Binding Of Transcription Factor Sp1 Research Articles

Functional SNP in an Sp1-binding site of AGTRL1 gene is associated with susceptibility to brain infarction

Brain infarction is one of the common causes of death and also a major cause of severe disability. To identify a gene(s) susceptible to brain infarction, we performed a large-scale association study of Japanese patients with brain infarction, using 52608 gene-based single nucleotide polymorphism (SNP) markers. Comparison of allele frequencies between 1112 cases with brain infarction and age- and sex-matched control subjects of the same number found an SNP in the 5'-flanking region of angiotensin receptor like-1 (AGTRL1) gene (rs9943582, - 154G/A) to have a significant association with brain infarction [odds ratio = 1.30, 95% confidence interval (CI) = 1.14-1.47, P = 0.000066]. We also found the binding of Sp1 transcription factor to the region including the susceptible G allele, but not the non-susceptible A allele. Luciferase assay and RT-PCR analysis demonstrated that exogenously introduced Sp1 induced transcription of AGTRL1 and its ligand, apelin, as well, indicating direct regulation of apelin/APJ pathway by Sp1. Furthermore, a 14 year follow-up cohort study in a Japanese community in Hisayama town, Japan revealed that the homozygote of the susceptible G allele of this particular SNP had significantly higher risk of brain infarction (hazard ratio = 2.00, 95% CI = 1.22-3.29, P = 0.006). Our results indicate that the SNP in the AGTRL1 gene is associated with the susceptibility to brain infarction.

PI 3-K signaling pathway suppresses PMA-induced expression of p21WAF1/Cip1 in human leukemia cells

Despite the understanding of the importance of phosphoinositide 3-kinase (PI 3-K) signaling pathway in the regulation of cellular proliferation, little is known about its role during phorbol 12-myristate 13-acetate (PMA)-induced differentiation in human leukemia cells. Here, we report a novel finding that PI 3-K inhibition by LY294002 significantly increases p21WAF1/Cip1 expression in PMA-stimulated human leukemia cells NB4 and THP1. LY294002 potentiated expression of p21WAF1/Cip1 via a p53-independent mechanism and did not affect mitogen activated protein kinase (MAPK) activation. Electrophoretic mobility shift (EMSA) experiments revealed that blocking of PI 3-K was associated with increased binding of transcription factor Sp1 to the PMA-responsive sites on the p21WAF1/Cip1 promoter. Pretreatment with rapamycin, an inhibitor of mTOR kinase, decreased the expression of p21WAF1/Cip1 protein in PMA-stimulated NB4 cells. The level of PMA-induced p21WAF1/Cip1 protein expression was lower in NB4 cells overexpressing wild type protein kinase C zeta (PKC zeta) compared to those transfected with empty vector or with kinase inactive PKC zeta. Sp1 binding to the p21WAF1/Cip1 promoter was completely lost in a wild type PKC zeta overexpressing and PMA-stimulated NB4 cells. We demonstrate that PI 3-K signaling pathway suppresses PMA-induced expression of p21WAF1/Cip1 in human leukemia cells, and that this effect is partly mediated by PKC zeta.

Loss of AP-2α results in deregulation of E-cadherin and MMP-9 and an increase in tumorigenicity of colon cancer cells in vivo

Activator protein-2 (AP-2) is a transcription factor that regulates proliferation and differentiation in mammalian cells and has been implicated in the acquisition of the metastatic phenotype in several types of cancer. Herein, we examine the role of AP-2alpha in colon cancer progression. We provide evidence for the lack of AP-2alpha expression in the late stages of colon cancer cells. Re-expression of the AP-2alpha gene in the AP-2alpha-negative SW480 colon cancer cells suppressed their tumorigenicity following orthotopic injection into the cecal wall of nude mice. The inhibition of tumor growth could be attributed to the increased expression of E-cadherin and decreased expression and activity of matrix-metalloproteinase-9 (MMP-9) in the transfected cells, as well as a substantial loss of their in vitro invasive properties. Conversely, targeting constitutive expression of AP-2alpha in AP-2-positive KM12C colon cancer cells with small interfering RNA resulted in an increase in their invasive potential, downregulation of E-cadherin and increased expression of MMP-9. In SW480 cells, re-expression of AP-2alpha resulted in a fourfold increase in the activity of E-cadherin promoter, and a 5-14-fold decrease in the activity of MMP-9 promoter, indicating transcriptional regulation of these genes by AP-2alpha. Chromatin immunoprecipitation assay showed that re-expressed AP-2alpha directly binds to the promoter of E-cadherin, where it has been previously reported to act as a transcriptional activator. Furthermore, chromatin immunoprecipitation assay revealed AP-2alpha binding to the MMP-9 promoter, which ensued by decreased binding of transcription factor Sp-1 and changes in the recruitment of transcription factors to a distal AP-1 element, thus, contributing to the overall downregulation of MMP-9 promoter activity. Collectively, our data provide evidence that AP-2alpha acts as a tumor suppressor gene in colon cancer..

