The interleukin (IL)-4, 1,25(OH)2D3 and retinoic acid, increase surface expression of functional integrin αvβ3 on murine osteoclast precursors. All three agonists stimulate transcription of the β3 ...gene, leading to increased steady-state levels of mRNA this protein. By contrast, mRNA levels of αv remain unchanged. In each instance, the increase in the surface expression of the integrin results in increased migration of the cells onto an αvβ3 substrate. Because β3 subunit, except platelet where β3 subunit conform a dimer with αIIb, associates solely with αv subunit monogamously, while promiscuous αv subunit combines with various subunit, our present data support the idea that the β3 subunit governs the surface-expressed functional integrin complex.
BACKGROUNDRegeneration of injured tissue is dependent on stem/progenitor cells, which can undergo proliferation and maturation processes to replace the lost cells and extracellular matrix (ECM). Bone ...has a higher regenerative capacity than other tissues, with abundant mesenchymal progenitor cells in the bone marrow, periosteum, and surrounding muscle. However, the treatment of bone fractures is not always successful; a marked number of clinical case reports have described nonunion or delayed healing for various reasons. Supplementation of exogenous stem cells by stem cell therapy is anticipated to improve treatment outcomes; however, there are several drawbacks including the need for special devices for the expansion of stem cells outside the body, low rate of cell viability in the body after transplantation, and oncological complications. The use of endogenous stem/progenitor cells, instead of exogenous cells, would be a possible solution, but it is unclear how these cells migrate towards the injury site.METHODSThe chemoattractant capacity of the elastin microfibril interface located protein 2 (Emilin2), generated by macrophages, was identified by the migration assay and LC-MS/MS. The functions of Emilin2 in bone regeneration were further studied using Emilin2-/- mice.RESULTSThe results show that in response to bone injury, there was an increase in Emilin2, an ECM protein. Produced by macrophages, Emilin2 exhibited chemoattractant properties towards mesenchymal cells. Emilin2-/- mice underwent delayed bone regeneration, with a decrease in mesenchymal cells after injury. Local administration of recombinant Emilin2 protein enhanced bone regeneration.CONCLUSIONEmilin2 plays a crucial role in bone regeneration by increasing mesenchymal cells. Therefore, Emilin2 can be used for the treatment of bone fracture by recruiting endogenous progenitor cells.
Macrophages are progenitors of osteoclasts as well as regulators of bone metabolism. Macrophages mediate not only bone formation by osteoblasts under physiological conditions, but also bone ...regeneration after fracture. The mechanisms of macrophages regulation of bone formation and regeneration remain unclear, however. Here, we demonstrate that the liposome-encapsulated Clodronate (Clod-lip) injected mouse model with cortical bone defect induced by drill-hole injury and targeted depletion of phagocytic macrophages exhibits impaired angiogenesis of type H vessels that couple angiogenesis and osteogenesis. Moreover, we identify Tgfbi (encoding TGFBI), Plau (encoding uPA) and Tgfb1 (encoding TGF-β1), through RNA-seq analysis, as genes of macrophage-secreted factors mediating angiogenesis and wound healing. The relevant mRNA was highly expressed in bone marrow-derived macrophages among bone cells, as determined through qRT-PCR. Finally, we disclose that treatment with uPA inhibitor or TGF-β receptor I, receptor II inhibitor impairs bone regeneration after injury, confirming the importance of uPA and TGF-β1 during bone regeneration. Our findings reveal a novel mechanism of bone regeneration mediated by macrophages.
•Clod-lip injection impairs type H vessel formation during bone regeneration.•Plau, Tgfb1 and Tgfbi are identified as macrophage-derived factors by RNA-seq.•uPA or TGF-β inhibitors injection impairs bone regeneration.
