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Wendy R Kam, Raheleh Rahimi Darabad, David A Sullivan; Membrane Steroid Receptors are Expressed by Human Meibomian Gland Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2014;55(13):17.
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© ARVO (1962-2015); The Authors (2016-present)
Recent research has shown that sex steroids can activate diverse signaling pathways to rapidly alter intracellular cyclic AMP (cAMP) and/or calcium (Ca2+) levels. These pathways are mediated by novel, membrane-bound receptors for 17β-estradiol (E2), dihydrotestosterone (DHT), and progesterone (P4), and serve to modulate many non-genomic cellular actions of these hormones. We hypothesize that such membrane receptors are present in human meibomian gland epithelial cells (HMGEC). This hypothesis is prompted by our recent discovery of rapid E2 effects on cAMP accumulation in these cells, as well as the recognition that sex steroids exert a significant regulatory influence on meibomian gland function. Our goal was to test this hypothesis, and also to determine whether [a] GPR30 receptors are present in human corneal epithelial cells (HCEC) and [b] E2 stimulates a rapid uptake of Ca2+ in HMGEC.
Near-confluent, immortalized HMGEC and HCEC (gift from Dr. James Jester) were lysed in SDS sample buffer, separated by gel electrophoresis, transferred to nitrocellulose, and incubated with antibodies specific for E2 (GPR30), DHT (GPRC6A), or P4 (PGRMC1) membrane receptor protein. Human breast cancer cell (MCF-7) lysate served as a positive control for GPR30 and PGRMC1 expression; human prostate cancer cell (LNCaP) lysate was a positive control for GPRC6A. Intracellular Ca2+ measurements in cells loaded with Fura-2 calcium indicator and treated with E2 were obtained in real-time using a ratio imaging system.
HMGEC express GPR30, GPRC6A, and PGRMC1 membrane receptor proteins for E2, DHT, and P4. Similarly, HCEC contain GPR30 receptors. In contrast, E2 had an inconsistent effect on rapid calcium accumulation in HMGEC.
Our study demonstrates the presence of sex steroid membrane receptors in HMGEC and HCEC. These receptors may mediate rapid signaling events. Our functional data indicate that E2 may act in HMGEC through pathways mediated by cAMP, rather than Ca2+.
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