Biomedical subjects

R D Polakiewicz

Publications and source records attributed to R D Polakiewicz.

17 recordsLinked to original sources

PubMed2001

Hierarchical phosphorylation of the translation inhibitor 4E-BP1.

In most instances, translation is regulated at the initiation phase, when a ribosome is recruited to the 5' end of an mRNA. The eIF4E-binding proteins (4E-BPs) interdict translation initiation by binding to the translation factor eIF4E, and preventing recruitment of the translation machinery to mRNA. The 4E-BPs inhibit translation in a reversible manner. Hypophosphorylated 4E-BPs interact avidly with eIF4E, whereas 4E-BP hyperphosphorylation, elicited by stimulation of cells with hormones, cytokines, or growth factors, results in an abrogation of eIF4E-binding activity. We reported previously that phosphorylation of 4E-BP1 on Thr 37 and Thr 46 is relatively insensitive to serum deprivation and rapamycin treatment, and that phosphorylation of these residues is required for the subsequent phosphorylation of a set of unidentified serum-responsive sites. Here, using mass spectrometry, we identify the serum-responsive, rapamycin-sensitive sites as Ser 65 and Thr 70. Utilizing a novel combination of two-dimensional isoelectric focusing/SDS-PAGE and Western blotting with phosphospecific antibodies, we also establish the order of 4E-BP1 phosphorylation in vivo; phosphorylation of Thr 37/Thr 46 is followed by Thr 70 phosphorylation, and Ser 65 is phosphorylated last. Finally, we show that phosphorylation of Ser 65 and Thr 70 alone is insufficient to block binding to eIF4E, indicating that a combination of phosphorylation events is necessary to dissociate 4E-BP1 from eIF4E.

Adaptor Proteins, Signal Transducing↗

PubMed2001

Phosphatidylinositol 3-kinase-dependent extracellular calcium influx is essential for CX(3)CR1-mediated activation of the mitogen-activated protein kinase cascade.

Fractalkine, the first member of the CX(3)C chemokine family, induces leukocyte chemotaxis through activation of its high affinity receptor, CX(3)CR1. Like other chemokine receptors, CX(3)CR1 is coupled to a pertussis toxin-sensitive heterotrimeric G(i) protein, which is necessary for rapid rise in the concentration of intracellular calcium. Using a Chinese hamster ovary cell line stably transfected with the CX(3)CR1 receptor, we show that the source of calcium mobilized by fractalkine stimulation is the extracellular pool. Calcium influx is blocked by extracellular calcium chelators, as well as by divalent heavy metals such as Ni(2+), Co(2+), and Cd(2+) without affecting the integrity of intracellular stores. Remarkably, selective phosphoinositide 3-kinase (PI3K) inhibitors, wortmannin and LY294002, abolish the wave extracellular calcium, suggesting that an active PI3K is necessary for this event. The influx of extracellular calcium is in turn required to trigger the activation of the p42/44 mitogen-activated protein/extracellular signal-regulated kinase pathway, but is not necessary for other signals downstream to PI3K, such as phosphorylation of Akt. The potential role of this signaling cascade in fractalkine-mediated chemotaxis is discussed.

Androstadienes↗

PubMed1999

Regulation of 4E-BP1 phosphorylation: a novel two-step mechanism.

The multisubunit eukaryotic translation initiation factor (eIF) 4F recruits 40S ribosomal subunits to the 5' end of mRNA. The eIF4F subunit eIF4E interacts directly with the mRNA 5' cap structure. Assembly of the eIF4F complex is inhibited by a family of repressor polypeptides, the eIF4E-binding proteins (4E-BPs). Binding of the 4E-BPs to eIF4E is regulated by phosphorylation: Hypophosphorylated 4E-BP isoforms interact strongly with eIF4E, whereas hyperphosphorylated isoforms do not. 4E-BP1 is hypophosphorylated in quiescent cells, but is hyperphosphorylated on multiple sites following exposure to a variety of extracellular stimuli. The PI3-kinase/Akt pathway and the kinase FRAP/mTOR signal to 4E-BP1. FRAP/mTOR has been reported to phosphorylate 4E-BP1 directly in vitro. However, it is not known if FRAP/mTOR is responsible for the phosphorylation of all 4E-BP1 sites, nor which sites must be phosphorylated to release 4E-BP1 from eIF4E. To address these questions, a recombinant FRAP/mTOR protein and a FRAP/mTOR immunoprecipitate were utilized in in vitro kinase assays to phosphorylate 4E-BP1. Phosphopeptide mapping of the in vitro-labeled protein yielded two 4E-BP1 phosphopeptides that comigrated with phosphopeptides produced in vivo. Mass spectrometry analysis indicated that these peptides contain phosphorylated Thr-37 and Thr-46. Thr-37 and Thr-46 are efficiently phosphorylated in vitro by FRAP/mTOR when 4E-BP1 is bound to eIF4E. However, phosphorylation at these sites was not associated with a loss of eIF4E binding. Phosphorylated Thr-37 and Thr-46 are detected in all phosphorylated in vivo 4E-BP1 isoforms, including those that interact with eIF4E. Finally, mutational analysis demonstrated that phosphorylation of Thr-37/Thr-46 is required for subsequent phosphorylation of several carboxy-terminal serum-sensitive sites. Taken together, our results suggest that 4E-BP1 phosphorylation by FRAP/mTOR on Thr-37 and Thr-46 is a priming event for subsequent phosphorylation of the carboxy-terminal serum-sensitive sites.

