Subunit Gene Transcription Research Articles

Regulation of Na,K-ATPase β1 Subunit Gene Transcription by Low External Potassium in Cardiac Myocytes: ROLE OF Sp1 AND Sp3

Expression of Na,K-ATPase activity is up-regulated in cells incubated for extended intervals in the presence of low external K(+). Our previous data showed that exposure of cardiac myocytes to low K(+) increased the steady-state abundance of Na,K-ATPase beta1 subunit mRNA. In the present study we determined that incubation of primary cultures of neonatal rat cardiac myocytes with low K(+) augmented Na,K-ATPase beta1 gene expression at a transcriptional level and that this effect required extracellular Ca(2+). The stimulatory effect of low K(+) on Na,K-ATPase beta1 gene transcription was not dependent on increased contractile activity of cardiac myocytes. Na,K-ATPase beta1 5'-flanking region deletion plasmids used in transient transfection analysis demonstrated that the region between nucleotides -62 to -42 of the beta1 promoter contained a low K(+) response element. Site-directed mutagenesis of a potential GC box core motif GCG in the -58/-56 region of the beta1 promoter decreased basal and low K(+)-mediated transcription. Mutation of the core sequence of a putative GC box element located between nucleotides -101 and -99 further decreased the low K(+) effect on beta1 gene transcription. Electrophoretic mobility shift assays using oligonucleotides spanning the proximal and distal GC box elements of the beta1 promoter showed enhanced binding of two complexes in response to low K(+). The inclusion of a consensus GC box sequence as a competitor in gel shift analysis reduced factor binding to the low K(+) response elements. Antibodies to transcription factors Sp1 and Sp3 interacted with components of both DNA-binding complexes and binding of nuclear factors was abolished in gel shift studies using GC box mutants. Together these data indicate that enhanced binding of Sp1 and Sp3 to two GC box elements in the rat Na,K-ATPase beta1 subunit gene promoter mediates beta1 gene transcription up-regulation in neonatal rat cardiac myocytes exposed to low external K(+).

Mechanism of action in thalidomide teratogenesis

In this commentary, we describe a model to explain the mechanism of the embryopathy of thalidomide. We propose that thalidomide affects the following pathway during development: insulin-like growth factor I (IGF-I) and fibroblast growth factor 2 (FGF-2) stimulation of the transcription of αv and β3 integrin subunit genes. The resulting αvβ3 integrin dimer stimulates angiogenesis in the developing limb bud, which promotes outgrowth of the bud. The promoters of the IGF-I and FGF-2 genes, the genes for their binding proteins and receptors, as well as the αv and β3 genes, lack typical TATA boxes, but instead contain multiple GC boxes (GGGCGG). Thalidomide, or a breakdown product of thalidomide, specifically binds to these GC promoter sites, decreasing transcription efficiency of the associated genes. A cumulative decrease interferes with normal angiogenesis, which results in truncation of the limb. Intercalation into G-rich promoter regions of DNA may explain why certain thalidomide analogs are not teratogenic while retaining their anti-tumor necrosis factor-α (TNF-α) activity, and suggests that we look elsewhere to explain the action of thalidomide on TNF-α. On the other hand, the anti-cancer action of thalidomide may be based on its antiangiogenic action, resulting from specific DNA intercalation. The tissue specificity of thalidomide and its effect against only certain neoplasias may be explained by the fact that various developing tissues and neoplasias depend on different angiogenesis or vasculogenesis pathways, only some of which are thalidomide-sensitive.

Divergent and composite gonadotropin-releasing hormone-responsive elements in the rat luteinizing hormone subunit genes.

