Nod2, a member of the Apaf1/Nod protein family, confers responsiveness to bacterial products and activates NF-κB, a transcription factor that plays a central role in innate immunity. Recently, genetic variation in Nod2 has been associated with susceptibility to Crohn's disease. Here, we report that expression of Nod2 is induced upon differentiation of CD34⁺hematopoietic progenitor cells into granulocyte or monocyte/macrophages. In peripheral blood cells, the highest levels of Nod2 were observed in CD14⁺ (monocytes), CD15⁺(granulocytes), and CD40⁺/CD86⁺ (dendritic cells) cell populations. Notably, stimulation of myeloblastic and epithelial cells with bacterial lipopolysaccharide or TNFα resulted in up-regulation of Nod2. A search for consensus sites within the Nod2 promoter revealed a NF-κB binding element that was required for transcriptional activity in response to TNFα. Moreover, ectopic expression of p65 induced transactivation, whereas that of dominant-negative IκBα blocked the transcriptional activity of the Nod2 promoter. Upon stimulation with TNFα or lipopolysaccharide, both p50 and p65 subunits of NF-κB were bound to the Nod2 promoter. Thus, Nod2 expression is enhanced by proinflammatory cytokines and bacterial components via NF-κB, a mechanism that may contribute to the amplification of the innate immune response and susceptibility to inflammatory disease.

Early growth response factor (Egr-1) is an inducible zinc finger transcription factor that binds specific GC-rich enhancer elements and impacts female reproduction. These studies document for the first time that FSH rapidly induces Egr-1 expression in granulosa cells of small growing follicles. This response is transient but is reinitiated in preovulatory follicles exposed to the LH analog, human chorionic gonadotropin. Immunohistochemical analysis also showed gonadotropin induced Egr-1 in theca cells. The Egr-1 gene regulatory region responsive to gonadotropin signaling was localized within −164 bp of the transcription initiation site. Binding of Sp1/Sp3 to a proximal GC-box at −64/−46 bp was enhanced by FSH in immature granulosa cells but reduced after human chorionic gonadotropin stimulation of preovulatory follicles despite constant protein expression. This dynamic regulation of Sp1 binding was dependent on gonadotropin-regulated mechanisms that modulate Sp1/3-DNA binding activity. Serum response factor was active in granulosa cells and bound a consensus CArG-box/serum response element site, whereas two putative cAMP response elements within the −164-bp region bound cAMP regulatory element (CRE) binding protein (CREB) and a second cAMP-inducible protein immunologically related to CREB. Transient transfection analyses using Egr-1 promoter-luciferase constructs and site-specific mutations show that the serum response element, GC-box, and CRE−131 are involved in gonadotropin regulation of Egr-1 expression in granulosa cells. Specific kinase inhibitors of Erk or protein kinase A antagonized this induction while exogenously expressed Egr-1 enhanced reporter expression. These observations indicate that the Egr-1 gene is a target of both FSH and LH action that may mediate molecular programs of proliferation and/or differentiation during follicle growth, ovulation, and luteinization.

The formation of various tissues requires close communication between two groups of cells, epithelial and mesenchymal cells. COUP-TFs are transcription factors which have been shown to have functions in embryonic development. COUP-TFI is expressed mainly in the nervous system, and its targeted deletion leads to defects in the central and peripheral nervous systems. COUP-TFII is highly expressed in the mesenchymal component of the developing organs. A null mutation of COUP-TFII results in the malformation of the heart and blood vessels. From their expression pattern, we proposed that COUP-TFs regulate paracrine signals important for mesenchymal cell-epithelial cell interactions. In order to identify genes regulated by COUP-TF in this process, a rat urogenital mesenchymal cell line was stably transfected with a COUP-TFI expression vector. We found that NGFI-A, a gene with important functions in brain, organ, and vasculature development, has elevated mRNA and protein levels upon overexpression of COUP-TFI in these cells. A study of the promoter region of this gene identified a COUP-TF-responsive element between positions −64 and −46. Surprisingly, this region includes binding sites for members of the Sp1 family of transcription factors but no COUP-TF binding site. Mutations that abolish the Sp1 binding activity also impair the transactivation of the NGFI-A promoter by COUP-TF. Two regions of the COUP-TF molecule are shown to be important forNGFI-A activation: the DNA binding domain and the extreme C terminus of the putative ligand binding domain. The C-terminal region is likely to be important for interaction with coactivators. In fact, the coactivators p300 and steroid receptor activator 1 can enhance the transactivation of the NGFI-A promoter induced by COUP-TFI. Finally, we demonstrated that COUP-TF can directly interact with Sp1. Taken together, these results suggest that NGFI-A is a target gene for COUP-TFs and that the Sp1 family of transcription factors mediates its regulation by COUP-TFs.

FA (Fanconi anaemia) is a hereditary disease characterized by congenital malformations, progressive bone marrow failure and an extraordinary elevated predisposition to develop cancer. In the present manuscript we describe an anomalous high level of the proinflammatory cytokine IL-1β (interleukin-1β) present in the serum of FA patients. The elevated levels of IL-1β were completely reverted by transduction of a wild-type copy of the FancA cDNA into FA-A (FA group A) lymphocytes. Although the transcription factor NF-κB (nuclear factor-κB) is a well established regulator of IL-1β expression, our experiments did not show any proof of elevated NF-κB activity in FA-A cells. However, we found that the overexpression of IL-1β in FA-A cells is related to a constitutively activated PI3K (phosphoinositide 3-kinase)-Akt pathway in these cells. We provide evidence that the effect of Akt on IL-1β activation is mediated by the inhibition of GSK3β (glycogen synthase kinase 3β). Finally, our data indicate that the levels of IL-1β produced by FA-A lymphoblasts are enough to promote an activation of the cell cycle in primary glioblastoma progenitor cells. Together, these results demonstrate that the constitutive activation of the PI3K-Akt pathway in FA cells upregulates the expression of IL-1β through an NF-κB-independent mechanism and that this overproduction activates the proliferation of tumour cells.

BMD has a strong heritable component. Estrogen activity depends on the aromatization of androgenic precursors in nongonadal tissues both in postmenopausal women and men. Therefore, aromatase is an appealing candidate gene to explain, in part, the genetic component of BMD. In fact, an association between aromatase polymorphisms and BMD has been previously reported in some relatively small groups. In this study, we explored the relationship between several SNPs in the aromatase region and hip BMD in 1163 postmenopausal women. We found significant differences across genotypes, particularly in older women. The BMD differences between homozygous women with opposing genotypes were 4.2% in the whole group and 7.3% in women >67 yr of age. Body weight was significantly associated with BMD also, but there was no evidence for a statistically significant interaction between body weight and aromatase polymorphisms. Electrophoretic mobility shift assays suggested the binding of the CEBPβ transcription factor to the C/G rs1062033 locus, located ∼12 kb upstream of the translation start site. Experiments of transient transfection of osteoblastic cells with luciferase reporters showed differences in the transcriptional activity of alleles C and G at this locus, with different responses to the co-transfection of a CEBPβ expression vector. Furthermore, evidence for differential allelic expression was found in bone tissue samples. In conclusion, polymorphisms in a 12-kb region of the aromatase gene are associated with BMD in postmenopausal women, particularly during the late postmenopausal period. In vitro functional studies point to rs1062033 as a true regulatory polymorphism.