Transfer RNA-derived small RNA tRF-Glu-CTC attenuates neointimal formation via inhibition of fibromodulin.

Neointimal hyperplasia is a pathological vascular remodeling caused by abnormal proliferation and migration of subintimal vascular smooth muscle cells (VSMCs) following intimal injury. There is increasing evidence that tRNA-derived small RNA (tsRNA) plays an important role in vascular remodeling. The purpose of this study is to search for tsRNAs signature of neointima formation and to explore their potential functions. The balloon injury model of rat common carotid artery was replicated to induce intimal hyperplasia, and the differentially expressed tsRNAs (DE-tsRNAs) in arteries with intimal hyperplasia were screened by small RNA sequencing and tsRNA library. A total of 24 DE-tsRNAs were found in the vessels with intimal hyperplasia by small RNA sequencing. In vitro, tRF-Glu-CTC inhibited the expression of fibromodulin (FMOD) in VSMCs, which is a negative modulator of TGF-ß1 activity. tRF-Glu-CTC also increased VSMC proliferation and migration. In vivo experiments showed that inhibition of tRF-Glu-CTC expression after balloon injury of rat carotid artery can reduce the neointimal area. In conclusion, tRF-Glu-CTC expression is increased after vascular injury and inhibits FMOD expression in VSMCs, which influences neointima formation. On the other hand, reducing the expression of tRF-Glu-CTC after vascular injury may be a potential approach to prevent vascular stenosis.

Differential MMP-14 targeting by biglycan, decorin, fibromodulin, and lumican unraveled by in silico approach.

Small leucine-rich proteoglycans (SLRPs) are major regulators of extracellular matrix assembly and cell signaling. Lumican, a member of the SLRPs family, and its derived peptides were shown to possess antitumor activity by interacting directly with the catalytic domain of MMP-14 leading to the inhibition of its activity. The aim of the present report was to characterize by in silico three-dimensional (3D) modeling the structure and the dynamics of four SLRPs including their core protein and their specific polysaccharide chains to assess their capacity to bind to MMP-14 and to regulate its activity. Molecular docking experiments were performed to identify the specific amino acids of MMP-14 interacting with each of the four SLRPs. The inhibition of each SLRP (100 nM) on MMP-14 activity was measured and the constants of inhibition (Ki) were evaluated. The impact of the number of glycan chains, structures, and dynamics of lumican on the interaction with MMP-14 was assessed by molecular dynamics simulations. Molecular docking analysis showed that all SLRPs bind to MMP-14 through their concave face, but in different regions of the catalytic domain of MMP-14. Each SLRPs inhibited significantly the MMP-14 activity. Finally, molecular dynamics showed the role of glycan chains in interaction with MMP-14 and shielding effect of SLRPs. Altogether, the results demonstrated that each SLRP exhibited inhibition of MMP-14 activity. However, the differential targeting of MMP-14 by the SLRPs was shown to be related not only to the core protein conformation but also to the glycan chain structures and dynamics.

Dark pigmentation and related low FMOD expression increase IL-3 and facilitateplasmacytoid dendritic cell maturation.

According to epidemiological research, skin autoimmune diseases are more prevalent among black Americans. We postulated that pigment-producing melanocytes may contribute to local immune regulation in the microenvironment. We examined murine epidermal melanocytes in vitro to determine the role of pigment production in immune responses mediated by dendritic cell (DC) activation. Our study revealed that darkly pigmented melanocytes produce more IL-3 and the pro-inflammatory cytokines, IL-6 and TNF-α, and consequently induce plasmacytoid DC (pDC) maturation. Additionally, we demonstrate that low pigment-associated fibromodulin (FMOD) interferes with cytokine secretion and subsequent pDC maturation.

Coupled fibromodulin and SOX2 signaling as a critical regulator of metastatic outgrowth in melanoma.

