A rapid decrease in epidermal growth factor-binding capacity accompanies the terminal differentiation of mouse myoblasts in vitro.

Specific mitogens stimulate the proliferation and repress the differentiation of mouse myoblasts (MM14). When mitogens are depleted, MM14 cells cease proliferation, commit to terminal differentiation, and become refractory to growth stimulation. The behavior of mitogen receptors during the transition from a proliferative to a permanently postmitotic state was examined using the epidermal growth factor receptor (EGFR) as a model system. Whereas proliferating myoblasts bound substantial amounts of EGF, their binding capacity declined rapidly upon exposure to low-mitogen medium. The decline became irreversible when a cell differentiated. Within 24 h, less than 5% of the original EGF binding capacity remained. Since the ability to internalize and degrade bound EGF was unaffected, the change presumably reflected a decrease in EGFR availability. Several observations indicated that loss of EGFR following mitogen removal is related to differentiation rather than the result of starvation or cell-cycle arrest. First, the decline is correlated with the absence of a single mitogen (fibroblast growth factor) and is independent of serum concentrations. Second, myoblasts that are either cycling through G1 or arrested at G0, but prevented from differentiating, all bind large amounts of EGF. These findings suggest that specific reduction in mitogen receptors could be part of a mechanism whereby terminally differentiating cells become refractory to mitogenic stimulation.

Induction of tumor promotor-inducible genes in murine 3T3 cell lines and tetradecanoyl phorbol acetate-nonproliferative 3T3 variants can occur through protein kinase C-dependent and -independent pathways.

We isolated a group of genes that are rapidly and transiently induced in 3T3 cells by tetradecanoyl phorbol acetate (TPA). These genes are called TIS genes (for TPA-inducible sequences). Epidermal growth factor (EGF), fibroblast growth factor (FGF), and TPA activated TIS gene expression with similar induction kinetics. TPA pretreatment to deplete protein kinase C activity did not abolish the subsequent induction of TIS gene expression by epidermal growth factor or fibroblast growth factor; both peptide mitogens can activate TIS genes through a protein kinase C-independent pathway(s). We also analyzed TIS gene expression in three TPA-nonproliferative variants (3T3-TNR2, 3T3-TNR9, and A31T6E12A). The results indicate that (i) modulation of a TPA-responsive sodium-potassium-chloride transport system is not necessary for TIS gene induction either by TPA or by other mitogens and (ii) TIS gene induction is not sufficient to guarantee a proliferative response to mitogenic stimulation.

Tumor promoter-inducible genes are differentially expressed in the developing mouse.

TIS genes are rapidly and transiently induced by tetradecanoyl phorbol acetate in 3T3 cells. We analyzed the developmental appearance of a number of the TIS genes to determine whether, in a normal physiological context, these genes have common or distinct mechanisms of regulation. Each TIS gene has a distinct tissue specificity and/or developmental profile.

Granulocyte-macrophage colony-stimulating factor and tetradecanoyl phorbol acetate induce a distinct, restricted subset of primary-response TIS genes in both proliferating and terminally differentiated myeloid cells.

Induction of early-response genes (tetradecanoyl phorbol acetate [TPA]-induced sequences, or TIS genes; R.W. Lim, B.C. Varnum, and H.R. Herschman, Oncogene 1:263-270, 1987) by granulocyte-macrophage colony-stimulating factor (GM-CSF) and TPA was examined both in a factor-dependent murine cell line, 32D clone 3, and in mature human neutrophils. When GM-CSF-deprived 32D clone 3 cells were exposed to GM-CSF or to TPA, four TIS mRNAs (TIS7, TIS8, TIS10, and TIS11) were rapidly and transiently induced. However, neither GM-CSF nor TPA could induce accumulation of TIS1 mRNA in 32D clone 3 cells, even under superinducing conditions. Both GM-CSF and TPA also elicited rapid, transient expression of TIS8 and TIS11 mRNA in postmitotic human neutrophils. However, neither agent could induce accumulation of TIS1 mRNA in human neutrophils. TIS1 is a member of the nuclear receptor supergene family that codes for ligand-dependent transcription factors. Cell-type restriction of inducible transcription factors may contribute to developmental specification.

