Glycoprotein Hormone Alpha-subunit Promoter Research Articles

The highly related LIM factors, LMO1, LMO3 and LMO4, play different roles in the regulation of the pituitary glycoprotein hormone α-subunit (αGSU) gene

LMO1, LMO3 and LMO4 were cloned from the adult porcine pituitary cDNA library. Amino acid sequences of porcine LMO1, LMO3 and LMO4 were highly conserved among mammalian species. Transfection assay of the pituitary-derived cell line L beta T2 was carried out using the pituitary alpha GSU (glycoprotein hormone alpha-subunit) promoter (-1059/+12 b) fused to pSEAP2-Basic vector as a reporter gene. The results demonstrated that, whereas LMO4 showed no apparent effect, alpha GSU promoter activity was markedly repressed by LMO1 but activated by LMO3, indicating the different roles of the three highly homologous proteins, LMO1, LMO3 and LMO4. Knockdown assay by LMO siRNAs (small interfering RNAs) confirmed the above results for LMO1 and LMO3, whereas that by LMO4 siRNA increased the expression, indicating different modes of action. RT-PCR (reverse transcription-PCR) for total RNAs of several cell lines showed that LMO1 and LMO4 mRNAs were present ubiquitously in all cell lines, except for LMO1 in L929 cells. In contrast, LMO3 mRNA was abundant only in L beta T4 and GH3 cells with only small amounts in L beta T2 and MtT/S cells, indicating the cell-type-specific function of this protein. Real-time analyses of porcine pituitary ontogeny revealed that the three LMO genes are expressed during the fetal period and decline immediately afterwards, followed by a remarkably low level of LMO3 and LMO4 after birth. RT-PCR of the porcine tissues examined showed ubiquitous expression of LMO4, whereas LMO1 and LMO3 are expressed tissue specifically. Thus the present study demonstrated that three highly related LIM cofactors, LMO1, LMO3 and LMO4, have different effects on alpha GSU gene expression in the pituitary glands.

Enhancement of Glycoprotein Hormone Alpha Subunit Promoter Reporter Gene Activity in Co-transfection Studies – A Cautionary Reminder

Reporter constructs have a wide range of application in determining eucaryotic gene regulation and expression. IN VITRO models frequently necessitate co-transfection and there have been reports of interference, predominantly inhibition, between promoters. Studies investigating the biological activity of a mutant thyrotropin receptor involved co-transfection with receptor constructs and a cAMP responsive luciferase reporter driven by the glycoprotein hormone alpha subunit promoter. We observed considerable enhancement of basal luciferase activity by co-transfecting the empty expression vector, which contained the SV40 late promoter. We have investigated the mechanism using different concentrations and several viral promoters in COS cells, following transient co-transfection. The increase was dose dependent but plateaued at 50 ng of vector DNA. This was not due to an adjuvant effect since luciferase activity was unchanged by adding increasing amounts of a promoterless plasmid. Enhancement was maintained when truncating the promoter to -346, but eliminated in the promoter truncated upstream of -244, indicating a binding site for a putative repressor of glycoprotein hormone alpha expression between -244 and -346. Enhancement was maintained with the addition of a second constitutive reporter (bos beta-gal) to correct for transfection efficiency, although this was the consequence of enhanced luciferase activity by the bos beta-gal, which in itself was inhibited by the SV40 promoter. Artefactual enhancement of reporter activity can occur and highlights the need for careful choice of controls when performing transient co-transfection experiments. In silico analysis of the promoters identified a possible shared forkhead transcription factor binding site.

An activator protein-1 complex mediates epidermal growth factor regulation of equine glycoprotein alpha subunit expression in trophoblast cells.

