A Pleiotrophin C-terminus peptide induces anti-cancer effects through RPTPβ/ζ

<p>Abstract</p> <p>Background</p> <p>Pleiotrophin, also known as HARP (Heparin Affin Regulatory Peptide) is a growth factor expressed in various tissues and cell lines. Pleiotrophin participates in multiple biological actions including the induction of cellular proliferation, migration and angiogenesis, and is involved in carcinogenesis. Recently, we identified and characterized several pleiotrophin proteolytic fragments with biological activities similar or opposite to that of pleiotrophin. Here, we investigated the biological actions of P(122-131), a synthetic peptide corresponding to the carboxy terminal region of this growth factor.</p> <p>Results</p> <p>Our results show that P(122-131) inhibits <it>in vitro </it>adhesion, anchorage-independent proliferation, and migration of DU145 and LNCaP cells, which express pleiotrophin and its receptor RPTPβ/ζ. In addition, P(122-131) inhibits angiogenesis <it>in vivo</it>, as determined by the chicken embryo CAM assay. Investigation of the transduction mechanisms revealed that P(122-131) reduces the phosphorylation levels of Src, Pten, Fak, and Erk¹/₂. Finally, P(122-131) not only interacts with RPTPβ/ζ, but also interferes with other pleiotrophin receptors, as demonstrated by selective knockdown of pleiotrophin or RPTPβ/ζ expression with the RNAi technology.</p> <p>Conclusions</p> <p>In conclusion, our results demonstrate that P(122-131) inhibits biological activities that are related to the induction of a transformed phenotype in PCa cells, by interacing with RPTPβ/ζ and interfering with other pleiotrophin receptors. Cumulatively, these results indicate that P(122-131) may be a potential anticancer agent, and they warrant further study of this peptide.</p

NEMO Prevents RIP Kinase 1-Mediated Epithelial Cell Death and Chronic Intestinal Inflammation by NF-kappaB-Dependent and -Independent Functions

Intestinal epithelial cells (IECs) regulate gut immune homeostasis, and impaired epithelial responses are implicated in the pathogenesis of inflammatory bowel diseases (IBD). IEC-specific ablation of nuclear factor kappaB (NF-kappaB) essential modulator (NEMO) caused Paneth cell apoptosis and impaired antimicrobial factor expression in the ileum, as well as colonocyte apoptosis and microbiota-driven chronic inflammation in the colon. Combined RelA, c-Rel, and RelB deficiency in IECs caused Paneth cell apoptosis but not colitis, suggesting that NEMO prevents colon inflammation by NF-kappaB-independent functions. Inhibition of receptor-interacting protein kinase 1 (RIPK1) kinase activity or combined deficiency of Fas-associated via death domain protein (FADD) and RIPK3 prevented epithelial cell death, Paneth cell loss, and colitis development in mice with epithelial NEMO deficiency. Therefore, NEMO prevents intestinal inflammation by inhibiting RIPK1 kinase activity-mediated IEC death, suggesting that RIPK1 inhibitors could be effective in the treatment of colitis in patients with NEMO mutations and possibly in IBD

Improved HSC reconstitution and protection from inflammatory stress and chemotherapy in mice lacking granzyme B.

The serine protease granzyme B (GzmB) is stored in the granules of cytotoxic T and NK cells and facilitates immune-mediated destruction of virus-infected cells. In this study, we use genetic tools to report novel roles for GzmB as an important regulator of hematopoietic stem cell (HSC) function in response to stress. HSCs lacking the GzmB gene show improved bone marrow (BM) reconstitution associated with increased HSC proliferation and mitochondrial activity. In addition, recipients deficient in GzmB support superior engraftment of wild-type HSCs compared with hosts with normal BM niches. Stimulation of mice with lipopolysaccharide strongly induced GzmB protein expression in HSCs, which was mediated by the TLR4-TRIF-p65 NF-κB pathway. This is associated with increased cell death and GzmB secretion into the BM environment, suggesting an extracellular role of GzmB in modulating HSC niches. Moreover, treatment with the chemotherapeutic agent 5-fluorouracil (5-FU) also induces GzmB production in HSCs. In this situation GzmB is not secreted, but instead causes cell-autonomous apoptosis. Accordingly, GzmB-deficient mice are more resistant to serial 5-FU treatments. Collectively, these results identify GzmB as a negative regulator of HSC function that is induced by stress and chemotherapy in both HSCs and their niches. Blockade of GzmB production may help to improve hematopoiesis in various situations of BM stress