Adaptor Proteins, Signal Transducing↗

PubMed1998

mu-Opioid receptor activates signaling pathways implicated in cell survival and translational control.

The mu-opioid receptor mediates the analgesic and addictive properties of morphine. Despite the clinical importance of this G-protein-coupled receptor and many years of pharmacological research, few intracellular signaling mechanisms triggered by morphine and other mu-opioid agonists have been described. We report that mu-opioid agonists stimulate three different effectors of a phosphoinositide 3-kinase (PI3K)-dependent signaling cascade. By using a cell line stably transfected with the mu-opioid receptor cDNA, we show that the specific agonist [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin (DAMGO) stimulates the activity of Akt, a serine/threonine protein kinase implicated in protecting neurons from apoptosis. Activation of Akt by DAMGO correlates with its phosphorylation at serine 473. The selective PI3K inhibitors wortmannin and LY294002 blocked phosphorylation of this site, previously shown to be necessary for Akt enzymatic activity. DAMGO also stimulates the phosphorylation of two other downstream effectors of PI3K, the p70 S6 kinase and the repressors of mRNA translation, 4E-BP1 and 4E-BP2. Upon mu-opioid receptor stimulation, p70 S6 kinase is activated and phosphorylated at threonine 389 and at threonine 421/serine 424. Phosphorylation of p70 S6 kinase and 4E-BP1 is also repressed by PI3K inhibitors as well as by rapamycin, the selective inhibitor of FRAP/mTOR. Consistent with these findings, DAMGO-stimulated phosphorylation of 4E-BP1 impairs its ability to bind the translation initiation factor eIF-4E. These results demonstrate that the mu-opioid receptor activates signaling pathways associated with neuronal survival and translational control, two processes implicated in neuronal development and synaptic plasticity.

Adaptor Proteins, Signal Transducing↗

PubMed1998

A mitogen-activated protein kinase pathway is required for mu-opioid receptor desensitization.

The mu-opioid receptor mediates not only the beneficial painkilling effects of opiates like morphine but also the detrimental effects of chronic exposure such as tolerance and dependence. Different studies have linked tolerance to opioid receptor desensitization. Agonist activation of the mu-opioid receptor stimulates a mitogen-activated protein kinase (MAPK) activity, but the functional significance of this pathway remains unclear. We have focused on the MAPK signaling cascade to study mu-opioid receptor desensitization. We report that inhibition of the MAPK pathway blocks desensitization of mu-opioid receptor signaling as well as the loss of receptor density due to internalization. Our results suggest that a feedback signal emanating from the MAPK cascade is required for mu-opioid receptor desensitization.

Analgesics, Opioid↗

PubMed1997

Zfx mutation results in small animal size and reduced germ cell number in male and female mice.

The zinc-finger proteins ZFX and ZFY, encoded by genes on the mammalian X and Y chromosomes, have been speculated to function in sex differentiation, spermatogenesis, and Turner syndrome. We derived Zfx mutant mice by targeted mutagenesis. Mutant mice (both males and females) were smaller, less viable, and had fewer germ cells than wild-type mice, features also found in human females with an XO karyotype (Turner syndrome). Mutant XY animals were fully masculinized, with testes and male genitalia, and were fertile, but sperm counts were reduced by one half. Homozygous mutant XX animals were fully feminized, with ovaries and female genitalia, but showed a shortage of oocytes resulting in diminished fertility and shortened reproductive lifespan, as in premature ovarian failure in humans. The number of primordial germ cells was reduced in both XX and XY mutant animals at embryonic day 11.5, prior to gonadal sex differentiation. Zfx mutant animals exhibited a growth deficit evident at embryonic day 12.5, which persisted throughout postnatal life and was not complemented by the Zfy genes. These phenotypes provide the first direct evidence for a role of Zfx in growth and reproductive development.

