Analysis of the role of the mitogen-activated protein kinase in mediating cyclic-adenosine 3',5'-monophosphate effects on prolactin promoter activity.

The mechanisms mediating cAMP effects to stimulate transcription of the PRL gene have been examined. Treatments that elevate intracellular cAMP concentrations were found to stimulate the mitogen-activated protein kinase (MAPK) in GH(3) cells. Elevated cAMP was also found to stimulate activation of the GTP-binding protein, Rap1. Rap1GAP1 reduced cAMP-induced phosphorylation of MAPK, offering evidence that Rap1 may play a role in mediating activation of MAPK. Treatment of GH(3) cells with PD98059, an inhibitor of the MAPK pathway, reduced the ability of forskolin to activate a PRL reporter gene, providing evidence that MAPK contributes to cAMP-mediated effects on the PRL promoter. As previous studies have implicated Ets factor binding sites within the PRL promoter in mediating responses to MAPK, we expected that the Ets sites would also play a role in cAMP responsiveness. Surprisingly, mutation of all of the consensus Ets factor binding sites in the proximal PRL promoter greatly reduced responsiveness to epidermal growth factor (EGF) and TRH but did not reduce cAMP responsiveness. Experiments using an expression vector for adenovirus 12S E1a provided evidence that the coactivators, CREB binding protein and/or p300, probably play a role in cAMP responsiveness of the PRL promoter. Interestingly, the ability of a GAL4-p300 fusion protein to enhance reporter gene activity was stimulated by cAMP in a MAPK-dependent manner. These findings provide evidence for a model for cAMP-induced PRL transcription involving Rap1-induced MAPK activity leading to stimulation of the transcriptional coactivators, CBP and p300.

A point mutation in the LIM domain of Lhx3 reduces activation of the glycoprotein hormone alpha-subunit promoter.

Lhx3, a member of the LIM homeodomain family of transcription factors, is required for development of the pituitary in mice. A recent report has described a point mutation in the human LHX3 gene that is associated with a combined pituitary hormone disorder. The mutation is predicted to lead to the replacement of a tyrosine residue with a cysteine in the second LIM domain of LHX3. We have characterized the effects of this point mutation (Y114C) when analyzed in the context of the mouse Lhx3 coding sequence. Mobility shift assays demonstrated that the Lhx3 Y114C mutant is capable of binding DNA, although a decrease in the formation of a specific complex was observed. Transfection assays using an expression vector for either full-length Lhx3 or a GAL4-Lhx3 LIM domain fusion provided evidence that the Lhx3 Y114C mutant has a decreased ability to stimulate transcription. In particular, a GAL4-Lhx3 Y114C LIM mutant was unable to support Ras responsiveness of a modified glycoprotein hormone alpha-subunit reporter gene. Protein interaction studies suggest that the Y114C mutation may modestly reduce binding to the POU transcription factor, Pit-1. Interestingly, the Y114C mutation essentially abrogated binding to the putative co-activator/adapter, selective LIM-binding protein. The findings provide insights into the mechanisms mediating transcriptional activation by Lhx3 and suggest that the observed phenotype of the human mutation probably involves reduced transcriptional activity of the mutant LHX3.

Thyrotropin-releasing hormone stimulates phosphorylation of the epidermal growth factor receptor in GH3 pituitary cells.

TRH has been found to stimulate tyrosine phosphorylation of the epidermal growth factor (EGF) receptor. A specific EGF receptor kinase inhibitor, tyrphostin AG1478, substantially reduced TRH-stimulated tyrosine phosphorylation of the EGF receptor. TRH-induced EGF receptor phosphorylation was found to lead to the recruitment of the adapter proteins Grb2 and Shc. TRH treatment also led to phosphorylation of the related receptor tyrosine kinase, HER2. HER2 activation likely contributes to downstream signaling events and enhances EGF receptor action. TRH-induced tyrosine phosphorylation of the EGF receptor was reduced by incubation with a protein kinase C (PKC) kinase inhibitor, GF109203X. EGF receptor phosphorylation was required for full TRH-induced activation of mitogen-activated protein kinase (MAPK) and stimulation of specific transcriptional responses.

