Prolactin signaling through the short isoform of the mouse prolactin receptor regulates DNA binding of specific transcription factors, often with opposite effects in different reproductive issues

<p>Abstract</p> <p>Background</p> <p>It has been well established that prolactin (PRL) signals through the long form of its receptor (PRL-RL) and activates the Jak/Stat pathway for transcription of PRL target genes. However, signaling pathways mediated through the short PRL-R isoform (PRL-RS) remains controversial. Our recent finding that PRL signaling through PRL-RS represses two transcription factors critical for follicular development lead us to examine other putative PRL/PRL-RS target transcription factors in the decidua and ovary, two well-known target tissues of PRL action in reproduction.</p> <p>Methods</p> <p>In this investigation we used mice expressing PRL-RS on a PRL-R knockout background and a combo protein/DNA array to study the transcription factors regulated by PRL through PRL-RS only.</p> <p>Results</p> <p>We show that PRL activation of the PRL-RS receptor either stimulates or inhibits the DNA binding activity of a substantial number of transcription factors in the decidua as well as ovary. We found few transcription factors to be similarly regulated in both tissues, while most transcription factors are oppositely regulated by PRL in the decidua and ovary. In addition, some transcription factors are regulated by PRL only in the ovary or only in the decidua. Several of these transcription factors are involved in physiological pathways known to be regulated by PRL while others are novel.</p> <p>Conclusion</p> <p>Our results clearly indicate that PRL does signal through PRL-RS in the decidua as well as the ovary, independently of PRL-RL, and activates/represses transcription factors in a tissue specific manner. This is the first report showing PRL/PRL-RS regulation of specific transcription factors. Many of these transcription factors were not previously known to be PRL targets, suggesting novel physiological roles for this hormone.</p

Generation of mice expressing only the long form of the prolactin receptor reveals that both isoforms of the receptor are required for normal ovarian function

Prolactin (PRL), a pleiotropic hormone essential for maintenance of corpus luteum (CL) function and pregnancy, transduces its signal through two types of receptors, a short form (PRLR-S) and a long form (PRLR-L). Both types of receptors are expressed in the CL, yet their individual roles are not well defined. We have shown previously that female transgenic mice expressing only PRLR-S display total infertility characterized by defective follicular development and early degeneration of CL, suggesting that expression of PRLR-L is a prerequisite for normal follicular development and maintenance of CL. To determine whether PRLR-L alone is the sole receptor required to maintain normal CL formation, differentiation, and progesterone secretion, we generated two transgenic mice which express only PRLR-L, either ubiquitously (Tg-RL) or in a CL-specific manner (CL-RL). To generate CL-specific expression, we used the HSD17B7 promoter. We found both transgenic mice models cycled normally, displayed no apparent defect in follicular development, and had normal ovulation rates. The STAT5 signaling pathway, considered essential for luteinization and progesterone production, was activated by PRL in both transgenic mice models. However, soon after mating, Tg-RL and CL-RL mice showed early regression of CL, lack of progesterone production, and implantation failure that rendered them totally infertile. Embryo transfer studies demonstrated no embryo abnormalities, and supplementation with progesterone rescued implantation failure in these mice. Close observation revealed lack of luteinization and reduced expression of proteins involved in progesterone biosynthesis despite normal levels of LHCGR (LHR), ESR1 (ER-alpha), CEBPB (C/EBP-beta) and CDKN1B (p27), proteins essential for luteinization. However, we found VEGFA, a key regulator of angiogenesis and vascularization, to be dramatically reduced in both Tg-RL and CL-RL mice. We also found collagen IV, a marker for the basal lamina of endothelial cells, aberrantly expressed and a discordant organization of endothelial cells in CL. Although luteinization did not occur in vivo, granulosa cells isolated from these mice luteinized in culture. Taken together, these results suggest that a vascularization defect in the CL may be responsible for lack of luteinization, progesterone production, and infertility in mice expressing only PRLR-L. This investigation therefore demonstrates that in contrast to earlier presumptions that PRLR-L alone is able to support normal CL formation and function, both isoforms of the PRL receptor are required in the CL for normal female fertility.Fil: Le, Jamie A.. University of Illinois; Estados UnidosFil: Wilson, Heather M.. University of Illinois; Estados UnidosFil: Shehu, Aurora. University of Illinois; Estados UnidosFil: Mao, Jifang. University of Illinois; Estados UnidosFil: Devi, Y. Sangeeta. University of Illinois; Estados UnidosFil: Halperin, Julia. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Aguilar, Tetley. University of Illinois; Estados UnidosFil: Seibold, Anita. University of Illinois; Estados UnidosFil: Maizels, Evelyn. University of Illinois; Estados UnidosFil: Gibori, Geula. University of Illinois; Estados Unido