Induction of transiently expressed genes in PC-12 pheochromocytoma cells.

Rat PC-12 pheochromocytoma cells, in response to nerve growth factor (NGF), stop proliferating and differentiate into cells resembling sympathetic neurons. This model of cell differentiation was used to investigate the expression of a previously isolated collection of mitogen-induced primary response sequences cloned from murine 3T3 cells; the TIS (tetradecanoyl phorbol acetate-induced sequences) genes (Lim et al., 1987). The TIS cDNAs were used to probe RNA isolated from PC-12 cells stimulated with NGF and other agents. Six of these messages were rapidly and transiently induced by NGF, tetradecanoyl phorbol acetate (TPA), or epidermal growth factor (EGF). Expression of these TIS genes generally resembled the NGF-stimulated induction of c-fos. In contrast, one TIS gene (TIS 10), induced by mitogens in 3T3 cells, was not induced by NGF, TPA, or EGF in PC-12 cells. Like c-fos, these TIS genes induced by NGF could also be superinduced by the combined administration of NGF and benzodiazepine. Elevated potassium ion, which leads to the induction of c-fos in PC-12 cells via activation of a voltage-dependent Ca2+ channel, also induces all TIS genes, with the notable exception of TIS 10. The induction of this family of genes may be involved in the general transduction of extracellular signals into biological responses.

Cloning of tetradecanoyl phorbol ester-induced 'primary response' sequences and their expression in density-arrested Swiss 3T3 cells and a TPA non-proliferative variant.

The tumor promoter tetradecanoyl phorbol acetate (TPA) is also a potent mitogen for murine 3T3 cells. We have previously described the isolation of variant Swiss 3T3 cell lines unable to proliferative in response to TPA. In this report we sought to identify genes that are stimulated by TPA as 'primary responses', i.e., without intervening protein synthesis. We constructed a cDNA library in lambda gt10, using RNA from quiescent 3T3 cells treated with TPA in the presence of cycloheximide (CHX). Of 50,000 recombinant phages, we identified 50 isolates that demonstrated preferential hybridization to cDNA probes generated from TPA-stimulated cells. One of the clones contains a fragment of the proto-oncogene c-fos. Twenty-nine of the remaining 49 clones fall into six cross-hybridization families. All the characterized clones detected mRNAs that are also inducible by epidermal growth factor, fibroblast growth factor or elevated serum. TPA induction of all the characterized messages is rapid and transient. All these mRNAs are superinduced by a combination of mitogens and CHX. Induction of these messages following TPA addition also occurs in subconfluent 3T3 cultures; expression of these genes is, therefore, not restricted to the G0/G1 transition. Expression of all six clones was also induced by TPA and other mitogens in 3T3-TNR9 cells, a TPA non-proliferative variant.

Nucleotide sequence of a cDNA encoding TIS11, a message induced in Swiss 3T3 cells by the tumor promoter tetradecanoyl phorbol acetate.

We report here the nucleic acid sequence and deduced amino acid sequence of a cDNA for TIS11, a gene induced in 3T3 cells by tetradecanoyl phorbol acetate.

Characterization of TIS7, a gene induced in Swiss 3T3 cells by the tumor promoter tetradecanoyl phorbol acetate.

We report here the nucleic acid sequence and deduced amino acid sequence of a cDNA for TIS7, a gene induced by mitogens in 3T3 cells and by nerve growth factor in PC12 pheochromocytoma cells. A fragment of the TIS7 gene has previously been cloned from murine fibroblast cells infected with Newcastle Disease Virus. The cDNA sequence suggests some similarities with murine interferons. TIS7 mRNA can also be transiently induced by double-stranded RNA. We suggest that the TIS7 protein may be an autocrine factor that attenuates or amplifies the initial ligand-induced signal.

