Prolactin Stimulation of Phosphoinositide Metabolism in CHO Cells Stably Expressing the PRL Receptor

doi:10.1006/bbrc.1997.7978

Biochemical and Biophysical Research Communications

Volume 243, Issue 1, 4 February 1998, Pages 127-130

https://doi.org/10.1006/bbrc.1997.7978 Get rights and content

Abstract

PRL receptor (PRL-R) activation by PRL triggers a cascade of intracellular events including homodimerization of the receptor, activation of cytoplasmic receptor-associated tyrosine kinase and tyrosine-phosphorylation of various signal transducers. In CHO cells, transfected with the long form of PRL-R, an increase in [Ca²⁺]i was observed following PRL stimulation whereas Ca²⁺is generally coupled with the phosphoinositide metabolism. In this study, we investigated phosphoinositide involvement in the PRL transduction pathway. We report that PRL induces rapid increases in two novel inositol phospholipids, almost certainly PtdIns(4,5)P2 and PtdIns(3,4,5)P3. Pre-traitment of CHO cells with wortmanin, a specific PtdIns3-kinase inhibitor, considerably reduces the PRL-induced increase in PtdInd(3,4,5)P3, thus suggesting an involvement of this enzyme in the cascade of activation of cytoplasmic kinase proteins. A pathway beginning with the activation of PtdIns3-kinase, phosphorylation of PtdIns(4,5)P2 and rapid synthesis of PtdIns(3,4,5)P3 is proposed. PtdIns(3,4,5)P3 may acts as a lipid second messenger, directly or indirectly responsible for some of the multiple cell changes attributed to PRL.

References (33)

J.N. Ihle et al.
TIBS
(1994)
M.J. Waters et al.
J. Biol. Chem.
(1995)
N. Tourkine et al.
J. Biol. Chem.
(1995)
C.V. Clevenger et al.
J. Biol. Chem.
(1994)
A.R. Buckley et al.
Biochem. Biophys. Res. Commun.
(1994)
J.J. Lebrun et al.
J. Biol. Chem.
(1994)
K.A. Al-Sakkaf et al.
Biochem. Biophys. Res. Commun.
(1996)
R.E. Vadnal et al.
Biochem. Biophys. Res. Commun.
(1989)
M. Nogaro et al.
Insect. Biochem. Molec. Biol.
(1995)
N.M. Dean et al.
Analyt. Biochem.
(1989)

L.A. Serunian et al.

Methods. Enzymol.

(1991)

M. Riddersträle et al.

J. Biol. Chem.

(1995)

M. Gold et al.

Am. Soc. Biochem. Mol. Biol.

(1994)

C.L. Carpenter et al.

J. Biol. Chem.

(1990)

M. Riddersträle et al.

Biochem. Biophys. Res. Commun.

(1994)

N. Sasakawa et al.

Biochem. Pharmacol.

(1995)

Cited by (24)

