Single cell RNA sequencing reveals emergent notochord-derived cell subpopulations in the postnatal nucleus pulposus.

Intervertebral disc degeneration is a leading cause of chronic low back pain. Cell-based strategies that seek to treat disc degeneration by regenerating the central nucleus pulposus (NP) hold significant promise, but key challenges remain. One of these is the inability of therapeutic cells to effectively mimic the performance of native NP cells, which are unique amongst skeletal cell types in that they arise from the embryonic notochord. In this study, we use single cell RNA sequencing to demonstrate emergent heterogeneity amongst notochord-derived NP cells in the postnatal mouse disc. Specifically, we established the existence of progenitor and mature NP cells, corresponding to notochordal and chondrocyte-like cells, respectively. Mature NP cells exhibited significantly higher expression levels of extracellular matrix (ECM) genes including aggrecan, and collagens II and VI, along with elevated transforming growth factor-beta and phosphoinositide 3 kinase-protein kinase B signaling. Additionally, we identified Cd9 as a novel surface marker of mature NP cells, and demonstrated that these cells were localized to the NP periphery, increased in numbers with increasing postnatal age, and co-localized with emerging glycosaminoglycan-rich matrix. Finally, we used a goat model to show that Cd9+ NP cell numbers decrease with moderate severity disc degeneration, suggesting that these cells are associated with maintenance of the healthy NP ECM. Improved understanding of the developmental mechanisms underlying regulation of ECM deposition in the postnatal NP may inform improved regenerative strategies for disc degeneration and associated low back pain.

Interleukin-35 protein inhibits osteoclastogenesis and attenuates collagen-induced arthritis in mice.

Rheumatoid arthritis (RA) is a chronic autoimmune disease. Its pathological features include synovial inflammation, bone erosion, and joint structural damage. Our previous studies have shown that interleukin (IL)-35 is involved in the pathogenesis of bone loss in RA patients. In this study, we are further evaluating the efficacy of IL-35 on collagen-induced arthritis (CIA) in the mouse model. Male DBA/1J mice (n = 10) were initially immunized, 2 µg/mouse IL-35 was injected intraperitoneally every week for 3 weeks after the establishment of the CIA model. Clinical arthritis, histopathological analysis, and three-dimensional micro-computed tomography (3D micro-CT) were determined after the mice were anesthetized on the 42th day. In vitro, RANKL/M-CSF induced mouse preosteoclasts (RAW264.7 cells line) was subjected to antiarthritis mechanism study in the presence of IL-35. The results of clinical arthritis, histopathological analysis, and 3D micro-CT, the expression of RANK/RANKL/OPG axis, inflammatory cytokines, and osteoclastogenesis-related makers demonstrated decreasing severity of synovitis and bone destruction in the ankle joints after IL-35 treatment. Furthermore, IL-35 attenuated inflammatory cytokine production and the expression of osteoclastogenesis-related makers in a mouse preosteoclasts cell line RAW264.7. The osteoclastogenesis-related makers were significantly reduced in IL-35 treated RAW264.7 cells line after blockage with the JAK/STAT1 signaling pathway. These results demonstrated that IL-35 protein could inhibits osteoclastogenesis and attenuates CIA in mice. We concluded that IL-35 can exhibit anti-osteoclastogenesis effects by reducing the expression of inflammatory cytokines and osteoclastogenesis-related makers, thus alleviating bone destruction in the ankle joint and could be a potential therapeutic target for RA.

SOX9 keeps growth plates and articular cartilage healthy by inhibiting chondrocyte dedifferentiation/osteoblastic redifferentiation.

