Growth factor-responsive genes in fibroblasts.
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Animals↗
Biomedical subjects
R Bravo
Publications and source records attributed to R Bravo.
Genes induced during the G0/G1 transition in mouse fibroblasts.
Growth factors trigger a complex genetic response in quiescent fibroblast which involves approximately 100 genes. The genes so far identified encode for nuclear proteins, secretory molecules and components of the cytoskeleton and extracellular matrix. Ten of the nuclear proteins so far found can be grouped into families of transcription factors according to common structural components. These include the families of JUN, FOS, zinc finger proteins and nuclear hormonal receptors. Two of the secretory proteins share structural homology with cytokines and one of them has been shown to have a mitogenic capacity. These observations demonstrate that several of the immediate early genes encode for molecules which could regulate the expression of other genes whose products are essential for the progress through G1.
Animals↗
c-fos mediated stimulation of an AP-1 DNA binding activity in undifferentiated teratocarcinoma cell lines.
Undifferentiated F9 and PCC4 embryonal carcinoma (EC) cells contain low levels of AP-1 DNA binding activity. Upon differentiation induced by retinoic acid and cyclic AMP or in differentiated cell lines, AP-1 DNA binding activity can be readily detected. Minute amounts of 3T6 cells extracts, that by themselves were unable to show any binding to an AP-1 site, stimulate AP-1 DNA binding activity when added to the EC cell extracts, suggesting that components of the 3T6 extracts stimulate this DNA binding activity in F9 and PCC4 cell extracts. This enhancement of DNA binding activity requires the presence in the donor fraction (3T6 cells) of a thermostable protein(s) that possesses neither protein kinase nor phosphatase activities. The proteins responsible for stimulation in 3T6 extracts can be separated from the ones responsible for AP-1 binding by chromatography. 3T6 c-fos immunodepleted fractions are unable to activate AP-1 DNA binding activity in EC cell extracts, while c-jun depleted fractions activate normally. Moreover, in vitro translated c-fos, but not c-jun proteins, are able to stimulate binding in EC extracts. These data suggest an important role for c-fos protein in activation of a specific DNA binding transcriptional factor during cellular differentiation and provide a convenient in vitro assay for c-fos function.
Cell Differentiation↗
The leucine repeat motif in Fos protein mediates complex formation with Jun/AP-1 and is required for transformation.
Cellular and viral Fos proteins form a tight complex with other nuclear proteins, including the transcription factor and proto-oncogene AP-1/Jun. We have mapped the c-Jun binding site in Fos to a region containing regularly spaced leucine residues recently suggested to interdigitate with a similar structure in Jun. Substitution of single or multiple leucine residues or the alteration of leucine phasing by insertion of additional amino acids reduces or abolishes the binding to Jun, while the substitution of other amino acids has no noticeable effect. These results strongly suggest that the formation of a "leucine zipper" mediates the interaction between Fos and Jun. We also show that the differential binding of the various Fos mutants correlates with their potential to trans-activate AP-1-dependent transcription and to induce morphological transformation.
Animals↗
Segment-specific expression of a zinc-finger gene in the developing nervous system of the mouse.
The process of segmentation, in which repeated homologous structures are generated along the anterior-posterior axis of the embryo is a widespread mechanism in animal development. In vertebrates, segmentation is most apparent in the somites and the peripheral nervous system, but the existence of repetitive bulges, termed neuromeres, in the early neural epithelium of vertebrates suggests that the CNS may also be segmented. Consistent with this, cranial ganglia and certain neurons are associated with specific hindbrain neuromeres. Here, we report that Krox-20, a zinc-finger gene, is expressed in two alternate neuromeres in the mouse early hindbrain. This pattern subsequently decays and Krox-20 is transiently expressed in specific hindbrain nuclei. In addition, Krox-20 is expressed in early neural crest cells, and then in the neural crest-derived boundary caps, glial components of the cranial and spinal ganglia. The demonstration that neuromeres are domains of gene expression provides molecular evidence for the segmentation of the CNS.
Animals↗
The product of a novel growth factor activated gene, fos B, interacts with JUN proteins enhancing their DNA binding activity.