Involvement of p27(kip1) and cyclin D3 in the regulation of cdk2 activity during skeletal muscle differentiation.

Terminal myogenic differentiation involves an irreversible transition from a proliferative state to a post-mitotic quiescent state. We showed here that in addition to the previously reported down regulation of G(1)-related cyclin-associated kinase activities, this transition was also accompanied by an extensive reorganization of the cyclin-cdk complexes, including a dramatic shift of cdk2 from cyclin A to cyclin D3. Moreover, the inhibition of cdk activity also correlated with an increase in the expression of the p27(kip1) cdk inhibitor and in its association with the cyclin-cdk2 complexes. Since depletion of p27 substantially reduced the cdk inhibitor activity present in differentiated muscle cells, we believe that the increase in p27 expression along with the reorganization of the cyclin-cdk2 complexes may play an important role in the inhibition of cdk2 activity during the differentiation process.

Differential regulation of primary response gene expression in skeletal muscle cells through multiple signal transduction pathways.

One of the earliest cellular responses to growth factors is the rapid induction of primary response genes. One group of such genes was originally isolated as tetradecanoyl phorbol acetate (TPA) inducible sequences (TIS genes) from mouse 3T3 cells. Proteins encoded by the TIS genes include two transcription factors: TIS8 (also known as egr1/NGFIA/zif268) and TIS1 (also known as NGFIB/nur77/N10). We have examined the inducibility of these two genes in a skeletal muscle cell line in response to agents that have been reported to block muscle differentiation. We report here that basic fibroblast growth factor (bFGF) induced the expression of both TIS1 and TIS8 in mouse C2C12cells. Both genes were also inducible by TPA while forskolin which activates the cAMP-dependent pathway induced TIS1 but not TIS8. Down-regulation of protein kinase C (PKC) activity by TPA pretreatment repressed the bFGF induction of TIS1 but had little effect on the bFGF-stimulated expression of TIS8. Moreover, while both TPA and bFGF stimulated the hyperphosphorylation of c-RAF and the activity of MAP kinase, TPA pretreatment failed to block RAF phosphorylation or the stimulation of MAP kinase activity by bFGF. Induction of the two TIS genes in skeletal myoblasts therefore appeared to be dependent to different extents on the activation of protein kinase A (PKA), PKC and MAP kinase.

Application of latex microspheres in the isolation of plasma membranes. Affinity density perturbation of erythrocyte membranes.

Immunolatex spheres, originally developed as visual markers for scanning electron microscopy, were employed as membrane density perturbation reagents. Methacrylate spheres were bound to antibody molecules and used to label antigens on erythrocytes. Ghosts prepared from labeled cells were subjected to isopycnic centrifugation on continuous sucrose and dextran gradients. It was found that the labeled erythrocyte membranes had a substantially higher density than unlabeled membranes. The extent to which the membrane density was shifted on a given gradient depended on the number, size and density of the latex spheres and could be closely predicted by theory. These results suggest that the reagents and techniques described here have potential application for the isolation of plasma membranes from more complex cell types.

Cloning, expression, and characterization of a novel guanylate-binding protein, GBP3 in murine erythroid progenitor cells.

