Cysteine-rich domain isoforms of the neuregulin-1 gene are required for maintenance of peripheral synapses.

Neuregulin-1 (NRG-1) signaling has been implicated in inductive interactions between pre- and postsynaptic partners during synaptogenesis. We used gene targeting to selectively disrupt cysteine-rich domain-(CRD-) containing NRG-1 isoforms. In CRD-NRG-1-/-mice, peripheral projections defasciculated and displayed aberrant branching patterns within their targets. Motor nerve terminals were transiently associated with broad bands of postsynaptic ACh receptor (AChR) clusters. Initially, Schwann cell precursors accompanied peripheral projections, but later, Schwann cells were absent from axons in the periphery. Following initial stages of synapse formation, sensory and motor nerves withdrew and degenerated. Our data demonstrate the essential role of CRD-NRG-1-mediated signaling for coordinating nerve, target, and Schwann cell interactions in the normal maintenance of peripheral synapses, and ultimately in the survival of CRD-NRG-1-expressing neurons.

Drosophila melanogaster H1 histone is phosphorylated stably.

Phosphorylation of histone H1 is developmentally regulated in Drosophila spp. It cannot be detected in preblastoderm embryos or polytene salivary gland cells, but in cellular blastoderm, postblastoderm embryo, and amitotic adult head nuclei, it occurs with a frequency of roughly 4 x 10(5) molecules per nucleus. We used pulse-labeling to study the relationship between H1 synthesis and modification in cultured cells. These results reveal that the H1-associated phosphate is stable and suggest that Drosophila H1 is synthesized, translocated to the nucleus, associated with chromatin, and then phosphorylated. Partial tryptic digestion of Drosophila H1 revealed that the phosphorylation site is located within the globular, central domain of the protein. Thus, the developmentally regulated phosphorylation of Drosophila H1 presents two contrasts with previously studied H1 phosphorylation. It is not correlated with DNA replication, and it is located in the central domain of the protein.

Polyomavirus middle T antigen induces ribosomal protein S6 phosphorylation through pp60c-src-dependent and -independent pathways.

Phosphorylation of ribosomal protein S6 is elevated in polyomavirus-infected cells. This elevation results only in part from activation of S6 kinase activity. These effects appear to reflect independent activities of wild-type middle T antigen. Hr-t mutant NG59, encoding a defective middle T protein, and mutant Py808A, encoding no middle T protein, were unable to induce S6 kinase activity or elevate S6 phosphorylation. Two other site-directed mutants encoding altered middle T proteins did elevate S6 phosphorylation while only weakly stimulating S6 kinase activity. These results suggest at least two independent pathways leading to elevation of S6 phosphorylation. One pathway leads to induction of S6 kinase activity following activation of pp60c-src by transformation-competent middle T antigen. Another pathway operates independently of S6 kinase induction and can be regulated by transformation-defective middle T mutants such as Py1387T. This mutant, encoding a truncated middle T protein that failed to associate with the plasma membrane and to activate pp60c-src, caused increased levels of S6 phosphorylation without detectably increasing S6 kinase activity. The ability of mutants such as Py1387T to induce S6 phosphorylation correlated with their ability to increase phosphorylation of VP1, an event linked to maturation of infectious virions.

Retinoic acid receptor alpha suppresses polyomavirus transformation and c-fos expression in rat fibroblasts.

