Daniel I. H. Linzer

Metallothionein is part of a zinc-scavenging mechanism for cell survival under conditions of extreme zinc deprivation

Metallothionein (MT) is a small cysteine-rich protein thought to play a critical role in cellular detoxification of inorganic species by sequestering metal ions that are present in elevated concentrations. We demonstrate here that metallothionein can play an important role at the other end of the homeostatic spectrum by scavenging an essential metal in a mouse fibroblast cell line that has been cultured under conditions of extreme zinc deprivation (LZA-LTK−). These cells unexpectedly produce constitutively high levels of metallothionein mRNA; however, the MT protein accumulates only when high concentrations of zinc are provided in the media. Until this MT pool is saturated, no measurable zinc remains in the external media. In this case, zinc deprivation leads to amplification of the MT gene locus in the LZA-LTK− cell line. Furthermore, the intracellular zinc levels in the fully adapted cells remain at the normal level of 0.4 fmol zinc/cell, even when extracellular zinc concentration is decreased by 2 orders of magnitude relative to normal media.

Reactivation of proliferin gene expression is associated with increased angiogenesis in a cell culture model of fibrosarcoma tumor progression

Proliferin (PLF) is an angiogenic placental hormone. We now report that PLF gene expression can also occur in a progressive fibrosarcoma mouse tumor cell model. PLF mRNA and protein are detectable at very low levels in cell lines derived from the mild noninvasive stage of tumor development. Expression is greatly augmented in cell lines from the aggressively invasive stage of development, a stage at which the tumor becomes highly angiogenic, and PLF expression remains high in cell lines from the end stage of fibrosarcoma. Activator protein 1 factors present at high levels in the more invasive stages of the tumor may in part allow for increased PLF expression, as cells from the mild stage in which c-jun and junB are stably expressed secrete levels of PLF comparable to that of the advanced stages. Secreted PLF protein is functionally important in tumor cell angiogenic activity, as demonstrated by the reduction of angiogenic activity in fibrosarcoma cell culture medium by immunodepletion of PLF. These results suggest that an extraembryonic genetic program, which has evolved to support fetal growth, may be reactivated in certain tumors and contribute to tumor growth.

A cloned human CCAAT-box-binding factor stimulates transcription from the human hsp70 promoter.

The basal promoter of the human hsp70 gene is predominantly controlled by a CCAAT element at position -70 relative to the transcriptional initiation site. We report the isolation of a novel cDNA clone encoding a 114-kDa polypeptide that binds to the CCAAT element of the hsp70 promoter. Expression of this CCAAT-binding factor (CBF) cDNA activated transcription from cotransfected hsp70 promoter-reporter gene constructs in a CCAAT-dependent manner. CCAAT-binding factor shows no homology to the previously identified human CCAAT transcription factor or rat CCAAT/enhancer-binding protein.

Induction of Megakaryocyte Differentiation by a Novel Pregnancy-specific Hormone

Maturation of megakaryocytes and subsequent platelet release are normally regulated by a network of cytokines, including thrombopoietin and various interleukins. Because abnormal platelet production and activation have been implicated in gestational pathologies, additional pregnancy-specific cytokines may play important roles in the regulation of megakaryocytopoiesis. Consistent with this hypothesis, we have found that the hormone prolactin-like protein E, a placental hormone that we have recently characterized, targets megakaryocytes through a specific cell surface receptor and induces megakaryocyte differentiation through a gp130-dependent signal transduction pathway.

Prolactin receptor expression in the developing mouse embryo

We have examined the developmental pattern of prolactin receptor expression in the mouse by reverse transcription‐polymerase chain reaction, in situ hybridization, and radioligand binding and have found two unexpected aspects of temporal regulation. First, high levels of prolactin receptor mRNA were detected in mouse embryos at day 8 and day 18, but levels decreased between these days to a minimum at ∼day 14. In contrast, placental prolactin receptor mRNA levels remained constant throughout this gestational period. Second, on embryonic day 16 the mRNA encoding the long form of the prolactin receptor is more abundant in the fetal liver than any of the short receptor form mRNAs, but by day 18 a switch occurs and the mRNA encoding one of the short receptor forms becomes the predominant receptor mRNA in that tissue. Expression of the receptor mRNA and protein is widespread throughout the fetus, with especially high levels in developing bone and cartilagenous structures, the thymus and pituitary, the tongue and skeletal muscle, and certain regions of the brain. The pattern of expression of prolactin receptor in the fetal mouse suggests an important role for the placental lactogens, the major ligands for fetal prolactin receptors, in fetal growth and development. Mol. Reprod. Dev. 48:45–52, 1997.

