Selective oestrogen receptor modulators differentially potentiate brain mitochondrial function.

The mitochondrial energy-transducing capacity of the brain is important for long-term neurological health and is influenced by endocrine hormone responsiveness. The present study aimed to determine the role of oestrogen receptor (ER) subtypes in regulating mitochondrial function using selective agonists for ERα (propylpyrazoletriol; PPT) and ERß (diarylpropionitrile; DPN). Ovariectomised female rats were treated with 17ß-oestradiol (E(2) ), PPT, DPN or vehicle control. Both ER selective agonists significantly increased the mitochondrial respiratory control ratio and cytochrome oxidase (COX) activity relative to vehicle. Western blots of purified whole brain mitochondria detected ERα and, to a greater extent, ERß localisation. Pre-treatment with DPN, an ERß agonist, significantly increased ERß association with mitochondria. In the hippocampus, DPN activated mitochondrial DNA-encoded COX I expression, whereas PPT was ineffective, indicating that mechanistically ERß, and not ERα, activated mitochondrial transcriptional machinery. Both selective ER agonists increased protein expression of nuclear DNA-encoded COX IV, suggesting that activation of ERß or ERα is sufficient. Selective ER agonists up-regulated a panel of bioenergetic enzymes and antioxidant defence proteins. Up-regulated proteins included pyruvate dehydrogenase, ATP synthase, manganese superoxide dismutase and peroxiredoxin V. In vitro, whole cell metabolism was assessed in live primary cultured hippocampal neurones and mixed glia. The results of analyses conducted in vitro were consistent with data obtained in vivo. Furthermore, lipid peroxides, accumulated as a result of hormone deprivation, were significantly reduced by E(2) , PPT and DPN. These findings suggest that the activation of both ERα and ERß is differentially required to potentiate mitochondrial function in brain. As active components in hormone therapy, synthetically designed oestrogens as well as natural phyto-oestrogen cocktails can be tailored to improve brain mitochondrial endpoints.

Mrp4, a new mitogen-regulated protein/proliferin gene; unique in this gene family for its expression in the adult mouse tail and ear.

Mitogen-regulated proteins (also known as proliferin; mrp/plf) are nonclassical members of the PRL/GH family. They are expressed at high levels during midgestation when they are thought to induce angiogenesis and uterine growth. There are between four and six mrp/plf genes, and three different complementary DNAs have been cloned. Here we identify a fourth mrp/plf gene (mrp4) that we have cloned and characterized. MRP4 is 91% identical in amino acid sequence with the other MRP/PLF proteins but is missing two glycosylation sites that are present in the other forms. Consistent with the loss of two of three glycosylation sites, the expressed form of MRP4 has a lower apparent molecular weight compared with other MRP/PLFs. In vivo, mrp4 is expressed in the placenta and the adult skin. Expression of mrp4 messenger RNA peaks in the placenta on day 12. In the skin, mrp4 expression is specific to the ears and tails of mice. Our results suggest that, as well as having growth and angiogenic effects during pregnancy, the MRP/PLFs may have functions in nonreproductive tissues. Unique among the members of the mrp/plf family for its expression in the hair follicles of the tail and ear, MRP4 is expected to have a singular role in the growth and development of these follicles.

Signaling between the placenta and the uterus involving the mitogen-regulated protein/proliferins.

The aim of this investigation was to examine signaling between the placenta and uterus during pregnancy. To do this, we determined the tissue messenger RNA and protein levels of members of a glycopeptide hormone family known to stimulate the proliferation of uterine cells and related these levels to the growth of the uterus during pregnancy in the mouse. This hormone family is known as mitogen-regulated protein (MRP); alternatively proliferin (PLF). Three mrp/plf genes, plf1, mrp3 and mrp4, are expressed by the placenta with different developmental profiles. The major increase of about 4-fold in DNA content of the uterus occurs between days 9 and 14 when MRP/PLFs are present in the placenta. By contrast, the gestational changes in estradiol-17beta levels in placental and uterine tissues and in circulation do not correlate with the period of uterine growth. The previously reported mitogenic activity of the MRP/PLFs and their gestational profiles suggest that one or more of these proteins stimulates uterine proliferation during gestation. Evidence is also presented that expression of MRP3 and/or PLF1, but not MRP4, is negatively regulated by feedback from the uterus. Our results are consistent with the hypothesis that MRP/PLFs stimulate uterine proliferation in vivo and that a uterine factor shuts off PLF1 and/or MRP3 synthesis in the latter half of gestation.