To explore the molecular mechanisms by which retinoic acid inhibits oncogenic transformation, we have examined the effects of retinoic acid on the polyomavirus-induced transformation of rat fibroblasts. Treatment of rat F111 fibroblasts with high concentrations of retinoic acid (10(-6) M) partially inhibited the ability of polyomavirus to induce dense focus formation (50-70%). This effect was not secondary to a retinoic acid-dependent block of cellular proliferation. To address the role of the retinoic acid receptor (RAR-alpha) in mediating the transformation-inhibitory effect of retinoic acid, we have overexpressed either RAR-alpha or cellular retinoic acid-binding protein I (CRABP) cDNAs in F111 cells. Introduction of a CRABP I expression vector did not alter the responsiveness of F111 cells to retinoic acid in any detectable fashion. In contrast, overexpression of RAR-alpha increased the sensitivity of F111 cells to the transformation-inhibitory action of retinoic acid by 10- to 100-fold. At high concentrations, retinoic acid inhibited transformation of F111-RAR cells by polyomavirus by about 90%. At near physiological concentrations, retinoic acid inhibited transformation by 25-50% in F111-RAR cells but not in control cells. Retinoic acid did not inhibit either the synthesis of polyoma middle T (mT) or pp60c-src, the cellular target for mT action, or the formation of mT:pp60c-src:PI-3 kinase (phosphatidylinositol-3 kinase) complexes. Therefore, RAR-alpha was not acting as a negative regulator of expression of either the polyomavirus middle T oncogene or the cellular proto-oncogene, c-src. It seems likely that RAR-alpha regulates the expression of cellular genes whose products interact in some way with mT-regulated signaling pathways, leading to a ligand-dependent suppression of polyoma transformation. In addition, RAR-alpha overexpression selectively inhibits the serum-stimulated expression of the c-fos gene, but does not affect the expression of a number of other serum- and polyomavirus-inducible genes including c-jun, junB, c-myc and actin.

Retinoic acid suppresses polyoma virus transformation by inhibiting transcription of the c-fos proto-oncogene.

In a previous paper, we predicted that retinoic acid suppressed polyoma virus transformation of rat F111 fibroblasts by affecting the expression of one or more genes that are involved in signalling pathways normally activated by the viral mT oncogene (Talmage & Lackey, Oncogene 7, 1837-1845, 1992). We had identified the cellular c-fos proto-oncogene as a possible candidate target for both polyoma virus mT and retinoic acid regulated expression. In this report we present the results of experiments that demonstrate that retinoic acid does indeed inhibit transcriptional transactivation of the c-fos promoter by polyoma virus, as well as by calf serum and purified serum growth factors. Further experiments demonstrate that inhibition of c-fos expression with antisense fos RNA also prevents polyoma virus induced transformation. Restoration of c-fos expression, even in the presence of retinoic acid, restored transformation, indicating that retinoic acid inhibition of c-fos expression is sufficient to explain the retinoid suppression of transformation. These results identify the c-fos proto-oncogene as a key nuclear target for mT-dependent transformation and show that the anticarcinogenic properties of retinoic acid can be brought about by inhibiting c-fos expression.

Retinoic acid inhibits transformation by preventing phosphatidylinositol 3-kinase dependent activation of the c-fos promoter.

Retinoic acid inhibits transformation of cells by polyoma virus middle T oncoprotein. Inhibition of transformation results from a retinoic acid-dependent failure of cells to fully express the c-fos proto-oncogene. Retinoic acid prevents transactivation of the c-fos promoter by disrupting signaling between tyrosine kinases at the plasma membrane and trans-acting factors at the c-fos promoter. We used complementary genetic, biochemical and molecular approaches to demonstrate that: (1) phosphatidylinositol 3-kinase signaling is the principle mechanism of polyoma virus middle T oncoprotein activation of c-fos expression; (2) middle T/phosphatidylinositol 3-kinase transactivation of the c-fos promoter and transformation of cells requires activation of both the small GTP-binding protein Rac and Jun N-terminal kinase; (3) retinoic acid inhibits activation of Jun N-terminal kinase, thereby preventing c-fos transactivation and transformation; and (4) middle T activation of c-fos transcription requires both the serum response element and the promoter proximal cyclic AMP response element. These studies identify a novel target through which retinoids prevent oncogenic transformation.

Decreased cellular retinol-binding protein expression coincides with the loss of retinol responsiveness in rat cervical epithelial cells.

