Glial-released proteins in clonal cultures and their modulation by hydrocortisone.

Rat glial C6 cells release into the culture medium a reproducible spectrum of soluble proteins of 12 major peaks over a broad molecular weight range as determined by fractionation on SDS-gel electrophoresis. Exposing C6 monolayers to hydrocortisone (HC) results in a selective alteration in the pattern of glial-released protein (GRP). The selective HC-induced increase or decrease in GRP peaks is specific to HC in that 17 beta-estradiol, dibutyryl cyclic AMP, isoproterenol and melatonin exert either no detectable or a qualitatively different influence on the GRP pattern. The HC influence is dose dependent over a physiological range of concentrations from 10(-9) to 10(-6) M. Differences in culture age and in subclones of C6 can influence both the normal and the HC-induced pattern of GRP. The origin of th GRP is unknown, but pattern reproducibility, viability tests, surface labeling studies and metabolic labeling studies of soluble and particulate compartment proteins and glycoproteins support the position that cell lysis is not an important source of GRP. More importantly, these studies indicate that GRP and HC-induced changes in GRP pattern are physiologically significant aspects of glial cell behavior.

Glial-released proteins: II. Two-dimensional electrophoretic identification of proteins regulated by hydrocortisone.

The proteins released into the culture medium (CM) by confluent C6 glioma cell monolayers were analyzed by two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (2-D SDS-PAGE). These cells synthesize and release a complex set of proteins which can be resolved on the basis of molecular charge and size. Over 400 spots on fluorograms corresponding to radio-labeled glial-released protein (GRP) were detected and classified according to their positions in 2-D gels. The marked species charge heterogeneity of the 2-D pattern was used as a criterion to assign the majority of GRP components to "series'. Series are composed of families of related proteins or glycoproteins distributed in a line of evenly spaced members in the isoelectric focusing dimension. Long-term exposure of monolayers to 2 microM hydrocortisone influenced the accumulation in the culture medium of half of the classified GRP species. Five classes of GRP were identified based on their steroid-responsiveness as seen in the deviation of GRP radiolabel ratios from control and hormone-treated culture CM. Hormonal response, verified by reverse-label experiments, showed a number of GRP in CM are either consistently increased 1- to 7- fold (Classes I and II) or decreased 1- to 3-fold (Classes IV and V). The remaining GRP (Class III) included those proteins which were found to be uninfluenced or to change in a week or inconsistent manner. Some GRP series were coordinately induced while other series gave graded responses. These results represent the first high-resolution classification of GRP by physical and biological properties.

Glial-released proteins: III. Influence on neuronal morphological differentiation.

In vitro model systems in neurobiology are available to detect and help characterize various intercellular development signals. The presence of such active substances in conditioned medium (CM) from rat C6 glioma cells (2BD clone) was examined using the pheochromocytoma-derived clonal cell lines PC12 and PCG2. Conditioned medium from C6 monolayers induces neurite outgrowth in PC12 cells and extensive alteration in PCG2 cell morphology by 24 h. This ability of CM was found (1) to remain after dialysis and lyophilization, (2) to be modulated by steroid treatment of C6 monolayers (PC12 cells only) and (3) to be distinct from the influence of NGF. Combinations of CM, nerve growth factor (NGF) and dibutyryl cyclic AMP when given together in pairs to PC12 or PCG2 cells revealed facilitation of neurite formation and cell morphology, respectively. These results suggest possible synergistic interaction between the three factors in the neuroglial regulation of neuronal morphological differentiation.

Induction of c-fos and TIS genes in cultured rat astrocytes by neurotransmitters.

