Separation of signal transduction and adaptation functions of the aspartate receptor in bacterial sensing.

In order to investigate the functions of stimulus recognition, signal transduction, and adaptation, the aspartate receptor gene for bacterial chemotaxis in Salmonella typhimurium has been sequenced and modified. A carboxyl-terminal truncated receptor was shown to bind aspartate and to transmit a signal to change motility behavior. However, the truncated receptor showed greatly reduced methyl-accepting capacity, and did not allow adaptation to the sensory stimulation. The separation of receptor functions by alteration of primary structure emphasizes that the receptor is directly involved in adaptation and is not solely a device for transmitting a signal across a membrane.

Neuronal expression of chimeric genes in transgenic mice.

Gene expression may occur in unexpected ectopic sites when diverse genetic elements are juxtaposed as chimeric genes in transgenic mice. To determine the specific contribution of the promoter and reporter gene in ectopic expression, we have analyzed the expression of 14 different fusion genes in transgenic mice. Chimeric genes containing the mouse metallothionein-I promoter linked to either the rat or human growth hormone gene or the calcitonin/CGRP gene are expressed in a very similar pattern of neuronal regions. This ectopic expression is not a unique feature of the metallothionein promoter, since transferring the human growth hormone gene to four other heterologous promoters resulted in varying degrees of ectopic expression in overlapping subsets of cortical and hypothalamic neurons. The novel pattern of ectopic expression suggests that these otherwise unrelated neurons share a common developmental regulatory machinery for activation of gene transcription.

Multifunctional role of the Pitx2 homeodomain protein C-terminal tail.

Pitx2 is a newly described bicoid-like homeodomain transcription factor that is defective in Rieger syndrome and shows a striking leftward developmental asymmetry. We have previously shown that Pitx2 (also called Ptx2 and RIEG) transactivates a reporter gene containing a bicoid enhancer and synergistically transactivates the prolactin promoter in the presence of the POU homeodomain protein Pit-1. In this report, we focused on the C-terminal region which is mutated in some Rieger patients and contains a highly conserved 14-amino-acid element. Deletion analysis of Pitx2 revealed that the C-terminal 39-amino-acid tail represses DNA binding activity and is required for Pitx2-Pit-1 interaction and Pit-1 synergism. Pit-1 interaction with the Pitx2 C terminus masks the inhibitory effect and promotes increased DNA binding activity. Interestingly, cotransfection of an expression vector encoding the C-terminal 39 amino acids of Pitx2 specifically inhibits Pitx2 transactivation activity. In contrast, the C-terminal 39-amino-acid peptide interacts with Pitx2 to increase its DNA binding activity. These data suggest that the C-terminal tail intrinsically inhibits the Pitx2 protein and that this inhibition can be overcome by interaction with other transcription factors to allow activation during development.

Retinoic acid repression of cell-specific helix-loop-helix-octamer activation of the calcitonin/calcitonin gene-related peptide enhancer.

We have investigated the mechanism underlying repression of calcitonin/calcitonin gene-related peptide (CT/CGRP) gene expression by retinoic acid. Retinoic acid treatment of the CA77 thyroid C-cell line decreased CT/CGRP promoter activity two- to threefold, which correlates well with the decrease in calcitonin and CGRP mRNA levels. Repression is mediated through the nuclear retinoic acid receptors (RAR) on the basis of the retinoid specificity, the sensitivity of repression (half-maximal repression at 0.2 nM), and the additional repression caused by cotransfection of an alpha-RAR expression vector. The sequences required for retinoic acid repression were localized to an 18-bp element containing cell-specific enhancer activity. The enhancer binds helix-loop-helix (HLH) and octamer transcription factors that act synergistically to activate transcription. Retinoic acid repression requires both these factors since mutations in either motif resulted in the loss of repression. Furthermore, repression was observed only in cell lines containing enhancer activity. We have used electrophoretic mobility shift assays to show that repression does not involve direct DNA binding of RAR or RAR-retinoid X receptor heterodimers. Instead, repression appears to involve interactions with the stimulatory enhancer factors. Following retinoic acid treatment, there was a specific decrease in an enhancer complex containing both HLH and octamer proteins. Formation of the HLH-octamer complex was also specifically blocked by the addition of exogenous RAR-retinoid X receptor protein. These results demonstrate that RAR can repress CT/CGRP gene transcription by interfering with combinatorial activation by cell-specific HLH and octamer proteins.

