Somatic gene therapies require targeted transfer of the therapeutic gene(s) into stem cells that proliferate and then differentiate and express the gene in a tissue-restricted manner. We have developed an approach for gene therapy using marrow cells that takes advantage of the osteoblast specificity of the osteocalcin promoter to confine expression of chimeric genes to bone. Adherent marrow cells, carrying a reporter gene [chloramphenicol acetyltransferase (CAT)] under the control of a 1.7-kilobase rat osteocalcin gene promoter, were expanded ex vivo. After transplantation by intravenous infusion, engrafted donor cells in recipient mice were detected by the presence of the transgene in a broad spectrum of tissues. However, expression of the transgene was restricted to osteoblasts and osteocytes, as established by biochemical analysis of CAT activity and immunohistochemical analysis of CAT expression at the single cell level. Our data indicate that donor cells achieved long-term engraftment in various tissues of the recipients and that the CAT gene under control of the osteocalcin promoter is expressed specifically in bone. Thus, transplantation of multipotential marrow cells containing the osteocalcin promoter-controlled transgene provides an efficacious approach to deliver therapeutic gene expression to osteoblasts for treatment of bone disorders or tumor metastasis to the skeleton.

A new concept in reproductive endocrinology is that the status of the ovary as a glucocorticoid target organ alters with follicular development. Evidence for a physiological role of glucocorticoids in the regulation of ovarian folliculogenesis has been strengthened by the discovery that 11beta-hydroxysteroid dehydrogenase (11betaHSD) mRNA expression in human granulosa cells is developmentally regulated. In this study, we quantified the pattern of expression and investigated the cellular location of 11betaHSD type 1 (11betaHSD1), 11betaHSD type 2 (11betaHSD2), glucocorticoid receptor (GR), and mineralocorticoid receptor (MR) mRNAs during follicular maturation in rat ovary. Immature female rats received treatment with eCG to induce preovulatory follicular development or eCG followed by hCG to induce luteinization. 11betaHSD1, 11betaHSD2, GR, and MR mRNAs were all detectable by ribonuclease protection assay in ovarian total RNA. Treatment with eCG alone caused an approximately 8-fold increase in the ovarian level of 11betaHSD1 mRNA, which rose to approximately 30-fold after additional treatment with hCG. Equine CG alone did not measurably affect the ovarian 11betaHSD2 mRNA level, but additional treatment with hCG reduced it to 34% of the control level. Expression of GR mRNA was unchanged by any gonadotropin treatment, while MR mRNA was down-regulated. A similar pattern of 11betaHSD1, 11betaHSD2, GR, and MR mRNA expression was observed in isolated granulosa cells. These results provide direct experimental evidence that 11betaHSD genes are gonadotropically regulated in the rat ovary, including granulosa cells, and are consistent with a shift in glucocorticoid metabolism from inactivation (due to oxidation by 11betaHSD2) to activation (reduction by 11betaHSD1) during hCG-induced granulosa cell luteinization.

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Granulosa cells from preovulatory follicles show increased expression of 11 -hydroxysteroid dehydrogenase type 1 (11 HSD1) at the time of ovulation. As ovulation may be an inflammatory process, this may be a mechanism of local enhancement of the activity of anti-inflammatory glucocorticoids. In this study, we examined direct effects of LH, the proinflammatory cytokine, interleukin-1 (IL-1 ), and pharmacological activators of protein kinase A (PKA) (forskolin and dibutyryl (db) cAMP) and PKC (LHreleasing hormone and phorbol 12-myristate 13-acetate (PMA)) signalling on the expression of 11 HSD1 mRNA in vitro. Granulosa cells from immature female rat ovaries were cultured (pretreatment) in serum-free medium 199 containing recombinant human (rh) FSH (1 ng/ml) for 48 h to induce responsiveness to LH. Cell monolayers were then washed and cultured (test treatment) for a further 12 h in the presence of rhLH (0-100 ng/ml), IL-1 (0-50 ng/ml), or both. Total RNA was extracted from granulosa cell monolayers and taken for quantitative ribonuclease protection analysis of 11 HSD1 mRNA. The low level of 11 HSD1 mRNA detectable in unstimulated (control) cultures was increased approximately twofold by the 48-h pretreatment with rhFSH. Subsequent exposure to rhLH (1-100 ng/ml) for a further 12 h dose-dependently increased 11 HSD1 mRNA expression by an additional two-to threefold. Forskolin (10 µM), db-cAMP (2 mM), LH-releasing hormone (LHRH; 1 µM) and PMA (200 nM) were also stimulatory. IL-1 (0·05-50 ng/ml) stimulated 11 HSD1 mRNA expression in a dose-related manner, both in the absence and in the presence of rhLH (3 ng/ml). The interaction between IL-1 (5 ng/ml) and rhLH (3 ng/ml) was additive. Cotreatment with a 50-fold excess of IL-1 receptor antagonist fully reversed the action of IL-1 . We conclude that 11 HSD1 mRNA expression in functionally mature granulosa cells is directly stimulated by gonadotrophins and IL-1 in vitro, potentially involving post-receptor signalling via PKA-and PKC-mediated pathways. Thus both LH and IL-1 may serve physiological roles in the upregulation of 11 HSD1 gene expression by granulosa cells in ovulatory follicles.