Biomedical subjects

K Grandien

Publications and source records attributed to K Grandien.

11 recordsLinked to original sources

PubMed2001

Functional comparison of the nematode Hox gene lin-39 in C. elegans and P. pacificus reveals evolutionary conservation of protein function despite divergence of primary sequences.

Hox transcription factors have been implicated in playing a central role in the evolution of animal morphology. Many studies indicate the evolutionary importance of regulatory changes in Hox genes, but little is known about the role of functional changes in Hox proteins. In the nematodes Pristionchus pacificus and Caenorhabditis elegans, developmental processes can be compared at the cellular, genetic, and molecular levels and differences in gene function can be identified. The Hox gene lin-39 is involved in the regulation of nematode vulva development. Comparison of known lin-39 mutations in P. pacificus and C. elegans revealed both conservation and changes of gene function. Here, we study evolutionary changes of lin-39 function using hybrid transgenes and site-directed mutagenesis in an in vivo assay using C. elegans lin-39 mutants. Our data show that despite the functional differences of LIN-39 between the two species, Ppa-LIN-39, when driven by Cel-lin-39 regulatory elements, can functionally replace Cel-lin-39. Furthermore, we show that the MAPK docking and phosphorylation motifs unique for Cel-LIN-39 are dispensable for Cel-lin-39 function. Therefore, the evolution of lin-39 function is driven by changes in regulatory elements rather than changes in the protein itself.

Amino Acid Sequence↗

PubMed2000

The human brain has distinct regional expression patterns of estrogen receptor alpha mRNA isoforms derived from alternative promoters.

The human estrogen receptor (ER) alpha gene is transcribed from multiple promoters, generating mRNA isoforms with unique 5' ends in the untranslated region. In the present study, alternative promoters were shown to regulate the ERalpha gene expression in different neuronal populations of the human brain. By using in situ hybridization histochemistry, the A and B promoters, but not the C promoter, in the ERalpha gene were found to be active in the human forebrain. The mRNA isoform transcribed from the A promoter was expressed in low levels in most of the brain areas where ERalpha mRNA was present. In contrast, the B promoter mRNA isoform was more restricted, localized predominantly in high-expressing ERalpha mRNA regions. The gross anatomical distribution of the different mRNA isoforms analyzed with RT-PCR generally supported the results obtained by the in situ hybridization. Estrogen is known to modulate many different brain functions, such as neuroendocrine events associated with reproduction, mood, and cognition, likely to be mediated by different neuronal populations. Thus, the current findings of alternative ERalpha promoter expression in distinct neuronal populations suggest that multiple promoter usage is a possible mechanism to achieve differentiated regulation of the ERalpha expression, dependent on the cell phenotype and consequently the functions mediated by the specific neuron.

Adolescent↗

PubMed1997

The estrogen receptor gene: promoter organization and expression.

The estrogen receptor (ER) is a ligand-activated transcription factor and a member of a large family of nuclear hormone receptors. As a mediator of estrogen hormone action, the ER is involved in many important physiological processes. ER gene expression has been demonstrated to be restricted to certain tissues and under complex hormonal control. However, the molecular mechanisms involved have remained largely unknown. Due to this lack of knowledge an investigation was undertaken to characterize the promoter organization of ER gene and investigate its expression. Approximately 3 kb of the 5' flanking region of the human ER (hER) gene was isolated and sequenced. By performing RT-PCR and RACE experiments it was shown that the hER gene is transcribed from three different promoters. Transcription of the hER gene from these promoters yields three different mRNA isoforms with unique 5' untranslated regions (5'UTRs), but identical coding regions. The expression pattern of the hER mRNA isoforms was investigated by RT-PCR. Both the A- and B-mRNA isoforms were found to be expressed in breast and uterus, whereas expression of the C-transcript was predominantly detected in liver. In bone cells only expression of the B-mRNA could be detected. The steady-state levels of the A- and B-transcripts in normal breast and uterus were quantified and compared with the hER mRNA levels in established cancer cell lines derived from the same tissues. This demonstrated approximately equal levels of the two transcripts in normal tissues whereas the A-mRNA was the most abundant isoform in the cancer cell lines investigated. Approximately 4.5 kb of the 5' flanking region of the rat ER (rER) gene were sequenced. Sequence analysis and PCR experiments suggested that the promoter organization of the rat and human ER genes is only partially conserved which might indicate species-specific differences in the regulation of ER expression. In conclusion, this work suggests tissue-specific alternative promoter usage as a mechanism in the regulation of human and rat ER gene expression.

Animals↗

PubMed1997

Tissue distribution and quantitative analysis of estrogen receptor-alpha (ERalpha) and estrogen receptor-beta (ERbeta) messenger ribonucleic acid in the wild-type and ERalpha-knockout mouse.

