Biomedical subjects

D I Linzer

Publications and source records attributed to D I Linzer.

At least 37 records · Page 2Linked to original sources

PubMed1997

Two novel members of the prolactin/growth hormone family are expressed in the mouse placenta.

Two novel members of the mouse PRL/GH family have been identified through a search of an expressed sequence tag database. The encoded proteins do not appear to be homologs of other known members of this hormone family. One of these proteins, designated PRL-like protein E (PLP-E), is predicted to be synthesized as a precursor of 265 amino acids, modified by N-linked glycosylation, and secreted as a mature glycoprotein of 236 residues. The second clone encodes a protein of 253 residues with consensus sites for N-linked glycosylation; the secreted form of the protein, designated PRL-like protein F (PLP-F), is predicted to be 223 amino acids in length. Both of these messenger RNAs are expressed specifically in the placenta, with peak levels of PLP-E on days 10-12 and of PLP-F on days 14-16. Expression of PLP-E is restricted to the trophoblast giant cells, whereas PLP-F is synthesized only in the spongiotrophoblasts. The genes for both of these proteins map to a 700-kilobase region of mouse chromosome 13 that includes other members of the PRL/GH family.

Amino Acid Sequence↗

PubMed1997

Proliferin induces endothelial cell chemotaxis through a G protein-coupled, mitogen-activated protein kinase-dependent pathway.

To investigate the mechanism of action of the placental angiogenic hormone proliferin (PLF), we analyzed the signaling components in endothelial cells that are required for PLF-induced chemotaxis. Pertussis toxin, which inactivates Gi proteins, inhibited PLF-induced chemotaxis of endothelial cells. Gi proteins can lead to activation of the mitogen-activated protein kinase (MAPK) pathway; PLF was found to stimulate MAPK activity, and this induction was blocked by both pertussis toxin and a specific inhibitor of MAPK kinase, PD 098059. Furthermore, a blockade of MAPK activation prevented endothelial cell movement in response to PLF. As PLF functionally interacts with the insulin-like growth factor II (IGF-II)/mannose 6-phosphate receptor, we also examined the effects of pertussis toxin and PD 098059 on another ligand for this receptor, a mutant form of IGF-II; both inhibitors also block the action of this factor on endothelial cells. These data suggest that chemotaxis initiated by PLF and mediated by the IGF-II/mannose 6-phosphate receptor occurs through a G protein-coupled pathway, and that MAPK activation is necessary for the chemotactic response.

Animals↗

PubMed1997

GATA-2 and GATA-3 regulate trophoblast-specific gene expression in vivo.

We previously demonstrated that the zinc finger transcription factors GATA-2 and GATA-3 are expressed in trophoblast giant cells and that they regulate transcription from the mouse placental lactogen I gene promoter in a transfected trophoblast cell line. We present evidence here that both of these factors regulate transcription of the placental lactogen I gene, as well as the related proliferin gene, in trophoblast giant cells in vivo. Placentas lacking GATA-3 accumulate placental lactogen I and proliferin mRNAs to a level 50% below that reached in the wild-type placenta. Mutation of the GATA-2 gene had a similar effect on placental lactogen I expression, but led to a markedly greater reduction (5- to 6-fold) in proliferin gene expression. Placentas lacking GATA-2 secrete significantly less angiogenic activity than wild-type placentas as measured in an endothelial cell migration assay, consistent with a reduction in expression of the angiogenic hormone proliferin. Furthermore, within the same uterus the decidual tissue adjacent to mutant placentas displays markedly reduced neovascularization compared to the decidual tissue next to wild-type placentas. These results indicate that GATA-2 and GATA-3 are important in vivo regulators of trophoblast-specific gene expression and placental function, and reveal a difference in the effect of these two factors in regulating the synthesis of related placental hormones.

Animals↗

PubMed1996

Cloning and characterization of an ovarian-specific protein that associates with the short form of the prolactin receptor.

Prolactin (PRL) is essential for progesterone biosynthesis and luteal cell hypertrophy of the rat corpus luteum during pregnancy. Both the long and short form of the PRL receptor have been identified in the corpus luteum of pregnant rat. The long form has been shown to transduce PRL signal in other cells, whereas no information is available on the role of the short form, especially in the corpus luteum. In the present study, we have cloned a rat ovarian-specific phosphoprotein, PRAP (PRL Receptor Associated Protein), which has no significant homology to other known proteins. We have demonstrated that this protein is immunoprecipitated by anti-PRL receptor and anti-phosphotyrosine antibodies. To determine whether PRAP associates with either the long or the short form of the PRL receptor, fusion proteins with glutathione S-transferase containing the cytoplasmic domain of the long or short form of the PRL receptor were produced, purified, and incubated with luteal proteins. Our results indicate that PRAP preferentially binds to the short form of the PRL receptor. Thus, the long form and short forms of the PRL receptor may signal through distinct pathways. These data provide evidence for the involvement of a novel protein in PRL signal transduction and suggest that PRAP may contribute to the luteotropic effects of PRL on the corpus luteum during pregnancy.