We have identified a gene, fos B, encoding a nuclear protein of 338 amino acids presenting a 70% homology with c-fos, whose expression is activated during G0/G1 transition. Growth factor stimulation of quiescent cells leads to a rapid and transient accumulation of fos B mRNA, with kinetics similar to those of c-fos. The induction of fos B mRNA levels is in part due to a dramatic increase in the transcription of the gene. The half-life of fos B mRNA is in the order of 10-15 min. Both transcriptional activation and mRNA stability are substantially increased in the presence of protein synthesis inhibitors. Immunoprecipitation studies showed that fos B as c-fos protein, forms a complex in vitro with c-jun and jun B proteins in the absence of a target binding sequence. Gel retardation assays demonstrated that fos B protein positively influences the binding of c-jun and jun B proteins to an AP-1 binding consensus sequence, suggesting that fos B protein plays a role in control of gene expression.
Amino Acid Sequence↗
Characterization of junD: a new member of the jun proto-oncogene family.
In an extensive screen of a cDNA library prepared from serum-stimulated mouse NIH 3T3 cells, we identified three distinct jun-related clones. Two of them were carrying c-jun and junB sequences respectively, whereas the sequence of the third group of clones (junD) was distinct from these two and from v-jun. The amino acid sequences derived from these jun-related clones are very well conserved in five distinct regions including the putative DNA binding domain. Truncated c-Jun and JunD proteins containing the C-terminus recognize the same DNA sequences which were defined as the PEA1/AP1 binding sequence or TPA response element (TRE). Furthermore, both can trans-activate a promoter including the TRE, and this activation is further enhanced by c-fos. Contrary to c-jun and junB transcription, which are strongly stimulated by serum or TPA treatment of quiescent 3T3 cells, junD transcription is not significantly stimulated in these conditions. The tissue distribution and levels of expression of junD mRNA differ from that of c-jun and junB mRNA. These observations suggest that each of these Jun-related gene products has a distinct role in the control of gene activity and growth in the organism.
Amino Acid Sequence↗
Structure, mapping and expression of a growth factor inducible gene encoding a putative nuclear hormonal binding receptor.
We have characterized a growth factor inducible gene, N10, encoding a nuclear protein of 601 amino acids with a significant similarity to members of the steroid and thyroid hormone receptor families. The gene is rapidly but transiently induced by several mitogens. Immunoprecipitation studies show that the N10 protein is transiently expressed after stimulation of quiescent cells, presenting a half-life of approximately 30 min. The N10 transcription unit is 8 kb in length, split into seven exons. The exon-intron distribution is in general similar to that of other members of the nuclear receptor superfamily, but presents some differences which suggest that N10 belongs to a new family of these molecules. The 5' flanking region contains one DSE which could explain its immediate response to external stimulus. The N10 gene is located in the [F1-F3] region of mouse chromosome 15.
Amino Acid Sequence↗
Coordinate induction of fibronectin, fibronectin receptor, tropomyosin, and actin genes in serum-stimulated fibroblasts.
From a collection of more than 80 nonoverlapping clones, isolated by differential screening of a lambda cDNA library prepared from serum-stimulated cells in the presence of cycloheximide, we have identified four clones that encoded for components of the cytoskeleton and extracellular matrix. DNA sequencing of clones B2, V58, TT1, and P38 demonstrated that they corresponded to beta-actin, alpha-tropomyosin, fibronectin, and the beta-subunit of fibronectin receptor. All four mRNA levels showed a detectable increase 30 min after stimulation and remained at high levels for at least 8 h. The half-lives of these mRNAs were found to be very long in contrast to those of other growth factor-inducible genes. An increase in transcription was observed for the four genes. Actin and fibronectin showed nearly maximal increase at 15 min, while fibronectin receptor and tropomyosin reached their maximum transcription at 1 h. These results demonstrated that four interacting components of the cytoskeleton and extracellular matrix are rapidly induced in stimulated quiescent cells, possibly reflecting part of the coordinate changes in gene expression that occur during embryogenesis and wound healing.
Actins↗
Expression, cloning and cDNA sequence of a fibroblast serum-regulated gene encoding a putative actin-associated protein (p27).
