Syndecan-1 enhances proliferation, migration and metastasis of HT-1080 cells in cooperation with syndecan-2

Syndecans are transmembrane heparan sulphate proteoglycans. Their role in the development of the malignant phenotype is ambiguous and depends upon the particular type of cancer. Nevertheless, syndecans are promising targets in cancer therapy, and it is important to elucidate the mechanisms controlling their various cellular effects. According to earlier studies, both syndecan-1 and syndecan-2 promote malignancy of HT-1080 human fibrosarcoma cells, by increasing the proliferation rate and the metastatic potential and migratory ability, respectively. To better understand their tumour promoter role in this cell line, syndecan expression levels were modulated in HT-1080 cells and the growth rate, chemotaxis and invasion capacity were studied. For in vivo testing, syndecan-1 overexpressing cells were also inoculated into mice. Overexpression of full length or truncated syndecan-1 lacking the entire ectodomain but containing the four juxtamembrane amino acids promoted proliferation and chemotaxis. These effects were accompanied by a marked increase in syndecan-2 protein expression. The pro-migratory and pro-proliferative effects of truncated syndecan-1 were not observable when syndecan-2 was silenced. Antisense silencing of syndecan-2, but not that of syndecan-1, inhibited cell migration. In vivo, both full length and truncated syndecan-1 increased tumour growth and metastatic rate. Based on our in vitro results, we conclude that the tumour promoter role of syndecan-1 observed in HT-1080 cells is independent of its ectodomain; however, in vivo the presence of the ectodomain further increases tumour proliferation. The enhanced migratory ability induced by syndecan-1 overexpression is mediated by syndecan-2. Overexpression of syndecan-1 also leads to activation of IGF1R and increased expression of Ets-1. These changes were not evident when syndecan-2 was overexpressed. These findings suggest the involvement of IGF1R and Ets-1 in the induction of syndecan-2 synthesis and stimulation of proliferation by syndecan-1. This is the first report demonstrating that syndecan-1 enhances malignancy of a mesenchymal tumour cell line, via induction of syndecan-2 expression.     

Modern Lineages of Mycobacterium tuberculosis Exhibit Lineage-Specific Patterns of Growth and Cytokine Induction in Human Monocyte-Derived Macrophages

Background

Strains of Mycobacterium tuberculosis vary in virulence. Strains that have caused outbreaks in the United States and United Kingdom have been shown to subvert the innate immune response as a potential immune evasion mechanism. There is, however, little information available as to whether these patterns of immune subversion are features of individual strains or characteristic of broad clonal lineages of M. tuberculosis.

Methods

Strains from two major modern lineages (lineage 2 [East-Asian] and lineage 4 [Euro-American]) circulating in the Western Cape in South Africa as well as a comparator modern lineage (lineage 3 [CAS/Delhi]) were identified. We assessed two virulence associated characteristics: mycobacterial growth (in liquid broth and monocyte derived macrophages) and early pro-inflammatory cytokine induction.

Results

In liquid culture, Lineage 4 strains grew more rapidly and reached higher plateau levels than other strains (lineage 4 vs. lineage 2 p = 0.0024; lineage 4 vs. lineage 3 p = 0.0005). Lineage 3 strains were characterized by low and early plateau levels, while lineage 2 strains showed an intermediate growth phenotype. In monocyte-derived macrophages, lineage 2 strains grew faster than lineage 3 strains (p<0.01) with lineage 4 strains having an intermediate phenotype. Lineage 2 strains induced the lowest levels of pro-inflammatory TNF and IL-12p40 as compared to other lineages (lineage 2: median TNF 362 pg/ml, IL-12p40 91 pg/ml; lineage 3: median TNF 1818 pg/ml, IL-12p40 123 pg/ml; lineage 4: median TNF 1207 pg/ml, IL-12p40 205 pg/ml;). In contrast, lineage 4 strains induced high levels of IL-12p40 and intermediate level of TNF. Lineage 3 strains induced high levels of TNF and intermediate levels of IL-12p40.

Conclusions

Strains of M. tuberculosis from the three major modern strain lineages possess distinct patterns of growth and cytokine induction. Rapid growth and immune subversion may be key characteristics to the success of these strains in different human populations.     