Serotonergic repression of mitogen-activated protein kinase control of the calcitonin gene-related peptide enhancer.

We have investigated the mechanisms underlying regulation of the calcitonin gene-related peptide (CGRP) cell-specific enhancer. Recently, we reported that this enhancer is inhibited by serotonin type-1 (5-HT1) agonists, similar to currently used antimigraine drugs. We have now tested whether this repression involves a mitogen-activated protein (MAP) kinase pathway. We first demonstrate that the CGRP enhancer is strongly (10-fold) activated by a constitutively active MAP kinase kinase (MEK1), yielding reporter activities 100-fold above the enhancerless control. The involvement of a MAP kinase pathway was confirmed by down-regulation of reporter activity upon cotransfection of a dominant negative Ras. Activation of the enhancer by MEK1 was blocked in a dose-dependent manner by the 5-HT1 receptor agonist CGS 12066A (CGS). Since it is not known whether the CGRP enhancer factors are immediate targets of MAP kinases, we then used EIk-1- and c-Jun-dependent reporter genes that are directly activated by the ERK (extracellular signal-regulated kinases) and JNK (c-Jun N-terminal kinase) MAP kinases. CGS treatment repressed the activation of both of these reporters, suggesting that at least two MAP kinases are the immediate targets of CGS-mediated repression. We further demonstrate that 5-HT1 agonists inactivate ERK by dephosphorylation, even in the presence of constitutively activated MEK1. This inactivation appears to be due to a marked increase in the level of MAP kinase phosphatase-1. These results have defined a novel and general mechanism by which 5-HT1 receptor agonists can repress MAP kinase activation of target genes, such as CGRP.

Differential regulation of mitogen-activated protein kinase-responsive genes by the duration of a calcium signal.

We have investigated the cellular mechanisms by which changes in intracellular calcium (Ca2+) can differentially regulate gene expression. Two Ca2+ paradigms, involving prolonged and transient Ca2+ increases, were used. As a starting point, we studied the slow, prolonged elevation of Ca2+ caused by activation of 5-HT1 receptors. We had previously shown that 5-HT1 agonists inhibit calcitonin gene-related peptide (CGRP) transcription and secretion. The Ca2+ ionophore, ionomycin, was used to produce a prolonged elevation of the Ca2+ signal similar to that generated by 5-HT1 receptor agonists. Ionomycin treatment of the neuronal-like CA77 cell line specifically inhibited mitogen-activated protein (MAP) kinase stimulation of the CGRP enhancer and two synthetic MAP kinase-responsive reporter genes (4- to 10-fold). We then showed that ionomycin repression of promoter activity involved selective induction of MAP kinase phosphatase-1 (MKP-1), but not MKP-2, and that overexpression of MKP-1 was sufficient to repress CGRP enhancer activity. These effects were then compared with a Ca2+ paradigm involving a transient elevation in Ca2+ as seen after depolarization. At 4 h after the transient increase in Ca2+, the CGRP enhancer and synthetic MAP kinase-responsive reporter genes were stimulated. In contrast, exposure to depolarizing stimuli overnight caused only a less than 2-fold inhibition of promoter activity. We propose that the duration of the Ca2+ signal can determine the magnitude of a negative feedback loop that leads to differential regulation of MAP kinase-responsive genes.

Neuronal properties of a thyroid C-cell line: partial repression by dexamethasone and retinoic acid.

We have analyzed the effect of extracellular stimuli on the differentiation state of the CA77 thyroid C-cell line as a model to understand the control of neural crest cell differentiation. In contrast to the endocrine C-cell phenotype, we found that CA77 cells have a neuronal phenotype characterized by laminin-induced neurites, neuronal antigens, and calcitonin gene-related peptide (CGRP) mRNA expression. Treatment with dexamethasone and retinoic acid reversibly repressed some of these neuronal characteristics to induce features more characteristic of the parental C-cells. In the case of dexamethasone treatment, there was a partial retraction and thinning of neurites, an increased number of secretory vesicles in the cell bodies, and about a 10-fold decrease in DNA synthesis. Treatment with retinoic acid alone or in combination with dexamethasone caused decreased cell adhesion and an even more extensive retraction of the neurites. Dexamethasone also biased the steady state levels of the alternatively spliced transcripts from the calcitonin/CGRP gene to favor calcitonin relative to CGRP mRNA. While retinoic acid treatment decreased both calcitonin and CGRP mRNA levels, the combination of dexamethasone and retinoic acid still yielded the increase in calcitonin relative to CGRP mRNA. These results suggest that glucocorticoids and retinoic acid may contribute to a late and reversible differentiation of thyroid C-cells by partly repressing neuronal properties.

