ABSTRACT Human T-cell leukemia virus type 1 is etiologically linked to the development of adult T-cell leukemia and various human neuropathies. The Tax protein of human T-cell leukemia virus type I has been implicated in cellular transformation. Like other oncoproteins, such as Myc, Jun, and Fos, Tax is a transcriptional activator. How it mechanistically dysregulates the cell cycle is unclear. Previously, it was suggested that Tax affects cell-phase transition by forming a direct protein-protein complex with p16INK4a, thereby inactivating an inhibitor of G1-to-S-phase progression. Here we show that, in T cells deleted for p16INK4a, Tax can compel an egress of cells from G0/G1 into S despite the absence of serum. We also show that in undifferentiated myocytes, expression of Tax represses cellular differentiation. In both settings, Tax expression was found to increase cyclin D-cdk activity and to enhance pRb phosphorylation. In T cells, a Tax-associated increase in steady-state E2F2 protein was also documented. In searching for a molecular explanation for these observations, we found that Tax forms a protein-protein complex with cyclin D3, whereas a point-mutated and transcriptionally inert Tax mutant failed to form such a complex. Interestingly, expression of wild-type Tax protein in cells was also correlated with the induction of a novel hyperphosphorylated cyclin D3 protein. Taken together, these findings suggest that Tax might directly influence cyclin D-cdk activity and function, perhaps by a route independent of cdk inhibitors such as p16INK4a.

The human T-cell leukemia virus type 1 (HTLV-1) Tax oncoprotein causes cellular transformation by deregulating important cellular processes such as DNA repair, transcription, signal transduction, proliferation, and growth. Although it is clear that normal cell cycle control is deregulated during HTLV-1-induced cellular transformation, the effects of Tax on cell cycle control are not well understood. Flow cytometric analyses of human T cells indicate that cell cycle arrest in late G1, at or before the G1/S restriction point, by p16INK4a is relieved by Tax. Furthermore, Tax-dependent stimulation of 5-bromo-2'-deoxyuridine incorporation and transcriptional activation is inhibited by p16INK4a. This result suggests that p16INK4a is able to block Tax-dependent stimulation of DNA synthesis and cell cycle progression into S phase. In vitro binding assays with recombinant glutathione S-transferase fusion proteins and [35S]methionine-labeled proteins indicate that Tax binds specifically with p16INK4a but not with either p21cip1 or p27kip1. Furthermore, sequential immunoprecipitation assays with specific antisera and [35S]methionine-labeled cell lysates subsequent to coexpression with Tax and p16INK4a indicate that the two proteins form complexes in vivo. Immunocomplex kinase assays with cyclin-dependent kinase 4 antiserum indicate that Tax blocks the inhibition of cdk4 kinase activity by p16INK4a. This study identifies p16INK4a as a novel cellular target for Tax and suggests that the inactivation of p16INK4a function is a mechanism of cell cycle deregulation by Tax.

Growth factors and cyclic AMP (cAMP) are known to activate distinct intracellular signaling pathways. Fibroblast growth factor (FGF) activates ras-dependent kinase cascades, resulting in the activation of MAP kinases, whereas cAMP activates protein kinase A. In this study, we report that growth factors and cAMP act synergistically to stimulate proenkephalin gene expression. Positive synergy between growth factor- and cAMP-activated signaling pathways on gene expression has not been previously reported, and we suggest that these synergistic interactions represent a useful model for analyzing interactions between these pathways. Transfection and mutational studies indicate that both FGF-dependent gene activation and cAMP-dependent gene activation require cAMP response element 2 (CRE-2), a previously characterized cAMP-dependent regulatory element. Furthermore, multiple copies of this element are sufficient to confer FGF regulation upon a minimal promoter, indicating that FGF and cAMP signaling converge upon transcription factors acting at CRE-2. Among many different ATF/AP-1 factors tested, two factors, ATF-3 and c-Jun, stimulate proenkephalin transcription in an FGF- or Ras-dependent fashion. Finally, we show that ATF-3 and c-Jun form heterodimeric complexes in SK-N-MC cells and that the levels of both proteins are increased in response to FGF but not cAMP. Together, these results indicate that growth factor- and cAMP-dependent signaling pathways converge at CRE-2 to synergistically stimulate gene expression and that ATF-3 and c-Jun regulate proenkephalin transcription in response to both growth factor- and cAMP-dependent intracellular signaling pathways.