GnRH pulses regulate gonadotropin subunit gene transcription in a frequency-dependent, subunit-specific manner. The alpha-subunit gene is stimulated by constant GnRH and by rapid to intermediate pulse frequencies, while stimulation of LHbeta subunit gene transcription requires intermediate frequency pulses. We have defined the GnRH-responsive elements of the rat LH subunit gene promoters by deletion/mutation analysis and transfection studies in rat pituitary cells and two clonal gonadotrope cell lines. The alpha-subunit gene GnRH-responsive region lies between -411 and -375 bp. The region contains two Ets-domain protein binding sites, and mutating either site obliterates the response. DNA protein binding studies demonstrate the two sites are not equivalent, and that Ets-1 does not mediate this response. Studies of the LHbeta promoter reveal a major GnRH-responsive region between -456 and -342 bp. Within this region, two Sp1 binding sites contribute to the GnRH response, and the 3'Sp1 site is also critical for basal expression. The 5'Sp1 site partially overlaps a CArG box, and mutating the CArG element specifically eliminates the response to pulsatile GnRH. DNA containing this mutation cannot form intermediate mobility complexes with nuclear proteins, but retains Sp1 binding. Mutation of the 3'Sp1 site and either the 5'Sp1 or CArG element partially restores GnRH stimulation, suggesting a downstream element contributes to the full GnRH response. These studies demonstrate that unique composite elements and transcription factors are responsible for GnRH stimulation of the LH subunit genes and may contribute to their differential responses to GnRH pulses.

Differential Role of Hepatocyte Nuclear Factor-1 in the Regulation of Glucose-6-phosphatase Catalytic Subunit Gene Transcription by cAMP in Liver- and Kidney-derived Cell Lines

In liver and kidney, the terminal step in gluconeogenesis is catalyzed by glucose-6-phosphatase. To examine the effect of the cAMP signal transduction pathway on transcription of the gene encoding the catalytic subunit of glucose-6-phosphatase (G6Pase), G6Pase-chloramphenicol acetyltransferase (CAT) fusion genes were transiently transfected into either the liver-derived HepG2 or kidney-derived LLC-PK cell line. Co-transfection of an expression vector encoding the catalytic subunit of cAMP-dependent protein kinase (PKA) markedly stimulated G6Pase-CAT fusion gene expression, and mutational analysis of the G6Pase promoter revealed that multiple regions are required for this PKA response in both the HepG2 and LLC-PK cell lines. A sequence in the G6Pase promoter that resembles a cAMP response element is required for the full PKA response in both HepG2 and LLC-PK cells. However, in LLC-PK cells, but not in HepG2 cells, a hepatocyte nuclear factor-1 (HNF-1) binding site was critical for the full induction of G6Pase-CAT expression by PKA. Changing this HNF-1 motif to that for the yeast transcription factor GAL4 reduces the PKA response in LLC-PK cells to the same degree as deleting the HNF-1 site. However, co-transfection of this mutated construct with chimeric proteins comprising the GAL4-DNA binding domain ligated to the coding sequence for HNF-1alpha, HNF-1beta, HNF-3, or HNF-4 completely restored the PKA response. Thus, we hypothesize that, in LLC-PK cells, HNF-1 is acting as an accessory factor to enhance PKA signaling through the cAMP response element by altering G6Pase promoter conformation or accessibility rather than specifically affecting some component of the PKA signal transduction pathway.

Thyroid hormone suppression of pituitary hormone gene expression.

T3 suppression of TSH subunit gene transcription is an important step in maintaining thyroid hormone homeostasis, and recent investigations have increased our understanding of this process. Thyrotrope-specific proteins play a critical role in TSH subunit gene expression, and influence T3-mediated regulatory mechanisms. The structure and placement of the TSH gene TREs define suppressive regulation by T3, and this process is favored by the TR isoforms expressed in the pituitary. Elimination of TR beta function compromises the pituitary response to T3. TR beta 2, the isoform specifically expressed in pituitary and neural tissue, contains a transferable domain that both increases T3-independent gene transcription and enhances T3-suppressed transcription. The functional interaction of TR beta 2 with other regulatory proteins is distinct from that of other TR isoforms, and likely plays a critical role in pituitary physiology and in pituitary resistance to thyroid hormone. The development of novel thyrotrope cell lines will allow investigators to define new proteins and molecular mechanisms that distinguish negative from positive T3 transcriptional regulation.

Glucocorticoids and Acidification Independently Increase Transcription of Branched-Chain Ketoacid Dehydrogenase Subunit Genes