Cartilage is essential throughout vertebrate life. It starts developing in embryos when osteochondroprogenitor cells commit to chondrogenesis, activate a pancartilaginous program to form cartilaginous skeletal primordia, and also embrace a growth-plate program to drive skeletal growth or an articular program to build permanent joint cartilage. Various forms of cartilage malformation and degeneration diseases afflict humans, but underlying mechanisms are still incompletely understood and treatment options suboptimal. The transcription factor SOX9 is required for embryonic chondrogenesis, but its postnatal roles remain unclear, despite evidence that it is down-regulated in osteoarthritis and heterozygously inactivated in campomelic dysplasia, a severe skeletal dysplasia characterized postnatally by small stature and kyphoscoliosis. Using conditional knockout mice and high-throughput sequencing assays, we show here that SOX9 is required postnatally to prevent growth-plate closure and preosteoarthritic deterioration of articular cartilage. Its deficiency prompts growth-plate chondrocytes at all stages to swiftly reach a terminal/dedifferentiated stage marked by expression of chondrocyte-specific (Mgp) and progenitor-specific (Nt5e and Sox4) genes. Up-regulation of osteogenic genes (Runx2, Sp7, and Postn) and overt osteoblastogenesis quickly ensue. SOX9 deficiency does not perturb the articular program, except in load-bearing regions, where it also provokes chondrocyte-to-osteoblast conversion via a progenitor stage. Pathway analyses support roles for SOX9 in controlling TGFß and BMP signaling activities during this cell lineage transition. Altogether, these findings deepen our current understanding of the cellular and molecular mechanisms that specifically ensure lifelong growth-plate and articular cartilage vigor by identifying osteogenic plasticity of growth-plate and articular chondrocytes and a SOX9-countered chondrocyte dedifferentiation/osteoblast redifferentiation process.

Correlation between Serum IL-35 Levels and Bone Loss in Postmenopausal Women with Rheumatoid Arthritis.

OBJECTIVE: IL-35 was reported as a crucial anti-inflammatory cytokine and could efficiently regulate bone metabolism in murine collagen-induced arthritis model. However, the relationship between IL-35 and bone health in human rheumatoid arthritis (RA) has not been clarified. In this study, the aim was to explore the correlations between IL-35 and bone loss in postmenopausal women with RA. METHODS: The study included 76 postmenopausal women with RA and 53 healthy postmenopausal women as healthy controls (HCs). Serum IL-35 levels were detected by enzyme-linked immunosorbent assay. Bone mineral density (BMD) at lumbar spine 1-4 and at total hip was measured using dual-energy X-ray absorptiometry. Alkaline phosphatase (ALP), ß-isomerised carboxy-terminal cross-linking telopeptide of type I collagen (ß-CTX), and 25-(OH) VitD3 were measured by turbidimetric inhibition immunoassay. RESULTS: Serum IL-35 levels were increased compared with HCs, and it positively correlated with BMD and 25-(OH) VitD3 and negatively correlated with ß-CTX in postmenopausal women with RA. Furthermore, serum IL-35 levels in the increased ALP group were higher than those in the normal ALP group. CONCLUSIONS: IL-35, an important anti-inflammatory cytokine, may participate in the pathogenesis of bone loss in postmenopausal women with RA.

Interleukin-35 inhibits angiogenesis through STAT1 signalling in rheumatoid synoviocytes.

OBJECTIVES: We studied the anti-angiogenic effect of interleukin-35 (IL-35) by investigating its effects on signal transmission through the Janus kinase signal transducer and activator of transcription (JAK-STAT) pathway in fibroblast-like synoviocytes (FLS). METHODS: Using the collagen-induced arthritis (CIA) model of rheumatoid arthritis (RA), we derived and cultured FLS, stimulated FLS with IL-35 at different concentrations and examined the expression levels of mRNA and protein of both vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2), endostatin, TNF-α, and IL-6 using reverse transcription polymerase chain reaction (RT-PCR) and immunoblotting. We used Western blotting to study the effects of IL-35 on the function of the JAK-STAT pathway in FLS. RESULTS: IL-35 treatment inhibited the expression of VEGF, FGF-2, TNF-α and IL-6, and increased the expression of endostatin in FLS. Western blotting showed that IL-35 treatment of CIA-derived FLS resulted in signalling through STAT1, but not through STAT3 or STAT5. CONCLUSIONS: IL-35 signalling through STAT1 and inhibition of the expression of mediators of angiogenesis and inflammation in FLS provide a likely mechanism for anti-angiogenic effects seen in experimental models of RA. Our data suggest that IL-35 and its signalling pathway represent a therapeutic target for the treatment of RA and other angiogenesis-related diseases.

MiR-338-5p Promotes Inflammatory Response of Fibroblast-Like Synoviocytes in Rheumatoid Arthritis via Targeting SPRY1.

