key: cord-0007508-tkoqx9l3
authors: Sawada, Makoto; Suzumura, Akio; Ohno, Kazushige; Marunouchi, Tohru
title: Regulation of astrocyte proliferation by prostaglandin E(2) and the α subtype of protein kinase C
date: 1993-06-04
journal: Brain Res
DOI: 10.1016/0006-8993(93)90455-v
sha: d8172a2a6371134b3128daffebd064ef33cd7eb5
doc_id: 7508
cord_uid: tkoqx9l3

We found that astrocytes expressed the α subtype of protein kinase C. Treatment with12-O-tetradecanoylphorbol 13-acetate (TPA) caused cultured astrocytes to proliferate. This effect of TPA was blocked by staurosporine, a potent protein kinase C inhibitor, suggesting the involvement of protein kinase C in astrocyte proliferation. Indomethacin, an inhibitor of prostaglandin formation, enhanced both the normal and TPA-induced proliferation of astrocytes. Authentic prostaglandin E(2) blocked this effect of indomethacin and also partially blocked the effect of TPA, suggesting that the intracellular mechanisms involved in prostaglandin E(2)-regulated astrocyte growth might differ from those acting in protein kinase-dependent growth. The effect of prostaglandin E(2) was blocked by a specific anti-prostaglandin E(2) polyclonal antibody. Cultured astrocytes and microglia produced and released prostaglandin E(2) in response to stimulants such as lipopolysaccharide, TPA, and lymphokines. Since the sensitivity of astrocytes and microglia to these stimuli was different, prostaglandin E(2) may differentially regulate astrocyte proliferation under different physiological conditions, acting in an autocrine fashion for astrocytes and in a paracrine fashion for microglia.

Astrocytes are a type of glial cell and they provide structural support for neurons. There is also growing evidence that astrocytes have additional functions since they have been shown to synthesize and/or respond to a variety of growth factors 2° and cytokines, including interleukin 111, interleukin 6 7'43, granulocyte-macrophage colony stimulating factor 19'31, and tumor necrosis factor O~ 40 '43. It has also been demonstrated that certain factors control both the activity and proliferation of astrocytes via intracellular signaling mechanisms. Protein kinase C is one of the key enzymes involved in intracellular signaling and its activation has been implicated in a wide range of cellular processes 27'28. One of the most important roles of protein kinase C is modulation of the process of cell growth and division ~'27. Brain tissue has the highest protein kinase C content in the body ~5'23 and the density and distribution of phorbol ester binding sites in the fetal brain suggest a role for this enzyme in both developmental processes and cell growth 25. The existence of protein kinase C has also been demonstrated in primary astrocyte cultures 37 and it has been shown that mediators such as neurotransmitters or growth factors can stimulate protein kinase C activity 26. A phorbol ester was recently shown to induce the proliferation of cultured astrocytes 2. Therefore, protein kinase C seems to be involved in the promotion of astrocyte growth both in the developmental stage and under certain pathological conditions. Molecular cloning of the cDNA for protein kinase C has recently clarified the existence of multiple subtypes of this enzyme 5'16'18'3°'32'36, and there appear to be at least seven subtypes (a-~', including two /3 subspecies) 29. The mammalian brain has been shown to contain at least four subtypes, a, /3I, /311, and ,~33, by immunohistochemical analysis; they were localized in neurons 38 and in glial cells 1°.

Like the cells of the immune system, brain cells such as astrocytes 8 and microglia 47 have also been shown to produce prostaglandin E2, although its function in the central nervous system is as yet unknown. An attractive hypothesis is that growth factors or cytokines may activate astrocyte proliferation and subsequently induce prostaglandin E 2 production, which then downregulates astrocyte growth in an autoregulatory circuit. This hypothesis has been supported by a number of studies on lymphocytes, which have demonstrated that prostaglandin E 2 suppresses the activity or growth of both macrophages and T cells 9'12'13. However, the effects of prostaglandin E 2 on astrocyte proliferation have not yet been investigated directly.

In this study, we showed that a phorbol ester 12-0tetradecanoylphorbol 13-acetate (TPA) could induce astrocyte proliferation, most likely via activation of the a subtype of protein kinase C, and that prostaglandin E 2 inhibited both the normal and TPA-stimulated proliferation of cultured astrocytes.

Bovine insulin, indomethacin, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTr), and lipopolysaccharide (LPS) were obtained from Sigma (St. Louis, MO, USA). Prostaglandin E 2 and anti-prostaglandin E 2 antibody were obtained from Funakoshi (Tokyo, Japan). Staurosporine was purchased from Kyowa Medex (Tokyo, Japan). All other reagents used were of the highest purity commercially available. A crude lymphokine preparation was obtained by the method of Suzumura et al. 46 .

