Nachum Reiss

Mechanism of Mitogen-Activated Protein Kinase Activation by Gonadotropin-Releasing Hormone in the Pituitary αT3–1 Cell Line: Differential Roles of Calcium and Protein Kinase C1

The mechanism of mitogen-activated protein kinase (MAPK, ERK) stimulation by the GnRH analog [d-Trp6]GnRH (GnRH-a) was investigated in the gonadotroph-derived αT3–1 cell line. GnRH-a as well as the protein kinase C (PKC) activator 12-O-tetradecanoyl phorbol-13-acetate (TPA) stimulated a sustained response of MAPK activity, whereas epidermal growth factor (EGF) stimulated a transient response. MAPK kinase (MEK) is also activated by GnRH-a, but in a transient manner. GnRH-a and TPA apparently activated mainly the MAPK isoform ERK1, as revealed by Mono-Q fast protein liquid chromatography followed by Western blotting as well as by gel kinase assay. GnRH-a and TPA stimulated the tyrosine phosphorylation of several proteins, and this effect as well as the stimulation of MAPK activity were inhibited by the PKC inhibitor GF 109203X. Similarly, down-regulation of TPA-sensitive PKC subspecies nearly abolished the effect of GnRH-a and TPA on MAPK activity. Furthermore, the protein tyrosine kinase (PTK) inhibitor ge...

Identification of Extracellular Signal-regulated Kinase 1/2 and p38 MAPK as Regulators of Human Sperm Motility and Acrosome Reaction and as Predictors of Poor Spermatozoan Quality

Mature spermatozoa acquire progressive motility only after ejaculation. Their journey in the female reproductive tract also includes suppression of progressive motility, reactivation, capacitation, and hyperactivation of motility (whiplash), the mechanisms of which are obscure. MAPKs are key regulatory enzymes in cell signaling, participating in diverse cellular functions such as growth, differentiation, stress, and apoptosis. Here we report that ERK1/2 and p38 MAPK are primarily localized to the tail of mature human spermatozoa. Surprisingly, c-Jun N-terminal kinase 1/2, which is thought to be ubiquitously expressed, could not be detected in mature human spermatozoa. ERK1/2 stimulation is downstream to protein kinase C (PKC) activation, which is also present in the human sperm tail (PKCβI and PKCϵ). ERK1/2 stimulates and p38 inhibits forward and hyperactivated motility, respectively. Both ERK1/2 and p38 MAPK are involved in the acrosome reaction. Using a proteomic approach, we identified ARHGAP6, a RhoGAP, as an ERK substrate in PMA-stimulated human spermatozoa. Inverse correlation was obtained between the relative expression level of ERK1 or the relative activation level of p38 and sperm motility, forward progression motility, sperm morphology, and viability. Therefore, increased expression of ERK1 and activated p38 can predict poor human sperm quality.

Differential Activation of Protein Kinase C δ and ε Gene Expression by Gonadotropin-releasing Hormone in αT3-1 Cells AUTOREGULATION BY PROTEIN KINASE C

The effect of gonadotropin-releasing hormone (GnRH) upon protein kinase C (PKC) δ and PKCε gene expression was investigated in the gonadotroph-derived αT3-1 cell line. Stimulation of the cells with a stable analog [d-Trp6]GnRH (GnRH-A) resulted in a rapid elevation of PKCε mRNA levels (1 h), while PKCδ mRNA levels were elevated only after 24 h of incubation. The rapid elevation of PKCε mRNA by GnRH-A was blocked by pretreatment with a GnRH antagonist or actinomycin D. The PKC activator 12-O-tetradecanoylphorbol-13-acetate (TPA), but not the Ca2+ ionophore ionomycin, mimicked the rapid effect of GnRH-A upon PKCε mRNA elevation. Additionally, the rapid stimulatory effect of GnRH-A was blocked by the selective PKC inhibitor GF109203X, by TPA-mediated down-regulation of endogenous PKC, or by Ca2+ removal. Interestingly, serum-starvation (24 h) advanced the stimulation of PKCδ mRNA levels by GnRH-A and the effect could be detected at 1 h of incubation. The rapid effect of GnRH-A upon PKCδ mRNA levels in serum-starved cells was mimicked by TPA, but not by ionomycin, and was abolished by down-regulation of PKC or by Ca2+ removal. Preactivation of αT3-1 cells with GnRH-A for 1 h followed by removal of ligand and serum resulted in elevation of PKCδ mRNA levels after 24 h of incubation. Western blot analysis revealed that GnRH-A and TPA stimulated (within 5 min) the activation and some degradation of PKCδ and PKCε. We conclude that Ca2+ and PKC are involved in GnRH-A elevation of PKCδ and PKCε mRNA levels, with Ca2+ being necessary but not sufficient, while PKC is both necessary and sufficient to mediate the GnRH-A response. A serum factor masks PKCδ but not PKCε mRNA elevation by GnRH-A, and its removal exposes preactivation of PKCδ mRNA by GnRH-A which can be memorized for 24 h. PKCδ and PKCε gene expression evoked by GnRH-A is autoregulated by PKC, and both isotypes might participate in the neurohormone action.