Hemodialysis Removes Uremic Toxins That Alter the Biological Actions of Endothelial Cells

Chronic kidney disease is linked to systemic inflammation and to an increased risk of ischemic heart disease and atherosclerosis. Endothelial dysfunction associates with hypertension and vascular disease in the presence of chronic kidney disease but the mechanisms that regulate the activation of the endothelium at the early stages of the disease, before systemic inflammation is established remain obscure. In the present study we investigated the effect of serum derived from patients with chronic kidney disease either before or after hemodialysis on the activation of human endothelial cells in vitro, as an attempt to define the overall effect of uremic toxins at the early stages of endothelial dysfunction. Our results argue that uremic toxins alter the biological actions of endothelial cells and the remodelling of the extracellular matrix before signs of systemic inflammatory responses are observed. This study further elucidates the early events of endothelial dysfunction during toxic uremia conditions allowing more complete understanding of the molecular events as well as their sequence during progressive renal failure

Contribution on the stydy of the biological action and signalling of the growth factor HARP (Heparin Affin Regulatory Peptide) on endothelial cells

Heparin affin regulatory peptide (HARP) is an 18 kDa growth factor that has a high affinity for heparin. HARP is highly conserved among species and shares 50% homology with Midkine and RI-HBP. The above proteins constitute a relatively new family of growth factors with high affinity for heparin. HARP has been originally purified from perinatal rat brain as a molecule that induces neurite outgrowth. HARP is also expressed in uterus, cartilage and bone extracts. Several reports have established a strong correlation between HARP expression and tumour growth and angiogenesis. High levels of this protein were found in many human cancers and cell lines derived from human tumours. HARP has been reported to be mitogenic for different types of endothelial cells and angiogenic in vivo and in vitro. HARP exerts its biological activity through interactions with cell surface proteoglycans, such as N-syndecan, or binding to more specific cell surface receptors. Receptor-type protein tyrosine-phosphatase β/ζ (RPTPβ/ζ) and its secreted variant phosphacan, as well as ALK, have been recently reported to bind HARP and to be implicated in its signalling.HARP has been previously shown to activate both the MAPK and PI3K - Akt signalling axes. Inhibitors of Erk½ or PI3K inhibit DNA synthesis stimulated by HARP. Additionally, analysis of tyrosine phosphorylated proteins following HARP stimulation, revealed induction of Shc and Erk ½ phosphorylation. Nevertheless, the signals from specific receptors to PI3K or MAPK are not well documented. In the present work, we examined the effect of HARP on migration and tube formation on matrigel of HUVEC and investigated the signalling pathway induced by HARP. We report that HARP induces migration and differentiation of endothelial cells through binding to RPTPβ/ζ, leading to activation of Src, FAK, PI3K and Erk½. Sodium orthovanadate, chondroitin sulfate-C, PP1, wortmannin, LY294002 and U0126 inhibit HARP-mediated signalling and HARP-induced HUVEC migration and differentiation. In addition, RPTPβ/ζ suppression using siRNA technology, interrupts intracellular signals, as well as HUVEC migration and differentiation that are induced by HARP. These results establish the role of RPTPβ/ζ as a receptor of HARP in HUVEC and elucidate the HARP signalling pathway in endothelial cells.