Estradiol downregulates NF-κb translocation by Ikbkg transcriptional repression in dendritic cells.

To reconcile immunity and reproduction, females must allow spermatozoa to survive and control the presence of commensal microbiota and sexually transmitted pathogens during ovulation. Female steroid sex hormones exert a powerful effect on the immune system, as do the hormonal changes associated with the ovarian cycle. Dendritic cells (DCs) are immunological sentinels that link innate immunity to adaptive immunity. Upon exposure to microbial invaders in tissue, they undergo a maturational process that culminates in the lymph nodes and activates T-cell-specific immune responses. Estradiol, which is highly expressed during ovulation, has an effect on the maturation of DCs, although the molecular mechanism remains elusive. We detected that estradiol regulates expression of Ikbkg in DCs and modulates nuclear factor-κb translocation to the nucleus, thus explaining the reduced DC function observed during ovulation. This change may be an adaptive mechanism to reconcile control of infection and reproductive functions.

Biogenic amines in fermented foods.

Food-fermenting lactic acid bacteria (LAB) are generally considered to be non-toxic and non-pathogenic. Some species of LAB, however, can produce biogenic amines (BAs). BAs are organic, basic, nitrogenous compounds, mainly formed through decarboxylation of amino acids. BAs are present in a wide range of foods, including dairy products, and can occasionally accumulate in high concentrations. The consumption of food containing large amounts of these amines can have toxicological consequences. Although there is no specific legislation regarding BA content in many fermented products, it is generally assumed that they should not be allowed to accumulate. The ability of microorganisms to decarboxylate amino acids is highly variable, often being strain specific, and therefore the detection of bacteria possessing amino acid decarboxylase activity is important to estimate the likelihood that foods contain BA and to prevent their accumulation in food products. Moreover, improved knowledge of the factors involved in the synthesis and accumulation of BA should lead to a reduction in their incidence in foods.

AM3, a natural glycoconjugate, induces the functional maturation of human dendritic cells.

BACKGROUND AND PURPOSE: Dendritic cells (DCs) are dedicated antigen-presenting cells able to initiate specific immune responses and their maturation is critical for the induction of antigen-specific T-lymphocyte responses. Here, we have investigated the effects of Inmunoferon-active principle (AM3), the active agent of a commercial immunomodulatory drug, on human monocyte-derived DCs (MDDCs). EXPERIMENTAL APPROACH: MDDCs derived from healthy and hepatitis C virus (HCV)-infected patients were stimulated with AM3. We analysed the expression of cell surface proteins by flow cytometry, that of cytokine production by ELISA, and the expression of chemokines and chemokine receptors by RNase protection assays. T-lymphocyte proliferation was assessed in mixed lymphocyte reactions, protein expression by western blot and luciferase-based reporter methods, and Toll-like receptor (TLR)-blocking antibodies were employed to analyse TLR activity. KEY RESULTS: In MDDCs, AM3 induced or enhanced expression of CD54, CD83, CD86, HLA-DR, chemokines and chemokine receptors, interleukin (IL)-12p70 and IL-10. Furthermore, AM3 stimulated MDDCs to increase proliferation of allogenic T cells. AM3 triggered nuclear translocation of NF-kappaB and phosphorylation of p38 mitogen-activated protein kinase. AM3 promoted NF-kappaB activation in a TLR-4-dependent manner, and blocking TLR-4 activity attenuated the enhanced expression of CD80, CD83 and CD86 induced by AM3. AM3 enhanced the expression of maturation-associated markers in MDDCs from HCV-infected patients and increased the proliferation of T lymphocytes induced by these MDDCs. CONCLUSIONS AND IMPLICATIONS: These results underline the effects of AM3 in promoting maturation of MDDCs and suggest that AM3 might be useful in regulating immune responses in pathophysiological situations requiring DC maturation.

Dendritic cells: still a promising tool for cancer immunotherapy.

