Ets transcription factors: nuclear effectors of the Ras-MAP-kinase signaling pathway.

The Ets family of transcription factors includes nuclear phosphoproteins that are involved in cell proliferation, differentiation and oncogenic transformation. The family is defined by a conserved DNA-binding domain (the ETS-DBD), which forms a highly conserved, winged, helix-turn-helix structural motif. As targets of the Ras-MAPK signaling pathway, Ets proteins function as critical nuclear integrators of ubiquitous signaling cascades. To direct signals to specific target genes, Ets proteins interact with (other) transcription factors that promote the binding of Ets proteins to composite Ras-responsive elements.

Epidermal growth factor and Ras regulate gene expression in GH4 pituitary cells by separate, antagonistic signal transduction pathways.

We have previously demonstrated that epidermal growth factor (EGF) produces activation of the rat prolactin (rPRL) promoter in GH4 neuroendocrine cells via a Ras-independent mechanism. This Ras independence of the EGF response appears to be cell rather than promoter specific. Oncogenic Ras also produces activation of the rPRL promoter when transfected into GH4 cells and requires the sequential activation of Raf kinase, mitogen-activated protein (MAP) kinase, and c-Ets-1/GHF-1 to mediate this response. In these studies, we have investigated the interaction between EGF and Ras in stimulating rPRL promoter activity and the role of Raf and MAP kinases in mediating the EGF response. We have also examined the role of several transcription factors and used various promoter mutants of the rPRL gene in order to better define the trans- and cis-acting components of the EGF response. EGF treatment of GH4 cells inhibits activation of the rPRL promoter produced by transfection of V12Ras from 24- to 4-fold in an EGF dose-dependent manner. This antagonistic effect of EGF and Ras is mutual in that transfection of V12Ras also blocks EGF-induced activation of the rPRL promoter in a Ras dose-dependent manner, from 5.5- to 1.6-fold. Transfection of a plasmid encoding the dominant-negative Raf C4 blocks Ras-induced activation by 66% but fails to inhibit EGF-mediated activation of the rPRL promoter. Similarly, transfection of a construct encoding an inhibitory form of MAP kinase decreases the Ras response by 50% but does not inhibit the EGF response. Previous studies have demonstrated that c-Ets-1 is necessary and that GHF-1 acts synergistically with c-Ets-1 in the Ras response of the rPRL promoter. In contrast, overexpression of neither c-Ets-1 nor GHF-1 enhanced EGF-mediated activation of the rPRL promoter, and dominant-negative forms of these transcription factors failed to inhibit the EGF response. Using 5' deletion and site-specific mutations, we have mapped the EGF response to two regions on the proximal rPRL promoter. One region maps between -255 and -212, near the Ras response element, and a second maps between -125 and -54. The latter region appears to involve footprint 2, a previously identified repressor site on the rPRL promoter. Neither footprint 1 nor 3, known GHF-1 binding sites, appears to be crucial to RGF-mediated rPRL promoter activation. The results of these studies indicate that in GH4 neuroendocrine cells, rPRL gene regulation by EGF is mediated by a signal transduction pathway that is separate and antagonistic to the Ras pathway. Hence, the functional role of the Ras/Raf/MAP kinase pathway in mediating transcriptional responses to EGF and other receptor tyrosine kinase may differ in highly specialized cell types.

Interaction of basal positive and negative transcription elements controls repression of the proximal rat prolactin promoter in nonpituitary cells.

The proximal rat prolactin (rPRL) promoter contains three cell-specific elements, designated footprints I, III, and IV, which restrict rPRL gene expression to anterior pituitary lactotroph cells. Footprint II (-130 to -120) binds a factor, which we have termed F2F, present in pituitary and nonpituitary cell types. Here we demonstrate that a key role of the footprint II site is to inhibit rPRL promoter activity in nonpituitary cells, specifically, by interfering with the basal activating function of a vicinal element. Gene transfer analysis revealed 20-fold activation of the rPRL promoter in nonpituitary cell types when footprint II was either deleted or specifically mutated. Similar activation of the intact rPRL promoter was obtained by in vivo F2F titration studies. In GH4 rat pituitary cells, the footprint II inhibitory activity was masked by the redundant, positively acting cell-specific elements and was inhibitory only if the two upstream sites, footprints III and IV, were deleted. Deletion of the -112 to -80 region in the footprint II site-specific mutant background resulted in complete loss of rPRL promoter activity in both pituitary and nonpituitary cell types, mapping a basal activating element that is operative irrespective of cell type to this region. While the basal activating element imparted an activating function in a heterologous promoter assay, the footprint II sequence did not display any inherent repressor function and actually induced several minimal heterologous promoters. However, the inhibitory activity of the footprint II site was detected only if it was in context with the basal activating element. These data underscore the importance of ubiquitous activating and inhibitory factors in establishing cell-specific gene expression and further emphasize the complexity of the molecular mechanisms which restrict gene expression to specific cell types. We provide a novel paradigm to study rPRL promoter function and hormone responsiveness independently of lactotroph cell-specific requirements.