Η Heparin affin regulatory peptide (HARP) είναι ένας αυξητικός παράγοντας με μοριακό βάρος 18 kDa, που έχει μεγάλη συγγένεια με την ηπαρίνη. Είναι συντηρημένη μεταξύ διαφόρων ειδών και παρουσιάζει 50% ομολογία με τη Midkine και την RI-HBP. Οι πρωτεΐνες αυτές συγκροτούν μια σχετικά νέα οικογένεια αυξητικών παραγόντων που έχουν συγγένεια με την ηπαρίνη. Η HARP απομονώθηκε για πρώτη φορά από τον εγκέφαλο νεογέννητου βοός ως ένα μόριο που μπορεί να επάγει την προέκταση των νευρικών κυττάρων. Επίσης, εκφράζεται στη μήτρα, στους χόνδρους και στα οστά. Αρκετές αναφορές αποδεικνύουν ότι υπάρχει μεγάλη συσχέτιση μεταξύ της έκφρασης της HARP και της ανάπτυξης καρκινικού όγκου και της αγγειογένεσης. Υψηλά επίπεδα της πρωτεΐνης έχουν ανιχνευθεί σε πολλούς καρκινικούς όγκους, αλλά και κυτταρικές σειρές που προέρχονται από διάφορους τύπους καρκίνου σε ανθρώπους. Η HARP αποτελεί μιτογόνο παράγοντα για διάφορους τύπους ενδοθηλιακών κυττάρων, ενώ μπορεί να επάγει την αγγειογένεση in vivo και in vitro. Ασκεί τη βιολογική της δράση μετά από αλληλεπίδραση με πρωτεογλυκάνες της επιφάνειας του κυττάρου, όπως η N-συνδεκάνη, ή μετά από δέσμευση σε πιο ειδικούς υποδοχείς. Η RPTPβ/ζ, η εκκρινόμενη μορφή της (φωσφακάνη), αλλά και η κινάση ALK, έχει αναφερθεί ότι μπορούν να δεσμεύουν τη HARP και να συμμετέχουν στη μεταγωγή του σήματός της. Παλαιότερες αναφορές έχουν δείξει ότι η HARP μπορεί να επάγει τις MAP-κινάσες και το μονοπάτι PI3K-Akt, ενώ αναστολείς των Erk½, ή της PI3K καταστέλλουν τη σύνθεση του DNA που επάγεται από τη HARP. Επιπλέον, η Shc και οι Erk ½ φωσφορυλιώνονται μετά από επώαση κυττάρων με HARP. Ωστόσο, τα ενδοκυτταρικά σήματα ειδικών υποδοχέων της HARP προς την PI3K ή τις MAPK δεν έχουν ακόμα χαρακτηριστεί ικανοποιητικά. Στην εργασία αυτή μελετήσαμε την επίδραση της HARP στη μετανάστευση κυττάρων HUVEC, στη δημιουργία αυλών σε υπόστρωμα matrigel, καθώς και το μονοπάτι μεταγωγής σήματος που ενεργοποιείται από τη HARP. Τα αποτελέσματά μας δείχνουν ότι η HARP επάγει τη μετανάστευση και τη διαφοροποίηση των ενδοθηλιακών κυττάρων HUVEC μετά από δέσμευσή της στην RPTPβ/ζ. Η δέσμευση αυτή οδηγεί σε ενεργοποίηση της Src, της FAK, της PI3K και των Erk ½. Το ορθοβαναδικό νάτριο, η θειική χονδροϊτίνη-C, το ΡΡ1, η wortmannin, το LY294002 και το U0126 αναστέλλουν τη μεταγωγή σήματος της HARP, καθώς και την επαγωγή της μετανάστευσης και διαφοροποίησης των HUVEC. Επιπλέον, η μείωση της έκφρασης της RPTPβ/ζ με τη χρησιμοποίηση παρεμβαλλόμενου RNA παρεμποδίζει τα ενδοκυτταρικά σήματα, καθώς και την επαγωγή της μετανάστευσης και της διαφοροποίησης που επάγεται από τη HARP. Τα αποτελέσματα αυτά δείχνουν ότι η RPTPβ/ζ αποτελεί υποδοχέα της HARP σε ενδοθηλιακά κύτταρα και αποσαφηνίζουν το μονοπάτι μεταγωγής σήματος της HARP στα κύτταρα αυτά

