Prolactin-releasing Peptide Activation of the Prolactin Promoter Is Differentially Mediated by Extracellular Signal-regulated Protein Kinase and c-Jun N-terminal Protein Kinase

Abstract

Regulation of the mitogen-activated protein kinase (MAPK) family by prolactin-releasing peptide (PrRP) in both GH3 rat pituitary tumor cells and primary cultures of rat anterior pituitary cells was investigated. PrRP rapidly and transiently activated extracellular signalregulated protein kinase (ERK) in both types of cells. Both pertussis toxin, which inactivates G i /G o proteins, and exogenous expression of a peptide derived from the carboxyl terminus of the ␤-adrenergic receptor kinase I, which specifically blocks signaling mediated by the ␤␥ subunits of G proteins, completely blocked the PrRPinduced ERK activation, suggesting the involvement of G i /G o proteins in the PrRP-induced ERK activation. Down-regulation of cellular protein kinase C did not significantly inhibit the PrRP-induced ERK activation, suggesting that a protein kinase C-independent pathway is mainly involved. PrRP-induced ERK activation was not dependent on either extracellular Ca 2؉ or intracellular Ca 2؉ . However, the ERK cascade was not the only route by which PrRP communicated with the nucleus. JNK was also shown to be significantly activated in response to PrRP. JNK activation in response to PrRP was slower than ERK activation. Moreover, to determine whether a MAPK family cascade regulates rat prolactin (rPRL) promoter activity, we transfected the intact rPRL promoter ligated to the firefly luciferase reporter gene into GH3 cells. PrRP activated the rPRL promoter activity in a time-dependent manner. Cotransfection with a catalytically inactive form of a MAPK construct or a dominant negative JNK, partially but significantly inhibited the induction of the rPRL promoter by PrRP. Furthermore, co-transfection with a dominant negative Ets completely abolished the response of the rPRL promoter to PrRP. These results suggest that PrRP differentially activates ERK and JNK, and both cascades are necessary to elicit rPRL promoter activity in an Ets-dependent mechanism.

Inhibition of Inhibitor of Nuclear Factor-κB Phosphorylation Increases the Efficacy of Paclitaxel in in Vitro and in Vivo Ovarian Cancer Models

“…Cells were seeded in 60-mm dishes and transfected with 2 g of NF B reporter plasmid (pElam-luc) for 24 hours with LipofectAMINE Plus (Life Technologies, Inc. Gaithersburg, MD) according to the manufacturer's protocol. Cells were treated with various agents, and then harvested and subjected to luciferase assays with the Luciferase Assay System (Promega) as described previously (37). A plasmid expressing the bacterial ␤-galactosidase gene was also cotransfected in each experiment to serve as an internal control for transfection efficiency.…”

Estrogen Induces the Akt-dependent Activation of Endothelial Nitric-oxide Synthase in Vascular Endothelial Cells

“…eNOS activity was determined as the conversion of radiolabeled L-arginine to L-citrulline by a method described previously (50, 51) with a minor modification. Briefly, 10 l of a sample was incubated for 10 min at 37°C in a solution consisting of 50 mM HEPES, 1 mM dithiothreitol, 1 mM CaCl 2 , 0.1 mM tetrahydro-L-biopterin, 1 mM NADPH, 10 g/ml calmodulin, 10 M FAD, and 1.55 M L-[guanidino- Assay of eNOS Activity Using a Transient Expression SystemTRLECs cultured in 100-mm dishes were transfected with 1 g of CMV-6, 1 g of CMV-6 containing the gene for HA-AktK179M, 1 g of pSG5, 1 g of ER␣ expression vector (pSG5-HEGO), or 1 g of ER␤ expression vector (pSG5-mER␤) using LipofectAMINE plus (Life Technologies, Inc.) as described previously (52,53). Seventy-two hours after transfection, serum-deprived cells were incubated with 10 Ϫ7 M 17␤-E2 for 15 min, and the eNOS activity was measured as described above.…”

“…Assay of Akt Activity Using a Transient Expression System-CHO cells cultured in 100-mm dishes were transfected with 1 g of pSG5, 1 g of ER␣ expression vector (pSG5-HEGO), or 1 g of ER␤ expression vector (pSG5-mER␤) using LipofectAMINE plus as described previously (52,53). Seventy-two hours after transfection, serum-deprived cells were incubated with 10 Ϫ7 M 17␤-E2 for 15 min, and the Akt activity was measured as described above.…”

“…Cell lysates were subjected to immunoprecipitation with anti-Akt antibody. For assay using a transient expression system, TRLECs cultured in 100-mm dishes were transfected with 1 g of HA-Akt, 1 g of HA-AktK179M, or 1 g of HA-m⌬4 -129Akt using LipofectAMINE plus (Life Technologies, Inc.) as described previously (52,53). Seventy-two hours after transfection, serum-deprived cells were incubated with 10 Ϫ7 M 17␤-E2 for 15 min, and lysates were immunoprecipitated with anti-HA antibody.…”

Signal transduction and the Ets family of transcription factors

“…The prolactin promoter contains a RRE that is composed of a Pit1 binding site juxtaposed to an Ets-binding site. Ets1 is necessary for maximal synergistic expression of prolactin and this expression is dependent upon Ras-MAPK phosphorylation of Ets1 (Bradford et al, 1995(Bradford et al, , 1996(Bradford et al, , 2000Kimura et al, 2000). Both factors serve to engender speci®city from a general signal transduction pathway, thus allowing for unique responses depending on the cell type.…”