External Ca2+ entry into myometrial smooth-muscle cells is important to uterine contraction and hence to labor progression and parturition. Proteins of the transient receptor potential (Trp) channel family are putative capacitative Ca2+ entry channels that respond to contractant-generated signals and intracellular Ca2+ store depletion. Quantitative reverse transcription-polymerase chain reaction was used to examine the relative expression of TrpC mRNAs in rat myometrium and determine their expression pattern during pregnancy and labor. rTrpC1, rTrpC2, rTrpC4, rTrpC5, rTrpC6, and rTrpC7 mRNAs, but not rTrpC3 mRNA, were expressed in nonpregnant rat myometrium. With the exception of rTrpC7, the resulting products were sequenced and found to be identical with published sequences; new rTrpC7 sequence exhibited >88% homology to mouse and human TrpC7 coding regions. Relative to beta-actin mRNA, rTrpC4 mRNA was expressed in the greatest abundance. rTrpC1, 5, and 6 mRNAs were expressed at lower levels, whereas rTrpC2 and 7 mRNAs were barely detectable. This relative expression pattern was also observed throughout the course of gestation. There were no major differences in expression of rTrpC1, 2, 4, or 7 mRNAs between Day 13 and Day 21 of gestation or labor. Rat TrpC5 and TrpC6 mRNA expression decreased in pregnancy but was not altered between Day 13 and Day 21 or in labor. Western blot analysis generally confirmed these observations with respect to protein expression. These data suggest that rTrpC4 may play a major role in regulated Ca2+ entry in myometrial cells and throughout pregnancy but do not rule out contributions from other Trp proteins.
Expression of pulmonary surfactant, a complex mixture of lipids and proteins that acts to reduce alveolar surface tension, is developmentally regulated and restricted to lung alveolar type II cells. The hydrophobic protein surfactant protein-B (SP-B) is essential in surfactant function, and insufficient levels of SP-B result in severe respiratory dysfunction. Glucocorticoids accelerate fetal lung maturity and surfactant synthesis both experimentally and clinically. Glucocorticoids act transcriptionally and post-transcriptionally to increase steady-state levels of human SP-B mRNA; however, the mechanism(s) by which glucocorticoids act post-transcriptionally is unknown. We hypothesized that glucocorticoids act post-transcriptionally to increase SP-B mRNA stability via sequence-specific mRNAprotein interactions. We found that glucocorticoids increase SP-B mRNA stability in isolated human type II cells and in nonpulmonary cells, but do not alter mouse SP-B mRNA stability in a mouse type II cell line. Deletion analysis of an artificially-expressed SP-B mRNA indicates that the SP-B mRNA 39-untranslated region (UTR) is necessary for stabilization, and the region involved can be restricted to a 126-nucleotide-long region near the SP-B coding sequence. RNA electrophoretic mobility shift assays indicate that cytosolic proteins bind to this region in the absence or presence of glucocorticoids. The formation of mRNA:protein complexes is not seen in other regions of the SP-B mRNA 39-UTR. These results indicate that a specific 126-nucleotide region of human SP-B 39-UTR is necessary for increased SP-B mRNA stability by glucocorticoids by a mechanism that is not lung cell specific and may involve mRNA-protein interactions.
Adequate expression of surfactant protein-B (SP-B) is critical in the function of pulmonary surfactant to reduce alveolar surface tension. Expression of SP-B mRNA is restricted to specific lung-airway epithelial cells, and human SP-B mRNA stability is increased in the presence of the synthetic glucocorticoid dexamethasone (DEX). Although the mechanism of SP-B mRNA stabilization by DEX is unknown, studies suggest involvement of the glucocorticoid receptor (GR). We developed a dual-cistronic plasmid-based expression assay in which steady-state levels of SP-B mRNA, determined by Northern analysis, reproducibly reflect changes in SP-B mRNA stability. Using this assay, we found that steady-state levels of SP-B mRNA increased greater than twofold in transfected human-airway epithelial cells (A549) incubated with DEX (10(-7) M). DEX-mediated changes in SP-B mRNA levels required the presence of the SP-B mRNA 3'-untranslated region but did not require ongoing protein synthesis. The effect of DEX on SP-B mRNA levels was dose dependent, with maximal effect at 10(-7) M. DEX increased levels of SP-B mRNA in cells lacking GR, and the presence of the GR antagonist RU486 did not interfere with the effect of DEX. Surprisingly, other steroid hormones (progesterone, estradiol, and vitamin D; 10(-7) M) significantly increased SP-B mRNA levels, suggesting a common pathway of steroid hormone action on SP-B mRNA stability. These results indicate that the effect of DEX to increase SP-B mRNA stability is independent of activated GR and suggests that the mechanism is mediated by posttranscriptional or nongenomic effects of glucocorticoids.
The ability of pulmonary surfactant to reduce alveolar surface tension requires adequate expression of surfactant protein B (SP-B). Dexamethasone (DEX, 10(-7) M) increases human SP-B mRNA stability by a mechanism that requires a 126-nt-long segment (the 7.6S region) of the 3'-untranslated region (3'-UTR). The objective of this study was to identify sequences in the 7.6S region that mediate regulation of SP-B mRNA stability. The 7.6S region was found to be sufficient for DEX-mediated stabilization of mRNA. Sequential substitution mutagenesis of the 7.6S region indicates that a 90-nt region is required for DEX-mediated stabilization and maintenance of intrinsic stability. In this region, one 30-nt-long element (002), predicted to form a stem-loop structure, is sufficient for DEX-mediated stabilization of mRNA and intrinsic mRNA stability. Cytosolic proteins specifically bind element 002, and binding activity is unaffected whether proteins are isolated from cells incubated in the absence or presence of DEX. While loop sequences of element 002 have no role in regulation of SP-B mRNA stability, the proximal stem sequences are required for DEX-mediated stabilization and specific binding of proteins. Mutation of the sequences that comprise the proximal or distal arm of the stem negates the destabilizing activity of element 002 on intrinsic SP-B mRNA stability. These results indicate that cytosolic proteins bind a single hairpin structure that mediates intrinsic and hormonal regulation of SP-B mRNA stability via mechanisms that involve sequences of the stems of the hairpin structure.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.