Phospholipase Cβ3 Mediates LH-Induced Granulosa Cell Differentiation

Previous studies showed that under certain conditions LH can stimulate not only adenylate cyclase (AC) but also phospholipase Cβ (PLCβ) signaling in target cells; however, the physiological involvement of PLCβ in LH-induced ovarian follicular cell differentiation has not been determined. To address this, ex vivo expression analyses and specific PLCβ targeting were performed in primary bovine granulosa cells. Expression analyses in cells from small (2.0–5.9 mm), medium (6.0–9.9 mm), and ovulatory-size (10.0–13.9 mm) follicles revealed an increase in mRNA and protein levels of heterotrimeric G protein subunits-αs, -αq, -α11, and -αi2 in ovulatory-size follicles, simultaneous with a substantial increase in LH receptor expression. Among the four known PLCβ isoforms, PLCβ3 (PLCB3) was specifically up-regulated in cells from ovulatory-size follicles, in association with a predominantly cytoplasmic location of PLCB3 in these cells and a significant inositol phosphate response to LH stimulation. Furthermore, RNA interference-mediated PLCB3 down-regulation reduced the ability of LH to induce hallmark differentiation responses of granulosa cells, namely transcriptional up-regulation of prostaglandin-endoperoxide synthase 2 and down-regulation of both aromatase expression and estradiol production. Responses to the AC agonist, forskolin, however, were not affected. In addition, PLCB3 down-regulation did not alter cAMP responses to LH in granulosa cells, ruling out a primary involvement of AC in mediating the effects of PLCB3. In summary, we provide evidence of a physiological involvement of PLCβ signaling in ovulatory-size follicles and specifically identify PLCB3 as a mediator of LH-induced differentiation responses of granulosa cells.

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A constitutively active mutant of the α1B-adrenergic receptor can cause greater agonist-dependent down-regulation of the G-proteins Gqα and G11α than the wild-type receptor

Biochemical Journal ◽

10.1042/bj3200079 ◽

1996 ◽

Vol 320 (1) ◽

pp. 79-86 ◽

Cited By ~ 10

Author(s):

Tae Weon LEE ◽

Alan WISE ◽

Susanna COTECCHIA ◽

Graeme MILLIGAN

Keyword(s):

G Proteins ◽

Cell Lines ◽

Adrenergic Receptor ◽

Inositol Phosphate ◽

Wild Type ◽

Down Regulation ◽

Eta Receptor ◽

Type Receptor ◽

In Cells ◽

Constitutively Active

Rat 1 fibroblasts transfected to express either the wild-type hamster α1B-adrenergic receptor or a constitutively active mutant (CAM) form of this receptor resulting from the alteration of amino acid residues 288–294 to encode the equivalent region of the human β2-adrenergic receptor were examined. The basal level of inositol phosphate generation in cells expressing the CAMα1B-adrenergic receptor was greater than for the wild-type receptor. The addition of maximally effective concentrations of phenylephrine or noradrenaline resulted in substantially greater levels of inositol phosphate generation by the CAMα1B-adrenergic receptor, although this receptor was expressed at lower steady-state levels than the wild-type receptor. The potency of both phenylephrine and noradrenaline to stimulate inositol phosphate production was approx. 200-fold greater at the CAMα1B-adrenergic receptor than at the wild-type receptor. In contrast, endothelin 1, acting at the endogenously expressed endothelin ETA receptor, displayed similar potency and maximal effects in the two cell lines. The sustained presence of phenylephrine resulted in down-regulation of the α subunits of the phosphoinositidase C-linked, pertussis toxin-insensitive, G-proteins Gq and G11 in cells expressing either the wild-type or the CAMα1B-adrenergic receptor. The degree of down-regulation achieved was substantially greater in cells expressing the CAMα1B-adrenergic receptor at all concentrations of the agonist. However, in this assay phenylephrine displayed only a slightly greater potency at the CAMα1B-adrenergic receptor than at the wild-type receptor. There were no detectable differences in the basal rate of Gqα/G11α degradation between cells expressing the wild-type or the CAMα1B-adrenergic receptor. In both cell lines the addition of phenylephrine substantially increased the rate of degradation of these G-proteins, with a greater effect at the CAMα1B-adrenergic receptor. The enhanced capacity of agonist both to stimulate second-messenger production at the CAMα1B-adrenergic receptor and to regulate cellular levels of its associated G-proteins by stimulating their rate of degradation is indicative of an enhanced stoichiometry of coupling of this form of the receptor to Gq and G11.

