Induction of LPL gene expression by sterols is mediated by a sterol regulatory element and is independent of the presence of multiple E boxes.

Overexpression of the adipocyte differentiation and determination factor-1 (ADD-1) or sterol regulatory element binding protein-1 (SREBP-1) induces the expression of numerous genes involved in lipid metabolism, including lipoprotein lipase (LPL). Therefore, we investigated whether LPL gene expression is controlled by changes in cellular cholesterol concentration and determined the molecular pathways involved. Cholesterol depletion of culture medium resulted in a significant induction of LPL mRNA in the 3T3-L1 preadipocyte cell line, whereas addition of cholesterol reduced LPL mRNA expression to basal levels. Similar to the expression of the endogenous LPL gene, the activity of the human LPL gene promoter was enhanced by cholesterol depletion in transient transfection assays, whereas addition of cholesterol caused a reversal of its induction. The effect of cholesterol depletion upon the human LPL gene promoter was mimicked by cotransfection of expression constructs encoding the nuclear form of SREBP-1a, -1c (also called ADD-1) and SREBP-2. Bioinformatic analysis demonstrated the presence of 3 potential sterol regulatory elements (SRE) and 3 ADD-1 binding sequences (ABS), also known as E-box motifs. Using a combination of in vitro protein-DNA binding assays and transient transfection assays of reporter constructs containing mutations in each individual site, a sequence element, termed LPL-SRE2 (SRE2), was shown to be the principal site conferring sterol responsiveness upon the LPL promoter. These data furthermore underscore the importance of SRE sites relative to E-boxes in the regulation of LPL gene expression by sterols and demonstrate that sterols contribute to the control of triglyceride metabolism via binding of SREBP to the LPL regulatory sequences.

Induction of c-fos, jun B and egr-1 expression by haloperidol in PC12 cells: involvement of calcium.

Acute injection of haloperidol, a dopamine D2 receptor antagonist, is known to increase immediate early gene expression of the fos and jun families in rodent striatal neurons. A set of gene induction, including c-fos, jun B and TIS8/egr-1, was found when haloperidol was added to PC12 cells in culture. Electrophoretic mobility-shift assays show that haloperidol-evoked gene induction was accompanied by a transient and dose-dependent increase in AP1 and EGR-1 binding activities in these cells. Gene expression is tentatively explained by the rapid and transient increase in cytosolic free Ca2+ concentration observed upon haloperidol addition. The cytosolic calcium rise and AP1 binding activation elicited by haloperidol were dependent on extracellular Ca2+, suggesting that haloperidol exerted its effects by promoting Ca2+ entry into PC12 cells. The haloperidol-induced increase in AP1 binding activity and intracellular Ca2+ was not reproduced by two other dopamine D2 receptor antagonists, sulpiride and (+)-butaclamol.

Okadaic acid induces activator protein 1 activity and immediate early gene transcription in rat pheochromocytoma cells. Mechanism of action.

The serine/threonine protein phosphatase inhibitor okadaic acid (OA) was found to enhance mRNA transcripts of c-fos and of the jun family of proto-oncogenes including c-jun, jun B and jun D in cultured pheochromocytoma PC12 cells. This expression remained elevated for more than 8 hr. An increase in the binding of the transcription factor activator protein 1 (AP1) to its DNA consensus sequence that occurred prior to early gene transcription was observed. Enhanced AP1 activity was still observed when OA was added to the cells together with the transcription inhibitor actinomycin D, or with the protein synthesis inhibitor cycloheximide, indicating that it is actually AP1 activation due to posttranslational modifications that triggers transcription of the fos and jun genes. AP1 was activated through serine/threonine phosphorylation since its activation was abolished when nuclear extracts of OA-treated cells were incubated with protein phosphatase-1 or, to a lesser extent, with protein phosphatase-2A. C-Jun and Jun D proteins are likely candidates for being phosphorylated, since they were shown to constitute the AP1 complex at the time when it was activated (2 hr after OA addition).

Inhibition of serine/threonine protein phosphatases promotes opening of voltage-activated L-type Ca2+ channels in insulin-secreting cells.

