Nancy E. Zorn

Prolactin activates protein kinase C and stimulates growth-related gene expression in rat liver

We have examined the effect of prolactin (PRL) on growth-related gene expression, protein kinase C (PKC) activity and diacylglycerol (DAG) mass in rat liver. Hepatic levels of messenger (m)RNA for c-myc, ornithine decarboxylase (ODC) and beta-actin increased in a dose-dependent manner within 1 h after PRL administration. Prolactin also caused a transient elevation of liver DAG levels and particulate-associated PKC activity. The PRL-provoked increases in DAG mass and particulate PKC activity were coincident and maximal at 20 min and began declining toward control levels by 30 min. These results suggest a temporal relationship between PRL-stimulated DAG accumulation and PKC activation. Furthermore, the subsequent rapid induction of growth-related gene expression provides new information on the role of PRL as a hepatic mitogen.

The HIV protein, GP120, activates nuclear protein kinase C in nuclei from lymphocytes and brain

Nuclear pool(s) of protein kinase C (PKC) may be a common target for hormones and growth factors which affect the trophic state of cells. The data presented demonstrate a time and dose-dependent activation of nuclear PKC by the HIV coat protein, gp120, in isolated nuclei from rat spleen and hippocampus. This gp120-stimulated PKC response was blocked by specific PKC inhibitors, a monoclonal antibody to PKC, and a monoclonal antibody directed against the murine T4 analog, L3T4. It is suggested that the gp120 interaction with the nuclear trophic factor-PKC system may impair normal gene expression, and thus result in the clinical symptoms associated in AIDS infection.

Prolactin and known modulators of rat splenocytes activate nuclear protein kinase C.

Prolactin (PRL) and other trophic factors rapidly activate a nuclear pool(s) of protein kinase C (nPKC) in purified splenocyte nuclei. The PRL also enhanced [2-3H]glycerol incorporation into nuclear mono- and triacylglycerol. An assay was devised which not only probed the ability of the hormone to activate protein kinase C (PKC) but also demonstrated the presence of nuclear substrates. Using this methodology, a biphasic concentration-response curve to PRL was observed. Heterologous species of PRL and various growth factors also activated nPKC. The PRL-induced nPKC stimulation was antagonized by various immunomodulators, G protein-coupling inhibitors, PKC inhibitors, a calmodulin inhibitor, and a peripheral benzodiazepine agonist and antagonist. A monoclonal antibody to PKC, anti-rat PRL antiserum and a monoclonal anti-rat PRL receptor antibody antagonized PRL-induced PKC-dependent nuclear phosphorylation, further implicating nPKC and a PRL receptor-mediated activation process. Nuclear PKC may be a major target for trophic regulation in response to both positive and negative growth signals.

Vasoactive intestinal peptide (VIP) activation of nuclear protein kinase C in purified nuclei of rat splenocytes

We have examined the actions of vasoactive intestinal peptide (VIP) and certain other known immune modulators on a nuclear pool(s) of protein kinase C (PKC) in isolated rat splenocyte nuclei. Rat splenocyte nuclei pure by enzymatic and electron microscope criteria demonstrated a time- and concentration-dependent activation of nuclear PKC (nPKC) by VIP. A biphasic pattern of three bell-shaped curves was observed with peak phosphorylation at 10(-15), 10(-9) and 10(-6)M VIP. The phosphorylation of endogenous nuclear substrates was characterized as a PKC-mediated event by use of three known PKC inhibitors, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), sphingosine, and staurosporine, which produced similar phosphate incorporation measurements. Also, this activity was blocked with the addition of a monoclonal antibody to PKC. Inhibitors of the ability of VIP to activate nPKC included somatostatin, 8-bromo-cAMP, peripheral benzodiazepine receptor modulators, and the PKC inhibitors, sphingosine and staurosporine. These data have direct relevance to our knowledge of cell-mediated immunity.