DNA methylation and Sp1 binding determine the tissue-specific transcriptional activity of the mouse Abcc6 promoter

The gene encoding the ABCC6 protein, an ABC transporter of the multidrug resistance-associated protein (MRP), is mainly expressed in liver and kidney. Mutations in ABCC6 are responsible for the development of the pseudoxanthoma elasticum (PXE) phenotype. PXE is a recessive disease characterized by the calcification of elastic fibers resulting in dermal, vascular, and ocular clinical manifestations. The physiological function of ABCC6 and the rodent orthologs Abcc6 is unknown and their precise relationship to elastic fibers is only a matter of speculation. Despite several studies focused on the transcriptional regulation of ABCC6/Abcc6, the molecular signals conferring the tissue-specificity to the ABCC6/Abcc6 expression are not well defined. In this report, we determined the level of the mouse Abcc6 promoter methylation in tissues with low level of expression (tail extremity and skin), intermediate (kidney), and high level of expression (liver). We observed that high and moderate levels of methylation correlated with low levels of Abcc6 expression. Moreover, we determined that CpG methylation of the Abcc6 proximal promoter region was interfering with the binding of the Sp1 transcription factor thereby inhibiting Sp1-dependent transactivation. Thus, our data provide the first direct evidence that an epigenetic mechanism regulates the binding of transcription factor Sp1 to the proximal promoter and participates in the tissue-specific expression control of the mouse Abcc6 gene.

P38MAPK mediates benzyl isothiocyanate-induced p21WAF1 expression in vascular smooth muscle cells via the regulation of Sp1

It has recently been reported that the transcription factors involved in p21WAF1 activation by certain signaling factors may vary in different cell types. However, the role and importance of the signaling pathway in the transcriptional regulation of p21WAF1 on vascular smooth muscle cells (VSMC) in response to benzyl isothiocyanate (BITC) has been unclear. In this report, we demonstrate that BITC induces the p21WAF1 expression at the transcriptional level. This increase in p21WAF1 gene expression was due to p38MAPK-dependent activation of the p21WAF1 promoter by BITC. Transcription factor Sp1 binding site was identified as the cis-element for the activation of p21WAF1 promoter by BITC, as determined by deletion and mutation analysis. In addition, gel shift and supershift assays demonstrated that this BITC-responsive element binds specifically to the transcription factor Sp1. Treatment with SB203580, an inhibitor of the p38MAPK, significantly downregulated transactivation of BITC-induced Sp1. Finally, the transient expression of VSMC with dominant negative p38MAPK plasmid suppressed BITC-stimulated Sp1 activity. In conclusion, we report that the transcription factor Sp1 involved in the p38MAPK-mediated control of p21WAF1 regulation on VSMC in response to BITC has now been identified.

The novel histone deacetylase inhibitor BML-210 exerts growth inhibitory, proapoptotic and differentiation stimulating effects on the human leukemia cell lines

Histone deacetylase inhibitors have a potent role in the strategy for the treatment of leukemias. BML-210 ( N-(2-Aminophenyl)- N′ phenyloctanol diamine) is the novel histone deacetylase inhibitor, and its mechanism of action has not been characterized. In this study, we examined the in vitro effects of BML-210 on the human leukemia cell lines (NB4, HL-60, THP-1, and K562). We found that BML-210 inhibits the growth of all cell lines and promotes apoptosis in a dose- and time-dependent manner. BML-210 alone induces HL-60 and K562 cell differentiation (up to 30%) to granulocytes and erythrocytes, respectively, and in combination with differentiation agents — all-trans retinoic acid and hemin, markedly potentates it. Those treatments cause G1 arrest and histone H4 acetylation, affects transcription factor NF-κB and Sp1 binding activity to their consensus sequences, the p21 or the FasL promoters, and influences expression of Sp1, NF-κB, p21 and FasL. These findings suggest that BML-210 could be a promising antileukemic agent to induce apoptosis and to modulate differentiation through the modulation of histone acetylation and gene expression.