A cDNA clone for a basic putative actin microfilament-associated protein, p27, highly induced in serum-stimulated NIH 3T3 cells, has been isolated by polyclonal antibodies and sequenced. p27 mRNA is a 1.2-kb molecule which is very low in resting NIH 3T3 cells but can be induced at least 100 times after 8 h of fetal calf serum stimulation. In contrast to other inducible mRNAs, p27 mRNA is stable, and its levels can be superinduced by cycloheximide mainly by prolonging transcription. The lack of expression of this messenger in mouse tissues, as well as in all cell lines so far tested, suggests that p27 may be an fibroblast-specific protein. One major open reading frame found in p27 cDNA codes for a 201 amino acid polypeptide not related to any previously described actin-binding protein. Interestingly, it shows alternative hydrophilic and hydrophobic domains of amino acids symmetrically arranged from the middle of the protein. The coordinate induction of p27 and actin mRNAs suggest that p27 may be involved in the cytoskeletal rearrangements induced early in cell growth and proliferation.
Amino Acid Sequence↗
Cloning and sequence of a secretory protein induced by growth factors in mouse fibroblasts.
From a collection of 80 cDNAs representing genes that are induced by serum growth factors in quiescent mouse fibroblasts we have characterized a clone, N51, and demonstrated that it codes for a precursor of a secretory protein. N51 mRNA is rapidly induced by serum, including in the presence of cycloheximide, demonstrating that its induction does not require de novo protein synthesis. The mRNA has a short half-life of approximately 10-15 min and is dramatically prolonged in the presence of protein synthesis inhibitors. Purified growth factors such as platelet-derived growth factor and bombesin are strong inducers of N51 expression. However, epidermal growth factor, fibroblast growth factor, and the tumor promoter tetradecanoyl phorbol acetate have a negligible effect. Transcriptional analysis demonstrates that the increase in N51 mRNA levels after serum stimulation is in part due to the activation of gene transcription.
Amino Acid Sequence↗
Ultrastructural immunolocalization of cyclin/PCNA in synchronized 3T3 cells.
The immunolocalization of cyclin/PCNA in synchronized 3T3 cells was performed with human autoantibodies using an immunogold technique performed on thin cryosections. Previous immunofluorescent studies demonstrated that the DNA replication sites correspond to the localization of bound cyclin. We have found that in the early periods of S phase, the DNA replication sites (or sites potentially ready for the replication during the hydroxyurea DNA synthesis block) are situated in the perichromatin region and correspond to clustered gold particles present frequently over a morphologically distinct small nuclear area. Heavily labeled chromocenters, including perinucleolar condensed chromatin, exhibiting several such distinct areas were found in later periods of S phase.
Animals↗
Structure, chromosome location, and expression of the mouse zinc finger gene Krox-20: multiple gene products and coregulation with the proto-oncogene c-fos.
We have analyzed the structure and the regulation of Krox-20, a mouse zinc finger-encoding gene which is transiently activated following serum stimulation of quiescent fibroblast cells in culture. The gene is localized on chromosome 10, band B5, in the mouse, and the homologous human gene also maps to chromosome 10 (region q21.1 to q22.1). Alternative splicing of the 5'-most intron of the Krox-20 gene gives rise to mRNAs encoding putative zinc finger proteins with different N termini. The first exon contains a sequence element with strong similarity to the c-fos proto-oncogene serum response element (SRE). This element can functionally substitute for the c-fos SRE, and it binds the same nuclear protein. It is probably responsible for the serum induction of Krox-20, possibly in combination with a weaker SRE located in the 5'-flanking region of the gene. Our findings suggest that c-fos, Krox-20, and a number of immediate-early serum response genes are coregulated and that the SRE and its cognate protein are essential components of this regulatory pathway.
Amino Acid Sequence↗
Tissue-specific expression of c-jun and junB during organogenesis in the mouse.
c-jun and junB are cellular genes related to the viral oncogene v-jun and encode members of the AP-1 transcription factor gene family. These genes have been implicated in the control of the G0/G1 transition in fibroblasts. Here, we have investigated the potential roles of c-jun and junB during fetal growth and organogenesis in the mouse by in situ hybridization analysis of their expression patterns. c-jun expression is detected throughout organogenesis, and transcripts are detected in many tissues, although in restricted cell populations within developing cartilage, gut and the central nervous system (CNS). In cartilage, c-jun expression is associated with rapidly proliferating perichondrial cells, but occurs in postmitotic motor neurones in the CNS. junB expression is initiated between 14.5 and 17.5 days of development, and is restricted to differentiating epidermal cells and endodermal gut epithelium. These data suggest that c-jun and junB have distinct, tissue-specific roles in cell proliferation and differentiation during fetal development.