Expression and development of a functional plasmalemmal 5-hydroxytryptamine transporter by thyroid follicular cells.

5-Hydroxytryptamine (5-HT) is synthesized and secreted by thyroid parafollicular (PF) cells. As all PF granules contain 5-HT, it is released whenever PF cells secrete. Because 5-HT stimulates follicular (F) cells and can modulate their response to TSH, 5-HT has been proposed to be a paracrine PF to F cell transmitter. This role would require a thyroid mechanism to rapidly inactivate 5-HT. A 5-HT transporter (SERT) in the plasma membrane of serotonergic neurons inactivates neuronal 5-HT. We thus tested the hypothesis that this molecule is expressed in the thyroid. Messenger RNA encoding SERT was demonstrated in both the human thyroid and a rat F cell line (FRTL-5). SERT immunoreactivity was detected in rat F, but not PF, cells. Transporter-mediated uptake of [3H]5-HT by F cells arose early in development (E13 in mice) and was maintained in adult life in mice, guinea pigs, bats, and rats (FRTL-5 cells). These observations indicate that a functional SERT is expressed in the thyroid, not by the 5-HT-secreting PF cells, but by their putative F cell targets.

Thyroid parafollicular cells. An accessible model for the study of serotonergic neurons.

Serotonergic neurons play key roles in modulating a wide variety of behavioral and homeostatic processes. However, there is a paucity of good model systems to study these neurons at a molecular level. In this review we will present evidence that cell lines derived from an unexpected source, thyroid parafollicular cells (PF) (also called C cells), fit the criteria for use as models for the study of serotonergic neurons. A strength of PF cell lines over other cell lines is that the parental PF cells have serotonergic properties and a neuronal potential that is consistent with their neural crest origin. Furthermore, PF cells and PF cell lines are capable of expressing the fundamental properties of serotonergic neurons, including: (1) serotonin (5-HT) biosynthesis by tryptophan hydroxylase (TPH), (2) vesicular 5-HT storage and regulated release, (3) expression of a 5-HT autoreceptor, and (4) expression of the 5-HT transporter. In this review, we will focus primarily on the serotonergic and neuronal properties of the rat CA77 PF cell line and the parental rat PF cells. The applicability of CA77 cells for molecular analyses will be described. First, their use for studies on the glucocorticoid regulation of the TPH gene will be discussed. Second, control of the calcitonin/calcitonin gene-related peptide (CT/CGRP) gene will be discussed, with particular emphasis on the application of serotonergic drugs in treating migraine headaches. These examples highlight the versatility of thyroid PF cell lines as a system for studying the control of both serotonin biosynthesis and physiological actions.

Developmental regulation of tryptophan hydroxylase messenger RNA expression and enzyme activity in the raphe and its target fields.

Tryptophan hydroxylase is the rate-limiting enzyme in the synthesis of serotonin and during development, brain serotonin levels and tryptophan hydroxylase activities increase. Increased tryptophan hydroxylase activity could result from alterations in tryptophan hydroxylase messenger RNA levels, translation, and/or post-translational regulation. Tryptophan hydroxylase messenger RNA levels in the dorsal raphe nucleus increased 35-fold between embryonic day 18 and postnatal day 22, measured by quantitative in situ hybridization, then decreased by 40% between postnatal days 22 and 61. These changes correlated with tryptophan hydroxylase enzyme activities in the raphe nuclei as expected, but not in cortical or hippocampal targets. Tryptophan hydroxylase messenger RNA expression in the nucleus raphe obscuris increased 2.5-fold between postnatal days 8 and 22 but did not correlate with enzyme activity in the spinal cord. Using an in vitro model of serotonergic raphe neuron differentiation, serotonergic differentiation was associated with an increase in both tryptophan hydroxylase promoter activity and protein expression. In vivo, tryptophan hydroxylase messenger RNA levels per single cell and per brain section were correlated during development up to postnatal day 22, but not beyond for both the dorsal raphe nucleus and nucleus raphe obscuris. Between postnatal days 22 and 61 single cell levels of tryptophan hydroxylase messenger RNA in the dorsal raphe nucleus did not change yet the levels per brain section significantly decreased by 40%. During the same period in the nucleus raphe obscuris, tryptophan hydroxylase messenger RNA levels per single cell signifcantly increased by 30% yet levels per brain section did not change. Comparison of tryptophan hydroxylase messenger RNA levels per cell and per brain section indicated a serotonergic loss between postnatal days 22 and 61 in both the dorsal raphe nucleus and nucleus raphe obscuris and may reflect either a loss of neurotransmitter phenotype or cell death. This study is the first to characterize the expression of brain tryptophan hydroxylase messenger RNA during rat development. In addition, this study is the first to report the activity of tryptophan hydroxylase in the spinal cord and hippocampus in the embryonic and neonatal rat. Together, the data provide a better understanding of the intricate relationship between patterns of tryptophan hydroxylase messenger RNA expression and enzyme activity.

