Protein kinase implication in prolactin signaling in bovine oocyte-cumulus complexes

The mechanisms of prolactin signal transduction in generative and somatic cells of mammalian ovarian follicles are poorly understood. In this work, participation of tyrosine kinases and protein kinase C in mediation of the previously revealed modulating effects of prolactin on the nuclear maturation of bovine oocytes and the morphologic and functional state of surrounding cumulus cells in vitro has been investigated. It was found that a tyrosine kinase inhibitor genistein suppresses the stimulating action of prolactin on the completion of oocyte nuclear maturation and cumulus expansion, whereas a protein kinase C inhibitor calpostin C does not affect the hormonal effect. Furthermore, both genistein and calpostin C inhibited the inducing influence of prolactin on the proliferative activity of cumulus cells. At the same time, the retarding action of prolactin on destructive processes in cumulus cells was blocked only in the presence of calpostin C. These results show that the stimulating influence of prolactin on oocyte nuclear maturation accompanied by cumulus expansion is achieved with participation of tyrosine kinases, whereas the modulating action of the hormone on the functional state of cumulus cells depends on activation both of tyrosine kinases and protein kinase C.     

Role of protein kinases in prolactin signaling in bovine oocyte-cumulus complexes

Mechanisms of prolactin signal transduction in generative and somatic cells of mammalian ovarian follicles have been studied only to a small extent. In the present work, the involvement oftyrosine kinases and protein kinase C in mediating of the previously revealed modulating effects of prolactin on the nuclear maturation of bovine oocytes and the morphologic-functional state of surrounding cumulus cells was investigated in vitro. Tyrosine kinase inhibitor, genistein, was found to suppress the stimulating action of prolactin on the completion of oocyte nuclear maturation and cumulus expansion, whereas protein kinase C inhibitor, calpostin C, did not affect these hormonal effects. Furthermore, both genistein and calpostin C inhibited the inducing influence of prolactin on the proliferative activity of cumulus cells. At the same time the retarding action ofprolactin on destructive processes in cumulus cells was blocked only in the presence of calpostin C. The results of the study suggest that the stimulatory influence of prolactin on oocyte nuclear maturation and attendant cumulus expansion is achieved with the participation of tyrosine kinases, whereas the modulating action of the hormone on the functional state of cumulus cells depends on activation of not only tyrosine kinases, but also protein kinase C.     

Specific binding of phorbol esters to nuclei of human promyelocytic leukemia cells

In this report, we demonstrate that HL-60 nuclei isolated in calcium but not EGTA containing buffers specifically bind PE and express approximately 37,000 receptor sites/nucleus. Nuclear phorbol ester binding is lost by isolation in the absence of calcium, but can be repleted by the addition of partially purified protein kinase C and calcium. When HL-60 cells are treated with bryostatin 1, a compound which activates protein kinase C in a similar fashion to phorbol esters but does not induce differentiation of HL-60 cells, and nuclei are isolated in the presence of EGTA, these nuclei continue to bind phorbol esters. These experiments suggest that HL-60 nuclei bind PE in vitro, and that compounds that activate protein kinase C may increase nuclear binding of PE in situ.     

Hepatic protein kinase C: translocation stimulated by prolactin and partial hepatectomy

Prolactin stimulates a hepatotrophic response similar to that caused by phorbol esters or partial hepatectomy in rats. Since phorbol esters, which activate protein kinase C, mimic prolactin action in liver, the relationship between prolactin administration and subsequent hepatic protein kinase C translocation was assessed. Prolactin administration rapidly stimulated a 4-fold elevation of membrane protein kinase C activity. The effect of prolactin on hepatic protein kinase C was specific for lactogenic hormones but could be duplicated by phorbol esters. Further, an increase in serum prolactin was demonstrated subsequent to partial hepatectomy and preceding hepatic protein kinase C translocation. Therefore, translocation of hepatic protein kinase C appears important for hepatic proliferation in response to prolactin administration and to partial hepatectomy.     

Signal transduction in erythropoiesis.