Astrocytes were prepared from primary mixed glial cell cultures of normal newborn ICR mouse and were purified by 3-4 cycles of trypsinization and replating, as described previously 4°. The purity of the astrocytes thus obtained was more than 95% as determined by indirect immunofluorescence using an anti-glial fibrillary acidic protein antibody 46. These astrocyte-enriched cultures did not contain neurofilament-positive neurons 44 and the contamination of microglia was negligible 45. Microglia were prepared as described previously 4°'42. The purity of microglia was more than 98% as determined by indirect immunofluorescence using an anti-MacI antibody.

Purified astrocytes were plated on 14 mm diameter glass coverslips at a density of 2.5 × 104 cells/ml. After 3 days of culture, they were immunolabeled with isozyme-specific anti-protein kinase C antibodies (directed against the a, /3 and 7 subtypes), using a commercially available protein kinase C staining kit (MBL, Nagoya, Japan).

Isolated astrocytes were seeded at a density of 2.5 × l04 cells per well in 24-well Falcon testplates. After 12 h of culture, they were stimulated with TPA, LPS, or a crude lymphokine preparation. For the inhibition test, a potent protein kinase inhibitor, staurosporine 48, was added to the medium in the presence of 100 ng/ml TPA. In addition, various concentrations of indomethacin (an inhibitor of prostaglandin production) were added to the medium in the presence or absence of 100 ng/ml TPA. Finally, prostaglandin E 2 with or without an anti-prostaglandin E 2 antibody was added to the medium in the presence or absence of TPA or indomethacin. Cul-69 tures were maintained in Eagle's MEM supplemented with 3.5 g/l glucose and 5 mg/l bovine insulin for 3 or 5 days at 37°C, and then assayed.

The proliferative activity of astrocytes was determined by a modification of the MTT colorimetric method 24. MTT is cleaved by living cells to yield a formazan product and this process requires active mitochondria. Thus, measurement of the formazan product by a colorimetric assay gives an indication of the number of surviving cells 4z. Cells in 24-well test plates were washed twice with phosphate-buffered saline (pH 7.2) and treated with 0.5 ml of a culture medium containing 0.5 mM MTT. After incubation at 37°C for 6 h, 0.04 M HCl-isopropanol was added, the amount of the formazan product in 0.2 ml of the culture fluid was measured at OD 620 nm with a J2000 Immuno Reader (Inter Med Japan Co., Tokyo, Japan).

Prostaglandin E e concentrations in the medium from cultured astrocytes and microglia were measured by radioimmunoassay with an anti-prostaglandin E 2 antibody (Amersham Japan, Tokyo, Japan).

Control and experimental values were compared using Student's t-test.

The purity of the cultured astrocytes was determined to be more than 95% by GFAP immunostaining with the anti-GFAP antibody (Fig. 1A) . These astrocytes contained the a subtype of protein kinase C (Fig.  1B ), but /3 and 7 subtypes were not detected in our experiment (data not shown).

The amount of formazan product detected at 620 nm showed a good correlation to astrocyte numbers ( Fig. 2) , so the colorimetric assay was validated for assessing astrocyte proliferation in the subsequent experiments.

When astroc~te cultures were treated with three stimulants (TPA, LPS and crude cytokine extract) that have been shown to activate these cells in different manners 39, only TPA was found to enhance astrocyte proliferation (Fig. 3A) . TPA enhanced astrocyte proliferation in a dose-dependent manner from a concentration of 0.1 to 100 ng/ml, but was toxic at higher concentrations (Fig. 313) . Since the cultures were not confluent, the astrocytes gradually increased in numbers, showing a 25% increase on day 3 and a 45% increase on day 5 (Fig. 3C) . This time-dependent increase was enhanced by TPA to about 40% on day 3 and 55% on day 5, when compared to the respective control (Fig. 3C) . The effect of TPA was inbibited by 10 nM staurosporine (Fig. 3C) .

Indomethacin (1 /xM) increased both the control and TPA-induced proliferation of astrocytes by 260% and 370%, respectively (Fig. 4) . This effect of indomethacin showed saturation at around 10 ~M. It also increased astrocyte proliferation in the presence of 10 nM staurosporine, which completely inhibited TPA-dependent growth; 10 /zM indomethacin increased astrocyte proliferation slightly but significantly (Fig. 4) .

Authentic prostaglandin E z inhibited both the indomethacin-induced and control proliferation of astrocytes (Fig. 5) , and it reduced the TPA-induced proliferative response of astrocytes to only 30% (Fig. 5) . These tion in the presence or absence of indomethacin to 112% and 140% of the respective prostaglandin E 2treated levels.