Metabolic acidosis and glucocorticoids act in concert to stimulate branched-chain amino acid (BCAA) oxidation in adrenalectomized rats. In muscles of normal rats, metabolic acidosis increases the maximal activity of the rate-limiting enzyme, branched-chain α-ketoacid dehydrogenase (BCKAD) and a genetic response to catabolic conditions like uremia is implicated by concurrently higher levels of BCKAD subunit mRNA. To determine if acidification or glucocorticoids increase transcription of BCKAD subunit genes, transfection studies were performed with BCKAD promoter-luciferase reporter minigenes in LLC-PK₁ cells which do not express gluco-corticoid receptors or LLC-PK₁ cells which express a rat glucocorticoid receptor gene (LLC-PK₁-GR101). Acidification significantly increased luciferase activity in LLC-PK₁ cells and LLC-PK₁-GR101 cells transfected with reporter plasmids containing 7.0 kb of E2 subunit or 0.8 kb of E1α subunit promoter region, respectively. Glucocorticoids in the form of dexamethasone induced transcription of these minigenes but only in LLC-PK₁-GR101 cells. Using promoter deletion analysis, independent transactivation response elements to acidification or glucocorticoids were localized in the E2 promoter. In summary, catabolic responses to low extracellular pH and glucocorticoids include enhanced expression of genes encoding BCKAD subunits.

The Balance between Glucocorticoids and Insulin Regulates Muscle Proteolysis via the Ubiquitin-Proteasome Pathway

In uremia, accelerated muscle protein degradation results from activation of the ATP-ubiquitin proteasome proteolytic pathway. Like uremia, other conditions (e.g., acidosis and diabetes) activate this pathway in rat muscles and are associated with excess glucocorticoids (GC) and impaired insulin action. To define the stimuli responsible for muscle wasting in IDDM, the roles of glucocorticoids, insulinopenia and acidosis in streptozotocin (STZ) – induced diabetes were studied. Proteolysis in isolated epitrochlearis muscles from acutely (3d) diabetic rats was 52% higher than pair-fed, sham-injected rats; this increase was eliminated by an inhibitor of the proteasome or by blocking ATP synthesis. In muscles of STZ-diabetic rats, the levels of ubiquitin-conjugated proteins and mRNAs encoding ubiquitin, the ubiquitin-carrier protein, E2_14k and the C3, C5 and C9 proteasome subunits were increased. Transcription of ubiquitin and C3 proteasome subunit genes in muscle was also increased by IDDM. Oral NaHCO₃ eliminated acidemia but did not prevent accelerated muscle proteolysis. Corticosterone excretion was higher in IDDM rats and adrenalectomy (ADX) prevented these catabolic responses; physiologic doses of glucorcoticoids restored the excessive protein catabolism in ADX-STZ rats. Giving IDDM rats replacement insulin also normalized protein degradation in muscles. In conclusion, reduced insulin together with physiologic levels of glucocorticoids activate the ubiquitin-proteasome pathway by a mechanism that includes enhancing ubiquitin conjugation and proteolysis by the proteasome. The balance between these stimuli could regulate muscle proteolysis in uremia.

Biologic and pharmacologic regulation of mammalian glutathione synthesis

Glutathione (L-γ-glutamyl-L-cysteinylglycine, GSH) is synthesized from its constituent amino acids by the sequential action of γ-glutamylcysteine synthetase (γ-GCS) and GSH synthetase. The intracellular GSH concentration, typically 1–8 mM, reflects a dynamic balance between the rate of GSH synthesis and the combined rate of GSH consumption within the cell and loss through efflux. The γ-GCS reaction is rate limiting for GSH synthesis, and regulation of γ-GCS expression and activity is critical for GSH homeostasis. Transcription of the γ-GCS subunit genes is controlled by a variety of factors through mechanisms that are not yet fully elucidated. Glutathione synthesis is also modulated by the availability of γ-GCS substrates, primarily L-cysteine, by feedback inhibition of γ-GCS by GSH, and by covalent inhibition of γ-GCS by phosphorylation or nitrosation. Because GSH plays a critical role in cellular defenses against electrophiles, oxidative stress and nitrosating species, pharmacologic manipulation of GSH synthesis has received much attention. Administration of L-cysteine precursors and other strategies allow GSH levels to be maintained under conditions that would otherwise result in GSH depletion and cytotoxicity. Conversely, inhibitors of γ-GCS have been used to deplete GSH as a strategy for increasing the sensitivity of tumors and parasites to certain therapeutic interventions.