Differentiation of Placental Trophoblast Giant Cells Requires Downregulation of p53 and Rb

Trophoblast giant cells in the rodent placenta form the outermost layer of the extraembryonic compartment, establish direct contact with maternal cells, and produce a number of pregnancy-specific cytokine hormones. Giant cells differentiate from proliferative trophoblasts as they exit the cell cycle and enter a genome-amplifying endocycle, a process we show involves decreased expression of the G1 checkpoint proteins p53 and Rb. Although p53 mRNA levels are unchanged in proliferative compared to differentiated trophoblasts, p53 protein levels are markedly reduced in giant cells. Forced expression of wild type p53 in trophoblasts inhibits differentiation, and expression of a dominant negative p53 peptide stimulates differentiation. Consistent with the loss of p53 protein, differentiated trophoblasts become resistant to apoptosis-inducing agents. Decreased expression of Rb is also detected during differentiation, and overexpression of Rb in trophoblasts inhibits giant cell differentiation. Although an increase in E2F activity would be expected with the loss of Rb, what is observed is an overall decrease in E2F DNA-binding complexes, a shift to new complexes, and a decrease in E2F-dependent gene expression in differentiating trophoblasts. Overall, these results indicate that the combination of a decrease in p53 and Rb represents a functionally important part of the transition of trophoblasts from a proliferative cell cycle to an endocycle in the giant cell differentiation programme.

GATA-2 Restricts Prolactin-Like Protein A Expression to Secondary Trophoblast Giant Cells in the Mouse

Abstract An analysis of the pattern of expression of the mouse placental hormone prolactin-like protein A (PLP-A) has revealed that this hormone is expressed exclusively in secondary trophoblast giant cells but not in primary giant cells. Thus, PLP-A serves as a marker for a subset of giant cells. Recent results have indicated that PLP-A binds to and inhibits the activity of natural killer cells, and thus, the localized expression of PLP-A may be important for regulating the activity of this class of T lymphocytes in a restricted region of the implantation site. Previous studies indicated that the transcription factor GATA-2 is required for the trophoblast giant cell-specific expression of two other hormones in the prolactin family, placental lactogen I and proliferin. In the absence of GATA-2, PLP-A continues to be expressed, but in this mutant background, PLP-A mRNA is detected in both primary and secondary giant cells. Thus, GATA-2 contributes both to positive and negative regulation of trophoblast giant cell-specific gene expression, and this factor apparently plays an important role in generating or maintaining the distinct functions of secondary, compared with primary, trophoblast giant cells.

Rodent Prolactin Family and Pregnancy

Rodents have exploited the ancestral PRL gene to evolve a large number of closely related genes encoding PRL-like proteins. These PRL-related genes are all expressed at the implantation site, either in the trophoblasts of the placenta or in the decidual cells of the uterus. Some of these hormones have bioactivities indistinguishable from PRL, whereas most others have distinct molecular and cellular targets, and as a consequence distinct activities. This chapter will review what is currently known about this family of hormones.

Identification of Three Prolactin-Related Hormones as Markers of Invasive Trophoblasts in the Rat

Abstract An expressed-sequence tag database search has identified three rat cDNA clones in the prolactin/growth hormone family, including a homologue of mouse proliferin-related protein (PRP). The encoded proteins of the two novel clones, designated prolactin-like proteins L (PLP-L) and M (PLP-M), are predicted to be synthesized as precursors of 229 and 227 amino acids, modified by N-linked glycosylation, and secreted as mature glycoproteins of 199 and 200 residues, respectively. Murine homologues to PLP-L and PLP-M were also identified. The open reading frame of rat PRP encodes a precursor protein of 245 amino acids and predicts a secreted 215-amino acid glycoprotein with 81% identity to mouse PRP. All three rat mRNAs are expressed in the placenta, and expression is not detected in other tissues. PLP-L mRNA expression is observed from Days 11–20, with highest levels at Day 13; highest levels of PLP-M are observed from Day 11 until parturition, with peak levels also on Day 13; and highest levels of PRP are also observed from Day 11 until term, with maximal expression on Day 17. All three genes are most highly expressed in invasive trophoblast cells lining the central placental vessel. The identification of molecular markers for endovascular trophoblasts serves to highlight the invasive nature of rodent placentation and may prove useful for future studies of placental function.

Biological activities of glycosylated and nonglycosylated porcine prolactin

Nonglycosylated and glycosylated porcine prolactin (PRL) were separated using concanavalin A-Sepharose CL-6B column chromatography and tested for mitogenic and lactogenic activities, as well as immunoaffinity and receptor binding characteristics compared to total (nonseparated) porcine PRL. Mitogenic activity, using Nb2 lymphoma cells, was 4- and 50-fold greater (P less than 0.01) for total PRL than nonglycosylated and glycosylated PRL, respectively. Glycosylated PRL had 64% higher (P less than 0.05) lactogenic activity than nonglycosylated or total PRL. In a homologous radioimmunoassay (RIA), displacement was greatest for total, followed by the nonglycosylated and glycosylated forms of PRL. Competitive inhibition of porcine [125I]-(total) PRL by radioinert total, nonglycosylated and glycosylated PRL in a homologous radioreceptor assay (RRA) indicated similar Ka values for total and nonglycosylated PRL, but different receptor numbers, while radioinert glycosylated PRL had a higher Ka, but bound fewer receptors. Therefore, glycosylated porcine PRL has greater lactogenic activity and higher binding affinity despite decreased mitogenicity, while nonglycosylated PRL had characteristics similar to total PRL. Results from the homologous RRA and the Nb2 assay suggest that both forms of PRL are necessary to achieve biological effects similar to those for total PRL. The two forms of PRL may have individual and collective effects, while changes in the ratio between these forms may influence physiologically diverse effects of PRL on target tissues.