Identification of a conserved protein that interacts with specific LIM homeodomain transcription factors.

Lhx3, a member of the LIM homeodomain family of transcription factors, is required for development of the pituitary and is implicated in the transcription of pituitary-specific hormone genes. In this report we describe a novel gene product, SLB, that selectively interacts with Lhx3 and the closely related LIM factor, Lhx4. The SLB cDNA encodes a 1749-residue protein that contains seven WD40 repeats near the amino terminus and a putative nuclear localization signal and does not contain other recognizable motifs. SLB is expressed in a tissue-specific manner with the highest concentrations of SLB mRNA in the testis and pituitary cells. We demonstrate that SLB specifically binds to Lhx3 and Lhx4 with high affinity both in vitro and in vivo. SLB has much lower affinity or no detectable affinity for other LIM domains. An expression vector for a fragment of SLB containing the LIM-interaction domain was shown to reduce expression of Lhx3-responsive reporter genes. The ability of the LIM-interacting domain of SLB to alter reporter gene activity as well as the tissue-specific expression and the specificity of SLB binding to LIM factors suggest a possible role in modulating the transcriptional activity of specific LIM factors.

MRG1 binds to the LIM domain of Lhx2 and may function as a coactivator to stimulate glycoprotein hormone alpha-subunit gene expression.

Tissue-specific expression of the alpha-subunit gene of glycoprotein hormones involves an enhancer element designated the pituitary glycoprotein basal element, which interacts with the LIM homeodomain transcription factor, Lhx2. In the present studies we have explored the function of the LIM domain of Lhx2 in stimulating alpha-subunit transcription. When fused to the GAL4 DNA-binding domain, the LIM domain of Lhx2 was shown to contain a transcriptional activation domain. Furthermore, in the context of an alpha-subunit reporter gene in which a GAL4-binding site replaced the pituitary glycoprotein basal element, the LIM domain enhanced both basal and Ras-mediated transcription. In addition, a synergistic response to Ras activation was observed when the Lhx2 LIM domain and the transactivation domain of Elk1 are directed to a minimal reporter gene. A yeast two-hybrid screen identified the recently described melanocyte-specific gene-related gene 1 (MRG1) as an Lhx2 LIM-interacting protein. MRG1 was shown to bind Lhx2 in vitro, and a co-immunoprecipitation assay provided evidence that endogenous MRG1 forms a complex with Lhx2 in alphaT3-1 cells. Expression of MRG1 in alphaT3-1 cells enhanced alpha-subunit reporter gene activity. MRG1 was also shown to bind in vitro to the TATA-binding protein and the transcriptional coactivator, p300. These data suggest a model in which the Lhx2 LIM domain activates transcription through interaction with MRG1 leading to recruitment of p300/CBP and the TATA-binding protein.

Regulation of glycoprotein hormone alpha-subunit gene expression.

The appropriate, regulated expression of the glycoprotein hormone subunit genes is required to enable the biosynthesis of luteinizing hormone, follicle-stimulating hormone, thyroid-stimulating hormone, and chorionic gonadotropin. We have focused our attention on mechanisms mediating regulated transcription of the common alpha-subunit gene. Our studies have examined both the signaling mechanisms and the DNA elements and transcription factors that are important for alpha-subunit expression. Our initial efforts involved characterization of DNA elements of the alpha-subunit gene important for basal and GnRH-stimulated expression. Clustered point mutation analysis identified two different, unrelated sequences that play a role in alpha-subunit transcription. When tested as multiple copies on a minimal promoter, one of these elements was sufficient to permit a response to GnRH, while the other enhanced basal expression. Therefore, we designated these DNA elements as the GnRH-response element (GnRH-RE) and the pituitary glycoprotein hormone basal element (PGBE), respectively. The GnRH-RE contains a consensus binding site for the Ets family of transcription factors. As several Ets factors have been shown to mediate transcriptional responses to the mitogen-activated protein kinase (MAPK) pathway, we investigated the possibility that GnRH effects on alpha-subunit transcription may involve the MAPK cascade. We found that GnRH can indeed activate MAPK and that MAPK activation is sufficient and necessary for transcriptional activation of the alpha-subunit gene. Efforts to further characterize proteins that interact with the PGBE led to the cloning of a LIM-homeodomain transcription factor designated LH-2. Recombinant LH-2 selectively binds to the PGBE in vitro. Transfection experiments have shown that an expression vector for LH-2 can activate the alpha-subunit promoter in heterologous cells. LH-2 appears to be a component of the endogenous factors that bind to the PGBE. Thus, LH-2 appears to be an excellent candidate as a factor responsible for basal expression of the alpha-subunit gene. Overall, these studies have contributed to identification of molecular components important for regulated expression of the glycoprotein hormone alpha-subunit gene.