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The Differential Effects of the Gonadotropin Receptors on Aromatase Expression in Primary Cultures of Immature Rat Granulosa Cells Are Highly Dependent on the Density of Receptors Expressed and the Activation of the Inositol Phosphate Cascade

Endocrinology ◽

10.1210/en.2005-0403 ◽

2005 ◽

Vol 146 (9) ◽

pp. 3907-3916 ◽

Cited By ~ 70

Author(s):

Francesc Xavier Donadeu ◽

Mario Ascoli

Keyword(s):

Signaling Pathways ◽

Granulosa Cells ◽

Inositol Phosphate ◽

Primary Cultures ◽

Active Form ◽

Aromatase Expression ◽

Immature Rat ◽

Differential Effects ◽

Akt Phosphorylation ◽

In Cells

Abstract Signaling pathways mediating the divergent effects of FSH and LH on aromatase in immature rat granulosa cells were studied by infecting cells with increasing amounts of adenoviral vectors for the human LH receptor (hLHR) or FSH receptor (hFSHR). Increasing amounts of Ad-hLHR, used at a multiplicity of infection (MOI) of 20 or 200 viable viral particles/cell, increased human chorionic gonadotropin (hCG) binding and hCG-induced cAMP and Akt phosphorylation, but inositol phosphates only increased in response to hCG in cells infected with 200 MOI Ad-hLHR. In contrast, hCG increased aromatase expression in cells infected with 20, but not in cells infected with 200, MOI Ad-hLHR. Cells infected with 20 or 200 MOI Ad-hFSHR showed increased hFSH binding and hFSH-induced Akt phosphorylation, but the hFSH-induced cAMP response was unchanged relative to control cells. However, hFSH was able to stimulate the inositol phosphate cascade in the Ad-hFSHR-infected cells, and the hFSH induction of aromatase was abolished. We also found that activation of C kinase or expression of a constitutively active form of Gαq inhibited the induction of aromatase by hFSH or 8Br-cAMP. We conclude that the differential effects of FSH and LH on aromatase in immature granulosa cells are highly dependent on gonadotropin receptor density and on the signaling pathways activated. We propose that aromatase is induced by common signals generated by activation of the FSHR and LHR (possibly cAMP and Akt) and that the activation of the inositol phosphate cascade in cells expressing a high density of LHR or FSHR antagonizes this induction.

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Regulation of platelet-activating-factor receptors and the desensitization response in polymorphonuclear neutrophils

Biochemical Journal ◽

10.1042/bj2880241 ◽

1992 ◽

Vol 288 (1) ◽

pp. 241-248 ◽

Cited By ~ 14

Author(s):

J T O'Flaherty ◽

D P Jacobson ◽

J F Redman

Keyword(s):