Cloning of the mink plasminogen activator inhibitor type-1 messenger RNA: an mRNA with a short half life.

In mink lung CCL64 epithelial cells the rate of synthesis of plasminogen activator inhibitor type I (PAI-1) increases 10-100-fold within 3 h in response to 12-O-tetradecanoyl phorbol-13-acetate (PMA). The PAI-1 gene is regulated transcriptionally. Parallel studies of the time-courses of PAI-1 synthesis and secretion and of mRNA accumulation indicate that the amount of secreted PAI-1 produced by the cells is tightly coupled to the level of its transcript. The half-life of the PAI-1 mRNA was found to be 25 min which is much shorter than previously reported for PAI-1 in other cells. Actinomycin D, which is commonly used to determine mRNA half-life, stabilized the PAI-1 mRNA. Cycloheximide also stabilized the mRNA. The short half-life and the superinducibility of PAI mRNA are properties shared with rapidly degraded mRNAs encoding protooncoproteins. A 2.97-kb cDNA clone containing the entire coding sequence of PAI-1 was isolated from a cDNA library made from mink lung CCL64 epithelial cells stimulated with PMA. The PAI-1 cDNA contains a long 3'-untranslated region (UTR) of 1720 bp whose sequence is highly conserved among PAI-1 mRNAs from different species. The PAI-1 mRNA also contains several AUUUA pentamer sequences which are the features of an A+U-rich regulatory element such as is found on the fos protooncogene mRNA. Upstream of one of these AUUUA pentamers are several highly conserved sequences that are also found in the 3' UTR of the fos and integrin receptor alpha-subunit mRNAs.(ABSTRACT TRUNCATED AT 250 WORDS)

Developing in a family way. Transforming Growth Factor Type beta and Related Proteins in Development: the Sixth Molecular, Cellular and Developmental Biology/Iowa State University Symposium, Ames, IA, USA September 20-23, 1991.

We are left with the impression that members of the TGF-beta family are critically positioned in a cascade of events regulating complex developmental processes. Roberts described the TGF-betas as providing the cells with cues to their temporal positions in a developmental program, that is, telling the cells "where they were, where they are, and where they're going." The broad diversity of cellular and tissue responses to TGF-betas and the widespread expression of their receptors suggest the TGF-betas may act as a "common currency," enabling diverse cell types to communicate with each other. Other members of the family are more restricted in their expression and the expression of their receptors, and may have a more limited and well-defined developmental role. The complex regulation of the members of the TGF-beta family is consistent with their importance as directors and coordinators of complicated physiological processes such as those occurring in the development of multicellular organisms. Their expression is highly regulated at the transcriptional and post-transcriptional levels, and their localization and activation can be affected by binding proteins and matrix proteins. We still have much to learn about the receptors for this family of growth factors, but the recent cloning of the activin and TGF-beta receptors and the discovery of their enzymatic nature has dramatically opened the way for future studies to resolve the signal transduction pathway(s) used by members of this family.(ABSTRACT TRUNCATED AT 250 WORDS)

Developmental expression of cathepsin L and c-rasHa in the mouse placenta.

An investigation is described of the expression of the cysteine proteinase cathepsin L during placental development. In addition, whether cathepsin L expression is linked to c-rasHa expression in development, as it is in metastatic cells, is examined. Large amounts of cathepsin L and its transcript are present in the mouse placenta, more than six times more than in adult kidney and liver. Throughout gestation, cathepsin L and its transcript are located in the giant cells and spongiotrophoblasts of the placenta. Several forms of different mobility on denaturing gels are found in the placenta. Their apparent molecular weights, as determined from the gels, are 43,000, 39,000, 29,000, and 20,000. The 39-kDa form is procathepsin L. The 29-kDa and 20-kDa forms are lysosomal cathepsin Ls. The 39-kDa procathepsin L and the 20-kDa mature cathepsin L are the most abundant species in the placenta and are present in about equal amounts throughout gestation. At any time during gestation, placental minces synthesize and secrete only procathepsin L. The amniotic fluid of the fetus contains the 43-kDa form of cathepsin L and procathepsin L, but no detectable amounts of mature cathepsin L. By contrast, serum from nonpregnant or pregnant mice contains three forms of cathepsin L (i.e., the 43-kDa form, procathepsin L, and mature cathepsin L). Cathepsin L and the rasHa oncogene are expressed in two coincident waves corresponding to periods during which the placenta is invasive and just before parturition. The presence of large amounts of cathepsin L in the placenta suggests that the proteinase has a significant function there. Expression of cathepsin L in the placenta is potentially under the control of the ras gene product p21; both are under developmental control.