The polypeptide hormone erythropoietin (Ep) is a growth factor whose actions on the erythroid progenitor cell induce proliferation and differentiation. The signal transduction system activated by Ep to mediate these cellular processes remains largely uncharacterized despite many years of research devoted to its elucidation. It is clear that an Ep receptor-mediated activation of adenylate cyclase or guanylate cyclase does not occur, although cAMP and cGMP may play modulatory roles. The role of calcium in the action of Ep is less clear. Although the presence of extracellular calcium seems to be an absolute requirement for Ep-induced proliferation, the positive changes induced by Ep in intracellular calcium occur with a time course suggestive of influx through ion channels opening within the cell membrane rather than release of intracellular stores by inositol trisphosphate. There is good evidence for the involvement of phospholipases A2 and C in the actions of Ep, including an early rise in lipoxygenase metabolites of arachidonic acid. Activation of phospholipase C can also result in the activation of protein kinase C in response to Ep. We present a model for the signal transduction pathway of Ep that is consistent with current knowledge and provides a framework for the coordinate actions of several intracellular mechanisms in the mediation of Ep-induced proliferation.     

Activation of protein kinase C and cAMP-dependent protein kinase in hormone-dependent mammary gland tumors during stimulation of their growth with progesterone and estrone

GR mouse mammary tumour growth is stimulated by simultaneous administration of progesterone and estrone. These hormones strongly activate cAMP-dependent protein kinases both in the cytosol and in humour cell nuclei by causing the elevation of PK-1 and PK-2 activities. Ovarian hormone action on the proliferation is similar to that of growth factors, i.e., the hormones significantly stimulate the calcium-activated, phospholipid-dependent protein kinase C. Protein kinase C has been discovered is growing tumour cell nuclei. In early periods after ovarian hormone administration protein kinase C is activated in a greater degree as compared to cAMP-dependent protein kinases. A hypothesis on the feasibility of simultaneous activation by steroid hormones of both second messenger systems, namely the cAMP system and the system of production of diacylglycerol from phosphtidylinositol diphosphate is proposed.     

Quantitation of specific binding of erythropoietin to human erythroid colony-forming cells

Highly purified human erythroid colony-forming cells (ECFC), which consist predominately of colony-forming units-erythroid (CFU-E), were prepared from human blood and used to study the binding and processing of erythropoietin (Ep). When radioiodinated human recombinant Ep (125I-rEp) was incubated with these cells, binding was specific and saturable. Specific binding was directly proportional to cell concentration and did not occur with other human cells. Saturation of specific binding at 3 degrees C occurred at 1 nM (3.9/U/ml), and Scatchard analysis revealed two classes of binding sites on the cell surface. Of a total of 1,050 binding sites per ECFC, one-fifth had a Kd of 0.10 nM, while the remainder had a Kd of 0.57 nM. Specific binding was twofold greater at 37 degrees C than at 3 degrees C, and removal of surface-bound Ep with acid indicated that 125I-rEp was internalized into the cells after incubation at 37 degrees C. Further incubation at this temperature showed a decline of cellular radioactivity, with a release of small molecular weight degradation fragments into the medium. These studies demonstrate two classes of receptors for Ep on normal human ECFC. Internalization and degradation of EP occur, and the biologic effect of the hormone is produced by a small number of Ep molecules, as demonstrated in murine erythroid progenitor cells.     

Activation of multifunctional Ca2+/calmodulin-dependent protein kinase in GH3 cells.