Actinomycin D ◽

Platelet Activating Factor ◽

Receptor Expression ◽

Polymorphonuclear Neutrophils ◽

Target Cells ◽

Surface Compound ◽

Down Regulation ◽

High Affinity ◽

Kinase C ◽

Paf Receptor

Platelet-activating factor (PAF) desensitizes as well as stimulates its various target cells, We find that human polymorphonuclear neutrophils (PMN) exposed to PAF became maximally unresponsive to a second PAF challenge within 15-90 s in assays of Ca2+ mobilization and degranulation. The cells regained full PAF-sensitivity over the ensuing 20-40 min. These effects correlated with changes in PAF receptor availability. PMN treated with PAF, washed in regular buffer and assayed for PAF binding exhibited falls (maximal in 15 s), followed by rises (reaching control levels by 60 min), in the number of high-affinity PAF receptors. However, tracking studies showed that [3H]PAF accumulated on the cell surface for approximately 2 min before being internalized. Regular-buffer washes did not remove this superficial PAF, whereas a washing regimen using excess albumin to adsorb PAF removed 99% of the surface compound. PMN washed by the latter regimen after PAF exposure lost PAF receptors relatively slowly (maximal at approximately 5 min), but the ultimate extent of this loss and the rate at which receptor expression normalized were similar to those of cells washed in regular buffer. Neither cycloheximide nor actinomycin D influenced the course of the receptor changes, but two protein kinase C (PKC) blockers, staurosporine and 1-(5-isoquinolinesulphonyl)piperazine, inhibited the receptor-receptor-depleting actions of PAF. Indeed, a phorbol diester activator of PKC also caused PMN to decrease high-affinity PAF receptor numbers, and the two PKC blockers antagonized this action at concentrations that inhibited PAF-induced PAF receptor losses. We conclude that: (a) PAF induces PMN to down-regulate and then to re-express PAF receptors independently of protein synthesis; (b) these changes are likely to underlie the later stages and reversal of desensitization; (c) the onset (t < or = 2 min) of desensitization, however, precedes receptor down-regulation and must be due to receptor uncoupling from transductional elements; and (d) down-regulation of receptors for PAF appears to be mediated by PKC and/or elements inhibited by PKC blockers.

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HIV infection does not alter Interferon α/β receptor expression on mucosal immune cells

10.1101/688440 ◽

2019 ◽

Author(s):

Julia Ickler ◽

Sandra Francois ◽

Marek Widera ◽

Mario L. Santiago ◽

Ulf Dittmer ◽

...

Keyword(s):

T Cells ◽

Hiv Infection ◽

Immune Cells ◽

Mononuclear Cells ◽

Ex Vivo ◽

Critical Role ◽

Receptor Expression ◽

Interferon Α ◽

Target Cells ◽

Type I

AbstractThe innate immune response induced by type I interferons (IFNs) play a critical role in the establishment of HIV infection. IFNs are induced early in HIV infection and trigger an antiviral defense program by signaling through the IFNa/b receptor (IFNAR), which consists of two subunits, IFNAR1 and IFNAR2. Changes in IFNAR expression in HIV target cells, as well as other immune cells, could therefore have important consequences for initial HIV spread. It was previously reported that IFNAR2 expression is increased in peripheral blood CD4+CXCR4+T cells of HIV+patients compared to HIV uninfected controls, suggesting that HIV infection may alter the IFN responsiveness of target cells. However, the earliest immune cells affected by HIVin vivoreside in the gut-associated lymphoid tissue (GALT). To date, it remains unknown if IFNAR expression is altered in GALT immune cells in the context of HIV infection and exposure to IFNs, including the 12 IFNa subtypes. Here, we analyzed the expression of surface bound and soluble IFNAR2 on Lamina propria mononuclear cells (LPMCs) isolated from the GALT of HIV−individuals and in plasma samples of HIV+patients. IFNAR2 expression varied between different T cells, B cells and natural killer cells, but was not altered following HIV infection. Furthermore, expression of the soluble IFNAR2a isoform was not changed in HIV+patients compared to healthy donors, nor in LPMCs after HIV-1 infectionex vivo. Even though the 12 human IFNα subtypes trigger different biological responses and vary in their affinity to both receptor subunits, stimulation of LPMCs with different recombinant IFNα subtypes did not result in any significant changes in IFNAR2 surface expression. Our data suggests that potential changes in the IFN responsiveness of mucosal immune cells during HIV infection is unlikely dictated by changes in IFNAR2 expression.