Basic fibroblast growth factor induces 3T3 fibroblasts to synthesize and secrete a cyclophilin-like protein and beta 2-microglobulin.

When stimulated by fibroblast growth factor (FGF) BALB/c 3T3 cells synthesize and secrete elevated amounts of five proteins called the 'superinducible proteins', or SIPs. The expression of these proteins is greatly enhanced if the cells are treated with cycloheximide during induction. The 24 kDa protein (SIP24) has been purified and antiserum raised against it. This protein is N-glycosylated and probably structurally constrained by one or more intramolecular disulfide bonds. The amino acid sequences of three of four peptides show significant identity with cyclophilin, an abundant cytoplasmic protein believed to mediate the immunosuppressive effects of cyclosporin A. Several members of the cyclophilin family have been identified, and cDNA clones of two cyclophilin-like proteins with signal sequences have been reported. Here we show that at least one cyclophilin-like protein is secreted and that its expression is regulated by growth factors. The 12.5 kDa protein (SIP12.5) was found to be immunoprecipitated by an antiserum raised to human beta 2-microglobulin. This protein is strongly induced by interferon, which is a characteristic of the beta 2-microglobulin gene. Thus, FGF stimulates mouse embryo 3T3 cells to produce two proteins related to immune regulatory molecules. This may reflect an interaction between immune cells and nonimmune cells that occurs in vivo during processes such as wound healing when growth factors are released locally.

Regulation of the expression of mitogen-regulated protein (MRP; proliferin) and cathepsin L in cultured cells and in the murine placenta.

The genes encoding mitogen-regulated protein (MRP; also called proliferin; PLF) and procathepsin L (CL; also called major excreted protein; MEP) are expressed to high levels in the mouse placenta. Although they are both regulated by epidermal growth factor (EGF) and fibroblast growth factor (FGF) in 3T3 cells, expression of these genes is differently regulated with growth state. The expression patterns of MRP and CL as a function of murine development are also different. Basal and growth factor-stimulated levels of MRP expression are much higher in growing than in quiescent 3T3 cells, whereas CL levels are similar. These changes in gene expression in cultured quiescent cells parallel the changes in MRP and CL expression observed in the late-gestational quiescent placenta. These results suggest growth factors may regulate the expression of these genes, but other influences also regulate the expression of MRP and CL in vivo.

Expression of the collagenolytic and Ras-induced cysteine proteinase cathepsin L and proliferation-associated oncogenes in synovial cells of MRL/I mice and patients with rheumatoid arthritis.

Based on the observation that rheumatoid joint destruction is related to the presence of transformed-appearing proliferating synovial lining cells attached to cartilage and bone at the site of early destruction, we searched for the expression of proliferation- and transformation-associated oncoproteins in synovial tissues from patients with early destructive rheumatoid arthritis (RA). Immunolocalization of Ras and Myc proteins was found in about 70% of the RA cases and was restricted to the proliferating synovial lining cells. The cysteine proteinase, cathepsin L, which has been shown to be the major ras-induced protein in ras-transformed murine NIH 3T3 cells, was detected in 50% of the RA cases, predominantly in synovial cells attached to cartilage and bone at the site of joint destruction. Moreover, utilizing cytoplastic dot hybridization analysis, we demonstrated the presence of RNA sequences complementary to human cathepsin L in primary cultures of human synovial cells from RA joints and complementary to murine cathepsin L in synovial lining cells derived from MRL/l mice developing spontaneously a RA-like disease. Significant levels of ras oncogene transcripts and products in human RA synovial cells associated with an increased expression of the cathepsin L gene indicate that this collagen-degrading enzyme may contribute to the destruction of cartilage and bone in RA.

Regulation of the production of a prolactin-like protein (MRP/PLF) in 3T3 cells and in the mouse placenta.

