Regulation of function of the murine luteinizing hormone receptor promoter by cis- and trans-acting elements in mouse Leydig tumor cells

El-Hefnawy, Talal; Krawczyk, Zdzisłlaw; Nikula, Hannu; Viherä, I; Huhtaniemi, Ilpo

doi:10.1016/0303-7207(96)03815-4

Cited by 25 publications

(9 citation statements)

References 35 publications

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“…The putative promoter area in the 5Ј-flanking region of the LHR gene has been sequenced from the rat, mouse, and human, and certain species differences have been observed (7)(8)(9)(10)(11)(12)(13). Rat and mouse studies have demonstrated that the TATA-less promoter has the basal transcriptional activity within the first 173 bp upstream of the translation initiation codon (8,9).…”

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confidence: 99%

Age- and Sex-Specific Promoter Function of a 2-Kilobase 5′-Flanking Sequence of the Murine Luteinizing Hormone Receptor Gene in Transgenic Mice1

1999

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A transgenic (TG) mouse model carrying a 2-kb murine LH receptor (LHR) promoter/beta-galactosidase (beta-GAL) fusion gene was created to study the regulatory function of the 5'-flanking region of the murine LHR gene. Of the five TG mouse lines produced, three displayed high beta-GAL expression in the testis, but none showed any expression in the ovary. In addition, all mouse lines of both sexes expressed beta-GAL consistently in the brain, most prominently in hippocampus, hypothalamus, midbrain, and cortex. Weak staining was found in a few pituitary samples. All other tissues examined were negative for transgene expression. In support of sex-specific gonadal expression of the transgene, transient transfection of the LHR/beta-GAL gene construct into immortalized mouse granulosa (KK-1) and Leydig (mLTC-1) tumor cells revealed a more than 5-fold higher expression level in the Leydig cells. Histological examination of the TG testes demonstrated strong beta-GAL expression in Leydig cells, but, unexpectedly, also in elongating spermatids of adult age and in some spermatogonia of the neonatal testis. The functional significance of the latter findings remains open. The transgene was only expressed in adult Leydig cells; no expression was found in the fetal population of these cells. Hence, these findings indicate that the immediate 2-kb fragment of the murine LHR 5'-flanking sequence is transcriptionally active only in adult Leydig cells and certain brain areas, but other promoter sequences are apparently needed for ovarian and fetal testicular expression of the LHR gene.

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Age- and Sex-Specific Promoter Function of a 2-Kilobase 5′-Flanking Sequence of the Murine Luteinizing Hormone Receptor Gene in Transgenic Mice1

1999

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“…TR is a member of the steroid/thyroid hormone receptor superfamily that binds to the hexameric motif AGGTCA, termed the nuclear receptor half-site, to activate gene transcription (55). The cloning and characterization of the 5Ј-flanking region of the mouse and rat LHR gene demonstrated that the basal promoter region (173 bp) is highly GC rich containing AP1 and several SP1 sites, which are involved in transcriptional regulation (36,37,56). The importance of this region was further confirmed, by expressing either the 173-bp fragment alone or its deletion from the full-length fragment (2040 bp) in mLTC-1 cells.…”

Section: Discussionmentioning

confidence: 99%

“…The murine LHR promoter/luciferase fusion genes were constructed as described previously (36). Briefly, the 2040-bp fragment of the 5Јflanking region (Ϫ1/Ϫ2040 bp), 173-bp deleted fragment from 2040-bp (Ϫ174/Ϫ2040 bp) and 173-bp (Ϫ1/Ϫ173 bp) fragment (in relation to translation initiation codon) of the mouse LHR gene, were linked to the coding sequence of the luciferase reporter construct (36,37).…”

Section: Plasmids Transfections and Determination Of Luciferase Actmentioning

confidence: 99%

“…The nuclear extracts (NE) from different experimental groups, were prepared as described previously (36), with slight modifications to improve their purity. Briefly, after washing the cells twice with PBS, they were collected (10 7 cells/300 l), and kept for 5 min in ice-cold lysis buffer A containing protease inhibitors (20 mmol/liter HEPES, 20 mm NaCl, 0.5% Nonidet P-40, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, 2 mg/liter leupeptin, 2 mg/liter aprotinin, pH 8.0).…”

Section: Preparation Of Nuclear Extracts and Electrophoretic Mobilitymentioning

confidence: 99%

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Assessment of Mechanisms of Thyroid Hormone Action in Mouse Leydig Cells: Regulation of the Steroidogenic Acute Regulatory Protein, Steroidogenesis, and Luteinizing Hormone Receptor Function**This investigation was supported in part by grants from the Sigrid Jusélius Foundation, Academy of Finland, Foundation for the Finnish Cancer Societies (to I.T.H.), and NIH Grant (HD-1-7481) (to D.M.S.).

