Matrix metalloproteinase-9 induces a pro-angiogenic profile in chronic lymphocytic leukemia cells

27 p.-3 fig.-2 tab.Increased angiogenesis is commonly observed in chronic lymphocytic leukemia (CLL) tissues in correlation with advanced disease. CLL cells express pro- and anti-angiogenic genes and acquire a pro-angiogenic pattern upon interaction with the microenvironment. Because MMP-9 (a microenvironment component) plays important roles in solid tumor angiogenesis, we have studied whether MMP-9 influenced the angiogenic pattern in CLL cells. Immunofluorescence analyses confirmed the presence of MMP-9 in CLL tissues. MMP-9 interaction with CLL cells increased their MMP-9 expression and secretion into the medium. Accordingly, the conditioned media of MMP-9-primed CLL cells significantly enhanced endothelial cell proliferation, compared to control cells. MMP-9 also increased VEGF and decreased TSP-1 and Ang-2 expression, all at the gene and protein level, inducing a pro-angiogenic pattern in CLL cells. Mechanistic analyses demonstrated that downregulation of the selected gene TSP-1 by MMP-9 involved α4β1 integrin, Src kinase family activity and the STAT3 transcription factor. Regulation of angiogenic genes is a novel contribution of MMP-9 to CLL pathology.This work was supported by grants SAF2015-69180R from the Ministerio de Economía y Competitividad (Spain)/Fondo Europeo de Desarrollo Regional (FEDER), European Union; S2010/BMD-2314 from the Comunidad de Madrid/European Union (AGP.); and by the Concerted Research Actions (KU Leuven C1 Grant C16/17/010) and the Research Foundation of Flanders (FWO-Vlaanderen, to EUB, PEVdS, and GO).Peer reviewe

ICAP-1 loss impairs CD8+ thymocyte development and leads to reduced marginal zone B cells in mice

ICAP-1 regulates β1-integrin activation and cell adhesion. Here, we used ICAP-1-null mice to study ICAP-1 potential involvement during immune cell development and function. Integrin α4β1-dependent adhesion was comparable between ICAP-1-null and control thymocytes, but lack of ICAP-1 caused a defective single-positive (SP) CD8+ cell generation, thus, unveiling an ICAP-1 involvement in SP thymocyte development. ICAP-1 bears a nuclear localization signal and we found it displayed a strong nuclear distribution in thymocytes. Interestingly, there was a direct correlation between the lack of ICAP-1 and reduced levels in SP CD8+ thymocytes of Runx3, a transcription factor required for CD8+ thymocyte generation. In the spleen, ICAP-1 was found evenly distributed between cytoplasm and nuclear fractions, and ICAP-1–/– spleen T and B cells displayed upregulation of α4β1-mediated adhesion, indicating that ICAP-1 negatively controls their attachment. Furthermore, CD3+- and CD19+-selected spleen cells from ICAP-1-null mice showed reduced proliferation in response to T- and B-cell stimuli, respectively. Finally, loss of ICAP-1 caused a remarkable decrease in marginal zone B- cell frequencies and a moderate increase in follicular B cells. Together, these data unravel an ICAP-1 involvement in the generation of SP CD8+ thymocytes and in the control of marginal zone B-cell numbers

RUNX3 Regulates Intercellular Adhesion Molecule 3 (ICAM-3) Expression during Macrophage Differentiation and Monocyte Extravasation

The adhesion molecule ICAM-3 belongs to the immunoglobulin gene superfamily and functions as a ligand for the β2 integrins LFA-1, Mac-1 and αdβ2. The expression of ICAM-3 is restricted to cells of the hematopoietic lineage. We present evidences that the ICAM-3 gene promoter exhibits a leukocyte-specific activity, as its activity is significantly higher in ICAM-3+ hematopoietic cell lines. The activity of the ICAM-3 gene promoter is dependent on the occupancy of RUNX cognate sequences both in vitro and in vivo, and whose integrity is required for RUNX responsiveness and for the cooperative actions of RUNX with transcription factors of the Ets and C/EBP families. Protein analysis revealed that ICAM-3 levels diminish upon monocyte-derived macrophage differentiation, monocyte transendothelial migration and dendritic cell maturation, changes that correlate with an increase in RUNX3. Importantly, disruption of RUNX-binding sites led to enhanced promoter activity, and small interfering RNA-mediated reduction of RUNX3 expression resulted in increased ICAM-3 mRNA levels. Altogether these results indicate that the ICAM-3 gene promoter is negatively regulated by RUNX transcription factors, which contribute to the leukocyte-restricted and the regulated expression of ICAM-3 during monocyte-to-macrophage differentiation and monocyte extravasation

