Scott Stone

Prolactin‐Stimulated Mitogenesis of Cultured Astrocytes Is Mediated by a Protein Kinase C‐Dependent Mechanism

Abstract: Prolactin (PRL) has been reported to activate cellular proliferation in nonreproductive tissue, such as liver, spleen, and thymus. Recently, we have extended the possible role of PRL as a mammalian mitogen by demonstrating a mitogenic effect of PRL in cultured astrocytes. Although the cellular mechanisms by which PRL regulates cell growth are not fully understood, protein kinase C (PKC) has been implicated as one of the transmembrane signaling systems involved in the regulation of PRL‐induced cell proliferation in Nb2 lymphoma cells and liver. In the present studies, we examined the possible role of PKC in PRL‐induced proliferation of cultured astrocytes. Incubation of cultured astrocytes with 1 nM PRL resulted in a rapid translocation of PKC from the cytosol to the membrane, with maximal PKC activity in the membrane occurring 30 min after exposure to PRL. Translocation of PKC activity occurred over a physiological range of PRL, with maximal PKC activation occurring at 1 nM. At concentrations greater than 10 nM PRL, there was a decrease in the amount of PKC activity associated with the membrane fraction compared with that of cells stimulated with 1 nM PRL. Incubation of astrocytes with PRL in the presence of the PKC inhibitors staurosporine, 1‐(‐5‐isoquinolinesulfonyl)‐2‐methylpiperazine, or polymyxin B blocked the PRL‐induced increase in cell number with IC50 values of approximately 2 nM, 10 μM, and 6 μM, respectively. PKC is the only known cellular receptor for 12‐O‐tetradecanoylphorbol 13‐acetate (TPA), which stimulates the translocation of PKC from the cytosol to the membrane. Incubation of astrocytes with 20 nM TPA resulted in an increase in the expression of proliferating cell nuclear antigen and cell number, whereas 4α‐phorbol 12,13‐didecanoate, an inactive phorbol ester, was ineffective. To examine further the effect of TPA and PRL on cellular proliferation, cultured astrocytes were incubated with increasing concentrations of TPA in the presence or absence of a minimal effective dose of PRL (100 pM). In the absence of PRL, incubation with TPA resulted in an inverted U‐shaped dose‐response curve, with 100 nM TPA resulting in a maximal increase in cell number. In the presence of 100 pM PRL, the TPA dose‐response curve was shifted to the left, with maximal activity occurring with 10 nM TPA. Chronic stimulation of astrocytes with 500 nM TPA depleted the cells of PKC and blocked the PRL‐induced increase in cell number. Finally, TPA treatment decreased cell‐surface binding of 125I‐PRL. These data indicate that the PKC is involved in the mitogenic effect of PRL in cultured astrocytes.

Prolactin induced expression of glial fibrillary acidic protein and tumor necrosis factor-alpha at a wound site in the rat brain.

To determine if PRL stimulates astrocyte proliferation and cytokine expression in vivo, we examined the effect of PRL on the wound-induced increase in the expression of glial fibrillary acid protein (GFAP) and tumor necrosis factor-alpha (TNF-alpha) in the CNS. Low levels of GFAP detected by Western blot analysis were identified in the non-wounded controls. Five days after the infliction of the wound, the relative abundance of GFAP in the tissue surrounding the wound site was greater than those of intact controls. Injection of PRL into the wound site markedly increased GFAP expression in the hypothalamus. Western blot analysis failed to detect TNF-alpha in the hypothalamus of non-wounded animals. In contrast, TNF-alpha was easily detected in the hypothalamus of wounded rats, and was markedly increased in PRL injected animals. To confirm the PRL-induced increase in TNF-alpha levels, TNF-alpha levels in hypothalamic extracts were measured by bioassay. In non-wounded controls, low but detectable TNF-alpha levels were found in the hypothalamus by bioassay (0.13 +/- 0.02 ng/mg protein). Infliction of a hypothalamic wound markedly increased TNF-alpha levels to 1.4 +/- 0.3 ng/mg protein. Injection of PRL into the wound site resulted in a further increase in TNF-alpha levels to 11.4 +/- 2.6 ng/mg protein. Further, infliction of the hypothalamic wound increased hypothalamic PRL content and PRL mRNA levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of prolactin receptor expression by estradiol in the female rat brain