Salmon gonadotropin IIβ subunit promoter contains multiple DNA elements responsible for stimulation by gonadotropin-releasing hormone through protein kinase C-dependent and -independent pathways

Gonadotropin-releasing hormone (GnRH) stimulates gonadotropin (GTH) production by activating GTH subunit gene transcription. In salmonid fish, the expression of the β subunit gene of GTH II (sGTH IIβ) is stimulated by GnRH at the final stages of reproduction. DNA elements required for the GnRH stimulation were examined by analyzing sGTH IIβ promoter activity by transfection studies in a gonadotrope-derived cell line, αT3-1. A GnRH analog (GnRHa) specifically stimulated the sGTH IIβ promoter (3358 bp) expression 3.6-fold, while phorbol myristate acid (PMA) stimulated it 6.2–9-fold. Analysis of a series of 5′-deletion mutants has revealed that a proximal region (−258 to −199) was important in GnRHa stimulation through protein kinase C (PKC)-independent signal transduction pathways, because an internal deletion mutant (Δ(246−217)/3358) showed a significant decrease in the level of GnRHa stimulation, but showed no change in stimulation by PMA. A large upstream region (−3358 to −1260) showed an enhancing activity of the GnRHa stimulation, and a far upstream 530 bp segment in this region (−3358 to −2829) may be responsible for this activity. The present results suggest that sGTH IIβ gene may be controlled by GnRH through multiple DNA elements including those responsive to PKC-dependent and -independent signal transduction pathways.

Constitutively Active MuSK Is Clustered in the Absence of Agrin and Induces Ectopic Postsynaptic-Like Membranes in Skeletal Muscle Fibers

In skeletal muscle fibers, neural agrin can direct the accumulation of acetylcholine receptors (AChR) and transcription of AChR subunit genes from the subsynaptic nuclei. Although the receptor tyrosine kinase MuSK is required for AChR clustering, it is less clear whether MuSK regulates gene transcription. To elucidate the role of MuSK in these processes, we constructed a constitutively active MuSK receptor, MuSKneuTMuSK, taking advantage of the spontaneous homodimerization of the transmembrane domain of neuT, an oncogenic variant of the neu/erbB2 receptor. In the extrasynaptic region of innervated muscle fibers, MuSKneuTMuSK formed highly concentrated aggregates that colocalized with AChR clusters. Associated with MuSK-induced AChR clusters was a normal complement of synaptic proteins. Moreover, transcription of the AChR-epsilon subunit gene was increased, albeit via an indirect mechanism by MuSK-induced aggregation of erbB receptors and neuregulin. Although neural agrin was not required, the activity of MuSKneuTMuSK was nevertheless potentiated by ectopic expression of a muscle agrin isoform inactive in AChR clustering. To define the role of the kinase domain in the formation of a postsynaptic-like membrane, a second fusion receptor, neuneuTMuSK, which included the MuSK kinase but not the MuSK extracellular domain, was expressed. Significantly, neuneuTMuSK induced AChR clusters that colocalized with aggregates of endogenous MuSK. Taken together, it was concluded that the MuSK kinase domain is sufficient to initiate the recruitment of additional MuSK receptors, which then develop into highly concentrated aggregates by means of a positive feedback loop to induce a postsynaptic membrane in the absence of neural agrin.

ERK MAP kinase activation is required for acetylcholine receptor inducing activity-induced increase in all five acetylcholine receptor subunit mRNAs as well as synapse-specific expression of acetylcholine receptor ε-transgene

The AChR is a pentamer of four different subunits in a stoichiometry of α 2βγδ in embryonic and α 2βεδ in adult animals. Transcription of AChR subunit genes is most active in synaptic nuclei in adult skeletal muscle cells, and is regulated by neural factors such as ARIA. We report here that ARIA up-regulated specifically the expression of all five AChR subunits in C2C12 cells. The mRNA level of erbB2, erbB3, rapsyn, MuSK, SHP-2 and β-actin remained unchanged in response to ARIA stimulation in C2C12 cells. The ARIA-induced increase in AChR subunit expression in C2C12 cells was inhibited by the erbB kinase inhibitor tyrphostin AG1478 and the MEK inhibitor PD98059, but not by the PI3 kinase inhibitor wortmannin, suggesting an important role of the erbB protein tyrosine kinases and MAP kinase in the regulation of the expression of the five different AChR subunits. To determine the signaling pathways in vivo, we studied the expression of reporter genes driven by the ε-promoter in injected muscles. The in vivo expression of the ε-transgene was inhibited by co-expression of dominant negative mutants of key components in the MAP kinase pathway including ras, raf and MEK, but not the dominant negative mutant of PI3 kinase. These results suggest that ERK MAP kinase activation is required for ARIA-induced increase in all five AChR subunit mRNAs as well as synapse-specific expression of AChR ε-transgene.