A role for the mitogen-activated protein kinase in mediating the ability of thyrotropin-releasing hormone to stimulate the prolactin promoter.

The hypothalamic hormone, TRH, stimulates PRL secretion and gene transcription. We have examined the possibility that the mitogen-activated protein kinase (MAPK) may play a role in mediating TRH effects on the PRL gene. TRH was found to stimulate sustained activation of MAPK in PRL-producing, GH3 cells, consistent with a possible role in transcriptional regulation. A kinase-defective, interfering MAPK kinase (MAPKK) mutant reduced TRH induction of the PRL promoter. Treatment with the MAPKK inhibitor, PD98059, blocked TRH-induced activation of MAPK and also reduced TRH induction of a PRL-luciferase reporter gene, confirming that MAPK activation is necessary for TRH effects on PRL gene expression. Previous studies have demonstrated that the PRL promoter contains binding sites for members of the Ets family of transcription factors, which are important for mediating MAPK responsiveness of the PRL promoter. Mutation of specific Ets sites within the PRL promoter reduced responsiveness to both TRH and MAPK. The finding that DNA elements required for MAPK responsiveness of the PRL gene colocalize with DNA elements required for TRH responsiveness further supports a role for MAPK in mediating TRH effects on the PRL gene. We also explored the signaling mechanisms that link the TRH receptor to MAPK induction. Occupancy of the TRH receptor results in activation of protein kinase C (PKC) as well as increases in the concentration of Ca2+ due to release from intracellular stores and entry of Ca2+ through Ca2+ channels. A PKC inhibitor, GF109203X, and an L-type Ca2+ channel blocker, nimodipine, both partially reduced TRH-induced MAPK activation and PRL promoter activity. The effects of the two inhibitors were additive. These studies are consistent with a signaling pathway involving PKC- and Ca2+-dependent activation of MAPK, which leads to phosphorylation of an Ets transcription factor and activation of the PRL promoter.

Characterization of DNA regions mediating the ability of Ca2+/calmodulin dependent protein kinase II to stimulate prolactin promoter activity.

The ability of Ca2+/calmodulin-dependent protein kinases (CaMKs) to regulate transcription of the rat prolactin (PRL) gene has been examined. We found that KN-62, a potent inhibitor of CaM kinases, blunted the ability of TRH to activate the prolactin promoter. Transfection experiments using expression plasmids for constitutively active forms of CaMKI, CaMKII, or CaMKIV show that CaMKII is the most effective activator of prolactin promoter expression. Deletion studies demonstrated that the upstream boundary of sequences necessary to respond to CaMKII is located within the distal enhancer of the prolactin gene. Neither the distal enhancer alone nor the proximal region of the prolactin gene are sufficient to mediate a response to CaMKII. Mutational analysis suggests that several Pit-1 binding sites contribute to CaMKII responsiveness. These findings suggest that CaMKII responsiveness of the prolactin promoter requires multiple factor binding sites in both the distal and proximal regions of the gene.

Regulation of activating transcription factor-1 and the cAMP response element-binding protein by Ca2+/calmodulin-dependent protein kinases type I, II, and IV.