Equids and primates are the only species known to express the placental hormone chorionic gonadotropin (CG). CG is a member of the heterodimeric glycoprotein family and is composed of an alpha subunit linked to a hormone-specific beta subunit. Previously, we have reported that epidermal growth factor (EGF) regulates the equine glycoprotein hormone alpha subunit promoter through a protein kinase C (PKC)/mitogen-activated protein kinase (MAPK) signal transduction pathway in trophoblasts. The current study investigates the regulatory element/factors involved in the induction of equine glycoprotein alpha subunit gene expression by EGF. Using 5' deletion mutagenesis, we have delineated the primary EGF/PKC responsive region of the equine alpha subunit gene to be located between -2039 to -2032 base pairs upstream of the transcriptional start site. The sequence within this region contains an activator protein 1 (AP-1)-like response element (TGAATCA) and is similar to a consensus AP-1 (TGAC/GTCA) response element. This element appeared to preferentially interact with a c-fos/JunD heterodimer. Stimulation by EGF induced the binding of c-fos and JunD to this element and subsequently elevated promoter activity. In conclusion, an EGF/PKC/MAPK signal transduction pathway regulates equine glycoprotein alpha subunit gene expression through a distinct regulatory element(s) that lies between -2039 to -2032 of the equine glycoprotein alpha subunit promoter in trophoblasts and involves an AP-1 complex.

Stereotactic injection of adenoviral vectors that target gene expression to specific pituitary cell types: implications for gene therapy.

Gene therapy is a potentially useful strategy for the treatment of pituitary adenomas or hormone deficiency disorders. We investigated the feasibility of targeting gene expression to specific pituitary cell types in vivo, using a combination of stereotactic injection and adenoviral vectors that carry pituitary-specific promoters. Recombinant adenoviruses containing the human growth hormone promoter (AdGHGal) or the human glycoprotein hormone alpha-subunit promoter (AdalphaGal) were used to drive expression of the beta-galactosidase gene. The expression of beta-galactosidase activity in the pituitary was analyzed after the administration of recombinant adenoviruses via the peripheral vein or the carotid artery, or by stereotactic injection into the rat pituitary. Double-label histology was used to evaluate cell-type expression in the pituitary. Intravascular injection of AdGHGal or AdalphaGal failed to deliver the marker gene to the pituitary. However, direct stereotactic injection of recombinant adenoviral vectors into the pituitary achieved a high level of transgene expression. In addition, immunohistochemical staining revealed selective expression of the AdGHGal or AdalphaGal transgenes in pituitary cells that normally produce the respective hormones. These findings indicate that adenoviral vectors carrying pituitary gland-specific promoters may be useful for targeted gene therapy of pituitary diseases. However, because of low transduction after peripheral administration, stereotactic injection or local administration of viruses at the time of pituitary surgery is probably required for efficient gene expression.

Activation of the p38 mitogen-activated protein kinase pathway by gonadotropin-releasing hormone.

Previous studies have shown that interaction of GnRH with its serpentine, G protein-coupled receptor results in activation of the extracellular signal regulated protein kinase (ERK) and the Jun N-terminal protein kinase (JNK) pathways in pituitary gonadotropes. In the present study, we examined GnRH-stimulated activation of an additional member of the mitogen-activated protein kinase (MAPK) superfamily, p38 MAPK GnRH treatment of alphaT3-1 cells resulted in tyrosine phosphorylation of several intracellular proteins. Separation of phosphorylated proteins by ion exchange chromatography suggested that GnRH receptor stimulation can activate the p38 MAPK pathway. Immunoprecipitation studies using a phospho-tyrosine antibody resulted in increased amounts of immunoprecipitable p38 MAPK from alphaT3-1 cells treated with GnRH. Immunoblot analysis of whole cell lysates using a phospho-specific antibody directed against dual phosphorylated p38 kinase revealed that GnRH-induced phosphorylation of p38 kinase was dose and time dependent and was correlated with increased p38 kinase activity in vitro. Activation of p38 kinase was blocked by chronic phorbol ester treatment, which depletes protein kinase C isozymes alpha and epsilon. Overexpression of p38 MAPK and an activated form of MAPK kinase 6 resulted in activation of c-jun and c-fos reporter genes, but did not alter the expression of the glycoprotein hormone alpha-subunit reporter. Inhibition of p38 activity with SB203580 resulted in attenuation of GnRH-induced c-fos reporter gene expression, but was not sufficient to reduce GnRH-induced c-jun or glycoprotein hormone alpha-subunit promoter activity. These studies provide evidence that the GnRH signaling pathway in alphaT3-1 cells includes protein kinase C-dependent activation of the p38 MAPK pathway. GnRH integration of c-fos promoter activity may include regulation by p38 MAPK.