Until recently, only a single type of estrogen receptor (ER) was thought to exist and mediate the genomic effects of the hormone 17beta-estradiol in mammalian tissues. However, the cloning of a gene encoding a second type of ER, termed ERbeta, from the mouse, rat, and human has prompted a reevaluation of the estrogen signaling system. Based on in vitro studies, the ERbeta protein binds estradiol with an affinity similar to that of the classical ER (now referred to as ERalpha) and is able to mediate the effects of estradiol in transfected mammalian cell lines. Essential to further investigations of the possible physiological roles of ERbeta, and its possible interactions with ERalpha, are data on the tissue distribution of the two ER types. Herein, we have described the optimization and use of an RNase protection assay able to detect and distinguish messenger RNA (mRNA) transcripts from both the ERalpha and ERbeta genes in the mouse. Because this assay is directly quantitative, a comparison of the levels of expression within various tissues was possible. In addition, the effect of disruption of the ERalpha gene on the expression of the ERbeta gene was also investigated using the ERalpha-knockout (ERKO) mouse. Transcripts encoding ERalpha were detected in all the wild-type tissues assayed from both sexes. In the female reproductive tract, the highest expression of ERbeta mRNA was observed in the ovary and showed great variation among individual animals; detectable levels were observed in the uterus and oviduct, whereas mammary tissue was negative. In the male reproductive tract, significant expression of ERbeta was seen in the prostate and epididymis, whereas the testes were negative. In other tissues of both sexes, the hypothalamus and lung were clearly positive for both ERalpha and ERbeta mRNA. The ERKO mice demonstrated slightly reduced levels of ERbeta mRNA in the ovary, prostate, and epididymis. These data, in combination with the several described phenotypes in both sexes of the ERKO mouse, suggest that the biological functions of the ERbeta protein may be dependent on the presence of ERalpha in certain cell types and tissues. Further characterization of the physiological phenotypes in the ERKO mice may elucidate possible ERbeta specific actions.

Animals↗

PubMed1997

Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors alpha and beta.

The rat estrogen receptor (ER) exists as two subtypes, ER alpha and ER beta, which differ in the C-terminal ligand binding domain and in the N-terminal transactivation domain. In this study we investigated the messenger RNA expression of both ER subtypes in rat tissues by RT-PCR and compared the ligand binding specificity of the ER subtypes. Saturation ligand binding analysis of in vitro synthesized human ER alpha and rat ER beta protein revealed a single binding component for 16 alpha-iodo-17 beta-estradiol with high affinity [dissociation constant (Kd) = 0.1 nM for ER alpha protein and 0.4 nM for ER beta protein]. Most estrogenic substances or estrogenic antagonists compete with 16 alpha-[125I]iodo-17 beta-estradiol for binding to both ER subtypes in a very similar preference and degree; that is, diethylstilbestrol > hexestrol > dienestrol > 4-OH-tamoxifen > 17 beta-estradiol > coumestrol, ICI-164384 > estrone, 17 alpha-estradiol > nafoxidine, moxestrol > clomifene > estriol, 4-OH-estradiol > tamoxifen, 2-OH-estradiol, 5-androstene-3 beta, 17 beta-diol, genistein for the ER alpha protein and dienestrol > 4-OH-tamoxifen > diethylstilbestrol > hexestrol > coumestrol, ICI-164384 > 17 beta-estradiol > estrone, genistein > estriol > nafoxidine, 5-androstene-3 beta, 17 beta-diol > 17 alpha-estradiol, clomifene, 2-OH-estradiol > 4-OH-estradiol, tamoxifen, moxestrol for the ER beta protein. The rat tissue distribution and/or the relative level of ER alpha and ER beta expression seems to be quite different, i.e. moderate to high expression in uterus, testis, pituitary, ovary, kidney, epididymis, and adrenal for ER alpha and prostate, ovary, lung, bladder, brain, uterus, and testis for ER beta. The described differences between the ER subtypes in relative ligand binding affinity and tissue distribution could contribute to the selective action of ER agonists and antagonists in different tissues.

Amino Acid Sequence↗

PubMed1997

Human estrogen receptor beta-gene structure, chromosomal localization, and expression pattern.

The estrogen receptor (ER) is a ligand-activated transcription factor that mediates the effects of the steroid hormone 17 beta-estradiol, in both males and females. Since the isolation and cloning of ER, the consensus has been that only one such receptor exists. The finding of a second subtype of ER (ER beta) has caused considerable excitement amongst endocrinologists. In this article, we present data regarding the genomic structure and chromosomal localization of the human ER beta gene, demonstrating that two independent ER genes do exist in the human. Furthermore, we present data regarding the tissue distribution of human ER beta, showing that this receptor is expressed in multiple tissues. For instance, ER beta is found in developing spermatids of the testis, a finding of potential relevance for the ongoing debate on the effects of environmental estrogens on sperm counts. In addition, we find ER beta in ovarian granulosa cells, indicating that estrogens also participate in the regulation of follicular growth in the human.

Amino Acid Sequence↗

PubMed1997

Mouse estrogen receptor beta forms estrogen response element-binding heterodimers with estrogen receptor alpha.