Amino Acid Sequence↗

PubMed1996

Cloning and characterization of mouse CCAAT binding factor.

Isolation of cDNA clones for the mouse CCAAT binding factor (mCBF) has revealed the expression of two distinct forms of mCBF that are generated by alternative splicing of a single primary transcript from a gene that maps to chromosome 17. The mCBF1 mRNA encodes a protein of 997 amino acids, whereas the mCBF2 protein is predicted to be only 461 amino acids in length; mCBF1 and human CBF (hCBF) share>80% amino acid sequence identity. Analysis of adult mouse tissue RNAs has revealed that the mCBF1 and mCBF2 mRNAs are ubiquitously expressed, but that mCBF1 mRNA is 5- to 10-fold more abundant than mCBF2 mRNA. Similarly, mCBF mRNA was detected through-out the placenta and in all tissues of the developing embryo from day 8 to day 18 of gestation. Overexpression of the two forms of mCBF in mammalian cells has demonstrated that the mCBF1 and mCBF2 proteins localize to different cellular compartments, with mCBF1 found predominantly in the nucleus and mCBF2 restricted to the cytoplasm. Co-expression of these two forms influences their localization, however, indicating that CBF activity can be regulated by the relative amounts of the two forms expressed in a cell.

3T3 Cells↗

PubMed1996

The decidual prolactin receptor and its regulation by decidua-derived factors.

Decidualization of the endometrial stroma in the rat gives rise to two different cell populations, located either mesometrially or antimesometrially in the uterus. We have previously shown that the rat decidua is the site of production and action of a PRL-like hormone. In this investigation we examined, using reverse transcription-PCR, whether and which type of PRL receptor (PRL-R) messenger RNA (mRNA) is expressed in the decidua, whether the receptor is confined only to one cell population, and whether the PRL-R expression is regulated by decidua-derived factors. The results indicate that the uterus of pseudopregnant rats does not express the PRL-R and that decidualization does not trigger a rapid appearance of PRL-R mRNA. It is only 3 days after the induction of decidualization that the long form of the PRL-R was first expressed. Thereafter, mRNAs for both the short (PRL-RS) and the long (PRL-RL) form became detectable in both antimesometrial and mesometrial decidua, although PRL-RL mRNA was much more abundant than PRL-RS. As development proceeded, PRL-R mRNA decreased and disappeared specifically from the antimesometrial decidua, whereas the mesometrial decidua continued to express this receptor mRNA. Concomitant with down-regulation of the PRL-R in the antimesometrial tissue was a rather abrupt expression of activin A. In contrast, the mesometrial tissue that maintained high levels of PRL-R mRNA expressed little activin A, but produced an activin-binding protein, alpha(2)-macroglobulin (alpha(2)MG). To determine whether activin A and alpha(2)MG regulate PRL-R expression, antimesometrial and mesometrial cells were separated by elutriation and maintained in culture in the presence or absence of activin A, alpha(2)MG, or follistatin. Just after cell separation, both cell populations expressed PRL-R, but not activin A. Within 6 h, activin A mRNA and protein became highly expressed in the mesometrial cells, whereas PRL-RL mRNA became undetectable. In contrast, activin A mRNA was at very low levels in the antimesometrial cells, and no activin A protein could be detected in the medium for at least 12 h. In these cells PRL-RL mRNA remained elevated. Addition of activin A to antimesometrial cells caused a marked down-regulation of PRL-RL mRNA expression, whereas addition of alpha(2)MG and follistatin to mesometrial cells prevented the disappearance of PRL-R. In summary, the results of this investigation 1) indicate that decidualization of the endometrial stroma induces the appearance of both forms of the PRL-R mRNAs; 2) show differential expression of the PRL-R mRNA in the two-cell population forming the decidua; 3) establish that this differential expression is due to two key decidual molecules, activin A and alpha(2) macroglobulin; and 4) demonstrate that activin A can cause the decidual cells to lose the PRL-R and that the disappearance of the decidual PRL-R can be prevented by addition to the culture of two activin binding proteins, follistatin and alpha(2) MG.