Our purpose is to study the roles of microRNA-338-5p (miR-338-5p) on the proliferation, invasion, and inflammatory response of fibroblast-like synoviocytes (SFs) in rheumatoid arthritis patients by regulating SPRY1. The target relationship between miR-338-5p and SPRY1 was validated through luciferase reporter system. The expression of miR-338-5p and SPRY1 in synovial tissues and synovial cells were detected using RT-PCR and western blot. The mimics and inhibitors of miR-338-5p were transfected into SFs. MTT, Transwell, and ELISA assays were used to analyze cell proliferation, invasiveness, and the secreted extracellular pro-inflammatory cytokines (such as IL-1a, IL-6, COX2) levels of SFs. MiR-338-5p was highly expressed in rheumatoid arthritis tissues and cells, and directly down-regulated the expression of SPRY1 in the SFs of rheumatoid arthritis patients. Cell proliferation, invasiveness and the expression level of pro-inflammatory cytokines in synovial cells increased after the transfection of miR-338-5p mimics, while the proliferation, invasion and expression level of pro-inflammatory cytokines decreased after the transfection of miR-338-5p inhibitors. In conclusion,miR-338-5p promoted the proliferation, invasion and inflammatory reaction in SFs of rheumatoid arthritis by directly down-regulating SPRY1 expression. J. Cell. Biochem. 118: 2295-2301, 2017. © 2017 Wiley Periodicals, Inc.

Interleukin-27 inhibits malignant behaviors of pancreatic cancer cells by targeting M2 polarized tumor associated macrophages.

Pancreatic cancer is characterized as inflammatory malignancy with a dismal prognosis. There is abundant intratumoral infiltration of macrophages, and most of these tumor associated macrophages (TAM) are induced to be M2 phenotype. The M2 polarized TAM has been demonstrated to promote progression and induce chemo-resistance of pancreatic cancer. Interleukin (IL)-27 is a novel member of IL-12 cytokine family and its roles in regulation of phenotypes and functions of TAM remain largely unknown. In this study, we demonstrated IL-27 significantly inhibited the M2 macrophages polarization and dampened the proliferation, migration and metastasis of pancreatic cancer cells and as well enhanced the efficacy of gemcitabine. IL-27 could be potential to improve the treatment of pancreatic cancer by targeting M2 polarized TAMs.

Changes in the Expression of Glucose-regulated Protein 78 in the Occurrence and Progression of Pancreatic Cancer in Mouse Models.

OBJECTIVE: To investigate the changes in the expression of glucose-regulated protein 78 (GRP78) in the occurrence and progression of pancreatic cancer in mouse models. METHODS: The mouse models of chronic pancreatitis,pancreatic intraepithelial neoplasia (PanIN), and pancreatic cancer were successfully established by dimethyl benzene and anthracene (DMBA) embedding in situ. GRP78 expression was detected in various stages by immunohistochemistry. RESULTS: Of these 60 mouse models, 18 mice (30%) died during the observation period. Two months after the embedding,the survived mice were sacrificed,and HE staining and IHC staining were performed. Among these mice, 9 (15%) developed chronic pancreatitis; 18 (30%) had PanIN [PanIN1,5 (8.3%);P anIN2,9 (15%); and PanIN 3,4 (6.7%)];15 (25%) developed pancreatic cancer. Immunohistochemistry showed that the expression of GRP78 in pancreatic cancer tissue was significantly higher than that in adjacent noncancerous duct cells (χ(2)=13.39,P =0.000). Also, GRP78 expression in pancreatic cancer tissue and high grade PanIN was significantly higher than that in low grade PanIN and chronic pancreatitis (χ(2)=17.84,P=0.000). CONCLUSION: The expression of GRP78 remarkably differs in different stages of pancreatic cancer and therefore is associated with the occurrence and progression of this disease.

Serum soluble ST2 is associated with ER-positive breast cancer.