Signal transduction of the gonadotropin releasing hormone (GnRH) receptor: cross-talk of calcium, protein kinase C (PKC), and arachidonic acid.

Summary1. The decapeptide neurohormone gonadotropin releasing hormone (GnRH) is the first key hormone of the reproductive system. Produced in the hypothalamus, GnRH is released in a pulsatile manner into the hypophysial portal system to reach the anterior pituitary and stimulates the release and synthesis of the gonadotropin hormones LH and FSH. GnRH, a Ca2+ mobilizing ligand, binds to its respective binding protein, which is a member of the seven transmembrane domain receptor family and activates a G-protein (Gq).2. Theα subunit of Gq triggers enhanced phosphoinositide turnover and the elevation of multiple second messengers required for gonadotropin release and biosynthesis.3. The messenger molecules IP3, diacylglycerol, Ca2+, protein kinase C, arachidonic acid and leukotriene C4 cross-talk in a complex networks of signaling, culminating in gonadotropin release and gene expression.

Estrogen responsive creatine kinase in normal and neoplastic cells.

Abstract Estrogen-responsive creatine kinase (uterine estrogen-induced protein, CK-BB) activity was compared during ontogeny of the rat uterus, in the human menstrual cycle and in mouse and human mammary tumors. Between days 2 and 26 of post-natal development of rat uterus, the specific activity of creatine kinase increases 4.5-fold. The glycolytic enzymes, phosphoglycerate kinase and phosphoglycerate mutase, show no increase and pyruvate kinase activity increases 1.5-fold, during this period. Only creatine kinase BB activity was increased by estrogen administration. In human endometrium, the specific activity of creatine kinase increases in the late-secretory stage of the menstrual cycle. During the progression from hormonal dependence to independence shown by the GR mouse mammary tumor in the course of successive transplantations, the total activity of creatine kinase increases. The increase in the MM (muscle type, non-estrogcn responsive) isozyme activity of creatine kinase exceeds the increase in creatine kinase BB activity. Normal human breast and breast tumors display both creatine kinase isozymes. Preliminary evidence for in vitro estrogen responsiveness of creatine kinase, in human breast explants, raises the possibility that creatine kinase BB may be a suitable marker for assessing the hormonal dependence of human tumors.

Biphasic modulation of protein kinase C and enhanced cell toxicity by amyloid beta peptide and anoxia in neuronal cultures

A major feature of Alzheimers disease is the deposition of the amyloid beta peptide (Aβ) in the brain by mechanisms which remain unclear. One hypothesis suggests that oxidative stress and Aβ aggregation are interrelated processes. Protein kinase C, a major neuronal regulatory protein is activated after oxidative stress and is also altered in the Alzheimers disease brain. Therefore, we examined the effects of Aβ1−40 peptide on the protein kinase C cascade and cell death in primary neuronal cultures following anoxic conditions. Treatment with Aβ1−40 for 48 h caused a significant increase in the content and activity of Ca2+‐dependent and Ca2+‐independent protein kinase C isoforms. By 72 h various protein kinase C isoforms were down‐regulated. Following 90 min anoxia and 6 h normoxia, a decrease in protein kinase C isoforms was noticed, independent of Aβ1−40 treatment. A combination of Aβ1−40 and 30‐min anoxia enhanced cytotoxicity as noticed by a marked loss in the mitochondrial ability to convert 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐tetrazolium bromide and by enhanced 4′,6‐diamidino‐2‐phenylindole nuclear staining. Phosphorylation of two downstream protein kinase C substrates of apparent molecular mass 80 and 43 kDa, tentatively identified as the mirystoyl alanine‐rich C‐kinase substrate (MARCKS), were gradually elevated up to 72 h upon incubation with Aβ1−40. Anoxia followed by 30 min normoxia enhanced MARCKS phosphorylation in the membrane but not in the cytosolic fraction. In the presence of Aβ1−40, phosphorylation of MARCKS was reduced. After 6 h normoxia, MARCKS phosphorylability was diminished possibly because of protein kinase C down‐regulation. The data suggest that a biphasic modulation of protein kinase C and MARCKS by Aβ1−40 combined with anoxic stress may play a role in Alzheimers disease pathology. 1