We report that the rat pituitary cell line GH3 contains a Ca2(+)- and calmodulin-dependent protein kinase with properties characteristic of multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) from rat brain. The GH3 kinase exhibits the hallmark of authentic CaM kinase: conversion from Ca2(+)-dependent to Ca2(+)-independent activity following a brief initial phosphorylation in vitro. This phosphorylation occurs at a site which is similar or identical to that of the "autonomy" site of the rat brain enzyme and thus may be an autophosphorylation event. GH3 CaM kinase is phosphorylated and becomes Ca2(+)-independent in situ. Depolarization of intact cells with K+ opens calcium channels and leads to the phosphorylation of CaM kinase at the autonomy site, and the kinase becomes significantly and persistently Ca2(+)-independent. Treatment of cells with thyrotropin-releasing hormone (TRH), which activates the phosphatidylinositol signaling pathway, also generates a Ca2(+)-independent CaM kinase in situ. The primary effect of TRH on CaM kinase activity is transient and correlates with the spike of Ca2+ released from intracellular stores and the rapid phase of prolactin release from GH3 cells. This study demonstrates that CaM kinase is able to detect and respond to both calcium that enters the cell through voltage-sensitive Ca2+ channels and calcium released from internal stores via the phosphatidylinositol pathway. We find that TRH, a hormone that causes release of prolactin and was previously believed to activate primarily protein kinase C, also significantly activates CaM kinase in intact cells.     

Three activators of protein kinase C, bryostatins, dioleins, and phorbol esters, show differing specificities of action on GH4 pituitary cells

Phorbol ester tumor promoters such as 12-O-tetradecanoylphorbol acetate (TPA) activate the calcium- and phospholipid-dependent protein kinase C and enhance three biological responses (prolactin release, prolactin synthesis, and cell stretching) in GH4C5 rat pituitary cells. We have examined several actions on GH4C5 cells of TPA and two other classes of protein kinase C activators, synthetic cell permeant dioleins and bryostatins isolated from the marine bryozoan Bugula neritina. Bryostatins 1 and 2 (B1 and B2, respectively) competed for [3H]phorbol 12,13-dibutyrate binding to the protein kinase C complex in intact cells nearly equipotently with TPA. B1 and B2, 1-oleoyl-2-acetylglycerol (OAG) and 1,2-dioctanoylglycerol (Di8) as well as TPA each activated partially purified protein kinase C from GH4C5 cells. B1, B2, and TPA each enhanced the acute release of prolactin from GH4C5 cells to a similar maximal extent. B1, B2, and TPA also enhanced prolactin synthesis. However, B1 and B2 were only partial agonists because they enhanced prolactin synthesis to a lesser maximal extent than did TPA and, given in combination, they reduced TPA-enhanced prolactin synthesis. OAG and Di8 stimulated prolactin release (to a lesser maximal extent than TPA) and did not stimulate prolactin synthesis. Pretreatment with OAG did not reduce TPA-stimulated prolactin release or synthesis. B2 and TPA induced cell stretching in GH4C5 cells, whereas B1, OAG, and Di8 induced little if any stretching. B1, but not B2, given in combination with TPA antagonized TPA-induced stretching but did not reduce thyrotropin-releasing hormone- or epidermal growth factor-induced stretching. We conclude that the bryostatins, phorbol esters, and dioleins bind to the same site on the protein kinase C complex to activate the enzyme, but they alter three biological responses in GH4C5 cells with selectivities and efficacies that differ. We propose that different activators of protein kinase C (such as bryostatins, dioleins, and phorbol esters) may elicit different cellular responses by altering the substrate specificity or activating multiple forms of the kinase.     

Effects of pretreatment with tetradecanoyl phorbol acetate on regulation of growth hormone and prolactin secretion from ovine anterior pituitary cells

Tetradecanoyl phorbol acetate (TPA) stimulates growth hormone (GH) and prolactin secretion from ovine anterior pituitary cells. Pretreatment of the cells with TPA abolishes this effect, presumably due to down-regulation of protein kinase C. Such pretreatment did not alter effects of thyrotropin-releasing hormone or dopamine on prolactin secretion, suggesting no involvement of protein kinase C. Pretreatment with TPA attenuated actions of GH-releasing hormone on GH release (but not actions on cyclic AMP levels), possibly due to depletion of cellular stores of GH. Such pretreatment also attenuated inhibition of GH release by somatostatin, possibly due to phosphorylation of receptors or associated proteins by protein kinase C.     

Mitogen-activated protein kinase activation by stimulation with thyrotropin-releasing hormone in rat pituitary GH3 cells.