Prolactin and epidermal growth factor stimulate adipophilin synthesis in HC11 mouse mammary epithelial cells via the PI3-kinase/Akt/mTOR pathway
2012, Biochimica et Biophysica Acta - Molecular Cell Research
Citation Excerpt :
It is interesting to note that at this concentration the inhibitor stimulated ADRP synthesis by 20%. The PI3-kinase/Akt/mTOR cascade is another important signaling pathway activated by PRL and EGF [43,44]. To determine whether one or both hormones regulate ADRP and β-casein synthesis through this signaling cascade, we examined the effect of inhibition of PI3-kinase and its downstream mediators Akt and the mammalian target of rapamycin (mTOR).
The aim of the present study is to estimate the role played by cortisol, prolactin (PRL) and epidermal growth factor (EGF) in the synthesis of adipocyte differentiation-related protein (ADRP) as compared to the well-studied regulation of β-casein synthesis by these hormones in the mammary epithelial cell line HC11. This comparison between a cytoplasmic lipid droplet-associated protein, which is strictly specific to both lipid accumulation and secretion by lactating mammary epithelial cells, and an archetypal milk protein is useful for evaluating the extent to which a mechanistic relationship exists between biosynthesis, transport and secretion of these two major milk components. We found that cortisol inhibits PRL-stimulated ADRP synthesis, as opposed to its known stimulating effect on β-casein synthesis. The involvement of PRL and EGF in ADRP synthesis was explored by means of a battery of inhibitors. The Jak2 inhibitor AG490 provoked a stimulation of ADRP synthesis whereas it totally suppressed that of β-casein. The use of AG1478, a specific inhibitor of EGF receptor phosphorylation, or of PD98059, a specific MEK inhibitor, revealed that the Ras/Raf/MEK/ERK1/2 pathway has no significant influence on ADRP levels. Inhibition of JNK was also ineffective. In contrast, incubation of the cells with SB 203580, a specific inhibitor of p38, slightly stimulated ADRP synthesis and induced a proportional dose–response inhibition of PRL-induced β-casein synthesis. Finally, cell treatment with wortmannin or LY294002 revealed that both PRL and EGF positively regulate ADRP and β-casein synthesis through PI3-kinase signaling. Because both the Akt inhibitor MK-2206 and the mTOR inhibitor rapamycin provoked a strong diminution of PRL-induced synthesis of the two proteins, and because oleate induced phosphorylation of Akt, we concluded that, in the mammary epithelial cell line HC11, the PI3-kinase/Akt/mTOR signaling pathway strongly participates in β-casein synthesis and is a main regulator of ADRP expression.
The role of nitric oxide in the biological activity of prolactin in the mouse mammary gland
2001, Molecular and Cellular Endocrinology
In mouse mammary epithelial cells, prolactin transiently elevates nitric oxide (NO) to a maximum of 6 nmol/mg protein at 15 min, after which levels fall rapidly. This stimulation can be achieved by as little as 100 ng prolactin/ml and can be mimicked by 100 μg sodium nitroprusside/ml. NO is both necessary and sufficient to mediate the prolactin-induced redistribution of its receptor from internal pools to the cell surface. NO can also enhance DNA synthesis stimulated by submaximal prolactin concentrations (50 ng/ml), but it is not necessary at pharmacological prolactin concentrations (1 μg/ml). In contrast, NO completely inhibits α-lactalbumin production. In summary, prolactin transiently elevates NO to enhance DNA synthesis and suppress premature differentiation; thereafter, NO declines, DNA synthesis ceases and differentiation proceeds. This data suggest that NO may mediate some of the effects of prolactin on growth in the mammary gland.
Effect of PRL on MAPK activation: Negative regulatory role of the C- terminal part of the PRL receptor
2000, Molecular and Cellular Endocrinology
Prolactin induces cell proliferation and cell differentiation through well-known MAPK Erk, and JAK2/STAT5 pathways depending on the cell line. The aim of the present study was to delineate the functional domains of the PRL receptor involved in PRL induced MAPK regulation. Using various PRL-R mutants of the cytoplasmic domain we found, that the membrane proximal domain is necessary for PRL induced MAPK activation and that the C-terminal part of the receptor exerts a negative regulatory role. A pharmacological approach, using different types of inhibitors, provided evidence that PRL induced MAPK activation requires both a MEK dependent pathway and a PI3K dependent pathway. The negative regulation induced by the carboxy-terminal part of the receptor involves a combination of tyrosine phosphatases and serine/threonine phosphatases as concluded from the actions of the phosphatase inhibitors: pervanadate, PAO and okadaı̈c acid. The mechanism by which these phosphatases are recruited or are induced by the last 141 cytoplasmic residues of the receptor remains to be determined. Finally the negative regulatory role of the carboxy-terminal part of the receptor, first demonstrated in the present study, is discussed in terms of the regulation of different effects of PRL on growth and differentiation.
Activation of PKC δ in the rat corpus luteum during pregnancy: Potential role of prolactin signaling
1999, Journal of Biological Chemistry
Maintenance of pregnancy in the rat requires the corpus luteum. At a time when rat placental lactogens (rPLs) are required to support progesterone production by the corpus luteum and when relaxin expression is initiated, expression of a specific protein kinase C (PKC) isoform, PKC δ, is dramatically increased. We therefore assessed whether prolactin (PRL) receptor activation promotes activation of PKC δ in a luteinized granulosa cell model. We also assessed the activation status of PKC δ in corpora lutea obtained when the corpus luteum is exposed to chronically high concentrations of rPLs. The activity of PKC δ was assessed by two means: an immune complex (IC) assay and Western blotting with a phospho-epitope-specific antibody that detects PKC δ phosphorylated on serine 662. PKC δ activation in the IC kinase assay was determined by the ability of immunoprecipitated PKC δ to phosphorylate the PKC δ-preferential substrate small heat shock protein (HSP-27). Treatment of luteinized rat granulosa cells with phorbol myristate acetate, a known activator of PKC, promoted a 7-fold increase in HSP-27 phosphorylation by PKC δ. Similarly, immunoreactivity with the phospho-epitope-specific PKC δ antibody was increased in extracts prepared from luteinized granulosa cells treated with phorbol myristate acetate or followingin vitro activation of recombinant PKC δ. Using these assays, we assessed whether PRL receptor agonists were capable of activating PKC δ in luteinized granulosa cells. PRL receptor agonists induced translocation PKC δ from the cytosolic to the Triton-soluble membrane fraction and increased PKC δ activity assessed by both IC kinase assay and Western blotting with phospho-epitope-specific PKC δ antibody. Analysis of PKC δ activity in corpora lutea obtained during pregnancy by both the IC kinase assay and Western blotting with the phospho-epitope-specific PKC δ antibody revealed that PKC δ activity was increased throughout the second half of pregnancy. These results demonstrate that PRL receptor activation promotes the acute activation of PKC δ in luteinized rat granulosa cells. At a time when the rat is exposed to chronically high concentrations of rPLs, PKC δ is increasingly expressed and active.
Distinct cytoplasmic regions of the prolactin receptor are required for prolactin-induced calcium entry
1998, Journal of Biological Chemistry
Two cytoplasmic regions of the prolactin (PRL) receptor are well documented for their participation in PRL signal transduction, the membrane proximal box 1 and the COOH-terminal region. In order to study the role of these regions in PRL-induced Ca²⁺ increase, we use Chinese hamster ovary (CHO) cells stably transfected with mutated PRL receptor cDNA. These cells express the long form of PRL receptor deleted from box 1 (CHO Δ1 cells) or the 141 amino acids of the COOH-terminal region (CHO H3 cells). The patch-clamp technique in “whole-cell” configuration and microfluorimetric techniques were used singly or in combination. Data obtained for these cells were compared with those we have recently published using CHO cells expressing the wild-type long form of the PRL receptor (CHO TSE32). In contrast to CHO TSE32 cells, exposure of CHO Δ1 or H3 cells to PRL (0.05–50 nm) did not modify [Ca²⁺]_i. We have previously shown that the PRL-induced calcium influx via voltage-insensitive, Ca²⁺ channels was due to the activation of tyrosine kinase-dependent K⁺ channels that hyperpolarize the CHO TSE32 cell membrane (hyperpolarization-driven Ca²⁺influx). Therefore, two events are involved in PRL-induced Ca²⁺ changes (i) JAK2-activation of K+ channels and (ii) intracellular messenger-opening of Ca²⁺ channels. In CHO Δ1 cells, PRL (0.05–50 nm) neither hyperpolarized the membrane potential nor stimulated the JAK2-dependent K⁺ current, confirming the pivotal role played by box 1/JAK2 in the PRL-induced activation of K⁺ channels. However, when these cells were voltage-clamped below the resting membrane potential, application of 5 nm PRL resulted in an increase in Ca²⁺ influx. Therefore, box 1/JAK2 was not involved in the opening of these Ca²⁺ channels. In CHO H3 cells, 5 nm PRL activated the K⁺ current and hyperpolarized the membrane potential without any effect on [Ca²⁺]_i. Moreover, PRL was also ineffective on CHO H3 cells voltage-clamped below the resting membrane potential. Therefore, the COOH-terminal region is involved in the production of the intracellular messenger that opens voltage-independent Ca²⁺ channels.
We conclude from these findings that box 1 and COOH-terminal regions are both needed for PRL-induced Ca²⁺ changes.
Mobilization of Ca<sup>2+</sup> from intracellular stores in Sus scrofa domesticus oocytes after vitrification and thawing
2018, Problems of Cryobiology and Cryomedicine

