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Estrogen is a major sex steroid that affects the growth, maintenance, and homeostasis of the skeleton. Two isoforms of the estrogen receptor (ERalpha and ERbeta) mediate the transcriptional effects of estrogen. Although both isoforms of ER are present and functional in some human osteoblast (OB) cell lines, there is minimal information on the differential regulation of transcription by ERalpha and ERbeta homo- or heterodimers. This report demonstrates that ERalpha and ERbeta coexpression decreases the transcriptional capacity (relative to each ER isoform alone) on an estrogen response element-dependent reporter gene in OBs but not in other non-osteoblastic cell lines. These data suggest that ERalpha and ERbeta coexpression can differentially influence the degree of transcriptional activation in certain cell types. Interestingly, the overexpression of the steroid hormone receptor coactivator-1 (SRC1) resulted in preferential transcriptional enhancement by ERbeta as well as coexpressed ERalpha and ERbeta, whereas SRC2 overexpression appeared to preferentially enhance ERalpha transactivation. SRC3 overexpression failed to enhance estrogen-dependent transcription of any ER combination in OBs. Similar overexpression experiments in COS7 cells exhibited preferential enhancement of ERalpha function with all SRCs, including SRC3. Our data also demonstrated that SRC3 mRNA is reduced in osteoblastic cells, suggesting that SRC3 may have only a minor role in these cells. These data suggest that the transactivation capacity of various ER isoforms is both SRC species and cell type dependent.

The prereceptor regulation of glucocorticoids (GCs) by 11β-hydroxysteroid dehydrogenase type-1 (11β-HSD1), a bidirectional isozyme that interconverts active (cortisol) and inactive (cortisone) GCs, is an established determinant of GC function in tissues such as liver, adipose and bone. Although the therapeutic use of GCs is abundant in ophthalmic practice, where GC interactions with nuclear receptors modulate gene transcription, the prereceptor regulation of endogenous cortisol is not well described in ocular tissues. Recent descriptive studies have localised 11β-HSD1 to the human corneal epithelium and non-pigmented epithelium (NPE) of the ciliary body, indicating a link to corneal epithelial physiology and aqueous humour production. In this study, we characterise the functional aspects of the autocrine regulation of GCs in the anterior segment of the rabbit eye. Using our in-house generated primary antibody to human 11β-HSD1, immunohistochemical analyses were performed on paraffin-embedded sections of whole New Zealand white albino rabbits, (NZWAR) eyes. As in human studies, 11β-HSD1 was localised to the corneal epithelium and the NPE. No staining was seen in the albino ‘pigmented’ ciliary epithelium. Specific enzyme assays for oxo-reductase (cortisone→cortisol) and dehydrogenase (cortisol→cortisone) activity indicated predominant 11β-HSD1 oxo-reductase activity from both the intact ciliary body tissue (n=12, median 2.1 pmol/mg per h and range 1.25–2.8 pmol/mg per h; P=0.006) and primary cultures of corneal epithelial cells (n=12, median 3.0 pmol/mg per h and range 1.0–7.4 pmol/mg per h, P=0.008) compared with dehydrogenase activity (median 1.0 pmol/mg per h and range 0.5–2.0 pmol/mg per h; median 0.5 pmol/mg per h and range 0.25–1.9 pmol/mg per h respectively). These findings were supported by expression of 11β-HSD1 protein as visualised by Western blotting of ciliary body tissue and immunocytochemistry of corneal epithelial cells. Reduction of corneal epithelial cell proliferation was seen after primary cultures were co-incubated with cortisol and cortisone. 11β-HSD1 activity was not demonstrated in naïve conjunctival fibroblasts or corneal stromal keratocytes. Our results indicate that the distribution of 11β-HSD1 in the rabbit resembles that of the human eye and activates cortisone to cortisol in both corneal and uveal tissues. The NZWAR provides a suitable in vivo model for the further evaluation of 11β-HSD1 activity in the eye, especially its role in corneal epithelial and ciliary body physiology.