Animals↗

PubMed1995

Mapping of ribosomal protein S3 and internally nested snoRNA U15A gene to human chromosome 11q13.3-q13.5.

The mammalian ribosome is a massive structure composed of 4 RNA species and about 80 different proteins. One of these ribosomal proteins, S3, appears to function not only in translation but also as an endonuclease in repair of UV-induced DNA damage. Moreover, the first intron of human RPS3 transcripts is processed to generate U15A, a small nucleolar RNA. We localized the nested RPS3/U15A genes to the immediate vicinity of D11S356 and D11S533 on human chromosome 11q13.3-q13.5 using a combination of somatic cell hybrid analysis, fluorescence in situ hybridization, and YAC/STS content mapping. These findings add to the evidence that genes encoding ribosomal proteins are scattered about the human genome.

Animals↗

PubMed1995

Developmental regulation of proenkephalin gene expression in osteoblasts.

Proenkephalin (PENK), a classically defined opioid gene, was originally thought to be expressed almost exclusively in the mature nervous and neuroendocrine systems. In the last few years, it was demonstrated, however, that high levels of PENK messenger RNA and PENK-derived peptides are expressed in embryonic mesenchymal tissues during differentiation into mature tissues and organs. Shortly after birth, as development progresses, PENK expression drops in those tissues to undetectable levels. Very little is known about the molecular mechanisms regulating this transient expression. To investigate those mechanisms, we used primary cell cultures of calvaria-derived osteoblasts. These cultures express PENK and exhibit a normal pattern of osteoblastic differentiation. In the present study we demonstrate that 1) a reciprocal interrelationship exists between PENK expression and osteoblastic differentiation in vivo, ex vivo, and in vitro; namely, PENK expression is down-regulated upon cellular differentiation; 2) PENK promoter usage and messenger RNA splicing function similarly in osteoblasts and in neural cells; 3) osteoblastic PENK expression is modulated by bone-targeting hormones; and 4) this down-regulation is inhibited by the serine/threonine kinase inhibitor H-8. The link between osteoblastic differentiation and down-regulation of PENK expression together with our preliminary findings indicating the existence of an osteoblastic opioid receptor suggest that opioids act in an autocrine/paracrine mechanism on undifferentiated osteoblasts and play a significant role in bone development.

1-Methyl-3-isobutylxanthine↗

PubMed1994

Lipopolysaccharide induces proenkephalin gene expression in rat lymph nodes and adrenal glands.

The proenkephalin gene encodes a family of neuropeptides that was originally identified in brain tissue and adrenal glands. Recently, it was shown that proenkephalin is also expressed in cultured lymphoid cells. To elucidate the physiological significance of this expression, we examined the in vivo expression of proenkephalin in lymphoid tissues. We show here that exposing rats to the endotoxin lipopolysaccharide induces an intense and transient expression of proenkephalin in adrenal glands and lymph nodes. By using combined in situ hybridization and immunohistochemistry on tissue slices, we identified proenkephalin expression in macrophages located within the lymph nodes and in chromaffin cells within the adrenal glands. This in vivo expression of proenkephalin was enhanced by adrenaline. The present observations demonstrate that the immune system is a site of significant expression of proenkephalin and provide a basis for neuroimmune interactions.

Adrenal Glands↗

PubMed1992

Regulation of proenkephalin expression in cultured skin mesenchymal cells.