We aimed to study mechanisms controlling metastatic outgrowth of melanoma into clinically relevant lesions, a critical process responsible for the majority of melanoma deaths. To this end, we developed novel in vivo models and identified molecular events that can be ascribed to their distinct phenotypes, indolent or highly metastatic. Induction of a proliferative state at distant sites was associated with high levels of the stem-like/progenitor marker, SOX2, and required the upregulation of FMOD, an extracellular matrix component, which modulates tumor-stroma interactions. Functional studies revealed a possible link between FMOD and SOX2; dual FMOD and SOX2 silencing nearly abolished brain metastasis and had a similar effect on distant metastasis to other sites. Our in vitro data suggests that FMOD and SOX2 cooperation plays an important role in tumor vasculogenic mimicry. Furthermore, we found that FMOD and SOX2 functional roles might converge at the activation of transcriptional co-factors YAP and TAZ, possibly via crosstalk with the tumor suppressor Hippo pathway. Finally, high expression of both genes in patient specimens predicted early development of brain metastasis. Thus, our study identifies FMOD and SOX2 cooperation as a novel regulatory mechanism that might be linked functionally to melanoma metastatic competence.

Involvement of small leucine-rich proteoglycans and telocytes in thin and thick human endometrium: immunohistochemical and ultrastructural examination.

Thin endometrium, defined as an endometrial thickness of less than 7 mm during the late follicular phase, is a common cause of frequent cancelation of embryo transfers or recurrent implantation failure during assisted reproductive treatment. Small proteoglycans regulate intracellular signaling cascades by bridging other matrix molecules and tissue elements, affecting cell proliferation, adhesion, migration, and cytokine concentration. The aim of the study is to investigate the role of small leucine-rich proteoglycans in the pathogenesis of thin and thick human endometrium and their differences from normal endometrium in terms of fine structure properties. Normal, thin, and thick endometrial samples were collected, and small leucine-rich proteoglycans (SLRPs), decorin, lumican, biglycan, and fibromodulin immunoreactivities were comparatively analyzed immunohistochemically. The data were compared statistically. Moreover, ultrastructural differences among the groups were evaluated by transmission electron microscopy. The immunoreactivities of decorin, lumican, and biglycan were higher in the thin endometrial glandular epithelium and stroma compared to the normal and thick endometrium (p < .001). Fibromodulin immunoreactivity was also higher in the thin endometrial glandular epithelium than in the normal and thick endometrium (p < .001). However, there was no statistical difference in the stroma among the groups. Ultrastructural features were not profoundly different among cases. Telocytes, however, were not seen in the thin endometrium in contrast to normal and thin endometrial tissues. These findings suggest a possible role of changes in proteoglycan levels in the pathogenesis of thin endometrium.

Melanocyte-secreted fibromodulin constrains skin inflammation in mice injected with lupus serum.

Skin pigmentation has been linked to the development, prevalence, and severity of several immune-mediated diseases such as SLE. Here, we asked whether fibromodulin (FMOD), which is highly expressed in skin with light complexion, can explain the known differences in the magnitude of inflammation. C57 mice with different levels of pigmentation and FMOD were injected with human lupus serum to induce skin inflammation. Histopathologic studies revealed that black C57 FMOD+/+ that produce low levels of FMOD and white C57 FMOD -/- mice develop more severe inflammation compared with white FMOD +/+ mice. This study also revealed that dark pigmentation and FMOD deletion correlates with the increased numbers of Langerhans cells. Altogether, we identify low pigmentation and FMOD are linked to low severity of inflammation and approaches to promote FMOD expression should offer clinical benefit.

MIF1 and MIF2 Myostatin Peptide Inhibitors as Potent Muscle Mass Regulators.

The use of peptides as drugs has progressed over time and continues to evolve as treatment paradigms change and new drugs are developed. Myostatin (MSTN) inhibition therapy has shown great promise for the treatment of muscle wasting diseases. Here, we report the MSTN-derived novel peptides MIF1 (10-mer) and MIF2 (10-mer) not only enhance myogenesis by inhibiting MSTN and inducing myogenic-related markers but also reduce adipogenic proliferation and differentiation by suppressing the expression of adipogenic markers. MIF1 and MIF2 were designed based on in silico interaction studies between MSTN and its receptor, activin type IIB receptor (ACVRIIB), and fibromodulin (FMOD). Of the different modifications of MIF1 and MIF2 examined, Ac-MIF1 and Ac-MIF2-NH2 significantly enhanced cell proliferation and differentiation as compared with non-modified peptides. Mice pretreated with Ac-MIF1 or Ac-MIF2-NH2 prior to cardiotoxin-induced muscle injury showed more muscle regeneration than non-pretreated controls, which was attributed to the induction of myogenic genes and reduced MSTN expression. These findings imply that Ac-MIF1 and Ac-MIF2-NH2 might be valuable therapeutic agents for the treatment of muscle-related diseases.