Identification and quantification of neuropeptides in naïve mouse spinal cord using mass spectrometry reveals [des‐Ser1]‐cerebellin as a novel modulator of nociception

Neuropeptide transmitters involved in nociceptive processes are more likely to be expressed in the dorsal than the ventral horn of the spinal cord. This study was designed to examine the relative distribution of neuropeptides between the dorsal and ventral spinal cord in naïve mice using liquid chromatography, high‐resolution mass spectrometry. We identified and relatively quantified 36 well‐characterized full‐length neuropeptides and an additional 168 not previously characterized peptides. By extraction with organic solvents we identified seven additional full‐length neuropeptides. The peptide [des‐Ser1]‐cerebellin (desCER), originating from cerebellin precursor protein 1 (CBLN1), was predominantly expressed in the dorsal horn. Immunohistochemistry showed the presence of CBLN1 immunoreactivity with a punctate cytoplasmic pattern in neuronal cell bodies throughout the spinal gray matter. The signal was stronger in the dorsal compared to the ventral horn, with most CBLN1 positive cells present in outer laminae II/III, colocalizing with calbindin, a marker for excitatory interneurons. Intrathecal injection of desCER induced a dose‐dependent mechanical hypersensitivity but not heat or cold hypersensitivity. This study provides evidence for involvement of desCER in nociception and provides a platform for continued exploration of involvement of novel neuropeptides in the regulation of nociceptive transmission.

     

The Strong In Vivo Anti-Tumor Effect of the UIC2 Monoclonal Antibody Is the Combined Result of Pgp Inhibition and Antibody Dependent Cell-Mediated Cytotoxicity

P-glycoprotein (Pgp) extrudes a large variety of chemotherapeutic drugs from the cells, causing multidrug resistance (MDR). The UIC2 monoclonal antibody recognizes human Pgp and inhibits its drug transport activity. However, this inhibition is partial, since UIC2 binds only to 10–40% of cell surface Pgps, while the rest becomes accessible to this antibody only in the presence of certain substrates or modulators (e.g. cyclosporine A (CsA)). The combined addition of UIC2 and 10 times lower concentrations of CsA than what is necessary for Pgp inhibition when the modulator is applied alone, decreased the EC₅₀ of doxorubicin (DOX) in KB-V1 (Pgp⁺) cells in vitro almost to the level of KB-3-1 (Pgp^-) cells. At the same time, UIC2 alone did not affect the EC₅₀ value of DOX significantly. In xenotransplanted severe combined immunodeficient (SCID) mice co-treated with DOX, UIC2 and CsA, the average weight of Pgp⁺ tumors was only ∼10% of the untreated control and in 52% of these animals we could not detect tumors at all, while DOX treatment alone did not decrease the weight of Pgp⁺ tumors. These data were confirmed by visualizing the tumors in vivo by positron emission tomography (PET) based on their increased ¹⁸FDG accumulation. Unexpectedly, UIC2+DOX treatment also decreased the size of tumors compared to the DOX only treated animals, as opposed to the results of our in vitro cytotoxicity assays, suggesting that immunological factors are also involved in the antitumor effect of in vivo UIC2 treatment. Since UIC2 binding itself did not affect the viability of Pgp expressing cells, but it triggered in vitro cell killing by peripheral blood mononuclear cells (PBMCs), it is concluded that the impressive in vivo anti-tumor effect of the DOX-UIC2-CsA treatment is the combined result of Pgp inhibition and antibody dependent cell-mediated cytotoxicity (ADCC).     

Novel Method to Load Multiple Genes onto a Mammalian Artificial Chromosome

Mammalian artificial chromosomes are natural chromosome-based vectors that may carry a vast amount of genetic material in terms of both size and number. They are reasonably stable and segregate well in both mitosis and meiosis. A platform artificial chromosome expression system (ACEs) was earlier described with multiple loading sites for a modified lambda-integrase enzyme. It has been shown that this ACEs is suitable for high-level industrial protein production and the treatment of a mouse model for a devastating human disorder, Krabbe’s disease. ACEs-treated mutant mice carrying a therapeutic gene lived more than four times longer than untreated counterparts. This novel gene therapy method is called combined mammalian artificial chromosome-stem cell therapy. At present, this method suffers from the limitation that a new selection marker gene should be present for each therapeutic gene loaded onto the ACEs. Complex diseases require the cooperative action of several genes for treatment, but only a limited number of selection marker genes are available and there is also a risk of serious side-effects caused by the unwanted expression of these marker genes in mammalian cells, organs and organisms. We describe here a novel method to load multiple genes onto the ACEs by using only two selectable marker genes. These markers may be removed from the ACEs before therapeutic application. This novel technology could revolutionize gene therapeutic applications targeting the treatment of complex disorders and cancers. It could also speed up cell therapy by allowing researchers to engineer a chromosome with a predetermined set of genetic factors to differentiate adult stem cells, embryonic stem cells and induced pluripotent stem (iPS) cells into cell types of therapeutic value. It is also a suitable tool for the investigation of complex biochemical pathways in basic science by producing an ACEs with several genes from a signal transduction pathway of interest.     