In response to estrogen the rat cervical epithelium undergoes squamous metaplastic changes, progressing from a resting state through a proliferating, secretory stage and finally to a cornified stage before sloughing or being reabsorbed. The transition from a secretory to a cornified epithelium is preceded by a dramatic reduction in the expression of the cellular retinol binding protein (CRBP). The associations among retinoids (retinol and retinoic acid), CRBP expression, and estrogen-induced keratinocyte differentiation were explored in cultured cervical epithelial cells. Retinoids supported proliferation of cervical epithelial cells expressing basal keratins. Alone, estrogen had no effect on proliferation and enhanced expression of keratins characteristic of stratified cervical epithelial cells. When added together, estrogen prevented retinoid effects on proliferation, whereas retinoids prevented the estrogen-enhanced expression of differentiation-associated cytokeratins. When CRBP expression was repressed by elevating intracellular cyclic AMP levels, the ability of retinol, but not retinoic acid, to block estrogen-induced changes in keratin expression was severely compromised. These results support a critical role for CRBP in cervical cell responsiveness to circulating retinoids (primarily retinol). We hypothesize that retinol inhibits estrogen-induced keratinization of the cervical epithelium, and the drop in CRBP level results in transient vitamin A deficiency within cervical epithelial cells, permitting the orderly transition from the secretory to the cornified stage.

Protein kinase Calpha expression confers retinoic acid sensitivity on MDA-MB-231 human breast cancer cells.

Retinoic acid activation of retinoic acid receptor alpha (RARalpha) induces protein kinase Calpha (PKCalpha) expression and inhibits proliferation of the hormone-dependent T-47D breast cancer cell line. Retinoic acid has no effect on proliferation or PKCalpha expression in a hormone-independent, breast cancer cell line (MDA-MB-231). To test the role of PKCalpha in retinoic acid-induced growth arrest of human breast cancer cells we established MDA-MB-231 cell lines stably expressing PKCalpha. Constitutive expression of PKCalpha did not affect proliferation of MDA-MB-231 cells but did result in partial retinoic acid sensitivity. Retinoic acid treatment of PKCalpha-MDA-MB-231 cells decreased proliferation (by approximately 40%) and inhibited serum activation of MAP kinases and induction of c-fos. Similar results were seen in MDA-MB-231 cells in which transcription of the transfected PKCalpha cDNA was reversibly induced by isopropyl beta-d-thiogalactoside. Expression of RARalpha in PKCalpha expressing MDA-MB-231 cells resulted in even greater retinoic acid responses, as measured by effects on cell proliferation, inhibition of serum signaling, and transactivation of an RARE-CAT reporter plasmid. In summary, PKCalpha synergizes with activated RARalpha to disrupt serum growth factor signaling, ultimately arresting proliferation of MDA-MB-231 cells.

Retinoic acid induced growth arrest of human breast carcinoma cells requires protein kinase C alpha expression and activity.

Retinoic acid inhibits proliferation of hormone-dependent, but not hormone-independent breast cancer cells. Retinoic acid-induced changes in cellular proliferation and differentiation are associated with disturbances in growth factor signaling and frequently with changes in protein kinase C expression. PKC delta, epsilon, and zeta are expressed in both hormone-dependent (T-47D) and hormone-independent (MDA-MB-231) cell lines. Retinoic acid arrested T-47D proliferation, induced PKC alpha expression and concomitantly repressed PKC zeta expression. The changes in PKC alpha and PKC zeta reflect retinoic acid-induced changes in mRNA. In contrast, retinoic acid had no effect on growth, or PKC expression in MDA-MB-231 cells. Growth arrest and the induction of PKC alpha, but not the reduction in PKC zeta, resulted from selective activation of RAR alpha. In total, these results support an important role for PKC alpha in mediating the anti-proliferative action of retinoids on human breast carcinoma cells.

Retinoic acid-induced transition from protein kinase C beta to protein kinase C alpha in differentiated F9 cells: correlation with altered regulation of proto-oncogene expression by phorbol esters.