BACKGROUND: ST2, a member of the interleukin (IL)-1receptor family, regulates Th1/Th2 immune responses in autoimmune and inflammatory conditions. However, the role of ST2 signaling in tumor growth and metastasis of breast cancers has not been investigated. This study investigated the possible role of soluble ST2 (sST2) in breast cancer. METHODS: The serum levels of IL-33, sST2, and vascular endothelial growth factor (VEGF) in 150 breast cancer patients and 90 healthy women were measured by enzyme-linked immunosorbent assay. Estrogen receptor(ER), progesterone receptor, human epithelial receptor (HER)-2, and cell cycle regulated protein Ki-67 were measured. Clinical stage, tumor size, lymph node metastasis, and histological type were also recorded. RESULTS: The serum levels of sST2, IL-33, and VEGF were significantly higher in breast cancer patients than in the control group (P < 0.05, each). Serum sST2 levels in ER-positive breast cancer patients were significantly associated with age, histological type, clinical stage, tumor size, and Ki-67 status (P < 0.05, each). Moreover, the serum levels of IL-33 and sST2 in breast cancers significantly correlated with VEGF levels (IL-33: r = 0.375, P < 0.0001; sST2: r = 0.164, P = 0.045). Serum levels of sST2, IL-33, and VEGF decreased after modified radical mastectomy in ER-positive breast cancers. Serum levels of IL-33, sST2, and VEGF and clinicopathological factors were not significantly correlated with disease-free survival and overall survival of ER-positive breast cancer women during follow-up. CONCLUSION: Serum sST2 levels in ER-positive breast cancer patients are significantly associated with factors that indicate poor prognosis.

The mediating role of self-efficacy in the relationship between Big five personality and depressive symptoms among Chinese unemployed population: a cross-sectional study.

BACKGROUND: Besides the rapid growth of economy, unemployment becomes a severe socio-economic problem in China. The huge population base in China makes the unemployed population a tremendously huge number. However, health status of unemployed population was ignored and few studies were conducted to describe the depressive symptoms of unemployed individuals in China. This study aims to examine the relationship between Big five personality and depressive symptoms and the mediating role of self-efficacy in this relationship. METHODS: This cross-sectional study was performed during the period of July to September 2011. Questionnaires consisting of the Center for Epidemiologic Studies Depression Scale (CES-D), the Big Five Inventory (BFI) and the General Self-efficacy Scale (GSE), as well as demographic factors, were used to collect information of unemployed population. A total of 1,832 individuals (effective response rate: 73.28%) became our subjects. Hierarchical linear regression analyses were performed to explore the mediating role of self-efficacy. RESULTS: The prevalence of depressive symptoms was 67.7% among Chinese unemployed individuals. After adjusting for demographic characteristics, extraversion, agreeableness and conscientiousness were all negatively associated with depressive symptoms whereas neuroticism was positively associated with depressive symptoms. The proportion of mediating effect of self-efficacy in the relationship between extraversion/agreeableness/conscientiousness/neuroticism and depressive symptoms was 25.42%, 10.91%, 32.21% and 36.44%, respectively. Self-efficacy is a mediator in the relationship between extraversion/agreeableness/conscientiousness/neuroticism and depressive symptoms. CONCLUSION: Self-efficacy partially mediated the relationship between Big five personality and depressive symptoms among Chinese unemployed individuals. Interventions that focus on both individuals' personality and self-efficacy may be most successful to reduce depressive symptoms of unemployed individuals.

Synovium and infrapatellar fat pad share common mesenchymal progenitors and undergo coordinated changes in osteoarthritis.

Osteoarthritis (OA) affects multiple tissues in the knee joint, including the synovium and intra-articular adipose tissue (IAAT) that are attached to each other. However, whether these two tissues share the same progenitor cells and hence function as a single unit in joint homeostasis and diseases is largely unknown. Single-cell transcriptomic profiling of synovium and infrapatellar fat pad (IFP), the largest IAAT, from control and OA mice revealed five mesenchymal clusters and predicted mesenchymal progenitor cells (MPCs) as the common progenitors for other cells: synovial lining fibroblasts (SLFs), myofibroblasts (MFs), and preadipocytes 1 and 2. Histologic examination of joints in reporter mice having Dpp4-CreER and Prg4-CreER that label MPCs and SLFs, respectively, demonstrated that Dpp4+ MPCs reside in the synovial sublining layer and give rise to Prg4+ SLFs and Perilipin+ adipocytes during growth and OA progression. After OA injury, both MPCs and SLFs gave rise to MFs, which remained in the thickened synovium at later stages of OA. In culture, Dpp4+ MPCs possessed mesenchymal progenitor properties, such as proliferation and multilineage differentiation. In contrast, Prg4+ SLFs did not contribute to adipocytes in IFP and Prg4+ cells barely grew in vitro. Taken together, we demonstrate that the synovium and joint fat pad are one integrated functional tissue sharing common mesenchymal progenitors and undergoing coordinated changes during OA progression.

