Expression and regulation of the LIM-class homeobox gene rlim-1 in neuronal progenitors of the rat cerebellum.

To investigate LIM gene function in the rat cerebellar system, we analyzed expression and regulation of the rat homologue of frog Xlim-1 (rlim-1) in vivo and in cultured cells. In developing cerebellum, peak levels of rlim-1 mRNA at postnatal day 8 (p8) are coincident with the peak period of granule cell proliferation. Analysis of rlim-1 protein with a specific antibody showed that expression was also maximal at p8. In situ hybridization showed that at p8 rlim-1 mRNA was expressed in Purkinje and granule cells. Both the proliferative and the premigratory granule cells in the external germinal zone displayed high levels of rlim-1 mRNA expression. Immunocytochemical staining demonstrated that at p8 rlim-1 protein was also present in proliferative and premigratory granule cells. In adult cerebellum (p30), rlim-1 mRNA and protein expression in granule cells was strongly attenuated. The down-regulation of rlim-1 mRNA occurred in granule cells just after the time of final division, coinciding with the onset of their migration. rlim-1 protein was detected in migratory granule neurons. The developmental decrease in rlim-1 mRNA and protein found in vivo was reproduced in pure cerebellar granule cell cultures. In these cultures, granule neurons were postmitotic 1 day after plating but still displayed high levels of rlim-1 protein expression up to 3 days in vitro. Our findings indicate that 1) rlim-1 is likely to act in concert with other genes to specify granule cell fate, 2) rlim-1 expression in granule neurons is regulated autonomously, and 3) rlim-1 protein may also play an important role in granule neuron differentiation and survival. Published 2001 Wiley-Liss, Inc.

Tyrosine phosphorylation of Vav stimulates IL-6 production in mast cells by a Rac/c-Jun N-terminal kinase-dependent pathway.

This study investigates whether the guanine nucleotide exchange activity of Vav is linked to cytokine production in mast cells. Overexpression of Vav in the RBL-2H3 mast cell line resulted in the constitutive tyrosine phosphorylation and activation of Vav. We analyzed the functional effect of Vav overexpression on cytokine production. IL-2 and IL-6 mRNA levels were dramatically increased in Vav-overexpressing cells and correlated with increased NF-AT activity. Little or no effect was observed on the mRNA levels of IL-3, IL-4, GM-CSF, TNF-alpha, and TGF-beta. FcepsilonRI engagement did not further enhance IL-2 and IL-6 mRNA levels and only slightly enhanced NF-AT activity, but dramatically increased the mRNA levels of other tested cytokines. To understand the signal transduction required, we focused primarily on IL-6 induction by measuring mitogen-activated protein kinase activity and analyzing the effects of mutant or dominant negative forms of Vav, Rac1, and c-Jun N-terminal kinase-1 (JNK1). Vav overexpression resulted in the constitutive activation of JNK1 with little or no effect on p38 mitogen-activated protein kinase and ERK2. This was dependent on Vav-mediated activation of Rac1 as a Dbl domain-mutated Vav, inactive Rac N17, and inactive JNK1 down-regulated the Vav-induced JNK1 or IL-6 responses. Vav expression, but not expression of domain-mutated Vav, increased IL-6 secretion from nonimmortalized bone marrow-derived mast cells upon FcepsilonRI engagement. We conclude that Vav phosphorylation contributes to IL-6 induction in mast cells.

Frog prohormone convertase PC2 mRNA has a mammalian-like expression pattern in the central nervous system and is colocalized with a subset of thyrotropin-releasing hormone-expressing neurons.

The prohormone convertase (PC2) is expressed in the mammalian central nervous system (CNS) and has been shown to play an important role in the processing of certain neuropeptide precursors and prohormones at paired basic residues. Amphibian PC2 cDNA was recently cloned for the frog Xenopus laevis, and both its sequence and its pituitary expression pattern were shown to be very similar to those of mammalian PC2. To investigate further the function of PC2 in the vertebrate CNS, we used in situ hybridization histochemistry to localize the distribution of cells expressing PC2 mRNA in the frog brain and the spinal cord. The distribution of PC2-expressing cells was also compared with that of cells expressing thyrotropin-releasing hormone (TRH) mRNA or peptide. PC2-expressing cells were detected in specific nuclei that were widely distributed in the frog CNS. In forebrain, telencephalic PC2 mRNA was found in the olfactory bulb, pallium, striatum, amygdala, and septum, and diencephalic PC2 mRNA was seen in the preoptic area, thalamus, and hypothalamus. More posteriorly, PC2 cells were localized to midbrain tegmentum, the torus semicircularis, and the optic tectum, as well as the cerebellum, brainstem, and spinal cord. Despite this wide distribution steady-state levels of PC2 mRNA were clearly different in various brain nuclei. Regions with higher levels showed good correspondence to areas shown by others in frog to contain large numbers of neuropeptide-expressing cells, including TRH cells. On the other hand, not all brain areas with high levels of TRH mRNA had high levels of PC2 mRNA. Localization studies combining in situ hybridization and immunocytochemistry showed that, at least in optic tectum and brainstem, PC2 mRNA and pro-TRH peptide coexist. These findings suggest that pro-TRH is processed by PC2 in some, but possibly not all, brain regions. Thus, different converting enzymes may be involved in pro-TRH processing in different brain regions.

Cloning and functional characterization of the rat glutamine synthetase gene.