Interaction of Ets-1 and the POU-homeodomain protein GHF-1/Pit-1 reconstitutes pituitary-specific gene expression.

The pituitary-specific, POU-homeodomain factor GHF-1/Pit-1 is necessary, but not sufficient, for cell-specific expression of prolactin (PRL), growth hormone (GH), and thyrotropin. Combinatorial interactions of GHF-1 with other factors are likely to be required; however, such factors and their mechanisms of action remain to be elucidated. Here we identify Ets-1 as a factor that functionally and physically interacts with GHF-1 to fully reconstitute proximal PRL promoter activity. In contrast, Ets-2 has no effect, and the alternatively spliced GHF-2/Pit-1beta variant fails to synergize with Ets-1. The Ets-1-GHF-1 synergy requires a composite Ets-1-GHF-1 cis element and is dependent on an Ets-1-specific protein domain. Furthermore, the ancestrally related and GHF-1-dependent GH promoter, which lacks this composite element, does not exhibit this response. Finally, Ets-1, but not Ets-2, binds directly to GHF-1 and GHF-2. These data show that a functional interaction of GHF-1 and Ets-1, acting via a composite DNA element, is required to establish lactotroph-specific PRL gene expression, thus providing a molecular mechanism by which GHF-1 can discriminate between the GH and PRL genes. These results underscore the importance of transcription factors that are distinct from, but interact with, homeobox proteins to establish lineage-specific gene expression.

Functional interaction of c-Ets-1 and GHF-1/Pit-1 mediates Ras activation of pituitary-specific gene expression: mapping of the essential c-Ets-1 domain.

The mechanism by which activation of common signal transduction pathways can elicit cell-specific responses remains an important question in biology. To elucidate the molecular mechanism by which the Ras signaling pathway activates a cell-type-specific gene, we have used the pituitary-specific rat prolactin (rPRL) promoter as a target of oncogenic Ras and Raf in GH4 rat pituitary cells. Here we show that expression of either c-Ets-1 or the POU homeo-domain transcription factor GHF-1/Pit-1 enhance the Ras/Raf activation of the rPRL promoter and that coexpression of the two transcription factors results in an even greater synergistic Ras response. By contrast, the related GHF-1-dependent rat growth hormone promoter fails to respond to Ras or Raf, indicating that GHF-1 alone is insufficient to mediate the Ras/Raf effect. Using amino-terminal truncations of c-Ets-1, we have mapped the c-Ets-1 region required to mediate the optimal Ras response to a 40-amino-acid segment which contains a putative mitogen-activated protein kinase site. Finally, dominant-negative Ets and GHF constructs block Ras activation of the rPRL promoter, and each blocks the synergistic activation mediated by the other partner protein, further corroborating that a functional interaction between c-Ets-1 and GHF-1 is required for an optimal Ras response. Thus, the functional interaction of a pituitary-specific transcription factor, GHF-1, with a widely expressed nuclear proto-oncogene product, c-Ets-1, provides one important molecular mechanism by which the general Ras signaling cascade can be interpreted in a cell-type-specific manner.

Lipopolysaccharide and Raf-1 kinase regulate secretory interleukin-1 receptor antagonist gene expression by mutually antagonistic mechanisms.

