Bernd Stein

Bone metabolism is regulated by a balance between bone resorption caused by osteoclasts and bone formation caused by osteoblasts. This balance is disturbed in postmenopausal women as a result of lower serum estrogen levels. Estrogen, which is used in hormone replacement therapy to prevent postmenopausal osteoporosis, downregulates expression of the interleukin 6 (IL-6) gene in osteoblasts and bone marrow stromal cells. IL-6 is directly involved in bone resorption by activating immature osteoclasts. We show here that NF-kappa B and C/EBP beta are important regulators of IL-6 gene expression in human osteoblasts. Importantly, the IL-6 promoter is inhibited by estrogen in the absence of a functional estrogen receptor (ER) binding site. This inhibition is mediated by the transcription factors NF-kappa B and C/EBP beta. Evidence is presented for a direct interaction between these two factors and ER. We characterized the protein sequence requirements for this association in vitro and in vivo. The physical and functional interaction depends in part on the DNA binding domain and region D of ER and on the Rel homology domain of NF-kappa B and the bZIP region of C/EBP beta. The cross-coupling between ER, NF-kappa B, and C/EBP beta also results in reduced activity of promoters with ER binding sites. We further show that the mechanism of IL-6 gene repression by estrogen is clearly different from that of activation of promoters with ER binding sites. Therefore, drugs that separate the transactivation and transrepression functions of ER will be very helpful for treatment of osteoporosis without causing undesirable side effects.

Genomic clones coding for human fibroblast collagenase were isolated. By constructing and transfecting mutants with 5' and 3' deletion mutations of the 5' control region of the gene into human or murine cells, we delimited a 32-base-pair sequence at positions -73 to -42 which is required for the induction of transcription by the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate. The DNA element behaves as a 12-O-tetradecanoyl-phorbol-13-acetate-inducible enhancer: it mediates the stimulation of transcription to the heterologous herpes simplex virus thymidine kinase promoter and acts in a position- and orientation-independent manner. Differences in enhancer efficiency in different cell lines are interpreted to indicate differences in the activity of a trans-acting factor.

UV irradiation of human and murine cells enhances the transcription of several genes. Here we report on the primary target of relevant UV absorption, on pathways leading to gene activation, and on the elements receiving the UV-induced signal in the human immunodeficiency virus type 1 (HIV-1) long terminal repeat, in the gene coding for collagenase, and in the cellular oncogene fos. In order to induce the expression of genes. UV radiation needs to be absorbed by DNA and to cause DNA damage of the kind that cannot be repaired by cells from patients with xeroderma pigmentosum group A. UV-induced activation of the three genes is mediated by the major enhancer elements (located between nucleotide positions -105 and -79 of HIV-1, between positions -72 and -65 of the collagenase gene, and between positions -320 and -299 of fos). These elements share no apparent sequence motif and bind different trans-acting proteins; a member of the NF kappa B family binds to the HIV-1 enhancer, the heterodimer of Jun and Fos (AP-1) binds to the collagenase enhancer, and the serum response factors p67 and p62 bind to fos. DNA-binding activities of the factors recognizing the HIV-1 and collagenase enhancers are augmented in extracts from UV-treated cells. The increase in activity is due to posttranslational modification. While AP-1 resides in the nucleus and must be modulated there, NF kappa B is activated in the cytoplasm, indicating the existence of a cytoplasmic signal transduction pathway triggered by UV-induced DNA damage. In addition to activation, new synthesis of AP-1 is induced by UV radiation.