Abstract
BackgroundThe left-right determination factor (LEFTY) is a novel member of the TGF-β/Smad2 pathway and belongs to the premenstrual/menstrual repertoire in human endometrium, but little is known about its functional role in endometrial carcinomas (Em Cas). Herein, we focused on LEFTY expression and its association with progesterone therapy in Em Cas.MethodsRegulation and function of LEFTY, as well as its associated molecules including Smad2, ovarian hormone receptors, GSK-3β, and cell cycle-related factors, were assessed using clinical samples and cell lines of Em Cas.ResultsIn clinical samples, LEFTY expression was positively correlated with estrogen receptor-α, but not progesterone receptor (PR), status, and was inversely related to phosphorylated (p) Smad2, cyclin A2, and Ki-67 levels. During progesterone therapy, expression of LEFTY, pSmad2, and pGSK-3β showed stepwise increases, with significant correlations to morphological changes toward secretory features and decreased Ki-67 values. In Ishikawa cells, an Em Ca cell line that expresses PR, progesterone treatment reduced proliferation and induced increased expression of LEFTY and pGSK-3β, although LEFTY promoter regions were inhibited by transfection of PR. Moreover, inhibition of GSK-3β resulted in increased LEFTY expression through a decrease in its ubiquitinated form, suggesting posttranslational regulation of LEFTY protein via GSK-3β suppression in response to progesterone. In addition, overexpression or knockdown of LEFTY led to suppression or enhancement of Smad2-dependent cyclin A2 expression, respectively.ConclusionUpregulation of LEFTY may serve as a useful clinical marker for the therapeutic effects of progesterone for Em Cas, leading to inhibition of tumor cell proliferation through alteration in Smad2-dependent transcription of cyclin A2.
Highlights
The left-right determination factor (LEFTY) is a novel member of the Transforming growth factor β (TGF-β)/Smad2 pathway and belongs to the premenstrual/menstrual repertoire in human endometrium, but little is known about its functional role in endometrial carcinomas (Em Cas)
LEFTY1/2 genes were transcriptionally upregulated by TGF-β1/Smad2 signaling in Ishikawa cells, while western blot analysis using Em Ca tissues revealed an inverse relation between LEFTY and pSmad2 expression
We found that LEFTY protein can be posttranslationally regulated by progesterone through alteration in GSK-3β activity in Em Cas from the following evidence: i) LEFTY protein contains five putative GSK-3β phosphorylation motifs and can be degraded through the ubiquitin-proteasome pathway; ii) inhibition of GSK-3β by LiCl resulted in increased expression of LEFTY, while the effects were abrogated by mutations in the five putative GSK-3β phosphorylation motifs in the protein; iii) pGSK-3β at Ser9 (pGSK-3β) score was significantly increased during progesterone therapy in Em Ca tissues, in line with the findings demonstrating an increase in pGSK-3β expression by progesterone in Ishikawa cells; and iv) LEFTY1/2 mRNA expression levels were significantly decreased during the therapy, probably through transcriptional repression of the promoter activities by progesterone signals, despite the increased
Summary
The left-right determination factor (LEFTY) is a novel member of the TGF-β/Smad pathway and belongs to the premenstrual/menstrual repertoire in human endometrium, but little is known about its functional role in endometrial carcinomas (Em Cas). Full list of author information is available at the end of the article factors including marked obesity, diabetes mellitus, and hypertension [3, 4]. In these younger patients who want to have children in the future, hormone therapy using highdose progesterone is frequently performed for the preservation of fertility [3,4,5]. TGF-β receptor I recruits and subsequently induces an intracellular downstream signaling cascade involving
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