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The FK506-binding protein 51 (FKBP51) binds progesterone receptor (PR), glucocorticoid receptor (GR), and androgen receptor (AR) to coregulate their transcriptional activity. We evaluated FKBP51 expression and function in human leiomyoma vs. myometrial tissues and primary cultures to discover FKBP51 role(s) in the pathogenesis of leiomyomas. Quantification of in situ FKBP51 mRNA and protein levels inpaired myometrial vs. leiomyoma tissues from proliferative and secretory phases were analyzed by qPCR (n = 14), immunoblotting (n = 20), and immunohistochemistry (n = 12). Control (scramble) vs. FKBP5 siRNA-transfected leiomyoma cell cultures were assessed for proliferation, apoptosis, and mRNA levels of genes involved in cell survival and extracellular matrix (ECM) formation. Significantly higher FKBP5 mRNA levels were detected in leiomyoma vs. paired myometrium (P 0.001). Immunoblot (P = 0.001) and immunostaining (P ≤ 0.001) confirmed increased FKBP51 levels in leiomyoma vs. paired myometrium. Compared to control siRNA transfection, FKBP5-silenced leiomyoma cell cultures displayed significantly decreased cell survival factors and reduced proliferation (P 0.05). Moreover, qPCR analysis revealed significantly lower mRNA levels of ECM, TIPM1, and TIPM3 proteins in FKBP5-silenced leiomyoma cell cultures (P 0.05). Increased FKBP51 expression in leiomyoma likely involves dysregulation of steroid signaling by blocking GR and PR action and promoting proliferation and ECM production. Evaluating the effect of FKBP51 inhibition in preclinical studies will clarify its significance as a potential therapeutic approach against leiomyoma.

Context Enhanced levels of catecholestradiols, 2-hydroxyestradiol (2-OHE2) or 4-hydroxyestradiol (4-OHE2), are reported in endometriosis. During gestation, catecholestradiol activation of adrenergic receptors (AR) elevates estrogen receptor (ER)-independent proliferation of uterine arterial endothelial cells. Objective To investigate β-AR-mediated catecholestradiol effects on human endometrial stromal cell (HESC) and epithelial cell survival in endometriosis. Design β-AR immunostaining of eutopic and ectopic endometria (n = 9). Assays for cell viability, 5-bromo-2′-deoxyuridine proliferation, apoptosis, quantitative PCR, and estrogenicity (alkaline phosphatase activity), as well as siRNA β-AR silencing and immunoblot analyses of cultured HESCs or Ishikawa cells treated with control or 2-OHE2 or 4-OHE2 ±β-AR antagonist or ±p38 MAPK inhibitor. Setting University research institution. Patients Women with or without endometriosis. Interventions None. Main Outcome Measures β-AR expression in eutopic vs ectopic endometria and regulation of HESC survival by 2-OHE2 and 4-OHE2. Results Eutopic and ectopic endometrial stromal and epithelial cells displayed β2-AR immunoreactivity with increased staining in the functionalis vs basalis layer (P Conclusions Catecholestradiols increase endometrial cell survival by an ER-independent β-AR-mediated p38 MAPK activation, suggesting that agents blocking β-AR (e.g., propranolol) or inhibiting 2-OHE2- or 4-OHE2-generating enzymes (i.e., CYP1A1/B1) could treat endometriosis.

Taste buds are aggregates of 50–100 cells, only a fraction of which express genes for taste receptors and intracellular signaling proteins. We combined functional calcium imaging with single-cell molecular profiling to demonstrate the existence of two distinct cell types in mouse taste buds. Calcium imaging revealed that isolated taste cells responded with a transient elevation of cytoplasmic Ca2+to either tastants or depolarization with KCl, but never both. Using single-cell reverse transcription (RT)-PCR, we show that individual taste cells express either phospholipase C β2 (PLCβ2) (an essential taste transduction effector) or synaptosomal-associated protein 25 (SNAP25) (a key component of calcium-triggered transmitter exocytosis). The two functional classes revealed by calcium imaging mapped onto the two gene expression classes determined by single-cell RT-PCR. Specifically, cells responding to tastants expressed PLCβ2, whereas cells responding to KCl depolarization expressed SNAP25. We demonstrate this by two methods: first, through sequential calcium imaging and single-cell RT-PCR; second, by performing calcium imaging on taste buds in slices from transgenic mice in which PLCβ2-expressing taste cells are labeled with green fluorescent protein. To evaluate the significance of the SNAP25-expressing cells, we used RNA amplification from single cells, followed by RT-PCR. We show that SNAP25-positive cells also express typical presynaptic proteins, including a voltage-gated calcium channel (α1A), neural cell adhesion molecule, synapsin-II, and the neurotransmitter-synthesizing enzymes glutamic acid decarboxylase and aromatic amino acid decarboxylase. No synaptic markers were detected in PLCβ2 cells by either amplified RNA profiling or by immunocytochemistry. These data demonstrate the existence of at least two molecularly distinct functional classes of taste cells: receptor cells and synapse-forming cells.