Glutamine synthetase catalyzes the formation of glutamine from glutamate and ammonia. It plays a central role in both amino acid neurotransmitter metabolism and ammonia detoxification in the central nervous system. Glutamine synthetase expression is regulated in developmental, hormonal, and in tissue- and cell-specific manners. We have cloned a full-length cDNA coding for rat glutamine synthetase, and have found an AT-rich area of conservation in the 3' untranslated regions between rat, mouse, and chicken, which may play a part in the regulation of the stability of the glutamine synthetase message. We have also cloned and mapped the gene coding for rat glutamine synthetase, and identified, by sequence analysis, areas potentially important for the regulation of glutamine synthetase transcription. Transient transfection of a variety of cell lines with deletion constructs of the glutamine synthetase promoter driving a chloramphenicol acetyltransferase reporter gene functionally demonstrates regions of the promoter containing elements important for transcriptional regulation.

Neuron-glial interactions involved in the regulation of glutamine synthetase.

Cocultures of rat cortical astrocytes with cerebellar granule cell neurons, but not a variety of other cell types tested, resulted in an induction of glutamine synthetase (GS) mRNA over the basal levels expressed in pure astrocyte cultures. This induction involved both contact- and noncontact-mediated events and may be a result of astroglial differentiation promoted by interactions with the primary neurons. Astrocytes grown in the presence of the granule neurons (but not the other cell types tested) exhibited a more complex, process-bearing morphology typical of more differentiated cells. In addition, glial cell proliferation was inhibited not only by the presence of live granule cells, but also by fixed neurons and neuronal membranes. Under the same experimental conditions, GS mRNA was increased (two- to threefold) compared with the expression observed in pure astrocyte cultures. Because of the role of GS in glutamate metabolism and the influence of the glutamatergic granule neurons on glial GS mRNA levels, the effect of exogenous glutamate was examined. The addition of 100 microM glutamate to the culture medium resulted in an increase in GS mRNA in the astrocyte cultures similar to that observed in the cocultures, where the addition of glutamate did not further increase GS mRNA levels. These results provide further evidence for the importance of neuron-glial interactions in the regulation of glial gene expression.

The ontogeny and localization of glutamine synthetase gene expression in rat brain.

A 2.4 kb cDNA clone containing the coding sequence for glutamine synthetase (GS) was isolated from a rat brain cDNA library, and a probe constructed from this cDNA was utilized in Northern analysis of total RNA to study the tissue distribution and the ontogeny of GS mRNA expression in the rat brain from embryonic day 14 (E14) to adulthood. The levels of GS mRNA were highest in the brain, followed by kidney and liver. In the brain, the GS message was detected as early as E14, earlier than it can be detected by either enzymatic assays of GS activity or by immunocytochemical localization of GS. The relatively low levels of GS mRNA seen at E14 increase to a peak around the time of birth, and in the second postnatal week another rise in GS message occurs approaching adult levels by P15. Localization of GS to astrocytes in the brain was confirmed using both immunocytochemistry and in situ hybridization.

Stabilization of tubulin mRNAs by insulin and insulin-like growth factor I during neurite formation.

Neurotrophic factors may increase axon and dendrite growth in part by regulating the content of cytoskeletal elements such as microtubules, which are comprised of tubulin subunits. The mechanism by which insulin, insulin-like growth factors (IGFs), and nerve growth factor (NGF) can increase the relative abundance of tubulin mRNAs as a prelude to neurite formation was studied. Insulin significantly increased the abundance of tubulin mRNAs relative to total RNA in cultured human neuroblastoma SH-SY5Y cells. This increase was not the result of a generalized elevation of all transcripts, because tubulin mRNAs were elevated relative to poly(A)+ RNA as well. Moreover, whereas polymerases I and III were elevated in activity, polymerase II was not. Tubulin mRNAs were stabilized against degradation in the presence of actinomycin D by both insulin and IGF-I. In contrast, actin and histone 3.3 mRNAs were neither increased nor stabilized. Insulin did not alter alpha- or beta-tubulin gene transcription rates in nuclear run-off experiments, and did increase the relative synthesis of tubulin proteins. These results suggest that tubulin mRNA levels are increased mainly through selective stabilization by insulin and IGFs. Because NGF is known to stabilize tubulin mRNA levels also, stabilization of tubulin mRNAs is suggested to be a common event in the pathway leading to neurite elongation directed by neuritogenic polypeptides.

Insulin, insulin-like growth factor II, and nerve growth factor effects on tubulin mRNA levels and neurite formation.

We have previously shown that insulin and the insulin-like growth factors share some important neurotrophic properties with nerve growth factor (NGF), including the capacity to enhance neurite formation. In this study, we have examined the effects of these neuritogenic agents on the expression of genes coding for important cytoskeletal proteins of axons and dendrites. Insulin specifically and coordinately increased the levels of alpha- and beta-tubulin mRNAs in human neuroblastoma SH-SY5Y cells. The dose-response curves for these increases were very similar to that for enhancement of neurite formation. Tubulin transcripts reached a transient maximum in approximately 1 day, suggesting that higher levels are important during initiation of neurites and that high levels are not required to sustain neurites once formed. Insulin-like growth factor II shared with insulin the capacity to substantially increase tubulin mRNA levels. NGF had but a small effect. Complementary mechanisms for these neurotrophic agents are suggested, because other studies show NGF and insulin can synergistically potentiate neurite formation. None of the factors altered the levels of actin mRNA. Thus, neurite formation does not seem to require a coordinate increase in actin and tubulin transcripts in SH-SY5Y cells.

Neurite formation modulated by nerve growth factor, insulin, and tumor promoter receptors.

Until recently, nerve growth factor could be considered the only neurotrophic factor with an established physiological role. We discuss the emerging evidence indicating that the insulinlike factors may constitute a family of related neurotrophic proteins, and the observations suggesting that the receptor for the phorbol ester tumor promoters is closely associated with neuronal differentiation. The emphasis of the discussion is placed on neurite formation under multiple modulation by insulinlike factors, nerve growth factor, and tumor promoter receptors in sensory, sympathetic and human neuroblastoma cells.