Astrocytes produced about 200 pg of prostaglandin E 2 per 2 × 105 cells under control culture conditions, an amount that was four times the production by microglia under the same conditions (Fig. 6 ). LPS and TPA increased prostaglandin E 2 production about 1.7-fold and 4.5-fold, respectively, when compared to the control astrocyte cultures. LPS and TPA also increased microglial prostaglandin E 2 production by about 40-fold and 5-fold, respectively. Thus, astrocytes were more sensitive to TPA than to LPS, while microglia were the opposite.

There are two major aspects to consider with regard to astrocyte proliferation, one being its contribution to normal brain ontogeny 17 and the other being its pathological role in causing gliosis in the mature brain 17 ' 22. In the latter situation, quiescent astrocytes re-enter the cell cycle like somatic cells such as skin fibroblasts, gut epithelial cells, and hepatocytes 34'35. Despite the physiological and pathological importance of this cell, relatively little is known about the intracellular and intercellular mechanisms regulating astrocyte proliferation.

We demonstrate here that TPA stimulated the proliferation of cultured astrocytes, and that this effect was blocked by staurosporine, a potent protein kinase C inhibitor 48 (Fig. 2) . Staurosporine also inhibited unstimulated astrocyte proliferation (Fig. 2) . These observations suggest that protein kinase C may be involved in astrocyte proliferation under both unstimulated and TPA-stimulated conditions. This hypothesis was supported by the result that astrocytes expressed protein kinase C (Fig. 1B) . Glial cells are reported to be rich in immunoreactive protein kinase C t° and a [3H]phorbol ester has been found to bind to glial ceils at a high level 4. In general, when cells are stimulated with TPA, protein kinase C activity is translocated from the cytosol to the membrane 2 or to the nucleus 27. Translocation of its activity to the nucleus seems to be necessary for DNA synthesis, suggesting that protein kinase C may be a key enzyme involved in cell proliferation. A similar mechanism may be involved in the astrocyte proliferation.

Indomethacin, a prostaglandin synthesis inhibitor, enhanced both unstimulated and TPA-stimulated astrocyte proliferation (Fig. 4) , suggesting that astrocytederived prostaglandins reduced its own proliferation in both unstimulated and TPA-stimulated conditions. To identify what type of prostaglandins inhibit astrocyte proliferation, we added the authentic prostaglandins to astrocyte cultures. An excess of prostaglandin E 2 reduced both unstimulated and stimulated proliferation of astrocytes (Fig. 5) . This inhibitory effect was blocked by an antibody to prostaglandin E 2 (Fig. 5) . These findings indicate that this prostaglandin apparently functions as an inhibitory regulator of astrocyte proliferation.

We also demonstrated that prostaglandin E 2 partially blocked the effect of TPA on astrocytes (Fig. 5) , suggesting that different intracellular signaling mecha-nisms are operative for these two agents. This hypothesis was supported by the following two lines of evidence: (1) the effects of indomethacin and TPA were additive, even in the presence of such a high concentration of indomethacin that its effect was almost saturated (Fig. 4) , and (2) indomethacin increased the proliferation of astrocytes even in the presence of staurosporine (Fig. 4) . The details of the relationship between the TPA-protein kinase C system and prostaglandin E 2 are not yet clear, however, with respect to the effect on astrocyte growth.

Astrocytes and microglia produced prostaglandin E 2 in response to several stimuli (Fig. 6) , a finding which was consistent with previous reports 8't4. The stimuli used in this experiment were designed to represent different physiological and pathological conditions 41. Thus, the addition of LPS, TPA, and the crude lymphokine preparation, respectively, represented the effect of bacterial antigens on the brain, the effect of protein kinase C activation by growth factors 42 and the effect of peripheral immune activation on the brain. Prostaglandin production by astrocytes and microglia differed in response to these stimuli (Fig. 6) , suggesting that this prostaglandin may regulate astrocyte proliferation in a differential manner. That is, in astrocytes it may act in an autocrine fashion and be sensitive to phorbol ester stimulation, while in microglia it appears to act in a paracrine fashion and be sensitive to LPS stimulation.

Prostaglandin E has inflammatory and immunomodulatory properties. Since astrocytes and microglia have been suggested to act as immunoregulatory cells in the central nervous system 6'49, the release of this prostaglandin from these cells may be a key element in the central nervous system immune response. An increased central nervous system level of prostaglandin E 2 has been reported in multiple sclerosis 21 and in experimental allergic encephalomyelitis 3. The production of prostaglandin E 2 by astrocytes in such conditions may have some role in their pathogenesis, since our study showed that this prostaglandin was not only involved in immunomodulation but also in regulating astrocyte growth.

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