Activins↗

PubMed1996

The insulin-like growth factor II/mannose 6-phosphate receptor is required for proliferin-induced angiogenesis.

Proliferin stimulates endothelial cell migration in culture and neovascularization in vivo. Previous studies have demonstrated that proliferin can bind to the insulin-like growth factor II/mannose 6-phosphate receptor, and that binding can be blocked by mannose 6-phosphate. We have now found that this receptor plays an essential role in proliferin-induced angiogenesis. Proliferin binding to endothelial cells is blocked by the addition of mannose 6-phosphate, as is the ability of both recombinant and placental-derived proliferin to stimulate the migration of capillary endothelial cells in vitro and to induce neovascularization in the rat cornea. Consistent with a direct role of this receptor in angiogenesis, insulin-like growth factor II, as well as a mutant form of insulin-like growth factor II that binds to the insulin-like growth factor II/mannose 6-phosphate receptor but not to the insulin-like growth factor I receptor, also stimulate endothelial cell migration and neovascularization.

Animals↗

PubMed1995

Cyclic adenosine 3',5'-monophosphate stimulation of placental proliferin and proliferin-related protein secretion.

To identify factors that regulate proliferin (PLF) and PLF-related protein (PRP) secretion by the mouse placenta, placental cells from day 9 of pregnancy were cultured for up to 5 days, and PLF and PRP release into the medium was assessed by RIA. Transforming growth factor-alpha, interleukin-1 alpha, and interleukin-6 did not regulate either PLF or PRP secretion. However, treatment of primary placental cell cultures with 8-bromo-cAMP, cholera toxin, or forskolin resulted in 2- to 3-fold increases in the percentages of PLF- and PRP-producing cells in the population and corresponding increases in both PLF and PRP messenger RNA and secreted protein. The increase in the number of PLF-producing cells was accompanied by an increase in the number of cells expressing both PLF and mouse placental lactogen-I. These data suggest that cAMP levels can regulate trophoblast giant cell differentiation and, consequently, the amount of PLF and PRP secretion.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1995

Role of the composite glucocorticoid response element in proliferin gene expression.

A binding site for the glucocorticoid receptor in the serum-inducible proliferin gene promoter has been reported to function as a composite glucocorticoid response element when fused to a minimal promoter. We now show that this element can also act as a glucocorticoid-independent negative regulator of transcription, both as an isolated element fused to a minimal promoter and within the context of the proliferin gene promoter. Furthermore, this element is recognized by a factor in mouse fibroblast cell extracts that is distinct from the glucocorticoid receptor and from AP-1, both of which have previously been shown to be able to bind to this site. The ability of this element to repress serum-inducible proliferin promoter activity is dependent on the position of this element with respect to the adjacent serum response region, and on the activity of a positive regulatory element located further upstream in the proliferin promoter.

Animals↗

PubMed1994

Stimulation and inhibition of angiogenesis by placental proliferin and proliferin-related protein.

In many mammalian species, the placenta is the site of synthesis of proteins in the prolactin and growth hormone family. Analysis of two such proteins, proliferin (PLF) and proliferin-related protein (PRP), revealed that they are potent regulators of angiogenesis; PLF stimulated and PRP inhibited endothelial cell migration in cell culture and neovascularization in vivo. The mouse placenta secretes an angiogenic activity during the middle of pregnancy that corresponds primarily to PLF, but later in gestation releases a factor that inhibits angiogenesis, which was identified as PRP. Incubation of placental tissue with PLF led to the specific binding of this hormone to capillary endothelial cells. Thus PLF and PRP may regulate the initiation and then the cessation of placental neovascularization.

Animals↗

PubMed1994

Cell aggregation in a Chinese hamster ovary cell microcarrier culture affects the expression rate and N-linked glycosylation of recombinant mouse placental lactogen-1.

A microcarrier culture of Chinese hamster ovary (CHO) cells, expressing the N-glycosylated recombinant protein mouse placental lactogen I (mPL-I), was found to form large cellular aggregates (400 to 600 microns in diameter). There was increased accumulation of lower molecular sized mPL-I glycoforms in cultures containing the large cellular aggregates at pH 7.3, but not at pH 7.6. Specific rates of mPL-I expression were found in the cultures with cellular aggregates at both pH values (7.3 and 7.6). These findings are interpreted in the light of our earlier studies that showed that extracellular pH and elevated ammonia concentrations affect both the glycosylation and the expression rates of mouse placental lactogen I.

