Transcriptional regulator CTCF controls human interleukin 1 receptor-associated kinase 2 promoter.

Immune responses to invading pathogens are mediated largely through a family of transmembrane Toll-like receptors and modulated by a number of downstream effectors. In particular, a family of four interleukin 1 receptor-associated kinases (IRAK) regulates responsiveness to bacterial endotoxins. Pharmacological targeting of particular IRAK components may be beneficial for treatment of bacterial infections. Here, we studied transcriptional regulation of the human IRAK2 gene. Analysis of the IRAK2 promoter region reveals putative binding sites for several transcriptional factors, including ZIP (EGR1 and SP1), CTCF and AP-2beta. Deletion of the ZIP or AP-2 sites did not significantly affect IRAK2 promoter activity in naive and endotoxin-treated mononuclear cells, in dormant and activated Jurkat T-cells, in lung and kidney cells. In contrast, we found that CTCF plays a major role in IRAK2 transcription. An electrophoretic mobility shift assay of the DNA fragments containing the IRAK2 CpG island, revealed a single high-affinity binding site for the transcriptional regulator and a chromatin insulator protein, CTCF. This assay revealed a CTCF-binding site within the mouse Irak2 promoter. The presence of the CTCF protein in human IRAK2 promoter was confirmed by chromatin immunoprecipitation assay. Specific residues that interacted with the CTCF protein, were identified by methylation interference assay. In all cell lines analyzed, including cells of lung, renal, monocytic and T-cell origin, the IRAK2 luciferase reporter construct, containing an intact CTCF-binding site, showed strong promoter activity. However, IRAK2 promoter activity was decreased dramatically for the constructs with a mutated CTCF-binding site.

Galectin-3 and oncofetal-fibronectin expression in thyroid neoplasia as assessed by reverse transcription-polymerase chain reaction and immunochemistry in cytologic and pathologic specimens.

Oncofetal fibronectin (onfFN) and galectin-3 (Gale-3) have been proposed as possible tools for the preoperative diagnosis of thyroid carcinomas, based on the finding that the expression of both onfFN and Gale-3 are significantly increased in papillary and anaplastic carcinomas, compared to normal thyroid tissues and follicular adenomas. In this study we analyzed the expression of these markers by immunochemical and molecular analysis of benign and malignant thyroid tumors. Sixty-five thyroid nodules, consisting of 20 follicular adenomas (FAs) and 45 papillary thyroid carcinomas (PTCs) at final histology were examined. At the molecular level, among the 45 PTCs, 44 (97.8%) showed a variable level of onfFN mRNA, while 8 of the 20 (40%) adenomas expressed the same marker. Similar results have been found analyzing Gale-3 expression: 97.8% of PTC and 55% of FAs were positive for this marker. Immunohistochemistry (IHC) for Gale-3 was positive in 42 of 45 (93.3%) PTC tissues. Staining was invariably confined to the cytoplasm, with a homogeneous distribution in the large majority of the neoplastic cells. The 3 negative cases (6.7%) were represented by 2 classic variants of PTC and 1 follicular variant of PTC. Eighteen of the 20 (90.0%) adenomas stained negative for Gale-3. A significant association was found between positive staining and malignant phenotype (p < 0.0001). Gale-3 protein expression was also performed on samples obtained by ex vivo fine-needle aspiration (FNA) in 35 PTCs by immunocytochemistry (ICC). Immunoreactivity was present in 32 (91.0%) and negative in 3 (8.8%) cases. With the exception of 1 case (negative by ICC and positive by IHC), ICC and IHC were fully concordant. In conclusion, our results indicate that a search for Gale-3 protein overexpression by IIC or ICC, but not by reverse transcription-polymerase chain reaction (RT-PCR), may yield an additional marker of malignant potential of thyroid nodular lesions, and may be a useful adjunct to the currently available diagnostic tools for the preoperative diagnosis of malignant thyroid tumors.

Role of the intracellular domain of IL-7 receptor in T cell development.

Signals from the IL-7R are uniquely required for T cell development and maintenance, despite the resemblance of IL-7R to other cytokine receptors and the apparent sharing of common signaling pathways. This unique requirement could either reflect unique expression of IL-7R or IL-7, or it could indicate that the IL-7R delivers unique signals. To determine whether the IL-7R provided unique signals, we exchanged its intracellular domain with that of other cytokine receptors: IL-4R, IL-9R, and prolactin receptor (PRLR). Chimeric receptors were used to reconstitute development of IL-7R(-/-) hemopoietic progenitors by transducing the receptors in retroviral vectors. Whereas IL-7R(-/-) thymocytes are arrested at the double-negative stage, IL-4R, IL-9R, or PRLR all imparted some progression to the double-positive stage. IL-4R and PRLR gave only small numbers of thymocytes, whereas IL-9R gave robust alphabeta T cell development and reconstitution of peripheral CD4 and CD8 cells, indicating that it can duplicate many of the functions of IL-7R. However, IL-9R failed to reconstitute rearrangement of the TCRgamma locus or development of gammadelta T cells. Thus, the IL-7R signals required in the alphabeta T cell lineage (such as survival and proliferation) are not unique to this receptor, whereas rearrangement of the TCRgamma locus may require a signal that is not shared by other receptors.