumor necrosis factor α

“…Because the Rbpj deletion enhanced the expression of Il6 , it is not possible to conclude from the experiments that the effect observed with the NOTCH2 gain-of-function was dependent, or not, on Notch canonical signaling. It is of interest that the deletion of Rbpj in the limb bud during development or in articular chondrocytes in postnatal life causes severe OA, and this could possibly be related to enhanced expression of Il6 , as shown in the present work ( 23 , 52 ). Indeed, IL6 plays a fundamental role in the OA that develops following the destabilization of medial meniscus surgeries ( 53 ).…”

Co-varying neighborhood analysis identifies cell populations associated with phenotypes of interest from single-cell transcriptomics

“…CNA identifies NAM-PC1 as the dominant signal in this dataset: NAM-PC 1 explains 39% of the variance in the NAM while no other NAM-PC explains more than 12%. NAM-PC1 reflects Notch activation: cells’ expression of PRG4 —an established Notch-response gene in the synovial joint tissue[ 15 ]—was most strongly correlated with their anchored neighborhoods’ NAM-PC1 loadings (Pearson r=0.79, p<1e-10), followed by expression of FN1 (Pearson r=0.71, p<1e-10), a signaling molecule shown to regulate Notch[ 16 ]. Further, two Notch gene sets were significantly enriched among all gene correlations to NAM-PC1 (“Vilimas NOTCH1 targets up” and “Reactome signalling by NOTCH”, FDR=0.0073 and FDR=0.019, respectively).…”

Molecular mechanisms underlying osteoarthritis development: Notch and NF-κB

“…Similar results have been reported by the same group using Acan-Cre ERT2 and Rbpj-flox mice [ 27 ]. Interestingly, fibrotic cells and high levels of Mmp13 are observed in the superficial zone of Rbpj-knockout cartilage [ 27 ].…”

“…In one study, Rbpj knockout in limb mesenchymal progenitor cells using Prx1-Cre, or in adult chondrocytes using Col2a1-Cre ERT2 , promoted OA development within 8 months without surgical induction [26]. Similar results have been reported by the same group using Acan-Cre ERT2 and Rbpj-flox mice [27]. Interestingly, fibrotic cells and high levels of Mmp13 are observed in the superficial zone of Rbpj-knockout cartilage [27].…”

NOTCH2 sensitizes the chondrocyte to the inflammatory response of tumor necrosis factor α

“…Because the Rbpj deletion enhanced the expression of Il6 , it is not possible to conclude from the experiments that the effect observed with the NOTCH2 gain-of-function was dependent, or not, on Notch canonical signaling. It is of interest that the deletion of Rbpj in the limb bud during development or in articular chondrocytes in postnatal life causes severe OA, and this could possibly be related to enhanced expression of Il6 , as shown in the present work ( 23 , 52 ). Indeed, IL6 plays a fundamental role in the OA that develops following the destabilization of medial meniscus surgeries ( 53 ).…”

Co-varying neighborhood analysis identifies cell populations associated with phenotypes of interest from single-cell transcriptomics

“…CNA identifies NAM-PC1 as the dominant signal in this dataset: NAM-PC 1 explains 39% of the variance in the NAM while no other NAM-PC explains more than 12%. NAM-PC1 reflects Notch activation: cells’ expression of PRG4 —an established Notch-response gene in the synovial joint tissue[ 15 ]—was most strongly correlated with their anchored neighborhoods’ NAM-PC1 loadings (Pearson r=0.79, p<1e-10), followed by expression of FN1 (Pearson r=0.71, p<1e-10), a signaling molecule shown to regulate Notch[ 16 ]. Further, two Notch gene sets were significantly enriched among all gene correlations to NAM-PC1 (“Vilimas NOTCH1 targets up” and “Reactome signalling by NOTCH”, FDR=0.0073 and FDR=0.019, respectively).…”

Molecular mechanisms underlying osteoarthritis development: Notch and NF-κB

“…Similar results have been reported by the same group using Acan-Cre ERT2 and Rbpj-flox mice [ 27 ]. Interestingly, fibrotic cells and high levels of Mmp13 are observed in the superficial zone of Rbpj-knockout cartilage [ 27 ].…”

“…In one study, Rbpj knockout in limb mesenchymal progenitor cells using Prx1-Cre, or in adult chondrocytes using Col2a1-Cre ERT2 , promoted OA development within 8 months without surgical induction [26]. Similar results have been reported by the same group using Acan-Cre ERT2 and Rbpj-flox mice [27]. Interestingly, fibrotic cells and high levels of Mmp13 are observed in the superficial zone of Rbpj-knockout cartilage [27].…”

NOTCH2 sensitizes the chondrocyte to the inflammatory response of tumor necrosis factor α

“…Because the Rbpj deletion enhanced the expression of Il6 , it is not possible to conclude from the experiments that the effect observed with the NOTCH2 gain-of-function was dependent, or not, on Notch canonical signaling. It is of interest that the deletion of Rbpj in the limb bud during development or in articular chondrocytes in postnatal life causes severe OA, and this could possibly be related to enhanced expression of Il6 , as shown in the present work ( 23 , 52 ). Indeed, IL6 plays a fundamental role in the OA that develops following the destabilization of medial meniscus surgeries ( 53 ).…”

Co-varying neighborhood analysis identifies cell populations associated with phenotypes of interest from single-cell transcriptomics

“…CNA identifies NAM-PC1 as the dominant signal in this dataset: NAM-PC 1 explains 39% of the variance in the NAM while no other NAM-PC explains more than 12%. NAM-PC1 reflects Notch activation: cells’ expression of PRG4 —an established Notch-response gene in the synovial joint tissue[ 15 ]—was most strongly correlated with their anchored neighborhoods’ NAM-PC1 loadings (Pearson r=0.79, p<1e-10), followed by expression of FN1 (Pearson r=0.71, p<1e-10), a signaling molecule shown to regulate Notch[ 16 ]. Further, two Notch gene sets were significantly enriched among all gene correlations to NAM-PC1 (“Vilimas NOTCH1 targets up” and “Reactome signalling by NOTCH”, FDR=0.0073 and FDR=0.019, respectively).…”