et al. 2001

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Recently, we demonstrated that triiodothyronine (T(3)) stimulated steroid hormone biosynthesis and steroidogenic acute regulatory (StAR) protein expression in mLTC-1 mouse Leydig tumor cells through the mediation of steroidogenic factor 1 (SF-1). We now report a dual response mechanism of T(3) on steroidogenesis and StAR expression, and on LH receptor (LHR) expression and binding in mLTC-1 cells. T(3) acutely (8 h), induced a 260% increase in StAR messenger RNA (mRNA) expression over the basal level which was coincident with an increase in progesterone (P) production. In contrast, chronic stimulation with T(3) (beyond 8 h), resulted in an attenuation of StAR expression and P production. This attenuation was most likely caused by a decrease in cholesterol delivery to the inner mitochondrial membrane as demonstrated by incubations with the hydrophilic steroid precursors, 22R hydroxycholesterol and pregnenolone, which restored P synthesis. In similar studies, chronic treatment with T(3) increased the levels of cytochrome P450scc mRNA by 83%, whereas those of cytochrome P450 17alpha-hydroxylase and 3ss-hydroxysteroid dehydrogenase decreased. The diminished response in steroidogenesis following chronic T(3) exposure was not a result of alterations in StAR mRNA stability, but rather was due to inhibition of transcription of the StAR gene. Similar acute stimulatory and chronic inhibitory responses to T(3) were found when LHR mRNA expression and LHR ligand binding were examined. Transfections with an LHR or StAR promoter/luciferase reporter construct demonstrated that a 173-bp fragment of the LHR promoter containing an SF-1 binding motif was involved in T(3) response, as was the SF-1 recognition site at -135 bp in the StAR promoter. Furthermore, the importance of SF-1 in T(3) function was also verified employing mutation in the bases of SF-1 sequences using electrophoretic mobility shift assays. The potential physiological relevance of these findings was demonstrated when similar responses were obtained in mice rendered hypo and hyperthyroid. Collectively, these observations further characterize the thyroid-gonadal connection and provide insights into the mechanisms for a dual regulatory role of thyroid hormone in Leydig cell functions.

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“…The mLTC-1 cells were grown in HEPES-buffered Waymouth's medium (Life Technologies, Inc., Paisley, Scotland), as previously described (27). For the binding experiments, 7 ϫ 10 4 cells/well were plated on 24-well culture dishes (Greiner, Frickenhausen, Germany), and 24 h later the binding experiments were performed on the living cells.…”

Section: Ligand Binding Assaysmentioning

confidence: 99%

Progesterone Action in a Murine Leydig Tumor Cell Line (mLTC-1), Possibly through a Nonclassical Receptor Type¹

et al. 2000

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In a recent report we demonstrated that a high (micromolar) concentration of progesterone (P) specifically down-regulates LH receptor (R) expression and function in murine Leydig tumor cells. The aim of the present study was to characterize further the putative novel R, mediating these P effects in the murine Leydig tumor cell line, mLTC-1. The binding of [3H]P to these cells revealed a high (Kd, approximately 9.3 nmol/liter) and a low affinity (Kd, approximately 284 nmol/liter) component, and the binding displayed with specificity (P > dehydroepiandrosterone > 17-OHP). The binding was apparently different from that of the classical nuclear PR in the following ways. 1) The P/glucocorticoid antagonist RU 486 did not compete with [3H]P binding to the mLTC-1 cells. 2) No expression of the classical PR messenger RNA was detected, despite clear P binding to these cells, by Northern hybridization or RT-PCR. 3) An antibody against the C-terminal end of the classical PR (alpha c-262) revealed in mLTC-1 cells several molecular size protein bands between 45-57 kDa on Western hybridization, whereas these immunoreactive proteins were faintly recognized by another antibody (alpha-PR) directed toward the NH2-terminal region of the classical PR. The sizes of the immunoreactive molecules were relatively similar to those detected using the same antibodies in human sperm lysates, but were at variance with the classical PR (120, 94, and 60 kDa), detected with these antibodies in human uterus. The immunoreactive proteins bound peroxidase-labeled-P, which could be displaced in the presence of a 10-fold excess of free P. 4) An immediate increase in the intracellular free calcium level was observed after P treatment in cultured mLTC-1 cells, whereas it also increased the 45Ca2+ entry within 15 min in these cells. 5) Increasing doses of P (0.1-10 micromol/liter) demonstrated significant inhibition of LH receptor messenger RNA levels in a dose-dependent manner in mLTC-1 cells. In conclusion, a nonclassical PR is expressed and functional in these cells, and it is clearly distinct from the classical nuclear PR. It is apparent that recently reported inhibitory effects of P on LH receptor gene expression and function are mediated through this novel type PR in mouse Leydig cells.

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Regulation of function of the murine luteinizing hormone receptor promoter by cis- and trans-acting elements in mouse Leydig tumor cells

Cited by 25 publications

References 35 publications

Age- and Sex-Specific Promoter Function of a 2-Kilobase 5′-Flanking Sequence of the Murine Luteinizing Hormone Receptor Gene in Transgenic Mice1

Age- and Sex-Specific Promoter Function of a 2-Kilobase 5′-Flanking Sequence of the Murine Luteinizing Hormone Receptor Gene in Transgenic Mice1

Progesterone Action in a Murine Leydig Tumor Cell Line (mLTC-1), Possibly through a Nonclassical Receptor Type¹

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Regulation of function of the murine luteinizing hormone receptor promoter by cis- and trans-acting elements in mouse Leydig tumor cells

Cited by 25 publications

References 35 publications

Age- and Sex-Specific Promoter Function of a 2-Kilobase 5′-Flanking Sequence of the Murine Luteinizing Hormone Receptor Gene in Transgenic Mice1

Age- and Sex-Specific Promoter Function of a 2-Kilobase 5′-Flanking Sequence of the Murine Luteinizing Hormone Receptor Gene in Transgenic Mice1

Progesterone Action in a Murine Leydig Tumor Cell Line (mLTC-1), Possibly through a Nonclassical Receptor Type1

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Progesterone Action in a Murine Leydig Tumor Cell Line (mLTC-1), Possibly through a Nonclassical Receptor Type¹