Dendritic cells are bone marrow-derived professional antigen-presenting cells that exert critical functions in innate and adaptive immune responses. Depending on their functional maturation status, dendritic cells trigger primary immune responses or promote immunological tolerance. This functional ambivalence has taken dendritic cells into the focus of attention of immunotherapy protocols for both vaccination and tolerance induction. The capacity of dendritic cells to generate anti-tumour immune responses has already been demonstrated, and numerous clinical trials are currently in progress to assess their therapeutic potential. In the present review we will briefly outline the types and effector functions of dendritic cells in the human system, and summarise the present state of anti-tumour immunotherapy protocols, emphasising the most relevant parameters currently evaluated in preclinical and clinical assays.

Dendritic cells: still a promising tool for cancer immunotherapy

Dendritic cells are bone marrow-derived professional antigen-presenting cells that exert critical functions in innate and adaptive immune responses. Depending on their functional maturation status, dendritic cells trigger primary immune responses or promote immunological tolerance. This functional ambivalence has taken dendritic cells into the focus of attention of immunotherapy protocols for both vaccination and tolerance induction. The capacity of dendritic cells to generate anti-tumour immune responses has already been demonstrated, and numerous clinical trials are currently in progress to assess their therapeutic potential. In the present review we will briefly outline the types and effector functions of dendritic cells in the human system, and summarise the present state of anti-tumour immunotherapy protocols, emphasising the most relevant parameters currently evaluated in preclinical and clinical assays (AU)

Extracellular signal-regulated protein kinase signaling pathway negatively regulates the phenotypic and functional maturation of monocyte-derived human dendritic cells.

Dendritic cells (DC) are highly specialized antigen-presenting cells that on activation by inflammatory stimuli (eg, tumor necrosis factor alpha [TNF-alpha] and interleukin-1beta [IL-1beta]) or infectious agents (eg, lipopolysaccharide [LPS]), mature and migrate into lymphoid organs. During maturation, DC acquire the capacity to prime and polarize resting naive T lymphocytes. Maturation of monocyte-derived DC (MDDC) is inhibited by the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580. This study found that in the presence of the mitogen-activated protein kinase kinase 1-extracellular signal-regulated kinase (ERK) inhibitors PD98059 or U0126, TNF-alpha- and LPS-induced phenotypic and functional maturation is enhanced. ERK pathway inhibitors increased expression of major histocompatibility complex and costimulatory molecules; loss of mannose-receptor-mediated endocytic activity; nuclear factor-kappaB DNA-binding activity; release of IL-12 p40; and allogeneic T-cell proliferation induced by LPS or TNF-alpha. Moreover, PD98059 and U0126 enhanced LPS-triggered production of IL-12 p70. In agreement with the effect of ERK inhibitors, maturation of MDDC was delayed in the presence of serum, an effect that was reversed by U0126. These results indicate that the ERK and p38 MAPK signaling pathways differentially regulate maturation of MDDC and suggest that their relative levels of activation might modulate the initial commitment of naive T-helper (Th) cells toward Th1 or Th2 subsets. The findings also suggest that maturation of MDDC might be pharmacologically modified by altering the relative levels of activation of both intracellular signaling routes.

Synergistic cooperation between hypoxia and transforming growth factor-beta pathways on human vascular endothelial growth factor gene expression.

Signaling by transforming growth factor (TGF)-beta family members is mediated by Smad proteins that regulate gene transcription through functional cooperativity and association with other DNA-binding proteins. The hypoxia-inducible factor (HIF)-1 is a transcriptional complex that plays a key role in oxygen-regulated gene expression. We demonstrate that hypoxia and TGF-beta cooperate in the induction of the promoter activity of vascular endothelial growth factor (VEGF), which is a major stimulus in the promotion of angiogenesis. This cooperation has been mapped on the human VEGF promoter within a region at -1006 to -954 that contains functional DNA-binding sequences for HIF-1 and Smads. Optimal HIF-1alpha-dependent induction of the VEGF promoter was obtained in the presence of Smad3, suggesting an interaction between these proteins. Consistent with this, co-immunoprecipitation experiments revealed that HIF-1alpha physically associates with Smad3. These results demonstrate that both TGF-beta and hypoxia signaling pathways can synergize in the regulation of VEGF gene expression at the transcriptional level.

Identification of a critical Sp1 site within the endoglin promoter and its involvement in the transforming growth factor-beta stimulation.