Reproductive actions of prolactin mediated through short and long receptor isoforms

Prolactin (PRL) is a polypeptide hormone with a wide range of physiological functions, and is critical for female reproduction. PRL exerts its action by binding to membrane bound receptor isoforms broadly classified as the long form and the short form receptors. Both receptor isoforms are highly expressed in the ovary as well as in the uterus. Although signaling through the long form is believed to be more predominant, it remains unclear whether activation of this isoform alone is sufficient to support reproductive functions or whether both types of receptor are required. The generation of transgenic mice selectively expressing either the short or the long form of PRL receptor has provided insight into the differential signaling mechanisms and physiological functions of these receptors. This review describes the essential finding that both long and short receptor isoforms are crucial for ovarian functions and female fertility, and highlights novel mechanisms of action for these receptors.Fil: Halperin, Julia. Universidad Maimónides. Área de Investigaciones Biomédicas y Biotecnológicas. Centro de Estudios Biomédicos, Biotecnológicos, Ambientales y de Diagnóstico; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Devi Sangeeta, Y.. Michigan State University; Estados Unido

Inhibition of IL-6 Signaling Pathway by Curcumin in Uterine Decidual Cells

<div><p>IL-6 is a multifunctional pro-inflammatory cytokine and has been implicated in many gestational disorders including preterm birth. Currently, there are no appropriate therapeutic interventions available to circumvent inflammatory-mediated gestational disorders. Therefore, the goal of this study was to identify a safe and effective pharmacological compound to counterbalance inflammatory responses in the uterus. Curcumin, a naturally-occuring polyphenolic compound, has been widely used in alternative medicine to treat inflammatory diseases. However, the anti-inflammatory effect of curcumin has not been explored in uterine decidual cells, a major source of IL-6. Therefore, we examined the effect of curcumin on IL-6 expression using two types of uterine decidual cells 1) HuF cells, primary human fibroblast cells obtained from the decidua parietalis; 2) UIII cells, a rodent non-transformed decidual cell line. Curcumin treatment completely abrogated the expression of IL-1β-induced IL-6 in these cells. Curcumin also strongly inhibited the expression of gp130, a critical molecule in IL-6 signaling, whereas expression of IL-6R and sIL-6R was not affected. Curcumin also inhibited phosphorylation and nuclear localization of STAT3, a well-known downstream mediator of IL-6 signaling. Furthermore, curcumin attenuated IL-1β-induced IL-6 promoter reporter activity suggesting transcriptional regulation. To further understand whether NF-ҡB is involved in this inhibition, we examined the effect of curcumin on the expression of p50 and p65 subunits of NF-ҡB in decidual cells. Expression of IL-1β-induced p50 mRNA was repressed by curcumin while p65 mRNA was not affected. However, curcumin treatment dramatically inhibited both p50 and p65 protein levels and prevented its nuclear localization. This effect is at least partly mediated through the deactivation of IKK, since IL-1β-induced IKKα/β phosphorylation is decreased upon curcumin treatment. Our results not only revealed molecular mechanisms underlying curcumin action in uterine decidual cells but also suggest that this compound may have therapeutic potential for the prevention of inflammation-mediated preterm birth and other gestational disorders.</p></div

Responsiveness of HuF and UIII cells to inflammatory stimuli.

