The IRF1/GBP5 axis promotes osteoarthritis progression by activating chondrocyte pyroptosis.

Background: Osteoarthritis (OA) is a chronic degenerative joint disease that primarily affects middle-aged and elderly individuals. The decline in chondrocyte function plays a crucial role in the development of OA. Inflammasome-mediated chondrocyte pyroptosis is implicated in matrix degradation and cartilage degeneration in OA patients. Guanylate binding protein 5 (GBP5), a member of the GTPase family induced by Interferon-γ (IFN-γ), significantly influences cellular inflammatory responses, including intracellular inflammasome activation and cytokine release. However, the role of GBP5 in chondrocyte pyroptosis and OA progression remains unclear. Methods: In this study, we used tumor necrosis factor-α (TNF-α) to induce inflammation and created an OA mouse model with surgically-induced destabilization of the medial meniscus (DMM). We isolated and cultured primary chondrocytes from the knee joints of suckling C57 mice. TNF-α-stimulated primary chondrocytes served as an in vitro model for OA and underwent RNA sequencing. Chondrocytes were transfected with GBP5-overexpression plasmids and small interfering RNA and were subsequently treated with TNF-α. We assessed the expression of cartilage matrix components (COL2A1 and aggrecan), catabolic factors (MMP9 and MMP13), and NLRP3 inflammasome pathway genes (NLRP3, Caspase1, GSDMD, Pro-IL-1ß, and Pro-Caspase1) using RT-qPCR and Western blotting. We analyzed the expression of GBP5, NLRP3, and Caspase1 in the cartilage of DMM-induced post-traumatic OA mice and human OA patients. Immunohistochemistry (IHC) was used to detect the expression of GBP5, NLRP3 and GSDMD in cartilage specimens from OA patients and mouse DMM models. Chondrocyte pyroptosis was assessed using flow cytometry, and the levels of interleukin-1ß (IL-1ß) and interleukin-18 (IL-18) were measured with ELISA. We conducted double luciferase reporter gene and chromatin immunoprecipitation (ChIP) assays to confirm the relationship between IRF1 and GBP5. Results: GBP5 expression increased in TNF-α-induced chondrocytes, as revealed by RNA sequencing. GBP5 inhibited COL2A1 and aggrecan expression while promoting the expression of MMP9, MMP13, NLRP3, Caspase1, GSDMD, Pro-IL-1ß, and Pro-Caspase1. GBP5 expression also increased in the cartilage of DMM-induced post-traumatic OA mice and human OA patients. Knockout of GBP5 reduced chondrocyte injury in OA mice. GBP5 promoted chondrocyte pyroptosis and the production of IL-1ß and IL-18. Additionally, we found that IRF1 bound to the promoter region of GBP5, enhancing its expression. After co-transfected with ad-IRF1 and siGBP5, the expression of pyroptosis-related genes was significantly decreased compared with ad-IRF1 group. Conclusions: The IRF1/GBP5 axis enhances extracellular matrix (ECM) degradation and promotes pyroptosis during OA development, through the NLRP3 inflammasome signaling pathway. The translational potential of this article: This study underscores the significance of the IRF1/GBP5 axis in NLRP3 inflammasome-mediated chondrocyte pyroptosis and osteoarthritic chondrocyte injury. Modulating IRF1 and GBP5 expression could serve as a novel therapeutic target for OA.

Osteoclast-derived exosomal miR-212-3p suppressed the anabolism and accelerated the catabolism of chondrocytes in osteoarthritis by targeting TGF-ß1/Smad2 signaling.

Osteoarthritis (OA) is a common aging-related disease affecting entire joint structures, encompassing articular cartilage and subchondral bone. Although senescence and dysfunction of chondrocytes are considered crucial factors in the occurrence of OA, the exact pathogenesis remains to be investigated. In our study, chondrocytes were incubated with a conditioned medium obtained from osteoclasts at different differentiation stages, suggesting that osteoclasts and osteoclast precursors suppressed anabolism and promoted the catabolism of chondrocytes in vitro. In contrast, the function of osteoclasts was more significant than osteoclast precursors. Further blocking of osteoclast exosome secretion by using GW4869 abolished the effect of osteoclasts on chondrocytes. Functionally, exosomal transfer of osteoclast-derived miR-212-3p inhibited Smad2 to mediate chondrocyte dysfunction, thus accelerating cartilage matrix degradation in OA via TGF-ß1/Smad2 signaling. The mechanism was also confirmed within the articular cartilage in OA patients and surgery-induced OA mice. Our study provides new information on intercellular interactions in the bone microenvironment within articular cartilage and subchondral bone during OA progression. The miR-212-3p/Smad2 axis is a potential target for the prevention and therapy of OA.

