IL-17A has recently emerged as a potential target that regulates the extensive inflammation and abnormal bone formation observed in ankylosing spondylitis (AS). Blocking IL-17A is expected to inhibit ...bony ankylosis. Here, we investigated the effects of anti IL-17A agents in AS.
TNFα, IL-17A, and IL-12/23 p40 levels in serum and synovial fluid from patients with ankylosing spondylitis (AS), rheumatoid arthritis (RA), osteoarthritis (OA), or healthy controls (HC) were measured by ELISA. Bone tissue samples were obtained at surgery from the facet joints of ten patients with AS and ten control (Ct) patients with noninflammatory spinal disease. The functional relevance of IL-17A, biological blockades, Janus kinase 2 (JAK2), and non-receptor tyrosine kinase was assessed in vitro with primary bone-derived cells (BdCs) and serum from patients with AS.
Basal levels of IL-17A and IL-12/23 p40 in body fluids were elevated in patients with AS. JAK2 was also highly expressed in bone tissue and primary BdCs from patients with AS. Furthermore, addition of exogenous IL-17A to primary Ct-BdCs promoted the osteogenic stimulus-induced increase in ALP activity and mineralization. Intriguingly, blocking IL-17A with serum from patients with AS attenuated ALP activity and mineralization in both Ct and AS-BdCs by inhibiting JAK2 phosphorylation and downregulating osteoblast-involved genes. Moreover, JAK2 inhibitors effectively reduced JAK2-driven ALP activity and JAK2-mediated events.
Our findings indicate that IL-17A regulates osteoblast activity and differentiation via JAK2/STAT3 signaling. They shed light on AS pathogenesis and suggest new rational therapies for clinical AS ankylosis.
While their function, as immune checkpoint molecules, is well known, B7-family proteins also function as regulatory molecules in bone remodeling. B7-H3 is a receptor ligand of the B7 family that ...functions primarily as a negative immune checkpoint. While the regulatory function of B7-H3 in osteoblast differentiation has been established, its role in osteoclast differentiation remains unclear. Here we show that B7-H3 is highly expressed in mature osteoclasts and that B7-H3 deficiency leads to the inhibition of osteoclastogenesis in human osteoclast precursors (OCPs). High-throughput transcriptomic analyses reveal that B7-H3 inhibition upregulates IFN signaling as well as IFN-inducible genes, including IDO. Pharmacological inhibition of type-I IFN and IDO knockdown leads to reversal of B7-H3-deficiency-mediated osteoclastogenesis suppression. Although synovial-fluid macrophages from rheumatoid-arthritis patients express B7-H3, inhibition of B7-H3 does not affect their osteoclastogenesis. Thus, our findings highlight B7-H3 as a physiologic positive regulator of osteoclast differentiation and implicate type-I IFN-IDO signaling as its downstream mechanism.
Background
Human gliomas are aggressive brain tumors characterized by uncontrolled cell proliferation. Differential expression of Polycomb repressive complex 2 (PRC2) has been reported in various ...subtypes of glioma. However, the role of PRC2 in uncontrolled growth in glioma and its underlying molecular mechanisms remain to be elucidated.
Objective
We aimed to investigate the functional role of PRC2 in human glioblastoma cell growth by silencing SUZ12, the non-catalytic core component of PRC2.
Methods
Knockdown of
SUZ12
was achieved by infecting T98G cells with lentivirus carrying sequences specifically targeting
SUZ12
(shSUZ12). Gene expression was examined by quantitative PCR and western analysis. The impact of shSUZ12 on cell growth was assessed using a cell proliferation assay. Cell cycle distribution was analyzed by flow cytometry, and protein stability was evaluated in cycloheximide-treated cells. Subcellular localization was examined through immunofluorescence staining and biochemical cytoplasmic-nuclear fractionation. Gene expression analysis was also performed on human specimens from normal brain and glioblastoma patients.
Results
SUZ12 knockdown (SUZ12 KD) led to widespread decrease in the PRC2-specific histone mark, accompanied by a slowdown of cell proliferation through G1 arrest. In SUZ12 KD cells, the degradation of CDKN1B protein was reduced, resulting from alterations in the MYC-SKP2-CDKN1B axis. Furthermore, nuclear localization of CDKN1B was enhanced in SUZ12 KD cells. Analysis of human glioblastoma samples yielded increased expression of EZH2 and MYC along with reduced CDKN1B compared to normal human brain tissue.
Conclusion
Our findings suggest a novel role for
SUZ12
in cell proliferation through post-translational regulation of CDKN1B in glioblastoma.
Major depressive disorder (MDD) is a devastating disease that arises in a background of environmental risk factors, such as chronic stress, that produce reactive oxygen species (ROS) in the brain. ...The chronic stress-induced ROS production involves Ca2+ signals; however, the mechanism is poorly understood. Transient receptor potential melastatin type 2 (TRPM2) is a Ca2+-permeable cation channel that is highly expressed in the brain. Here we show that in animal models of chronic unpredictable stress (CUS), deletion of TRPM2 (Trpm2
−/−) produces antidepressant-like behaviors in mice. This phenotype correlates with reduced ROS, ROS-induced calpain activation, and enhanced phosphorylation of two Cdk5 targets including synapsin 1 and histone deacetylase 5 that are linked to synaptic function and gene expression, respectively. Moreover, TRPM2 mRNA expression is increased in hippocampal tissue samples from patients with MDD. Our findings suggest that TRPM2 is a key agent in stress-induced depression and a possible target for treating depression.