Animals↗
[Women, development and population in Latin America].
Women's situation as defined according to cultural factors which are present both in the division of labor by sex and in the power relationships, has its own specificities in the different social groups of a society. This happens for material reasons as well as because prevalent ideologies vary, among the different groups, from very traditional models of sexual roles to others which allow a greater flexibility in these roles. The consequences are reflected in the reproductive behavior. A woman's social insertion affects her specific situation, through a set of closely related variables: the educational opportunities, the quantity and characteristics of the domestic work they must carry out and the opportunities to participate in paid work. These variables influence demographic behavior through the age at 1st marriage, infant mortality, the values assigned to children and the capacity of taking reproductive decisions. In order to improve the situation of poorer women it is necessary that the economic, social and cultural development give special benefits to that sector, greater educational opportunities which could lead to possibilities of stable and well remunerated work. In these conditions women could achieve a greater self-determination to establish more equitable conjugal relationships and to decide with more freedom the number of their children.
Developing Countries↗
Transcriptional activation of c-jun during the G0/G1 transition in mouse fibroblasts.
Before quiescent cells can respond to mitogens and progress through the G1 phase of cell growth, new messenger RNA synthesis is required. The G1 phase seems to be a critical point of control in the cell cycle, where normal cells deprived of growth factors halt cycling while transformed cells do not, suggesting that regulatory genes, uncontrolled in the neoplastic phenotype, are expressed during the G0 to G1 transition. Some of these may code for nuclear proteins that participate in the transactivation of genes required for the progression through G1. The observed changes in expression of the proto-oncogenes c-fos and c-myc, following stimulation of fibroblasts with growth factors, support this notion as recent evidence suggests that c-FOS and c-MYC proteins can function as transactivating factors. Moreover, the rapid induction of several genes in fibroblasts coding for putative transacting factors during the G0 to G1 transition has been recently reported. Here we present the nucleotide sequence of a mouse cDNA clone coding for a 334 residue protein which shows 80% similarity with v-JUN and more than 98% similarity with the human c-JUN sequence. We have demonstrated that in quiescent fibroblasts c-jun transcription is rapidly induced during the G0 to G1 transition.
Amino Acid Sequence↗
Role of nuclear material in the early cell cycle of Xenopus embryos.
Activated Xenopus eggs show periodic surface contraction waves and oscillations in endogenous protein phosphorylation, MPF, and kinase activities timed with the cleavage cycle of control fertilized eggs. In this paper, we show that in activated eggs lacking the material that originates from the oocyte nucleus, MPF and kinase oscillations occur in the absence of surface contraction waves. Two mitotic phosphoproteins (M116 and M46), previously described by 32P labeling in nucleated eggs, are no longer detected in the enucleated eggs. We conclude that a cytoplasmic temporal control of MPF and kinase activities is likely to be the essential cell cycle oscillator. The oocyte nuclear components normally stored in the cytoplasm of the embryos are not involved in the clock although they appear to be required for the generation of surface contraction waves.
Animals↗
A gene encoding a protein with zinc fingers is activated during G0/G1 transition in cultured cells.
Zinc fingers are DNA-binding domains present in several eukaryotic regulatory proteins. We have identified a mouse gene, Krox-20, encoding a protein with three zinc fingers and whose expression is activated during G0/G1 transition in cultured cells. Serum stimulation of quiescent cells leads to rapid and transient accumulation of Krox-20 mRNA, with kinetics similar to those of the c-fos proto-oncogene. The induction does not require de-novo protein synthesis. In the mouse, Krox-20 is expressed at low levels in tissues which contain rapidly dividing cells. These properties suggest that Krox-20 encodes a transcription control factor, possibly involved in the modulation of cell proliferation.
Amino Acid Sequence↗