Endoglin, a component of the transforming growth factor-beta (TGF-beta) receptor complex expressed on endothelial cells, is involved in cardiovascular morphogenesis and vascular remodeling, as exemplified by the fact that the endoglin gene is the target for the autosomal dominant disorder known as hereditary hemorrhagic telangiectasia type 1. Since haploinsufficiency is the underlying mechanism for hereditary hemorrhagic telangiectasia type 1, understanding the regulation of endoglin gene expression appears to be a crucial step to correct the disease. In this study we have identified an Sp1 site at -37 as a critical element for the basal transcription of the endoglin TATA-less promoter. Since endoglin promoter activity is stimulated by TGF-beta and this stimulation is located at the Sp1-containing proximal region, we have investigated the possible involvement of Sp1 in the TGF-beta-mediated induction. Mutation of the Sp1-binding sequence, or addition of the Sp1 inhibitor WP631, abolished both the basal transcription activity and the TGF-beta responsiveness of the endoglin promoter. Binding of Sp1 and Smad3 to the proximal promoter region -50/-29 was evidenced by electrophoretic mobility shift assays and DNA affinity precipitation studies. Furthermore, synergistic cooperation on the promoter activity between Sp1 and TGF-beta or Smad3 could be demonstrated by co-transfection experiments of reporter promoter constructs. The molecular mechanism underlying this cooperation appears to involve a direct physical interaction between Sp1 and Smad3/Smad4.

Maturation-dependent expression and function of the CD49d integrin on monocyte-derived human dendritic cells.

Dendritic cells (DC) are highly specialized APC that are critical for the initiation of T cell-dependent immune responses. DC exert a sentinel function while immature and, after activation by inflammatory stimuli or infectious agents, mature and migrate into lymphoid organs to prime T cells. We have analyzed integrin expression on monocyte-derived DC (MDDC) and found that expression of CD49d integrins (CD49d/CD29 and CD49d/beta7) was induced/up-regulated during TNF-alpha- or LPS-initiated MDDC maturation, reflecting the induction/up-regulation of CD49d and beta7 mRNA. CD49d mRNA steady-state level increased more than 10 times during maturation, with the highest levels observed 24 h after TNF-alpha treatment. CD49d integrin expression conferred mature MDDC with an elevated capacity to adhere to the CS-1 fragment of fibronectin, and also mediated transendothelial migration of mature MDDC. Up-regulation of CD49d integrin expression closely paralleled that of the mature DC marker CD83. CD49d integrin expression was dependent on cell maturation, as its induction was abrogated by N:-acetylcysteine, which inhibits NF-kappaB activation and the functional and phenotypic maturation of MDDC. Moreover, CD49d integrin up-regulation and MDDC maturation were prevented by SB203580, a specific inhibitor of p38 mitogen-activated protein kinase, but were almost unaffected by the mitogen-activated protein/extracellular signal-related kinase kinase 1/2 inhibitor PD98059. Our results support the existence of a link between functional and phenotypic maturation of MDDC and CD49d integrin expression, thus establishing CD49d as a maturation marker for MDDC. The differential expression of CD49d on immature and mature MDDC might contribute to their distinct motility capabilities and mediate mature DC migration into lymphoid organs.

c-Myc inhibits CD11a and CD11c leukocyte integrin promoters.

The c-Myc transcription factor is an important regulator of cell growth and differentiation, and its gene repression ability seems to play a key role in Myc-mediated cellular transformation. Since Myc overexpression has been associated with reduced expression of beta1 and beta2 integrins, we have investigated the role of c-Myc on CD11a and CD11c transcription. c-Myc inhibited CD11a and CD11c integrin promoter activity in co-transfection experiments, and similar repression was obtained in cells where c-Myc expression (KmycB) or activity (Rat-1 c-MycER) is inducible. The c-Myc repression on the CD11c promoter was independent of the USF-binding site (USF-150), other putative Myc-binding elements, or the integrity of the initiator (Inr)-like sequence present at the major transcriptional start site. Analysis of deletion and mutant promoter constructs revealed that, in the absence of additional upstream cisacting elements, an AP-1-binding site at -60 (AP1-60) is required for c-Myc repressor activity. The c-Myc repressor activity on both integrin promoters was abrogated by deletion of c-Myc residues 106-143, a domain involved in Inr-dependent transcriptional repression. These results demonstrate a direct effect of c-Myc on integrin gene transcription and suggest the existence of a c-Myc-dependent mechanism for coupling leukocyte integrin expression to the cell proliferative state.