The role of Sp1 and Sp3 in the constitutive DPYD gene expression

Dihydropyrimidine dehydrogenase (DPD), the initial and rate-limiting enzyme in the 5-fluorouracil (5-FU) catabolic pathway, has been implicated as one of the factors determining the efficacy and toxicity of the anticancer agent 5-FU. Studies have attributed variation in DPD activity partially to alterations at the transcriptional level of DPYD gene. We investigated the transcription factors implicated in the constitutive expression of DPYD by utilizing a 174-bp fragment of the DPYD promoter region in which three consensus Sp protein binding sites (SpA, SpB and SpC) were predicted. The binding of Sp1 and Sp3 transcription factors to this region was detected by electrophoretic mobility shift and chromatin immunoprecipitation assays. By ectopically expressing human Sp1 and Sp3 in Sp-deficient Drosophila S2 cells, we demonstrated that Sp1 is a strong activator, while Sp3 by its own is a weak activator of the DPYD promoter. Moreover, Sp3 may serve as a competitor of Sp1, thus decreasing the Sp1 induced promoter activity. SpA, SpB and SpC sites are all Sp1 inducible. In the full activation of the DPYD promoter in human cell lines, the SpB site is essential; the SpC site works cooperatively with SpB, while SpA has minor promoter activity. These studies provide further insight into the molecular mechanisms underlying the heterogeneity of DPD activity, and may facilitate the efficacy and safety of 5-FU-based chemotherapy.

Regulation of Human T-Cell Leukemia Virus Type 1 Gene Expression by Sp1 and Sp3 Interaction with TRE-1 Repeat III

Transcription factors of the Sp family are known to play key roles in the regulation of both constitutive as well as cell type- and differentiation stage-specific gene expression. Binding sites for factors of the Sp family (Sp1 and Sp3) have previously been identified within the U3 region of the human T-cell leukemia virus type 1 (HTLV-1) long terminal repeat (LTR). Although previous studies have demonstrated that Sp1 and Sp3 can interact with the Tax-responsive element 1 (TRE-1) repeat III, the sequences required for Sp1/Sp3 binding have not been mapped in detail. Herein, we demonstrate that the GC-rich regions flanking the viral cAMP-responsive element (CRE) within TRE-1 repeat III exhibit substantial affinity for both Sp1 and Sp3. We demonstrate that purified Sp1 competes with purified CREB for binding to TRE-1 repeat III due to the physical proximity of the Sp1/Sp3 and ATF/CREB binding sites, while purified Sp1 forms a multiprotein complex with purified CREB in the presence of Tax as demonstrated by electrophoretic mobility shift (EMS) analyses. Sp1 and Sp3 binding to the U3 region of the HTLV-1 LTR in the presence of Tax in vivo was confirmed by chromatin immunoprecipitation using HTLV-1-infected T cells (SLB-1 and C8166). Overexpression of Sp1 was modestly enhanced, while overexpression of Sp3 inhibited basal and Tax-mediated transactivation of the HTLV-1 LTR in U-937 cells (which express relatively low levels of endogenous Sp1 and Sp3). Furthermore, the modest upregulation of LTR activation caused by overexpression of Sp1 could be blocked by site-directed mutagenesis of the GC-rich Sp1/Sp3 binding sites within TRE-1 repeat III. These results suggest that both Sp1 and Sp3 transcription factor binding to TRE-1 repeat III participate in regulation of HTLV-1 viral gene expression.