PROLACTIN (PRL) receptors have been identified in many tissues, including the brain, but little is known about their distribution and regulation. In the female rat brain, ovariectomy significantly (p < 0.05) decreased PRL binding capacity, but not the affinity, in the hypothalamus and pons-medulla. Using reverse transcription and polymerase chain reaction (RT-PCR) amplification and Western blot analyses we found both the long and short forms of the PRL receptor mRNAs and proteins in the hypothalamus, pons-medulla and cortex in the female rat. Ovariectomy decreased the expression of short, but not the long form of the PRL receptor in the hypothalamus and pons-medulla, but not the cortex. Administration of estradiol (1.0 mg per 100 g b.w.) restored the PRL binding capacity, protein and mRNA levels of the short form of the receptor back to control levels. These results suggest that the expression and distribution of PRL receptors in the brain are differentially regulated in specific brain regions.

Stimulation of hypothalamic prolactin release by veratridine and angiotensin II in the female rat : effect of ovariectomy and estradiol administration

In the female rat immunoreactive prolactin (IR-PRL) has been identified in the hypothalamus and in other brain regions. Brain IR-PRL is not of pituitary origin and, based on polyacrylamide gel electrophoresis and peptide mapping, shares a high degree of sequence homology with its pituitary counterpart. We have previously shown that hypothalamic tissue can release IR-PRL in vitro when depolarized by potassium. In this study, we examined the release of IR-PRL from hypothalami obtained from intact and ovariectomized rats and incubated in the presence of veratridine (an alkaloid which depolarizes excitable membranes), angiotensin II, or thyrotropin-releasing hormone. Hypothalamic tissue spontaneously released IR-PRL, and this release was significantly increased by veratridine or angiotensin II in a dose-dependent manner. The specificity of the angiotensin-II-evoked IR-PRL release was demonstrated by the inhibitory effect of saralasin, an angiotensin II receptor antagonist, on hypothalamic IR-PRL release. Thyrotropin-releasing hormone (100 microM) had no effect on hypothalamic IR-PRL release. Ovariectomy decreased hypothalamic IR-PRL content and IR-PRL release in response to veratridine and angiotensin II. The effect of estradiol on hypothalamic IR-PRL content and release was also examined by obtaining hypothalami from ovariectomized rats injected with estradiol (1 microgram/day) or vehicle for 5 days. When compared with vehicle injected rats, administration of estradiol significantly increased the hypothalamic IR-PRL content (46 +/- 4 vs. 81 +/- 16 ng/mg protein).(ABSTRACT TRUNCATED AT 250 WORDS)

Ethanol inhibits prolactin-induced activation of the JAK/STAT pathway in cultured astrocytes.

Alcohol consumption has multiple effects in the central nervous system (CNS). Whereas, alcohol is an immunosuppressive drug the effect of alcohol on the neuroimmune system, remains unclear. In cultured astrocytes, prolactin (PRL) induces mitogenesis and the expression of inflammatory cytokines, including tumor necrosis factor‐α (TNFα). We have recently shown that whereas ethanol does not inhibit PRL receptor binding, it markedly inhibits PRL‐induced mitogenesis and TNFα secretion in cultured astrocytes. It is clear that PRL activates the tyrosine phosphorylation of several proteins, including members of a novel family of protein tyrosine kinases, the Janus Kinases (JAKs). The aims of this study were to characterize PRL‐induced activation of the JAK/STAT (signal transducers and activators of transcription) pathway, and to determine if ethanol affects JAK/STAT activation in cultured astrocytes. We found that PRL specifically increases the tyrosine phosphorylation of JAK2, but not JAK1, JAK3, or Tyk2, and the subsequent phosphorylation of STAT1α, STAT5a, and STAT5b. Preincubation of astrocytes with ethanol markedly inhibited phosphorylation of JAK2, STAT1α, STAT5a, and STAT5b. In PRL‐stimulated astrocytes, ethanol inhibited binding of nuclear proteins to oligonucleotides corresponding to the gamma‐interferon activated sequence (GAS). Further, ethanol blocked PRL‐induced increases in interferon regulatory factor‐1 (IRF‐1) mRNA, a PRL/cytokine inducible transcription factor involved in the regulation of a number of cytokine inducible genes. The inhibition of tyrosine phosphorylation by ethanol was not a general effect, however, as we found that ethanol increased basal and NGF‐induced tyrosine phosphorylation of extracellular signal‐activated protein kinase‐1 (ERK‐1). These data indicate that ethanol inhibits PRL‐induced tyrosine phosphorylation of the JAK/STAT pathway resulting in decreased nuclear GAS DNA binding and inhibition of the PRL inducible gene, IRF‐1. Thus, suggesting that ethanol‐induced inhibition of JAK2 phosphorylation may be one mechanism though which ethanol could alter the brains response to injury or infection. J. Cell. Biochem. 74:278–291, 1999.