Glucose induces glucose 6-phosphatase hydrolytic subunit gene transcription in an insulinoma cell line (INS-1)

Primer extension analysis and RNase protection assays revealed the identity of glucose 6-phosphatase gene transcripts in both the insulinoma cell line INS-1 and hepatic cells. In transient transfection assays of INS-1 cells, using constructs between the human glucose 6-phosphatase gene promoter and a luciferase reporter gene, the reporter gene activity was induced by dexamethasone and dibutyryl cAMP. Furthermore, the promoter was regulated by the glucose concentration in the medium. This effect was dependent on glucose metabolism. The data indicated that glucose 6-phosphatase gene transcription is regulated in a similar way in the insulinoma cell line and in liver.

Neuronal Nicotinic Receptors in the Locust Locusta migratoria: CLONING AND EXPRESSION

We have identified five cDNA clones that encode nicotinic acetylcholine receptor (nAChR) subunits expressed in the nervous system of the locust Locusta migratoria. Four of the subunits are ligand-binding alpha subunits, and the other is a structural beta subunit. The existence of at least one more nAChR gene, probably encoding a beta subunit, is indicated. Based on Northern analysis and in situ hybridization, the five subunit genes are expressed. localpha1, localpha3, and locbeta1 are the most abundant subunits and are expressed in similar areas of the head ganglia and retina of the adult locust. Because Loc<alpha3 binds alpha-bungarotoxin with high affinity, it may form a homomeric nAChR subtype such as the mammalian alpha7 nAChR. Localpha1 and Locbeta1 may then form the predominant heteromeric nAChR in the locust brain. localpha4 is mainly expressed in optic lobe ganglionic cells and localpha2 in peripherally located somata of mushroom body neurons. localpha3 mRNA was additionally detected in cells interspersed in the somatogastric epithelium of the locust embryo, suggesting that this isoform may also be involved in functions other than neuronal excitability. Transcription of all nAChR subunit genes begins approximately 3 days before hatching and continues throughout adult life. Electrophysiological recordings from head ganglionic neurons also indicate the existence of more than one functionally distinct nAChR subtype. Our results suggest the existence of several nAChR subtypes, at least some of them heteromeric, in this insect species.

Agrin can mediate acetylcholine receptor gene expression in muscle by aggregation of muscle-derived neuregulins.

The neural isoforms of agrin can stimulate transcription of the acetylcholine receptor (AChR) epsilon subunit gene in electrically active muscle fibers, as does the motor neuron upon the formation of a neuromuscular junction. It is not clear, however, whether this induction involves neuregulins (NRGs), which stimulate AChR subunit gene transcription in vitro by activating ErbB receptors. In this study, we show that agrin- induced induction of AChR epsilon subunit gene transcription is inhibited in cultured myotubes overexpressing an inactive mutant of the ErbB2 receptor, demonstrating involvement of the NRG/ErbB pathway in agrin- induced AChR expression. Furthermore, salt extracts from the surface of cultured myotubes induce tyrosine phosphorylation of ErbB2 receptors, indicating that muscle cells express biological NRG-like activity on their surface. We further demonstrate by RT-PCR analysis that muscle NRGs have Ig-like domains required for their immobilization at heparan sulfate proteoglycans (HSPGs) of the extracellular matrix. In extrasynaptic regions of innervated muscle fibers in vivo, ectopically expressed neural agrin induces the colocalized accumulation of AChRs, muscle-derived NRGs, and HSPGs. By using overlay and radioligand-binding assays we show that the Ig domain of NRGs bind to the HSPGs agrin and perlecan. These findings show that neural agrin can induce AChR subunit gene transcription by aggregating muscle HSPGs on the muscle fiber surface that then serve as a local sink for focal binding of muscle-derived NRGs to regulate AChR gene expression at the neuromuscular junction.

Identification of cis-acting deoxyribonucleic acid elements that mediate gonadotropin-releasing hormone stimulation of the rat luteinizing hormone beta-subunit gene.