Mitogen-regulated protein (MRP), a heterogeneously glycosylated mouse protein of Mr 34,000, is in the same protein family as prolactin, growth hormone, and placental lactogen. We show here that the level of translatable MRP mRNA is increased in response to fibroblast growth factor. Also, the amount of MRP secreted by 3T3 cells is modulated by the rate of degradation of newly synthesized MRP in the lysosomes. This is indicated by several results. First, agents that inhibit protein degradation by lysosomal proteases selectively increased by 2- to 6-fold the incorporation of [35S]methionine into MRP. These agents are ammonium chloride, the carboxylic ionophores, monensin and nigericin, and two thiol protease inhibitors, leupeptin and antipain. MRP that has already been secreted is not degraded by 3T3 cells. We examined the developmental appearance of MRP using immunofluorescence microscopy and found MRP localized in the mouse placenta between days 9 and 13 of development. The amount of MRP in the placenta drops suddenly after day 13. Whereas the appearance of MRP in the placenta follows the reported appearance of its mRNA, MRP disappears from the placenta more rapidly than its mRNA. On the basis of the results of our studies with cells in culture we propose that the production of MRP in the placenta is regulated similarly to prolactin. Thus we propose that the initial increase in MRP production in the placenta is due to pretranslational regulation by growth factors, and the later rapid decline is due to posttranslational regulation through degradation in the lysosomes.

Relationship between mitogen-regulated protein (MRP) and proliferin (PLF), a member of the prolactin/growth hormone family.

Mitogen-regulated protein (MRP) is a glycoprotein secreted by Swiss murine 3T3 cells whose levels are increased 63-fold or more over the controls by growth factors. The sequence of a 226-bp MRP cDNA clone showed that a region close to the C terminus of MRP is identical to a sequence found in the cDNA-encoding proliferin (PLF). PLF, cloned from Balb/c 3T3 cells, is a member of the prolactin/growth-hormone family. Here we show that MRP and PLF are also antigenically identical. Antiserum raised against purified MRP specifically immunoprecipitated PLF secreted by CV-1 cells that had been transfected with PLF cDNA in an SV40 vector. Also, fibroblast growth factor (FGF) specifically increased the amount of PLF poly(A)+ RNA in Swiss 3T3 cells. We have previously shown that FGF increases the amount of MRP and MRP mRNA synthesized by the same cells. The anti-MRP antiserum recognized both unglycosylated and glycosylated forms of MRP and PLF. The unglycosylated and glycosylated forms of PLF had the same Mr values as those of the unglycosylated (21,500) and glycosylated (34,000) forms of MRP. However, the anti-MRP antiserum did not recognize mouse prolactin and anti-mouse prolactin antibody did not recognize MRP. Evidently, MRP/PLF is an immunologically distinct member of the prolactin/growth-hormone family of secreted, intercellular regulators.

Cysteine proteinase cathepsin L expression correlates closely with the metastatic potential of H-ras-transformed murine fibroblasts.

A set of H-ras-transfected mouse C3H 10T1/2 cell lines that vary in their experimental and spontaneous metastatic potential has been analysed for the level of expression of the cysteine proteinase cathepsin L, also known as the major excreted protein (MEP). We found a good positive correlation between the extent of ras expression, the metastatic potential, and the amount of the secreted protease in nine independently isolated lines. There was an increased abundance of the MEP mRNA in cells with increased ras expression, suggesting that this gene is under ras control. Expression of glyceraldehyde phosphate dehydrogenase mRNA was also elevated.

Relation between the regulation of DNA synthesis and the production of two secreted glycoproteins by 12-O-tetradecanoylphorbol-13-acetate in 3T3 cells and in phorbol ester nonresponsive 3T3 variants.

12-O-tetradecanoylphorbol-13-acetate (TPA), a potent tumor promoter, acts similarly to growth factors by selectively increasing the rate of production of the secreted proteins, mitogen regulated protein (MRP) and major excreted protein (MEP) by murine 3T3 cells. MRP, a 34 kilodalton (kDa) glycoprotein, is a member of the prolactin-growth hormone family of proteins. MEP, a 39 kDa glycoprotein, is a lysosomal thiol protease that is also secreted. The aim of our investigation was to determine the relation between increases in MRP and MEP production and the initiation of DNA synthesis in response to mitogens. The TNR-9 cell line is a variant of 3T3 cells in which growth factors, but not TPA and teleocidin, stimulate DNA synthesis and cell division. Using [35S]methionine to metabolically label proteins and SDS polyacrylamide gel electrophoresis to resolve the proteins, we found that growing cultures of 3T3 and TNR-9 cells responded equally well to TPA and teleocidin with increased rates of production of MRP and MEP. By contrast, the responses of quiescent TNR-9 cells to these tumor promoters in the increased production of MRP and MEP was greatly diminished compared with quiescent 3T3 cells. The changes in production of MRP in response to tumor promoters in quiescent and growing cells paralleled similar changes in the level of MRP mRNA. In summary, the ability to TPA and teleocidin to increase the rate of production of MRP and MEP correlated with the ability of these tumor promoters to stimulate DNA synthesis in quiescent 3T3 and TNR-9 cells. Evidently the biochemical condition that distinguishes TNR-9 from 3T3 cells and that limits the ability of tumor promoters to stimulate the production of MEP and MRP, and perhaps also DNA synthesis in TNR-9 cells occurs only when the cells are quiescent.