The −318 C>G Single-Nucleotide Polymorphism in GNAI2 Gene Promoter Region Impairs Transcriptional Activity through Specific Binding of Sp1 Transcription Factor and Is Associated with High Blood Pressure in Caucasians from Italy

Inhibiting Galpha subunit 2 protein, which is encoded by the GNAI2 gene, is suggested to be pathogenic for essential hypertension and/or insulin resistance. The aim of this study was to determine whether GNAI2 variations modulate the risk for these abnormalities. Seven single-nucleotide polymorphisms (SNP) at the GNAI2 locus were identified. Because of either low allelic frequency or unlikely biologic relevance (i.e., synonymous or intronic), six SNP were not studied further. The -318C>G SNP (allelic frequency 6%) in the promoter region was studied for association with adiposity, systolic BP (SBP) and diastolic BP, fasting insulin and glucose, and lipids levels in 655 nondiabetic Caucasians from Italy. As compared with individuals who carry the C/C genotype, G carriers (i.e., individuals who carry either the G/G or the C/G genotype) had higher SBP (117.8 +/- 16 versus 113.6 +/- 12.6 mmHg; P = 0.010) and were at increased risk for hypertension (odds ratio 2.2; 95% confidence interval 1.1 to 4.5). Compared with the C, the G allele had 2.5-fold reduced transcriptional activity in transfected HEK293 cells. As predicted by the TRANSFAC database, competition with YY1 or Sp1 transcription factors specifically reduced the binding of HeLa cell nuclear proteins to -318C or -318G allele, respectively, as indicated by shifted electrophoretic mobility. A "supershift" of the nuclear proteins/-318G allele complex was observed after anti-Sp1 was added but not anti-YY1 antibody. The GNAI2 -318 C>G SNP impairs transcriptional activity through specific binding of Sp1 and is associated with high SBP in Caucasians from Italy.

Activation of the human pregnancy-specific glycoprotein PSG-5 promoter by KLF4 and Sp1

Pregnancy-specific glycoproteins (PSGs) are major placental proteins thought to be essential for the maintenance of gestation. Little is known about the regulation of expression of the 11 genes encoding these proteins. It was previously demonstrated that Krüppel-like factor 6 (KLF6) and specific-protein 1 (Sp1) bind to conserved sequence within the PSG-5 gene promoter. Informatics analysis revealed the presence of one potential binding site for Krüppel-like factor 4 (KLF4), in the PSG-5 promoter, suggesting a potential transcriptional regulator role for KLF4. Using gene promoter-reporter transfections and X-ChIP assays, we demonstrated that KLF4 is an activator of the PSG-5 promoter by binding to a KLF consensus like binding which includes the Core Promoter Element region (−147/−140). Furthermore, we used previous data showing the binding of Sp1 transcription factor to a GT-box (−443/−437) and co-transfection assays with KLF4 and Sp1 to demonstrate the strong synergic activity of these two factors on the PSG-5 promoter.

TNF Family Protein Regulation in Megakaryocytes and Platelets.

Activated platelets express and release several TNF family proteins with diverse immunological and inflammatory functions. While the biological effects of these potent immunological modifiers are an area of intense study, there are no studies to date that have investigated the mechanism of TNF protein regulation in megakaryocytes. Understanding the mechanisms of TNF protein regulation in megakaryocytes may allow for the design of new therapeutic strategies for the treatment of chronic inflammatory and autoimmune diseases. Our data show that CD40 Ligand, Fas Ligand and TNF-Related Apoptosis Inducing Ligand (TRAIL) expression are induced during megakaryocyte-differentiation of hematopoietic precursor cells. Promoter analysis and expression studies reveal multiple mechanisms of TNF family protein regulation. Megakaryocyte-specific regulation of TRAIL and Fas Ligand are primarily dependent on Sp1 and Sp3 transcription factor binding to proximal Sp1 binding elements within the respective promoter regions. Our preliminary data demonstrates differentiation-induced expression of CD40 Ligand on the other hand, is mediated via an Ets-1 site within 50 nucleotides of the transcription start site. In addition, endogenous CD40 Ligand but not TRAIL or Fas Ligand is constitutively expressed with ectopic expression of Fli-1, in the absence of differentiation-stimuli. Fli-1 is an Ets-1 family transcription factor that modulates several megakaryocyte-restricted genes. Further regulation studies reveal megakaryocyte expression of CD40 Ligand is diminished by the peroxisome proliferator-activated receptor (PPAR) agonist prostaglandin J2 and enhanced by 9-cis retinoic acid. Conversely, TRAIL and Fas Ligand expression are enhanced by prostaglandin J2 and diminished by retinoic acid. Taken from these data is there are differential mechanisms of differentiation-driven and induced regulation of the TNF family proteins. Continuing studies include the determination of the mechanisms of PPAR agonist, retinoic acid and differentiation-modulated TNF protein expression. Our studies demonstrate novel TRAIL and Fas Ligand regulatory pathways in the context of megakaryocyte differentiation and represent the first investigation into megakaryocyte-associated regulatory mechanisms for the platelet-derived CD40 Ligand, Fas Ligand, and TRAIL.

