Phosphate‐Dependent Regulation of MGP in Osteoblasts: Role of ERK1/2 and Fra‐1

Inorganic phosphate (Pi) and the matrix Gla protein (MGP) are key regulators of bone formation. We have recently shown that Pi upregulates MGP in growth plate chondrocytes, which may represent a negative feedback loop for the control of mineralization. Osteoblasts from Fra‐1‐deleted mice express low levels of MGP, whereas the expression of MGP is elevated in Fra‐1 transgenic osteoblasts, suggesting a role for Fra‐1 in MGP expression and bone formation. In this study, we aimed at deciphering the relationships between Pi and MGP in osteoblasts to determine the molecular mechanisms involved in the Pi‐dependent regulation of MGP. In MC3T3‐E1 cells and primary calvaria‐derived osteoblasts, Pi increased MGP and Fra‐1 expression at both the mRNA and protein levels. We also found that Pi enhanced the phosphorylation of ERK1/2. U0126 (MEK1/2 inhibitor) suppressed Pi‐stimulated MGP and Fra‐1 expression, indicating that ERK1/2 is required for Pi‐dependent regulation of MGP and Fra‐1. In addition, using in vitro DNA binding and chromatin immunoprecipitation assays, we showed that Fra‐1 interacts with the MGP promoter in response to Pi in MC3T3‐E1 cells. Finally, we found that in fra‐1 knockdown MC3T3‐E1 osteoblasts, the level of MGP expression is no more significantly upregulated by Pi. We further showed that primary osteoblasts from Fra‐1‐deficient mice failed to exhibit a Pi‐dependent stimulation of MGP expression. These data show, for the first time, that Pi regulates MGP expression in osteoblasts through the ERK1/2‐Fra‐1 pathway.     

Growth differentiation factor-15: induction in liver injury through p53 and tumor necrosis factor-independent mechanisms

Expression of macrophage inhibitory cytokine-1 (MIC-1), a divergent transforming growth factor-beta family member, and its murine ortholog, growth/differentiation factor-15 (GDF-15), is induced in hepatocytes by surgical and chemical injury and heat shock. Here, we demonstrate that the regulation of GDF-15/MIC-1 expression may be evolutionarily conserved because MIC-1 was induced in diseased human livers. Gdf15 induction was independent of protein synthesis, a hallmark of immediate-early gene regulation. Although tumor necrosis factor (TNF) induced GDF-15 expression, injury-elicited Gdf15 expression was not reduced in mice deficient for both TNF receptor subtypes. Furthermore, although the stress sensor p53 is known to induce GDF-15/MIC-1 expression, injury-elicited Gdf15 expression was unchanged in p53 null mice. Our results demonstrate that GDF-15 induction is an immediate early response to liver injury that can occur through TNF and p53 independent pathways.     

Cartilage‐Specific Autophagy Deficiency Promotes ER Stress and Impairs Chondrogenesis in PERK‐ATF4‐CHOP–Dependent Manner

Autophagy is activated during nutritionally depleted or hypoxic conditions to facilitate cell survival. Because growth plate is an avascular and hypoxic tissue, autophagy may have a crucial role during chondrogenesis; however, the functional role and underlying mechanism of autophagy in regulation of growth plate remains elusive. In this study, we generated ^TamCartAtg7^–/– (Atg7cKO) mice to explore the role of autophagy during endochondral ossification. Atg7cKO mice exhibited growth retardation associated with reduced chondrocyte proliferation and differentiation, and increased chondrocyte apoptosis. Meanwhile, we observed that Atg7 ablation mainly induced the PERK‐ATF4‐CHOP axis of the endoplasmic reticulum (ER) stress response in growth plate chondrocytes. Although Atg7 ablation induced ER stress in growth plate chondrocytes, the addition of phenylbutyric acid (PBA), a chemical chaperone known to attenuate ER stress, partly neutralized such effects of Atg7 ablation on longitudinal bone growth, indicating the causative interaction between autophagy and ER stress in growth plate. Consistent with these findings in vivo, we also observed that Atg7 ablation in cultured chondrocytes resulted in defective autophagy, elevated ER stress, decreased chondrocytes proliferation, impaired expression of col10a1, MMP­13, and VEGFA for chondrocyte differentiation, and increased chondrocyte apoptosis, while such effects were partly nullified by reduction of ER stress with PBA. In addition, Atg7 ablation‐mediated impaired chondrocyte function (chondrocyte proliferation, differentiation, and apoptosis) was partly reversed in CHOP^–/– cells, indicating the causative role of the PERK‐ATF4‐CHOP axis of the ER stress response in the action of autophagy deficiency in chondrocytes. In conclusion, our findings indicate that autophagy deficiency may trigger ER stress in growth plate chondrocytes and contribute to growth retardation, thus implicating autophagy as an important regulator during chondrogenesis and providing new insights into the clinical potential of autophagy in cartilage homeostasis. © 2017 American Society for Bone and Mineral Research.     