GnRH plays a critical role in reproductive development and function by regulating the biosynthesis and secretion of the pituitary gonadotropins, LH and FSH. Although it is known that GnRH induces gonadotropin subunit gene transcription, the mechanism by which this occurs has not been elucidated. Studies have been hindered by the lack of available cell lines that express the LH and FSH subunit genes and respond to GnRH. We have transfected the rat pituitary GH3 cell line with the rat GnRH receptor complementary DNA. These cells, when cotransfected with regulatory regions of the LH or FSH subunit genes fused to a luciferase reporter gene, respond to GnRH with an increase in promoter activity comparable to that seen in primary rat pituitary cells. In this study, we have used this cell model to identify cis-acting elements of the LHbeta gene that mediate stimulation by GnRH. Analysis of a series of 5'-deletion and internal deletion constructs has revealed two regions of the rat LHbeta gene promoter involved in mediating the response to GnRH, region A (-490/-352) and region B (-207/-82). Fusion of region A upstream of a heterologous minimal promoter linked to the luciferase gene conferred GnRH responsiveness to the promoter, whereas region B did not. However, the presence of both regions A and B conferred a greater GnRH response than region A alone. Electrophoretic mobility shift assay revealed the presence of a protein(s) binding to region A using GH3 as well as alphaT3-1 nuclear extracts. Thus, region A (-490/-352) confers GnRH responsiveness to the LHbeta subunit gene and binds to a protein(s) present in pituitary cell lines. DNA sequences in region B (-207/-82) also contribute to GnRH responsiveness. The identification of putative GnRH response elements in the rat LHbeta gene promoter will aid in elucidation of the mechanisms of regulation of gene expression by GnRH.

Mechanism of action of pituitary adenylate cyclase-activating polypeptide on human glycoprotein hormone alpha-subunit transcription in alphaT3-1 gonadotropes.

Pituitary adenylate cyclase activating polypeptide (PACAP) has been shown to increase glycoprotein hormone alpha-subunit synthesis and release from pituitary cells. We have used alphaT3-1 clonal gonadotropes to investigate the intracellular mechanisms involved in PACAP regulation of alpha-subunit gene transcription; and using deletion, mutation, and heterologous constructs of the alpha-promoter linked to a luciferase reporter gene, we have defined DNA sequences responsive to PACAP. Stimulation of alphaT3-1 cells for 24 h with PACAP, GnRH, or vasoactive intestinal peptide (VIP) resulted in a time- and concentration-dependent increase in alpha-promoter transcription at 100 nM for GnRH (17.5-fold, P < 0.001), PACAP (12.7-fold, P < 0.01), and VIP (4.1-fold, P < 0.05). Incubation of alphaT3-1 cells in calcium-depleted medium suggested that the transcriptional response to PACAP was less dependent on changes in intracellular calcium concentration, in contrast to the results seen with GnRH or VIP, where alpha-subunit transcription was significantly reduced. Transfection of an alpha-promoter construct containing a mutant cAMP response element (CRE) suggested that the CRE region is involved in PACAP and VIP responsiveness, with stimulatory effects on the mutant construct by PACAP (11.1-fold) and VIP (7.6-fold) being significantly (P < 0.001) reduced, compared with their stimulatory effects (PACAP: 25.6-fold, VIP: 23.1-fold) on the native alpha-promoter. In the same experiment, the transcriptional response of the mutant CRE construct and the native CRE construct to GnRH was not significantly different. Both PACAP and VIP enhanced GnRH-stimulated alpha-subunit gene transcription, but this additive effect was lost when their combined effects on the mutant CRE were examined. Deletion analysis indicated that sequences between -244 and -195 bp were involved in mediating the response to PACAP, with a dramatic reduction in fold-stimulation by PACAP (2.0-fold) of the -195-bp construct, compared with the -244-bp construct (15.8-fold). Constructs containing only upstream alpha-promoter sequences from -517 bp to -98 bp, fused to the heterologous thymidine kinase promoter, exhibited a similar loss of responsiveness to PACAP below -298 bp. Thus, our studies show that, unlike GnRH, PACAP stimulation of alpha-subunit gene transcription in alphaT3-1 cells is less dependent on changes in intracellular calcium concentration; and full transcriptional activation of the alpha-subunit by PACAP requires an intact CRE. PACAP responsiveness involves sequences between -244 and -195 bp of the alpha-promoter. These sequences have been implicated also in GnRH-responsiveness and may thus provide a mechanism for coordinated regulation of the alpha-subunit gene by PACAP and GnRH in alphaT3-1 cells.