<p>Primary human uterine fibroblasts (HuF) cells were isolated from the decidua parietalis of placenta as described in materials and methods. UIII cells, a rat uterine stromal cell line that decidualizes in culture, were cultured as described previously [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125627#pone.0125627.ref027" target="_blank">27</a>]. Cells were treated with vehicle, or different doses of IL-1β or TNF-α for 24 hr. Total mRNA was analyzed for IL-6 expression in HuF cells (A), and UIII cells (B) by qPCR. RPS17 or L19 expression was used as control. The values are expressed as the means ± S.E. (<i>n</i> = 3). *, <i>p</i> < 0.05; **, <i>p</i> < 0.01; ***, <i>p</i> < 0.001.</p

Curcumin mediated inhibition of IL-6 downstream signaling.

<p>HuF cells were treated with either vehicle (control), IL-1β (10ng/ml), or with a combination of IL-1β (10ng/ml) and curcumin (30μM), or curcumin alone (30μM) for 24 hr. (A) Phosphorylation of STAT3 (p-STAT3) was analyzed by Western blot using a phospho-specific antibody (<i>left panel</i>). β-actin was used as loading control. The band intensity of p-STAT3 was plotted against β-actin and normalized to percentage of control (<i>right panel</i>). The values are expressed as the means ± S.E. (<i>n</i> = 3). *, <i>p</i> < 0.05; **, <i>p</i> < 0.01; ***, <i>p</i> < 0.001. (B) UIII cells were treated with IL-1β (10ng/ml), or with a combination of IL-1β (10ng/ml) and curcumin (30μM) for 24 hr. Cells were fixed in paraformaldehyde and processed for immunocytochemistry to analyze p-STAT3 (<i>green</i>) cellular localization.</p

Inhibition of IKK activation by curcumin in decidual/stromal cells.

<p>HuF cells were treated with either vehicle (control), IL-1β (10ng/ml), or with a combination of IL-1β and curcumin (30μM), or curcumin alone for different time points (1–24 hr). (A) Phosphorylation of IKKα/β (p-IKKα/β) was analyzed by Western blot using phospho-specific antibody and β-actin was used as loading control (<i>upper panel</i>). The band intensity of p-IKKα/β was plotted against β-actin and normalized to percentage of control (<i>lower panel</i>). (B) Expression of IL-6 was analyzed by Western blot and β-actin was used as loading control (<i>upper panel</i>). The band intensity of IL-6 was plotted against β-actin and normalized to percentage of control (<i>lower panel</i>). The values are expressed as the means ± S.E. (<i>n</i> = 3). *, <i>p</i> < 0.05; **, <i>p</i> < 0.01; ***, <i>p</i> < 0.001.</p

Transcriptional repression of IL-6 and regulation of NF-κB by curcumin.

<p>UIII and HuF cells were transfected with an IL-6 promoter reporter construct spanning -276 to +20 bp region. Cells were then treated with vehicle (control), IL-1β (10ng/ml), or with a combination of IL-1β (10ng/ml) and 30μM curcumin, or curcumin alone for 24hr. (A) IL-6 promoter activity was measured by dual luciferase assay in UIII cell lysates and normalized to Renilla. (B, C) Expression of p65 and p50 subunits of NF-κB was analyzed in HuF cells by qPCR. The values are expressed as the means ± S.E. (<i>n</i> = 3). ***, <i>p</i> < 0.001. (D, E) UIII cells were treated with IL-1β (10ng/ml), or with a combination of IL-1β and curcumin (30μM) for 24 hr. Cells were fixed in paraformaldehyde and processed for immunocytochemistry to analyze p65 and p50 NF-κB cellular localization. <i>Red</i>, p65/p50; <i>blue</i>, DAPI.</p

Regulation of IL-6 signaling pathway molecules by curcumin.

<p>HuF cells were treated with either vehicle (control), IL-1β (10ng/ml), or with a combination of IL-1β (10ng/ml) and curcumin (30μM), or curcumin alone (30μM). Total mRNA was analyzed for IL-6 (A), IL-6R (C) and gp130 (D) expression by qPCR. (B) Expression of IL-6 at protein level was examined by Western blot analysis. The values are expressed as the means ± S.E. (<i>n</i> = 3). ***, <i>p</i> < 0.001.</p