Phospholipase C-type beta2 (PLCbeta2) is expressed in a subset of cells within mammalian taste buds. This enzyme is involved in the transduction of sweet, bitter, and umami stimuli and thus is believed to be a marker for gustatory sensory receptor cells. We have developed transgenic mice expressing green fluorescent protein (GFP) under the control of the PLCbeta2 promoter to enable one to identify these cells and record their physiological activity in living preparations. Expression of GFP (especially in lines with more than one copy integrated) is strong enough to be detected in intact tissue preparations using epifluorescence microscopy. By immunohistochemistry, we confirmed that the overwhelming majority of cells expressing GFP are those that endogenously express PLCbeta2. Expression of the GFP transgene in circumvallate papillae occurs at about the same time during development as endogenous PLCbeta2 expression. When loaded with a calcium-sensitive dye in situ, GFP-positive taste cells produce typical Ca2+ responses to a taste stimulus, the bitter compound cycloheximide. These PLCbeta2 promoter-GFP transgenic lines promise to be useful for studying taste transduction, sensory signal processing, and taste bud development.

After ovulation, there is a shift in ovarian steroidogenesis from an estrogen-producing ovarian follicle to a progesterone-producing corpus luteum. The first step in human ovarian steroidogenesis is catalyzed by cholesterol side-chain cleavage cytochrome P450 (CYP11A1) enzyme. Steroidogenic factor-1 is an orphan nuclear receptor that regulates several steroidogenic enzymes, including CYP11A1. Liver receptor homolog-1 (LRH-1) is another orphan nuclear receptor that is expressed in the human ovary. After ovulation there is a down-regulation in steroidogenic factor-1, which is associated with an up-regulation of LRH-1 expression. These changes coincide with increased level of CYP11A1 expression in human corpus luteum. In this study, we examined the role of LRH-1 in the regulation of human granulosa cell CYP11A1 expression. Cotransfection of human granulosa cell tumor cells with CYP11A1 promoter and LRH-1 expression vector resulted in a significant increase in CYP11A1 expression. Deletion analysis revealed two putative LRH-1 binding sites at -1580 and -40, which was confirmed by EMSA. Dosage-sensitive sex-reversal-adrenal hypoplasia congenita critical region on the X chromosome, gene-1 inhibited LRH-1 stimulated CYP11A1 expression, and that was not overcome by the presence of PKA agonist. We conclude that CYP11A1 expression in human granulosa cells is regulated by LRH-1. We propose that LRH-1 could be the major transcription factor for the post-ovulatory surge in human ovarian steroidogenesis.

The introduction and expression of exogenous DNA in neurons is valuable for analyzing a range of cellular and molecular processes in the periphery, e.g., the roles of transduction-related proteins, the impact of growth factors on development and differentiation, and the function of promoters specific to cell type. However, sensory receptor cells, particularly chemosensory cells, have been difficult to transfect. We have successfully introduced plasmids expressing green and Discosoma Red fluorescent proteins (GFP and DsRed) into rat taste buds in primary culture. Transfection efficiency increased when delaminated taste epithelium was redigested with fresh protease, suggesting that a protective barrier of extracellular matrix surrounding taste cells may normally be present. Because taste buds are heterogeneous aggregates of cells, we used α-gustducin, neuronal cell adhesion molecule (NCAM), and neuronal ubiquitin carboxyl terminal hydrolase (PGP9.5), markers for defined subsets of mature taste cells, to demonstrate that liposome-mediated transfection targets multiple taste cell types. After testing eight commercially available lipids, we identified one, Transfast, that is most effective on taste cells. We also demonstrate the effectiveness of two common “promiscuous” promoters and one promoter that taste cells use endogenously. These studies should permit ex vivo strategies for studying development and cellular function in taste cells. © 2005 Wiley Periodicals, Inc. J. Neurobiol, 2005