The ability of activating transcription factor-1 (ATF1) or the cAMP response element-binding protein (CREB) to enhance transcription can be stimulated by increases in intracellular Ca2+ concentrations. To identify protein kinases which may mediate the ability of Ca2+ to activate these transcription factors, we compared the ability of constitutively active forms of several Ca2+/calmodulin-dependent protein kinases (CaM kinases) to activate ATF1 or CREB. We find that constitutively active CaM kinase I and IV can activate both ATF1 and CREB. In addition, expression vectors for full-length CaM kinase I and IV were able to augment the ability of Ca2+ influx to activate ATF1 or CREB consistent with a role for these kinases in mediating transcriptional responses to Ca2+ signaling. In contrast, CaM kinase II was unable to activate either ATF1 or CREB. These findings provide a potential mechanism that may permit variation in the ability of ATF1 and CREB to respond to changes in intracellular Ca2+ concentrations depending on differences in the relative concentrations of specific CaM kinases.

Two enzymes, both of which process recombination intermediates, have opposite effects on adaptive mutation in Escherichia coli.

Reversion of a lac- frameshift allele carried on an F' episome in Escherichia coli occurs at a high rate when the cells are placed under lactose selection. Unlike Lac+ mutations that arise during nonselective growth, the production of these adaptive mutations requires the RecA-RecBCD pathway for recombination. In this report, we show that enzymes that process recombination intermediates are involved in the mutagenic process. RuvAB and RecG, E. coli's two enzymes for translocating Holliday junctions, have opposite effects: RuvAB is required for RecA-dependent adaptive mutations, whereas RecG inhibits them.

In vivo mutational analysis of the DNA binding domain of the tissue-specific transcription factor, Pit-1.

Pit-1 is a member of the POU family of transcription factors, which contain a bipartite DNA binding domain. The DNA binding domain consists of a POU-specific domain and a POU homeodomain. Each of the subdomains can interact with DNA independently, but both subdomains are required for high affinity, sequence-specific DNA binding. To examine the contributions of individual amino acids to the function of the DNA binding domain of Pit-1, we developed an approach involving random, in vitro mutagenesis followed by functional screening in Saccharomyces cerevisiae. Using this strategy, we identified a number of point mutations that altered the function of the Pit-1 DNA binding domain. Mutations that altered Pit-1 function were found in both the POU-specific and the POU homeodomain. Most of the mutations involve amino acid residues that are conserved in POU factors. One of the more frequent kinds of mutation affected residues located in the hydrophobic core of the protein. Another common mutation involved amino acids that are thought to make specific contacts with DNA. These mutations define a number of amino acid residues that are important for the function of the DNA binding domain of Pit-1.

A composite Ets/Pit-1 binding site in the prolactin gene can mediate transcriptional responses to multiple signal transduction pathways.

Binding sites for the tissue-specific transcription factor, Pit-1, are required for basal and hormonally induced prolactin gene transcription. Although Pit-1 is phosphorylated in response to several signaling pathways, the mechanism by which Pit-1 contributes to hormonal induction of gene transcription has not been defined. Recent reports suggest that phosphorylation of Pit-1 may not be required for hormonal regulation of the prolactin promoter. Analysis of the contribution of individual Pit-1 binding sites has been complicated due to the fact that some of the elements appear to be redundant. To better understand the role of Pit-1 sites in mediating hormonal regulation of the prolactin gene, we have performed enhancer tests using the three most proximal Pit-1 binding sites of the rat prolactin gene which are designated the 1P, 2P, and 3P sites. The results demonstrate that multimers of the 3P Pit-1 binding site are much more responsive to several hormonal and intracellular signaling pathways than multimers of the 1P or 2P sites. The 3P DNA element was found to contain a consensus binding site for the Ets family of proteins. Mutation of the Ets binding site greatly decreased the ability of epidermal growth factor, phorbol esters, Ras, or the Raf kinase to induce reporter gene activity. Mutation of the Ets site had little effect on basal enhancer activity. In contrast, mutation of the consensus Pit-1 binding site in the 3P element essentially abolished all basal enhancer activity. Overexpression of Ets-1 in GH3 pituitary cells enhanced both basal and Ras induced activity from the 3P enhancer. These data describe a composite element in the prolactin gene containing binding sites for two different factors and the studies suggest a mechanism by which Ets proteins and Pit-1 functionally cooperate to permit transcriptional regulation by different signaling pathways.