Polyomavirus enhancer-binding protein 2/core binding factor/acute myeloid leukemia factors contribute to the cell type-specific activity of the CD11a integrin gene promoter.

The CD11a/CD18 leukocyte integrin (LFA-1; also known as alphaL/beta2) mediates leukocyte transendothelial migration during immune and inflammatory responses and participates in lymphoma metastasis. CD11a/CD18 leukocyte-restricted expression is controlled by the CD11a gene promoter, which confers tissue-specific expression to reporter genes in vitro and in vivo. DNase I protection analysis of the CD11a proximal gene promoter revealed DNA-protein interactions centered at position -110 (CD11a-110). Disruption of CD11a-110 reduced CD11a promoter activity in a cell type-specific manner, as it reduced its activity by 70% in Jurkat lymphoid cells, whereas the effect was considerably lower in K562 and HepG2 cells. Electrophoretic mobility shift assays showed evidence of cell type-specific differences in CD11a-110 binding and indicated its specific recognition by members of the polyomavirus enhancer-binding protein 2/core binding factor (CBF)/acute myeloid leukemia (AML) family of transcription factors. AML1B/CBFbeta transactivated the CD11a promoter, with AML1B/CBFbeta-mediated transactivation being completely dependent on the integrity of the CD11a-110 element. Therefore, CBF/AML factors play a role in the cell type-restricted transcription of the CD11a integrin gene through recognition of CD11a-110. The involvement of CBF/AML factors in CD11a expression raises the possibility that CD11a/CD18 expression might be deregulated in acute myeloid and B-lineage acute lymphoblastic leukemias, thus contributing to their altered adhesion and metastatic potential.

[Life events, quality of life and social support prior to acute cerebral vascular disease, as predictive factors of the quality of life and functional state following stroke]. / Acontecimientos vitales, calidad de vida y apoyo social previos a la enfermedad vascular cerebral aguda, como factores predictores de la calidad de vida y situación funcional posteriores al ictus.

INTRODUCTION: The repercussion of stroke on quality of life has been evaluated but not the possible relation between the quality of life before and months after an acute stroke. OBJECTIVE: To study the possible relation between quality of life, social support, stressful life events prior to the stroke and quality of life, social support and functional state months after. PATIENTS AND METHODS: A prospective study was made of 34 patients (71.7 +/- 8 years; 19 (56%) men; 15 (44%) women with stroke, by means of two evaluations: personal interview within the first 36 hours (quality of life--Nothingham health profile (NHP)-, perception of social support and stressful life events--Holmes and Rake inventory-) and an interview over the phone 16.5 +/- 5.3 months after the stroke (NHP, perception of social support and functional state--Rankin scale-). RESULTS: Following the stroke there was deterioration in perception of social support (19.8 +/- 3 vs 12.5 +/- 8; p = 0.000) and in the degree of social isolation of the NHP (9.4 +/- 20 vs 21.1 +/- 30; p = 0.03). The only relation found was between the following variables: pain at the first evaluation and pain (r = 0.45; p = 0.007) at the second evaluation; mobility at the first evaluation and emotional state (r = 0.39; p = 0.029) and social support (r = 0.37; p = 0.027) at the second evaluation; sleepiness at the first evaluation and energy (r = 0.55; p = 0.0006), pain (r = 0.39; p = 0.022), emotional state (r = 0.35; p = 0.038), mobility (r = 0.34, p = 0.048) and sleepiness (r = 0.51; p = 0.001) at the second evaluation. CONCLUSION: Our results indicate that there is little relationship between the previous state and that following stroke, and that the deterioration in perception of support and social isolation is due to the stroke itself.

The alpha2 and alpha5 integrin genes: identification of transcription factors that regulate promoter activity in epidermal keratinocytes.