We report the molecular cloning of a novel guanylate-binding protein (GBP), termed mouse GBP3 (mGBP3) in Friend virus-induced mouse erythroid progenitor (FVA) cells. The 71-kDa mGBP3 belongs to a family of known GBPs that contain the first two consensus motifs, GXXXXGK(S/T) and DXXG, but lack the third element, (N/T)KXD, found in typical GTP-binding proteins. Recombinant mGBP3 protein, expressed using a baculovirus expression system, binds to agarose-immobilized guanine nucleotides (GTP, GDP and GMP). Moreover, mGBP3 has been found to have an intrinsic GTPase activity with K(m) and Vmax values of 77 +/- 4 microM and 21 +/- 0.5 pmol min-1 microgram-1 of protein, respectively. The mGBP3 is distinct from the other GBPs, in that it does not have an isoprenylation/methylation motif CAAX at the carboxyl terminus. The mGBP3 appears to be localized in the cytosol based on immunofluorescence staining. Although the mGBP3 transcript is expressed to a varying degree in numerous mouse tissues, the message is most abundant in FVA cells. The mGBP3 transcript increases in FVA cells undergoing differentiation to a maximum within a few hours and then decreases to an undetectable level by 24 h. These results, taken together, suggest that mGBP3 is a novel member of a family of guanylate-binding proteins, which plays a role in the erythroid differentiation. The nucleotide sequence reported in this paper has been submitted to the GenBank with accession number U44731.

Genomic organization and promoter analysis of the murine Id3 gene.

Overexpression of the helix-loop-helix motif-containing transcription inhibitor Id3 has been shown to repress muscle-specific gene expression. Consistent with its putative negative regulatory role in the myogenic process, Id3 is highly expressed in proliferating myoblasts but down regulated when myoblasts are induced to differentiate. To investigate how Id3 expression may be transcriptionally regulated, we isolated a mouse Id3 genomic DNA fragment and characterized its organization and promoter activity. Comparison of the Id3 gene from human and mouse demonstrated a conserved exon-intron organization in which the first intron interrupts the C-terminal protein coding region and the second intron interrupts the 3' untranslated region at analogous positions in the two species. Sequence analysis of the 5'-flanking region revealed an unexpected mouse strain-specific genetic polymorphism due to a single base substitution. Deletion analysis revealed that as little as 180 base pairs of the mouse Id3 promoter upstream of the transcription start site is sufficient for a high level of gene expression in proliferating C2C12 myoblasts. In particular, the region between the nucleotide position -180 and -34 appeared to be crucial for maximal reporter gene activity and interacted specifically with C2C12 nuclear proteins. Finally, we showed that, despite the creation of a putative transcription factor-binding site, the genetic polymorphism observed did not affect Id3 promoter activity in proliferating C2C12 cells.

Platelet activating factor induces expression of early response genes c-fos and TIS-1 in human epidermoid carcinoma A-431 cells.

The effect of platelet activating factor (PAF) on the induction of early response genes was investigated in A-431 cells (human epidermal carcinoma cells). PAF induced a transient expression of c-fos and TIS-1 mRNA in a time- and dose-dependent manner. As low as 10(-10) M PAF caused detectable expression of these genes with a maximum observed at 10(-7) M. In the presence of cycloheximide, increases in the gene expression were noticeable at 20 min and peaked between 30-60 min. A lack of induction with lyso-PAF, an inactive PAF metabolite, confirmed the specificity of PAF towards this expression. The cells pretreated with CV-6209, a PAF receptor antagonist, did not show any induction of these genes by PAF. It is concluded that PAF causes induction of the early response genes c-fos and TIS-1 in a structurally specific and receptor dependent manner. This finding offers a new role for PAF at the nuclear level and may have important implications in the long term effects of PAF in pathophysiological conditions.

Interactive risk factors for treatment adherence in a chronic psychotic disorders population.