Both synovium and intra-articular adipose tissue (IAAT) in knee joint play a critical role in joint health and osteoarthritis (OA) progression. Recent single-cell RNA-sequencing studies have been performed on the mouse and human synovium. However, IAATs residing in close proximity to the synovium have not been studied yet. Our study reveals mesenchymal cell heterogeneity of synovium/infrapatellar fat pad (Syn/IFP) tissue and their OA responses. We identify Dpp4+ multipotent progenitors as a source that give rise to Prg4+ lining layer fibroblasts in the synovium, adipocytes in the IFP, and myofibroblasts in the OA Syn/IFP tissue. Our work demonstrates that Syn/IFP is a functionally connected tissue that shares common mesenchymal progenitors and undergoes coordinated OA changes. This novel insight advances our knowledge of previously understudied joint tissues and provides new directions for drug discovery to treat joint disorders.

Single-cell atlas of human infrapatellar fat pad and synovium implicates APOE signaling in osteoarthritis pathology.

The infrapatellar fat pad (IPFP) and synovium play essential roles in maintaining knee joint homeostasis and in the progression of osteoarthritis (OA). The cellular and transcriptional mechanisms regulating the function of these specialized tissues under healthy and diseased conditions are largely unknown. Here, single-cell and single-nuclei RNA sequencing of human IPFP and synovial tissues were performed to elucidate the cellular composition and transcriptional profile. Computational trajectory analysis revealed that dipeptidyl peptidase 4+ mesenchymal cells function as a common progenitor for IPFP adipocytes and synovial lining layer fibroblasts, suggesting that IPFP and synovium represent an integrated tissue unit. OA induced a profibrotic and inflammatory phenotype in mesenchymal lineage cells with biglycan+ intermediate fibroblasts as a major contributor to OA fibrosis. Apolipoprotein E (APOE) signaling from intermediate fibroblasts and macrophages was identified as a critical regulatory factor. Ex vivo incubation of human cartilage with soluble APOE accelerated proteoglycan degeneration. Inhibition of APOE signaling by intra-articular injection of an anti-APOE neutralizing antibody attenuated the progression of collagenase-induced OA in mice, demonstrating a detrimental effect of APOE on cartilage. Our studies provide a framework for designing further therapeutic strategies for OA by describing the cellular and transcriptional landscape of human IPFP and synovium in healthy versus OA joints.

Targeting YAP1-regulated Glycolysis in Fibroblast-Like Synoviocytes Impairs Macrophage Infiltration to Ameliorate Diabetic Osteoarthritis Progression.

The interplay between immune cells/macrophages and fibroblast-like synoviocytes (FLSs) plays a pivotal role in initiating synovitis; however, their involvement in metabolic disorders, including diabetic osteoarthritis (DOA), is largely unknown. In this study, single-cell RNA sequencing (scRNA-seq) is employed to investigate the synovial cell composition of DOA. A significant enrichment of activated macrophages within eight distinct synovial cell clusters is found in DOA synovium. Moreover, it is demonstrated that increased glycolysis in FLSs is a key driver for DOA patients' synovial macrophage infiltration and polarization. In addition, the yes-associated protein 1 (YAP1)/thioredoxin-interacting protein (TXNIP) signaling axis is demonstrated to play a crucial role in regulating glucose transporter 1 (GLUT1)-dependent glycolysis in FLSs, thereby controlling the expression of a series of adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) which may subsequently fine-tune the infiltration of M1-polarized synovial macrophages in DOA patients and db/db diabetic OA mice. For treatment, M1 macrophage membrane-camouflaged Verteporfin (Vt)-loaded PLGA nanoparticles (MVPs) are developed to ameliorate DOA progression by regulating the YAP1/TXNIP signaling axis, thus suppressing the synovial glycolysis and the infiltration of M1-polarized macrophages. The results provide several novel insights into the pathogenesis of DOA and offer a promising treatment approach for DOA.

Potential contribution of interleukin-33 to the development of interstitial lung disease in patients with primary Sjogren's Syndrome.