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Regulation of Human Growth Hormone Receptor Expression by MicroRNAs

Molecular Endocrinology ◽

10.1210/me.2014-1183 ◽

2014 ◽

Vol 28 (9) ◽

pp. 1448-1459 ◽

Cited By ~ 12

Author(s):

Samar Elzein ◽

Cynthia Gates Goodyer

Keyword(s):

Gene Expression ◽

Growth Hormone Receptor ◽

Human Growth ◽

Receptor Expression ◽

Hek293 Cells ◽

Luciferase Reporter ◽

Target Cells ◽

Protein Levels ◽

Coding Regions ◽

Hormone Receptor Expression

Human GH binds to its receptor (GHR) on target cells and activates multiple intracellular pathways, leading to changes in gene expression, differentiation, and metabolism. GHR deficiency is associated with growth and metabolic disorders whereas increased GHR expression has been reported in certain cancers, suggesting that the GHR gene requires tight controls. Several regulatory mechanisms have been found within its 5′-untranslated region (UTR) promoter and coding regions. However, the 3′-UTR has not been previously examined. MicroRNAs (miRNAs) are small (19–22 nucleotides) noncoding RNAs that downregulate gene expression mainly through targeting the 3′-UTR of mRNAs and enhancing their degradation or inhibiting translation. In the present study, we investigated whether miRNAs regulate GHR expression. To define putative miRNA binding sites in the GHR 3′-UTR, we used multiple in silico prediction tools, analyzed conservation across species and the presence of parallel sites in GH/IGF axis-related genes, and searched for reports linking miRNAs to GHR-related physiological or pathophysiological activities. To test prioritized sites, we cotransfected a wild-type GHR 3′-UTR luciferase reporter vector as well as miRNA binding site mutants into HEK293 cells with miRNA mimics. Furthermore, we tested whether the miRNAs altered endogenous GHR mRNA and protein levels in HEK293 cells and in 2 cancer cell lines (MCF7 and LNCaP). Our experiments have identified miRNA (miR)-129–5p, miR-142–3p, miR-202, and miR-16 as potent inhibitors of human GHR expression in normal (HEK293) and cancer (MCF7 and LNCaP) cells. This study paves the way for the development of miRNA inhibitors as therapeutic agents in GH/GHR-related pathophysiologies, including cancer.

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Dynamic changes in E-protein activity regulate T reg cell development

Journal of Experimental Medicine ◽

10.1084/jem.20132681 ◽

2014 ◽

Vol 211 (13) ◽

pp. 2651-2668 ◽

Cited By ~ 13

Author(s):

Ping Gao ◽

Xiaojuan Han ◽

Qi Zhang ◽

Zhiqiong Yang ◽

Ivan J. Fuss ◽

...

Keyword(s):

Cell Differentiation ◽

Signaling Pathways ◽

Cell Lineage ◽

Foxp3 Expression ◽

Protein Activity ◽

E Proteins ◽

Tcr Signaling ◽

Down Regulation ◽

Protein Levels ◽

E Protein

E-proteins are TCR-sensitive transcription factors essential for intrathymic T cell transitions. Here, we show that deletion of E-proteins leads to both enhanced peripheral TGF-β–induced regulatory T (iT reg) cell and thymic naturally arising T reg cell (nT reg cell) differentiation. In contrast, deletion of Id proteins results in reduced nT reg cell differentiation. Mechanistic analysis indicated that decreased E-protein activity leads to de-repression of signaling pathways that are essential to Foxp3 expression. Decreased E-protein binding to an IL-2Rα enhancer locus facilitated TCR-induced IL-2Rα expression. Similarly, decreased E-protein activity facilitated TCR-induced NF-κB activation and generation of c-Rel. Consistent with this, microarray analysis indicated that cells with E-protein depletion that are not yet expressing Foxp3 exhibit activation of the IL-2 and NF-κB signaling pathways as well as enhanced expression of many of the genes associated with Foxp3 induction. Finally, studies using Nur77-GFP mice to monitor TCR signaling showed that TCR signaling strength sufficient to induce Foxp3 differentiation is accompanied by down-regulation of E-protein levels. Collectively, these data suggest that TCR stimulation acts in part through down-regulation of E-protein activity to induce T reg cell lineage development.

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Down-regulation of cellular platelet-derived growth factor receptors induced by an activated neu receptor tyrosine kinase.

