Galanin, a novel neuropeptide/hypothalamic hormone originally identified and isolated by virtue of its carboxy-terminal amide group, has recently been shown to have a diverse range of biological activities, including potent effects on the secretion of insulin and growth hormone. The physiological role of galanin remains unclear, with different effects being observed when porcine and rat galanin have been used in various animal model systems and in human studies. Molecular cloning of cDNA encoding human galanin and galanin mRNA associated peptide (GMAP) from both pituitary and neuroblastoma sources has revealed a unique and unexpected structure. In contrast to porcine, bovine and rodent galanin, human galanin lacks a carboxy-terminal amide. By analogy to other neurohormones, the absence of carboxy-terminal amidation would be expected to have significant effects on functional properties such as affinity for different receptor subtypes and physiological half life, and may be responsible for the species specificity observed in the action of galanin.

Bone loss observed in postmenopausal women is clearly associated with a decrease in estrogen levels. Interleukin 6 (IL-6), a multifunctional cytokine involved in osteoclast differentiation, is secreted by osteoblasts and appears to be a key molecule in the osteoporotic process. As previous reports have shown that the human IL-6 promoter is inhibited by estradiol, we investigated the mechanism of estradiol (E2)-mediated IL-6 inhibition in human cells. Analysis of the IL-6 secretion as a function of time in osteoblastoma Saos-2 cells, using an IL-6 ELISA test, showed that a maximal E2 inhibition of tumor necrosis factor-alpha (TNF alpha) induction could be monitored between 2 and 24 h of treatment. IL-6 inhibition was clearly estrogen agonist-specific in Saos-2 and MCF7 cells. Transient transfections of HeLa cells with a pIL-6/CAT plasmid and an estrogen receptor (human ER) expression vector, confirmed the role of the human ER in inhibition of the IL-6 promoter. Deletion and mutational analysis of the promoter highlighted the role of the -185/-60 region and showed that in both MCF7 and HeLa cells, the nuclear factor-IL 6 (NF-IL6) site cooperates with the nuclear factor-kappa B (NF-kappa B) motif to produce maximal induction by TNF alpha, whereas the CCAAT/enhancer-binding protein (C/EBP) site displayed different cooperative effects toward NF-kappa B depending on the cell line used. In HeLa cells, but not in MCF7 cells, we defined an essential role for the C/EBP site by showing that the E2 sensitivity was clearly dependent on its integrity. In these cell lines, the NF-kappa B site mutation abrogated both the TNF alpha-and E2- sensitivity of the construct.

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