Proenkephalin, a classically defined opioid encoding gene, is transiently expressed in nondifferentiated mesodermal cells during organogenesis. We examined the hypothesis that this expression is associated with mesenchymal cell proliferation. For this purpose, we established a cell culture derived from fetal skin mesenchyme that specifically expresses proenkephalin mRNA in correlation with hypodermis development. These mesenchymal cells also produce and secrete significant amounts of proenkephalin-derived peptides. Using this model system, we observed a marked increase in proenkephalin mRNA expression in response to serum. This effect is time dependent and reaches peak levels during the G1/S transition. Similarly, 12-O-tetradecanoyl-phorbol-13-ester, whose biological actions have been shown to be mediated by the activity of protein kinase C (PKC), up-regulates proenkephalin expression. Desensitization of PKC by prolonged exposure of cells to 12-O-tetradecanoyl-phorbol-13-ester attenuates the serum induction of proenkephalin. The results presented in this report demonstrate that proenkephalin expression in mesenchymal cells is regulated by serum factors via mechanisms that involve PKC activity. A possible association between proenkephalin expression and cell proliferation is suggested.

Animals↗

PubMed1992

Presence of prion protein in peripheral tissues of Libyan Jews with Creutzfeldt-Jakob disease.

The prion protein (PrP) gene on chromosome 20 encodes a protein designated PrPC. An abnormal, protease-resistant isoform of PrPC, denoted PrPCJD or PrPSc, is present in the brains of patients with Creutzfeldt-Jakob disease (CJD). In Libyan Jews, CJD segregates with a point mutation at codon 200 of the PrP gene, resulting in the substitution of lysine for glutamate. In the present study, we examined the presence of PrP in fibroblasts and leukocytes derived from eight CJD patients with the codon 200 mutation. In cultured fibroblasts as well as in leukocytes, there was a significant increase in PrP as judged by immunocytochemistry in addition to immunoblotting. Most of the PrP in fibroblasts and leukocytes could be released from the external surface by phosphatidylinositol-specific phospholipase C, a property characteristic of PrPC. In leukocytes only, part of the protein was protease resistant, resembling PrPCJD. The concentration of PrP mRNA was similar in fibroblast lines derived from controls and CJD patients. These results suggest that in CJD patients carrying a mutation at codon 200 of the PrP gene, the metabolism of PrP, rather than PrP synthesis, is abnormal.

Animals↗

PubMed1991

Proenkephalin A in bone-derived cells.

Enkephalins, a group of small peptides with opiate-like activity, have been defined originally as neuropeptides. Recent reports showed, using in situ hybridization, that the enkephalin-encoding gene, proenkephalin A (pEnkA), is expressed in nondifferentiated cells of diverse mesodermal lineages. The transient expression of pEnkA in these tissues during organogenesis suggests that this gene is involved in processes such as differentiation and/or cell proliferation. In situ hybridization revealed that bone and cartilage are among the tissues that express pEnkA most actively during organogenesis. Here we show that pEnkA mRNA is abundant in normal calvaria-derived cells and in osteosarcoma-derived cell lines ROS 17/2.8 and ROS 25/1. In addition, pEnkA-derived peptides are synthesized and secreted by these cells, as revealed by specific RIA. pEnkA expression in ROS cells is decreased by osteogenin, an osteoinductive factor, and by the calcium-regulating hormone, 1,25-dihydroxyvitamin D3, whereas the osteoblastic phenotype marker, alkaline phosphatase, is increased by these factors. These results together with the inhibitory effects of pEnkA-derived peptides on alkaline phosphatase activity in ROS 17/2.8 cells suggest that pEnkA is involved in bone development and provide a model system for further analysis of pEnkA expression during this process.

Alkaline Phosphatase↗

PubMed1991

Regulation of proenkephalin A messenger ribonucleic acid levels in normal B lymphocytes: specific inhibition by glucocorticoid hormones and superinduction by cycloheximide.

Proenkephalin A (PEA) encodes a group of small peptides known to function as neurotransmitters, neuromodulators, and neurohormones in the nervous and neuroendocrine systems. This gene has been shown to be expressed in lymphoid cells, supporting the concept of bidirectional communication between the immune system and the central nervous system. In the present study, we investigated the effect of steroids and the inhibition of protein and RNA syntheses on the regulation of PEA expression in normal rat B cells. The transient expression of PEA messenger (m) RNA levels occurring normally in B cells was markedly inhibited by the presence of either 50 nM prednisolone or dexamethasone, both of which are glucocorticoids; other steroids, such as testosterone or the steroid-inactive metabolite androsterone, were ineffective. In the presence of cycloheximide, a protein synthesis inhibitor, PEA mRNA was superinduced by a factor of 15-fold. Sorting by flow cytometry of cycloheximide-treated cells followed by in situ hybridization analysis revealed that the expression of PEA mRNA was exclusively confined to a small fraction of B cells. These results indicate that the mechanisms regulating PEA gene expression in B cells differ from those previously described in cells of the neuroendocrine and the nervous systems.