OBJECTIVE: To determine whether interleukin (IL)-33 and soluble ST2 (sST2) are associated with primary Sjogren's Syndrome (pSS). METHODS: Serum levels of IL-33 and sST2 in 110 pSS patients and 78 healthy controls were measured by enzyme-linked immunosorbent assay (ELISA). Immunoglobulins, rheumatoid factors (RF), antinuclear antibody (ANA), anti-SSA/RO-52 antibody, anti-SSB antibody and erythrocyte sedimentation rate (ESR) were measured by standard laboratory techniques. Interstitial lung disease (ILD) was identified on high-resolution computed tomography (HRCT). Disease activity in pSS was scored with the European League Against Rheumatism Sjogren's Syndrome Disease Activity Index (ESSDAI). RESULTS: Serum levels of IL-33 and sST2 were significantly elevated in pSS patients, especially in patients with ILD. There was significant positive correlation between IL-33 and RF, anti-SSB antibody. CONCLUSION: IL-33/sST2 may be involved in the pathogenesis of pSS and partly contribute to the ILD in pSS patients.

Serum levels of soluble ST2 and interleukin-33 in patients with dermatomyositis and polymyositis.

OBJECTIVES: The purpose of this study is to determine whether soluble ST2 (sST2) and interleukin (IL)-33 is involved in dermatomyositis (DM) and polymyositis (PM). METHODS: Serum sST2 and IL-33 levels in 49 DM and 21 PM were detected by enzyme-linked immunosorbent assay (ELISA). Erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), creatine kinase (CK), lactate dehydrogenase (LDH) and antinuclear antibody (ANA), anti-Jo-1 antibody and anti-Mi-2 antibody were tested by standard laboratory techniques. Interstitial lung disease (ILD) was identified on high-resolution computed tomography (HRCT). The visual analogue scale (VAS) of the disease activity, muscle strength, lung functional parameters and other clinical features of DM/PM patients were recorded as well. RESULTS: Sera sST2 levels were significantly higher in DM and PM patients and correlated with CRP, CK, LDH and VAS. The level of serum sST2 decreased after therapy. Conversely, serum levels of IL-33 in patients with PM and DM were not significantly higher than those from HC. CONCLUSIONS: The level of sST2 is elevated in sera of DM and PM patients. sST2 levels were correlated with other markers of disease activity. This data support that sST2 may play a role in DM and PM.

IL-35 inhibits adipogenesis via PPARγ-Wnt/ß-catenin signaling pathway by targeting Axin2.

Interleukin (IL)-35, a member of the IL-12 family, functions as an immunosuppressive cytokine that plays a crucial role in the regulation of immune-related disorders and inflammatory diseases. Adipose tissue, which is now recognized as an immune organ, is regulated by immunocytes through various signaling pathways, including the peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein α (C/EBPα) pathway and the Wnt/ß-actin pathway. However, there is limited research regarding the effects of IL-35 on adipogenesis. Our current findings indicated that IL-35 impedes the proliferation and promotes the cytotoxicity of 3T3-L1 preadipocytes. Furthermore, IL-35 inhibited the adipogenic differentiation, as well as suppressed triglyceride and lipid accumulation. Additionally, the expression of PPARγ and C/EBPα, two key regulators of adipogenesis, were both down-regulated with IL-35 treatment. In order to explicate the mechanisms underlying the effects of IL-35, we conducted an investigation into the expression of Axin2, an intracellular inhibitor of Wnt/ß-catenin signaling, in 3T3-L1 preadipocyte cells. Gene silencing of Axin2 through small interfering RNAs (siRNAs) enhanced PPARγ and C/EBPα expression while decreasing nuclear ß-catenin levels in the presence of IL-35. Furthermore, in IL-35-treated cells, Axin2 knockdown boosted adipogenic differentiation (as measured by increased Oil Red O staining). These findings imply that IL-35 regulates Axin2 expression and thereby plays an important role in adipocyte development.

Palovarotene Action Against Heterotopic Ossification Includes a Reduction of Local Participating Activin A-Expressing Cell Populations.