New Perspectives in the Renin-Angiotensin-Aldosterone System (RAAS) II: Albumin Suppresses Angiotensin Converting Enzyme (ACE) Activity in Human

About 8% of the adult population is taking angiotensin-converting enzyme (ACE) inhibitors to treat cardiovascular disease including hypertension, myocardial infarction and heart failure. These drugs decrease mortality by up to one-fifth in these patients. We and others have reported previously that endogenous inhibitory substances suppress serum ACE activity, in vivo, similarly to the ACE inhibitor drugs. Here we have made an effort to identify this endogenous ACE inhibitor substance. ACE was crosslinked with interacting proteins in human sera. The crosslinked products were immunoprecipitated and subjected to Western blot. One of the crosslinked products was recognized by both anti-ACE and anti-HSA (human serum albumin) antibodies. Direct ACE-HSA interaction was confirmed by binding assays using purified ACE and HSA. HSA inhibited human purified (circulating) and human recombinant ACE with potencies (IC₅₀) of 5.7±0.7 and 9.5±1.1 mg/mL, respectively. Effects of HSA on the tissue bound native ACE were tested on human saphenous vein samples. Angiotensin I evoked vasoconstriction was inhibited by HSA in this vascular tissue (maximal force with HSA: 6.14±1.34 mN, without HSA: 13.54±2.63 mN), while HSA was without effects on angiotensin II mediated constrictions (maximal force with HSA: 18.73±2.17 mN, without HSA: 19.22±3.50 mN). The main finding of this study is that HSA was identified as a potent physiological inhibitor of the ACE. The enzymatic activity of ACE appears to be almost completely suppressed by HSA when it is present in its physiological concentration. These data suggest that angiotensin I conversion is limited by low physiological ACE activities, in vivo.     

GEF-H1 Mediates Tumor Necrosis Factor-��-induced Rho Activation and Myosin Phosphorylation: ROLE IN THE REGULATION OF TUBULAR PARACELLULAR PERMEABILITY*