Extracellular matrix components and elasticity regulate mouse vaginal epithelial differentiation induced by mesenchymal cells†.

Oviduct, uterus, and vagina are derived from Müllerian ducts. But only in the vagina, the epithelium differentiates into stratified layers. Organ-specific secreted factors derived from the stroma of a neonatal mouse induce epithelial differentiation in the female reproductive tracts. However, the effects of the components and mechanical property of extracellular matrix (ECM) on the regulation of gene expression in the mesenchymal cells of neonatal stroma and differentiation of epithelium in the female reproductive tracts have been overlooked. In the present study, we have developed a simple 3D neonatal vaginal model using clonal cell lines to study the effect of ECM's components and stiffness on the epithelial stratification. Transcriptome analysis was performed by DNA-microarray to identify the components of ECM involved in the differentiation of vaginal epithelial stratification. The knockdown experiment of the candidate genes relating to vaginal epithelial stratification was focused on fibromodulin (Fmod), a collagen cross-linking protein. FMOD was essential for the expression of Bmp4, which encodes secreted factors to induce the epithelial stratification of vaginal mesenchymal cells. Furthermore, stiffer ECM as a scaffold for epithelial cells is necessary for vaginal epithelial stratification. Therefore, the components and stiffness of ECM are both crucial for the epithelial stratification in the neonatal vagina.

Influence of microcurrent on the modulation of remodelling genes in a wound healing assay.

The literature has shown the beneficial effects of microcurrent (MC) therapy on tissue repair. We investigated if the application of MC at 10 µA/90 s could modulate the expression of remodeling genes transforming growth factor beta (Tgfb), connective tissue growth factor (Ctgf), insulin-like growth factor 1 (Igf1), tenascin C (Tnc), Fibronectin (Fn1), Scleraxis (Scx), Fibromodulin (Fmod) and tenomodulin in NIH/3T3 fibroblasts in a wound healing assay. The cell migration was analyzed between days 0 and 4 in both fibroblasts (F) and fibroblasts + MC (F+MC) groups. On the 4th day, cell viability and gene expression were also analyzed after daily MC application. Higher expression of Ctgf and lower expression of Tnc and Fmod, respectively, were observed in the F+MC group in relation to F group (p < 0.05), and no difference was observed between the groups for the genes Tgfb, Fn1 and Scx. In cell migration, a higher number of cells in the scratch region was observed in group F+MC (p < 0.05) compared to group F on the 4th day, and the cell viability assay showed no difference between the groups. In conclusion, MC therapy at an intensity/time of 10 µA/90 s with 4 daily applications did not affect cell viability, stimulated fibroblasts migration with the involvement of Ctgf, and reduced the Tnc and Fmod expression.

Identification of Lumican and Fibromodulin as Hub Genes Associated with Accumulation of Extracellular Matrix in Diabetic Nephropathy.

INTRODUCTION: Diabetic nephropathy (DN) remains a major cause of end-stage renal disease. The development of novel biomarkers and early diagnosis of DN are of great clinical importance. The goal of this study was to identify hub genes with diagnostic potential for DN by weighted gene co-expression network analysis (WGCNA). METHODS: Gene Expression Omnibus database was searched for microarray data including distinct types of CKD. Gene co-expression network was constructed, and modules specific for DN were identified by WGCNA. Gene ontology (GO) analysis was performed, and the hub genes were screened out within the selected gene modules. In addition, cross-validation was performed in an independent dataset and in samples of renal biopsies with DN and other types of glomerular diseases. RESULTS: Dataset GSE99339 was selected, and a total of 179 microdissected glomeruli samples were analyzed, including DN, normal control, and 7 groups of other glomerular diseases. Twenty-three modules of the total 10,947 genes were grouped by WGCNA, and a module was specifically correlated with DN (r = 0.54, p = 9e-15). GO analysis showed that module genes were mainly enriched in the accumulation of extracellular matrix (ECM). LUM, ELN, FBLN1, MMP2, FBLN5, and FMOD were identified as hub genes. Cross verification showed LUM and FMOD were higher in the DN group and were negatively correlated with estimated glomerular filtration rate (eGFR). In renal biopsies, expression levels of LUM and FMOD were higher in DN than IgA nephropathy, membranous nephropathy, and normal controls. CONCLUSION: By using WGCNA approach, we identified LUM and FMOD related to ECM accumulation and were specific for DN. These 2 genes may represent potential candidate diagnostic biomarkers of DN.