Smooth muscle cell specific NEMO deficiency inhibits atherosclerosis in ApoE(-/-) mice

The development of atherosclerotic plaques is the result of a chronic inflammatory response coordinated by stromal and immune cellular components of the vascular wall. While endothelial cells and leukocytes are well-recognised mediators of inflammation in atherosclerosis, the role of smooth muscle cells (SMCs) remains incompletely understood. Here we aimed to address the role of canonical NF-kappa B signalling in SMCs in the development of atherosclerosis. We investigated the role of NF-kappa B signalling in SMCs in atherosclerosis by employing SMC-specific ablation of NEMO, an IKK complex subunit that is essential for canonical NF-kappa B activation, in ApoE(-/-) mice. We show that SMC-specific ablation of NEMO (NEMOSMCiKO) inhibited high fat diet induced atherosclerosis in ApoE(-/-) mice. NEMOSMCiKO/ApoE(-/-) mice developed less and smaller atherosclerotic plaques, which contained fewer macrophages, decreased numbers of apoptotic cells and smaller necrotic areas and showed reduced inflammation compared to the plaques of ApoE(-/-) mice. In addition, the plaques of NEMOSMCiKO/ApoE(-/-) mice showed higher expression of alpha-SMA and lower expression of the transcriptional factor KLF4 compared to those of ApoE(-/-) mice. Consistently, in vitro, NEMO-deficient SMCs exhibited reduced proliferation and migration, as well as decreased KLF4 expression and lower production of IL-6 and MCP-1 upon inflammatory stimulus (TNF or LPS) compared to NEMO-expressing SMCs. In conclusion, NEMO-dependent activation of NF-kappa B signalling in SMCs critically contributes to the pathogenesis of atherosclerosis by regulating SMC proliferation, migration and phenotype switching in response to inflammatory stimuli

Smooth muscle cell specific NEMO deficiency inhibits atherosclerosis in ApoE−/− mice

Abstract The development of atherosclerotic plaques is the result of a chronic inflammatory response coordinated by stromal and immune cellular components of the vascular wall. While endothelial cells and leukocytes are well-recognised mediators of inflammation in atherosclerosis, the role of smooth muscle cells (SMCs) remains incompletely understood. Here we aimed to address the role of canonical NF-κB signalling in SMCs in the development of atherosclerosis. We investigated the role of NF-κB signalling in SMCs in atherosclerosis by employing SMC-specific ablation of NEMO, an IKK complex subunit that is essential for canonical NF-κB activation, in ApoE −/− mice. We show that SMC-specific ablation of NEMO (NEMOSMCiKO) inhibited high fat diet induced atherosclerosis in ApoE −/− mice. NEMOSMCiKO/ApoE −/− mice developed less and smaller atherosclerotic plaques, which contained fewer macrophages, decreased numbers of apoptotic cells and smaller necrotic areas and showed reduced inflammation compared to the plaques of ApoE −/− mice. In addition, the plaques of NEMOSMCiKO/ApoE −/− mice showed higher expression of α-SMA and lower expression of the transcriptional factor KLF4 compared to those of ApoE −/− mice. Consistently, in vitro, NEMO-deficient SMCs exhibited reduced proliferation and migration, as well as decreased KLF4 expression and lower production of IL-6 and MCP-1 upon inflammatory stimulus (TNF or LPS) compared to NEMO-expressing SMCs. In conclusion, NEMO-dependent activation of NF-κB signalling in SMCs critically contributes to the pathogenesis of atherosclerosis by regulating SMC proliferation, migration and phenotype switching in response to inflammatory stimuli

Differential role of MyD88 and TRIF signaling in myeloid cells in the pathogenesis of autoimmune diabetes