Replenishing decoy extracellular vesicles inhibits phenotype remodeling of tissue-resident cells in inflammation-driven arthritis.

The interleukin 6 (IL6) signaling pathway plays pleiotropic roles in regulating the inflammatory milieu that contributes to arthritis development. Here, we show that activation of IL6 trans-signaling induces phenotypic transitions in tissue-resident cells toward an inflammatory state. The establishment of arthritis increases the serum number of extracellular vesicles (EVs), while these EVs express more IL6 signal transducer (IL6ST, also known as gp130) on their surface. Transferring these EVs can block IL6 trans-signaling in vitro by acting as decoys that trap hyper IL6 and prevent inflammatory amplification in recipient arthritic mice. By genetically fusing EV-sorting domains with extracellular domains of receptors, we engineered EVs that harbor a higher quantity of signaling-incompetent decoy receptors. These exogenous decoy EVs exhibit significant potential in eliciting efficient anti-inflammatory effects in vivo. Our findings suggest an inherent resistance of decoy EVs against inflammation, highlighting the therapeutic potential of efficient decoy EVs in treating inflammatory diseases.

GASC1 promotes hepatocellular carcinoma progression by inhibiting the degradation of ROCK2.

Hepatocellular carcinoma (HCC) is a devastating malignancy without targeted therapeutic options. Our results indicated that the histone demethylase GASC1 signature is associated with later tumor stage and poorer survival in HCC patients. GASC1 depletion led to diminished HCC proliferation and tumor growth. A distinct heterogeneity in GASC1 levels was observed among HCC cell populations, predicting their inherent high or low tumor-initiating capacity. Mechanistically, GASC1 is involved in the regulation of several components of the Rho-GTPase signaling pathway including its downstream target ROCK2. GASC1 demethylase activity ensured the transcriptional repression of FBXO42, a ROCK2 protein-ubiquitin ligase, thereby inhibiting ROCK2 degradation via K63-linked poly-ubiquitination. Treatment with the GASC1 inhibitor SD70 impaired the growth of both HCC cell lines and xenografts in mice, sensitizing them to standard-of-care chemotherapy. This work identifies GASC1 as a malignant-cell-selective target in HCC, and GASC1-specific therapeutics represent promising candidates for new treatment options to control this malignancy.

IL-11 is essential in promoting osteolysis in breast cancer bone metastasis via RANKL-independent activation of osteoclastogenesis.

A variety of osteolytic factors have been identified from breast cancer cells leading to osteolysis, but less is known about which factor plays an essential role in the initiation process prior to the overt vicious osteolytic cycle. Here, we present in vitro and in vivo evidences to clarify the role of interleukin-11 (IL-11) as an essential contributor to breast cancer bone metastasis mediated osteolysis. Animal studies showed that bone specific metastatic BoM-1833 cells induce earlier onset of osteolysis and faster tumor growth compared with MCF7 and parental MDA-MB-231 cells in BALB/c-nu/nu nude mice. IL-11 was further screened and identified as the indispensable factor secreted by BoM-1833 cells inducing osteoclastogenesis independently of receptor activator of nuclear factor κB ligand (RANKL). Mechanistic investigation revealed that the JAK1/STAT3 signaling pathway as a downstream effector of IL-11, STAT3 activation further induces the expression of c-Myc, a necessary factor required for osteoclastogenesis. By inhibiting STAT3 phosphorylation, AG-490 was shown effective in reducing osteolysis and tumor growth in the metastatic niche. Overall, our results revealed the essential role and the underlying molecular mechanism of IL-11 in breast cancer bone metastasis mediated osteolysis. STAT3 targeting through AG-490 is a potential therapeutic strategy for mitigating osteolysis and tumor growth of bone metastatic breast cancer.

