Fas ligand pathway

Wednesday, October 16, 2002 4:15 P.M. O-252 Mutation analysis of the GREAT (G Protein-Coupled Receptor Affecting Testis Descent) gene in cryptorchid patients. Aparna A. Kamat, Natalia Bogatcheva, Alexander I. Agoulnik. Baylor Coll of Medicine, Houston, TX. Objective: We have recently identified a novel gene, Great, which causes infertile bilateral intra-abdominal cryptorchidism in homozygous Great knockout mice. Here, we performed mutation screening of the human GREAT gene using denaturing high-pressure liquid chromatographic (DHPLC) analysis of cryptorchid patients. Relaxin is known to activate the GREAT receptor through the adenosine 3,5-monophosphate (cAMP)-dependent pathway. We used relaxin to analyze the functional properties of the mutant receptor that was identified. Design: Basic study. Materials/Methods: Samples from 61 patients with idiopathic cryptorchidism and 193 controls were analyzed for mutations in all eighteen exons of the GREAT gene using the automated WAVE Nucleic Acid Fragment Analysis System. Polymerase Chain Reaction was used to amplify the exons. Chromatograms of the elution profiles showing deviation from the wild-type profile were subjected to direct sequencing by the dye terminator method on an Applied Biosystems 373 DNA sequencer. For functional analysis of the mutant receptor, we produced mutant GREAT cDNA using site-directed mutagenesis. 293T cells derived from human embryonic kidney (HEK) fibroblast were transfected with plasmid of interest and reporter plasmid (pAP-tag5). Cells were stimulated with 0 –30 nM porcine relaxin in the presence of 0.25 mM 3-isobutyl-1-methylxanthine for 30 min, then harvested by flow and centrifuged. cAMP concentration in each well was measured in quadruplicate using enzyme immunoassay. Production of cAMP was normalized based on the efficiency of transfection estimated by the activity of secreted alkaline phosphatase produced by the co-transfected reporter pAP-tag5 plasmid. All experiments were repeated three times. Statistical analysis was performed using Student’s t-test. Results: In exon 12, two silent mutations (A/G transversions at nucleotide position 957 and 993) were detected in both cryptorchid patients (40/61) and controls. An A/G transversion was observed at nucleotide position 1810 in exon 17. A unique mutation detected in exon 8 causes an A to C nucleotide change at position 664 in heterozygous condition in one patient. This causes a missense substitution of threonine to proline (T222P). Analysis of 193 controls did not reveal a similar variation. Functional analysis revealed that relaxin stimulates dose-dependent cAMP production in HEK 293T cells transfected with wild-type GREAT. In contrast, cells expressing mutant T222P GREAT showed significantly lower cAMP production when stimulated with relaxin (p ⬍0.001). Relaxin does not affect cAMP production in cells transfected with an empty pCR3.1 vector. Conclusions: Mutation analysis of 61 cryptorchid patients with DHPLC identified a unique T222P mutation in the fourth leucine-rich repeat (LRR) of the receptor ectodomain, a structure essential for glycoprotein hormone binding. The mutant receptor is ineffective in activating relaxin-induced cAMP production suggesting that a mutation in one of the LRR could impair binding of the receptor with a ligand and/or downstream transmission of the signal. Thus, the GREAT gene may be involved in the etiology of some cases of cryptorchidism in humans. Supported by: NIH grants R01 HD37067 and P01 HD36289.

MENOPAUSE SPECIAL INTEREST GROUP Wednesday, October 16, 2002 2:00 P.M. O-253 Estradiol induces apoptosis in endothelial cells by up-regulating Fas/ Fas ligand pathway. Emre Seli, Umit Ali Kayisli, Belgin Selam, Murat Berkkanoglu, Aydin Arici. Dept of Obstetrics and Gynecology, Yale Univ Sch of Medicine, New Haven, CT. Objective: Atherosclerosis develops as a response to vascular injury. In response to atherogenic injury endothelial cells secrete chemoattractant molecules and increase leukocyte adhesion molecule expression on their

