Most cultured cell types secrete small latent transforming growth factor-␤ (TGF-␤) as a disulfide-bonded complex with a member of the latent TGF-␤ binding protein (LTBP) family. Using the baculovirus expression system, we have mapped the domain of LTBP-1 mediating covalent association with small latent TGF-␤1. Coexpression in Sf9 cells of small latent TGF-␤1 with deletion mutants of LTBP-1 showed that the third eight-cysteine repeat of LTBP-1 is necessary and sufficient for covalent interaction with small latent TGF-␤1. Analysis by mass spectrometry of this eight-cysteine repeat, produced as a recombinant peptide in Sf9 cells, confirmed that it was N-glycosylated, as expected from the primary sequence. No other post-translational modifications of this domain were detected. Alkylation of the recombinant peptide with vinyl pyridine failed to reveal any free cysteines, indicating that, in the absence of small latent TGF-␤, the eight cysteines of this domain are engaged in intramolecular bonds. These data demonstrate that the third LTBP-1 eight-cysteine repeat recognizes and associates covalently with small latent TGF-␤1 through a mechanism that does not require any specific post-translational modification of this domain. They also suggest that this domain adopts different conformations depending on whether it is free or bound to small latent TGF-␤. First identified as a transforming agent on cultured fibroblasts, transforming growth factor-␤ (TGF-␤)1 is a potent, ubiquitous regulator of cell growth and differentiation and a modulator of the immune system (1-3). TGF-␤ also stimulates extracellular matrix production both by increasing the rate of synthesis of extracellular matrix components like fibronectin, collagen I, and biglycan, and by down-regulating the overall proteolytic activity. Three isoforms of TGF-␤, TGF-␤1, TGF-␤2, and TGF-␤3, have been identified in mammals. These isoforms have similar, but distinct, activities and are differently distributed among tissues. Translation of TGF-␤ mRNA results in the formation of a 105-kDa dimeric proform, which is proteolytically cleaved in the Golgi apparatus to mature TGF-␤ (4). Upon secretion, the mature growth factor (m ϭ 25 kDa), remains noncovalently associated with the propeptide (also known as latency-associated peptide or LAP, m ϭ 80 kDa), thus forming an inactive, latent precursor or small latent complex. In order to interact with specific cell surface serine/threonine kinase receptors, TGF-␤ must dissociate from LAP, a process known as TGF-␤ activation. Most TGF-␤ produced by cultured cells and found in tissues is latent, suggesting that the activation process is a key regulatory step in modulating TGF-␤ activity.In most cell types and tissues, the small latent complex is disulfide-linked during secretion to a 160 -240-kDa glycoprotein called the latent TGF-␤ binding protein (LTBP). This complex is known as the large latent complex. Three LTBP family members have been described, LTBP-1, LTBP-2, and LTBP-3, which have been cloned respectively from human and ra...