Lipopolysaccharide (LPS) treatment of monocytic cells has been shown to activate the Raf-1/mitogen-activated protein kinase (MAPK) signaling pathway and to increase secretory interleukin-1 receptor antagonist (sIL-1Ra) gene expression. The significance of the activation of the Raf-1/MAPK signaling pathway to LPS regulation of sIL-1Ra gene expression, however, has not been determined. This study addresses the role of the Raf-1/MAPK signaling pathway in regulation of sIL-1Ra gene expression by LPS. Cotransfection of the murine macrophage cell line RAW 264.7 with a 294-bp sIL-1Ra promoter/luciferase construct (pRA-294-luc) and a constitutively active Raf-1 kinase expression vector (pRSV-Raf-BXB) resulted in induction of sIL-1Ra promoter activity, indicating that Raf-1, like LPS, can regulate sIL-1Ra promoter activity. An in vitro MAPK analysis indicated that both LPS treatment and pRSV-Raf-BXB transfection of RAW 264.7 cells increases p42 MAPK activity. An in vitro Raf-1 kinase assay, however, failed to detect LPS-induced Raf-1 kinase activity in RAW 264.7 cells, suggesting that in RAW 264.7 cells, Raf-1 kinase is not an activating component of the LPS signaling pathway regulating MAPK activity or sIL-1Ra promoter activity. This observation was supported by results from transfection studies which demonstrated that expression of a dominant-inhibitory Raf-1 mutant in RAW 264.7 cells does not inhibit LPS-induced MAPK activity or sIL-1Ra promoter activity, indicating that LPS-induced sIL-1Ra promoter activation occurs independent of the Raf-1/MAPK signaling pathway. In additional studies, cotransfection of RAW 264.7 cells with pRA-294-luc and increasing amounts of pRSV-Raf-BXB caused a dose-dependent inhibition of LPS-induced sIL-1Ra promoter activity, indicating that the role of the Raf-1 pathway in the regulation of sIL-1Ra promoter activity by LPS is as an antagonizer. Interestingly, LPS treatment of RAW 264.7 cells, cotransfected with pRA-294-luc and pRSV-Raf-BXB, also inhibited pRSV-Raf-BXB-induced sIL-1Ra promoter activity, suggesting that inductions of sIL-1Ra promoter activity by LPS and Raf-1 actually occur by mutually antagonistic mechanisms. In support of this conclusion, sIL-1Ra promoter mapping studies indicated that LPS and Raf-1 responses localized to different regions of the sIL-1Ra promoter. Further studies demonstrated that mutual antagonism between the LPS and Raf-1 kinase pathways is not promoter specific, as the same phenomenon is observed in assays using a c-fos enhancer/thymidine kinase promoter/luciferase construct (pc-fos-TK81-luc). Additionally, mutual antagonism with regard to sIL-1Ra promoter activity also was observed between the LPS and MEK kinase pathways, indicating that mutual antagonism can occur in more than one MAPK activation pathway.

Identification of the functional components of the Ras signaling pathway regulating pituitary cell-specific gene expression.

Ras, a small GTP-binding protein, is required for functional receptor tyrosine kinase signaling. Ultimately, Ras alters the activity of specific nuclear transcription factors and regulates novel patterns of gene expression. Using a rat prolactin promoter construct in transient transfection experiments, we show that both oncogenic Ras and activated forms of Raf-1 kinase selectively stimulated the cellular rat prolactin promoter in GH4 rat pituitary cells. We also show that the Ras signal is completely blocked by an expression vector encoding a dominant-negative Raf kinase. Additionally, using a molecular genetic approach, we determined that inhibitory forms of p42 mitogen-activated protein kinase and an Ets-2 transcription factor interfere with both the Ras and the Raf activation of the rat prolactin promoter. These findings define a functional requirement for these signaling constituents in the activation of the prolactin gene, a cell-specific gene which marks the lactotroph pituitary cell type. Further, this analysis allowed us to order the components in the Ras signaling pathway as it impinges on regulation of prolactin gene transcription as Ras-->Raf kinase-->mitogen-activated protein kinase-->Ets. In contrast, we show that intact c-Jun expression inhibited the Ras-induced activation of the prolactin promoter, defining it as a negative regulator of this pathway, whereas c-Jun was able to enhance the Ras activation of an AP-1-driven promoter in GH4 cells. These data show that c-Jun is not the nuclear mediator of the Ras signal for the highly specialized, pituitary cell-specific prolactin cellular promoter. Thus, we have defined a model system which provides an ideal paradigm for studying Ras/Raf signaling pathways and their effects on neuroendocrine cell-specific gene regulation.

Pituitary Ets-1 and GABP bind to the growth factor regulatory sites of the rat prolactin promoter.

