Lung Krüppel-like factor contains an autoinhibitory domain that regulates its transcriptional activation by binding WWP1, an E3 ubiquitin ligase.

Lung Krüppel-like factor (LKLF/Krüppel-like factor 2), a member of the Krüppel-like factor family of transcription factors, is expressed predominantly in the lungs, with low levels of expression in other organs such as heart, spleen, skeletal muscle, and testis. LKLF is essential during pulmonary development and single-positive T-cell development and is indispensable during mouse embryogenesis. In this study, we performed a series of experiments to define the activation domain of LKLF as a means to further advance the understanding of the molecular mechanisms underlying transcriptional regulation by this transcription factor. Using deletion analysis, it is shown that LKLF contains a transcriptional activation domain as well as a strong autoinhibitory subdomain. The inhibitory subdomain is able to independently suppress transcriptional activation of other strong activators such as viral protein 16, VP16. This occurs either when the inhibitory domain is fused directly to VP16 or when the inhibitory domain is independently bound to DNA by GAL4 DNA-binding domain independent of the VP16 activator. Overexpression of the LKLF autoinhibitory domain alone potentiates transactivation by wild type LKLF, suggesting that the inhibitory domain binds a cofactor that prevents LKLF from transactivating. A yeast-two hybrid screen identified WWP1, an E3 ubiquitin ligase that binds specifically to the LKLF inhibitory domain but not to other transcription factors. In mammalian cells, WWP1 functions as a cofactor by binding LKLF and suppressing transactivation. These data demonstrate that LKLF contains multiple domains that either potentiate or inhibit the ability of this factor to function as an activator of transcription; moreover, regulation of LKLF transactivation is attenuated by an E3 ubiquitin ligase, WWP1.

cDNA isolation, genomic structure, regulation, and chromosomal localization of human lung Kruppel-like factor.

Lung Kruppel-like factor (LKLF) is a zinc finger transcription factor critical for embryonic development. We have previously identified and isolated the mouse LKLF gene and examined its role using gene targeting. In this report, we describe the isolation and molecular characterization of the human homolog of murine LKLF. The human and mouse LKLF homologs exhibit an 85% nucleotide identity and share 90% amino acid similarity. Furthermore, the 5' sequence in the proximal promoter region and 3' untranslated region are also conserved between the two species. Of particular interest is the finding that while sequences in the proximal promoter have diverged between mouse and human, a region of 75 nucleotides is essentially identical. Site-directed mutagenesis in this region impairs the ability of the LKLF promoter to drive reporter gene expression, indicating that it represents a novel transcriptional element important in the regulation of LKLF gene expression. The activation domain is highly proline-rich and, similar to mouse LKLF, contains 22% proline residues. The human LKLF transcriptional unit is located in a genomic region of approximately 3 kb on chromosome 19p13.1. This region of chromosome 19 is known to contain genes involved in various human diseases. Like mouse LKLF, human LKLF consists of three exons that are interrupted by two small introns. The locations of intron/exon boundaries and splice sites are conserved between two homologs. Northern analysis shows that LKLF is expressed in lung in addition to heart, skeletal muscle, placenta, and pancreas. The isolation and chromosomal mapping of human LKLF will make it possible to initiate studies devoted to assess the involvement of this gene in human disease(s).

A gene encoding an intestinal-enriched member of the Krüppel-like factor family expressed in intestinal epithelial cells.

The Krüppel-like factors make up a multigene family of transcription factors that have discrete patterns of expression, implying they play important biological roles in the tissues in which they are expressed. We have identified and characterized the cDNA for a novel murine transcription factor that is an additional member of the Krüppel-like family of transcription factors, named intestinal-enriched Krüppel-like factor (IKLF). This gene appears to be a homolog of the human BTEB-2 gene, although it exhibits a different pattern of tissue expression and the translated product is larger. IKLF is expressed in a limited number of tissues; the highest levels of IKLF expression are found in the digestive tract. IKLF shows temporal changes in expression during embryogenesis indicating that this gene is developmentally regulated. In addition, IKLF expression is limited to the epithelial lining of the intestine and is localized primarily to the base of the crypts in the adult intestine. The IKLF cDNA encodes for a 446 amino acid protein and is able to transactivate by binding specific DNA elements that are also recognized by other members of the Krüppel-like family. In addition, mutations in the activation domain attenuate the ability of this protein to function as a transcription factor. Collectively, these findings show that we have identified a transcription factor that is expressed predominantly in the epithelial crypt cells of the gastrointestinal tract and is a member of the Krüppel-like family of transcription factors.

Role of progesterone in luteinizing hormone-induced fibronectin production and deposition by chicken granulosa cells in vitro.

The role of progesterone in luteinizing hormone- (LH) induced fibronectin production and deposition by chicken ovarian granulosa cells was examined in vitro. Granulosa cells isolated from pre-ovulatory follicles of the domestic hen ovary were incubated in serum-free Medium 199, and the total amount of fibronectin (deposited, secreted into the medium and associated with cells) produced was measured by ELISA. LH increased the amount of fibronectin deposited by granulosa cells. Similarly, it increased the quantity of fibronectin secreted into the medium or associated with cells. Cyanoketone (an inhibitor of progesterone synthesis) suppressed dose dependently basal and LH-induced fibronectin deposition. Cyanketone also attenuated the total amount of fibronectin produced by control or LH-stimulated granulosa cells. Exogenous progesterone reversed the inhibitory effects of cyanoketone on basal and LH-induced fibronectin production and deposition. The non-degradable synthetic progestin R5020 stimulated fibronectin production in a dose-dependent manner. R5020 also reversed the inhibitory effects of cyanoketone on LH-induced fibronectin production and deposition. The antiprogestin, ZK 98.299, inhibited basal and LH-stimulated fibronectin production. The data demonstrate that endogenous progesterone regulates fibronectin production and deposition perhaps in an intracrine/autocrine manner. They indicate that LH-stimulated fibronectin production and deposition by chicken granulosa cells is mediated (at least in part) by progesterone.