Retinoic acid (RA) induced differentiation of F9 embryonal carcinoma cells is accompanied by changes in cellular responsiveness to extracellular signals. These changes include an increase in the AP1 transcription factor that is associated with the expression of differentiation markers (e.g., cytokeratin 18 and plasminogen activator). Since AP1 activity is a target for protein kinase C (PKC)-regulated changes in gene expression, we have examined the effects of RA on the expression and function of the PKC isozymes. F9 stem cells express PKC beta, delta, epsilon, and zeta. RA-induced differentiation to primitive endoderm led to a transition from PKC beta to PKC alpha expression. Additional treatment with dibutyryl cyclic AMP (dbcAMP), required for terminal differentiation into parietal endoderm, further increased PKC alpha expression and total PKC activity. RA and dbcAMP had negligible effects on the expression of PKC delta, epsilon, and zeta. The PKC beta to PKC alpha transition was specific for parietal endoderm; aggregation of RA-treated F9 cells induced visceral endoderm differentiation with elevated expression of PKC beta. The PKC activation with phorbol esters induced the expression of c-fos, c-jun, and junB proto-oncogenes in F9 stem cells. In the presence of either RA or RA and dbcAMP, phorbol ester treatment enhanced the expression of type IV collagen, a parietal endoderm marker. It also increased the expression of c-jun gene but not c-fos. The specific involvement of PKC beta in c-fos induction and PKC alpha in type IV collagen induction was confirmed in each PKC isozyme-transfected F9 cells. Together, our data demonstrate that the RA-induced (and dbcAMP-induced) changes in conventional PKC expression alters gene expression during parietal endoderm formation.

Synergistic activation of simian immunodeficiency virus and human immunodeficiency virus type 1 transcription by retinoic acid and phorbol ester through an NF-kappa B-independent mechanism.

The activation of human immunodeficiency virus type 1 (HIV-1) expression in latently infected cells by exogenous agents is believed to be important in the progression of AIDS. Most factors that are known to activate HIV-1 gene expression increase the binding of NF-kappa B or NF-kappa B-like transcription factors to the HIV-1 core enhancer region. In this report, we demonstrate that retinoic acid (RA) treatment of promonocytic U937 cells stimulates expression from the simian immunodeficiency virus (SIVmac) long terminal repeat (LTR). Furthermore, RA and phorbol 12-myristate 13-acetate (PMA) synergistically stimulated both SIVmac and HIV-1 LTRs to levels of expression comparable to that achieved by the viral transactivator Tat. The cis-acting elements required for a response to RA and PMA cotreatment are located between nucleotides -50 and +1 of SIVmac and between nucleotides -83 and +80 of HIV-1. Thus, the synergistic stimulation induced by RA and PMA is NF-kappa B independent. Analysis of deletion mutants of the SIVmac LTR demonstrates that RA and PMA stimulation cooperates with NF-kappa B and Sp1. An SIVmac LTR-reporter gene construct [pLTR(-50/+466)CAT] lacking NF-kappa B and Sp1 binding sites was not activated by Tat in untreated cells but was activated in cells that were cotreated with RA and PMA. Furthermore, gel retardation assays demonstrated that RA treatment causes a change in the pattern of a cellular factor(s) which binds to the -50 through +1 region of the SIVmac LTR. These data suggest that RA induces a PMA-activatable cellular factor that cooperates with NF-kappa B, Sp1, or Tat to stimulate LTR-directed transcription.

Regulation of pp60c-src expression in rat and mouse fibroblasts by an inducible antisense gene: effects on serum regulation of growth and polyoma virus middle T function.

Expression of antisense c-src RNAs in rat and mouse fibroblasts had a dramatic effect on the function of polyoma virus middle T (mT). Antisense c-src RNA decreased the amount of mT:pp60c-src complexes in de novo virus-infected cells and prevented expression of the transformed phenotype in rat F111 cells. Expression of antisense c-src RNA in infected NIH3T3 cells also reduced the formation of mT:pp60c-src complexes but did not affect the ability of polyoma virus to carry out a productive infection. Further analysis of the effects of antisense c-src RNA in uninfected cells revealed that pp60c-src is required for cell growth. When pp60c-src synthesis was reduced, F111 cells stopped proliferating and showed decreased S6 phosphorylation in response to serum. However, F111 cells expressing reduced pp60c-src could be efficiently transformed by v-rasHa, even in the presence of low serum. Thus, pp60c-src appears to function as a component of a signal transduction pathway which regulates cell proliferation in response to serum.

Distinct functions of protein kinase Calpha and protein kinase Cbeta during retinoic acid-induced differentiation of F9 cells.

