No abstract

We performed deletion analysis of WT1-reporter constructs containing up to 24 kilobases of 5-flanking and first intron WT1 sequence in stably transfected cultured cells as an unbiased approach to identify cis elements critical for WT1 transcription. Although not a tissuespecific element, a proximate 9-base pair CTC repeat accounted for ϳ80% of WT1 transcription in this assay. Enhancer activity of the element and mutated versions correlated completely with their ability to form a DNAprotein complex in gel shifts. Antibody supershift, oligonucleotide competition, and Southwestern studies indicated that the CTC-binding factor is the transcriptional activator Sp1. Sp1 binds the CTC repeat with an affinity, K D ‫؍‬ 0.37 nM, at least as high as the consensus GC box. Similar CTC repeats are found in promoters of other growth-related genes. Because Sp1 is important for WT1 expression, we examined Sp1 immunohistochemistry in fetal and adult kidney. In a pattern that precedes that of WT1 message, Sp1 immunostaining was highest in uninduced mesenchyme, early tubules, developing podocytes, and mature glomeruli, but was minimal in mature proximal tubules. This work suggests abundant Sp1 may be a prerequisite for WT1 expression, and that Sp1 may have a wider role in nephrogenesis.

Most nonpeptide antagonists for CC-chemokine receptors share a common pharmacophore with a centrally located, positively charged amine that interacts with the highly conserved glutamic acid (Glu) located in position 6 of transmembrane helix VII (VII:06). We present a novel CCR8 nonpeptide agonist, -268), and N-(1-(3-(2-methoxyphenoxy)benzyl)piperidin-4-yl)-1,2,3,4-tetrahydro-2-oxoquinoline-4-carboxamide (LMD-174)] included several key-residues for nonpeptide antagonists targeting CCR1, -2, and -5. It is noteworthy that a decrease in potency of nearly 1000-fold was observed for all five compounds for the Ala substitution of the anchor-point GluVII:06 (Glu 286 ) and a gain-of-function of 19-fold was observed for LMD-009 (but not the four other analogs) for the Ala substitution of PheVI:16 (Phe 254 ). These structural hallmarks were particularly important in the generation of a model of the molecular mechanism of action for LMD-009. In conclusion, we present the first molecular mapping of the interaction of a nonpeptide agonist with a chemokine receptor and show that the binding pocket of LMD-009 and of analogs overlaps considerably with the binding pockets of CCchemokine receptor nonpeptide antagonists in general.Chemokine receptors belong to the superfamily of rhodopsin-like G protein-coupled 7TM receptors (Murphy et al., 2000). The chemokine ligands (chemotactic cytokines) are a family of large peptides (70 -80 amino acids in length) composed of around 50 members. The CC-chemokines are characterized by the absence of an amino acid between the first two of four conserved cysteines and constitute the largest group (CCL1-28), whereas the CXC-chemokines constitute the other major group (CXCL1-16). Two additional chemokines, the XCL1 and the CX3CL1, have been described previously (Murphy et al., 2000). The chemokine system regulates the development, activation, and recruitment of leukocytes and plays important roles outside the immune system (for instance, on organogenesis, angiogenesis, and carcinogenesis) (Gerard and Rollins, 2001). CCR8 is selectively expressed on a subset of T-helper-2 (Th2) and regulatory T cells and is upregulated on Th2 cells upon activation (Soler et al

Most nonpeptide antagonists for CC-chemokine receptors share a common pharmacophore with a centrally located, positively charged amine that interacts with the highly conserved glutamic acid (Glu) located in position 6 of transmembrane helix VII (VII:06). We present a novel CCR8 nonpeptide agonist, 8-[3-(2-methoxyphenoxy)benzyl]-1-phenethyl-1,3,8-triaza-spi-ro[4.5]decan-4-one (LMD-009), that also contains a centrally located, positively charged amine. LMD-009 selectively stimulated CCR8 among the 20 identified human chemokine receptors. It mediated chemotaxis, inositol phosphate accumulation, and calcium release with high potencies (EC 50 from 11 to 87 nM) and with efficacies similar to that of the endogenous ago-nist CCL1, and it competed for 125 I-CCL1 binding with an affinity of 66 nM. A series of 29 mutations targeting 25 amino acids broadly distributed in the minor and major ligand-binding pockets of CCR8 uncovered that the binding of LMD-009 and of four analogs [2-(1-(3-(2-methoxyphenoxy)benzyl)-4-hy-droxypiperidin-4-yl)benzoic acid (LMD-584), N-ethyl-2-4-methoxybenzenesulfonamide (LMD-902), N-(1-(3-(2-meth-oxyphenoxy)benzyl)piperidin-4-yl)-2-phenyl-4-(pyrrolidin-1-yl)butanamide (LMD-268), and N-(1-(3-(2-methoxyphen-oxy)benzyl)piperidin-4-yl)-1,2,3,4-tetrahydro-2-oxoquinoline-4-carboxamide (LMD-174)] included several key-residues for nonpeptide antagonists targeting CCR1,-2, and-5. It is noteworthy that a decrease in potency of nearly 1000-fold was observed for all five compounds for the Ala substitution of the anchor-point GluVII:06 (Glu 286) and a gain-of-function of 19-fold was observed for LMD-009 (but not the four other analogs) for the Ala substitution of PheVI:16 (Phe 254). These structural hallmarks were particularly important in the generation of a model of the molecular mechanism of action for LMD-009. In conclusion, we present the first molecular mapping of the interaction of a nonpeptide agonist with a chemokine receptor and show that the binding pocket of LMD-009 and of analogs overlaps considerably with the binding pockets of CC-chemokine receptor nonpeptide antagonists in general. Chemokine receptors belong to the superfamily of rhodop-sin-like G protein-coupled 7TM receptors (Murphy et al., 2000). The chemokine ligands (chemotactic cytokines) are a family of large peptides (70-80 amino acids in length) composed of around 50 members. The CC-chemokines are characterized by the absence of an amino acid between the first two of four conserved cysteines and constitute the largest group (CCL1-28), whereas the CXC-chemokines constitute the other major group (CXCL1-16). Two additional chemo-kines, the XCL1 and the CX3CL1, have been described previously (Murphy et al., 2000). The chemokine system regulates the development, activation, and recruitment of leuko-cytes and plays important roles outside the immune system (for instance, on organogenesis, angiogenesis, and carcinogenesis) (Gerard and Rollins, 2001). CCR8 is selectively expressed on a subset of T-helper-2 (Th2) and regulatory T cells and is ...