Ammonia↗

PubMed1994

Characterization of a delayed early serum response region.

The proliferin (PLF) gene promoter provides a relatively simple model system for the study of growth-regulated gene expression in mouse cells. The promoter elements required for this serum-induced regulation have been identified and include an AP-1 site as well as an adjacent element comprised of three imperfect repeats that are similar in sequence to the simian virus 40 (SV40) Sph motif. Distinct protein complexes bound independently to the AP-1 and Sph elements, and both of these juxtaposed sites could be occupied simultaneously. Furthermore, serum stimulation of mouse fibroblasts resulted in similar increases in protein binding to the AP-1 and Sph elements. Consistent with this increase in AP-1 and Sph binding activity, the PLF AP-1 and Sph elements were independently able to confer serum responsiveness to a minimal promoter, and together these two elements acted synergistically in response to serum. Although several members of the AP-1 family were able to activate the PLF gene promoter in transient cotransfection experiments, the predominant AP-1 components interacting with the PLF gene promoter in serum-stimulated cells were Fra-1, JunB, and JunD. Analysis of the Sph element revealed that mutation of Sph repeats I or III abolished serum responsiveness of the PLF gene promoter, and mutation of Sph repeat III decreased protein binding to this element. Although the Sph element is similar in sequence to the SV40 element, the PLF Sph-binding factor is distinct from TEF-1, the factor that binds to the SV40 Sph motif.

Animals↗

PubMed1994

GATA factor activity is required for the trophoblast-specific transcriptional regulation of the mouse placental lactogen I gene.

The molecular determinants governing tissue-specific gene expression in the placenta are at present only poorly defined, particularly with respect to the regulation of specific hormone genes whose products are vital to embryonic development and the maintenance of a nurturing maternal environment. In continuing our analysis of the trophoblast-specific expression of the mouse placental lactogen I gene, we now demonstrate that the transcription factors GATA-2 and GATA-3 regulate the activity of this gene promoter. These factors are expressed in placental trophoblast cells, with peak levels of the GATA-2, GATA-3 and placental lactogen I mRNAs each accumulating at midgestation. Analysis of a region of the placental lactogen I gene promoter, previously shown to be sufficient for directing trophoblast-specific transcription, revealed the presence of three consensus binding sites for GATA-2 or GATA-3. Both GATA-2 and GATA-3 bind to these sites in vitro and mutation of these sites results in a significant decrease in promoter activity as assayed by transient transfection into the choriocarcinoma-derived cell line Rcho-1, which expresses endogenous GATA-2 and GATA-3. Furthermore, overexpression of GATA factors in Rcho-1 cells stimulates transcription from a co-transfected placental lactogen I gene promoter. Most significantly, expression of GATA-2 or GATA-3 was found to induce transcription from this promoter in transfected non-trophoblast (fibroblast) cells. These data indicate that GATA factors are both limiting and required transcriptional regulatory molecules in placental trophoblasts, and that the tissue specificity of the placental lactogen I gene is determined, at least in part, by GATA-2 and/or GATA-3.

Animals↗

PubMed1993

Regulation of the human hsp70 promoter by p53.

The tumor suppressor p53 is a nuclear phosphoprotein with characteristics of a transcription factor. It displays sequence-specific DNA binding, contains a potent transactivation domain, and has been implicated as both a transcriptional activator and a repressor. Transcription of the human hsp70 gene is stimulated by adenovirus E1a protein. This E1a transactivation of the hsp70 promoter is mediated by CCAAT binding factor (CBF). It is demonstrated here that p53 both represses transcription from the human hsp70 promoter and also interacts with CBF. Thus, the repression of the hsp70 promoter by p53 may be mediated by direct protein-protein interaction with CBF. These results suggest that protein-protein interaction between p53 and specific transcription factors may be an additional mechanism by which p53 regulates gene expression.

Adenovirus E1A Proteins↗

PubMed1993

Culture pH affects expression rates and glycosylation of recombinant mouse placental lactogen proteins by Chinese hamster ovary (CHO) cells.

Glycosylation patterns and specific expression rates of the recombinant protein mouse placental lactogen-I (mPL-I) by Chinese hamster ovary (CHO) cells varied significantly over the extracellular pH (pHe) range of 6.1 to 8.7. The maximum specific mPL-I expression rates occurred between pHe 7.6 and 8.0. The pHe effect on protein expression was confirmed using a different CHO cell expressing the unglycosylated recombinant protein mouse placental lactogen-II (mPL-II). Decreases in the extent of glycosylation of mPL-I were observed at low (below 6.9) and high (above 8.2) pHe values. The pHe dependent variations in mPL-I accumulation in the supernatant as well as in glycosylation patterns were not the result of enzymatic degradation in the culture medium.