This study identified the unique and primary contributions of several concurrent risk factors for poor adherence to treatment recommendations in a clinic population of individuals with chronic psychotic disorders, i.e. 48% had DSM-IV diagnoses of schizoaffective disorder, 38% had schizophrenia, paranoid type, 12% had schizophrenia, undifferentiated type, and 2% had affective disorder with psychotic features. The target cohort consisted of 87 consecutive admissions to a continuing day treatment program. As part of a services-oriented quality assurance program, clinical staff completed rating scales for all patients. These included the BASIS-32 rating scale, which consisted of the following five subscales: psychosis; depression/anxiety; impulsive/addictive behavior; relation to self and others; and daily living and role functioning, and the Working Alliance Inventory-short form (therapist version), which consisted of the following three subscales: goal; task; and bond. These data were used to identify risk factors that weaken a patient's adherence to medication and non-medication treatment during the first 2 weeks of treatment in the clinic. Medication treatment consisted of both typical and atypical neuroleptic medications, with most patients being on multiple medications. Correlational analyses suggested that many of the risk factor variables were significantly associated with poor treatment adherence. Regression analyses suggested that the degree of psychoticism was most strongly associated with poor adherence to medication treatment and that difficulties relating to self and others were the strongest predictor of poor adherence to non-medication treatment. A large-sample services research design such as this can begin to determine patterns of associations between previous identified risk factors and poor treatment adherence in individuals with chronic psychotic disorders.

Glucocorticoid effects on the skeletal muscle differentiation program: analysis of clonal proliferation, morphological differentiation and the expression of muscle-specific and regulatory genes.

We examined the effect of glucocorticoids on the proliferation and differentiation of skeletal muscle cells using the C2C12 cell line. We found that treatment with glucocorticoids enhanced muscle cell differentiation but had only minor effects on the clonal growth rate of C2C12 cells. The stimulatory effect of glucocorticoids on myogenic differentiation was reflected in the increased expression of muscle-specific genes, creatine kinase (CK) and acetylcholine receptor gamma subunit (AChR). Dexamethasone had no effect on CK and AChR mRNA stability and enhanced transcription from a CAT reporter genes containing the 3.3kb 5' flanking region of the murine CK gene (-3300MCK-CAT). Since dexamethasone did not affect the expression levels of the myogenic regulatory genes such as myoD and myogenin, the enhancement of muscle-specific transcription might reflect an increase in the functional activity of the regulatory proteins. Other possible mechanisms involved in the differentiation-enhancing effect of glucocorticoids are discussed.

Physical and functional interactions between the transcriptional inhibitors Id3 and ITF-2b. Evidence toward a novel mechanism regulating muscle-specific gene expression.

We have used an interaction cloning strategy to identify an inhibitory isoform of the ITF-2 transcription factor, ITF-2b, that interacts with the transcriptional inhibitor Id3/HLH462. The interaction was confirmed in vitro, and inside intact myogenic C2C12 cells. As expected, overexpression of either Id3/HLH462 or ITF-2b effectively inhibited the activation of the muscle-specific creatine kinase promoter by the myogenic transcription factor MyoD. However, when overexpressed simultaneously, ITF-2b and Id3/HLH462 counteracted each other's inhibitory effect to produce a reduced overall inhibition. Moreover, while ITF-2b inhibited the creatine kinase promoter, it acted as a weak transactivator on an artificial promoter consisting of three tandem copies of the consensus myogenic factor DNA binding site. Further investigation indicated that the ITF-2b/MyoD heterodimer bound to its specific DNA binding site in vitro, and the DNA binding was effectively blocked by Id3/HLH462. Additional analysis revealed the presence of transcripts for both the activating (ITF-2a) and inhibitory (ITF-2b) isoforms in differentiating C2C12 cultures, suggesting that both isoforms might participate in regulating the differentiation process. Taken together, this study reveals a more complex pattern of regulatory interactions involving the helix-loop-helix proteins than was previously anticipated.

Modulation of muscle creatine kinase promoter activity by the inducible orphan nuclear receptor TIS1.