Ets factors play a critical role in oncogenic Ras- and growth factor-mediated regulation of the proximal rat prolactin (rPRL) promoter in pituitary cells. The rPRL promoter contains two key functional Ets binding sites (EBS): a composite EBS/Pit-1 element located at -212 and an EBS that co-localizes with the basal transcription element (BTE, or A-site) located at -96. Oncogenic Ras exclusively signals to the -212 site, which we have named the Ras response element (RRE); whereas the response of multiple growth factors (FGFs, EGF, IGF, insulin and TRH) maps to both EBSs. Although Ets-1 and GA binding protein (GABP) have been implicated in the Ras and insulin responses, respectively, the precise identity of the pituitary Ets factors that specifically bind to the RRE and BTE sites remains unknown. In order to identify the Ets factor(s) present in GH4 and GH3 nuclear extracts (GH4NE and GH3NE) that bind to the EBSs contained in the RRE and BTE, we used EBS-RRE and BTE oligonucleotides in electrophoretic mobility shift assays (EMSAs), antibody supershift assays, western blot analysis of partially purified fractions and UV-crosslinking studies. EMSAs, using either the BTE or EBS-RRE probes, identified a specific protein-DNA complex, designated complex A, which contains an Ets factor as determined by oligonucleotide competition studies. Using western blot analysis of GH3 nuclear proteins that bind to heparin-Sepharose, we have shown that Ets-1 and GABP, which are MAP kinase substrates, co-purify with complex A, and supershift analysis with specific antisera revealed that complex A contains Ets-1, GABPalpha and GABPbeta1. In addition, we show that recombinant full-length Ets-1 binds equivalently to BTE and EBS-RRE probes, while recombinant GABPalpha/beta preferentially binds to the BTE probe. Furthermore, comparing the DNA binding of GH4NE containing both Ets-1 and GABP and HeLa nuclear extracts devoid of Ets-1 but containing GABP, we were able to show that the EBS-RRE preferentially binds Ets-1, while the BTE binds both GABP and Ets-1. Finally, UV-crosslinking experiments with radiolabeled EBS-RRE and BTE oligonucleotides showed that these probes specifically bind to a protein of approximately 64 kDa, which is consistent with binding to Ets-1 (54 kDa) and/or the DNA binding subunit of GABP, GABPalpha (57 kDa). These studies show that endogenous, pituitary-derived GABP and Ets-1 bind to the BTE, whereas Ets-1 preferentially binds to the EBS-RRE. Taken together, these data provide important insights into the mechanisms by which the combination of distinct Ets members and EBSs transduce differential growth factor responses.

The ras and protein kinase A pathways are mutually antagonistic in regulating rat prolactin promoter activity.

In an attempt to characterize the ras signaling pathway, we studied the effects of expression vectors encoding the valine 12 mutant ras oncogene on rat prolactin (rPRL) promoter activity. Using this approach we have been able to dissect the interplay between the ras and the protein kinase A (PKA) pathways as they relate to neuroendocrine gene activation. Here we show that the ras oncogene product induces rPRL promoter activity selectively from 5- to 14-fold in GH4 rat pituitary tumor cells, whereas it has a minimal effect on the SV40 early promoter and no effect on the Rous sarcoma virus (RSV) or rat growth hormone promoters. By contrast, an inactivated form of ras (N-17 ras) did not stimulate the rPRL promoter, but rather inhibited it to 40% of control. Of note, activation of the PKA pathway by two different methods decreased the fold activation mediated by ras by at least 50%, whereas inhibition of the PKA pathway accentuated ras activation of the rPRL promoter. Although rPRL promoter activity is consistently induced by PKA activation in control GH4 cells, acute ras oncogene expression inhibited forskolin induction of rPRL promoter activity. Moreover, this ras-mediated interference of the forskolin activation of rPRL promoter activity was also noted in GH4 cells stably expressing ras. Taken together, these data show that the valine 12 ras oncogene activates the rPRL promoter selectively and, more importantly, that the ras and PKA signaling pathways are mutually antagonistic with respect to specific transcriptional activation of a neuroendocrine gene.

Conserved mechanisms of Ras regulation of evolutionary related transcription factors, Ets1 and Pointed P2.

Cell transformation by the Ras oncogene is mediated by members of the ets gene family. To analyse the mechanisms of regulation, we have studied activation of several ets factors by Ras expression. We show that expression of Ha-Ras strongly activates the Ets1 p68 and p54 isoforms and Ets2 in F9 EC cells. We have mapped the Ras responsive elements of Ets1 p68 to two domains, RI+II and RIII. Mutation of threonine 82 to alanine in RI+II abolishes both Ras activation and phosphorylation by MAP kinase. Threonine 82 is part of a sequence that is conserved in Drosophila Pointed P2, an ets protein that has been shown both genetically and biochemically to mediate Ras signalling in Drosophila cells. We extend the comparison of these evolutionary related proteins by showing that Pointed P2 is activated by Ras in mammalian cells and mutation of the homologous threonine abolishes activation. Furthermore, we show that Pointed P2 resembles Ets1, in that it has conserved sequences in a similar position adjacent to the ets DNA binding domain that negatively auto-regulates DNA binding. These results go towards showing that the Drosophila Pointed and vertebrate Ets1 are evolutionary related proteins that have remarkably conserved Ras regulatory mechanisms downstream from MAP kinase.