The biological activity of many proteins, including voltage-sensitive ion channels, is controlled by their state of phosphorylation. Ca2+ influx through voltage-activated L-type Ca2+ channels serves as the major stimulatory signal in insulin-secreting cells. We have now investigated the extent to which Ca2+ handling in clonal insulin-secreting RiNm5F cells was affected by okadaic acid, an inhibitor of various serine/threonine protein phosphatases. Whole-cell patch-clamp experiments showed that okadaic acid generated an increase in membrane current, suggesting that it promotes Ca2+ influx through L-type voltage-gated Ca2+ channels probably by modifying their phosphorylation state. Okadaic acid was found to provoke a transient rise in the cytoplasmic free Ca2+ concentration ([Ca2+]i) but had no further effect on the K(+)-induced increase. The Ca2+ transient induced by okadaic acid was dependent on the presence of extracellular Ca2+ and was abolished by D600, a blocker of voltage-activated L-type Ca2+ channels. Concomitant with the rise in [Ca2+]i, okadaic acid induced insulin secretion, a phenomenon that was also dependent on extracellular Ca2+. It is proposed that hyperphosphorylation of voltage-activated L-type Ca2+ channels in insulin-secreting cells lowers the threshold potential for their activation.

Stimulation of the cyclic GMP pathway by NO induces expression of the immediate early genes c-fos and junB in PC12 cells.

Stimulation of several second messenger pathways induces the expression of immediate early genes such as c-fos, c-jun, junB, and junD, but little is known about their induction via the stimulation of the cyclic GMP pathway. Here we looked at the expression of early genes in pheochromocytoma PC12 cells after activation of cytosolic guanylate cyclase by sodium nitroprusside. This compound spontaneously releases NO, a molecule known to be involved in cell communication. We found that expression of c-fos and junB but not of c-jun or junD is increased upon activation of cyclic GMP pathway. c-fos mRNA expression was the most activated (fourfold at 30 min), whereas junB response was more modest (2.2-fold activation at 60 min). Nuclear extracts of stimulated cells show increased binding capacity to the AP1 binding site consistent with the dose-response curve. The activating effect of nitroprusside could be reproduced by dipyridamole, a selective cyclic GMP phosphodiesterase inhibitor and by 8-p-chlorophenylthio-cyclic GMP, a permeant selective cyclic GMP-dependent protein kinase activator, and abolished by KT5823, an inhibitor of that kinase. The results show that NO promotes early gene activation and AP1 binding enhancement through the stimulation of the cyclic GMP pathway.

Chronic administration of NMDA antagonists induces D2 receptor synthesis in rat striatum.

Dopamine D2 receptor gene expression was examined in rat striatum after chronic treatment with N-methyl-D-aspartate (NMDA) receptor antagonists (ketamine at 15 mg/kg/day or MK-801 at 0.1, 0.2 and 0.4 mg/kg/day per os, for 50 days). The long-isoform mRNA, as well as the total D2 mRNA expression were induced. No change was noticed in striatal dopamine release or turnover. D2 binding studies carried out in MK-801 chronically treated (0.3 mg/kg/day per os, for 50 days) and control rats revealed an increased receptor density in treated animals without a significant change in receptor affinity. These results suggest that the synthesis of both striatal D2 receptor isoforms is postsynaptically regulated at the transcriptional level, by events triggered by glutamate through the NMDA-type receptor.

Induction of D2 dopamine receptor mRNA synthesis in a 6-hydroxydopamine parkinsonian rat model.

By injecting 6-hydroxydopamine into the pars compacta of the substantia nigra, we produced a hemiparkinsonian rat model in which there is almost complete destruction of the dopaminergic nigrostriatal pathway but sparing of the dopaminergic mesolimbic system. The lesion has been characterized by several criteria: a rotational behavior in response to apomorphine, a complete loss of tyrosine hydroxylase immunoreactivity in the lesioned substantia nigra, a near total depletion of dopamine and metabolites in the striatum ipsilateral to the lesion, and a supersensitivity of the dopamine D2 receptors in the ipsilateral striatum. Dopaminergic striatal deafferentation was accompanied by an increase of D2 receptor mRNA synthesis in the striatum ipsilateral to the lesion, suggesting that the increased D2 receptor density observed after the lesion is due to an increase of the synthesis of receptor molecules. This synthesis appears to be regulated at the transcriptional level.