The recent discovery that an additional estrogen receptor subtype is present in various rat tissues has advanced our understanding of the mechanisms underlying estrogen signaling. Here we report on the cloning of the cDNA encoding the mouse homolog of estrogen receptor-beta (ER beta) and the functional characterization of mouse ER beta protein. ER beta is shown to have overlapping DNA-binding specificity with that of the estrogen receptor-alpha (ER alpha) and activates transcription of reporter gene constructs containing estrogen-response elements in transient transfections in response to estradiol. Using a mammalian two-hybrid system, the formation of heterodimers of the ER beta and ER alpha subtypes was demonstrated. Furthermore, ER beta and ER alpha form heterodimeric complexes with retained DNA-binding ability and specificity in vitro. In addition, DNA binding by the ER beta/ER alpha heterodimer appears to be dependent on both subtype proteins. Taken together these results suggest the existence of two previously unrecognized pathways of estrogen signaling; I, via ER beta in cells exclusively expressing this subtype, and II, via the formation of heterodimers in cells expressing both receptor subtypes.

Amino Acid Sequence↗

PubMed1996

Determination of transcription start sites in the human estrogen receptor gene and identification of a novel, tissue-specific, estrogen receptor-mRNA isoform.

The human estrogen receptor (ER) gene has previously been shown to be transcribed from two different promoters which are expressed in a cell- and tissue-specific manner. Whereas the transcriptional start sites of the proximal promoter are known, the transcriptional initiation site(s) of the distal promoter has not been identified. In this study, a PCR-based technique was used to isolate the 5' ends of ER cDNAs from different human cell lines and tissues. Using this technique the positions of the transcription initiation sites in the distal promoter were determined. In addition a novel 5' untranslated exon was isolated from human liver. These results demonstrate that the human ER gene contains a third, previously not described promoter which appears to be predominantly expressed in liver.

Base Sequence↗

PubMed1996

The 5' flank of the rat estrogen receptor gene: structural characterization and evidence for tissue- and species-specific promoter utilization.

Published data on the rat estrogen receptor (rER) gene have so far provided evidence for only one promoter. In contrast, the human ER (hER) gene has been reported to be transcribed from three different promoters A, B and C. In order to facilitate a more in-depth analysis of the promoter organization in the rER gene 4.5 kb of its 5' flanking region were isolated. Sequence analysis suggests that the rER gene is transcribed from a promoter homologous to promoter B in the hER gene. However, the region corresponding to the human promoter A is much more divergent between the species and rapid amplification of cDNA ends (RACE)-PCR as well as RT-PCR experiments support the notion that the rER gene does not contain a functional A promoter. Interestingly, a novel rER mRNA isoform containing a previously unknown 5' untranslated exon was isolated by RACE-PCR, suggesting the existence of a second, distal promoter in the rER gene. Our results show that this ER mRNA isoform C is the only one expressed in rat liver. It thus appears that the rER gene contains two promoters that are utilized in a tissue-specific fashion.

Animals↗

PubMed1995

Estrogen target tissue determines alternative promoter utilization of the human estrogen receptor gene in osteoblasts and tumor cell lines.

The estrogen receptor (ER) mediates the effects of its cognate ligand on important cellular processes such as development of female secondary sexual characteristics, establishment and maintenance of pregnancy, progression of breast cancer, and maintenance of bone mass. We have previously demonstrated that the human ER (hER) gene is transcribed from two promoters, suggesting that tissue- and cell-specific expression patterns of this gene may, at least in part, be regulated by differential promoter usage. Here we show, by using a reverse transcriptase coupled polymerase chain reaction assay, that transcripts initiated from both hER gene promoters were expressed in breast and uterus. In contrast, only transcripts originating from the distal promoter could be detected in human primary osteoblasts. Furthermore, determination of the expression levels of the two hER transcripts by quantitative polymerase chain reaction demonstrated an almost 30-fold increase of the transcripts originating from the proximal promoter in breast cancer cell lines over that detected in normal breast tissue. Taken together, our results demonstrate a previously unrecognized mechanism for regulation of hER gene expression by tissue-specific differential promoter utilization. In addition, our results suggest that estrogen-dependent cell transformation may be accompanied by a change in the relative expression levels of the two hER transcripts.

Adult↗

PubMed1993

Protein-protein interactions between the DNA-binding domains of nuclear receptors: influence on DNA-binding.

The glucocorticoid and thyroid hormone receptors have the capacity to bind as dimers to palindromic DNA-binding sites. Protein-protein interactions between the DNA-binding domains of glucocorticoid receptor dimers restrict the DNA-binding to elements where the half-sites are separated by three base pairs, whereas DNA-binding by the thyroid hormone receptor does not appear to require a strict half-site spacing. We have previously shown that a five amino-acid segment close the the C-terminal zinc-binding site (D-box) was involved in dimerization of the glucocorticoid receptor (GR) DNA-binding domain (Dahlman-Wright et al., 1991, J. Biol. Chem., 266, 3107-3112). Here we provide functional evidence, using mutated thyroid hormone receptor DNA-binding domains, that this five amino acid segment (D-box) of the GR interacts with the equivalent segment on the second DNA-binding domain in the dimer. In contrast, the thyroid hormone receptor DNA-binding domain binds to palindromic thyroid hormone response elements in a weakly co-operative manner, independent of the D-box.

Amino Acid Sequence↗