Pit-1beta reduces transcription and CREB-binding protein recruitment in a DNA context-dependent manner.

Many transcription factors are expressed as multiple isoforms with distinct effects on the regulation of gene expression, and the functional consequences of structural differences between transcription factor isoforms may allow for precise control of gene expression. The pituitary transcription factor isoforms Pit-1 and Pit-1beta differentially regulate anterior pituitary hormone gene expression. Pit-1 is required for the development of and appropriate hormone expression by anterior pituitary somatotrophs and lactotrophs. Pit-1beta differs structurally from Pit-1 by the splice-insertion of the 26-residue beta-domain in the trans-activation domain, and it differs functionally from Pit-1 in that it represses expression of the prolactin promoter in a cell-type specific manner. In order to identify signal and promoter context requirements for repression by Pit-1beta, we examined its function in the presence of physiological regulatory signals as well as wild-type and mutant Pit-1-dependent target promoters. Here, we demonstrate that Pit-1beta impairs recruitment of cAMP response element-binding protein (CREB)-binding protein to the promoters that it represses. In addition, we show that repression of target promoter activity, reduction in promoter histone acetylation, and decrease of CREB-binding protein recruitment all depend on promoter context. These findings provide a mechanism for promoter-specific repression by Pit-1beta.

Analysis of rat prolactin promoter sequences that mediate pituitary-specific and 3',5'-cyclic adenosine monophosphate-regulated gene expression in vivo.

To determine the rat PRL (rPRL) promoter sequences that mediate pituitary-specific and cAMP-induced gene expression in vivo, various lengths of the rPRL promoter were ligated to the luciferase reporter gene and introduced into pituitary and non-pituitary cell lines. A 30-fold increase in rPRL promoter activity was observed in GH4 rat pituitary tumor cells compared to nonpituitary Rat2 fibroblast and HeLa cervical carcinoma cells. About 45% of this cell-specific promoter activity was competed by a plasmid containing the -67 to -45 rPRL promoter region, which is the most proximal binding site for a lactotroph-specific factor. Compared to a -425 rPRL construct, transfection with rPRL 5'-end points of -212, -178, and -127 contained 23%, 45%, and 1%, respectively, of luciferase activity. Forskolin stimulation resulted in a 10-fold induction of all the rPRL promoter fragments tested. Of note, a -127 deletion which was devoid of any basal promoter activity was also induced 10-fold by forskolin. The forskolin effect was abolished when GH4 rat pituitary cells were cotransfected with a plasmid encoding a protein kinase A inhibitor, indicating protein kinase A is involved in the activation mechanism. These data document that both positive and negative effectors influence basal rPRL promoter activity. Furthermore, the minimum sequences required for pituitary-specific rPRL promoter activity are altered by intracellular cAMP levels. Taken together, the data indicate that hormone-activated and cell-specific factors may interact to establish a particular setpoint for rPRL gene expression.

Reconstitution of the protein kinase A response of the rat prolactin promoter: differential effects of distinct Pit-1 isoforms and functional interaction with Oct-1.

PRL gene transcription is primarily regulated by dopamine, which lowers cAMP levels and inhibits protein kinase A (PKA) activity. Current data indicate that the cAMP/PKA response maps to the most proximal Pit-1/Pit-1beta binding site footprint I (FP I) on the rat PRL (rPRL) promoter. Pit-1, a POU-homeo domain transcription factor, is specifically expressed in the anterior pituitary and is required both for the normal development of anterior pituitary cell types, somatotrophs, lactotrophs, and thyrotrophs, and for the expression of their hormones: GH, PRL, and TSHbeta. Pit-1 has been shown to functionally interact, via FP I, with several transcription factors, including Oct-1, a ubiquitous homeobox protein, and thyrotroph embryonic factor, which is found in lactotrophs, to activate basal rPRL promoter activity. Pit-1beta/GHF-2, a distinct splice isoform of Pit-1, acts to inhibit Ras-activated transcription from the rPRL promoter, which is mediated by a functional interaction between Pit-1 and Ets-1 at the most distal Pit-1 binding site (FP IV). In this manuscript we show 1) that the Pit-1beta isoform not only fails to block PKA activation, but is, in fact, a superior mediator of the PKA response; 2) that the PKA response requires intact POU-specific and POU-homeo domains of Pit-1; and 3) that Oct-1, but not thyrotroph embryonic factor, functions as a Pit-1-interacting factor to mediate an optimal PKA response.

