Control by fibroblast growth factor of differentiation in the BC3H1 muscle cell line

The regulation of creatine phosphokinase (CPK) expression by polypeptide growth factors has been examined in the clonal mouse muscle BC3H1 cell line. After arrest of cell growth by exposure to low concentrations of serum, BC3H1 cells accumulate high levels of muscle-specific proteins including CPK. The induction of this enzyme is reversible in the presence of high concentrations of fetal calf serum, which cause quiescent, differentiated cells to reenter the cell cycle. Under these conditions, the rate of CPK synthesis is drastically reduced. We show in the present communication that either pituitary-derived fibroblast growth factor (FGF) or brain-derived FGF are as effective as serum in repressing the synthesis of CPK when added to quiescent, differentiated cells. The decrease in the rate of synthesis of CPK occurs within 22 h after the addition of pituitary FGF to the cells. Pituitary FGF had very little effect, if any, on the rate CPK degradation. The overall rate of protein synthesis and the pattern of synthesis of the major polypeptides made by these cells was not altered by the addition of FGF. Although pituitary FGF was mitogenic for BC3H1 cells, the rate of cell growth was not absolutely correlated with the extent of repression of CPK. Brain-derived FGF fully repressed CPK induction under conditions where it showed no significant mitogenic activity. These results show that the expression of a muscle-specific protein, CPK, can be controlled by a single defined polypeptide growth factor in fully differentiated cultures, and that initiation of cell division is not required for their regulation to take place.     

Nitric oxide-dependent synthesis of vascular endothelial growth factor is impaired by high glucose

Synthesis of vascular endothelial growth factor (VEGF), the major angiogenic molecule, is induced by nitric oxide (NO) in various cell types, including vascular smooth muscle cells (VSMC). Therefore, compounds which inhibit NO generation can also influence VEGF synthesis. Here we investigated the effect of increased glucose concentration (25 mM vs. 5.5 mM) on cytokine-induced VEGF synthesis in rat VSMC. The cells growing in the medium containing 5.5 mM glucose and exposed to IL-1-beta, TNF-alpha and IFN-gamma induced expression of an inducible isoform of nitric oxide synthase (NOS II). This is followed by generation of NO and the concomitant expression of VEGF gene and release of VEGF protein. In contrast, 25 mM glucose impaired induction of NOS II expression and thus NO synthesis was lower than in 5.5 mM glucose. Consequently, the VEGF promoter activation was attenuated, resulting in decreased mRNA synthesis and lower production of VEGF protein. The results indicate that abnormally high concentrations of glucose can impair generation of NO and the NO-dependent VEGF synthesis. This may play a role in the development and progression of vascular dysfunctions in cardiovascular diseases.     

Regulation of alpha 1 (I)-collagen gene expression in response to cell adhesion in Swiss 3T3 fibroblasts

Nonadhesive conditions cause Swiss 3T3 fibroblasts to enter a quiescent state that is reversed upon reattachment to a surface. Previously, we demonstrated that adhesion in serum-free conditions is sufficient to activate suspension-arrested cells out of Go, with the induction of the growth-associated genes, c-fos, c-myc, and actin. In this study, we have employed this system to identify programs of gene expression that respond primarily to the adhesive state of the cell, rather than the growth state. We show that cells in different adhesive states can be distinguished by their patterns of protein synthesis. Analysis of one adhesion-responsive protein led to its identification as pro-alpha 1 (I)-collagen. Pro-alpha 1 (I)-collagen synthesis and mRNA levels are decreased up to 6-fold in suspension-arrested fibroblasts, but are enhanced up to 5-fold as cells approach confluence. This suggests that the reduced expression in suspension-arrested cells is not simply a result of quiescence. In addition, reattachment of suspended cells in serum-free conditions caused a 7-fold induction of collagen mRNA levels and a greater than 20-fold rise in the rate of procollagen synthesis. The expression of c-myc was induced during adhesion in serum-free medium as well as by serum addition to suspension-arrested cells. However, alpha 1 (I)-collagen gene expression was unaffected by serum in the absence of adhesion. These results indicate that collagen gene expression is directly responsive to cell adhesion, independent of the growth state.     