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Abstracts

membranes. This is followed by leukocyte recruitment and vascular smooth muscle cell proliferation resulting in intimal thickening. Estrogen delays the development of atherosclerosis and down-regulates intimal thickening in response to arterial injury in animal models, but the molecular mechanism of its actions is not well understood. We hypothesized that the vascular effects of estrogen may be partly mediated by a pro-apoptotic effect facilitating endothelial cell turnover and decreasing atherogenic signals from injured endothelial cells. We have previously shown that estrogen increases apoptotic activity in vascular cells in vitro and in vivo. In this study, we aimed to identify the mechanism by which estrogen promotes apoptosis in endothelial cells and evaluated the effect of estrogen on the activity of a major apoptotic pathway: Fas/Fas ligand (FasL) in human coronary artery endothelial cells (HCAEC) in culture. Design: HCAEC obtained from women were treated with different concentrations of 17-estradiol for 4 to 72 hours and cellular FasL mRNA and protein expression and secreted soluble FasL were quantified. Materials/Methods: HCAEC were grown in medium supplemented with growth factors and 5% fetal bovine serum. Prior to experiments, cells were incubated in phenol red-free media prepared with 2% charcoal-treated fetal bovine serum for 24 hours. Apoptosis was induced by growth factor and serum deprivation. Culture supernatants were collected and soluble FasL production by HCAEC was evaluated using ELISA. Total cellular protein was extracted and intracellular FasL was detected using Western analysis. Total RNA was also extracted and FasL mRNA expression was determined using semi-quantitative RT-PCR. FasL mRNA and protein expression was normalized by comparing to the expression of G3PDH and GAPDH, respectively. Results: FasL mRNA expression in HCAEC as detected by semi-quantitative RT-PCR increased with estradiol treatment in a concentrationdependent manner. By 8 hours, a 65% increase in FasL mRNA was observed in HCAEC treated with 10⫺8M 17-estradiol compared to controls. This was paralleled by a later increase in soluble FasL in culture supernatants (p ⬍0.05). On the other hand, cellular FasL protein detected by Western analysis decreased in response to estrogen treatment and a 50% decrease was observed by 48 hours. Conclusions: We had previously shown that estrogen induces apoptosis in vascular cells and that this increase is associated with an increase in Fas expression. Fas expressing cells undergo apoptosis when bound to FasL. In this study we have shown that in cultured HCAEC, under conditions that promote apoptosis, estrogen causes an increase in FasL mRNA expression and soluble FasL secretion resulting in a decrease in cellular FasL protein. These findings suggest that injured endothelial cells promote apoptotic cell death in response to estrogen by activating Fas/FasL pathway. We speculate that this would increase endothelial cell turnover and decrease atherogenic signals from injured endothelial cells. A similar effect in vascular smooth muscle cells would further inhibit intimal thickening and atherogenesis.

Wednesday, October 16, 2002 2:15 P.M. O-254 Analysis of endometrial RNA transcripts before and after treatment with raloxifene, tamoxifen, conjugated estrogen or placebo using polymerase chain reaction. Michael D. Vardy, Robert Lindsay, David S. Loose-Mitchell, Gregory L. Shipley, Jeri Nieves, Felicia Cosman. Albert Einstein Coll of Medicine, Bronx, NY; Columbia Univ and the Clin Reseach Ctr at Helen Hayes Hosp, West Haverstraw, NY; Univ of Texas, Houston, TX. Objective: To determine the feasibility of utilizing real time quantitative polymerase chain reaction (RT-QPCR) for extraction of messenger ribonucleic acid (mRNA) from paraffin fixed endometrial samples. To determine the effect of raloxifene (RLX), tamoxifen (TAM) and estrogen (CEE) on the level of expression of estrogen receptor alpha (ER), progesterone receptor (PR), c-fos and insulin-like growth factor-1 (IGF-1). Design: A randomized, triple blind, placebo(PBO)-controlled study was undertaken in healthy postmenopausal women to compare effect of 20 weeks of CEE 0.625 mg (n⫽15), TAM 20 mg (n⫽15), RLX 60 mg, (n⫽15) and PBO (n⫽13) on the endometrium. Materials/Methods: Total RNA was extracted from endometrial biopsies obtained by pipelle sampling before and after 20 weeks of drug therapy. Endometrial samples were fixed in paraffin blocks. RT-QPCR assaysspe-

Vol. 78, No. 3, Suppl. 1, September 2002