Human basic fibroblast growth factor (FGF-2) occurs in four isoforms: a low molecular weight (LMW FGF-2, 18 kDa) and three high molecular weight (HMW FGF-2, 22, 22.5, and 24 kDa) forms. LMW FGF-2 is primarily cytoplasmic and functions in an autocrine manner, whereas HMW FGF-2s are nuclear and exert activities through an intracrine, perhaps nuclear, pathway. Selective overexpression of HMW FGF-2 forms in fibroblasts promotes growth in low serum, whereas overexpression of LMW FGF-2 does not. The HMW FGF-2 forms have two functional domains: an amino-terminal extension and a common 18-kDa amino acid sequence. To investigate the role of these regions in the intracrine signaling of HMW FGF-2, we produced stable transfectants of NIH 3T3 fibroblasts overexpressing either individual HMW FGF-2 forms or artificially nucleartargeted LMW FGF-2. All of these forms of FGF-2 localize to the nucleus/nucleolus and induce growth in low serum. The nuclear forms of FGF-2 trigger a mitogenic stimulus under serum starvation conditions and do not specifically protect the cells from apoptosis. These data indicate the existence of a specific role for nuclear FGF-2 and suggest that LMW FGF-2 represents the biological messenger in both the autocrine/paracrine and intracrine FGF-2 pathways. INTRODUCTIONBasic fibroblast growth factor (FGF-2) 1 is a member of a large family of heparin-binding growth factors. Thus far 19 members (Nishimura et al., 1999) have been described. These proteins affect various biological processes ranging from cell proliferation to plasminogen activation, integrin expression, cell migration, embryonic development, and cell differentiation. FGFs also may be involved in tumor angiogenesis and malignant transformation (Burgess and Maciag, 1989;Rifkin and Moscatelli, 1989;Basilico and Moscatelli, 1992;Mason, 1994).The biological functions of the FGFs are mediated by their interaction with both high-and low-affinity plasma membrane receptors (Baird, 1994). A family consisting of four high-affinity tyrosine kinase FGFreceptors has been identified (Basilico and Moscatelli, 1992;Jaye et al., 1992). The interaction of FGF-2 with its plasma membrane high-affinity receptors induces autophosphorylation of the receptor and initiates the phosphorylation of tyrosine residues in cytosolic substrates (Fantl et al., 1993). The low-affinity receptors consist of heparan sulfate proteoglycans and are thought to provide a mechanism both to concentrate ligand and to present FGF dimers to tyrosine kinase receptors Baird, 1994) The prototypic members of the FGF family, FGF-1 and -2, lack a signal sequence for secretion, although the proteins are released and have been visualized in ‡ Corresponding author. E-mail address: aresem01@mcrcr.med. nyu.edu. 1 Abbreviations used: FGF-2, basic fibroblast growth factor; HMW FGF-2, high molecular weight FGF-2; LMW FGF-2, low molecular weight FGF-2.© 1999 by The American Society for Cell Biology 1429 the ECM of different tissues (Abraham et al., 1986;Jaye et al., 1986). The mechanism of FGF-1 and FGF-2 r...

Intra-articular injection of miRNA-140 can alleviate OA progression by modulating ECM homeostasis in rats, and may have potential as a new therapy for OA.

BackgroundAccumulating evidence suggests the pro-inflammatory cytokine interleukin-6 (IL-6) in tumor microenvironment may promote the development of hepatocellular carcinoma (HCC). However, the underlying mechanism remains largely unknown.MethodsThe expression and promoter activity of lncTCF7 were measured by quantitative real-time polymerase chain reaction (qRT-PCR) and luciferase reporter assay. The function of the STAT3 binding site in the lncTCF7 promoter region was tested by luciferase reporter assay with nucleotide substitutions. The binding of STAT3 to the lncTCF7 promoter was confirmed by chromatin immunoprecipitation assay (CHIP) in vivo. The effects of decreasing STAT3 with small interference RNA and inhibiting STAT3 activation by small molecular inhibitor on lncTCF7 expression were also determined.ResultsWe demonstrate that IL-6 could induce lncTCF7 expression in a time- and dose-dependent manner, and we showed that IL-6 transcriptionally activated the expression of lncTCF7 in HCC cells by activating STAT3, a transcription activator which binds to promoter regions of lncTCF7. Furthermore, knocking-down STAT3 and inhibiting STAT3 activation reduced lncTCF7 expression. Importantly, RNA interference-based attenuation of lncTCF7 prevented IL-6-induced EMT and cell invasion.ConclusionThus, these data provides evidence to the existence of an aberrant IL-6/STAT3/ lncTCF7 signaling axis that leads to HCC aggressiveness through EMT induction, which could be novel therapeutic targets in malignancies.

Oxygen deprivation (hypoxia) is a common feature of various human maladies, including cardiovascular diseases and cancer; however, the effect of hypoxia on Ad-based gene therapies has not been described. In this study, we evaluated how hypoxia (1% pO 2 ) affects different aspects of Ad-based therapies, including attachment and uptake, transgene expression, and replication, in a series of cancer cell lines and primary normal cells. We found that hypoxia had no significant effect on the expression or function of the Ad5 attachment (Coxsackievirus and Adenovirus Receptor) and internalization (a v integrins) proteins, nor on the human cytomegalovirus-driven expression of an exogenous gene carried by a replication-incompetent Ad. Viral replication, however, was compromised by hypoxic conditions. Our studies revealed hypoxia-induced reductions in E1A levels that were mediated at the post-transcriptional level. E1A drives cells into the viral replication optimal S phase of the cell cycle; consequently, the combination of reduced E1A protein and hypoxia-induced G1 arrest of cells may be responsible for the lack of efficient viral replication under hypoxic conditions. Consequently, while traditional replication-incompetent Ad-based vectors appear to be viable delivery systems for hypoxia-associated disease indications, our studies suggest that Oncolytic Ads may need additional factors to efficiently treat hypoxic regions of human tumors. Gene Therapy (2005) 12, 902-910.