The epidermal growth factor-induced cell migration and expression of the 47,000 Mr secreted glycoprotein EIP-1 of rat liver epithelial cells are down-modulated by cyclic AMP

The migration of rat liver epithelial cells induced by epidermal growth factor (EGF) was inhibited by cyclic AMP (cAMP) and cholera toxin, but not by cGMP, cAMP and cholera toxin also inhibited the expression of the EGF/transforming growth factor (TGF) alpha-inducible protein EIP-1 (Mr 47,000), but not that of other proteins induced by the growth factor. cAMP therefore specifically and selectively represses the EGF-induced expression of this protein, which by synthesis in the presence of tunicamycin and by enzymatic treatments was shown to be N-glycosylated and sialylated. The close correlation of the expression of EIP-1 with the growth factor-induced migration suggests that this glycoprotein is involved in the cellular translocation process. Modulation of cell migration and of EIP-1 expression through increased intracellular concentrations of cAMP indicate that factors operating through this signal system can modulate the phenotypic and gene expression changes mediated by the EGF-receptor. Identification of the ligand(s) that can cause the cAMP-mediated effects might be an important step towards understanding the regulation of liver cell migration in vivo.     

Effect of a dominant inhibitory Ha-ras mutation on mitogenic signal transduction in NIH 3T3 cells.

We used a dominant inhibitory mutation of c-Ha-ras which changes Ser-17 to Asn-17 in the gene product p21 [p21(Asn-17)Ha-ras] to investigate ras function in mitogenic signal transduction. An NIH 3T3 cell line [NIH(M17)] was isolated that displayed inducible expression of the mutant Ha-ras gene (Ha-ras Asn-17) via the mouse mammary tumor virus long terminal repeat and was growth inhibited by dexamethasone. The effect of dexamethasone induction on response of quiescent NIH(M17) cells to mitogens was then analyzed. Stimulation of DNA synthesis by epidermal growth factor (EGF) and 12-O-tetradecanoylphorbol-13-acetate (TPA) was completely blocked by p21(Asn-17) expression, and stimulation by serum, fibroblast growth factor, and platelet-derived growth factor was partially inhibited. However, the induction of fos, jun, and myc by EGF and TPA was not significantly inhibited in this cell line. An effect of p21(Asn-17) on fos induction was, however, demonstrated in transient expression assays in which quiescent NIH 3T3 cells were cotransfected with a fos-cat receptor plasmid plus a Ha-ras Asn-17 expression vector. In this assay, p21(Asn-17) inhibited chloramphenicol acetyltransferase expression induced by EGF and other growth factors. In contrast to its effect on DNA synthesis, however, Ha-ras Asn-17 expression did not inhibit fos-cat expression induced by TPA. Conversely, downregulation of protein kinase C did not inhibit fos-cat induction by activated ras or other oncogenes. These results suggest that ras proteins are involved in at least two parallel mitogenic signal transduction pathways, one of which is independent of protein kinase C. Although either pathway alone appears to be sufficient to induce fos, both appear to be necessary to induce the full mitogenic response.     

Overexpression of cloned genes using recombinant Escherichia coli regulated by a T7 promoter: II. Two-stage continuous cultures and model simulations

A two-stage culture strategy was studied for continuous high-level production of a foreign protein in the chemically inducible T7 expression system. The first stage is dedicated to the maintenance of plasmid-bearing cells and the second stage to the target protein synthesis by induction of cells coming from the first stage. On entering the second stage, recombinant cells undergo a gradual induction of the target gene expression. These plasmid-bearing cells experience dynamic changes in intracellular compositions and specific growth rates with their individual residence times. Therefore, the overall cultural characteristics in the production stage are really averages of the contributions from the various cells with different residence times. The behavior of the two-stage culture is described by a model, which accounts for dynamic variations of cell growth and protein synthesis rates with cell residence times. Model simulations were compared with experimental results at a variety of operating conditions such as inducer concentration and dilution rate. This model is useful for understanding the behavior of two-stage continuous cultures. (c) 1993 John Wiley & Sons, Inc.     

Overexpression of cloned genes using recombinant Escherichia coli regulated by a T7 promoter: I. Batch cultures and kinetic modeling

A novel Eschericha coli expression system directed by bacteriophage T7 RNA Polymerase utilized for overexpression of the cloned gene. The recombinant cell contains the plasmid with a bacteriophage promoter, the T7 promoter, to regulate the expression of the target gene. This promoter is recongnized only by T7 RNA polymerase, whose gene has been fused into the host chromosome and is under control of the lacUV5 promoter. Therefore, the target gene on the plasmid can be expressed only in the presence of T7 RNA polymerase, which is induced by isopropyl-beta-D-thiogalactopyranoside (IPTG). The batch cultures were performed to investigate the effect of induction on kinetics of cell growth and foreign protein formation and to determine the optimal induction strategy. It was observed that the specific growth rates of the recombinant cells dramatically decrease after induction, and that there is an optimal induction time for maximizing the accumulated intracellular foreign protein. This optimal induction time varies singificantly with inducer concentration. To better understand the optimal behavior, a lumped mechanistic model was constructed to analyze the induced cell growth and foreign protein formation rates. (c) 1992 John Wiley & Sons, Inc.     