Somatostatin acts by inhibiting the cyclic 3',5'-adenosine monophosphate (cAMP)/protein kinase A pathway, cAMP response element-binding protein (CREB) phosphorylation, and CREB transcription potency.

Somatostatin (SRIF) was discovered as an inhibitor of GH secretion from pituitary somatotroph cells. SRIF analogs are very effective agents used to treat neuroendocrine tumors and are now being used with increasing frequency in clinical trials to treat more aggressive malignancies. However, the cellular components mediating SRIF signal transduction remain largely unknown. We have stably overexpressed the SRIF type 2 receptor (SST2) in GH4 rat somatomammotroph cells, establishing a physiologically relevant model system. In this model, the SRIF analog, BIM23014, inhibited forskolin-induced cAMP accumulation, protein kinase A activation, cAMP response element-binding protein phosphorylation, and Pit-1/GHF-1 promoter activation in an okadaic acid-insensitive manner. Pertussis toxin inhibited the effects of BIM23014, documenting that SST2 signaling was coupled to Gi. Moreover, the inhibitory effects of BIM23014 were reversed by overexpression of protein kinase A catalytic subunit, indicating that SRIF does not act via serine/threonine phosphatases, but, rather, by lowering protein kinase A activity. These data define the components of the SRIF/SST2 receptor signaling pathway and provide important mechanistic insights into how SRIF controls neuroendocrine tumors. As SRIF analogs are effective antitumor agents, and many other related compounds are in development, the knowledge gained here will further our understanding of their mechanism of action in other malignancies as well.

Structure-function analysis of the rat prolactin promoter: phasing requirements of proximal cell-specific elements.

Expression of PRL, a member of the GH family of genes, is restricted to the lactotroph cells of the anterior pituitary. The proximal promoter of the rat PRL (rPRL) gene contains four factor-binding sites. Three nonadjacent elements, footprints (FP) I, III, and IV, are separated by an integral number of helical turns and bind a pituitary-specific factor, LSF-1. FP II binds another factor present in pituitary and nonpituitary cells. The mechanisms by which DNA-bound proteins influence RNA polymerase-II activity over large distances are not fully understood, but protein-protein interactions, with looping of intervening DNA, may bring distant sites into close proximity. Here, we demonstrate, using protein titration studies, that LSF-1 binds to the most proximal FP I element with the highest affinity, whereas it binds the more distal elements, FP III and FP IV, with progressively lower affinities. Time-course and salt-sensitivity studies reveal that binding of LSF-1 to all three pituitary-specific rPRL promoter sites occurs rapidly (less than or equal to 1 min) and requires fairly high salt concentrations (greater than or equal to 300 mM KCl) to destabilize protein-DNA interactions. Moreover, once bound, the pituitary nuclear factor(s) induces a conformational change in rPRL DNA structure with greatly delayed kinetics (greater than 15 min) and at a different salt concentration than are required for simply factor binding. Taken together, these data suggest a model in which LSF-1 initially binds fairly rapidly to multiple nonadjacent elements and then interacts with itself or other DNA-bound proteins much more slowly, possibly looping or bending the rPRL promoter.(ABSTRACT TRUNCATED AT 250 WORDS)

Upstream stimulatory factor, a basic-helix-loop-helix-zipper protein, regulates the activity of the alpha-glycoprotein hormone subunit gene in pituitary cells.

In an effort to determine whether basic-helix-loop-helix (bHLH) proteins are important in pituitary-specific expression of the alpha-glycoprotein hormone subunit gene, we examined the effect of the dominant negative HLH protein, Id, on the activity of the alpha-subunit gene promoter in pituitary cells. Id over-expression reduces the expression of alpha-subunit reporter genes in either alpha T3-1 gonadotrope-derived or alpha TSH thyrotrope-derived cells. A deletion fragment containing nucleotides from -131 to +44 of the human alpha-subunit promoter is inhibited to a similar degree as a -244 to +44 fragment in alpha T3-1 cells. Nuclear proteins in alpha T3-1 cells bind two potential bHLH protein binding sites (E-boxes, alpha EB1 and alpha EB2) present in this fragment but not to mutations that specifically alter only these sequences. An antibody-specific for upstream stimulatory factor, a widely expressed bHLH-leucine zipper protein, is able to inhibit factor binding to the alpha EB2 sequences but not the alpha EB1 site. Mutating the alpha EB1 element of the alpha-subunit promoter decreases basal activity of this promoter to about 42% of control levels in alpha T3-1 cells. A mutation that abolishes upstream stimulatory factor binding, either alone or in combination with the alpha EB1 mutation, reduces basal activity of the promoter to approximately 21% of control levels in alpha T3-1 cells and abolishes the decrease in promoter activity seen when Id is overexpressed. These results demonstrate that the bHLH family of proteins are important regulators of alpha-subunit gene expression in pituitary cells.