Close relationship of the major excreted protein of transformed murine fibroblasts to thiol-dependent cathepsins.

Complementary DNA clones corresponding to 638 nucleotides of the messenger RNA encoding the major portion of murine major excreted protein have been isolated and sequenced. The amino acid sequence of a part of the murine major excreted protein deduced from the DNA sequence reveals substantial and significant homology with the cysteine proteases actinidin, rat cathepsin H, and papain. Since the amount of murine major excreted protein secreted by cultured cells is often enhanced by transformation, it is implicated in oncogenic phenomena and may play a role in the metastatic process by virtue of its proteolytic activity.

Characterization of a cDNA clone encoding murine mitogen-regulated protein: regulation of mRNA levels in mortal and immortal cell lines.

Mitogen-regulated protein (MRP) is secreted by certain immortal murine cell lines (Swiss 3T3, BNL) stimulated with serum or particular growth factors. We have identified a cDNA clone that encodes part of the protein and have confirmed that MRP is closely related to, if not identical to, the prolactin-related protein designated proliferin. MRP is not produced by primary mouse embryo fibroblasts to nearly the same extent as it is produced by many immortal or transformed lines. Control of expression of this protein by growth factors is achieved both by regulating the extent of transcription and by regulating the processing of the protein.

Superinduction by cycloheximide of mitogen-induced secreted proteins produced by Balb/c 3T3 cells.

We describe here some of the characteristics of the regulation of a group of secretory proteins whose secreted levels rise within 2-4 h of adding fibroblast growth factor (FGF), epidermal growth factor (EGF), or serum to quiescent Balb/c 3T3 cells. The levels of these secretory proteins are regulated similarly to the interferons. When cycloheximide is present during the induction period, the amounts of [35S]methionine incorporated into five of these proteins that we have called "superinducible proteins" (SIPs) is increased 2-5-fold. Superinduction of the SIPs is seen also in response to polyribol-polyriboC, the classical inducer of interferons. None of the SIPs, however, are immuno-precipitated by anti-beta-interferon antibody. Induction and superinduction of the SIPs is inhibited by actinomycin D. Superinduction occurs at concentrations of cycloheximide that inhibit protein synthesis by at least 85%. The SIPs are not major intracellular proteins; they are barely detectable in cellular fractions. Their induction is, however, correlated with the ability of the polypeptide growth factor to stimulate DNA synthesis; EGF, FGF, and serum induce the SIPs, whereas insulin does not, and insulin alone weakly stimulates DNA synthesis in these cells. Because FGF, EGF, and serum cause the SIPs to be produced at concentrations of cycloheximide that inhibit 85% of bulk protein and DNA synthesis, it follows that the SIPs are produced directly from the action of the growth factor and not as a consequence of increased growth. Although probably not interferons, in analogy to the lymphokines, the SIPs could be a set of autocrine or paracrine factors that rapidly convey the growth or differentiation signal between cells.

Synergistic stimulation of S6 ribosomal protein phosphorylation and DNA synthesis by epidermal growth factor and insulin in quiescent 3T3 cells.

Using an improved method to quantify the level of phosphorylation of the S6 ribosomal protein, we have analyzed the effect of growth stimuli on S6 phosphorylation in quiescent murine Swiss/3T3 cells to see if it can be dissociated from the later increase in DNA synthesis. Saturating concentrations of epidermal growth factor (EGF), insulin and serum each stimulate phosphorylation of the S6 ribosomal protein to the same maximal level; this is not so for DNA synthesis. Subsaturating concentrations of EGF and insulin act synergistically to stimulate both S6 phosphorylation and DNA synthesis, but qualitatively the two synergistic interactions are expressed differently. Insulin increases the maximal response of DNA synthesis to EGF, whereas it decreases the concentration of EGF required for half-maximal stimulation of S6 phosphorylation. We conclude that S6 phosphorylation is not a principal regulator of DNA synthesis, and that insulin and EGF regulate both S6 phosphorylation and DNA synthesis through different, but interacting, pathways of action.