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^☆: Abbreviations: Ins. inositol; InsPs. inositol phosphates; InsP1. inositol monophosphate; InsP2. inositol bisphosphate; Ins(1,3,4)P3. Inositol 1,3,4-trisphosphate; Ins(1,4,5)P3. inositol 1,4,5-trisphosphate; Ins(1,3,4,5)P4. inositol 1,3,4,5-tetrakisphosphate; InsP5. inositol pentakisphosphate; InsP6. inositol hexakisphosphate; PtdIns. phosphatidylinositol; PtdIns4P. phosphatidylinositol 4-monophosphate; PtdInsP2. phosphatidylinositol 4,5-bisphosphate; PtdIns(3,4,5)P3. phosphatidylinositol 3,4,5-trisphosphate.

¹: Please address all correspondence to: Laboratoire de Neurophysiologie, Centre National de la Recherche Scientifique, UMR 5543, Université de Bordeaux 2, 146 rue Léo Saignat 33076 Bordeaux cedex, France. Fax: 33-556.90.14.21. E-mail:[email protected]. bordeaux2.fr.

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Regular ArticleProlactin Stimulation of Phosphoinositide Metabolism in CHO Cells Stably Expressing the PRL Receptor☆

Abstract

TIBS

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

Insect. Biochem. Molec. Biol.

Analyt. Biochem.

Methods. Enzymol.

J. Biol. Chem.

Am. Soc. Biochem. Mol. Biol.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Biochem. Pharmacol.

Regular Article
Prolactin Stimulation of Phosphoinositide Metabolism in CHO Cells Stably Expressing the PRL Receptor☆