Tumor necrosis factor-α (TNF-α), an inflammatory cytokine, has been shown to activate the small GTPase Rho, but the underlying signaling mechanisms remained undefined. This general problem is particularly important in the kidney, because TNF-α, a major mediator of kidney injury, is known to increase paracellular permeability in tubular epithelia. Here we aimed to determine the effect of TNF-α on the Rho pathway in tubular cells (LLC-PK₁ and Madin-Darby canine kidney), define the upstream signaling, and investigate the role of the Rho pathway in the TNF-α-induced alterations of paracellular permeability. We show that TNF-α induced a rapid and sustained RhoA activation that led to stress fiber formation and Rho kinase-dependent myosin light chain (MLC) phosphorylation. To identify new regulators connecting the TNF receptor to Rho signaling, we applied an affinity precipitation assay with a Rho mutant (RhoG17A), which captures activated GDP-GTP exchange factors (GEFs). Mass spectrometry analysis of the RhoG17A-precipitated proteins identified GEF-H1 as a TNF-α-activated Rho GEF. Consistent with a central role of GEF-H1, its down-regulation by small interfering RNA prevented the activation of the Rho pathway. Moreover GEF-H1 and Rho activation are downstream of ERK signaling as the MEK1/2 inhibitor PD98059 mitigated TNF-α-induced activation of these proteins. Importantly TNF-α enhanced the ERK pathway-dependent phosphorylation of Thr-678 of GEF-H1 that was key for activation. Finally the TNF-α-induced paracellular permeability increase was absent in LLC-PK₁ cells stably expressing a non-phosphorylatable, dominant negative MLC. In summary, we have identified the ERK/GEF-H1/Rho/Rho kinase/phospho-MLC pathway as the mechanism mediating TNF-α-induced elevation of tubular epithelial permeability, which in turn might contribute to kidney injury.Tumor necrosis factor-α (TNF-α)² is a pleiotropic proinflammatory cytokine that is synthesized as a membrane protein in response to inflammation, infection, and injury (1). Subsequently it is cleaved by the metalloprotease TNF-α convertase enzyme to release a 17-kDa soluble peptide (for a review, see Ref. 2). TNF-α has two receptors, the constitutively expressed, ubiquitous TNF receptor 1 and the inducible TNF receptor 2.An increasing body of evidence supports a key role for TNF-α in both acute renal injury and chronic kidney diseases (for reviews, see Refs. 3 and 4). Although TNF-α is almost undetectable in normal kidneys, elevated intrarenal, serum, or urine concentrations have been reported in various pathological states including ischemia-reperfusion, endotoxinemia, and early diabetic nephropathy (5–8). Moreover kidney injury in various pathological states was prevented or mitigated by inhibition of TNF-α production, by addition of neutralizing antibodies, or in TNF receptor knock-out mice (for a review, see Ref. 3). The central role of TNF-α in mediating kidney injury is therefore well established. Importantly TNF-α can be produced in the kidney not only by infiltrating macrophages and lymphocytes but by resident cells including the tubular epithelium. For example, in reperfusion injury TNF-α expression precedes macrophage infiltration and localizes mostly to the tubules (3, 7). Tubular TNF-α production is also enhanced by endotoxin and hypoxia (9–12). Although effects of locally released TNF-α on the tubular epithelium could contribute to its deleterious actions, the underlying mechanisms have been incompletely explored.Although a large number of studies have focused on the inflammatory and apoptotic signaling initiated by TNF-α in various cells, its cytoskeletal effects remain much less explored. In recent years Rho and its effector, Rho kinase (ROK), key regulators of both the actin cytoskeleton and myosin phosphorylation (13), have emerged as important mediators of TNF-α effects in endothelial cells (14–18). Similar effects in the tubular epithelium, however, have not been established. Even more importantly, the upstream signaling that connects the TNF receptor to activation of the Rho pathway remains completely unknown. Like other small GTPases, Rho cycles between an inactive (GDP-bound) and active (GTP-bound) form (13). The exchange of GDP to GTP during activation is stimulated by GDP-GTP exchange factors (GEFs). The diverse family of Rho GEFs contains >70 members in humans (19), making it challenging to identify the specific factors involved in mediating Rho activation through receptor-mediated stimuli. In the case of TNF-α, neither the particular Rho GEF involved nor the mechanism of its regulation has been identified in any of the cell systems studied.A rise in epithelial paracellular permeability through the intercellular junctions is a prominent event during inflammation (“leaky epithelium”) (for reviews, see Refs. 20 and 21). In addition, the junctions maintain the polarized phenotype of epithelial cells that is necessary for directional transport processes and constitute an important signaling platform that transmits environmental cues to the cells. Therefore, the consequences of junction disruption during inflammation might go beyond the compromised barrier functions. Interestingly TNF-α has been reported to affect the permeability of the tubular epithelium. Mullin et al. (22) have reported that in a tubular cell line TNF-α induced a temporary elevation in transepithelial resistance followed by a drop in transepithelial resistance and increased paracellular permeability. The transepithelial resistance decrease was blocked by genistein, a general tyrosine kinase inhibitor; however, the exact mechanism underlying the observed permeability changes remained incompletely explored.The actin cytoskeleton and especially phosphorylation of myosin light chain (MLC) was shown to be essential for the permeability increase caused by pathogens, cytokines, and growth factors in various epithelial and endothelial systems (for reviews, see Refs. 21, 23, and 24). Interestingly although myosin phosphorylation mediates the TNF-α-elicited permeability changes in intestinal cells (25, 26), phospho-MLC was reported not to be involved in the TNF-α-induced permeability rise in endothelial cells (17). The possible role of the Rho pathway and myosin phosphorylation in the TNF-α-induced permeability changes in the tubular epithelium therefore remains to be established.The aim of this study was to explore the signaling pathways through which TNF-α causes cytoskeleton remodeling and elevates paracellular permeability in kidney tubular cells. Our findings show that TNF-α induces rapid activation of RhoA that leads to Rho/Rho kinase-dependent actin remodeling and myosin phosphorylation. Using an affinity precipitation assay followed by mass spectrometry, we identified GEF-H1 as a TNF-α-activated GEF. We showed that GEF-H1 mediates the TNF-α-induced stimulation of Rho and its effectors. In addition, activation of the GEF-H1/Rho pathway by TNF-α was downstream of ERK signaling and required GEF-H1 phosphorylation on Thr-678. Finally using a dominant negative MLC mutant, we showed that myosin phosphorylation is essential for the TNF-α-induced elevation in paracellular permeability.