We analysed the activity of the proximal promoters of the alpha2 and alpha5 integrin genes in human keratinocytes. An AP-1 site, found in the alpha5 but not the alpha2 promoter, bound c-Jun/c-Fos dimers and contributed strongly to promoter activity. Both promoters had a CCAAT/enhancer binding protein (C/EBP) binding site: the alpha5 C/EBP element enhanced activity, while the alpha2 site was a negative regulatory element. C/EBP overexpression repressed the activity of both promoters, but the effect was independent of occupancy of the identified C/EBP binding sites, suggesting interactions with additional transcription factors. We propose that upregulation of C/EBPs contributes to the inhibition of integrin transcription during keratinocyte terminal differentiation, while AP-1 factors play a role in the selective induction of the alpha5 gene during wound healing.

Acontecimientos vitales, calidad de vida y apoyo social previos a la enfermedad vascular cerebral aguda, como factores predictores de la calidad de vida y situación funcional posteriores al ictus / Live events quality of life and social support prior to acute cerebral vascular disease, as predictive factors of the quality of life and functional state following stroke

Introducción. Se ha valorado la repercusión del ictus en la calidad de vida pero no la posible relación entre la calidad de vida previa y la que tienen los pacientes meses después del episodio agudo. Objetivo. Estudiar la posible relación entre calidad de vida, apoyo social y acontecimientos vitales estresantes previos al ictus y calidad de vida, apoyo social y situación funcional meses después del mismo. Pacientes y métodos. Estudio prospectivo de 34 pacientes (71,7 ± 8 años; 19 (56%) varones; 15 (44%) mujeres) con ictus mediante dos valoraciones: entrevista personal en las primeras 36 horas (calidad de vida –perfil de salud de Nottingham (PSN)–, percepción de apoyo social y acontecimientos vitales estresantes –inventario de Holmes y Rake–) y entrevista telefónica 16,5 ± 5,3 meses después del ictus (PSN, percepción de apoyo social y situación funcional –escala de Rankin–). Resultados. Después del ictus se produce un deterioro en la percepción del apoyo social (19,8 ± 3 frente a 12,5 ± 8; p= 0,000) y en la dimensión de aislamiento social del PSN (9,4 ± 20 frente a 21,1 ± 30; p= 0,03). Sólo se encontró relación entre las siguientes variables: dolor en la primera evaluación y dolor (r= 0,45; p= 0,007) en la segunda evaluación; movilidad en la primera evaluación y estado emocional (r= 0,39; p= 0,029) y apoyo social (r= 0,37; p= 0,027) en la segunda evaluación; sueño en la primera evaluación y energía (r= 0,55; p= 0,0006), dolor (r= 0,39; p= 0,022), estado emocional (r= 0,35; p= 0,038), movilidad (r= 0,34; p= 0,048) y sueño (r= 0,51; p= 0,001) en la segunda evaluación. Conclusión. Nuestros resultados sugieren que la relación existente entre la situación previa y la posterior al ictus es escasa, y que el deterioro en la percepción del apoyo y el aislamiento social se deben al ictus en sí mismo (AU)

Introduction. The repercussion of stroke on quality of life has been evaluated but not the possible relation between the quality of life before and months after an acute stroke. Objective. To study the possible relation between quality of life, social support, stressful life events prior to the stroke and quality of life, social support and functional state months after. Patients and methods. A prospective study was made of 34 patients (71.7 ± 8 years; 19 (56%) men; 15 (44%) women with stroke, by means of two evaluations: personal interview within the first 36 hours (quality of life –Nothingham health profile (NHP)–, perception of social support and stressful life events –Holmes and Rake inventory–) and an interview over the phone 16.5 ± 5.3 months after the stroke (NHP, perception of social support and functional state –Rankin scale–). Results. Following the stroke there was deterioration in perception of social support (19.8 ± 3 vs 12.5 ± 8; p= 0.000) and in the degree of social isolation of the NHP (9.4 ± 20 vs 21.1 ± 30; p= 0.03). The only relation found was between the following variables: pain at the first evaluation and pain (r= 0.45; p= 0.007) at the second evaluation; mobility at the first evaluation and emotional state (r= 0.39; p= 0.029) and social support (r= 0.37; p= 0.027) at the second evaluation; sleepiness at the first evaluation and energy (r= 0.55; p= 0.0006), pain (r= 0.39; p= 0.022), emotional state (r= 0.35; p= 0.038), mobility (r= 0.34, p= 0.048) and sleepiness (r= 0.51; p= 0.001) at the second evaluation. Conclusion. Our results indicate that there is little relationship between the previous state and that following stroke, and that the deterioration in perception of support and social isolation is due to the stroke itself (AU)

Identification of a functional NF-kappa B site in the platelet endothelial cell adhesion molecule-1 promoter.