The Ras and protein kinase C signaling pathways are functionally antagonistic in GH4 neuroendocrine cells.

Oncogenic Ras appears to act via protein kinase C (PKC)-dependent and PKC-independent pathways. In several systems, oncogenic Ras cooperates with c-Jun to activate gene transcription from promoters containing an AP-1 site by augmenting phosphorylation of the transcriptional activation domain of c-Jun. We have previously shown that oncogenic valine 12 Ras and PKA each separately activate the rat PRL (rPRL) promoter but together are mutually antagonistic. The goal of this study was to determine whether oncogenic Ras acts through PKC and c-Jun to activate transcription of an rPRL-luciferase reporter construct transiently transfected into GH4 rat pituitary cells. Our results show that phorbol 12-myristate 13-acetate (TPA) activates rPRL promoter activity through PKC, and that TPA activation of PKC diminishes the Ras response in a dose-dependent manner. Additionally, inhibition of PKC with staurosporine does not block the oncogenic Ras effect. Similarly, rPRL promoter activity in GH4 cells expressing oncogenic Ras fails to respond to TPA activation of PKC. Finally, cotransfection of a c-Jun expression vector results in inhibition of basal, TPA, and oncogenic Ras-stimulated activity of the rPRL promoter. Thus, we show that the mechanism of Ras signaling does not involve PKC, and that PKC does not signal via Ras. Taken together, these results verify that the Ras and PKC signaling pathways are separate and mutually antagonistic, and that c-Jun is not the nuclear mediator of either the Ras or PKC signal. These findings emphasize the possibility that the roles and/or functions of specific components in signaling pathways may be different in distinct cell types.

Reconstitution of protein kinase A regulation of the rat prolactin promoter in HeLa nonpituitary cells: identification of both GHF-1/Pit-1-dependent and -independent mechanisms.

Expression of the rat PRL (rPRL) gene is highly restricted to pituitary lactotroph cells and is induced by the cAMP-dependent protein kinase A (PKA) pathway. Current data indicate that this PKA effect requires at least one of the redundant pituitary-specific elements of the proximal rPRL promoter, suggesting the involvement of the pituitary-specific transcription factor, GHF-1/Pit-1. To directly determine whether GHF-1 is necessary and sufficient to mediate the PKA activation of the rPRL promoter, we established a cotransfection reconstitution assay whereby the activity of an intact and site-specific mutants of the (-425 to +73) rPRL promoter-luciferase reporter gene was reconstituted by cotransfecting expression vectors encoding for either the PKA beta catalytic subunit, GHF-1, or both, into HeLa nonpituitary cells. Cotransfection of PKA beta alone significantly stimulated rPRL promoter activity in HeLa cells in a GHF-1-independent manner, and this PKA beta effect was mapped to the most proximal GHF-1 site [footprint (FP) I; -67/-36]. Site-specific alterations of either FP II (-130/-120), or of the basal transcription element (BTE; -112/-80), did not significantly affect the PKA beta response. As expected, the transactivation effect of cotransfected GHF-1 mapped to the GHF-1/Pit-1 binding sites, FP I and/or FP III, of the rPRL promoter. Finally, cotransfection of PKA beta and GHF-1 resulted in a marked synergistic response of the rPRL promoter, and this response also localized to the FP I site. These data confirm not only that GHF-1/Pit-1 and the FP I site are involved in mediating the PKA response, but also imply that a distinct and possibly ubiquitous factor is involved by binding to FP I and functionally interacting with GHF-1 to modulate PKA beta regulation of the rPRL promoter.

Modification of DNA topoisomerase II activity via direct interactions with the cyclic adenosine-3',5'-monophosphate response element-binding protein and related transcription factors.