We examined whether mitogen-activated protein (MAP) kinase is activated by thyrotropin-releasing hormone (TRH) in GH3 cells, and whether MAP kinase activation is involved in secretion of prolactin from these cells. Protein kinase inhibitors--such as PD098059, calphostin C, and genistein--and removal of extracellular Ca2+ inhibited MAP kinase activation by TRH. A cAMP analogue activated MAP kinase in these cells. Effects of cAMP on MAP kinase activation were inhibited by PD098059. TRH-induced prolactin secretion was not inhibited by levels of PD098059 sufficient to i activation but was inhibited by wortmannin (1 microM) and KN93. Treatment of GH3 cells with either TRH or cAMP significantly inhibited DNA synthesis and induced morphological changes. The effects stimulated by TRH were reversed by PD098059 treatment, but the same effects stimulated by cAMP were not. Treatment of GH3 cells with TRH for 48 h significantly increased the prolactin content in GH3 cells and decreased growth hormone content. The increase in prolactin was completely abolished by PD098059, but the decrease in growth hormone was not. These results suggest that TRH-induced MAP kinase activation is involved in prolactin synthesis and differentiation of GH3 cells, but not in prolactin secretion.     

Nuclear sphingomyelin-synthase and protein kinase C delta in melanoma cells

The aim of this research is to study the influence of protein kinase C delta on the nuclear phospholipids metabolism. Murine and human melanoma cells, in which overexpression of protein kinase delta was induced, were used. After purification of the nuclei, the phosphatidylcholine-dependent phospholipase C, sphingomyelin-synthase, and sphingomyelinase activities were measured. The results showed that the nuclear sphingomyelin-synthase activity increased and sphingomyelinase activity decreased in the protein kinase C delta overexpressive cells with respect to the controls. As a consequence, the ceramide pool decreased and diacylglycerol pool increased; this effect was not due to the phosphatidylcholine-dependent phospholipase C activity that did not change. The inhibition of sphingomyelinase could be due to protein kinase C delta as well as to existence of a sort of nuclear self-regulation between sphingomyelin-synthase and sphingomyelinase. The possible role of nuclear sphingomyelin-synthase in cell proliferation is discussed.     

Nuclear accumulation of multiple protein kinases during prolactin-induced proliferation of Nb2 rat lymphoma cells

Intracellular kinases play important roles in signal transduction and are involved in the surface receptor-mediated regulation of cellular functions, including mitogenesis. In the present study, we examined the possible involvement of various protein kinases in the passage of a mitogenic signal from the cell surface to the nucleus of Nb₂ cells, a rat nodal lymphoma cell line in which prolactin is a mitogen. Following a prolactin challenge, various kinase activities were monitored at short intervals in different cellular fractions over a 60 min period. Protein kinase C (PKC) activity in the cytosolic fraction rapidly declined to 50% of its original activity within the first 30 min, while PKC activity in the nuclear fractions increased sharply, reaching its highest level by 30 min following a prolactin challenge. There were also increases in both casein kinase and protein tyrosine kinase (PTK) activities in the nuclear fractions during the first 30 min following a prolactin challenge that paralleled PKC activity. The activities of all three kinases declined thereafter, reaching levels close to their respective basal values by 60 min following initiation of prolactin treatment. These observations suggest the possibility that multiple protein kinases may be involved in mitogenic signal transduction for prolactin in Nb₂ cells. © 1996 Wiley-Liss, Inc.     

Differential regulation of pituitary hormone secretion and gene expression by thyrotropin-releasing hormone. A role for mitogen-activated protein kinase signaling cascade in rat pituitary GH3 cells