Caracterización de linfocitos T y células dentríticas en pacientes con enfermedad inflamatoria intestinal

Este trabajo recoge un análisis fenotípico y funcional de linfocitos T, de sangre periférica y de mucosa intestinal, de pacientes con Enfermedad Inflamatoria Intestinal (EII), así como de líneas de linfocitos T transformados con el "Herpesvirus saimiri" (T-HVS), procedentes de células de sangre periférica y de mucosa de estos pacientes. Al analizar células mononucleares de sangre periférica (CMSP), se ha encontrado una disminución de células CD45RA+ y CD28+ en pacientes con Colitis Ulcerosa (CU), pero no con enfermedad de Crohn (EC), con respecto a individuos sanos. Sin embargo, en "lamina propria" existe un menor procentaje de células CD3+, CD4+, CD25+, y CD86+, y un mayor porcentaje de células CD45RA+, CD28+ y CD80+ en pacientes con EII. Los estudios de proliferación celular con líneas T-HVS indican un estado de hiperreactividad de los linfocitos T CD8+ de sangre periférica de pacientes con EC, que no se parecia en CMSP sin infectar, mientras que en mucosa se observa una mayor capacidad proliferativa en pacientes con EC y CU, en respuesta a los estímulos utilizados. Por otra parte, se ha realizado un estudio de las distintas subpoblaciones de células dendríticas (DC) en diferentes localizaciones intestinales, tanto en individuos sanos como en pacientes con EII, encontrándose en placas de Peyer de pacientes con EII mayores porcentajes de DC CD11c+ y CD123+, y un menor porcentaje de DC CD11c+ en mucosa de colon

Gene expression profile induced by arsenic trioxide in chronic lymphocytic leukemia cells reveals a central role for heme oxygenase-1 in apoptosis and regulation of matrix metalloproteinase-9

19 p.-9 fig.-1 tab.CLL remains an incurable disease in spite of the many new compounds being tested. Arsenic trioxide (ATO) induces apoptosis in all CLL cell types and could constitute an efficient therapy. To further explore this, we have studied the gene expression profile induced by ATO in CLL cells. ATO modulated many genes, largely involved in oxidative stress, being HMOX1 the most upregulated gene, also induced at the protein level. ATO also increased MMP-9, as we previously observed, both at the mRNA and protein level. Using specific inhibitors, qPCR analyses, and gene silencing approaches we demonstrate that upregulation of MMP-9 by ATO involved activation of the p38 MAPK/AP-1 signaling pathway. Moreover, gene silencing HMOX1 or inhibiting HMOX1 activity enhanced p38 MAPK phosphorylation and c-jun expression/activation, resulting in transcriptional upregulation of MMP-9. Overexpression of HMOX1 or enhancement of its activity, had the opposite effect. Cell viability analyses upon modulation of HMOX1 expression or activity demonstrated that HMOX1 had a proapoptotic role and enhanced the cytotoxic effect of ATO in CLL cells. We have therefore identified a new mechanism in which HMOX1 plays a central role in the response of CLL cells to ATO and in the regulation of the anti-apoptotic protein MMP-9. Thus, HMOX1 arises as a new therapeutic target in CLL and the combination of HMOX1 modulators with ATO may constitute an efficient therapeutic strategy in CLL.This work was supported by grants SAF2012-31613 and SAF2015-69180R (AGP) and RTICC (Red Temática de Investigación Cooperativa en Cáncer) RD12/0036/0061 (AGP), from the Ministry of Economy and Competitiveness (MINECO), Spain; S2010/BMD-2314-Neoplasbim (AGP) from the Comunidad de Madrid/European Union; and a grant from the Fundación para la Investigación Biomédica Hospital Universitario Puerta de Hierro, Madrid (JAGM).Peer reviewe

Proteomic characterization of human proinflammatory M1 and anti-inflammatory M2 macrophages and their response to Candida albicans

