Tissue-specific enhancer of the human glycoprotein hormone alpha-subunit gene: dependence on cyclic AMP-inducible elements.

Abstract

We identified and characterized elements which confer tissue specificity and cyclic AMP (cAMP) responsiveness to the human glycoprotein a-subunit gene. An enhancer containing an 18-base-pair repeat conferred cAMP responsiveness in a non-tissue-specific fashion. DNase I protection assays revealed DNA-binding factors that bound to this element in both placental and nonplacental cells. It also enhanced the a-subunit promoter in a tissue-specific manner but had a negligible effect on a heterologous promoter. A unique element found upstream of this enhancer had no independent activity but, in combination with the cAMP-responsive enhancet-, distinctly increased the tissue-specific activity of both the a-subunit promoter and a heterologous promoter. A factor that bound to this upstream element was found in placental but not nonplacental cells. We conclude that this novel element acts, perhaps through a specific trans-acting factor, in concert with a cAMP-responsive enhancer to confer tissue specificity to the a-subunit gene.The a-subunit gene of human glycoprotein hormones is expressed coordinately with each of four separate ,B-subunit genes. Their combined products comprise the four glycoprotein hormones: chorionic gonadotropin, luteinizing hormone, follicle-stimulating hormone, and thyroid-stimulating hormone (14,36). These hormones direct the synthesis of several classes of hormones and control aspects of reproductive and metabolic function. They are produced in three separate cell types: placental trophoblasts produce chorionic gonadotropin, pituitary gonadotropes produce folliclestimulating hormone and luteinizing hormone and pituitary thyrotropes produce thyroid-stimulating hormone (36). In each of these cell types, production of the a subunit is regulated differently in coordination with the a subunit with which it becomes associated (16,33,37). For example, thyroid-stimulating hormone expression is induced by thyrotropin-releasing hormone and inhibited by thyroid hormones, while luteinizing hormone and follicle-stimulating hormone are induced by gonadotropin-releasing hormone and inhibited by estrogens (12). Thus, the a-subunit gene must contain regulatory sequences which direct its expression in several different tissues and mediate its differential hormonal responsiveness.Some information is available concerning the mechanisms which control these complex responses. Nuclear transcription assays have denmonstrated that thyroid hormones have direct effects on the transcription of both the a(-and 1B-subunit genes in pituitary thyrotropes (40). In addition, cyclic AMP (cAMP) increases the transcription rate of the axand ,B-chorionic gonadotropin genes in placental cells (21), and the cAMP responsiveness of the ox-subunit gene has been shown by gene transfer experiments to reside in its 5'-flanking sequences (8,21,41 mally involved in transcription in order to modify their activity.In the current studies we analyzed two elements important for the expression of the human a-subunit getne. The 5'-flanking region conta...

Interleukin-6-induced STAT3 transactivation and Ser727 phosphorylation involves Vav, Rac-1 and the kinase SEK-1/MKK-4 as signal transduction components

“…Schuringa, L. J. C. Jonk, W. H. A. Dokter, E. Vellenga and W. Kruijer, unpublished work). These results indicate that IL-6-induced STAT3 Ser(#( phosphorylation in HepG2 cells is ERKindependent, in agreement with previously published reports [53]. In quiescent Swiss 3T3 cells, however, EGF-induced STAT3 Ser(#( phosphorylation has been shown to be dependent on activation of ERK-1 [53].…”

“…These results indicate that IL-6-induced STAT3 Ser(#( phosphorylation in HepG2 cells is ERKindependent, in agreement with previously published reports [53]. In quiescent Swiss 3T3 cells, however, EGF-induced STAT3 Ser(#( phosphorylation has been shown to be dependent on activation of ERK-1 [53]. At present, we have no explanation for the different involvement of ERKs in STAT3 Ser(#( phosphorylation by these different factors.…”

LIF-Induced STAT3 Signaling in Murine versus Human Embryonal Carcinoma (EC) Cells

“…When necessary, pUC18 was added to the transfection mixture to obtain a total of 10 g of DNA. Cells were incubated with precipitate for 24 h, washed with phosphate-buffered saline (PBS), and stimulated for an additional 24 h. Cells were collected in 200 l reporter lysis buffer (Promega) and subjected to the assays for luciferase [32] and ␤-galactosidase [33] as previously described. The data represent two independent experiments using different batches of DNA, and in each experiment transient transfections were performed in triplicate.…”

Interleukin‐6‐induced serine phosphorylation of transcription factor APRF: evidence for a role in interleukin‐6 target gene induction

“…Briefly, HepG2 cells were transfected using calcium phosphate precipitates [26] containing 6 gg of reporter plasmids and 4 ktg of the expression vector for fl-galactosidase pSV-LacZ [23] as an internal control for transfection efficiency, were stimulated without or with IL-6 one day after transfection for an additional 4 h, and harvested thereafter. Protein extracts prepared by freeze-thawing [27] were assayed for fl-galactosidase [28], CAT [29], and luciferase activities [30] as described. Units of CAT or luciferase activities were normalized to fl-galactosidase activity.…”