Animals↗

PubMed1990

Expression of proenkephalin A mRNA and enkephalin-containing peptides in cultured fibroblasts.

Proenkephalin A (PEA) gene was found to be expressed in primary, secondary and tertiary cultures of rat fibroblasts. The 1.4 kb PEA mRNA was detected by Northern blot analysis. The same cultures do not express detectable amounts of proenkephalin B (prodynorphin) or (POMC) mRNAs. Acidic cell extracts were purified on a C18 octadecyl Amprep column and analysed with a specific methionine enkephalin radioimmunoassay to detect whether PEA mRNA is translated. A significant amount of enkephalin immunoreactivity (178-185 fmol/mg protein) was observed upon trypsin and carboxypeptidase B digestion of fibroblast cell extracts, whereas only 3-5% of this amount was free enkephalin. It is therefore indicated that the PEA mRNA expressed in fibroblasts is indeed translated to the proenkephalin precursor protein, but the cells accumulate only a small quantity of the processed pentapeptides. The implication of these observations to the possible developmental role of PEA in various non-neuronal cells, including mesodermal lineages, is discussed.

Animals↗

PubMed1990

Regulated expression of proenkephalin A during ontogenic development of mesenchymal derivative tissues.

Proenkephalin A (PEA), a neuropeptide-encoding gene, is widely expressed in the nervous and endocrine systems. Recently, we demonstrated that in addition to its abundance in fetal brain tissue; PEA is markedly expressed in nondifferentiated mesodermal cells of developing fetuses. To evaluate the implication of these findings for the normal development of tissues of mesodermal origin, we examined the expression of PEA in rat mesenchymal tissues during pre- and postnatal development. Using in situ hybridization analysis combined with RNA blots and a Met-enkephalin-specific radioimmunoassay, we showed that (i) PEA mRNA levels in embryonic and newborn mesenchymal derivative tissues were as high as in the developing brain, (ii) PEA mRNA concentrations in these tissues dropped to undetectable levels shortly after birth, and (iii) this mRNA was translated and processed differentially among different mesenchymal tissues, yielding a tissue-specific pattern of PEA-derived peptides. Our results demonstrate multilevel regulation of PEA gene expression during ontogenic development of mesenchymal derivative tissues. The transient expression and the correlation between PEA mRNA and tissue maturation support the notion that peptides encoded by PEA play a significant role in normal development of these tissues. These findings provide a framework for examination of the mechanisms and roles of PEA gene expression during mesenchymal ontogeny.

Aging↗

PubMed1989

Increase in hypothalamic pro-opiomelanocortin gene expression in response to prolonged low level lead exposure.

Lead, an environmental pollutant, has been long recognized as a neurotoxic agent. Several reports have recently described behavioral disturbances caused by low level lead exposure. Lead-induced changes in neurotransmitter metabolism in the brain may help to elucidate the molecular basis for the observed behavioral alterations. In this study, the influence of low level lead exposure on the hypothalamic expression of rat opioid genes was examined. Determination of relative opioid mRNA levels revealed a dramatic increase of pro-opiomelanocortin (POMC) mRNA concentrations in response to lead. This increase was found to be specific, developmentally regulated, reversible and dependent on prolonged lead exposure. These findings may provide substantial evidence for the possible connections between early environmental inputs and the expression of neuropeptide genes in the brain.

Animals↗

PubMed1989

Proenkephalin A is expressed in mesodermal lineages during organogenesis.

Proenkephalin A (PEA) encodes several neuropeptides with an opioid activity, as well as other peptides with as yet unknown functions. As an initial step toward finding possible roles for PEA gene products in non-neuronal tissues, we have determined sites of PEA expression during mouse embryonic development, employing in situ hybridization. We report here the unexpected observation that in addition to its abundance in brain, PEA RNA is expressed in non-differentiated mesodermal cells of diverse lineages in the process of their development into several adult tissues and organs; it drops to undetectable levels upon terminal differentiation of these tissues. In a particular example of differentiating mesoderm, the developing kidney, the transient expression of PEA mRNA and of its encoded peptide Met-enkephalin was demonstrated by both in situ and Northern blot hybridizations, as well as by a radioimmunoassay. These findings suggest a novel role for PEA-derived peptide(s) in mesoderm growth or differentiation during organogenesis.

Animals↗