Heterotopic ossification (HO) consists of extraskeletal bone formation. One form of HO is acquired and instigated by traumas or surgery, and another form is genetic and characterizes fibrodysplasia ossificans progressiva (FOP). Recently, we and others showed that activin A promotes both acquired and genetic HO, and in previous studies we found that the retinoid agonist palovarotene inhibits both HO forms in mice. Here, we asked whether palovarotene's action against HO may include an interference with endogenous activin A expression and/or function. Using a standard mouse model of acquired HO, we found that activin A and its encoding RNA (Inhba) were prominent in chondrogenic cells within developing HO masses in untreated mice. Single-cell RNAseq (scRNAseq) assays verified that Inhba expression characterized chondroprogenitors and chondrocytes in untreated HO, in addition to its expected expression in inflammatory cells and macrophages. Palovarotene administration (4 mg/kg/d/gavage) caused a sharp inhibition of both HO and amounts of activin A and Inhba transcripts. Bioinformatic analyses of scRNAseq data sets indicated that the drug had reduced interactions and cross-talk among local cell populations. To determine if palovarotene inhibited Inhba expression directly, we assayed primary chondrocyte cultures. Drug treatment inhibited their cartilaginous phenotype but not Inhba expression. Our data reveal that palovarotene markedly reduces the number of local Inhba-expressing HO-forming cell populations. The data broaden the spectrum of HO culprits against which palovarotene acts, accounting for its therapeutic effectiveness. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Osteoblast-intrinsic defect in glucose metabolism impairs bone formation in type II diabetic mice.

Skeletal fragility is associated with type 2 diabetes mellitus (T2D), but the underlying mechanism is not well understood. Here, in a mouse model for youth-onset T2D, we show that both trabecular and cortical bone mass are reduced due to diminished osteoblast activity. Stable isotope tracing in vivo with 13 C-glucose demonstrates that both glycolysis and glucose fueling of the TCA cycle are impaired in diabetic bones. Similarly, Seahorse assays show suppression of both glycolysis and oxidative phosphorylation by diabetes in bone marrow mesenchymal cells as a whole, whereas single-cell RNA sequencing reveals distinct modes of metabolic dysregulation among the subpopulations. Metformin not only promotes glycolysis and osteoblast differentiation in vitro, but also improves bone mass in diabetic mice. Finally, targeted overexpression of Hif1a or Pfkfb3 in osteoblasts of T2D mice averts bone loss. The study identifies osteoblast-intrinsic defects in glucose metabolism as an underlying cause of diabetic osteopenia, which may be targeted therapeutically.

Tnfa , Il6 , Cxcl1 , and Adam8 Genes Are the Early Markers After Mouse Tail Intervertebral Disc Injury.

OBJECTIVES: The early molecular events after intervertebral disc injury remain unclear. In this study, we aimed to compare inflammatory markers from 1 day to 4 wks after injury to have a comprehensive understanding of the intervertebral disc response to injury. DESIGN: Mouse tail intervertebral disc injury was induced by a needle puncture. Inflammatory marker gene expression and morphological changes were recorded at 1 day, 1 wk, and 4 wks after injury. RESULTS: Tnfa , Il6 , and Cxcl1 gene expression peaked at day 1 post-needle puncture of the mouse intervertebral disc, Adam8 gene expression peaked at 1-wk time point, while Tipe2 gene expression was upregulated at week 4 postinjury. F4/80 positive cells, likely to be macrophages, are present as early as day 1 in the injured intervertebral discs and consistently present at week 4 postinjury. Loss of Safranin O staining and increased histological scores of the injured intervertebral discs are consistent with progressive degeneration after injury. CONCLUSIONS: Inflammatory cytokines including Tnfa precede Tipe2 , suggesting that Tipe2 is likely induced by Tnfa . Upregulation of Adam8 and Cxcl1 gene expression persisted at week 4, suggesting that they play a role in the transition to chronic phase of intervertebral disc degeneration.

Osteoblast-intrinsic defect in glucose metabolism impairs bone formation in type II diabetic male mice.

Skeletal fragility is associated with type 2 diabetes mellitus (T2D), but the underlying mechanism is not well understood. Here, in a mouse model for youth-onset T2D, we show that both trabecular and cortical bone mass is reduced due to diminished osteoblast activity. Stable isotope tracing in vivo with 13C-glucose demonstrates that both glycolysis and glucose fueling of the TCA cycle are impaired in diabetic bones. Similarly, Seahorse assays show suppression of both glycolysis and oxidative phosphorylation by diabetes in bone marrow mesenchymal cells as a whole, whereas single-cell RNA sequencing reveals distinct modes of metabolic dysregulation among the subpopulations. Metformin not only promotes glycolysis and osteoblast differentiation in vitro, but also improves bone mass in diabetic mice. Finally, osteoblast-specific overexpression of either Hif1a, a general inducer of glycolysis, or Pfkfb3 which stimulates a specific step in glycolysis, averts bone loss in T2D mice. The study identifies osteoblast-intrinsic defects in glucose metabolism as an underlying cause of diabetic osteopenia, which may be targeted therapeutically.