Steroidogenic acute regulatory protein (StAR) plays a critical role in the initial step of steroid hormone synthesis. In the present study, we investigated the role of liver receptor homolog-1 (LRH-1) and dosage-sensitive sex reversal, adrenal hypoplasia congenital critical region on the X chromosome, gene 1 (DAX-1) in the regulation of StAR gene expression in human granulosa cell tumor cells. We also examined the effect of protein kinase A (PKA) signaling pathway on the expression of StAR in the presence of LRH-1 and DAX-1. Cell transfection, mutation analysis, and EMSA were performed. LRH-1 significantly induced StAR promoter activity in a concentration-dependent manner. This induction was further augmented in the presence of PKA agonist. Using deletion analysis, we demonstrated LRH-1 binding site at 105/95. Mutation of this site resulted in a significant decrease in the StAR promoter activity. Using EMSA, the ability of this ciselement to bind LRH-1 was confirmed. DAX-1 inhibited LRH1-stimulated StAR promoter activity in a concentration-dependent manner. This inhibition was also maintained in the presence of PKA stimulation. Our results demonstrated that LRH-1 plays a critical role in the induction of StAR gene expression. We hypothesize that LRH-1 could be the major transcription factor responsible for the rapid and significant increase in ovarian StAR gene expression after ovulation. (J Clin Endocrinol Metab 89: 3042–3047, 2004)

Cyclic AMP (cAMP) and Ca(2+) are two ubiquitous second messengers in transduction pathways downstream of receptors for hormones, neurotransmitters and local signals. The availability of fluorescent Ca(2+) reporter dyes that are easily introduced into cells and tissues has facilitated analysis of the dynamics and spatial patterns for Ca(2+) signaling pathways. A similar dissection of the role of cAMP has lagged because indicator dyes do not exist. Genetically encoded reporters for cAMP are available but they must be introduced by transient transfection in cell culture, which limits their utility. We report here that we have produced a strain of transgenic mice in which an enhanced cAMP reporter is integrated in the genome and can be expressed in any targeted tissue and with tetracycline induction. We have expressed the cAMP reporter in beta-cells of pancreatic islets and conducted an analysis of intracellular cAMP levels in relation to glucose stimulation, Ca(2+) levels, and membrane depolarization. Pancreatic function in transgenic mice was normal. In induced transgenic islets, glucose evoked an increase in cAMP in beta-cells in a dose-dependent manner. The cAMP response is independent of (in fact, precedes) the Ca(2+) influx that results from glucose stimulation of islets. Glucose-evoked cAMP responses are synchronous in cells throughout the islet and occur in 2 phases suggestive of the time course of insulin secretion. Insofar as cAMP in islets is known to potentiate insulin secretion, the novel transgenic mouse model will for the first time permit detailed analyses of cAMP signals in beta-cells within islets, i.e. in their native physiological context. Reporter expression in other tissues (such as the heart) where cAMP plays a critical regulatory role, will permit novel biomedical approaches.

After ovulation, ovarian 3β-hydroxysteroid dehydrogenase type II (HSD3B2) expression increases to enhance the shift of steroidogenesis toward progesterone biosynthesis. Steroidogenic factor-1 (SF-1) is a transcription factor for several genes encoding steroidogenic enzymes. However, the level of SF-1 expression decreases in the human corpus luteum (CL) after ovulation. Liver receptor homolog-1 (LRH-1) is another member of the orphan nuclear receptor family. We hypothesize that LRH-1, rather than SF-1, plays an essential role in the regulation of corpus luteum steroidogenesis. Semiquantitative RT-PCR and real-time PCR were performed to quantify the level of LRH-1 expression and correlate with HSD3B2 level. Cell transfection, mutation analysis, and EMSA were performed to examine the role of LRH-1 in the regulation of HSD3B2. LRH-1 expression was higher in CL, compared with mature ovarian follicles. Cotransfection of granulosa cells with HSD3B2 and LRH-1 resulted in a 10-fold increase of transcription. DAX-1 inhibited LRH-1-stimulated HSD3B2, which was maintained in the presence of dibutyryl cAMP. Mutation of the either of the two putative LRH-1 binding sites, which were confirmed by EMSA, in the HSD3B2 promoter decreased LRH-1 stimulation. Our findings suggest that LRH-1 is highly expressed in CL, and it plays an essential role in the regulation of HSD3B2.