TIS1, an inducible orphan nuclear receptor, was originally isolated as a tumour-promoter-inducible gene in mouse 3T3 cells and later shown to be induced by growth factors and other extracellular stimuli. We show here that TIS1 mRNA was expressed in proliferating C2C12 mouse skeletal muscle cells out that the level of TIS1 expression increased during muscle differentiation. Overexpression of TIS1 transactivated muscle creatine kinase (MCK) reporter genes containing as little as 80 bp of the proximal 5' flanking region. In contrast, a promoterless TIS1 construct and a frameshift mutant TIS1 construct were unable to transactivate the MCK reporter gene. Moreover, the effect exerted by TIS1 appeared to be selective for the MCK promoter. Treatment of C2C12 cells with forskolin, which is known to induce TIS1 expression, also stimulated MCK reporter gene activity. Interestingly, in vitro translated TIS1 protein failed to bind to the MCK promoter region, suggesting that the transactivation effect of TIS1 may be mediated without direct interaction of the protein with the MCK promoter DNA. Collectively, these results suggest that changing levels of TIS1 may help to modulate the expression of MCK, and perhaps other muscle-specific genes, in response to physiological changes.

EGF responsiveness and receptor regulation in normal and differentiation-defective mouse myoblasts.

The interrelationship between cell proliferation and terminal myogenic differentiation has been analyzed by studying a differentiation-defective subclone (DD-1) of the permanent mouse myoblast line MM14. Parental MM14 myoblasts withdraw irreversibly from the cell cycle and initiate terminal differentiation when they are deprived of certain mitogens. In contrast, DD-1 cells become quiescent in a mitogen-depleted environment and less than 0.4% of the cells differentiate. When refed with mitogen-rich medium quiescent DD-1 cells resume proliferation. Expression of this differentiation-defective phenotype is apparently coupled to an alteration in mitogen sensitivity: MM14 myoblasts require horse serum plus either chick embryo extract or fibroblast growth factor (FGF) to sustain cell growth: DD-1 variants are responsive to FGF, but also proliferate in response to serum alone or to reduced serum plus epidermal growth factor (EGF). Interestingly, EGF also appears to retard DD-1 cell differentiation in a manner similar to the FGF repression of differentiation in normal myoblasts. Normal and differentiation-defective myoblasts which have been maintained under growth-promoting conditions exhibit similar EGF binding, internalization, and degradation. However, whereas the EGF binding capacity of MM14 myoblasts declines to less than 5% of its initial level within 24 hr of FGF removal, DD-1 variants exhibit an increase in EGF binding when switched to an FGF-depleted medium. The relationship of altered EGF receptor regulation to changes in mitogen sensitivity and differentiation capacity of the DD-1 variant is discussed, and implications for general in vivo processes governing cell proliferation and differentiation are considered.

Regulated association of microtubule-associated protein 2 (MAP2) with Src and Grb2: evidence for MAP2 as a scaffolding protein.

Microtubule-associated protein 2 (MAP2) and tau, which is involved in Alzheimer's disease, are major cytoskeletal proteins in neurons. These proteins are involved in microtubule assembly and stability. To further characterize MAP2, we took a strategy of identifying potential MAP2 binding partners. The low molecular weight MAP2c protein has 11 PXXP motifs that are conserved across species, and these PXXP motifs could be potential ligands for Src homology 3 (SH3) domains. We tested for MAP2 interaction with SH3 domain-containing proteins. All neuronal MAP2 isoforms bound specifically to the SH3 domains of c-Src and Grb2 in an in vitro glutathione S-transferase-SH3 pull-down assay. Interactions between endogenous proteins were confirmed by co-immunoprecipitation using brain lysate. All three proteins were also found co-expressed in neuronal cell bodies and dendrites. Surprisingly, the SH3 domain-binding site was mapped to the microtubule-binding domain that contains no PXXP motif. Src bound primarily the soluble, non-microtubule-associated MAP2c in vitro. This specific MAP2/SH3 domain interaction was inhibited by phosphorylation of MAP2c by the mitogen-activated protein kinase extracellular signal-regulated kinase 2 but not by protein kinase A. This phosphorylation-regulated association of MAP2 with proteins of intracellular signal transduction pathways suggests a possible link between cellular signaling and neuronal cytoskeleton, with MAP2 perhaps acting as a molecular scaffold upon which cytoskeleton-modifying proteins assemble and dissociate in response to neuronal activity.