Characterization of putative cis‐regulatory elements that control the transcriptional activity of the human Oct4 promoter

Octamer-binding transcription factor-4 (Oct4), a member of the POU domain transcription factors, is crucial for both early embryonic development and the maintenance of stem cell pluripotency. The human Oct4 (hOct4) 5' upstream sequence contains four conserved regions (CR1, 2, 3, 4) that are homologous in the murine. In this study, we constructed a series of deletion mutants of the hOct4 5' upstream region and identified cis-regulatory elements that may be important determinants for the transcriptional activity of the hOct4 promoter. Our studies showed that CR2, 3, and 4 each acted as positive cis-regulatory elements in hOct4 promoter activity. We also newly identified a putative negative cis-acting element located between CR1 and CR2. In addition, the sequence -380/-1 at CR1 that contains a GC box was sufficient to provide the minimal promoter activity. Site-directed mutagenesis and electrophoretic mobility shift assays revealed the GC box located in the -380/-1 region may play a critical role in controlling the transcriptional activity of hOct4 by the direct binding of Sp1 or Sp3 transcription factors to the GC box. An overexpression study showed that Sp1 and Sp3 positively and negatively regulate hOct4 promoter activity. Thus, the hOct4 promoter upstream region contains multiple regulatory elements, one of which, the GC box, may be an important cis-regulatory element that regulates the transcription of the hOct4 promoter by the binding of Sp family transcription factors.

Vascular Endothelial Growth Factor Induction by Prostaglandin E2 in Human Airway Smooth Muscle Cells Is Mediated by E Prostanoid EP2/EP4 Receptors and SP-1 Transcription Factor Binding Sites

Prostaglandin E2 (PGE2) can increase endothelial vascular endogrowth factor A (VEGF-A) production but the mechanisms involved are unclear. Here we characterized the transcriptional mechanisms involved in human airway smooth muscle cells (HASMC). PGE2 increased VEGF-A mRNA and protein but not mRNA stability. PGE2 stimulated the activity of a transiently transfected 2068-bp (-2018 to +50) VEGF-A promoter-driven luciferase construct. Functional 5' deletional analysis mapped the PGE2 response element to the 135-bp sequence (-85/+50) of the human VEGF-A promoter. PGE2-induced luciferase activity was reduced in cells transfected with a 135-bp VEGF promoter fragment containing mutated Sp-1 binding sites but not in cells transfected with a construct containing mutated EGR-1 binding sites. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay confirmed binding of Sp-1 to the VEGF promoter. PGE2 increased phosphorylation of Sp-1 and luciferase activity of a transfected Sp-1 reporter construct. PGE receptor agonists EP2 (ONO-AE1 259) and EP4 (ONO-AE1 329) mimicked the effect of PGE2, and reverse transcription-PCR, Western blotting, and flow cytometry confirmed the presence of EP2 and EP4 receptors. VEGF protein release and Sp-1 reporter activity were increased by forskolin and isoproterenol, which increase cytosolic cAMP, and the cAMP analogue, 8-bromoadenosine-3',5'-cyclophosphoric acid. These studies suggest that PGE2 increases VEGF transcriptionally and involves the Sp-1 binding site via a cAMP-dependent mechanism involving EP2 and EP4 receptors.

The binding of the ubiquitous transcription factor Sp1 at the locus control region represses the expression of β-like globin genes