GABA A-receptor assembly in vivo: Lessons from subunit mutant mice

The rules governing the assembly of GABA A receptors in vivo were assessed in subunit mutant mice. The transcription of individual subunit genes was regulated independently. The lack of a particular subunit did not result in a molecular rescue by an enhanced transcription of other subunits. In addition, the availability of an α- and β-subunit was essential for receptor formation. Finally, highly selective recognition processes directed the subcellular targeting of receptors. The loss of a particular receptor subtype ( α 5) did not lead to a subcellular redistribution of the remaining subtype (α2) present in the same cell.

Glutamate receptors in dysplasic cortex: an in situ hybridization and immunohistochemistry study in rats with prenatal treatment with methylazoxymethanol

Injection of the antimitotic drug methylazoxymethanol (MAM) in the pregnant rat at E14 leads in the offsprings to a severe malformation with microcephaly and cortical heterotopiae in the white matter and in the CA1 field of the hippocampus. These animals suffer cognitive and epileptic disorders. Since these pathologies have been associated with glutamatergic transmission abnormalities, we have examined by in situ hybridization and immunohistochemistry the distribution and expression levels of several glutamate receptors subunits in these rats. Examination of the GluR2 flip and flop, NR1, NR2A and NR2B subunit gene transcripts showed a qualitatively similar distribution in both the neocortex and hippocampus of MAM and control rats. Quantitative analysis revealed an altered proportion of the GluR2 flip and flop subunits in the CA1 region of MAM animals as compared to controls. Moreover, a 26% reduction in the expression of the NR1 subunit and a 40% increase in the expression of the GluR2 flip subunit were noted in cortical heterotopiae, as compared to the adjacent neocortex. Immunostaining for GluR2/3, NR1 or NR2 showed, in both MAM and control animals, that glutamate receptors were mainly concentrated in the soma and dendrites of neocortical and hippocampal pyramidal cells, including in heterotopiae, and in the apical dendrites of hippocampal granule cells. Abnormalities in the expression of glutamate receptor subtypes in cortical heterotopiae and in the hippocampal CA1 region could contribute to functional disorders previously reported in MAM animals such as memory impairments and epilepsy.

Induction by agrin of ectopic and functional postsynaptic-like membrane in innervated muscle.

Two factors secreted from the nerve terminal, agrin and neuregulin, have been postulated to induce localization of the acetylcholine receptors (AChRs) to the subsynaptic membrane in skeletal muscle fibers. The principal function ascribed to neuregulin is induction of AChR subunit gene expression and to agrin is the aggregation of AChRs. Here we report that when myoblasts engineered to secrete an agrin fragment were placed into the nerve-free region of denervated rodent muscle, the host muscle fibers expressed AChR epsilon-subunit gene transcripts, characteristic of the neuromuscular synapse in adult muscle. Transcripts were colocalized with agrin deposits and AChR clusters that were resistant to electrical muscle activity. More directly, single innervated muscle fibers injected intracellularly with agrin expression plasmids in their extrasynaptic region developed a functional ectopic postsynaptic membrane with clusters of adult-type AChR channels and acetylcholinesterase and accumulation of myonuclei. The results demonstrate that agrin is the principal neural signal that induces the formation of the subsynaptic apparatus in the muscle fiber and controls locally, either indirectly or directly, the transcription of AChR subunit genes and the aggregation of AChRs.

Gonadal hormone feedback on pituitary gonadotropin genes

The pituitary gonadotropins LH and FSH, which act on the ovaries and testes to promote gametogenesis and sex steroid production, are regulated by changes in the levels of steroids and gonadal peptides. Steroid feedback can be positive, as demonstrated by the estrogen and LH surge at ovulation, or negative, as demonstrated by the rise in LH and FSH after gonadectomy or reductions in steroid synthesis. Modulatory effects of the steroids estrogen and testosterone may be mediated directly at the level of the pituitary cells, or by alterations in the release of the hypothalamic releasing factor GnRH. Gonadal peptides, including activin and inhibin, have been shown to have direct effects on pituitary cells to alter FSH synthesis specifically, with no effects on LH. Changes in gonadotropin subunit gene transcription and mRNA levels occur very rapidly and have profound effects on physiological levels of the hormones. In this article, the effects of the gonadal steroids and peptides as modifiers of the rat gonadotropin genes in a subunit specific manner are reviewed, and the physiological implications discussed.