Characterization of the calcitonin/CGRP gene in Williams syndrome.

We have investigated the possibility of mutations in the calcitonin/calcitonin gene related peptide (CGRP) gene in children with Williams syndrome. Involvement of the calcitonin/CGRP gene in Williams syndrome is postulated on the basis that Williams syndrome children often have infantile hypercalcemia and deficient expression of calcitonin, a hormone that lowers serum calcium levels. To test the hypothesis that mutations in the calcitonin/CGRP gene might be responsible for the reduced calcitonin levels, we examined the calcitonin/CGRP gene structure in Williams syndrome children. Analysis of white blood cell DNA by Southern blot hybridizations in 5 individuals did not show any detectable large deletions or rearrangements in the calcitonin/CGRP gene locus. The possibility of small deletions or point mutations within the exon encoding the mature calcitonin hormone is unlikely based on ribonuclease protection assays with patient DNA amplified by the polymerase chain reaction (PCR) technique. These findings suggest that the calcitonin deficiency might be due either to mutations elsewhere in the gene or to defects in the cellular machinery needed for calcitonin synthesis and/or secretion.

Tissue-specific glucocorticoid regulation of tryptophan hydroxylase mRNA levels.

A potential long-term target of glucocorticoid modulation of serotonin (5-HT) production is tryptophan hydroxylase (TPH) gene expression. However, studies on TPH gene expression have been hampered by the extremely low levels of TPH mRNA in the brain, and there have been contradictory reports on the effects of glucocorticoids on 5-HT levels. To overcome these obstacles, we have developed a sensitive competitive RT-PCR assay to directly measure TPH mRNA levels from the rat brain. We observed a tissue-specific modulation of TPH mRNA levels in the melatonin producing pineal gland and the serotonin producing raphe nuclei of the brain. Following chronic treatment of adrenalectomized rats with the synthetic glucocorticoid dexamethasone for 1 week, there was a 16-fold increase in TPH mRNA in the pineal gland that was contrasted by a decrease in TPH mRNA to 16% of the control levels in the brain. To address the mechanism of dexamethasone repression of TPH mRNA levels, we then tested a serotonergic neuronal-like cell line derived from rat thyroid C cells. Dexamethasone caused a rapid decrease in TPH mRNA levels to approximately 20% of control values in CA77 C cells. This was measured by both competitive RT-PCR and a standard hybridization assay, which confirmed the validity of the RT-PCR assay. Furthermore, the reduction of TPH mRNA levels was associated with a decrease in 5-HT levels in the CA77 C cells. Hence, glucocorticoids may alter serotonin and melatonin biosynthetic capacity by cell-specific modulation of the TPH gene.

Antagonistic regulation of Dlx2 expression by PITX2 and Msx2: implications for tooth development.