Ceria nanoparticles enhance endochondral ossification-based critical-sized bone defect regeneration by promoting the hypertrophic differentiation of BMSCs via DHX15 activation.

Central ischemic necrosis is one of the biggest obstacles in the clinical application of traditional tissue-engineered bone (TEB) in critical-sized bone defect regeneration. Because of its ability to promote vascular invasion, endochondral ossification-based TEB has been applied for bone defect regeneration. However, inadequate chondrocyte hypertrophy can hinder vascular invasion and matrix mineralization during endochondral ossification. In light of recent studies suggesting that ceria nanoparticles (CNPs) improve the blood vessel distribution within TEB, we modified TEB scaffold surfaces with CNPs and investigated the effect and mechanism of CNPs on endochondral ossification-based bone regeneration. The CNPs used in this study were synthesized by the microemulsion method and modified with alendronate-anchored polyethylene glycol 600. We showed that CNPs accelerated new bone formation and enhanced endochondral ossification-based bone regeneration in both a subcutaneous ectopic osteogenesis model and a mouse model of critical-sized bone defects. Mechanistically, CNPs significantly promoted endochondral ossification-based bone regeneration by ensuring sufficient hypertrophic differentiation via the activation of the RNA helicase, DEAH (Asp-Glu-Ala-His) box helicase 15, and its downstream target, p38 MAPK. These results suggested that CNPs could be applied as a biomaterial to improve the efficacy of endochondral ossification-based bone regeneration in critical-sized bone defects.-Li, J., Kang, F., Gong, X., Bai, Y., Dai, J., Zhao, C., Dou, C., Cao, Z., Liang, M., Dong, R., Jiang, H., Yang, X., Dong, S. Ceria nanoparticles enhance endochondral ossification-based critical-sized bone defect regeneration by promoting the hypertrophic differentiation of BMSCs via DHX15 activation.

LncRNA AK077216 promotes RANKL-induced osteoclastogenesis and bone resorption via NFATc1 by inhibition of NIP45.

Osteoclasts derived from the monocyte/macrophage hematopoietic lineage regulate bone resorption, a process balanced by bone formation in the continual renewal of the skeletal system. As dysfunctions of these cells result in bone metabolic diseases such as osteoporosis and osteopetrosis, the exploration of the mechanisms regulating their differentiation is a priority. A potential mechanism may involve long noncoding RNAs (lncRNAs), which are known to regulate various cell biology activities, including proliferation, differentiation, and apoptosis. The expression of the lncRNA AK077216 (Lnc-AK077216) is significantly upregulated during osteoclastogenesis identified by microarray and verified by qPCR. Up- and downregulation of Lnc-AK077216, respectively promotes and inhibits osteoclast differentiation, bone resorption, and the expression of related genes on the basis of tartrate-resistant acid phosphatase staining, qPCR, and western blot results. In addition, Lnc-AK077216 suppresses NIP45 expression and promotes the expression of NFATc1, an essential transcription factor during osteoclastogenesis. Besides, it was found that the expression of Lnc-AK077216 and Nfatc1 is upregulated, whereas Nip45 expression is downregulated in bone marrow and spleen tissues of ovariectomized mice. The results suggest that Lnc-AK077216 regulates NFATc1 expression and promotes osteoclast formation and function, providing a novel mechanism of osteoclastogenesis and a potential biomarker or a new drug target for osteoporosis.

Proliferating cell nuclear antigen interacts with the CRL4 ubiquitin ligase subunit CDT2 in DNA synthesis-induced degradation of CDT1.