To investigate the function of transcription factor Sp1 in beta-like globin gene activation, we analyzed the recruitment of Sp1, fetal Krüppel-like factor 2 (FKLF2), and related factors at the human beta-globin locus in a human fetal liver and mouse erythroleukemia hybrid cell (A181gamma cell) that contains a single copy of human chromosome 11. Sp1 binds at the GT boxes of the cis-elements throughout the beta-locus, but it is phosphorylated and lost over DNase I hypersensitive site (HS)2, HS3, HS4, and the human beta-globin gene promoter after A181gamma cell differentiation. The binding of FKLF2 at HS2 and HS3 was unchanged. Histone deacetylase 1, which could be recruited by Sp1, is also lost over HS2 and HS3 after differentiation, resulting in the acetylation of histones 3 and 4 across the human beta-globin locus. We previously detected in vivo GT footprints over the beta-globin locus after A181gamma differentiation. Here, we report that after differentiation, the p300/CREB-binding protein-associated factor is recruited by FKLF2 to the locus control region to acetylate histones 3 and 4 at the human beta-globin gene locus. Our results suggest that Sp1 is an inhibitor of beta-like globin gene transcription during erythroid terminal differentiation. Its phosphorylation and release allow the erythroid-specific FKLF2 or erythroid Krüppel-like factor to interact with other erythroid-specific transcription factors to initiate the transcription of beta-like globin genes.

A single nucleotide polymorphism in the HDM-2 gene regulates the p53 apoptotic response and influences the age of onset of cancers in humans: the SNP 309 HDM-2 polymorphism

The HDM-2 gene in humans has two promoters for transcription. 5' to the first exon is a maintenance promoter providing low levels of HDM-2 in the cell. In the first intron are the P53 DNA binding sites and the p53 inducible promoter that yields threefold to 10-fold more HDM-2 mRNA after a p53 activation and response. When this intronic promoter is employed, transcriptional initiation starts at the second exon and this mRNA is translated more efficiently than mRNA that starts at the first exon. The coding region of the HDM-2 protein starts in the third exon. At residue 309 in this first intron is a single nucleotide polymorphism, with 12% of people being a G/G homozygote, 40% being a G/T heterozygote and 48% of people being T/T wild-type homozygotes (the G/G genotype is lower in black Americans and the sample size is now over 300 people). We have found that the G/G genotype creates a better SP-1 transcription factor binding site, raises the level of m-RNA in unstressed cells and produces threefold to sixfold more HDM-2 protein in cells (cancer cells in culture) with the G/G genotype. This mRNA starts at the second exon, and is probably translated better in unstressed cells. After DNA damage or other stresses, P53 activity in cells with the G/G genotype is lower and the percentage of cells undergoing apoptosis is lower when compared with cells in culture with T/T genotypes. We have reproduced these observations with lymphocytes taken from human volunteers and placed in culture, with EBV-immortalized B cells in culture, with primary fibroblasts in cell culture and with cancer cell lines in culture. In 92 individuals that have donated lymphocytes we see individuals forming a distribution of apoptotic responses between 20% and 60% after gamma radiation, with individuals being quite reproducible in repeated experiments. The lower half of the distribution is heavily weighted with the G/G genotype, while the upper half of the distribution has mainly the T/T genotype. The higher HDM-2 levels in cells thus result in a lower apoptotic index in cells from these volunteers. It has become clear in recent studies that SNP 309 has a clinical impact. We have genotyped two cancer cohorts, one at MD Anderson and one in Germany, containing patients with sarcomas and breast cancers. The results have been statistically significant (P = 0.01-0.02) and clear in both cohorts, and the average age of onset of these cancers is 10–15 years earlier in people with the G/G genotype than in people with the same cancer with the T/T genotype. The interpretation is then that the probability of eliminating pre-cancerous clones of cells via a p53 mechanism is lower in people with a G/G genotype (high HDM-2 levels) and the probability of developing a cancer at an earlier time in life is higher. In addition, in patients that have a germline mutation in the p53 gene (this yields one-half of the p53 protein level in a cell) those individuals that have a G/G genotype or a G/T genotype develop multiple cancers (three, four or five cancers) over their lifetimes, while no T/T homozygotes develop that many independent cancers.

