D van Agoston | PubMed Health

PubMed1994

Subnanomolar concentration of VIP induces the nuclear translocation of protein kinase C in neonatal rat cortical astrocytes.

At subnanomolar concentrations, vasoactive intestinal peptide (VIP) can act as an astroglial mitogen and as a secretagogue for neurotrophic substances released from glia (Brenneman et al.: J Neurosci Res 25:386-394, 1990). Here we report that treatment with subnanomolar (0.1 nM) VIP, that does not produce an increase in intracellular cAMP levels, induced the translocation of protein kinase C (PKC) from the cytoplasm to the nucleus in neonatal cortical astrocytes, as revealed by immunohistochemistry, Western blot analysis, and measurements of the enzyme activity. Western blot analysis of subcellular fractions, using PKC isotype-specific antisera, showed PKC alpha as well as the two novel PKC isotypes, delta and zeta immunoreactivities, whereas PKC beta or gamma immunoreactivities were not detected. PKC alpha was associated predominantly with the cytosolic compartment, while PKC delta was found in the plasma membrane and in nuclear fractions. In contrast, PKC zeta was distributed ubiquitously within the major subcellular fractions. Treatment of the cells with 0.1 nM VIP caused a marked increase in nuclear PKC alpha and, to a lesser extent, PKC delta and PKC zeta immunoreactivities. Western blot analysis showed that a low (1 nM) concentration of phorbol, 12-myristate, 13 acetate also caused the subcellular redistribution of PKC immunoreactivities from the cytoplasm to the nuclear fraction, similar to VIP treatment. Exposure of astrocytes to high concentrations (1 microM) of phorbol, 12-myristate, 13 acetate resulted in the down-regulation of PKC alpha and PKC delta, while distribution of PKC zeta immunoreactivities were only slightly altered.(ABSTRACT TRUNCATED AT 250 WORDS)

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Protein-DNA interactions during phenotypic differentiation.

We have been studying the molecular mechanism of neuronal differentiation through which the multipotent precursor becomes limited to the final transmitter phenotype. Here we focused on the role of the 5' proximal regulatory cassette (-190; +53 bp) of the rat enkephalin (rENK) gene in the developmental regulation of the enkephalin phenotype. Several well characterized cis-elements, including AP2, CREB, NF1, and NFkB, reside on this region of the rENK gene. These motifs were sufficient to confer activity-dependent expression of the gene during neurodifferentiation when it was tested using transient transfection assays of primary developing spinal cord neurons treated with tetrodotoxin (TTX). This region was then used as a DNA probe in mobility shift assays, with nuclear proteins derived from phenotypically and ontogenetically distinct brain regions. Only a few low abundance protein-DNA complexes were detected and only with nuclear proteins derived from developing but not from adult brain. The spatiotemporal pattern of these complexes did not show correlation with enkephalin expression which was assessed by RT-PCR. We employed synthetic probes corresponding to consensus as well as ENK-specific sequences of the individual motifs to identify the nature of the observed bands. Although both consensus NF1 and enkCRE1(NF1) formed complexes with nuclear proteins derived from the striatum and cortex at various ages, the appearance of the bands was not correlated with ENK expression. Surprisingly, no complexes were detected if other ENK-specific motifs were used as probes. We also tested nuclear extracts derived from forskolin-induced and control C6 glioma cells, again using the whole proximal regulatory cassette as well as individual motifs. These experiments showed the formation of elaborate protein-DNA bands. There was no direct correlation between the appearance of bands and forskolin-induced ENK expression. Unexpectedly, all ENK-specific motifs formed specific and highly abundant protein-DNA complexes when nuclear extracts from the human tumor cell line (HeLa), which does not express ENK, were used. Based on these observations, we concluded that: 1. Interactions between the proximal regulatory cassette and additional probably far distant regions of the rENK gene and their binding proteins may be necessary to confer developmentally regulated, cell-specific expression of the ENK gene; and 2. Inducibility of the gene by common cis-elements can be governed by this region; however, the cell-specificity of the induction remains elusive.

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