Intracrine role of progesterone in follicle-stimulating hormone- and cyclic adenosine 3',5'-monophosphate-induced fibronectin production and deposition by chicken granulosa cells: influence of follicular development.

The role of progesterone in FSH- and 8-bromo-cAMP (8-Br-cAMP)-induced fibronectin production by chicken ovarian granulosa cells was examined. Granulosa cells isolated from the third largest (F3; developing; 15-20 mm in diameter) preovulatory follicle and a pool of immature small yellow follicles (SYF; 6-8 mm in diameter) were incubated in serum-free medium 199, and the total amount of fibronectin produced (deposited, secreted into the medium, and associated with cells) was measured by enzyme-linked immunosorbent assay. Unstimulated F3 cells deposited greater amounts of fibronectin than unstimulated SYF cells. FSH or 8-Br-cAMP significantly increased fibronectin deposition. Similarly, both agents increased the quantity of fibronectin secreted into the medium and that associated with cells. The magnitude of FSH- and 8-Br-cAMP-enhanced fibronectin deposition or secretion into medium by SYF cells was greater than that by F3 cells. Cyanoketone (an inhibitor of progesterone synthesis) significantly suppressed basal fibronectin production by F3 cells, but not that by SYF cells. Cyanoketone completely blocked FSH- or 8-Br-cAMP-induced fibronectin production by F3 cells, but caused only a modest inhibition (nonsignificant) of agonist-induced fibronectin production by SYF cells. Exogenous progesterone completely reversed the inhibitory effects of cyanoketone on agonist-induced fibronectin production. The nondegradable synthetic progestin R5020 also reversed the inhibitory effects of cyanoketone on agonist-induced fibronectin production. The antiprogestin, ZK 98.299, inhibited basal and FSH-stimulated fibronectin production. The data demonstrate that FSH- and cAMP-stimulated fibronectin production by chicken granulosa cells is dependent (at least in part) on de novo progesterone synthesis. Furthermore, they indicate that fibronectin production and deposition by these cells are stimulated by progesterone, perhaps in an intracrine/autocrine manner, and that the role of progesterone increases with advancing stages of follicular development.

Intracrine role of progesterone in fibronectin production and deposition by chicken ovarian granulosa cells in vitro: effect of extracellular calcium.

The role of extracellular Ca2+ in progesterone-induced fibronectin production and deposition by hen granulosa cells was studied. Granulosa cells isolated from preovulatory follicles of the chicken ovary were incubated in Ca(2+)-deficient or Ca(2+)-containing medium 199, and the amount of total fibronectin produced (fibronectin deposited in the matrix, secreted into the medium, or associated with cells) was measured by ELISA. The quantity of fibronectin deposited as well as the total amount of fibronectin produced in the presence or absence of exogenous progesterone was suppressed in Ca(2+)-deficient medium. It required greater concentrations of exogenous progesterone to significantly increase fibronectin production in Ca(2+)-deficient medium than in Ca(2+)-replete medium. Cyanoketone, an inhibitor of progesterone synthesis, suppressed total fibronectin produced by unstimulated cells both in the absence and presence of Ca2+. However, the inhibitory effect of cyanoketone was significantly less in Ca(2+)-replete medium than in Ca(2+)-deficient medium. Exogenous progesterone reversed completely the inhibitory effects of cyanoketone. In the presence of cyanoketone, progesterone caused a greater (3-fold) increase in fibronectin production in Ca(2+)-replete medium than in the absence of Ca2+. Thapsigargin, an agent that mobilizes Ca2+ from internal stores, suppressed basal and progesterone-induced fibronectin production in the absence and presence of cyanoketone. However, the inhibitory effect of thapsigargin was significantly reduced in Ca(2+)-replete medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Progesterone stimulates fibronectin production by chicken granulosa cells in vitro.

Experiments were conducted in vitro to examine the effect of progesterone on fibronectin production by chicken ovarian granulosa cells. Granulosa cells isolated from the largest (F1; mature) and third-largest (F3; developing) preovulatory follicles as well as form a pool of immature small yellow follicles (SYF) of the domestic chicken ovary were incubated in serum-free Medium-199 and the amounts of fibronectin and progesterone produced were quantified by enzyme-linked immunosorbent assay and radioimmunoassay respectively. The amounts of basal fibronectin and progesterone produced by granulosa cells from F1, F3 and SYF follicles increased with advancing stages of follicular development. Thus, the quantity of basal fibronectin secreted by granulosa cells was directly proportional to the amount of progesterone produced by them. Exogenously supplied progesterone increased the amount of fibronectin secreted by F1 and F3 cells in a dose-dependent manner, but its effect on SYF cells was marginal. Cyanoketone (an inhibitor of progesterone synthesis) suppressed basal fibronectin production by F1 and F3 granulosa cells and its inhibitory action was reversed by exogenous progesterone. The progesterone antagonist RU 486 also attenuated basal fibronectin production by F1 and F3 granulosa cells, but only the highest concentration affected SYF cells. The inhibitory effect of RU 486 was diminished in the presence of exogenous progesterone. These data show that progesterone regulates fibronectin production by chicken granulosa cells. They suggest that in avian granulosa cells, endogenous progesterone can stimulate fibronectin synthesis in an intracrine or autocrine manner.