Type I collagen α1 Sp1 transcription factor binding site polymorphism is associated with reduced risk of hip osteoarthritis defined by severe joint space narrowing in elderly women

A common G/T substitution at an Sp1 binding site in intron 1 of the COL1A1 gene has been reported to be associated with reduced bone mineral density and increased risk of osteoporotic fracture. The purpose of this study was to examine whether there is an association between COL1A1 Sp1 polymorphism and radiographic osteoarthritis (OA) of the hip in elderly women in the Study of Osteoporotic Fractures. Radiographic hip OA status of subjects was defined by the presence of 1 of the following criteria in either hip: a joint space narrowing (JSN) score of >/=3, a Croft summary grade of >/=3, or both definite (score >/=2) osteophytes and JSN in the same hip. Cases of radiographic OA of the hip were further subdivided into those with JSN score >/=3 and those with a femoral osteophyte score >/=2 and JSN score </=2. The COL1A1 Sp1 polymorphism was genotyped using allele-specific kinetic polymerase chain reaction in 4,746 women. Multivariate logistic regression was performed to estimate odds ratios (ORs) and 95% confidence intervals (95% CIs). Radiographic OA of the hip was present in 571 women (12%). Of these patients, 325 (57%) had severe JSN (score >/=3), and 131 (23%) had moderate or moderate-to-severe femoral osteophytosis (score >/=2). There was no association of the T/T genotype with either radiographic hip OA or radiographic hip OA characterized by osteophytosis. For radiographic OA of the hip characterized by moderate-to-severe JSN, the odds of disease were significantly reduced among subjects with the T/T compared with the G/G genotype (OR 0.30, 95% CI 0.11-0.81, P = 0.02) and did not change after adjustment for potential confounders (OR 0.36, 95% CI 0.13-0.99, P = 0.048). The T/T genotype of the COL1A1 Sp1 polymorphism was associated with a reduced risk of radiographic OA of the hip characterized by JSN. This association should be confirmed in other populations to determine if mechanistic studies are warranted.

Carbon Monoxide Differentially Modulates STAT1 and STAT3 and Inhibits Apoptosis via a Phosphatidylinositol 3-Kinase/Akt and p38 Kinase-dependent STAT3 Pathway during Anoxia-Reoxygenation Injury

Carbon monoxide (CO), previously considered a toxic waste product of heme catabolism, is emerging as an important gaseous molecule. In addition to its important role in neurotransmission, exogenous CO protects against vascular injury, transplant rejection, and acute lung injury. However, little is known regarding the precise signaling mechanisms of CO. We have recently shown that CO attenuates endothelial cell apoptosis during anoxia-reoxygenation injury by activating MKK3/p38alpha mitogen-activated protein kinase (MAPK) pathways. Our current study is the first to demonstrate that CO can differentially modulate STAT1 and STAT3 activation and, specifically, that STAT3 activation by CO is responsible for the anti-apoptotic effect in endothelial cells. In addition, we show that the anti-apoptotic effects of CO depend upon both phosphatidylinositol 3-kinase/Akt and p38 MAPK signaling pathways in endothelial cells, whereas previous reports have implicated only the MKK3/p38 MAPK pathway. Using chemical inhibitors and dominant negative constructs, we show that CO enhances STAT3 activation via phosphatidylinositol 3-kinase/Akt and p38 MAPK pathways with subsequent attenuation of Fas expression and caspase 3 activity. These data highlight the anti-apoptotic signaling mechanisms of CO and, importantly, delineate potential therapeutic strategies to prevent ischemia-reperfusion or anoxia-reoxygenation injury in the vasculature.

V-Jun downregulates the alpha 2 (I) collagen target gene indirectly through Sp1/3

Transformation of chick embryo fibroblasts (CEFs) by the v-Jun oncoprotein correlates with a downregulation of the alpha 2 (I) collagen gene. To investigate whether this gene constitutes a direct target of v-Jun, an analysis of a large proximal fragment of the promoter, extending from position -1080 to +109, was performed. Transient transfections with -1080/+109 and deleted derivatives revealed that a short proximal fragment, -433/+11, is the target for repression by v-Jun. Extensive analysis, conducted in CEFs and in Sp1/3-deficient Drosophila SL2 cells, further showed that (i) high constitutive activity of -433/+11 requires a direct binding of the ubiquitous Sp1 and/or Sp3 transcription factors acting on two distinct motifs, that is, a proximal TCC-rich region and an upstream GC box, and that (ii) repression by v-Jun does not require any direct binding of the oncoprotein to the DNA, but an indirect binding within a v-Jun-Sp1/3-DNA chromatin-associated complex. This situation is reminiscent of a situation previously reported with the tata-less, SPARC (secreted protein, acidic, and rich in cysteine) target promoter that regulates the expression of another extracellular matrix component in the same model of cell transformation. Taken together, these data reinforce the view that, at least in CEFs, v-Jun downregulates a family of direct target genes by binding to the DNA indirectly through Sp1/3.