Human proenkephalin gene transcription is transactivated by human T-cell leukemia virus type I (HTLV-I) Tax in human Jurkat T lymphocytes. This transactivation was further enhanced in Jurkat cells treated with concanavalin A, cyclic AMP, or 12-O-tetradecanoylphorbol-13-acetate. Deletion and cis-element transfer analyses of the human proenkephalin promoter identified a cyclic AMP-responsive AP-1 element (-92 to -86) as both necessary and sufficient to confer Tax-dependent transactivation. Different AP-1 or cyclic AMP-responsive element-binding protein (CREB)/activating transcription factor (ATF) proteins which bind this element were expressed in murine teratocarcinoma F9 cells to identify those capable of mediating Tax-dependent transactivation of human proenkephalin gene transcription. Although CREB, c-Fos, c-Jun, and JunD did not have significant effects, JunB inhibited the Tax-dependent transactivation. In contrast, ATF3 dramatically induced Tax-dependent transactivation, which was further enhanced by protein kinase A. Electrophoretic mobility shift assays with recombinant fusion proteins expressed and purified from bacteria indicate that the DNA-binding activity of ATF3 is also dramatically enhanced by Tax. Chimeric fusion proteins consisting of the DNA-binding domain of the yeast transcription factor Gal4 and the amino-terminal domain (residues 1 to 66) of ATF3 were able to mediate Tax-dependent transactivation of a Gal4-responsive promoter, which suggests a direct involvement of this region of ATF3. Recombinant fusion proteins of glutathione S-transferase with either the amino- or carboxy-terminal (residues 139 to 181) domain of ATF3 were able to specifically interact with Tax. Furthermore, specific antisera directed against Tax coimmunoprecipitated ATF3 only in the presence of Tax.

Prodynorphin is expressed by neurons of the hypothalamus and gonadotrophs of the anterior pituitary gland (AP) and plays a role in the negative feedback regulation of the reproductive neuroendocrine axis. The present study examined whether gonadal steroid hormones are capable of modulating pituitary prodynorphin expression in immature, female rats. Steroids were administered via subcutaneous Silastic implants and rats were killed at 29 days of age. Northern blot analysis was used to measure AP prodynorphin, luteinizing hormone-/gb (LH/gb), follicle-stimulating hormone-/gb (FSH/gb), and common α-subunit mRNA levels (normalized to 18S ribosomal RNA). Treatment groups (n = 5–6) consisted of control (CNT; empty implants), estradiol (E2; 4 days), E2 + progesterone (E2 + P4; 8 days and 4 days, respectively), and dihydrotestosterone (DHT; 4 days). Pituitary prodynorphin mRNA was significantly suppressed in only the DHT-treated animals (26 ± 10% of CNT, p < 0.01). LH/gb mRNA was suppressed by all steroid treatments (p < 0.01), FSH/gb was lower in only the E2 group, and α-subunit was reduced in both the E2 + P4 and DHT groups (p < 0.01). Serum LH was suppressed by all steroid treatments but FSH was reduced in only the E2 and E2 + P4 groups (p < 0.01). Treatment of prepubescent rats with continuous high levels of gonadal steroids is known to severely reduce endogenous hypothalamic gonadotropin releasing hormone (GnRH) release and this is supported by our observation of reduced gonadotropin-subunit gene expression. Since only DHT produced a significant reduction in AP prodynorphin expression, these data suggest that androgens can act directly on the pituitary to suppress prodynorphin expression.