Osteoclasts are multinucleated cells with bone resorption activity. Excessive osteoclast activity has been implicated in osteoporosis, rheumatoid arthritis, and bone destruction due to bone ...metastases from cancer, making osteoclasts essential target cells in bone and joint diseases. C-terminal domain nuclear envelope phosphatase 1 (Ctdnep1, formerly Dullard) is a negative regulator of transforming growth factor (TGF)-β superfamily signaling and regulates endochondral ossification in mesenchymal cells during skeletal development. In this study, we investigated the role of Ctdnep1 in the Receptor activator of nuclear factor-kappa B ligand (RANKL)-induced RAW264.7 osteoclast differentiation. Expression of Ctdnep1 did not change during osteoclast differentiation; Ctdnep1 protein localized to the cytoplasm before and after osteoclast differentiation. Small interfering RNA-mediated knockdown of Ctdnep1 increased tartrate-resistant acid phosphatase-positive multinucleated osteoclasts and the expression of osteoclast marker genes, including Acp5, Ctsk, and Nfatc1. Interestingly, the knockdown of Ctdnep1 increased the protein level of Nfatc1 in cells unstimulated with RANKL. Knockdown of Ctdnep1 also enhanced calcium-resorbing activity. Mechanistically, the knockdown of Ctdnep1 increased the phosphorylation of RANKL signaling components. These results suggest that Ctdnep1 negatively regulates osteoclast differentiation by suppressing the RANKL signaling pathway.
•Ctdnep1 knockdown results in a significant increase in osteoclast differentiation.•Ctdnep1 knockdown enhances the calcium-resorbing activity of osteoclasts.•Ctdnep1 knockdown increases Nfatc1protein levels.•Ctdnep1knockdown enhances the RANKL signaling pathway.
Teriparatide is a peptide derived from a parathyroid hormone (PTH) and an osteoporosis therapeutic drug with potent bone formation-promoting activity. To identify novel druggable genes that act ...downstream of PTH signaling and are potentially involved in bone formation, we screened PTH target genes in mouse osteoblast-like MC3T3-E1 cells. Here we show that Gprc5a, encoding an orphan G protein-coupled receptor, is a novel PTH-inducible gene and negatively regulates osteoblast proliferation and differentiation. PTH treatment induced Gprc5a expression in MC3T3-E1 cells, rat osteosarcoma ROS17/2.8 cells, and mouse femurs. Induction of Gprc5a expression by PTH occurred in the absence of protein synthesis and was mediated primarily via the cAMP pathway, suggesting that Gprc5a is a direct target of PTH signaling. Interestingly, Gprc5a expression was induced additively by co-treatment with PTH and 1α, 25-dihydroxyvitamin D3 (calcitriol), or retinoic acid in MC3T3-E1 cells. Reporter analysis of a 1 kb fragment of human GPRC5A promoter revealed that the promoter fragment showed responsiveness to PTH via the cAMP response element, suggesting that GPRC5A is also a PTH-inducible gene in humans. Gprc5a knockdown promoted cell viability and proliferation, as demonstrated by MTT and BrdU assays. Gprc5a knockdown also promoted osteoblast differentiation, as indicated by gene expression analysis and mineralization assay. Mechanistic studies showed that Gprc5a interacted with BMPR1A and suppressed BMP signaling induced by BMP-2 and constitutively active BMP receptors, ALK2 (ACVR1) Q207D and ALK3 (BMPR1A) Q233D. Thus, our results suggest that Gprc5a is a novel gene induced by PTH that acts in an inhibitory manner on both cell proliferation and osteoblast differentiation and is a candidate for drug targets for osteoporosis.
In the groove of Ranvier (GOR), osteoblast lineages form bone bark, which develops into endosteal cortical bone. This ossification process is thought to be regulated by the microenvironment in the ...GOR. Type VI collagen (Col VI), an extracellular matrix (ECM) protein found in the periosteum/perichondrium, mediates osteoblast differentiation via the cell-surface receptor neural/glial antigen 2 (NG2) chondroitin sulfate proteoglycan. In order to clarify the function of Col VI during osteoblast differentiation in the GOR, in the present study, we examined the distribution of Col VI and osteoblast lineages expressing NG2 in the rat tibia proximal end during postnatal growing periods by immunohistochemistry. Our data revealed that Col VI accumulated in the ECM of the GOR middle layer and that Col VI accumulation was reduced and disappeared in the inner and middle lower regions. Runt-related transcription factor 2-immunoreactive pre-osteoblasts expressed NG2 in Col VI-immunopositive areas. However, Osterix-immunoreactive mature osteoblasts were only found in the Col VI-immunonegative area. These findings indicate that Col VI provided a characteristic microenvironment in the GOR and that NG2-Col VI interactions may regulate the differentiation of osteoblast lineages prior to terminal maturation.
