Functional characterization of SDF-1 proximal promoter.

Stromal-cell derived factor 1 (SDF1) is a CXC chemokine that binds and signals through the CXCR4 receptor, playing an essential role in embryonic B lymphopoiesis, myelopoiesis and organogenesis. The CXCR4/SDF1 pathway is associated with several pathologies. CXCR4 serves as a fusion cofactor for lymphotropic strains of human immunodeficiency virus type 1 and SDF1 inhibits viral entry. Moreover, recent works suggest an important role for SDF1 in metastasis progression and autoimmune diseases such as rheumatoid arthritis. To understand the molecular mechanisms that regulate SDF1 expression, we have cloned and functionally analysed its 5' flanking regulatory region. An SDF1-promoter luciferase construct showed high levels of reporter gene activity in transient transfection experiments. DNase I footprinting analysis revealed that the proximal promoter was occupied by six putative Sp1-binding motifs. Binding of Sp1 to the promoter was confirmed by electrophoretic mobility shift assay, and its importance in SDF1 gene expression verified by in vitro mutagenesis. Particularly, mutation of an Sp1 motif located between -57 and -39 upstream of the main transcription start-site resulted in a marked reduction in promoter activity. It has been shown that the SDF1 expression could be induced by mitogenic stimuli, X-ray radiation or treatment with IL1beta, depending on cell environment. We have analysed the effect of these stimuli on SDF1 promoter transactivation in three different cell lines. Phorbol myristated acetate plus ionomycin increased promoter activity in U373 and LC5 but repressed it in MS5 cells. On the contrary, gamma irradiation promoted SDF1 transcription in MS5 cells but not in the other cell lines. Interferon-gamma acted as a transcriptional repressor in U373 and LC5 but not in MS5 cells. Finally, IL1beta functions as mild activator only in U373 cells. The present study demonstrates that these stimuli mediate SDF1 production through promoter activation in a cell-specific manner.

Immunophenotype of vitamin D receptor polymorphism associated to risk of HIV-1 infection and rate of disease progression.

Vitamin-D-receptor (VDR) mediates immunomodulatory effects of vitamin-D3 (VD3). The VDR-rs1544410_GG polymorphism has been associated with delayed progression rates to AIDS and resistance to HIV-1 infection. The aim of the present study was to investigate differences in VD3 mediated effects on rs1544410 genotyped dendritic cells (DCs) and macrophages (MDM), key cells involved in HIV-1 infection. Immature DCs exhibited lower b-actin-normalized VDR mRNA expression in rs1544410_GG compared to cells with a rs1544410_AA genotype. VD3 response on cell differentiation markers (CD14 inhibition and CD209 induction) was two-fold higher in rs1544410_AA (CD209, p=0.012; CD14, p=0.02). HIV-1-LTR reporter gene activity in MDM was boosted by VD3; however, the effect was up to 50% higher in rs1544410_AA. We conclude that the rs1544410_AA association with progression to AIDS and resistance to HIV-1 appears to be linked to an enhanced response to VD3.

Molecular phenotype of CXCL12beta 3'UTR G801A polymorphism (rs1801157) associated to HIV-1 disease progression.

OBJECTIVE: To investigate the molecular phenotype of the AIDS-onset delaying polymorphism in CXCL12beta 3'UTR (rs1801157). METHODS: The 3 UTRs of the CXCL12beta isoform containing the A or G polymorphic variants were cloned downstream of the Luciferase gene under the control of the CXCL12 promoter. The plasmids were transfected in U373 and LC5 cells and the polymorphism phenotype was evaluated in terms of Luciferase activity and mRNA stability. RESULTS: The 3'A genotype compared to 3'G leads to an increased luciferase activity in unstimulated and PMA+Ionomycin treated cells both in astrocytes (p = 0,0002, p = 0,02) and fibroblasts (p = 0,002, p = 0,03). The mRNA containing the 3'A variant have two-fold longer half-life compared to the 3'G variant (p = 6,99E(-7)). CONCLUSIONS: CXCL12beta 3'A polymorphism, previously associated with resistance to AIDS progression and other diseases, leads to increased levels of CXCL12 mRNA, the results presented here demonstrate that this effect is a consequence of an enhanced mRNA stability. Our data contribute to characterize the CXCL12 as a potential pharmacological target in AIDS, autoimmune diseases and cancer.

Transforming growth factor-beta1 down-regulates expression of chemokine stromal cell-derived factor-1: functional consequences in cell migration and adhesion.

Chemokine stromal cell-derived factor-1 (SDF-1) is expressed by bone marrow (BM) stromal cells and plays key roles in BM cell migration. Modulation of its expression could affect the migratory capacity of cells trafficking the BM, such as hematopoietic progenitor and leukemic cells. Transforming growth factor-beta1 (TGF-beta1) is present in the BM environment and constitutes a pivotal molecule controlling BM cell proliferation and differentiation. We used the BM stromal cell line MS-5 as a model to investigate whether SDF-1 expression constitutes a target for TGF-beta1 regulation and its functional consequences. We show here that TGF-beta1 down-regulates SDF-1 expression, both at the mRNA level, involving a decrease in transcriptional efficiency, and at the protein level, as detected in lysates and supernatants from MS-5 cells. Reduction of SDF-1 in supernatants from TGF-beta1-treated MS-5 cells correlated with decreased, SDF-1-dependent, chemotactic, and transendothelial migratory responses of the BM model cell lines NCI-H929 and Mo7e compared with their responses to supernatants from untreated MS-5 cells. In addition, supernatants from TGF-beta1-exposed MS-5 cells had substantially lower efficiency in promoting integrin alpha4beta1-mediated adhesion of NCI-H929 and Mo7e cells to soluble vascular cell adhesion molecule-1 (sVCAM-1) and CS-1/fibronectin than their untreated counterparts. Moreover, human cord blood CD34+ hematopoietic progenitor cells displayed SDF-1-dependent reduced responses in chemotaxis, transendothelial migration, and up-regulation of adhesion to sVCAM-1 when supernatants from TGF-beta1-treated MS-5 cells were used compared with supernatants from untreated cells. These data indicate that TGF-beta1-controlled reduction in SDF-1 expression influences BM cell migration and adhesion, which could affect the motility of cells trafficking the bone marrow.