OPG-Fc treatment partially rescues low bone mass phenotype in mature Bgn/Fmod deficient mice but is deleterious to the young mouse skeleton.

Biglycan (Bgn) and Fibromodulin (Fmod) are small leucine rich proteoglycans (SLRPs) which are abundant in the extra-cellular matrix (ECM) of mineralized tissues. We have previously generated a Bgn/Fmod double knock-out (DKO) mouse model and found it has a 3-fold increase in osteoclastogenesis compared with Wild type (WT) controls, resulting in a markedly low bone mass (LBM) phenotype. To try and rescue/repair the LBM phenotype of Bgn/Fmod DKO mice by suppressing osteoclast formation and activity, 3- and 26-week-old Bgn/Fmod DKO mice and age/gender matched WT controls were treated with OPG-Fc for 6 weeks after which bone parameters were evaluated using DEXA, micro-computed tomography (µCT) and serum biomarkers analyses. In the appendicular skeleton, OPG-Fc treatment improved some morphometric and geometric parameters in both the trabecular and cortical compartments in Bgn/Fmod DKO female and male mice, especially in the repair module. For many of the skeletal parameters analyzed, the Bgn/Fmod DKO mice were more responsive to the treatment than their WT controls. In addition, we found that OPG-Fc treatment was not able to prevent or ameliorate the formation of ectopic ossification, which are common lesions seen in aged joints and are one of the phenotypical hallmarks of our Bgn/Fmod DKO model. Analysis of skull bones, specifically the occipital bone, showed the treatment recovered some parameters of LBM phenotype in the craniofacial skeleton, more so in the younger rescue module. Using OPG-Fc as treatment alleviated, yet did not completely restore, the severe osteopenia and mineralized tissue structural abnormalities that Bgn/Fmod DKO mice suffer from.

Fibromodulin is upregulated by oxidative stress through the MAPK/AP-1 pathway to promote pancreatic stellate cell activation.

BACKGROUND/OBJECTIVES: Fibromodulin (FMOD) expression in chronic pancreatitis (CP) tissues and its effect on PSC was unknown. Our aim was to investigate the role of FMOD in regulating PSC profibrogenic phenotype and the molecular mechanism of CP. METHODS: Rat CP models were induced by dibutyltin dichloride. Pancreatic fibrosis was evaluated by Sirius Red staining. The expression of FMOD and α-SMA was measured, the correlation between FMOD expression and fibrosis was investigated in CP models and CP patients. The effects of FMOD on PSCs were examined by CCK-8 and migration assays. We investigated the mechanisms underlying FMOD expression using MND and a MAPK pathway inhibitor. Luciferase reporter and chromatin immunoprecipitation assays were used to investigate the effects of AP-1 on FMOD expression. RESULTS: Sirius Red staining revealed high collagen deposition in model rats. Higher expression of FMOD and α-SMA was observed in fibrotic tissues, and the expression of FMOD was correlated with that of α-SMA and the areas of Sirius Red staining. Upregulation of FMOD increased the expression of collagen I and α-SMA and the proliferation and migration of PSCs. MND induced FMOD and α-SMA expression, and knockdown of FMOD abated α-SMA expression. ERK and JNK inhibitors attenuated FMOD expression as induced by MND. AP-1 upregulated the expression of FMOD. AP-1 binds to the FMOD promoter and transcriptionally regulates FMOD expression. CONCLUSION: FMOD levels are upregulated in fibrosis tissues in CP and it is a critical downstream mediator of oxidative stress. FMOD induces PSC activation and maintains the fibrosis phenotype of PSCs.

[Effect of Silencing FMOD on Proliferation and Migration of H322 Cells and Its Mechanism].

