Abstract
Purpose: :
The nuclear receptor superfamily is comprised of ligand-activated and orphan nuclear factors involved in the transcriptional regulation of genes controlling cell fate, steroidogenic endocrine functions, and neurogenesis. The purpose of this work was to study the gene and protein expression profiles which confer endocrine genotype/phenotype to the human ciliary epithelium (CE).
Methods: :
ESTs clones (CBS-533, CBS-375, CBS-180 and CBS-321) sharing 100% homology with orphan and steroid/thyroid hormone nuclear receptors [COUP-TFI; RXRβ; LXRβ; and TRβ] were isolated by subtractive hybridization from a human ciliary body (CB) cDNA library. Additional clones were identified by PCR amplification including CYP19 (aromatase), SF-1, and chorionic gonadotropin β subunit (hCGβ). Northern and Western blot analysis was used to determine their patterns of expression (mRNA and protein) in ocular tissues. Indirect immunofluorescence on cryostat sections was used to determine the cellular distribution of the encoded proteins. The detection of hCG in ciliary processes and cultured ciliary epithelial cells was determined by a chemiluminescent immunoassay system (Immulite analyzer).
Results: :
The patterns of expression of the above genes within the human eye, confirmed that they were expressed in the CB. The COUP-TFI gene, in particular, was very abundant and when compared to other ocular tissues (i.e., retina) it was most restricted to the CE. The underlying endocrine genotype/phenotype of the CE was also supported by the expression of the proendocrine transcription factor Neurogenin 3 (Ngn3), and Lgr5, a marker of adult stem cells. hCG+ cells were immunodetected within the inner cell layer of the CE. Under serum-free culture medium, human NPE cells secreted and accumulated hCG (38mIU/ml) over a period of 24h. During the same period of time when the NPE cells were exposed to galanin (20ng/ml), a modulator of neuroendocrine activities, hCG was released with up to a 2.5-fold increase.
Conclusions: :
Collectively, these results provide new evidence on the restricted expression of transcription factors linked to steroidogenesis in the CE, and the expression of hCG, a hormone that has been shown to lower intraocular pressure. These results also demonstrate the expression, in the adult CE, of molecular factors known to direct differentiation of multipotent secretory progenitors to endocrine cells.
Keywords: ciliary body • gene screening • gene/expression