Estrogen-induced genes,WISP-2 and pS2, respond divergently to protein kinase pathway

Recently, we identified WISP-2 (Wnt-1 inducible signaling pathway protein 2) as a novel estrogen-inducible gene in the MCF-7 human breast cancer cell line. In this study, we examined whether WISP-2 expression is modulated by PK activators. Treatment with protein kinase A (PKA) activators [cholera toxin plus 3-isobutyl-1-methylxanthine (CT/IBMX)] induced WISP-2 expression. CT/IBMX induced expression of the other estrogen-responsive gene, pS2, more dramatically than maximum stimulation by 17beta-estradiol (E2). Treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA), which directly stimulates protein kinase C (PKC) activity, completely prevented WISP-2 mRNA induction by E2, whereas it increased pS2 mRNA expression more dramatically than maximum stimulation by E2. Results of treatments with the protein synthesis inhibitor cycloheximide and the pure antiestrogen ICI182,780 suggest that these PK pathways modulate WISP-2 gene expression via different molecular mechanisms than those for pS2. Because TPA inhibits cell proliferation, we investigated whether WISP-2 induction was dependent on cell growth. Cells were treated with insulin-like growth factor-1 (IGF-1) or interleukin-1alpha (IL-1alpha) to stimulate or inhibit cell growth, respectively. These treatments had no effect on WISP-2 mRNA expression either alone or in combination with E2, suggesting that WISP-2 induction is independent of cell growth.     

Multihormonal induction of alpha 2u-globulin in an established rat hepatoma cell line

A subclone of the FU5-5 rat hepatoma cell line has been isolated which is inducible more than several hundred fold for the 20,000 dalton form of the major rat urinary protein alpha 2u-globulin. The basal relative synthetic rate (RSR) in growth medium containing 10% fetal calf serum was less than 2 X 10(-6) of total protein synthesis. Both dexamethasone and insulin were necessary for induction, and yielded a maximum induced RSR of 4-8 X 10(-3). Triiodothyronine (T3), dihydrotestosterone (DHT), rat growth hormone (GH), and estrogen, all of which have been shown to influence the induction of alpha 2u-globulin in the intact rat, were without effect on the cell line. A factor present in fetal calf serum was also necessary for maximum induction, since dexamethasone plus insulin in serum-free medium raised the RSR to only 3 X 10(-5); exogenous T3, GH, and DHT could not substitute for this serum factor. The kinetics of induction by dexamethasone were slow, with a lag of approximately 48 hr followed by a period of increasing RSR for 6-20 days. Removal of dexamethasone from induced cells led to an exponential decline in the RSR (t 1/2 15 hr). The concentrations of dexamethasone and insulin that could yield half maximum induction were 5 X 10(-8)M and 3 X 10(-11)M, respectively. Higher concentrations of insulin, although still in physiological range (10(-9)M), inhibited induction. At yet higher insulin levels, beyond the physiological range, alpha 2u-globulin synthesis returned to maximum values. The lack of DHT, T3, and GH requirement for alpha 2u-globulin induction in this cell line may mean that a regulatory aberrancy has occurred in this transformed cell line, or, alternatively, that these hormones act indirectly in the intact animal. This cell line should prove useful for the study of the molecular events associated with alpha 2u-globulin induction and for genetic approaches to the problem of multihormonal regulation of gene expression.     

Regulation of Exocellular Proteases in Neurospora crassa: Induction and Repression of Enzyme Synthesis

Neurospora crassa strain 74A grown on Vogel's medium containing bovine serum albumin (BSA) as principal carbon source secretes proteolytic enzymes which appear in the culture filtrate. Low concentrations of sucrose (0.1%) are necessary for growth from conidia, as conidia will not germinate on BSA alone. Once growth is initiated, however, protease production begins and at 5 to 6 hr growth and enzyme production are parallel. Higher concentrations of sucrose (0.5-2%) repress protease synthesis. Other metabolizable materials (sugars, amino acids, peptide mixtures) also repress protease synthesis. Some sugars will not sustain growth but allow germination and full induction of protease in the presence of protein. A material found in culture fluids of cells during induction of protease synthesis when added to repressed cultures causes a five-fold increase in the amount of protease production, although this is still approximately half that of normally induced cells. This material appears to be produced by induced cells in as little as 2 hr of culture, which is before detectable levels of protease can be found. It is heat-stable, of low molecular weight, and is not a simple product of protein digestion by the N. crassa proteases.     