Animals↗

PubMed1993

Changes in prolactin receptor expression during pregnancy in the mouse ovary.

We have isolated the cDNA encoding the cytoplasmic domain of a long form of the mouse PRL receptor (PRL-R). The mRNA for this long form PRL-R and the three mRNAs encoding short mouse PRL-R that have been previously characterized are all expressed in both the liver and ovary. The relative amounts of these receptor forms differ between tissues, however. In addition, the structure of one of the short receptor forms may not be identical in the liver and ovary. Within the ovary, the abundance and sites of synthesis of the four PRL-R mRNAs vary during pregnancy. Expression of the two most abundant PRL-R mRNAs increases significantly at midgestation. Expression of PRL-R mRNA is detected in the corpus luteum throughout pregnancy, while increased receptor mRNA levels are evident in the granulosa cells of a subset of Graafian follicles toward the end of pregnancy and during lactation. Some differences are also observed in the expression patterns of the individual receptor forms. Most notably, one of the short form PRL-R mRNAs is uniquely detected in atretic follicles in early to midgestation.

Amino Acid Sequence↗

PubMed1993

Prolactin receptor messenger ribonucleic acid expression in the ovary during the rat estrous cycle.

We have investigated the relative amounts and sites of synthesis during the rat estrous cycle of the two ovarian mRNAs encoding the long and short PRL receptors (PRL-R). Quantitative analysis has revealed that the mRNA encoding the short PRL-R is consistently present throughout the cycle in lower quantities than the long receptor mRNA. Both receptor mRNAs are at maximal levels during proestrus, decline to their lowest level of expression during estrus, then gradually rise in metestrus and diestrus. By in situ hybridization, both receptor mRNAs are present during early proestrus in corpora lutea, in the granulosa cell layers of large Graafian follicles, and in the interstitial cells closely associated with these follicles. The short PRL-R mRNA was detected at significant levels in the granulosa-derived cumulus oophorus and in the thecal cell region at this time, whereas the long PRL-R mRNA was only weakly expressed in these cell types. In contrast, the long PRL-R mRNA was present at higher levels, compared to the short receptor mRNA, in the granulosa cells of preantral follicles in the interior of the ovary. On late proestrus, the long PRL-R mRNA was found predominantly in the mural granulosa cells of large Graafian follicles and in corpora lutea, but by estrous morning this mRNA appeared to be mostly restricted to the corpora lutea. This distribution was maintained through estrous evening and metestrous morning. On diestrus, both mRNAs were present in some corpora lutea and in the granulosa cell layer in a subset of the larger Graafian follicles, but were detected at even higher levels in the interstitial cells surrounding these follicles; again, the long receptor mRNA appeared to be only weakly expressed in the thecal cell region of these follicles. These results indicate that the levels and locations of PRL-R mRNA expression in the ovary, and therefore, the potential responsiveness of the ovary to PRL, change throughout the reproductive cycle. Furthermore, the presence of both receptor mRNAs in several different ovarian cell types suggests that both of these receptor forms play important roles in PRL physiology in the ovary.

Animals↗

PubMed1993

Trophoblast-specific transcription from the mouse placental lactogen-I gene promoter.

We have isolated the gene encoding mouse placental lactogen-I and characterized the promoter region of this gene by transient and stable transfection. Promoter sequences extending 274 basepairs (bp) up-stream from the start site of transcription contain all of the elements necessary for maximal expression upon transient transfection into the rat choriocarcinoma Rcho-1 cell line; these Rcho-1 cultures contain both proliferative trophoblast stem cells and terminally differentiated trophoblast giant cells. In stably transfected cell lines, expression from this promoter increases as the percentage of differentiated cells in the culture increases. In contrast to these results in trophoblast cells, the 274-bp promoter as well as a promoter region extending 2700 bp up-stream of the transcriptional start site are unable to drive transcription in a variety of other cell types. Mutational and protein binding analyses indicate that two AP-1 sites are required for maximal expression in Rcho-1 cells, and that the composition of the AP-1 transcription factor may vary as differentiation in the cell culture increases. In addition to these two AP-1 sites, at least one other element appears to be critical for promoter activity in trophoblast cells.

Animals↗