Alterations in splenocyte protein kinase C (PKC) activity by 2,3,7,8-tetrachlorodibenzo-p-dioxin in vivo

The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on growth factor-coupled activation of nuclear protein kinase C (nPKC) and on the subcellular distribution of PKC activity in rat splenocytes were investigated. Seven days after a single injection of TCDD (50 micrograms/kg body weight), cytosolic and particulate PKC activity was significantly higher in splenocytes from TCDD-treated rats or pair-fed control rats compared to ad libitum-fed animals. In a separate experiment, purified splenocyte nuclei from TCDD-treated animals and controls were used to study activation of nPKC by growth factors and other trophic agents. Growth factor-stimulated nPKC activation was attenuated in splenic nuclei from TCDD-treated rats compared to vehicle-treated controls. Evidence presented here suggests that the cellular mechanism of TCDD toxicity leading to immunosuppression in rodents may be mediated in part by uncoupling of growth factor receptors linked to PKC activation at the level of the nucleus. However, changes in total splenocyte PKC activity appear to be correlated with hypophagia since cytosolic and particulate PKC levels were elevated in TCDD-treated rats and their pair-fed partners.

Retinoic acid induces translocation of protein kinase C (PKC) and activation of nuclear PKC (nPKC) in rat splenocytes

Retinoic acid (RA), a vitamin A metabolite, has marked effects on growth of normal and malignant cells; however, the exact mechanism of action remains unclear. The effect of two RA analogs, 13-cis-RA and all-trans-RA, on transmembrane signalling processes was investigated in rat splenocytes. Treatment of rat splenic cells with these retinoic acid analogs resulted in translocation of protein kinase C (PKC) from the cytosol to the membrane. Previous studies have described nuclear RA receptors (RARs and RXRs) for several species and the biologic activity of RA has been shown to be mediated by specific interaction with these nuclear receptors. Thus, activation of nuclear pool(s) of protein kinase C (nPKC) by RA analogs was also studied. Rat splenocyte nuclei pure by enzymatic and electron microscope criteria demonstrated a biphasic pattern of bell-shaped curves for both cis- and trans-RA with maximum statistically significant peak of phosphate incorporation into endogenous substrates at 10(-16) M cis-RA and 10(-16)-10(-17) M trans-RA. A monoclonal antibody to PKC and the PKC inhibitors, H-7, sphingosine, and staurosporine, blocked the RA-stimulated nuclear phosphorylation. The ability of RA to activate cell membrane PKC resulting in an increase in particulate PKC activity correlates well with the activation of nPKC since the particulate fraction would include nuclear enzyme systems. This ability of RA to activate nPKC and possibly affect the growth status of a cell may provide a missing link to our understanding of the cellular sites of action for this vitamin.

Prolactin induces growth-related gene expression in rat aortic smooth muscle in vivo☆

We examined the effects of in vivo administration of prolactin on growth-related gene expression in aorta. Optimal mRNA expression for both the proto-oncogene, c-myc and ornithine decarboxylase occurred at 22 mg/kg prolactin. For c-myc, this was seen as early as 15 min. Prolactin-induced ornithine decarboxylase mRNA expression began at 1 h. The results confirm work showing induction of these genes by prolactin in tissues where it is mitogenic and supports a role for prolactin in the trophic response of vascular smooth muscle.

Prolactin Receptors in the Nervous and Immune Systems

Prolactin, an anterior pituitary hormone with a multitude of roles and sites of action, is produced in lactrotrophs and is one of a family of four to six somatomammotropic hormones. A great diversity of functions has been ascribed to prolactin in many different species. Of particular recent importance has been the emergence of substantial evidence indicating that prolactin acts as a modulator of the immune response in mammals (1,2; see also Carr, this volume). Furthermore, prolactin has been identified in various areas of the brain and appears to play a role in neural function. Thus, prolactin appears to function as an immunomodulator and may in fact influence some events within the nervous system of mammals. It is very likely that bidirectional communication exists between the nervous and immune systems (3), and prolactin seems to play an important role in this process.

Adenosine activation of a nuclear pool of protein kinase C in rat splenocytes

The stimulatory effects of adenosine analogues on a nuclear pool of protein kinase C (PKC) were examined using isolated rat splenocyte nuclei. Nuclear receptors met pharmacological criteria of A1 adenosine receptors including a potency profile in which cyclopentyladenosine (CPA), a selective A1 agonist, was more potent than 2-phenylaminoadenosine (2PAA), a selective A2 agonist. The selective A1 receptor agonist N6-1-(phenyl-2R-propyl) adenosine (R-PIA) activated PKC whereas the S diastereomer did not. The adenosine-induced PKC response could be attenuated using a monoclonal antibody to PKC, an A1 receptor antagonist, three known PKC inhibitors and pertussis toxin (PTX). The results suggest that adenosine may exert immunomodulatory effects through the activation of nuclear PKC.