OBJECTIVE: To investigate the regulation of fibromodulin (FMOD) on proliferation, adhesion and migration of non-small cell lung cancer cell line H322, and discuss its action mechanism. METHODS: H322 cells were randomly divided into control group, small interfering RNA (siRNA) silencing FMOD ( FMOD siRNA) group and control siRNA (Con siRNA) group. FMOD siRNA and Con siRNA were transfected into H322 cells. The cell viability of each group was detected by CCK-8 method. The adhesion ability of cells was detected by fluorescein diacetate (FDA) fluorescent staining. The cell migration ability was detected by Transwell method. Real time-PCR was used to detect the mRNA expressions of Cyclin D1, intercellular adhesion molecule -1 （ICAM-1）, E-cadherin, FMOD, transforming growth factor-ß (TGF-ß), Smad2, Smad3, Smad4 and Smad7 in cells. The protein expressions of Cyclin D1, ICAM-1, E-cadherin, FMOD, TGF-ß1, Smad2, Smad3, Smad4 and Smad7 were detected by Western blot. RESULTS: Compared with the Con siRNA group, the cell viability, cell adhesion and migration ability of the FMOD siRNA group were decreased, and the difference was statistically significant ( P<0.01). There was no significant difference between the control group and the Con siRNA group. Real time-PCR and Western blot results showed that the mRNA and protein expression levels of Cyclin D1, ICAM-1, TGF-ß1, Smad2, Smad3 and Smad4 were decreased in FMOD siRNA group, compared with Con siRNA group, while the mRNA and protein expression levels of E-cadherin and Smad7 are elevated. CONCLUSION: Silencing of the FMOD gene significantly reduces the proliferation, adhesion and migration of H322 cells, which may be conducted by inhibiting the TGF-ß/Smad signaling pathway.

The endoplasmic reticulum-resident collagen chaperone Hsp47 interacts with and promotes the secretion of decorin, fibromodulin, and lumican.

The build-up of diversified and tissue-specific assemblies of extracellular matrix (ECM) proteins depends on secreted and cell surface-located molecular arrays that coordinate ECM proteins into discrete designs. The family of small leucine-rich proteins (SLRPs) associates with and dictates the structure of fibrillar collagens, which form the backbone of most ECM types. However, whether SLRPs form complexes with proteins other than collagens is unclear. Here, we demonstrate that heat shock protein 47 (Hsp47), a well-established endoplasmic reticulum-resident collagen chaperone, also binds the SLRPs decorin, lumican, and fibromodulin with affinities comparable with that in the Hsp47-type I collagen interaction. Furthermore, we show that a lack of Hsp47 inhibits the cellular secretion of decorin and lumican. Our results expand the understanding of the concerted molecular interactions that control the secretion and organization of a functional collagenous ECM.

Fibromodulin and regulation of the intricate balance between myoblast differentiation to myocytes or adipocyte-like cells.

Interactions between myoblasts and the surrounding microenvironment led us to explore the role of fibromodulin (FMOD), an extracellular matrix protein, in the maintenance of myoblast stemness and function. Microarray analysis of FMODkd myoblasts and in silico studies were used to identify the top most differentially expressed genes in FMODkd, and helped establish that FMOD-based regulations of integral membrane protein 2a and clusterin are essential components of the myogenic program. Studies in knockout, obese, and diabetic mouse models helped characterize the operation of a novel FMOD-based regulatory circuit that controls myoblast switching from a myogenic to a lipid accumulation fate. FMOD regulation of myoblasts is an essential part of the myogenic program, and it offers opportunities for the development of therapeutics for the treatment of different muscle diseases.-Lee, E. J., Jan, A. T., Baig, M. H., Ahmad, K., Malik, A., Rabbani, G., Kim, T., Lee, I.-K., Lee, Y. H., Park, S.-Y., Choi, I. Fibromodulin and regulation of the intricate balance between myoblast differentiation to myocytes or adipocyte-like cells.

Catabolism of Fibromodulin in Developmental Rudiment and Pathologic Articular Cartilage Demonstrates Novel Roles for MMP-13 and ADAMTS-4 in C-terminal Processing of SLRPs.