Conjunctival epithelial and goblet cell P2Y2 nucleotide receptors regulate ion transport and secretory function. Diquafosol is a P2Y2 purinergic receptor agonist that stimulates secretion of aqueous ...tear components from conjunctival epithelial cells and secretion of mucin from conjunctival goblet cells. In humans suffering from keratoconjunctivitis sicca (dry eye), topical administration of diquafosol improves corneal epithelial integrity and stabilises the tear film. The aim of the present study was to investigate P2Y2 receptor expression and to determine the effect of topical administration of diquafosol on mucin and aqueous tear production in dogs.
Canine conjunctival P2Y2 receptor expression was evaluated by Western blotting and immunohistochemical analysis. The effect of diquafosol on mucin secretion was evaluated by examining mucin-5 subtype AC (MUC5AC) concentration in tears. The effect of diquafosol on aqueous secretions was evaluated by performing the Schirmer tear test (STT) and phenol red thread test. Expression of the P2Y2 receptor was confirmed in canine bulbar and palpebral conjunctivae and receptors were identified at the conjunctival epithelial and goblet cell surface. Tear MUC5AC concentration significantly increased after administration of 3% diquafosol ophthalmic solution, although neither STT nor phenol red thread test values showed any significant change after diquafosol instillation. Topical ocular administration of 3% diquafosol might improve corneal epithelial disorders in dogs through stabilisation of the tear film, by virtue of an increase in MUC5AC secretion.
A 37-year-old woman with systemic lupus erythematosus presented with gait disturbance and cognitive dysfunction. Brain magnetic resonance imaging (MRI) revealed small, punctate, T2-/fluid-attenuated ...inversion recovery-hyperintense and T1-hypointense lesions without gadolinium enhancement, which is atypical for progressive multifocal leukoencephalopathy (PML). On a pathological examination of biopsied brain tissues, JC virus-infected cells were hardly detected via immunohistochemistry but were certainly detected via in situ hybridization, conclusively verifying the PML diagnosis. After tapering off the immunosuppressant and mefloquine administration, the MRI findings revealed gradual improvement, and she has been stable for over 18 months. A punctate MRI pattern is not specific to natalizumab-associated PML but may be a ubiquitous early sign useful for the early diagnosis of PML.
Muscle mass might be a possible predictor for walking function in patients with stroke; however, evidence is limited.
To investigate whether skeletal muscle mass is associated with walking function ...at discharge during the acute phase.
In this observational cohort study, we assessed skeletal muscle mass in patients with acute ischemic stroke using the noninvasive and portable multifrequency bio-impedance device. This device can easily be used in bedridden patients. Appendicular skeletal muscle mass was converted to skeletal muscle index (SMI) standardizing by height squared (kg/m2). The primary outcome was walking function assessed by the modified Rankin Scale score at acute phase hospital discharge. Logistic regression analysis was used to determine the association between skeletal muscle mass and walking function.
Of the 107 patients enrolled, low SMI (SMI: male <7.0 kg/m2, female <5.7 kg/m2) was identified in 29.9% (19.7% in men, 48.6% in women). Logistic regression analysis showed that low SMI OR: 4.02, 95% confidence interval (CI): 1.38-11.7, p = 0.001 independently associated with walking function at discharge. Further, patients with mild and moderate severity had significant difficulty in walking when they had low SMI (p = 0.039).
Low skeletal muscle mass at the onset of ischemic stroke is an independent predictor of walking function at discharge during the acute phase. Our findings highlight the importance of detecting skeletal muscle mass in patients with acute ischemic stroke.
Cerebral air embolism is a rare cause of cerebral infarction. In cerebral air embolism, T2 star-weighted imaging shows numerous spotty hypointense signals. Previous reports have suggested that these ...signals represent air in the brain and are gradually diminished and absorbed. We experienced two cases of cerebral air embolism, and in one of them, we conducted an autopsy.
Case 1 was a 76-year-old Japanese man with lung cancer and emphysema. A spasmodic cough induced massive cerebral and cardiac air embolisms and the patient died because of cerebral herniation. T2 star-weighted imaging of brain magnetic resonance imaging showed multiple spotty low signals. Brain autopsy showed numerous spotty hemorrhagic infarcts in the area of T2 star-weighted imaging signals. Case 2 was an 85-year-old Japanese man with emphysema who suffered from acute stroke. Similar spotty T2 star-weighted imaging signals were observed and remained unchanged 2 months after the onset.
These findings indicate that T2 star-weighted imaging in cerebral air embolism partially represents micro-hemorrhagic infarction caused by air bubbles that have migrated into the brain.
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