Prolactin stimulation of protein kinase C activity in the rat hypothalamus

Stimulation of cultured hypothalamic slices with PRL causes a rapid translocation of a Ca2+/phospholipid dependent protein kinase from the cytosol to the membrane fraction. The translocation of PKC from the cytosol to the membrane occurred at physiological concentrations of PRL with a maximal response occurring at 10(-10) M. At concentrations above this, there was less PKC activity translocated from the cytosol to the membrane. When injected into the medial preoptic area of the hypothalamus, PRL resulted in a similar translocation of PKC activity. These data clearly indicate that PRL can activate PKC in the rat hypothalamus, and suggest that PKC may be one of the transmembrane signaling mechanisms involved in the regulation of brain function by prolactin.

Prenatal alcohol exposure increases TNFα-induced cytotoxicity in primary astrocytes

We examined the effect of prenatal alcohol exposure (PAE) on tumor necrosis factor-alpha-(TNFalpha) induced cell death in primary astrocyte cultures. Flow cytometry revealed that PAE increased the sensitivity of astrocytes to the cytotoxic effects of TNFalpha when compared to astrocytes prepared from pair-fed and chow-fed controls. In a number of cell types, TNFalpha regulates cell growth or death, in part, by the hydrolysis of sphingomyelin to ceramide and sphingosine-1-phosphate (SPP). Using a 3-(4. 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxic assay we found that PAE increased the sensitivity of astrocytes to the cytotoxic effects of TNFalpha, sphingomyelinase (SMase), and C(2)- and C(6)-ceramide. The increasing cellular concentrations of SPP, a sphingolipid metabolic that induces cell growth, protected the cells from TNFalpha-induced cell death. N, N-dimethylsphingosine (DMS), which inhibits SPP production, and N-oleoylethanolamine, which inhibits acid ceramidases, increased TNFalpha-induced cytotoxicity in astrocytes prepared from PAE rats. These studies suggest that PAE shifts the balance of sphingolipid metabolism in favor of a pathway that increases the susceptibility of astrocytes to the cytotoxic effect of TNFalpha.

Ethanol inhibits prolactin- and tumor necrosis factor-?-, but not gamma interferon-induced expression of intercellular adhesion molecule-1 in human astrocytoma cells