A role for mitogen-activated protein kinase in mediating activation of the glycoprotein hormone alpha-subunit promoter by gonadotropin-releasing hormone.

Gonadotropin-releasing hormone (GnRH) interacts with a G protein-coupled receptor and increases the transcription of the glycoprotein hormone alpha-subunit gene. We have explored the possibility that mitogen-activated protein kinase (MAPK) plays a role in mediating GnRH effects on transcription. Activation of the MAPK cascade by an expression vector for a constitutively active form of the Raf-1 kinase led to stimulation of the alpha-subunit promoter in a concentration-dependent manner. GnRH treatment was found to increase the phosphorylation of tyrosine residues of MAPK and to increase MAPK activity, as determined by an immune complex kinase assay. A reporter gene assay using the MAPK-responsive, carboxy-terminal domain of the Elk1 transcription factor was also consistent with GnRH-induced activation of MAPK. Interference with the MAPK pathway by expression vectors for kinase-defective MAPKs or vectors encoding MAPK phosphatases reduced the transcription-stimulating effects of GnRH. The DNA sequences which are required for responses to GnRH include an Ets factor-binding site. An expression vector for a dominant negative form of Ets-2 was able to reduce GnRH effects on expression of the alpha-subunit gene. These findings provide evidence that GnRH treatment leads to activation of the MAPK cascade in gonadotropes and that activation of MAPK contributes to stimulation of the alpha-subunit promoter. It is likely that an Ets factor serves as a downstream transcriptional effector of MAPK in this system.

An inactivating point mutation demonstrates that interaction of cAMP response element binding protein (CREB) with the CREB binding protein is not sufficient for transcriptional activation.

The cAMP response element binding protein (CREB) mediates transcriptional activation in response to the cAMP signaling pathway. Several recent studies have suggested that phosphorylation-dependent interaction of CREB with a co-activator designated CREB binding protein (CBP) is a crucial step in mediating transcriptional responses to cAMP. In the present study we have determined that replacement of Ser142 of CREB with Asp greatly decreases the ability of the cAMP-dependent protein kinase to activate CREB. As Ser142 is located within the region of CREB that interacts with CBP, it seemed quite likely that mutations at this site might interfere with binding to CBP. However, both in vitro and in vivo protein-protein interaction assays revealed that replacement of Ser142 with Asp does not interfere with the binding of CREB to CBP. These studies argue strongly that although the binding of CREB to CBP is necessary, it is not sufficient for transcriptional responses to cAMP.

Multiple Pit-1-binding sites facilitate estrogen responsiveness of the prolactin gene.

Previous studies have shown that estrogen responsiveness of the rat PRL gene requires the presence of both the estrogen receptor and the tissue-specific transcription factor, Pit-1. To examine the contribution of individual Pit-1-binding sites in permitting an estrogen response, we mutated specific sites in both the proximal and distal regions of the rat PRL gene. The studies reveal that mutation of Pit-1-binding sites in either the proximal or the distal region can have an effect on estrogen responsiveness. The most important Pit-1-binding site appears to be the site in the distal enhancer, which is adjacent to the estrogen receptor-binding site. However, mutation of combinations of other Pit-1-binding sites reveals that these sites also contribute to the estrogen response of the PRL gene. The binding sequences for another transcription factor cannot substitute for Pit-1 sites in bringing about a wild-type estrogen response, as shown by replacement of Pit-1-binding sites with a consensus cAMP-responsive element. Conversion of the imperfect palindromic estrogen response element of the PRL gene to a perfect palindrome eliminated the positive effects of an intact 1D Pit-1-binding site. To examine potential physical interactions between the estrogen receptor and Pit-1, a protein interaction assay was performed. The results demonstrate that labeled estrogen receptor can bind to Pit-1 immobilized on glutathione agarose beads. However, most of the interaction between Pit-1 and the estrogen receptor appears to be DNA dependent.(ABSTRACT TRUNCATED AT 250 WORDS)

Pit-1 binding sites mediate transcriptional responses to cyclic adenosine 3',5'-monophosphate through a mechanism that does not require inducible phosphorylation of Pit-1.