We examined the possible involvement of mitogen-activated protein (MAP) kinase activation in the secretory process and gene expression of prolactin and growth hormone. Thyrotropin-releasing hormone (TRH) rapidly stimulated the secretion of both prolactin and growth hormone from GH3 cells. Secretion induced by TRH was not inhibited by 50 microM PD098059, but was completely inhibited by 1 microM wortmannin and 10 microM KN93, suggesting that MAP kinase does not mediate the secretory process. Stimulation of GH3 cells with TRH significantly increased the mRNA level of prolactin, whereas expression of growth hormone mRNA was largely attenuated. The increase in prolactin mRNA stimulated by TRH was inhibited by addition of PD098059, and the decrease in growth hormone mRNA was also inhibited by PD098059. Transfection of the cells with a pFC-MEKK vector (a constitutively active MAP kinase kinase kinase), significantly increased the synthesis of prolactin and decreased the synthesis of growth hormone. These data taken together indicate that MAP kinase mediates TRH-induced regulation of prolactin and growth hormone gene expression. Reporter gene assays showed that prolactin promoter activity was increased by TRH and was completely inhibited by addition of PD098059, but that the promoter activity of growth hormone was unchanged by TRH. These results suggest that TRH stimulates both prolactin and growth hormone secretion, but that the gene expressions of prolactin and growth hormone are differentially regulated by TRH and are mediated by different mechanisms.     

Inhibitory effects of tetradecanoylphorbol acetate and diacylglycerol on erythropoietin production in human renal carcinoma cell cultures

A human renal carcinoma from a patient with an erythrocytosis, serially transplanted into athymic nude mice, was grown in primary monolayer cell cultures. After reaching confluency the cultured cells formed multicellular hemicysts (domes) which became more abundant as the cultures approached saturation density. Erythropoietin (Ep) production by this renal carcinoma in culture was only slightly increased at the time of semiconfluency but showed a marked increase in Ep levels in the culture medium after the cultures reached confluency, in parallel with an increase in dome formation. The phorbol ester tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) showed a significant dose-related inhibitory effect on Ep production and dome formation in the renal carcinoma cell cultures, suggesting an important role of protein kinase C, the only known receptor for TPA, in inhibiting the expression of differentiated phenotypes in the renal carcinoma cells. TPA also suppressed Ep secretion over a period of 96 h, indicating a time course of suppression of this differentiated function of the renal carcinoma cells in culture. This hypothesis was further supported by the observation that diacylglycerol, the endogenous activator of protein kinase C, likewise inhibited Ep production and dome formation in the renal carcinoma cell cultures. These studies suggest a role of the inositol-lipid second messenger path and protein kinase C in the regulation of Ep production.     

Role of cadmium in activating nuclear protein kinase C and the enzyme binding to nuclear protein.

Specific effects of cadmium on nuclear protein kinase C activity were found with 3T3/10T1/2 mouse fibroblast and rat liver nuclei. Treatment of the mouse fibroblasts in culture with 12-O-tetradecanoylphorbol-13-acetate resulted in the stimulation of nuclear protein kinase C activity in a "fixed" pool which is defined by its resistance to chelator extraction, whereas the chelator extractable enzyme activity, defined as the "labile" pool was unaffected. Cadmium was found to potentiate the effect of the phorbol ester, directed specifically to nuclei, since the particulate protein kinase C activity was not changed under similar treatment. In a reconstituted system consisting of rat liver nuclei and rat brain protein kinase C, cadmium stimulated the binding of the enzyme to a 105-kDa nuclear protein. The binding of a 105-kDa protein to protein kinase C is attributed strictly due to the cadmium effect, whereas a 50-kDa protein binding to protein kinase C was only enhanced by cadmium. We propose a mechanistic model, where cadmium substitutes zinc in the regulatory domain of protein kinase C rendering the putative protein-protein binding site exposed.     

Analysis of the role of the Shc and Grb2 proteins in signal transduction by the v-ErbB protein.