In response to different stimuli, macrophages can differentiate into either a pro-inflammatory subtype (M1, classically activated macrophages) or acquire an anti-inflammatory phenotype (M2, alternatively activated macrophages). Candida albicans is the most important opportunistic fungus in nosocomial infections, and it is contended by neutrophils and macrophages during the first steps of the invasive infection. Murine macrophages responses to C. albicans have been widely studied, whereas the responses of human-polarized macrophages remain less characterized. In this study, we have characterized the proteomic differences between human M1- and M2-polarized macrophages, both in basal conditions and in response to C. albicans, by quantitative proteomics (2DE). This proteomic approach allowed us to identify metabolic routes and cytoskeletal rearrangement components that are the most relevant differences between M1 and M2 macrophages. The analysis has revealed fructose-1,6-bisphosphatase 1, a critical enzyme in gluconeogenesis, up-regulated in M1, as a novel protein marker for macrophage polarization. Regarding the response to C. albicans, an M1-to-M2 switch in polarization was observed. This M1-to-M2 switch might contribute to Candida pathogenicity by decreasing the generation of specific immune responses, thus enhancing fungal survival and colonization, or instead, may be part of the host attempt to reduce the inflammation and limit the damage of the infection

Identification and characterization of RUNX and C/EBP-binding elements within the ICAM-3 gene proximal regulatory region.

<p><b>A.</b> EMSA was performed on the indicated oligonucleotides spanning the −157/−14 region of the ICAM-3 promoter using nuclear extracts from THP-1, K-562 and Jurkat cells. The position of the major retarded species is indicated. <b>B.</b> EMSA was performed on the ICAM3.3 and ICAM3.5 oligonucleotides using nuclear extracts from the indicated COS-7 cells transfected with an empty expression vector (pCDNA3) or with either RUNX1 or RUNX3 together with CBF-β expression vector. The position of the RUNX1- and RUNX3-containing complex is shown. <b>C.</b> EMSA was performed on the ICAM3.5 and ICAM3.3 oligonucleotides using nuclear extracts from Jurkat cells in the absence (−) or presence of unlabeled competitor oligonucleotides (ICAM3.5, ICAM3.5mutRUNX, ICAM3.3, ICAM3.3mutRUNX, AMLcons) or polyclonal antisera against CD209 (Control antibody, Cnt Ab) or RUNX1 proteins (R-3034). The position of RUNX1-containing complexes are shown. Unlabeled competitor oligonucleotides were added at a 100-fold molar excess. <b>D.</b> EMSA was performed on the ICAM3.4 oligonucleotide using nuclear extracts from THP-1 cells in the absence (−) or presence of unlabeled competitor oligonucleotides (ICAM3.4, ICAM3.4mutCEBP, C/EBPcons) or polyclonal antibody against CD209 (Control antibody, Cnt Ab) or C/EBPα proteins (α-C/EBPα). The position of C/EBPα-containing complexes are shown. Unlabeled competitor oligonucleotides were added at a 100-fold molar excess. In A–D, EMSA's were performed twice with similar result and a representative experiment is shown. <b>E.</b> ICAM-3 promoter-based oligonucleotides with mutated nucleotides in lowercase and their relative positions. <b>F. </b><i>In vivo</i> occupancy of the ICAM-3 promoter by RUNX1. Chromatin immunoprecipitation on Jurkat cells was performed with an affinity-purified polyclonal antisera specific for RUNX1 or purified rabbit IgG. Immunoprecipitated chromatin was analyzed by PCR using a pair of ICAM-3 promoter-specific primers that amplify a 234-bp fragment flanking the RUNX-binding sites at −80 and −29. ChIP experiment was performed twice with similar results, and a representative experiment is shown.</p

Restricted expression of ICAM-3 and cell-specific activity of the ICAM-3 promoter.

<p><b>A.</b> Determination of ICAM-3 expression in BLM, Jurkat, K-562 and THP-1 cell lines by Western blot. As a control, β-actin expression levels were also determined. The experiment was performed twice and one of the experiments is shown. <b>B.</b> The ICAM-3 promoter-based constructs pCD50-1000, pCD50-500 and pCD50-200 were transfected in Jurkat (ICAM-3+), THP-1 (ICAM-3+), BLM (ICAM-3−) and K-562 (ICAM-3−) cell lines. After 48 hours cells were lysed and luciferase activity determined. For each reporter construct, promoter activity is expressed relative to the activity produced by the reporter plasmid in BLM cells, arbitrarily set to 1, after normalization for transfection efficiency. Data represent mean ± SD of 4 independent experiments using two different DNA preparations. (*p<0,05 for pCD50-1000Luc in THP-1 and Jurkat and p = 0.8 for K-562 when compared with the activity of pCD50-1000Luc in BLM cells; **p<0.005 for pCD50-500Luc in THP-1 and Jurkat and p = 0.5 for K-562 when compared with the activity of pCD50-500Luc in BLM cells; and * p<0.05 for pCD50-200Luc in THP-1 and Jurkat cells and p = 0.2 for K-562 cells when compared with the activity of pCD50-200Luc in BLM cells). <b>C.</b> Nucleotide sequence of the 5′-regulatory region of the ICAM-3 gene. The transcriptional initiation sites are identified by * and the major transcription initiation site is denoted by +1. First exon nucleotides are shown in boldface type and are underlined. The derived amino acid sequence is shown under the coding region of the first exon. Underlined areas correspond to consensus sequences for RUNX, C/EBP and Ets transcription factors.</p

Expression of ICAM-3, RUNX1 and RUNX3 during macrophage differentiation, monocyte transendothelial migration and DC maturation.