Adverse effect of cadmium on binding of transcription factor Sp1 to the GC-rich regions of the mouse sodium-glucose cotransporter 1, SGLT1, promoter

Exposure of the kidney to cadmium can cause glucosuria. Effect of cadmium on sodium-glucose cotransporter 1, (SGLT1) mRNA molecules in cultured mouse kidney cortical cells was determined by quantitative competitive RT-PCR. SGLT1 mRNA molecules decreased from 58 x 10(4) microg(-1) total RNA in untreated cells to 29 x 10(4) microg(-1) total RNA in cells exposed to 5 microM cadmium. Increasing cadmium to 7.5 and 10 microM, reduced mRNA molecules to 21 x 10(4) and 12 x 10(4) microg(-1) total RNA, respectively. The half-life of SGLT1 mRNA in control and in cells exposed to 7.5 microM cadmium were almost the same and calculated to be 9.1 h (S.E.+/-2.7) for the former and 8.5 h (S.E.+/-2.2) for the latter. We also analyzed mouse SGLT1 promoter sequences and identified two conserved Sp1 binding sites. The Sp1 binding sequences were used as probes in electrophoretic mobility shift assay (EMSA) with nuclear proteins from cultured cells. Intensity of complexes of the 5' and the 3' Sp1 probes with nuclear Sp1 from cells treated with 7.5 microM cadmium were 84% (S.E.+/-4) and 61% (S.E.+/-14) of controls, respectively. Cadmium had no effect on expression of Sp1 mRNA or protein level. Cadmium-induced inhibition of glucose uptake in kidney may be the result of transcriptional down-regulation of SGLT1 mediated through modification of Sp1 binding to its promoter.

FAS Promoter Polymorphism and Etiology and Outcome of Childhood Acute Myeloid Leukemia.

FAS (TNFRSF6/CD95/APO-1) is a cell surface receptor involved in apoptotic signal transmission. Dysregulation of this pathway is believed to result in down regulation of apoptosis and subsequent persistence of a malignant clone. A single nucleotide polymorphism resulting in guanine-to-adenine transition in the FAS promoter region occurs at position −1377, affecting an SP1 transcription factor binding site; an adenine residue at position −1377 of FAS promoter significantly reduces SP1 binding compared to guanine residue causing a decrease in FAS expression. Recent data in adult leukemia indicate that a variant allele at this site increases susceptibility to AML (Morgan et al; Cancer Research 63, 4327–4330). We hypothesized that FAS genotype would also increase risk of childhood AML and, by altering susceptibility to apoptosis might impact outcome of AML therapy. Methods: 442 children treated for AML on CCG studies 2941 and 2961 (intensive induction with daunorubicin, idarubicin, Ara-C, 6-thioguanine, etoposide, dexamethasone, +/− fludarabine, Ara-C, idarubicin; consolidation with high-dose Ara-C and L-asparaginase or HLA-matched sibling BMT) were genotyped for the −1377 FAS promoter polymorphism using mismatch amplification coupled with real time PCR (MAMA assay). Genotype frequencies were compared with published normal control frequencies. In addition patient outcomes were analyzed according to genotype.Results: Comparison of gene frequencies in AML patients and reported normal controls showed similar frequencies (78.7% GG, 18.6% GA, 2.7% AA in patients vs. 78% GG, 20% GA and 2% AA in controls; p=0.6). Stratification of cases by age at diagnosis, white blood count at diagnosis, AML subtype, or cytogenetics revealed no difference in genotype frequencies. Outcome data showed no significant difference in OS (p=0.22), EFS (p=0.31), TRM (p=0.42) or relapse rate (p=0.57) between patients with 1377GG genotype vs. 1377GA/1377AA genotypes. However, there was trend towards improved survival (OS at 5 years 75% AA, 55% GA, 49% GG; p=0.16) and EFS (66% AA, 43% GA, 39% GG; p=0.19) in AA cases, but small numbers (n=12 AA) may have contributed to the results not being statistically significant. Conclusion: These data, representing the only data in pediatric AML, suggest that FAS genotype does not affect the etiology of childhood AML and that children with AML differ from adults in terms of biology of their disease. However, further studies are needed to evaluate a potentially important role of FAS genotype in outcome of AML therapy.