As F9 embryonal carcinoma cells differentiate into parietal endoderm-like cells, expression of conventional protein kinase C (PKC) changes. Undifferentiated stem cells express PKCbeta but not PKCalpha, whereas differentiated parietal endoderm cells express PKCalpha but not PKCbeta. To determine whether changes in PKCalpha and/or PKCbeta expression control retinoic acid (RA)- and dibutyryl cyclic AMP-induced F9 cell differentiation, we established cell lines stably expressing PKCalpha, PKCbeta, antisense PKCalpha, or antisense PKCbeta RNAs. Constitutive expression of PKCalpha or inhibition of PKCbeta expression in F9 stem cells enhanced RA induced differentiation, both by increasing total expression and accelerating RA-induced expression of laminins A, B1, B2, and type IV collagen. In addition, expressing PKCbeta in a parietal endoderm cell line caused these cells to retrodifferentiate into stem cells. Based on these results, we conclude that PKCbeta and PKCalpha are key targets for RA-regulated gene expression, that PKCalpha plays an important, active role in inducing and maintaining the parietal endoderm phenotype, and that PKCbeta activity is incompatible with maintaining the differentiated state of these cells.

Separation of host range from transformation functions of the hr-t gene of polyomavirus.

hr-t mutants of polyomavirus are defective in virus growth as well as in cell transformation, and have genetic alterations that invariably affect both the middle and small T proteins. We have examined the growth properties of three site-directed mutants that either eliminate or alter the middle T without affecting the small T protein. Mutant 808A encodes large and small T proteins but no middle T; it grew poorly in NIH 3T3 cells. In contrast, mutants 1387T and 1178T which express altered middle T along with normal large and small T proteins grew nearly as well as wild-type virus. Thus, although the altered middle T proteins encoded by 1387T and 1178T are defective for cell transformation, they retained the ability to induce expression of a cellular permissivity factor(s) required for virus production. At the biochemical level, the induction of permissivity by middle T was manifested primarily in terms of phosphorylation of VP1 on threonine and in efficient encapsidation of viral DNA to form infectious virus. The natural role of middle T involves regulation of phosphorylation events, and can be enacted, at least in part, independently of interactions with pp60c-src.

Retinoid-induced repression of human immunodeficiency virus type 1 core promoter activity inhibits virus replication.

The rates of mother-to-child transmission of human immunodeficiency virus type 1 (HIV-1), progression to AIDS following HIV-1 infection, and AIDS-associated mortality are all inversely correlated with serum vitamin A levels (R. D. Semba, W. T. Caiaffa, N. M. H. Graham, S. Cohn, and D. Vlahov, J. Infect. Dis. 171:1196-1202, 1995; R. D. Semba, N. M. H. Graham, W. T. Caiaffa, J. B. Margolik, L. Clement, and D. Vlahov, Arch. Intern. Med. 153:2149-2154, 1993; R. D. Semba, P. G. Miotti, J. D. Chiphangwi, A. J. Saah, J. K. Canner, G. A. Dallabetta, and D. R. Hoover, Lancet 343:1593-1596, 1994). Here we show that physiological concentrations of vitamin A, as retinol or as its metabolite, all-trans retinoic acid, repressed HIV-1Ba-L replication in monocyte-derived macrophages (MDMs). Repression required retinoid treatment of peripheral monocytes during their in vitro differentiation into MDMs. Retinoids had no repressive effect if they were added after virus infection. Retinol, as well as all-trans retinoic acid and 9-cis retinoic acid, also repressed HIV-1 long terminal repeat (LTR)-directed expression up to 200-fold in transfected THP-1 monocytes. Analysis of HIV-1 LTR deletion mutants demonstrated that retinoids were able to repress activation of HIV-1 expression by both NF-kappaB and Tat. A cis-acting sequence required for retinoid-mediated repression of HIV-1 transcription was localized between nucleotides -51 and +12 of the HIV-1 LTR within the core promoter. Protein-DNA cross-linking experiments identified four proteins specific to retinoid-treated cells that bound to the core promoter. We conclude that retinoids render macrophages resistant to virus replication by modulating the interaction of cellular transcription factors with the viral core promoter.