The ability of Pit-1 to mediate transcriptional responses to cAMP has been explored. To test the ability of Pit-1 to mediate transcriptional responses to cAMP, an expression vector was prepared for a mutant Pit-1 in which the major sites of phosphorylation by the cAMP-dependent protein kinase were eliminated. Before using the mutant Pit-1 to study transcriptional regulation, we first examined the ability of the protein to be phosphorylated in vivo in response to cAMP. Transfection and in vivo labeling experiments confirmed that the mutant Pit-1 did not support cAMP-inducible phosphorylation. The ability of the wild type or mutant Pit-1 to mediate transcriptional responses to cAMP was assessed in cotransfection experiments using reporter genes containing either the proximal region of the rat PRL gene or seven copies of a Pit-1 binding site placed upstream of a minimal promoter. Surprisingly, the wild type and mutant Pit-1 expression vectors supported similar responses to cAMP. To further assess the ability of Pit-1 to mediate responses to cAMP, a GAL4-Pit-1 fusion gene was prepared. Although a GAL4-cAMP response element binding protein fusion gene was found to permit transcriptional responses to cAMP, the GAL4-Pit-1 gene was unresponsive. These findings demonstrate that although Pit-1 can facilitate the ability of the PRL promoter to respond to cAMP, phosphorylation of Pit-1 is not required for this response. It seems likely that additional factors that interact with Pit-1 binding sites are important for mediating transcriptional responses to cAMP.

Thyrotropin releasing hormone stimulates transient phosphorylation of the tissue-specific transcription factor, Pit-1.

The hypothalamic hormone, thyrotropin releasing hormone (TRH), stimulates prolactin (PRL) secretion and gene transcription in the GH3 pituitary cell line. Several studies have provided indirect evidence that phosphorylation of the pituitary-specific transcription factor Pit-1 may mediate TRH effects on PRL transcription. In the present study we have investigated the ability of TRH to alter the phosphorylation of Pit-1. In vivo 32P labeling experiments demonstrated that TRH stimulated a transient phosphorylation of Pit-1, reaching a maximum in 5 min and returning to basal levels within 30 min. Phosphopeptide mapping experiments demonstrated that TRH induced the transient phosphorylation of specific sites in Pit-1. TRH-stimulated phosphorylation of Pit-1 was blocked by treatments that deplete the cellular content of protein kinase C. Metabolic labeling and Western blot analysis demonstrate that TRH does not alter the total cellular content or nuclear concentration of Pit-1. TRH-mediated stimulation of a PRL promoter-luciferase fusion gene occurred under conditions that blocked the transient phosphorylation of Pit-1. These studies suggest that phosphorylation of Pit-1 may not be necessary for TRH mediated enhancement of PRL gene transcription.

Differential activation of CREB by Ca2+/calmodulin-dependent protein kinases type II and type IV involves phosphorylation of a site that negatively regulates activity.

The cAMP response element-binding protein (CREB) has been shown to mediate transcriptional activation of genes in response to both cAMP and calcium influx signal transduction pathways. The roles of two multifunctional calcium/calmodulin-dependent protein kinases, CaMKIV and CaMKII, were examined in transient transfection studies that utilized either the full-length or the constitutively active forms of these kinases. The results indicate that CaMKIV is much more potent than CaMKII in activating CREB in three different cell lines. It was also found in these studies that Ser133 of CREB is essential for its activation by CaMKIV. Because both CaMKII and CaMKIV can phosphorylate CREB, we pursued further the mechanism by which CaMKII and CaMKIV differentially regulate CREB activity. Mutagenesis studies and phosphopeptide mapping analysis demonstrated that in vitro, CaMKIV phosphorylates CREB at Ser133 only, whereas CaMKII phosphorylates CREB at Ser133 and a second site, Ser142. Transient transfection studies revealed that phosphorylation of Ser142 by CaMKII blocks the activation of CREB that would otherwise occur when Ser133 is phosphorylated. When Ser142 was mutated to alanine, CREB was activated by CaMKII, as well as by CaMKIV. Furthermore, mutation of Ser142 to alanine enhanced the ability of Ca2+ influx to activate CREB, suggesting a physiological role for the phosphorylation of Ser142 in modulation of CREB activity. These data provide evidence for a new mechanism for regulation of CREB activity involving phosphorylation of a negative regulatory site in the transcriptional activation domain. The studies also provide new insights into possible interactions between the cAMP and Ca2+ signaling pathways in the regulation of transcription. In particular, changes in intracellular Ca2+ have the potential to either inhibit or augment the ability of cAMP to stimulate transcription, depending on the presence of specific forms of Ca2+/calmodulin-dependent protein kinases.