Type 1 diabetes (T1D) is caused by the autoimmune destruction of the insulin-producing pancreatic beta cells. While the role of adaptive immunity has been extensively studied, the role of innate immune responses and particularly of Toll-like Receptor (TLR) signaling in T1D remains poorly understood. Here we show that myeloid cell-specific MyD88 deficiency considerably protected mice from the development of streptozotocin (STZ)-induced diabetes. The protective effect of MyD88 deficiency correlated with increased expression of the immunoregulatory enzyme indoleamine 2,3-dioxygenase (IDO) in pancreatic lymph nodes from STZ-treated mice and in bone marrow-derived dendritic cells (BMDC) stimulated with apoptotic cells. Mice with myeloid cell specific TIR-domain-containing adapter-inducing interferon-beta (TRIF) knockout showed a trend towards accelerated onset of STZ-induced diabetes, while TRIF deficiency resulted in reduced IDO expression in vivo and in vitro. Moreover, myeloid cell specific MyD88 deficiency delayed the onset of diabetes in Non-Obese Diabetic (NOD) mice, whereas TRIF deficiency had no effect. Taken together, these results identify MyD88 signaling in myeloid cells as a critical pathogenic factor in autoimmune diabetes, which is antagonized by TRIF-dependent responses. This differential function of MyD88 and TRIF depends at least in part on their opposite effects in regulating IDO expression in phagocytes exposed to apoptotic cells

Conditional Targeting of Tumor Necrosis Factor Receptor-Associated Factor 6 Reveals Opposing Functions of Toll-Like Receptor Signaling in Endothelial and Myeloid Cells in a Mouse Model of Atherosclerosis

Background-Previous studies implicated Toll-like receptor signaling as a critical pathogenic pathway in atherosclerosis, but the cell-specific mechanisms by which Toll-like receptors act to control atherosclerotic plaque development remain poorly understood. Methods and Results-To study the cell-specific role of tumor necrosis factor receptor-associated factor 6 (TRAF6) in atherosclerosis, we generated ApoE(-/-) mice with endothelial cell-or myeloid cell-specific TRAF6 deficiency using Cre/LoxP-mediated gene targeting. Endothelial TRAF6 deficiency reduced atherosclerosis in female ApoE(-/-) mice by inhibiting nuclear factor-kappa B-dependent proinflammatory gene expression and monocyte adhesion to endothelial cells. In contrast, myeloid cell-specific TRAF6 deficiency caused exacerbated atherosclerosis, with larger plaques containing more necrotic areas in both male and female ApoE(-/-) mice. TRAF6-deficient macrophages showed impaired expression of the antiinflammatory and atheroprotective cytokine interleukin-10, elevated endoplasmic reticulum stress, increased sensitivity to oxidized low-density lipoprotein-induced apoptosis, and reduced capacity to clear apoptotic cells. Thus, the reduced antiinflammatory properties, coupled with increased sensitivity to apoptosis and impaired efferocytosis capacity of TRAF6-deficient macrophages, result in exacerbated atherosclerosis development in TRAF6(MYKO)/ApoE(-/-) mice. Conclusion-Toll-like receptor-mediated TRAF6 signaling acts in endothelial cells to promote atherosclerosis but displays atheroprotective, antiinflammatory and prosurvival functions in myeloid cells. (Circulation. 2012;126:1739-1751.

Development of autoimmune diabetes in NOD mice with myeloid- specific MyD88 or TRIF deficiency.

<p>(A and C) Graphs depicting the incidence of diabetes in mice with the indicated genotypes. (B and D) Representative images and quantification of H&E stained paraffin sections of pancreatic tissue from 10-week-old mice with the indicated genotypes. Graph depicts the percentage of mice with a given histology score per genotype: 0 or I, no islet infiltrates or only small peri-islet infiltrates; II, invasive insulitis (<50% of islet area); III, severe invasive insulitis (>50% of islet area). 20–30 islets per mouse were counted. NOD.MyD88^CD11c-KO (n = 8), NOD.MyD88^LysM-KO (n = 9), NOD.TRIF^CD11c-KO (n = 7) and NOD.TRIF^LysM-KO (n = 9), <i>NOD</i>.<i>Myd88</i>^FL (n = 13), and <i>NOD</i>.<i>Trif</i>^FL (n = 12).</p