SDF‐1/CXCR4 Axis in Tie2‐Lineage Cells Including Endothelial Progenitor Cells Contributes to Bone Fracture Healing

CXC chemokine receptor 4 (CXCR4) is a specific receptor for stromal‐derived‐factor 1 (SDF‐1). SDF‐1/CXCR4 interaction is reported to play an important role in vascular development. On the other hand, the therapeutic potential of endothelial progenitor cells (EPCs) in fracture healing has been demonstrated with mechanistic insight of vasculogenesis/angiogenesis and osteogenesis enhancement at sites of fracture. The purpose of this study was to investigate the influence of the SDF‐1/CXCR4 pathway in Tie2‐lineage cells (including EPCs) in bone formation. We created CXCR4 gene conditional knockout mice using the Cre/loxP system and set two groups of mice: Tie2‐Cre^ER CXCR4 knockout mice (CXCR4^?/?) and wild‐type mice (WT). We report here that in vitro, EPCs derived from of CXCR4^?/? mouse bone marrow demonstrated severe reduction of migration activity and EPC colony‐forming activity when compared with those derived from WT mouse bone marrow. In vivo, radiological and morphological examinations showed fracture healing delayed in the CXCR4^?/? group and the relative callus area at weeks 2 and 3 was significantly smaller in CXCR4^?/? group mice. Quantitative analysis of capillary density at perifracture sites also showed a significant decrease in the CXCR4^?/? group. Especially, CXCR4^?/?group mice demonstrated significant early reduction of blood flow recovery at fracture sites compared with the WT group in laser Doppler perfusion imaging analysis. Real‐time RT‐PCR analysis showed that the gene expressions of angiogenic markers (CD31, VE‐cadherin, vascular endothelial growth factor [VEGF]) and osteogenic markers (osteocalcin, collagen 1A1, bone morphogenetic protein 2 [BMP2]) were lower in the CXCR4^?/? group. In the gain‐of‐function study, the fracture in the SDF‐1 intraperitoneally injected WT group healed significantly faster with enough callus formation compared with the SDF‐1 injected CXCR4^?/? group. We demonstrated that an EPC SDF‐1/CXCR4 axis plays an important role in bone fracture healing using Tie2‐Cre^ER CXCR4 conditional knockout mice. © 2014 American Society for Bone and Mineral Research.     

Inflammatory Cytokines Locally Elevated in Chronic Subdural Haematoma

Summary The involvement of inflammation in the development and propagation of chronic subdural haematoma (CSH) was investigated by measuring the levels of inflammatory cytokines (tumour necrosis factor [TNF]α, interleukin [IL]-1β, IL-6, and IL-8). Peripheral venous blood and subdural fluid were obtained at the time of burr hole surgery from 34 patients with CSH and from 9 with subdural effusion. The levels of the inflammatory cytokines were analysed by enzyme-linked immunosorbent assay. The blood levels of TNFα, IL-1β, IL-6, and IL-8 in both CSH and subdural effusion groups were almost within the range of normal subjects, and no differences were observed between the two groups. IL-6 and IL-8 in the subdural fluid were much higher than in the blood of both groups, and the levels in CSH patients were significantly higher (10 times) than in subdural effusion patients. Local elevation of inflammatory cytokines in the subdural space of both CSH and subdural effusion without systemic change suggests the presence of local inflammation in the two diseases. The same behavioural patterns of cytokines for these and higher levels of cytokines in the CSH also suggest that inflammatory cytokines may be involved in the continuous development from subdural effusion to CSH and propagation of CSH.     