DNA topoisomerase II (topo II) is an essential nuclear enzyme which catalyzes the interconversions of various forms of DNA. As predicted from the human topo II cDNA, the enzyme contains a potential leucine zipper protein dimerization motif. We therefore tested whether topo II could enter protein-protein interactions with other better characterized leucine zipper-containing proteins and determined if these interactions could modify topo II enzymatic activity in vitro. By far Western analyses, a large C-terminal fragment of human topo II was shown to interact with the DNA binding and dimerization regions of either cAMP response element binding protein (CREB) or the activating transcription factor-2. The C-terminal topo II fragment also interacted with full-length c-Jun, but not with full-length c-Fos. Using CREB as a prototype, the effect of this interaction on various topo II catalytic activities was assessed in vitro. CREB, at a 1- to 10-fold molar excess relative to topo II, inhibited site-specific DNA cleavage activity on a 242-base pair fragment of the human alpha-glycoprotein hormone subunit gene promoter. Very high CREB concentrations (400-fold excess) apparently inhibited topo II DNA relaxation activity, but this result was likely a direct effect of CREB on the topology of the DNA substrate. More interestingly, a 10-fold molar excess of CREB stimulated topo II decatenation activity, the essential function of this enzyme in cell division. This stimulatory effect could also be elicited by c-Jun, which interacts with topo II, but not by c-Fos, which does not bind topo II in our in vitro assay. Since similar amounts of CREB reduced the abundance of topo II DNA cleavage products from the human alpha-CG promoter yet also stimulated decatenation activity, it can be concluded that either: 1) CREB stimulated the religation rate of topo II; or 2) CREB directed topo II to a new cleavage site present on the decatenation substrate but not present on the limited alpha-CG promoter. The structural requirements for topo II protein-protein interactions were also investigated. Site-directed mutations which destroyed the putative topo II leucine zipper did not disrupt topo II protein-protein interactions. Since the putative leucine zipper in topo II does not appear to mediate protein-protein interactions, we propose that an alternate as yet uncharacterized structure is involved in the association of topo II with itself and other regulatory proteins.

Cyclic adenosine 3',5'-monophosphate activation of the rat prolactin promoter is restricted to the pituitary-specific cell type.

Pituitary lactotroph cell function and PRL gene expression are highly regulated by the cAMP-protein kinase-A (PKA) pathway. To further our understanding of the molecular mechanisms by which cAMP/PKA regulates rat (r) PRL promoter activity and to determine whether cAMP regulation is cell type specific, we 1) transected intact (-425), internal and 5'-deletion, and site-specific mutants of the rPRL promoter ligated to the firefly luciferase reporter gene into both pituitary and nonpituitary cell lines; and 2) assessed the role of the cAMP-cAMP response element-binding protein (CREB) pathway in GH4 rat pituitary cells. The data show that deleting the rPRL promoter from -425 to -116 did not abolish cAMP regulation, implying that proximal elements, such as the basal transcription element (-112/-80) or the pituitary-specific footprint (FP) I (-67/-45), mediate the cAMP response. However, nucleotide changes within FP I or FP II (-130/-120) did not alter the rPRL promoter response to 1 microM forskolin (FSK), despite the 77% and 26% reductions in basal rPRL promoter activity caused by these mutations, respectively. Furthermore, internal deletion of either the basal transcription element of FP I element also failed to affect cAMP regulation of the rPRL promoter, again despite the 90% and 93% reductions in basal promoter activity by these deletions, respectively. Since these internal deletion constructs otherwise contain rPRL promoter sequences from -425 to +73, including the up-stream pituitary-specific FPs III and IV, the data suggest that any one of these cell-specific elements is capable of imparting cAMP regulation to the proximal rPRL promoter. To directly test the implication that the cAMP response of the rPRL promoter is restricted to the pituitary-specific cell type, we took advantage of a 5'-deletion mutant truncated at position -116 and a FP II site-specific mutant, since constructs containing these rPRL promoters are active in nonpituitary cells. Despite the 6.6- and 18.5-fold stimulations over wild-type rPRL promoter activity in nonpituitary cells, respectively, these mutations remained completely unresponsive to FSK treatment. To document that the cAMP-CREB pathway was functional in GC/GH4 rat pituitary cells, CREB was affinity purified from GC rat pituitary cells, and DNase-I protection studies showed that it does not bind to the proximal rPRL promoter. Also, the human glycoprotein alpha-subunit promoter was induced 10-fold by FSK in GH4 rat pituitary cells.(ABSTRACT TRUNCATED AT 400 WORDS)