During DNA replication or repair, the DNA polymerase cofactor, proliferating cell nuclear antigen (PCNA), homotrimerizes and encircles the replicating DNA, thereby acting as a DNA clamp that promotes DNA polymerase processivity. The formation of the PCNA trimer is also essential for targeting the replication-licensing protein, chromatin-licensing, and DNA replication factor 1 (CDT1), for ubiquitin-dependent proteolysis to prevent chromosomal DNA re-replication. CDT1 uses its PCNA-interacting peptide box (PIP box) to interact with PCNA, and the CRL4 E3 ubiquitin ligase subunit CDT2 is recruited through the formation of PCNA-CDT1 complexes. However, it remains unclear how CDT1 and many other PIP box-containing proteins are marked for degradation by the CRL4CDT2 ubiquitin ligase during DNA replication or damage. Here, using recombinant protein expression coupled with site-directed mutagenesis, we report that CDT2 and PCNA directly interact and this interaction depends on the presence of a highly conserved, C-terminal PIP box-like region in CDT2. Deletion or mutation of this region abolished the CDT2-PCNA interaction between CDT2 and PCNA both in vitro and in vivo Moreover, PCNA-dependent CDT1 degradation in response to DNA damage and replication during the cell cycle requires an intact PIP box in CDT2. The requirement of the PIP boxes in both CDT2 and its substrate CDT1 suggests that the formation of the PCNA trimeric clamp around DNA during DNA replication and repair may bring together CDT1 and CRL4CDT2 ubiquitin E3 ligase to target CDT1 for proteolysis in a DNA synthesis-dependent manner.

Mangiferin enhances endochondral ossification-based bone repair in massive bone defect by inducing autophagy through activating AMP-activated protein kinase signaling pathway.

Endochondral ossification is crucial for bone formation in both adult bone repair process and embryo long-bone development. In endochondral ossification, bone marrow-derived mesenchymal stem cells (BMSCs) first differentiate to chondrocytes, then BMSC-derived chondrocytes endure a hypertrophic process to generate new bone. Endochondral ossification-based bone repair is a promising strategy to cure massive bone defect, which is a major clinical issue in orthopedics. However, challenges still remain for this novel strategy. One challenge is to ensure the sufficient hypertrophic differentiation. Another is to maintain the survival of the above hypertrophic chondrocytes under the hypoxic environment of massive bone defect. To solve this issue, mangiferin (MAG) was introduced to endochondral ossification-based bone repair. In this report, we proved MAG to be a novel autophagy inducer, which promoted BMSC-derived hypertrophic chondrocyte survival against hypoxia-induced injury through inducing autophagy. Furthermore, MAG enhances hypertrophic differentiation of BMSC-derived chondrocytes via upregulating key hypertrophic markers. Mechanistically, MAG induced autophagy in BMSC-derived chondrocytes by promoting AMPKα phosphorylation. Additionally, MAG balanced the expression of sex-determining region Y-box 9 and runt-related transcription factor 2 to facilitate hypertrophic differentiation. These results indicated that MAG was a potential drug to improve the efficacy of endochondral ossification-based bone repair in massive bone defects.-Bai, Y., Liu, C., Fu, L., Gong, X., Dou, C., Cao, Z., Quan, H., Li, J., Kang, F., Dai, J., Zhao, C., Dong, S. Mangiferin enhances endochondral ossification-based bone repair in massive bone defect by inducing autophagy through activating AMP-activated protein kinase signaling pathway.

Long noncoding RNA expression profiles in chondrogenic and hypertrophic differentiation of mouse mesenchymal stem cells.

Long noncoding RNAs (lncRNAs) are important regulators for a variety of biological processes. Chondrogenic differentiation of mesenchymal stem cells (MSCs) is a crucial stage in chondrogenesis while chondrocyte hypertrophy is related to endochondral ossification and osteoarthritis. However, the effects of lncRNAs on chondrogenic and hypertrophic differentiation of mouse MSCs are unclear. To explore the potential mechanisms of lncRNAs during chondrogenesis and chondrocyte hypertrophy, microarray was performed to investigate the expression profiles of lncRNA and mRNA in MSCs, pre-chondrocytes, and hypertrophic chondrocytes. Then, we validated microarray data by RT-PCR and screened three lncRNAs from upregulating groups during chondrogenesis and chondrocyte hypertrophy respectively. After downregulating any of the above lncRNAs, we found that the expression of chondrogenesis-related genes such as Sox9 and Col2a1 and hypertrophy-related genes including Runx2 and Col10a1 was inhibited, respectively. Furthermore, the target genes of above lncRNAs were predicted by bioinformatics approaches. Gene ontology and Kyoto encyclopedia of genes and genome biological pathway analysis were also made to speculate the functions of above lncRNAs. In conclusion, the study first revealed the expression profile of lncRNAs in chondrogenic and hypertrophic differentiations of mouse MSCs and presented a new prospect for the underlying mechanisms of chondrogenesis and endochondral ossification.