The transcriptional mechanisms underlying tooth development are only beginning to be understood. Pitx2, a bicoid-like homeodomain transcription factor, is the first transcriptional marker observed during tooth development. Because Pitx2, Msx2, and Dlx2 are expressed in the dental epithelium, we examined the transcriptional activity of PITX2 in concert with Msx2 and the Dlx2 promoter. PITX2 activated while Msx2 unexpectedly repressed transcription of a TK-Bicoid luciferase reporter in a tooth epithelial cell line (LS-8) and CHO cell line. Surprisingly, Msx2 binds to the bicoid element (5'-TAATCC-3') with a high specificity and competes with PITX2 for binding to this element. PITX2 binds to bicoid and bicoid-like elements in the Dlx2 promoter and activates this promoter 45-fold in CHO cells. However, it is only modestly activated in the LS-8 tooth epithelial cell line that endogenously expresses Msx2 and Pitx2. RT-PCR and Western blot assays reveal that two Pitx2 isoforms are expressed in the LS-8 cells. We further demonstrate that PITX2 dimerization can occur through the C-terminus of PITX2. Msx2 represses the Dlx2 promoter in CHO cells and coexpression of both PITX2 and Msx2 resulted in transcriptional antagonism of the Dlx2 promoter. Electrophoretic mobility shift assays demonstrate that factors in the LS-8 cell line specifically interact with PITX2. Thus, Dlx2 gene transcription is regulated by antagonistic effects between PITX2, Msx2, and factors expressed in the tooth epithelia.

Measurement of tryptophan hydroxylase mRNA levels by competitive RT-PCR.

Tryptophan hydroxylase (TPH) is the rate limiting enzyme in serotonin biosynthesis [D.G. Grahame-Smith, Tryptophan hydroxylation in brain, Biochem. Biophys. Res. Commun. 16 (1964) 586-592 [19]]. As such, the TPH gene is a likely target for modulation of serotonergic function, which has been associated with several psychiatric disorders [E.C. Azmitia, P.M. Whitaker-Azmitia, Awakening the sleeping giant: anatomy and plasticity of the brain serotonergic system, J. Clin. Psychiatry 52 (12, Suppl.) (1991) 4-16 [1]; R.P. Hart, R. Yang, L.A. Riley., T.L. Green, Post-transcriptional control of tryptophan hydroxylase gene expression in rat brain stem and pineal gland, Mol. Cell. Neurosci. 2 (1991) 71-77 [20]; M.J. Owens, C.B. Numeroff, Role of serotonin in the pathophysiology of depression: focus on the serotonin transporter, Clin. Chem. 40 (1994) 288-295 [24]]. Unfortunately, it has been technically difficult to measure TPH mRNA levels in central serotonergic neurons due to its low levels. For example, detection with ribonuclease protection assays requires pooling of 5-10 dissected brainstems [M.C. Darmon, B. Guibert, V. Leviel, M. Ehret, M. Maitre, J. Mallet, Sequence of two mRNAs encoding active rat tryptophan hydroxylase, J. Neurochem. 51 (1988) 312-316 [15]; B.L. Jacobs, E.C. Azmitia, Structure and function of the brain serotonin system, Physiol. Rev. 72 (1992) 165-229 [21]]. This protocol describes the use of competitive RT-PCR to measure TPH mRNA levels from rat brain. First described in 1988, competitive RT-PCR has become an accepted method of measuring RNA abundance [M. Clementi, S. Menzo, P. Bagnarelli, A. Manzin, A. Valenza, P.E. Varaldo, Quantitative PCR and RT-PCR in virology, PCR Methods Appl. 2 (1994) 191-196 [12]; N.C.P. Cross, Quantitative PCR techniques and applications, Br. J. Haematol. 89 (1995) 693-697 [14]; K.P. Foley, M.W. Leonard, J.D. Engel, Quantitation of RNA using the polymerase chain reaction, Trends Genet. 9 (1993) 380-385 [17]; P.D. Siebert, J.W. Larrick, Competitive PCR, Nature 359 (1992) 558 [27]]. Competitive RT-PCR uses co-amplification with a known quantity of an in vitro transcribed RNA which amplifies using the same primers and thus competes for reactants with the product of interest. As the two products amplify with the same efficiency, the relative abundance of the two amplification products remains constant, and thus can be used to determine initial tissue TPH mRNA levels [G. Gilliland, S. Perrin, K. Blanchard, H.F. Bunn, Analysis of cytokine mRNA and DNA: detection and quantitation by competitive polymerase chain reaction, Proc. Natl. Acad. Sci. U.S.A. 87 (1990) 2725-2729 [18]; A.M. Wang, M. V. Doyle, D.F. Mark, Quantitation of mRNA by the polymerase chain reaction, Proc. Natl. Acad. Sci. U.S.A. 86 (1989) 9717-9721 [31]]. We first demonstrate equivalent results between RNA slot blots and competitive RT-PCR using the CA77 thyroid C cell line [M.S. Clark, A. F. Russo, Tissue-specific glucocorticoid regulation of tryptophan hydroxylase mRNA levels, Mol. Brain Res. 48 (1997) 346-354 [9]]. We then describe the use of competitive RT-PCR to measure TPH mRNA levels in RNA isolated from rat brain poly-A+ RNA.