Novel in vitro model for studying ureteric stent‐induced cell injury

OBJECTIVE

To develop a novel in vitro model for the study of bladder and kidney epithelial cell injury akin to stent movement, as ureteric stents are associated with urinary tract complications that can significantly add to patient morbidity. These sequelae may be linked to inflammation triggered by stent‐mediated mechanical injury to the urinary tract.

MATERIALS AND METHODS

T24 bladder and A498 kidney cell line monolayers were damaged mechanically by segments of either Percuflex Plus® (PP) or Triumph® (triclosan‐eluting) stents (both from Boston Scientific Corporation Inc. Natick, MA, USA) and the resulting expression profiles of several pro‐inflammatory cytokines and growth factors were analysed.

RESULTS

After control injury using the PP stent, supernatants of both cell lines had significantly increased levels of interleukin (IL)‐6, IL‐8, basic fibroblast growth factor and platelet‐derived growth factor BB, and A498 cells also had increased tumour necrosis factor α. In almost all cases, the presence of triclosan within the media abrogated the pro‐inflammatory cytokine increases, while its effects on growth factors varied.

CONCLUSION

This study suggests that stent‐related symptoms in the bladder and kidney may be partially due to a local inflammatory response to epithelial damage caused by the presence and movement of the stent. Future stent design should take these inflammatory responses, with respect to physical injury, into consideration, using either more biocompatible materials or anti‐inflammatory compounds such as triclosan.     

Loss of Gi G‐Protein‐Coupled Receptor Signaling in Osteoblasts Accelerates Bone Fracture Healing

G‐protein‐coupled receptors (GPCRs) are key regulators of skeletal homeostasis and are likely important in fracture healing. Because GPCRs can activate multiple signaling pathways simultaneously, we used targeted disruption of G_i‐GPCR or activation of G_s‐GPCR pathways to test how each pathway functions in the skeleton. We previously demonstrated that blockade of G_i signaling by pertussis toxin (PTX) transgene expression in maturing osteoblastic cells enhanced cortical and trabecular bone formation and prevented age‐related bone loss in female mice. In addition, activation of G_s signaling by expressing the G_s‐coupled engineered receptor Rs1 in maturing osteoblastic cells induced massive trabecular bone formation but cortical bone loss. Here, we test our hypothesis that the G_i and G_s pathways also have distinct functions in fracture repair. We applied closed, nonstabilized tibial fractures to mice in which endogenous G_i signaling was inhibited by PTX, or to mice with activated G_s signaling mediated by Rs1. Blockade of endogenous G_i resulted in a smaller callus but increased bone formation in both young and old mice. PTX treatment decreased expression of Dkk1 and increased Lef1 mRNAs during fracture healing, suggesting a role for endogenous G_i signaling in maintaining Dkk1 expression and suppressing Wnt signaling. In contrast, adult mice with activated G_s signaling showed a slight increase in the initial callus size with increased callus bone formation. These results show that G_i blockade and G_s activation of the same osteoblastic lineage cell can induce different biological responses during fracture healing. Our findings also show that manipulating the GPCR/cAMP signaling pathway by selective timing of G_s and G_i‐GPCR activation may be important for optimizing fracture repair. © 2015 American Society for Bone and Mineral Research.     