Developmental expression of protein kinase C subspecies in rat brain-pituitary axis

We have examined the neonatal developmental expression of protein kinase C subspecies (PKCs) in rat brain, pituitary glands and cells by enzymatic activity assays, immunohistochemistry and Western blot analysis with type-specific antibodies. A very large increase (455%) was noticed in brain PKC activity during the first week of life with the particulate fraction (22% of total enzyme activity on day 1) increasing dramatically (900%) during the first week to 50% of enzyme activity. In contrast, the pituitary gland showed high activity on day 1 that decreased progressively to reach the lowest levels at 1 year of age. Paradoxically, the number of pituitary cells immunolabeled for PKC increases as a function of age. Western blot analysis showed only small changes in PKC alpha, PKC beta and PKC epsilon when brains from 6-day-old and 3-month-old female rats were compared, whereas PKC tau and PKC delta increased markedly during this period. On the other hand, brain PKC zeta decreased between 6 days and 3 months of age. Western blot analysis showed no major changes in pituitary PKC alpha, PKC beta and PKC zeta when 6-day-old and 3-month-old female rats were compared, while PKC tau was not detected. The major band of pituitary PKC delta (76 kDa) decreased markedly between 6 days and 3 months of age whereas the minor band (68 kDa) did not change.(ABSTRACT TRUNCATED AT 250 WORDS)

Phosphatidylserine directs differential phosphorylation of actin and glyceraldehyde-3-phosphate dehydrogenase by protein kinase C: possible implications for regulation of actin polymerization.

The phospholipid‐dependent protein kinase C is implicated in the regulation of cellular motility and energy metabolism. Phosphatidylserine, a main cofactor of protein kinase C, is involved in the regulation of glyceraldhehyde‐3‐phosphate dehydrogenase, which as actin, was shown to be phosphorylated by purified protein kinase C. Here, we study the effect of phosphatidylserine on the enzyme‐substrate interaction of protein kinase C with glyceraldhehyde‐3‐phosphate dehydrogenase and actin. The stoichiometry of glyceraldhehyde‐3‐phosphate dehydrogenase phosphorylation is not affected by varying the level of phosphatidylserine. However, actin phosphorylation is dependent on phosphatidylserine level, peaking at high phosphatidylserine concentration. Moreover, if actin and glyceraldhehyde‐3‐phosphate dehydrogenase are cophosphorylated at high phosphatidylserine concentration, actin phosphorylation is favored, despite lower affinity for protein kinase C. Hence, phosphatidylserine directs differential phosphorylation of these key proteins of glycolysis and cellular motility and might be capable of recruiting protein kinase C for preferential actin phosphorylation. The sedimentation of phosphorylated actin is increased 3.8 fold and total actin 1.7 fold, suggesting that phosphorylation promotes actin polymerization.

Hormonal regulation of creatine kinase BB.

The induction of increased synthesis of creatine kinase BB (EC 2.7.3.2) now appears to be a response produced by a variety of hormones. The list includes steroid and polypeptide hormones, prostaglandin E2, bone derived growth factor, and dibutyryl cAMP. This survey is the first summary of the breadth of the phenomenon and will concentrate on examples from work which is still in press or is yet unpublished.

The 'estrogen-induced protein': quantitation by autoradiography of polyacrylamide gels.

Abstract The increased rate of synthesis of the “estrogen-induced protein” (IP) first detected by Notides and Gorski is one of the earliest macromolecular responses to estrogen by the rat uterus. The IP detected by autoradiography of sodium dodecyl sulfate polyacrylamide gel electropherograms was shown to correspond to a stainable protein band in electropherograms of cytosol from uteri of both untreated and estrogen-treated rats. The uteri of 9–10-day-old rats showed a maximum rate of IP synthesis 1 h after estrogen-treatment, followed by a decrease to constitutive levels by 24 h. The rate of synthesis of IP was doubled by estrogen administration to 10–30-day-old rats. The improved sensitivity of the autoradiographic method permitted the detection of some stimulation of IP synthesis in uteri of 5-day-old rats, in contrast to our previous findings in Wistar rats using a double isotope labelling technique.