Activation of the glycoprotein hormone alpha-subunit promoter by a LIM-homeodomain transcription factor.

Recently, a pituitary-specific enhancer was identified within the 5' flanking region of the mouse glycoprotein hormone alpha-subunit gene. This enhancer is active in pituitary cells of the gonadotrope and thyrotrope lineages and has been designated the pituitary glycoprotein hormone basal element (PGBE). In the present studies, we sought to isolate and characterize proteins which interact with the PGBE. Mutagenesis experiments identified a 14-bp imperfect palindrome that is required for binding of a factor which is present in cells of gonadotrope and thyrotrope lineages but not in other cells. Screening of a mouse cDNA library with a DNA probe containing the imperfect palindrome resulted in the isolation of a LIM-homeodomain transcription factor. The cDNA predicts a mouse protein which is 94% identical to the recently described rat LIM-homeodomain protein LH-2. LH-2 contains two zinc fingers (LIM domain) and a consensus homeodomain. Hybridization analysis revealed relatively high expression of LH-2 mRNA in the central nervous system and in pituitary cells of the gonadotrope and thyrotrope lineages. Lower or nondetectable levels of LH-2 mRNA were found in other pituitary cells and tissues, including placental cells. Recombinant LH-2 homeodomain was found to selectively bind to the previously identified imperfect palindrome in the PGBE. Point mutations in the PGBE resulted in parallel losses in the binding of a nuclear factor from a cell line of the gonadotrope lineage and recombinant LH-2-binding activity. Use of an antibody to LH-2 provided evidence that endogenous PGBE-binding activity from cells of the gonadotrope lineage involves a protein which is immunologically related to LH-2. Expression of LH-2 in two heterologous cell types resulted in activation of a reporter gene containing the mouse alpha promoter. These data suggest that the LIM-homeodomain factor LH-2 plays a role in stimulating tissue-specific expression of the mouse glycoprotein hormone alpha subunit. The finding that a LIM-homeodomain protein can stimulate expression of one of the earliest markers of pituitary differentiation raises the possibility that this factor plays a role in cell lineage determination in the pituitary.

Involvement of a cAMP-responsive DNA element in mediating TRH responsiveness of the human thyrotropin alpha-subunit gene.

TRH is known to stimulate the transcription of the TSH gene in pituitary cells. To examine TRH-responsive elements of the human TSH alpha-subunit gene, we have used transient transfection of GH3 rat pituitary tumor cells. Using this system, TRH treatment stimulated expression of a reporter gene containing 846 base pairs from the 5'-flanking region of the human glycoprotein hormone alpha-subunit gene linked to luciferase. Analysis of 5'-deletions of the alpha-subunit sequence revealed that at least two DNA regions with upstream limits between positions -223 to -190 and positions -151 to -135 are important for regulation by TRH. The more proximal region includes a previously defined cAMP-response element (CRE) while the more upstream region contains an element with sequence similarity to the binding site for the pituitary transcription factor, Pit-1. The TRH responsiveness of each individual region was tested by inserting fragments upstream of a thymidine kinase-luciferase reporter gene. The -151 to -100 region had basal enhancer activity and permitted a 3.4-fold response to TRH. The -223 to -168 region did not permit a TRH response, but possessed basal enhancer activity. The combination of both regions resulted in a 5-fold stimulation by TRH. To assess the contributions of different signal transduction pathways, various combinations of treatments were examined. Combined treatment with TRH and forskolin led to an additive activity. Treatment with TRH plus phorbol 12-myristate-13-acetate resulted in the same level of reporter gene activity as with either agent alone.(ABSTRACT TRUNCATED AT 250 WORDS)