Msx1 is present in thyrotropic cells and binds to a consensus site on the glycoprotein hormone alpha-subunit promoter.

Our studies are aimed at identifying the transcription factors that activate the glycoprotein hormone alpha-subunit promoter. Therefore, we performed a Southwestern screening of a thyrotropic (alphaTSH) cDNA expression library, using the region of the promoter from -490 to -310 that contains sequences critical for expression in thyrotrope cells. A clone was isolated corresponding to part of the coding sequence of Msx1, which is a homeodomain-containing transcription factor that has been found to play an important role in the development of limb buds and craniofacial structures. Northern blot analysis, using the cloned Msx1 cDNA fragment as a probe, demonstrated that alpha-subunit-expressing thyrotrope cells (alphaTSH cells and TtT97 tumors) contained Msx1 RNA transcripts of 2.2 kb, while somatomammotrope (GH3) cells that do not produce the alpha-subunit had barely detectable levels. The presence of Msx1 protein was demonstrated by Western blot analysis in alphaTSH cells. We also demonstrated that transcripts encoding the closely related Msx2 factor were not detectable by Northern blot analysis in either thyrotrope or somatomammotrope-derived cells. Subfragments of the region from -490 to -310 of the alpha-subunit promoter were used in a Southwestern blot assay using bacterially produced Msx1 and demonstrated that binding was localized specifically to the region from -449 to -421. Deoxyribonuclease I protection analysis, using purified Msx1 homeodomain, demonstrated structurally induced differences in DNA digestion patterns between -436 and -413, and sequence analysis of this region revealed a direct repeat of the sequence GXAATTG, which is similar to the Msx1 consensus-binding site. When nucleotides at both sites were mutated, Msx1 binding was dramatically reduced, and the activity of an alpha-subunit promoter construct decreased by approximately 50% in transfected thyrotrope (alphaTSH) cells. These studies suggest that Msx1 may play a role in the expression of the alpha-subunit gene in thyrotrope cells.

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.

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.

Identification of a gonadotropin-releasing hormone-responsive region in the glycoprotein hormone alpha-subunit promoter.

Transcriptional regulation of the glycoprotein hormone alpha-subunit gene has been studied extensively in placental cells, but much less is known about its regulation in the pituitary gland. In this study, transcriptional control of the human alpha-subunit gene by GnRH was analyzed using transient expression assays in primary cultures of rat pituitary cells using alpha promoter constructs linked to a luciferase reporter gene. Deletion mutants between -846 and -156 basepairs (bp) had little effect on basal expression, but further deletion to -132 bp reduced basal activity by approximately 50%. Deletion of a cAMP response element between -132 and -99 bp caused a marked loss of basal activity, reducing expression to that of background luciferase activity. The same constructs were analyzed for cAMP responsiveness in primary pituitary cells. The degree of stimulation with 1 mM 8-bromo-cAMP (3.6- to 6.0-fold) was relatively unaffected by deletions from -846 to -132 bp, whereas cAMP stimulation was decreased by further deletion to -99 bp, consistent with the presence of previously defined cAMP response elements in this region of the alpha promoter. GnRH stimulation of alpha promoter activity was highly dependent upon the time of hormone addition after transfection, being most effective when added soon after transfection. Under optimal conditions, GnRH stimulated -846 alpha LUC expression by 20-fold. GnRH responsiveness was retained with deletion to -346 bp, but it was decreased by 55% after deletion to -280 bp and by 79% with deletion to -244 bp.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of a gonadotropin-releasing hormone-responsive region in the glycoprotein hormone alpha-subunit promoter

Identification of a gonadotropin-releasing hormone-responsive region in the glycoprotein hormone alpha-subunit promoter