B‐Cell Infiltrates Induce Endosteal Bone Formation in Inflammatory Arthritis

The objective of this study was to investigate the function of inflammatory bone marrow infiltrates found in vicinity to joints affected by inflammatory arthritis. These bone marrow infiltrates are rich in B cells and emerge at the interphase between bone marrow and synovial inflammatory tissue, where cortical bone has been broken. We deleted an essential molecule of B‐cell development, Brutońans tyrosine kinase (Btk), in arthritic TNF‐transgenic mice and studied its effect on bone marrow inflammation. Although antigen responses, immunoglobulin levels, and autoantibody production were diminished in Btk^?/?hTNFtg mice, synovial inflammation developed normally. However, bone marrow infiltrates were significantly diminished in Btk^?/?hTNFtg mice, which lead to impaired bone formation at endosteal sites underneath bone erosions and an increased invasion of synovial inflammatory cells into the bone marrow. Expression of bone morphogenic protein‐7 was dramatically decreased in Btk^?/?hTNFtg mice. These results do not only indicate that bone formation at endosteal regions next to bone marrow infiltrates is driven by B cells but also show that bone marrow aggregates in the vicinity of inflamed joint appear as an attempt to counter the invasion of inflammatory tissue into the bone marrow.     

Reduced COX‐2 Expression in Aged Mice Is Associated With Impaired Fracture Healing

The cellular and molecular events responsible for reduced fracture healing with aging are unknown. Cyclooxygenase 2 (COX‐2), the inducible regulator of prostaglandin E₂ (PGE₂) synthesis, is critical for normal bone repair. A femoral fracture repair model was used in mice at either 7–9 or 52–56 wk of age, and healing was evaluated by imaging, histology, and gene expression studies. Aging was associated with a decreased rate of chondrogenesis, decreased bone formation, reduced callus vascularization, delayed remodeling, and altered expression of genes involved in repair and remodeling. COX‐2 expression in young mice peaked at 5 days, coinciding with the transition of mesenchymal progenitors to cartilage and the onset of expression of early cartilage markers. In situ hybridization and immunohistochemistry showed that COX‐2 is expressed primarily in early cartilage precursors that co‐express col‐2. COX‐2 expression was reduced by 75% and 65% in fractures from aged mice compared with young mice on days 5 and 7, respectively. Local administration of an EP4 agonist to the fracture repair site in aged mice enhanced the rate of chondrogenesis and bone formation to levels observed in young mice, suggesting that the expression of COX‐2 during the early inflammatory phase of repair regulates critical subsequent events including chondrogenesis, bone formation, and remodeling. The findings suggest that COX‐2/EP4 agonists may compensate for deficient molecular signals that result in the reduced fracture healing associated with aging.     

Osteoblast‐Derived TGF‐β1 Stimulates IL‐8 Release Through AP‐1 and NF‐κB in Human Cancer Cells

Introduction: The bone marrow microenvironment is further enriched by growth factors released during osteoclastic bone resorption. It has been reported that the chemokine interleukin (IL)‐8 is a potent and direct activator of osteoclastic differentiation and bone resorption. However, the effect of bone‐derived growth factors on the IL‐8 production in human cancer cells and the promotion of osteoclastogenesis are largely unknown. The aim of this study was to investigate whether osteoblast‐derived TGF‐β1 is associated with osteolytic bone diseases. Materials and Methods: IL‐8 mRNA levels were measured using RT‐PCR analysis. MAPK phosphorylation was examined using the Western blot method. siRNA was used to inhibit the expression of TGF‐β1, BMP‐2, and IGF‐1. DNA affinity protein‐binding assay and chromatin immunoprecipitation assays were used to study in vitro and in vivo binding of c‐fos, c‐jun, p65, and p50 to the IL‐8 promoter. A transient transfection protocol was used to examine IL‐8, NF‐κB, and activator protein (AP)‐1 activity. Results: Osteoblast conditioned medium (OBCM) induced activation of IL‐8, AP‐1, and NF‐κB promoter in human cancer cells. Osteoblasts were transfected with TGF‐β1, BMP‐2, or IGF‐1 small interfering RNA, and the medium was collected after 48 h. TGF‐β1 but not BMP‐2 or IGF‐1 siRNA inhibited OBCM‐induced IL‐8 release in human cancer cells. In addition, TGF‐β1 also directly induced IL‐8 release in human cancer cells. Activation of AP‐1 and NF‐κB DNA‐protein binding and MAPKs after TGF‐β1 treatment was shown, and TGF‐β1–induced IL‐8 promoter activity was inhibited by the specific inhibitors of MAPK cascades. Conclusions: In this study, we provide evidence to show that the osteoblasts release growth factors, including TGF‐β1, BMP‐2, and IGF‐1. TGF‐β1 is the major contributor to the activation of extracellular signal‐related kinase (ERK), p38, and c‐Jun N‐terminal kinase (JNK), leading to the activation of AP‐1 and NF‐κB on the IL‐8 promoter and initiation of IL‐8 mRNA and protein release, thereby promoting osteoclastogenesis.     