BACKGROUND: Cartilage regeneration requires a balance of anabolic and catabolic processes. AIM: To examine the susceptibility of fibromodulin (FMOD) and lumican (LUM) to degradation by MMP-13, ADAMTS-4 and ADAMTS-5, the three major degradative proteinases in articular cartilage, in cartilage development and in osteoarthritis (OA). METHODS: Immunolocalization of FMOD and LUM in fetal foot and adult knee cartilages using an FMOD matrix metalloprotease (MMP)-13 neoepitope antibody (TsYG11) and C-terminal anti-FMOD (PR184) and anti-LUM (PR353) antibodies. The in vitro digestion of knee cartilage with MMP-13, A Disintegrin and Metalloprotease with Thrompospondin motifs (ADAMTS)-4 and ADAMTS-5, to assess whether FMOD and LUM fragments observed in Western blots of total knee replacement specimens could be generated. Normal ovine articular cartilage explants were cultured with interleukin (IL)-1 and Oncostatin-M (OSM) ± PGE3162689, a broad spectrum MMP inhibitor, to assess FMOD, LUM and collagen degradation. RESULTS AND DISCUSSION: FMOD and LUM were immunolocalized in metatarsal and phalangeal fetal rudiment cartilages and growth plates. Antibody TsYG11 localized MMP-13-cleaved FMOD in the hypertrophic chondrocytes of the metatarsal growth plates. FMOD was more prominently localized in the superficial cartilage of normal and fibrillated zones in OA cartilage. TsYG11-positive FMOD was located deep in the cartilage samples. Ab TsYG11 identified FMOD fragmentation in Western blots of normal and fibrillated cartilage extracts and total knee replacement cartilage. The C-terminal anti-FMOD, Ab PR-184, failed to identify FMOD fragmentation due to C-terminal processing. The C-terminal LUM, Ab PR-353, identified three LUM fragments in OA cartilages. In vitro digestion of human knee cartilage with MMP-13, ADAMTS-4 and ADAMTS-5 generated FMOD fragments of 54, 45 and 32 kDa similar to in blots of OA cartilage; LUM was less susceptible to fragmentation. Ab PR-353 detected N-terminally processed LUM fragments of 39, 38 and 22 kDa in 65⁻80-year-old OA knee replacement cartilage. FMOD and LUM were differentially processed in MMP-13, ADAMTS-4 and ADAMTS-5 digestions. FMOD was susceptible to degradation by MMP-13, ADAMTS-4 and to a lesser extent by ADAMTS-5; however, LUM was not. MMP-13-cleaved FMOD in metatarsal and phalangeal fetal rudiment and growth plate cartilages suggested roles in skeletogenesis and OA pathogenesis. Explant cultures of ovine cartilage stimulated with IL-1/OSM ± PGE3162689 displayed GAG loss on day 5 due to ADAMTS activity. However, by day 12, the activation of proMMPs occurred as well as the degradation of FMOD and collagen. These changes were inhibited by PGE3162689, partly explaining the FMOD fragments seen in OA and the potential therapeutic utility of PGE3162689.

Fibromodulin reduces scar size and increases scar tensile strength in normal and excessive-mechanical-loading porcine cutaneous wounds.

Hypertrophic scarring is a major postoperative complication which leads to severe disfigurement and dysfunction in patients and usually requires multiple surgical revisions due to its high recurrence rates. Excessive-mechanical-loading across wounds is an important initiator of hypertrophic scarring formation. In this study, we demonstrate that intradermal administration of a single extracellular matrix (ECM) molecule-fibromodulin (FMOD) protein-can significantly reduce scar size, increase tensile strength, and improve dermal collagen architecture organization in the normal and even excessive-mechanical-loading red Duroc pig wound models. Since pig skin is recognized by the Food and Drug Administration as the closest animal equivalent to human skin, and because red Duroc pigs show scarring that closely resembles human proliferative scarring and hypertrophic scarring, FMOD-based technologies hold high translational potential and applicability to human patients suffering from scarring-especially hypertrophic scarring.

Downregulated microRNA-340-5p promotes proliferation and inhibits apoptosis of chondrocytes in osteoarthritis mice through inhibiting the extracellular signal-regulated kinase signaling pathway by negatively targeting the FMOD gene.

