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.