ObjectivesEmerging evidence suggests that the microbiome plays an important role in the pathogenesis of osteoarthritis (OA). We aimed to test the two-hit model of OA pathogenesis and potentiation in which one ‘hit’ is provided by an adverse gut microbiome that activates innate immunity; the other ‘hit’ is underlying joint damage.MethodsMedical history, faecal and blood samples were collected from human healthy controls (OA-METS-, n=4), knee OA without metabolic syndrome (OA+METS-, n=7) and knee OA with metabolic syndrome (OA+METS+, n=9). Each group of human faecal samples, whose microbial composition was identified by 16S rRNA sequencing, was pooled and transplanted into germ-free mice 2 weeks prior to meniscal/ligamentous injury (MLI) (n≥6 per group). Eight weeks after MLI, mice were evaluated for histological OA severity and synovitis, systemic inflammation and gut permeability.ResultsHistological OA severity following MLI was minimal in germ-free mice. Compared with the other groups, transplantation with the OA+METS+ microbiome was associated with higher mean systemic concentrations of inflammatory biomarkers (interleukin-1β, interleukin-6 and macrophage inflammatory protein-1α), higher gut permeability and worse OA severity. A greater abundance of Fusobacterium and Faecalibaterium and lesser abundance of Ruminococcaceae in transplanted mice were consistently correlated with OA severity and systemic biomarkers concentrations.ConclusionThe study clearly establishes a direct gut microbiome-OA connection that sets the stage for a new means of exploring OA pathogenesis and potentially new OA therapeutics. Alterations of Fusobacterium, Faecalibaterium and Ruminococcaceae suggest a role of these particular microbes in exacerbating OA.

Background: Osteoarthritis (OA) is a disease characterized by progressive degeneration, joint hyperplasia, narrowing of joint spaces, and extracellular matrix metabolism. Recent studies have shown that the pathogenesis of OA may be related to non-coding RNA, and its pathological mechanism may be an effective way to reduce OA. Objective: The purpose of this review was to investigate the recent progress of miRNA, long noncoding RNA (lncRNA) and circular RNA (circRNA) in gene therapy of OA, discussing the effects of this RNA on gene expression, inflammatory reaction, apoptosis and extracellular matrix in OA. Methods: The following electronic databases were searched, including PubMed, EMBASE, Web of Science, and the Cochrane Library, for published studies involving the miRNA, lncRNA, and circRNA in OA. The outcomes included the gene expression, inflammatory reaction, apoptosis, and extracellular matrix. Results and Discussion: With the development of technology, miRNA, lncRNA, and circRNA have been found in many diseases. More importantly, recent studies have found that RNA interacts with RNA-binding proteins to regulate gene transcription and protein translation, and is involved in various pathological processes of OA, thus becoming a potential therapy for OA. Conclusion: In this paper, we briefly introduced the role of miRNA, lncRNA, and circRNA in the occurrence and development of OA and as a new target for gene therapy.

BackgroundRisk factors for venous thromboembolism (VTE) of total joint arthroplasty (TJA) have been examined by many studies. A comprehensive systematic review of recent findings of high evidence level in this topic is needed.MethodsWe conducted a PubMed search for papers published between 2003 and 2013 that provided level-I and level-II evidences on risk factors for VTE of TJA. For each potential factors examined in at least three papers, we summarize the the number of the papers and confirmed the direction of statistically significant associations, e.g. “risk factor” “protective factor” or “controversial factor”.ResultsFifty-four papers were included in the systematic review. Risk factors found to be associated with VTE of both total hip arthroplasty and total knee arthroplasty included older age, female sex, higher BMI, bilateral surgery, surgery time > 2 hours. VTE history was found as a VTE risk factor of THA but an controversial factor of TKA. Cemented fixation as compared to cementless fixation was found as a risk factor for VTE only of TKA. TKA surgery itself was confirmed as a VTE risk factor compared with THA surgery.ConclusionsThis systematic review of high level evidences published in recent ten years identified a range of potential factors associated with VTE risk of total joint arthroplasty. These results can provide informations in this topic for doctors, patients and researchers.