Acetate-inducible protein overexpression from the glnAp2 promoter of Escherichia coli.

The Ntr regulon in Escherichia coli has previously been engineered to control the expression of a heterologous metabolic pathway. In this study, we reengineered the same system for protein production. In the absence of NRII (glnL gene product), we showed that glnAp2 can be an effective promoter for protein production that is inducible by exogenous acetate, but both the induction ratio and the range of modulation are low. To deal with this issue, we inactivated phosphotransacetylase (pta gene product), which disrupts the acetate pathway and denies the cell the ability to synthesize acetate. With this additional modification, gene expression from glnAp2 can be controlled by directly adding acetate into the growth medium. Using a lacZ reporter fusion, we found that glnAp2 induction was modulatable over a range of potassium acetate concentrations, and the induction/noninduction ratio increased to 77 in the absence of pta. The extracellular acetate required for maximal induction is lower than the concentration that causes toxicity, and thus growth inhibition by acetate addition was not a matter of concern. Furthermore, compared to the P(tac) promoter, overexpression of a model protein using the modified glnAp2 promoter system did not cause significant growth inhibition, although a higher level of protein expression was achieved.     

Mechanisms of induction of human tissue inhibitor of metalloproteinases-1 (TIMP-1) gene expression by all-trans retinoic acid in combination with basic fibroblast growth factor.

The addition of all-trans retinoic acid (ATRA) in combination with basic fibroblast growth factor (bFGF) to human fibroblasts results in a synergistic induction of tissue inhibitor of metalloproteinases-1 (TIMP-1) protein production. The synergistic stimulation of TIMP-1 protein by ATRA and bFGF increased across 72 h. An incubation of 10 min to 12 h with bFGF alone followed by ATRA gave a similar synergistic induction of TIMP-1 protein to that seen with both agents together. Treatment of cells with ATRA first followed by bFGF was ineffective. Expression of RARbeta mRNA was induced by ATRA alone, but not further induced by ATRA and bFGF; expression of RARgamma mRNA was induced by both ATRA or bFGF alone, and further induced by both reagents together; expression of RXRgamma was repressed by ATRA alone, but not by ATRA in combination with bFGF. Steady-state levels of TIMP-1 mRNA were induced 14 to 40-fold above control by ATRA and bFGF. Treatment with ATRA and bFGF did not alter the stability of TIMP-1 mRNA. The induction of TIMP-1 mRNA by ATRA and bFGF was greatly diminished by cycloheximide and therefore required new protein synthesis. The tyrosine kinase inhibitor genistein caused a dose-dependent inhibition of TIMP-1 protein induction by ATRA and bFGF. A MEK1 inhibitor (PD98059) inhibited both basal and induced levels of TIMP-1. At high concentrations, p38 MAP kinase inhibitors further enhanced the synergistic stimulation of TIMP-1 protein by ATRA and bFGF, but at these concentrations, p42/44 MAP kinase was strongly activated. These data begin to elucidate the mechanisms by which TIMP-1 gene expression can be upregulated.     

Investigating autocatalytic gene expression systems through mechanistic modeling.

A structured model of gene expression, which incorporates the stochastic behavior of cellular processes, was developed to examine the "all-or-none" phenomenon observed in autocatalytic systems (e.g. the lac operon). Autocatalytic expression systems typically have the genes encoding the inducer transport proteins controlled by internal inducer levels, so that transport of the inducer increases production of the transport protein. The model was able to predict the unique behaviors of autocatalytic expression systems that have been experimentally observed and provided valuable insight into the role of population heterogeneity in these systems. The simulations substantiate the importance of stochastic processes on induction of gene expression in autocatalytic systems. The simulation results show that the all-or-none phenomenon is governed largely by random cellular events, and that population-averaged variations in gene expression are due to changes in the frequency of full gene induction in individual cells rather than to uniform variations in gene expression across the entire population. In addition, the model shows how concentrations of inducer too low to induce expression in uninduced cells can maintain induction in pre-induced cultures. A comparison of induction behaviors from an autocatalytic system and a system having constitutive synthesis of the transport protein showed that transport protein levels must be decoupled from inducer control to achieve homogeneous expression of a gene of interest in all cells of a culture.     