PURPOSE: Osteoarthritis (OA) is a degenerative joint disease that leads to the destruction of joint function. The aim of this study is to investigate the effects of microRNA-340-5p (miR-340-5p) and its target gene, FMOD, on the proliferation and apoptosis of chondrocytes in mice with OA through the extracellular signal-regulated kinase (ERK) signaling pathway. MATERIALS: Twenty healthy C57BL/6J mice aged 15 months with a weight of 50 ± 2 g were selected. Ten mice were treated using a unilateral knee anterior cruciate ligament transection as well as a medial meniscectomy to establish the OA model. Besides, another 10 mice were used as the control group. METHODS: A reverse transcription quantitative polymerase chain reaction and Western blot analysis methods were used to examine the expressions of related genes in cells of each group. A 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide assay and flow cytometry were also conducted to evaluate the cell function after transfection had been completed. RESULTS: The expressions of fibromodulin (FMOD), type II collagen (Col II), B-cell lymphoma-2 (Bcl-2), sex-determining region of Y chromosome (SRY)-related high-mobility group-box gene 9 (Sox9), and proliferating cell nuclear antigen (PCNA) were decreased, whereas the expressions of miR-340-5p, runt-related transcription factor-2 (Runx2), Bcl-2-associated X protein (Bax), and ERK1/2 were elevated in the OA mice. Downregulation of miR-340-5p and upregulation of FMOD decreased the expressions of Runx2, Bax, and ERK1/2, and cell apoptosis of chondrocytes, and increased the expressions of FMOD, Col II, Bcl-2, Sox9, and PCNA, and cell proliferation. CONCLUSION: This study suggests that downregulation of miR-340-5p plays a role in promoting cell proliferation and suppressing cell apoptosis of chondrocytes in OA mice through inhibition of the ERK signaling pathway via the FMOD gene.

Fibromodulin modulates myoblast differentiation by controlling calcium channel.

Fibromodulin (FMOD) is a proteoglycan present in extracellular matrix (ECM). Based on our previous findings that FMOD controls myoblast differentiation by regulating the gene expressions of collagen type I alpha 1 (COL1α1) and integral membrane protein 2 A (Itm2a), we undertook this study to investigate relationships between FMOD and calcium channels and to understand further the mechanism by which they control myoblast differentiation. Gene expression studies and luciferase reporter assays showed FMOD affected calcium channel gene expressions by regulating calcium channel gene promoter, and patch-clamp experiments showed both L- and T-type calcium channel currents were almost undetectable in FMOD knocked down cells. In addition, gene knock-down studies demonstrated the COL1α1 and Itm2a genes both regulate the expressions of calcium channel genes. Studies using a cardiotoxin-induced mouse muscle injury model demonstrated calcium channels play important roles in the regeneration of muscle tissue, possibly by promoting the differentiation of muscle stem cells (MSCs). Summarizing, the study demonstrates ECM components secreted by myoblasts during differentiation provide an essential environment for muscle differentiation and regeneration.

Glial Cell Line-Derived Neurotrophic Factor (GDNF) Promotes Angiogenesis through the Demethylation of the Fibromodulin (FMOD) Promoter in Glioblastoma.

BACKGROUND Angiogenesis plays an important role in the progression of glioblastoma, with a high degree of malignancy. Previous studies have proved that glial cell line-derived neurotrophic factor (GDNF) and fibromodulin (FMOD) are strongly expressed in human glioblastoma. The purpose of this study was to explore the roles of GDNF and FMOD in angiogenesis and the molecular mechanisms underlying these roles in human glioblastoma. MATERIAL AND METHODS The effects of GDNF on the expression and secretion of vascular endothelial growth factor (VEGF) in human glioblastoma cell line U251 and angiogenesis in human umbilical vein endothelial cells (HUVECs) were investigated. The molecular mechanism of GDNF-induced expression of FMOD was explored. The roles of FMOD in GDNF-induced expression and secretion of VEGF and angiogenesis were also examined. RESULTS In the present study, we showed that GDNF promoted the expression and secretion of VEGF in U251 cells. VEGF mediated GDNF-induced angiogenesis in human glioblastoma. In addition, GDNF significantly upregulated the expression of FMOD in U251 cells. Mechanistically, the results of luciferase reporter assay and methylation-specific PCR (MSP) demonstrated that GDNF facilitated the demethylation of the FMOD promoter. More importantly, we found that FMOD acted as an important mediator in VEGF expression and angiogenesis induced by GDNF in human glioblastoma. CONCLUSIONS Collectively, our data show that GDNF promotes angiogenesis through demethylation of the FMOD promoter in human glioblastoma, indicating that GDNF and FMOD may be potential therapeutic targets for glioblastoma.