Binding of upstream stimulatory factor and a cell-specific activator to the calcitonin/calcitonin gene-related peptide enhancer.

The calcitonin/calcitonin gene-related peptide (CT/CGRP) gene is selectively transcribed in thyroid C cells and neurons. We have previously shown that the rat CT/CGRP cell-specific enhancer is synergistically regulated by a helix-loop-helix (HLH) protein and the OB2 octamer-binding protein. In this report, we show that the HLH-OB2 enhancer is required for full promoter activity, even in the context of other HLH elements. Since this enhancer appears to be a major controlling element, we have characterized the HLH and OB2 DNA binding proteins. We have identified the major HLH complex as a heterodimer of the ubiquitous upstream stimulatory factor (USF)-1 and USF-2 proteins. USF bound the enhancer with a reasonably high affinity (KD 1.6 nM), comparable to other genes. Characterization of a series of mutations revealed that a portion of the HLH motif is also recognized by OB2 and confirmed that HLH activity requires OB2. We have shown that OB2 is a single DNA binding protein based on UV cross-linking studies. The 68-kDa protein-DNA complex was detected only in C cell lines, including a human C cell line that has robust HLH-OB2 enhancer activity. These results suggest that the calcitonin/CGRP gene is controlled by the combinatorial activity of a ubiquitous USF HLH heterodimer and an associated cell-specific activator.

Cell-specific glucocorticoid repression of calcitonin/calcitonin gene-related peptide transcription. Localization to an 18-base pair basal enhancer element.

We have investigated the mechanisms underlying cell-specific glucocorticoid repression of calcitonin/calcitonin gene-related peptide (CGRP) gene expression. Treatment with the synthetic glucocorticoid dexamethasone has been shown to decrease mRNA levels in the 44-2C thyroid C cell line. Nuclear run-on assays showed that dexamethasone repressed transcription 2-3-fold in 44-2C cells. In contrast, dexamethasone stimulated calcitonin/CGRP transcription 4-6-fold in the CA77 thyroid C cell line. Transient transfection assays were used to map repression of reporter gene activity in 44-2C cells to a neuroendocrine cell-specific enhancer located between -920 and -1125 base pairs (bp). Within this region, an 18-bp element was found that conferred both full basal enhancer activity and dexamethasone-dependent repression in 44-2C cells. The 18-bp region contains possible binding sites for AP-1 and helix-loop-helix transcription factors as well as a glucocorticoid receptor half-site. Colocalization of repression and enhancer activity was then investigated in other cell lines. In CA77 cells, while the 920-1125 region strongly enhanced transcription, the 18-bp region conferred only partial activation and dexamethasone had little effect on reporter gene activity. Dexamethasone did not repress the calcitonin/CGRP activity in the heterologous HeLa and Rat1 fibroblast cell lines. These results suggest that glucocorticoids repress transcription of the calcitonin/CGRP gene by inhibiting cell-specific transcription factor activity.

Identification of the tip-encoded receptor in bacterial sensing.

A chemotaxis gene encoding a protein with receptorlike properties has been identified in Salmonella typhimurium and termed tip for taxis-involved protein. Based on the stringency of DNA hybridization, the tip gene has about 75% homology with a region of the tar gene encoding the cytoplasmic domain of the aspartate receptor. Introduction of the tip gene into a smooth-swimming Escherichia coli receptor mutant (tar tsr tap) restored both chemotaxis ability on soft-agar-tryptone plates and a wild-type swimming phenotype. We have shown, by overexpressing the CheY protein, that shifting of the mutant swimming bias in the absence of receptors is insufficient to restore chemotaxis ability. This suggests that in addition to resetting the swimming bias, the tip gene product functions as a receptor. By functional criteria, we found that Tip is not a duplicate aspartate (Tar) or serine (Tsr) receptor gene. Based on behavioral properties, the S. typhimurium Tip receptor provides functional features similar to those of the E. coli Tap receptor.

Autoregulation of cell-specific MAP kinase control of the tryptophan hydroxylase promoter.