Platelet endothelial cell adhesion molecule-1 (PECAM-1) is a type I transmembrane adhesion protein of 130 kDa that belongs to a subgroup of the Ig gene superfamily, characterized by the presence of immunoreceptor tyrosine-based inhibitory motifs. PECAM-1 is expressed in circulating platelets, monocytes, neutrophils, a selective subgroup of T cells, and in endothelial cells, where it is preferentially located at intercellular junctions and participates in leukocyte transmigratory processes. The identification of two consensus NF-kappa B sites within the PECAM-1 promoter led us to analyze their possible involvement in the PECAM-1 expression regulated by inflammatory stimuli. We found that surface expression and promoter activity of PECAM-1 in myeloid cells are regulated by modulators of NF-kappa B, including TNF-alpha, PMA, and pyrrolidine dithiocarbamate. Mobility shifts assays identified a specific NF-kappa B-binding element at +110/+120, whose mutation abolished the basal promoter activity of PECAM-1 and decreased NF-kappa B-dependent responses of the PECAM-1 gene promoter. Furthermore, cotransfection experiments with an expression vector encoding the p65 subunit of NF-kappa B showed transactivation of the PECAM-1 promoter. These results demonstrate that NF-kappa B can regulate the transcriptional activity of PECAM-1.

Icodextrin effluent leads to a greater proliferation than glucose effluent of human mesothelial cells studied ex vivo.

OBJECTIVE: To compare the effect of glucose (Glu) and icodextrin (Ico) dialysate on in vitro culture of mesothelial cells (MC) from peritoneal dialysis (PD) patients. DESIGN: Prospective, controlled comparative study on the effects of two PD solutions. SETTING: A tertiary-care public university hospital. PATIENTS: Sixteen PD patients regularly using Glu dialysate were asked to collect an 8-hour dwell peritoneal effluent on 2 different days, with an interval shorter than 7 days. In the first collection, 2.27% Glu solution and in the last, 7.5% Ico solution was infused. Human MC were isolated from the nocturnal peritoneal effluent bags and grown ex vivo. MAIN OUTCOME MEASURES: Mesothelial cell proliferative capacity ex vivo. RESULTS: Mesothelial cells were present in all patient dialysates except that of a single patient's Glu dialysate. The number of MC drained was similar with both solutions. After the initial culture reached confluence, MC were identified in 14 and 12 patients receiving Ico and Glu, respectively. However, in 1 patient using Ico and in 2 using Glu, the MC count at this stage was so low that further subculture could not be performed. Cells from Ico-derived solutions exhibited a higher degree of proliferation than cells from Glu-derived solutions. The morphology of MC was also different. Cells from drained effluent were typical in 11 patients using Glu solution in contrast with 14 patients using Ico. At confluence, the percentages of typical appearance were 50% and 92.9% (p < 0.05) in Glu and Ico respectively. CONCLUSIONS: Mesothelial cells taken from icodextrin effluent show a greater proliferation ex vivo than those taken from glucose effluent.

CCAAT-enhancer-binding proteins (C/EBP) regulate the tissue specific activity of the CD11c integrin gene promoter through functional interactions with Sp1 proteins.