Dickkopf-1 (DKK1) is a secreted protein that acts as an antagonist of the canonical WNT/β-catenin pathway, which regulates osteoblast differentiation. However, the role of DKK1 on osteoblast ...differentiation has not yet been fully clarified. Here, we investigate the functional role of DKK1 on osteoblast differentiation. Primary osteoprogenitor cells were isolated from human spinal bone tissues. To examine the role of DKK1 in osteoblast differentiation, we manipulated the expression of DKK1, and the cells were differentiated into mature osteoblasts. DKK1 overexpression in osteoprogenitor cells promoted matrix mineralization of osteoblast differentiation but did not promote matrix maturation. DKK1 increased Ca + influx and activation of the Ca + /calmodulin-dependent protein kinase II Alpha (CAMK2A)-cAMP response element-binding protein 1 (CREB1) and increased translocation of p-CREB1 into the nucleus. In contrast, stable DKK1 knockdown in human osteosarcoma cell line SaOS2 exhibited reduced nuclear translocation of p-CREB1 and matrix mineralization. Overall, we suggest that manipulating DKK1 regulates the matrix mineralization of osteoblasts by Ca + -CAMK2A-CREB1, and DKK1 is a crucial gene for bone mineralization of osteoblasts. BMB Reports 2022; 55(12): 627-632
Ankylosing spondylitis (AS) is characterized by excessive bone formation with syndesmophytes, leading to bony ankylosis. The contribution of osteoblasts to the pathogenesis of ankylosis is poorly ...understood. The aim of this study was to determine molecular differences between disease controls (Ct) and AS bone-derived cells (BdCs) during osteogenic differentiation with or without inflammation using AS patient serum. We confirmed osteoblastic differentiation of Ct and AS BdCs under osteogenic medium by observing morphological changes and measuring osteoblastic differentiation markers. Osteoblast differentiation was detected by alkaline phosphatase (ALP) staining and activity, and alizarin red and hydroxyapatite staining. Osteoblast-specific markers were analyzed by quantitative reverse-transcriptase-polymerase chain reaction, immunoblotting, and immunostaining. To examine the effects of inflammation, we added AS and healthy control serum to Ct and AS BdCs, and then analyzed osteoblast-specific markers. AS BdCs showed elevated basal intercellular and extracellular ALP activity compared to Ct. When osteoblast differentiation was induced, AS BdCs exhibited higher expression of osteoblast-specific marker genes and faster mineralization than Ct, indicating that these cells differentiated more rapidly into osteoblasts. ALP activity and mineralization accelerated when serum from AS patients was added to Ct and AS BdCs. Our results revealed that AS BdCs showed significantly increased osteoblastic activity and differentiation capacity by regulating osteoblast-specific transcription factors and proteins compared to Ct BdCs. Active inflammation of AS serum accelerated osteoblastic activity. Our study could provide useful basic data for understanding the molecular mechanism of ankylosis in AS.
Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease linked to oxidative stress, which is associated with significant morbidity. The NADPH oxidase complex (NOX) produces reactive ...oxygen species (ROS) that are among the key markers for determining RA's pathophysiology. Therefore, understanding ROS-regulated molecular pathways and their interaction is necessary for developing novel therapeutic approaches for RA. Here, by combining mouse genetics and biochemistry with clinical tissue analysis, we reveal that in vivo Rubicon interacts with the p22phox subunit of NOX, which is necessary for increased ROS-mediated RA pathogenesis. Furthermore, we developed a series of new aryl propanamide derivatives consisting of tetrahydroindazole and thiadiazole as p22phox inhibitors and selected 2-(tetrahydroindazolyl)phenoxy-N-(thiadiazolyl)propanamide 2 (TIPTP, M.W. 437.44), which showed considerably improved potency, reaching an IC
value up to 100-fold lower than an inhibitor that we previously synthesized reported N8 peptide-mimetic small molecule (blocking p22phox-Rubicon interaction). Notably, TIPTP treatment showed significant therapeutic effects a mouse model for RA. Furthermore, TIPTP had anti-inflammatory effects ex vivo in monocytes from healthy individuals and synovial fluid cells from RA patients. These findings may have clinical applications for the development of TIPTP as a small molecule inhibitor of the p22phox-Rubicon axis for the treatment of ROS-driven diseases such as RA.
Objective
To demonstrate the immune landscape of blood and synovial cells in the setting of ankylosing spondylitis (AS) through the analysis of both single‐cell transcriptome and surface protein ...expression, and to unveil the molecular characteristics of pathogenic Th17 cells.
Methods
This study included 40 individuals with active AS, 20 individuals with stable AS, 40 patients with active rheumatoid arthritis, and 20 healthy controls. Surface phenotype and intracellular staining were assessed using flow cytometry after peripheral blood mononuclear cells and synovial fluid mononuclear cells were stimulated with T cell receptor. Single‐cell transcriptomes of 6 patients with active AS were studied along with cellular indexing of transcriptomes and epitopes by sequencing. We also assessed the outcome of targeting OX40 and glucocorticoid‐induced tumor necrosis factor receptor (GITR) on the surface of Th17 cells in a mouse model of curdlan‐injected SKG mice in which anti‐GITR ligand and/or anti‐OX40 ligand were used.
Results
We identified pathogenic Th17 cells as polyfunctional interleukin‐17A (IL‐17A)– and interferon‐γ (IFNγ)–producing memory CD4+ T cells, with clinically supportive evidence for their pathogenic roles at sites of inflammation in AS. Transcriptome and flow cytometric analyses revealed that the coexpression of TNFRSF4 (OX40) and TNFRSF18 (GITR) is increased in pathogenic Th17 cells. Suppression of ligand receptor interactions in vivo through OX40 and GITR effectively suppressed clinical arthritis and decreased pathogenic Th17 cells in the curdlan‐injected SKG mouse model.
Conclusion
Our results have implications for the understanding of pathogenic Th17 cells in AS patients and suggest potential therapeutic targets.
PDF