<p><b>A.</b> Left, <i>ICAM-3</i> and <i>FOLR2</i> mRNA expression levels along M-CSF monocyte-derived-macrophages, as determined by qRT-PCR at the indicated time points. Results are expressed as Relative mRNA levels (relative to <i>GAPDH</i> mRNA levels and the <i>ICAM-3</i> and <i>FOLR2</i> mRNA level on monocytes). Data represent mean ± SD of 3 independent donors (*P<0.05 compared with ICAM-3 mRNA level of monocytes). Right, ICAM-3, RUNX1 and RUNX3 expression on monocytes and M-CSF-polarized macrophages, as determined by Western blot at the indicated time points. As a control, GAPDH expression levels were also determined. The experiment was performed twice and one of the experiments is shown. <b>B.</b> Left, <i>ICAM-3</i> mRNA expression levels of monocytes (Mo.) and transendothelial migrated monocytes (Migrated Mo.), as determined by qRT-PCR. Results are expressed as Relative mRNA levels (relative to <i>GAPDH</i> mRNA levels and the <i>ICAM-3</i> mRNA level on monocytes). Data represent mean ± SD of 3 independent donors. Right, ICAM-3, RUNX1 and RUNX3 expression on monocytes (Mo. and transendothelial migrated monocytes (Migrated Mo.), as determined by Western blot at the indicated time points. As a control, GAPDH expression levels were also determined. The experiment was performed twice and one of the experiments is shown. <b>C.</b> Left, <i>ICAM-3</i> mRNA expression levels of DC either untreated (−) or treated with 10 ng/ml of LPS during 24 h (+) as determined by qRT-PCR. Results are expressed as Relative mRNA levels (relative to <i>GAPDH</i> mRNA levels and the <i>ICAM-3</i> mRNA level on untreated DC). Data represent mean ± SD of 3 independent donors. Right, ICAM-3 and RUNX3 expression on DC either untreated (−) or treated with 10 ng/ml of LPS during 24 h (+), as determined by Western blot. As a control, GAPDH expression levels were also determined. The experiment was performed twice and one of the experiments is shown.</p

RUNX factors regulate the activity of the ICAM-3 promoter through the recognition of both RUNX-binding sites.

<p><b>A.</b> K-562 cells were transfected with 1 µg of the indicated reporter plasmid in the presence of CMV-0 (empty expression vector), pCMV-RUNX1 or pCDNA3-RUNX3, and luciferase activity determined after 24 h. For each individual reporter construct, fold induction represents the luciferase activity yielded by an expression vector relative to the activity produced by a similar amount of CMV-0 plasmid. Data represent mean ± SD of 4 independent experiments using distinct DNA preparations. (*<i>P</i><0.005 compared with the activity of pCMV-0–transfected cells). <b>B.</b> K-562 cells were transfected with 1 µg of the indicated reporter plasmids in the presence of CMV-0, RUNX1/CBF-β or RUNX3/CBF-β expression plasmids, and luciferase activity determined after 24 h. (*<i>P</i><0.05 compared with the activity of pCD50-200Luc–in the presence of RUNX1/CBF-β or RUNX3/CBF-β, respectively). <b>C.</b> Schematic representation of the proximal regulatory region of the ICAM-3 gene and reporter plasmids used for its functional dissection. <b>D.</b> COS-7 cells were transfected with the indicated reporter plasmids in the presence of CMV-0, RUNX1/CBF-β or Ets-1 expression plasmids, and luciferase activity determined after 24 h. <b>E.</b> K-562 cells were transfected with 1 µg of the indicated reporter plasmids in the presence of CMV-0, RUNX1/CBF-β or C/EBPα42 expression plasmids, and luciferase activity determined after 24 h. In <b>B, D, E,</b> for each individual reporter construct, fold induction represents the luciferase activity yielded by an expression vector relative to the activity produced by a similar amount of CMV-0 plasmid. Data represent mean ± SD of 3 independent experiments.</p