The interaction of neurotransmitters with their specific receptors initiates a cascade of intracellular biochemical events which lead to induction of specific genes. Included in this cascade is the rapid and transient induction of a family of primary early response genes we term TIS genes (Lim et al.: Oncogene 1: 263-270, 1987). Expression of six TIS gene, including c-fos, was examined in secondary cultures of rat neocortical astrocytes exposed to muscarinic and adrenergic agonists and antagonists to study the early genomic responses which accompany neurotransmitter-induced alteration of glial morphology and physiology. Carbachol induced accumulation of mRNA for c-fos and the other TIS genes. Carbachol-mediated induction of TIS mRNA expression was sensitive to atropine blockade and was potentiated by lithium. Norepinephrine (NE), isoproterenol, or phenylephrine also induced TIS mRNA accumulation. In order to determine which second-messenger pathways mediate NE induction of TIS gene expression, the influences of the beta(B) antagonist propranolol (PR), the alpha I(AI) antagonist prazosin (PZ), and the alpha 2(A2) antagonist yohimbine (YB) were examined. The induction of TIS1 mRNA by NE was partially blocked by PR or PZ alone, and completely abolished by both antagonists in combination. YB had no effect on TIS1 mRNA expression. These results suggest that NE induces TIS1 mRNA through both B- and A 1-adrenergic, but not A2, pathways. The lack of effect of inhibitors of phospholipase A2 and cyclooxygenase suggests that the A1 component is mediated through a protein kinase C pathway. The induction of transient gene expression by neurotransmitters may mediate the secondary genomic responses and phenotypic changes occurring in astrocytes in response to alterations in neuronal neurotransmitter release.

TIS gene expression in cultured rat astrocytes: induction by mitogens and stellation agents.

The expression of a number of TIS genes (Lim et al.: Oncogene 1:263-270, 1987) was examined in secondary cultures of rat neocortical astrocytes treated with mitogens and stellation agents, to study the early nuclear events which accompany the induction of glial proliferation and/or differentiation. Tetradecanoyl phorbol acetate (TPA), epidermal growth factor, and fibroblast growth factor, three mitogens for astrocytes, stimulated marked, rapid, and transient increases in TIS gene mRNAS. TIS10, which is not expressed in rat PC12 pheochromocytoma cells, could be induced by these mitogens in rat astrocytes. Dibutyryl cyclic adenosine monophosphate and forskolin, which induce rapid stellation in astrocytes, and ganglioside GM1, a potent mitogen as well as an antagonist of the induction and maintenance of stellation, all induced TIS gene expression. Thus, a broad range of agents which elicit both proliferative and differentiation responses in astrocytes are capable of inducing a family of genes that may play a role in the early events of signal transduction.

TIS gene expression in cultured rat astrocytes: multiple pathways of induction by mitogens.

Accumulation of TIS1 and TIS11 (Lim et al.: Oncogene 1:263-270, 1987) mRNAs in secondary cultures of rat neocortical astrocytes was much greater in response to tetradecanoyl phorbol acetate (TPA) than in response to either epidermal growth factor (EGF) or fibroblast growth factor (FGF). In contrast, EGF, FGF, and TPA were equally effective in inducing accumulation of TIS8 and TIS28/c-fos mRNAs. These data suggested that TPA and the polypeptide mitogens might induce TIS gene expression by distinct pathways. When maximally inducing concentrations of EGF and FGF were co-administered to astrocyte cultures, TIS mRNA accumulations were no greater than those observed for the individual growth factors, suggesting that EGF and FGF saturate a common, limiting step in their induction pathways. In contrast, when either EGF or FGF was presented to astrocytes in combination with maximally inducing levels of TPA, the resulting levels of accumulation of TIS mRNAs were at least as great as the sum of the levels induced by the individual mitogens. Stimulation of [3H]-thymidine incorporation demonstrated an identical pattern of interaction; EGF and FGF co-administration was no more effective than either polypeptide mitogen alone, but, when presented to astrocyte cultures along with maximally inducing concentrations of TPA, either EGF or FGF was able to increase incorporation of [3H]-thymidine. Superinduction of all the TIS genes occurred if cycloheximide (CHX) was present during TPA exposure. Once again, two distinct classes of responses of the various TIS genes occurred; superinduction of TIS1, TIS7, TIS11, and TIS28/c-fos mRNA accumulation ranged from 10- to 20-fold, while CHX superinduction of TIS8 and TIS10 was far more modest, ranging from 2- to 3-fold.(ABSTRACT TRUNCATED AT 250 WORDS)