Inhibition of miR‐92a Enhances Fracture Healing via Promoting Angiogenesis in a Model of Stabilized Fracture in Young Mice

MicroRNAs (miRNAs) are endogenous small noncoding RNAs regulating the activities of target mRNAs and cellular processes. Although no miRNA has been reported to play an important role in the regulation of fracture healing, several miRNAs control key elements in tissue repair processes such as inflammation, hypoxia response, angiogenesis, stem cell differentiation, osteogenesis, and chondrogenesis. We compared the plasma concentrations of 134 miRNAs in 4 patients with trochanteric fractures and 4 healthy controls (HCs), and the levels of six miRNAs were dysregulated. Among these miRNAs, miR‐92a levels were significantly decreased 24 hours after fracture, compared to HCs. In patients with a trochanteric fracture or a lumbar compression fracture, the plasma concentrations of miR‐92a were lower on days 7 and 14, but had recovered on day 21 after the surgery or injury. To determine whether systemic downregulation of miR‐92a can modulate fracture healing, we administered antimir‐92a, designed using locked nucleic acid technology to inhibit miR‐92a, to mice with a femoral fracture. Systemic administration of antimir‐92a twice a week increased the callus volume and enhanced fracture healing. Enhancement of fracture healing was also observed after local administration of antimir‐92a. Neovascularization was increased in mice treated with antimir‐92a. These results suggest that plasma miR‐92a plays a crucial role in bone fracture healing in human and that inhibition of miR‐92a enhances fracture healing through angiogenesis and has therapeutic potential for bone repair. © 2014 American Society for Bone and Mineral Research.     

Analysis of αSMA‐Labeled Progenitor Cell Commitment Identifies Notch Signaling as an Important Pathway in Fracture Healing

Fracture healing is a regenerative process that involves coordinated responses of many cell types, but characterization of the roles of specific cell populations in this process has been limited. We have identified alpha smooth muscle actin (αSMA) as a marker of a population of mesenchymal progenitor cells in the periosteum that contributes to osteochondral elements during fracture healing. Using a lineage tracing approach, we labeled αSMA‐expressing cells, and characterized changes in the periosteal population during the early stages of fracture healing by histology, flow cytometry, and gene expression profiling. In response to fracture, the αSMA‐labeled population expanded and began to differentiate toward the osteogenic and chondrogenic lineages. The frequency of mesenchymal progenitor cell markers such as Sca1 and PDGFRα increased after fracture. By 6 days after fracture, genes involved in matrix production and remodeling were elevated. In contrast, genes associated with muscle contraction and Notch signaling were downregulated after fracture. We confirmed that activating Notch signaling in αSMA‐labeled cells inhibited differentiation into osteogenic and adipogenic lineages in vitro and ectopic bone formation in vivo. By characterizing changes in a selected αSMA‐labeled progenitor cell population during fracture callus formation, we have shown that modulation of Notch signaling may determine osteogenic potential of αSMA‐expressing progenitor cells during bone healing. © 2014 American Society for Bone and Mineral Research.