Expression of cellular retinol- and cellular retinoic acid-binding proteins in the rat cervical epithelium is regulated by endocrine stimuli during normal squamous metaplasia.

To determine the potential roles of retinoids in the growth and differentiation of the reproductive tract epithelium, we have studied the expression of the cellular retinol- and retinoic acid-binding proteins, CRBP I and CRABP I, in the reproductive tract of female rats. CRBP I and CRABP I gene expression have been examined in the oviduct, ovary, uterus, and particularly in the cervix, which normally undergoes a cyclical squamous metaplasia during the estrus cycle. CRBP I was expressed in all four tissues examined, whereas CRABP I was expressed predominantly in cervix and uterus. In the cervix, CRBP I was detected in all epithelial layers including the columnar epithelium but was greatly reduced in the superficial, cornified layers of the stratified squamous epithelium. CRABP I was localized to the basement membrane region of the epithelium with the strongest expression in the basal layer of epithelial cells. While the expression of CRBP I and CRABP I in the keratinizing exocervix changed during the estrus cycle, it remained constant in the incompletely keratinized endocervix. The highest levels of CRBP I were seen during anestrus and proestrus, and for CRABP I during proestrus. Both CRBP I and CRABP I levels fell to barely detectable levels during estrus and metestrus. Using estrogen repletion of ovariectomized rats, we found that CRABP I levels transiently increased during the early proliferative response to estrogen, whereas CRBP I levels gradually declined, becoming barely detectable by 24 to 48 hours. These results suggest that CRBP I and CRABP I play different roles in the cyclical squamous metaplasia normally occurring in this tissue and that hormonal control of CRBP I and CRABP I expression might modulate the retinoid responsiveness of the epithelium during this process.

Regulation of neurogenesis by interactions between HEN1 and neuronal LMO proteins.

Basic-helix-loop-helix transcription factors regulate neurogenesis and neuronal differentiation by as yet unknown mechanisms. We show that an embryonic neuronal-specific basic-helix-loop-helix protein, HEN1 (also known as NSCL1 or NHLH), interacts with 'LIM only' proteins. Examination of the expression patterns of XHEN1 and XLMO-3, the Xenopus homologues of these human genes, reveals extensive overlap during early neurogenesis: at the onset of gastrulation on the dorsal side of the blastopore lip and, subsequently, in the prospective neural plate. Binding of XLMO-3 increases the transcriptional activity of XHEN1 in vivo. Co-expression of these two genes in Xenopus embryos induces a cascade of expression of neuronal-specific basic-helix-loop-helix proteins that leads to neuronal differentiation. We propose that XHEN1, in concert with XLMO-3, is a critical regulator of neurogenesis.

Apolipoprotein E inhibits serum-stimulated cell proliferation and enhances serum-independent cell proliferation.

Independently of its role in lipid homeostasis, apolipoprotein E (apoE) inhibits cell proliferation. We compared the effects of apoE added to media (exogenous apoE) with the effects of stably expressed apoE (endogenous apoE) on cell proliferation. Exogenous and endogenous apoE increased population doubling times by 30-50% over a period of 14 days by prolonging the G(1) phase of the cell cycle. Exogenous and endogenous apoE also decreased serum-stimulated DNA synthesis by 30-50%. However, apoE did not cause cell cycle arrest; both apoE-treated and control cells achieved equivalent saturation densities at 14 days. Further analyses demonstrated that exogenous and endogenous apoE prevented activation of MAPK but not induction of c-fos expression in response to serum growth factors. Endogenous (but not exogenous) apoE altered serum concentration-dependent effects on proliferation. Whereas control (non-apoE-expressing) cell numbers increased with increasing serum concentrations (1.6-fold for every 2-fold increase in serum), apoE-expressing cell numbers did not differ as serum levels were raised from 2.5 to 10%. In addition, in low serum (0.1%), apoE-expressing cells had elevated DNA synthesis levels compared with control cells. We conclude that apoE does not simply inhibit cell proliferation; rather, the presence of apoE alters the response to and requirement for serum mitogens.