The epidermal growth factor receptor, EGFR, has been implicated in cell transformation in both mammalian and avian species. The v-ErbB oncoprotein is an oncogenic form of the chicken EGFR. The tyrosine kinase activity of this oncoprotein is required for transformation, but no transformation-specific cellular substrates have been described to date. Recently activation of the ras signal transduction pathway by the EGFR has been shown to involve the Shc and Grb2 proteins. In this communication, we demonstrate that the Shc proteins are phosphorylated on tyrosine residues and are complexed with Grb2 and the chicken EGFR following ligand activation of this receptor. In fibroblasts and erythroid cells transformed by the avian erythroblastosis virus (AEV) strains H and ES4, the Shc proteins are found to be constitutively phosphorylated on tyrosine residues. The tyrosine-phosphorylated forms of the AEV strain H v-ErbB protein are found in a complex with Shc and Grb2, but the Shc proteins do not bind to the AEV strain ES4 v-ErbB protein. Mutant forms of the v-ErbB protein (in which several of the tyrosines that become autophosphorylated have been deleted by truncation) are unable to transform erythroid cells but can still transform fibroblasts. Analysis of cells transformed by one of these mutants revealed that the truncated v-ErbB protein could no longer bind to either Shc or Grb2, but this oncoprotein still gave rise to tyrosine-phosphorylated Shc proteins that complexed with Grb2 and led to activation of mitogen-activated protein (MAP) kinase. The results suggest that stable binding of Grb2 and Shc to the v-ErbB protein is not necessary to activate this signal transduction pathway and assuming that the mutant activate MAP kinase in erythroid cells in a manner similar to that of fibroblasts, that activation of this pathway is not sufficient to transform erythroid cells.     

Subnuclear Trafficking of Glucocorticoid Receptors In Vitro: Chromatin Recycling and Nuclear Export

We have used digitonin-permeabilized cells to examine in vitro nuclear export of glucocorticoid receptors (GRs). In situ biochemical extractions in this system revealed a distinct subnuclear compartment, which collects GRs that have been released from chromatin and serves as a nuclear export staging area. Unliganded nuclear GRs within this compartment are not restricted in their subnuclear trafficking as they have the capacity to recycle to chromatin upon rebinding hormone. Thus, GRs that release from chromatin do not require transit through the cytoplasm to regain functionality. In addition, chromatin-released receptors export from nuclei of permeabilized cells in an ATP- and cytosol-independent process that is stimulated by sodium molybdate, other group VI-A transition metal oxyanions, and some tyrosine phosphatase inhibitors. The stimulation of in vitro nuclear export by these compounds is not unique to GR, but is restricted to other proteins such as the 70- and 90-kD heat shock proteins, hsp70 and hsp90, respectively, and heterogeneous nuclear RNP (hnRNP) A1. Under analogous conditions, the 56-kD heat shock protein, hsp56, and hnRNP C do not export from nuclei of permeabilized cells. If tyrosine kinase inhibitors genistein and tyrphostin AG126 are included to prevent increased tyrosine phosphorylation, in vitro nuclear export of GR is inhibited. Thus, our results are consistent with the involvement of a phosphotyrosine system in the general regulation of nuclear protein export, even for proteins such as GR and hnRNP A1 that use distinct nuclear export pathways.     

A murine recombinant retrovirus containing the src oncogene transforms erythroid precursor cells in vitro.

A murine retrovirus (MRSV) containing the src gene of Rous sarcoma virus has been shown to cause an erythroproliferative disease in mice (S. M. Anderson and E. M. Scolnick, J. Virol. 46:594-605, 1983). We now demonstrate that this same virus can transform erythroid progenitor cells in vitro. Infection of fetal liver cells or spleen and bone marrow cells from phenylhydrazine-treated adult mice gave rise to colonies of erythroid cells which grew in methylcellulose under conditions not favorable for the growth of normal erythroid cells. The presence of pp60src in the transformed erythroid cells was demonstrated by an immune complex protein kinase assay. The time course of appearance and subsequent differentiation of erythroid colonies indicated that the target cell for MRSV was a 6- to 8-day burst-forming unit. Differentiation of the erythroid progenitors was not blocked by the presence of pp60src, and the cells retained sensitivity to the hormone erythropoietin. In fact, the transformed cells exhibited increased hormone sensitivity since the number, the size, and the extent of hemoglobinization of the colonies were all increased by the addition of small amounts of erythropoietin. MRSV was not susceptible to restriction by the Fv-2 locus, as MRSV could transform hematopoietic cells from C57BL/6 mice. These results indicate that (i) the erythroid proliferation observed in vivo is caused by a direct effect of MRSV on erythroid progenitors and (ii) the transformed erythroid precursors acquire a growth advantage over uninfected cells without losing the ability to differentiate and respond to physiologic regulators.