The neurotransmitter serotonin controls a wide range of biological systems, including its own synthesis and release. As the rate-limiting enzyme in serotonin biosynthesis, tryptophan hydroxylase (TPH) is a potential target for this autoregulation. Using the serotonergic neuron-like CA77 cell line, we have demonstrated that treatment with a 5-hydroxytryptamine autoreceptor agonist, CGS 12066A, can lower TPH mRNA levels and promoter activity. We reasoned that this repression might involve inhibition of MAP kinases, since 5-HT1 receptors can increase mitogen-activated protein (MAP) kinase phosphatase levels. To test this hypothesis, we first showed that the TPH promoter can be activated 20-fold by mitogen-activated extracellular-signal regulated kinase kinase kinase (MEKK), an activator of MAP kinases. This activation was then blocked by CGS 12066A. The maximal MAP kinase and CGS repression regulatory region was mapped to between -149 and -45 base pairs upstream of the transcription start site. The activation by MEKK appears to be cell-specific, because MEKK did not activate the TPH promoter in nonneuronal cell lines. At least part, but not all, of the MAP kinase responsiveness was mapped to an inverted CCAAT box that binds the transcription factor NF-Y. These data suggest a model for the autoregulation of serotonin biosynthesis by repression of MAP kinase stimulation of the TPH promoter.

Regulation of the calcitonin/calcitonin gene-related peptide gene by cell-specific synergy between helix-loop-helix and octamer-binding transcription factors.

The calcitonin/calcitonin gene-related peptide (CGRP) gene is transcribed in thyroid C-cells and a subset of neurons. We have localized sequences required for cell-specific enhancement of calcitonin/CGRP transcription in rat thyroid C-cell lines. An 18-base pair element approximately 1 kilobase pair upstream of the transcriptional start site stimulated expression of a luciferase reporter gene 50-fold in 44-2C C-cells. There was less than 2-fold stimulation in HeLa and Rat-1 cells, which do not express the endogenous calcitonin/CGRP gene. The enhancer contains potential binding sites for helix-loop-helix (HLH) and octamer transcription factors based on sequence homologies. The functional significance of these sites was shown by point mutations in the HLH and octamer motifs and by separation of the two motifs, all of which decreased enhancer activity greater than 10-fold. The involvement of HLH proteins was further shown by co-expression of the mammalian achaete-scute homologue-1 HLH protein, which activated the enhancer severalfold in HeLa cells. Electrophoretic mobility shift analyses revealed several DNA-protein complexes containing HLH and octamer-binding proteins. One octamer-binding complex (OB1) most likely contains the ubiquitous Oct-1 protein, whereas a second complex (OB2) was cell-specific. In contrast to OB1, OB2 had lower affinity for a consensus octamer motif, and its DNA binding was not affected by addition of antiserum that recognizes Oct-1 and Oct-2 proteins. In addition, we observed a large complex that appears to contain both an HLH protein and OB2. These results demonstrate that calcitonin/CGRP enhancer activity is controlled by a cell-specific synergistic activation involving HLH and octamer-binding factors.

Regulation of calcitonin gene-related peptide secretion by a serotonergic antimigraine drug.

We have investigated the regulation of calcitonin gene-related peptide (CGRP) release from trigeminal neurons by the serotonergic antimigraine drug sumatriptan. Serum levels of the neuropeptide CGRP are elevated during migraine. Treatment with the drug sumatriptan returns CGRP levels to normal coincident with the alleviation of headache. However, despite this clinical efficacy, the cellular target and mechanism of sumatriptan action are not well understood beyond the pharmacology of its recognition of the 5-HT1 class of serotonin receptors. We have used cultured trigeminal neurons to demonstrate that sumatriptan can directly repress CGRP secretion from sensory neurons. The stimulated secretion in response to depolarization or inflammatory agents was inhibited, but not the basal secretion rate. Unexpectedly, sumatriptan did not lower cAMP levels, in contrast to the classical role ascribed to the 5-HT1 receptors. Instead, activation of 5-HT1 receptors caused a slow and remarkably prolonged increase in intracellular calcium. The inhibition of CGRP secretion is attenuated by the phosphatase inhibitor okadaic acid, suggesting that sumatriptan action is mediated by calcium-recruited phosphatases. These results suggest that 5-HT1 agonists may block a deleterious feedback loop in migraine at the trigeminal neurons and provide a general mechanism by which this class of drugs can attenuate stimulated neuropeptide release.