In humans, alcohol consumption has multiple effects on the immune system. Despite an increase in our understanding of the effects of alcohol on the immune system, little is known about the effect of alcohol on the neuroimmune response. In the central nervous system (CNS), astrocytes and microglial function as immune effector cells. In response to infection of injury, astrocytes increase in number and size, express several proinflammatory cytokines, MHC class I and II antigens, and several adhesion molecules, including intercellular adhesion molecule‐1 (ICAM‐1). Interactions between ICAM‐1 and its counter‐receptors play an important role in the regulation of neuroimmune response. In this study, cultured human astrocytoma cells were used to examine the effect of ethanol on ICAM‐1 expression. Western blot analyses show that quiescent astrocytes express, at least, four immunoreactive ICAM‐1 proteins with apparent molecular weights 55, 67, 82, and 90 kDa. Incubation of human astrocytoma cells with tumor necrosis factor‐α (TNF‐α) or prolactin (PRL) resulted in marked increases in all four immunoreactive ICAM‐1 proteins. In the presence of ethanol, however, PRL‐ and TNF‐α‐induced increases in all four immunoreactive ICAM‐1 proteins were markedly inhibited. ICAM‐1 is a cell surface transmembrane glycoprotein. Using a cell surface specific ICAM‐1 adhesion assay we found that in human astrocytoma cells TNF‐α, interferonγ (IFN‐γ) and PRL increased cell surface ICAM‐1 expression. Consistent with our Western blot analyses, ethanol significantly inhibited TNF‐α‐ and PRL‐induced cell surface ICAM‐1 expression. By contrast, IFN‐γ‐induced ICAM‐1 expression was not inhibited by exposure of the cells to ethanol. Expression of ICAM‐1 is regulated predominantly at the transcriptional level. In the present report, we show that TNF‐α increased ICAM‐1 mRNA levels in human astrocytoma cells and that ethanol markedly blocked TNF‐α‐induced increases in ICAM‐1 mRNA levels. Further, we found that PRL‐induced ICAM‐1 expression was, at least in part, due to a PRL‐induced increase in TNF‐α syntheses and secretion. Our results clearly indicate that ethanol has a pronounced effect on ICAM‐1 expression in human astrocytoma cells, thus suggesting that ETOH exposure may impair the immune response in the CNS by blocking leukocytes adhesion and migration into the CNS in response to injury or infection. J. Cell. Biochem. 77:455–464, 2000.

Prenatal exposure to ethanol alters the neuroimmune response to a central nervous system wound in the adult rat.

We examined the long-term effects of in utero ethanol exposure on the expression of tumor necrosis factor-alpha (TNF-alpha), glial fibrillary acidic protein (GFAP), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and ED1 in the tissue at the site of a central nervous system (CNS) wound. Adult rats obtained from dams fed control diets or an ethanol diet were fed either control diets or an ethanol diet 5 days before and after infliction of a CNS wound. In pair-fed controls, the expression of TNF-alpha, GFAP, ICAM-1, VCAM-1, and ED1 immunoreactive proteins was increased in the tissue at the wound site when compared with that in nonlesioned tissues. In adult rats previously exposed to ethanol in utero and then fed a liquid diet before and after infliction of a CNS wound, however, expression of TNF-alpha, GFAP, and ICAM-1 was markedly decreased when compared with findings in pair-fed controls. In contrast, VCAM-1 levels and ED1 immunoreactive proteins were markedly increased when compared with findings for pair-fed controls. Furthermore, in adult rats exposed to ethanol in utero, re-exposure to ethanol before and after sustaining a CNS wound resulted in further decreases in TNF-alpha, GFAP, and ICAM-1 levels and marked increases in VCAM-1 levels and ED1 immunoreactive proteins. Results of these studies suggest to us that prenatal exposure to ethanol has a long-term immunoteratogenic effect in the CNS, resulting in altered responses of key components of the neuroimmune response, which could leave the animal immunocompromised as an adult.

Proteolytic modification of prolactin by the female rat brain.

Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analyses we have identified immunoreactive prolactin (PRL) proteins with molecular weights of 24 and 16 kD in the female rat brain. Because PRL target tissues have been shown to contain enzymes which, in vitro, cleave PRL into a 16-kD PRL fragment, studies were performed to characterize PRL proteolysis in the female rat brain. In vitro proteolysis of PRL was examined by incubating [125]I-PRL with 25,000 g subcellular fractions followed by SDS-PAGE under reducing conditions. At acidic pHs, incubation of PRL with 25,000 g hypothalamic fractions consistently resulted in the generation of a 16-kD fragment. The generation of the 16-kD fragment was time and tissue concentration dependent. Enzyme inhibitor analysis indicated that PRL proteolysis could be blocked by aspartate and serine protease inhibitors, but not sulfhydryl, metalloenzyme or trypsin protease inhibitors. Subcellular localization of hypothalamic PRL proteolytic activity by equilibrium density centrifugation revealed a bimodal distribution of proteolytic activity with modal densities of 1.12 and 1.24 g/ml. Homogenization of the tissue in a hypo-osmotic medium disrupted the high density peak resulting in a single low-density peak at the top of the gradient. These data indicate that subcellular fractions of the rat brain contain enzymes which can cleave PRL into a 16-kD fragment under acidic conditions. The majority of the enzymatic activity is localized in membrane-bound particles with a density similar to subcellular particles which contain PRL.