Astrocytes have previously been shown to respond to cytokines such as interleukin-1 beta, tumor necrosis factor-alpha, and gamma-interferon from multiple sources including microglia and astrocytes. Recently, astrocytes have also been shown to express the opioid precursor gene proenkephalin and proenkephalin-derived peptides. The objectives of the current study were to determine if immune cytokines regulate proenkephalin gene expression in primary cultures of neonatal rat cerebral astrocytes. Northern analysis of RNA from primary cultures of neonatal rat cerebral astrocytes indicated that proenkephalin transcript levels were decreased by approximately 50% with gamma-interferon treatment and increased approximately 100% by treatment with both tumor necrosis factor-alpha and interleukin-1 beta relative to untreated controls. Tumor necrosis factor-alpha treatment was unable to reverse the inhibitory effect of gamma-interferon pretreatment on proenkephalin messenger RNA levels in the astrocytes. In contrast, expression of the constitutively expressed glutamine synthetase gene was not altered by either tumor necrosis factor-alpha or gamma-interferon treatment. These cytokines also regulate the secretion of proenkephalin-derived peptides from astrocytes. The levels of immunoreactive Met-enkephalin-Arg6-Phe7 were increased by approximately 50% with tumor necrosis factor-alpha and decreased by approximately 40% with gamma-interferon relative to untreated controls. Tumor necrosis factor-alpha was again unable to reverse the inhibitory effect of gamma-interferon pretreatment on the secretion of proenkephalin-derived peptides. These results provide additional support for the hypothesis that rapidly proliferating astrocytes may serve an important and pivotal role in mediating the bi-directional neuroimmune interactions during central nervous system disease, infection, or trauma.

Previous results have shown that the relative abundance of proenkephalin mRNA in the rat heart is comparable to the levels found in the brain; however, the extractable enkephalin-containing peptide levels are much lower in the heart. This lack of correspondence between the levels of transcript and peptide could arise from either the inefficient translation of proenkephalin transcripts or the translation of proenkephalin transcripts into peptides that are rapidly secreted or degraded. To distinguish between these possibilities, the translational status of proenkephalin mRNA in the rat heart was established by Northern blot analysis of sucrose density gradient-sedimented polysomal fractions and compared to the striatum, which is known to efficiently translate proenkephalin transcripts. In both tissues, we detected 1.5-kilobase transcripts, but an additional larger transcript of approximately 3.6 kilobases was detected in the heart. Both transcripts were associated primarily with polyribosomes, suggesting active translation of proenkephalin mRNA in the rat heart. RIA of the culture media and extracts from primary cultures of neonatal rat cardiomyocytes indicated the presence of immunoreactive Met-enkephalin-Arg6-Phe7, which was stimulated by 8-(4-chlorophenylthio)cAMP. These results suggest that proenkephalin transcripts are translated in the heart and that detectable levels of immunoreactive Met-enkephalin-Arg6-Phe7 are present in the media and cell extracts of primary cultures of neonatal rat cardiomyocytes.

Annals of the New York Academy of SciencesVolume 594, Issue 1 p. 475-478 Expression of High Levels of Proenkephalin in an Isolated Glial Cell Type. Inhibition by γ-Interferona KENNETH G. LOW, KENNETH G. LOW Oregon Regional Primate Research Center Beaverton, Oregon 97006Search for more papers by this authorMICHAEL H. MELNER, MICHAEL H. MELNER Oregon Regional Primate Research Center Beaverton, Oregon 97006Search for more papers by this author KENNETH G. LOW, KENNETH G. LOW Oregon Regional Primate Research Center Beaverton, Oregon 97006Search for more papers by this authorMICHAEL H. MELNER, MICHAEL H. MELNER Oregon Regional Primate Research Center Beaverton, Oregon 97006Search for more papers by this author First published: June 1990 https://doi.org/10.1111/j.1749-6632.1990.tb40533.xCitations: 3 a Supported in part by Grants DK-41035 and RR00163-28 from the National Institutes of Health, and Grant N00014-88-0030 R & T Code 4411724 from the Office of Naval Research. AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume594, Issue1Neuropeptides and Immunopeptides: Messengers in a Neuroinunune AxisJune 1990Pages 475-478 RelatedInformation