Cell Regulation ◽

10.1091/mbc.2.8.651 ◽

1991 ◽

Vol 2 (8) ◽

pp. 651-661 ◽

Cited By ~ 3

Author(s):

L Lehtola ◽

M Nistér ◽

E Hölttä ◽

B Westermark ◽

K Alitalo

Keyword(s):

Growth Factor ◽

Tyrosine Kinase ◽

Receptor Expression ◽

Platelet Derived Growth Factor ◽

Target Cells ◽

Mrna Levels ◽

Pdgf Receptor ◽

Down Regulation ◽

Beta Receptors ◽

Neu Oncogene

The functional integration of growth factor signaling occurs at several levels in target cells. One of the most proximal mechanisms is receptor transmodulation, by which one activated receptor can regulate the expression of other receptors in the same cells. Well-established transregulatory loops involve platelet-derived growth factor (PDGF) down-regulation of epidermal growth factor (EGF) receptors and beta-type transforming growth factors modulation of PDGF receptors. We have studied the relationship between neu tyrosine kinase activation and the expression of the PDGF receptors in transfected NIH/3T3 cells. Expression of the neu oncogene, but not of the neu proto-oncogene, was associated with a decrease of PDGF alpha- and beta-receptors on the cell surface, as measured by [125-I]PDGF-AA and -BB binding. These results were corroborated by metabolic labeling and immunoprecipitation of the PDGF beta-receptors. PDGF alpha- and beta-receptor mRNAs were strongly decreased in the neu oncogene-transformed cells in comparison with control cells expressing the neu proto-oncogene. Down-regulation of the PDGF receptors and their mRNAs was also observed after EGF treatment of cells expressing a chimeric EGF receptor/neu receptor, where the neu tyrosine kinase is activated by EGF binding. These results show that the neu tyrosine kinase can down-modulate PDGF receptor expression, and the effect is mediated via decreased PDGF receptor mRNA levels.

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Transcriptional and post-transcriptional regulation of FSH receptor in rat granulosa cells by cyclic AMP and activin

Journal of Endocrinology ◽

10.1677/joe.0.1530465 ◽

1997 ◽

Vol 153 (3) ◽

pp. 465-473 ◽

Cited By ~ 40

Author(s):

M Tano ◽

T Minegishi ◽

K Nakamura ◽

S Karino ◽

Y Ibuki ◽

...

Keyword(s):

Granulosa Cells ◽

Receptor Expression ◽

Northern Blot Hybridization ◽

Fsh Receptor ◽

Mrna Levels ◽

Mrna Transcript ◽

Receptor Mrna ◽

Degradation Rates ◽

Mrna Transcripts ◽

In Cells

Abstract The effect of FSH on the induction of FSH receptors in granulosa cells is believed to be mediated, at least in part, by the cAMP second messenger system. We examined the effect of activin and cAMP on FSH receptor expression in this culture system. Steady-state levels of FSH receptor mRNA, analyzed by Northern blot hybridization, increased 3·5-fold in response to 24-h incubation with activin and 1·7-fold with 12-h incubation with 8-bromoadenosine 3,5-cyclic monophosphate (8-Br-cAMP; 0·2 mm). We have investigated whether 8-Br-cAMP- and/or activin-induced increases in FSH receptor mRNA levels are the result of increased transcription and/or altered mRNA stability. The rates of FSH receptor mRNA gene transcription, assessed by nuclear run-on transcription assay, increased 3-fold in cells treated with activin and 1·5-fold in cells treated with 8-Br-cAMP for 2 h. To examine the degradation rates of FSH receptor mRNA transcripts, granulosa cells were preincubated with 8-Br-cAMP, activin, or medium alone for 6 h. After the preincubation period, 5 μm actinomycin-D or 200 μm 5,6-dichloro-1-β-ribofuranosyl benzimidazole were added to arrest new RNA synthesis. The decay curves for the 2·4 kb FSH receptor mRNA transcript in granulosa cells were not significantly different in the absence or presence of 8-Br-cAMP. Activin, on the other hand, significantly altered the slope of the FSH receptor mRNA decay curve and increased the half-life of the 2·4 kb FSH receptor mRNA transcript. These data provide evidence that cAMP induces FSH receptor mRNA levels by stimulating the transcription rate and that activin increases FSH receptor mRNA levels both by stimulating transcription rates and by stabilizing the FSH receptor mRNA transcripts. Journal of Endocrinology (1997) 153, 465–473

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