The CD11c/CD18 integrin binds lipopolysaccharide, fibrinogen, and heparin, and mediates leukocyte adhesion, spreading, and migration. CD11c/CD18 is primarily found on myeloid cells and its expression is regulated during myeloid differentiation by transcriptional mechanisms acting on the CD11c gene promoter. We now describe that CCAAT/enhancer-binding proteins (C/EBP) contribute to the basal, tissue-specific and developmentally regulated activity of the CD11c promoter. A C/EBP-binding site within the CD11c promoter (CEBP-80) is bound by CEBPalpha in undifferentiated U937 cells and by C/EBPalpha- and C/EBPbeta-containing dimers in phorbol 12-myristate 13-acetate-differentiating cells, and its disruption decreased the CD11c promoter activity in a cell type-dependent manner. C/EBPalpha transactivated the CD11c promoter through the CEBP-80 element, and C/EBPalpha transactivation was also dependent on the Sp1-70- and Sp1-120 Sp1-binding sites. The -90/-50 fragment from the CD11c promoter, containing the adjacent CEBP-80, Sp1-70, and AP1-60 sites, differentially enhanced the activity of the minimal prolactin promoter in hematopoietic and epithelial cells. Altogether, these results demonstrate that C/EBP factors participate in the tissue-restricted and regulated expression of the CD11c/CD18 integrin through functional interactions with Sp1, suggest that Sp1-related factors modulate C/EBPalpha transcriptional activity on the CD11c promoter, and demonstrate the existence of a composite regulatory element recognized by C/EBP, Sp1, and AP-1 factors and whose enhancing effects are cell-type dependent.

PU.1 negatively regulates the CD11c integrin gene promoter through recognition of the major transcriptional start site.

CD11c integrin expression is restricted to myeloid cells and activated B lymphocytes, mainly through the collaborative action of Sp1 and members of the AP-1 and C/EBP transcription factor families on the proximal region of the CD11c gene promoter. While analyzing the role of an initiator-like sequence at the major transcriptional start site, an inverted consensus GGAA Ets binding site was identified as a negative regulatory element whose disruption increases the activity of the CD11c promoter. The GGAA element was specifically recognized by PU.1 in THP-1 monocytic cells and by PU.1 and GABP-related proteins in U937 promonocytic cells. Mutational analysis indicated that PU.1 recognition depends not only on the GGAA consensus element but also on flanking sequences. The functional relevance of PU.1 binding was assayed in transactivation experiments in HeLa cells, where PU.1 co-expression led to a significant decrease in the activity of the CD11c promoter, demonstrating that PU.1 inhibits the activity of the CD11c promoter through a PU.1 binding site located at the major transcriptional start site (PU1-5). The inhibitory action of PU.1 on CD11c is in contrast with its positive regulatory effect on the CD11b and CD18 integrin gene promoters, which might contribute to the differentially regulated expression of CD11b/CD18 and CD11c/CD18 during monocyte extravasation and terminal maturation. In addition, since PU.1 transcriptional activity correlates with macrophage proliferation, PU.1 might modulate CD11c gene transcription according to the proliferative state of the cell.

An octamer element functions as a regulatory element in the differentiation-responsive CD11c integrin gene promoter: OCT-2 inducibility during myelomonocytic differentiation.

The integrin CD11c/CD18 mediates leukocyte adhesion to endothelium and other cell types and is a receptor for LPS, iC3b, and fibrinogen. CD11c expression is restricted to myeloid and activated B cells, is regulated during leukocyte differentiation, and constitutes a diagnostic tool for hairy cell leukemia. Mapping of in vivo DNA-protein interactions in the CD11c proximal promoter revealed three adjacent myeloid-specific interactions, one of which lies on an octamer consensus sequence, ATTT GCAT (Oct185). Oct185 disruption increased the CD11c promoter activity while decreasing its myeloid differentiation responsiveness, indicating that Oct185 contributes to the activity of the CD11c promoter and suggesting that Oct185 is a negative regulatory element whose function changes during myeloid differentiation. Oct185 is recognized by the ubiquitous Oct-1 factor in all cell lineages and by Oct-2 in B lymphoid lineage cells. Unexpectedly, Oct-2 binding to Oct185 was induced de novo upon monocytic differentiation of U937 and HL-60 cells but not during HL-60 granulocytic differentiation, as determined by electrophoretic mobility shift assays and immunochemical studies, and Oct-2 complexes were also observed in cultured adherent monocytes. Western blotting showed that the pattern of Oct-2 isoforms in myeloid cells is similar to that seen in B cells. The Oct-2 up-regulated expression in differentiating myeloid cells and its binding to the Oct185 negative regulatory element suggests its involvement in the differentiation-regulated activity of the CD11c promoter and might represent an important parameter for the myeloid- and B cell-restricted expression of the CD11c/CD18 integrin and other molecules with similar patterns of expression.