The expression of opioid genes was examined in isolated populations of glial cells in primary culture. Northern blot analysis of purified type I astrocytes, oligodendrocytes and mixed oligodendrocyte-type-2-astrocyte lineage cells derived from cerebral cortex demonstrated robust expression of proenkephalin mRNA exclusively in type I astrocytes. The expression of proenkephalin mRNA was stimulated by the beta-adrenergic agonist isoproterenol, and 8-(4-chlorophenyl thio)adenosine 3'-5'-cyclic monophosphate (cpt-cAMP). Both of these compounds regulated a proenkephalin-chloramphenicol acetyltransferase fusion gene transiently transfected into type I astrocytes. HPLC and immunoassay of the cell culture media revealed significant levels of unprocessed proenkephalin secreted by the cell and this secretion was stimulated by isoproterenol and cpt-cAMP. The relatively high levels of proenkephalin expressed suggest that enhanced expression in astrocytes may be important during neural development, in trauma-induced gliosis and in neuroimmune interactions.

Proenkephalin mRNA has previously been shown to be expressed in the rodent uterus with varying levels during the estrous cycle. To examine for the potential regulation of proenkephalin gene expression by steroid hormones in a primate displaying a menstrual cycle and to define the functional tissue within the uterus expressing this transcript, we have used Northern blot analysis of extracted RNA from isolated uterine tissue subtypes from normal adult rhesus macaques obtained during the menstrual cycle and from ovariectomized females under different physiological steroid hormone treatments. A strong band of proenkephalin mRNA of 1.3 kilobases was detected almost exclusively in the proliferative endometrium from monkeys in the follicular phase of the cycle. No proenkephalin mRNA was detected in secretory endometrium obtained from monkeys in the luteal phase. When ovariectomized macaques were implanted with silastic capsules of 17 beta-estradiol, proenkephalin mRNA was detected in the endometrium but not the myometrium of the estradiol-treated animals. No proenkephalin mRNA was detected in ovariectomized control animals. Under these conditions, we were unable to detect proenkephalin mRNA in ovariectomized macaques implanted with separate silastic capsules of 17 beta-estradiol and progesterone or in decidual tissue from early or late pregnancy. These results suggest that in the primate uterus 1) proenkephalin mRNA is expressed primarily in the endometrium of the uterus, 2) expression of the proenkephalin gene is regulated by 17 beta-estradiol in the endometrium, and 3) this effect of estradiol is antagonized by progesterone.

The expression of myelin basic protein by the oligodendrocyte is an integral event in the maturation of central nervous sytem function. Although much is known concerning the various myelin basic protein species, their temporal expression, and processing of RNA transcripts, little is known about the epigenetic factors responsible for the regulation of myelin basic protein (MBP) expression. In this study, we present evidence that insulin/insulin‐like growth factor‐I can increase the levels of MBP protein in isolated oligodendrocyte progenitor cells cultured in a serumless, chemically defined medium (ODM). Insulin was found to increase MBP protein in a dose‐responsive manner, reaching a maximal level at 72 hr of exposure. Both insulin‐like growth factor‐I (IGF‐I) and insulin were demonstrated to have no effect on MBP RNA levels. These data indicate that insulin/IGF‐I increased MBP protein levels at a level distal to transcription The dose response of insulin action suggests that it may have a MBP regulatory function, distinct from IGF‐I. When added individually, the other supplements of ODM, transferrin (500 ng/ml), and basic fibroblast growth factor (5 ng/ml) had no effect on MBP expression. However, when all three components were combined, a synergistic effect resulting in increased MBP protein and total RNA levels was found. The phorbol ester 12‐O‐tetradecanoyl phorbol acetate was found to reduce intracellular MBP RNA levels. The cAMP analogue/dibutyryl cAMP had contrasting effects on MBP RNA levels; no effect occurred in cultures grown in fetal calf serum, but a reduction in RNA levels was found in cultures grown in ODM. These data suggest that only a select range of extrinsic factors may be involved in MBP regulation, and depending on the environmental milieu, epigenetic agents may modulate gene activity differently.