Using a kinetic model that considers cell segregation to optimize hEGF expression in fed-batch cultures of recombinant Escherichia coli

Growth inhibition of recombinant Escherichia coli during the expression of human epidermal growth factor was observed. The recombinant cells could be segregated into three populations based on their cell division and plasmid maintenance abilities: dividing and plasmid-bearing cells, dividing and plasmid-free cells, and viable-but-non-culturable (VBNC) cells. Fed-batch fermentations were performed to investigate the effect of cell segregation on the kinetics of growth and foreign protein production. The results showed that a low concentration of inducer caused weak induction, whereas high levels cause strong induction, resulting in cells segregating into VBNC bacteria and producing a low foreign protein yield. A kinetic model for cell segregation was proposed and its predictions correlated well with experimental data for cell growth and protein expression. The optimal induction strategy could then be predicted by the model, and this prediction was then verified by experimentally deriving the conditions necessary for maximum expression of recombinant protein.     

Bcl-XL induction during terminal differentiation of friend erythroleukaemia cells correlates with delay of apoptosis and loss of proliferative capacity but not with haemoglobinization

Friend murine erythroleukaemia (F-MEL) cells are a useful model for studying the processes that regulate erythroid differentiation since exposure of these cells to chemical inducers (DMSO or HMBA) results in commitment to terminal cell division and synthesis of haemoglobin. This study examined the relationship between differentiation and apoptosis in DMSO sensitive and resistant F-MEL cells. Clear apoptosis was not observed in DMSO-treated sensitive F-MEL (strain 745A) cells during the induction of differentiation. In contrast, DMSO-induced 745A cells exhibited delayed apoptosis compared to uninduced cells. Since the Bcl-2 family members play a major role in the control of apoptosis and/or differentiation, we determined their expression before and after DMSO or HMBA treatment. Neither untreated nor chemically-induced 745A cells expressed the Bcl-2 protein. The levels of Bax and Bad proteins remained relatively constant during DMSO-induced differentiation. DMSO or HMBA treatment of 745A cells induced a marked increase of Bcl-XL expression during the late phase of differentiation which persisted even when the cells began to die. This upregulation of Bcl-XL was independent of cell density but was correlated with cell arrest in G0/G1. DMSO treatment induced a similar delay of apoptosis and enhancement of Bcl-XL expression in F-MEL (strain TFP10) cells which fail to synthesize haemoglobin in the presence of DMSO. Dexamethasone, which blocks DMSO-induced differentiation of F-MEL cells, prevented the induction of Bcl-XL. Inhibitors such as imidazole or succinylacetone, which inhibit haemoglobin synthesis but not commitment to terminal cell division, did not suppress Bcl-XL induction in DMSO-induced cells. Taken together, these results indicate that DMSO treatment of F-MEL cells induces a marked increase in Bcl-XL expression suggesting a role for this anti-apoptotic protein in the process of erythroid differentiation in F-MEL cells. Moreover, induction of Bcl-XL during this process seems to be associated with loss of proliferative capacity rather than with haemoglobin synthesis.     

Stimulation and inhibition of growth by EGF in different A431 cell clones is accompanied by the rapid induction of c-fos and c-myc proto-oncogenes

Stimulation of quiescent fibroblasts to growth by polypeptide growth factors is accompanied by the rapid induction of c-fos and c-myc proto-oncogenes. In contrast to fibroblasts, A431 cells respond to epidermal growth factor (EGF) with a decreased growth rate. Here we report that, in spite of its growth inhibitory effect, EGF rapidly induces transient expression of c-fos mRNA, followed by the synthesis of nuclear c-fos protein. In addition, EGF treatment resulted in elevated levels of c-myc expression. Practically identical results were obtained with variant A431 clones that are resistant to the inhibitory effect of EGF on cell proliferation. These observations suggest that in A431 cells c-fos and c-myc induction is a primary consequence of growth factor-receptor interaction. Indeed, efficient induction of both genes was also observed with cyanide bromide-cleaved EGF, which has previously been shown to be non-mitogenic but able to trigger early events induced by EGF. We observed strong induction of c-fos and to a lesser extent of c-myc also by TPA, and by the calcium ionophore A23187, indicating an important role for kinase C in proto-oncogene activation by growth factors.     

Growth factor-deprived BALB/c 3T3 murine fibroblasts can enter the S phase after induction of c-myc gene expression.

Induction of quiescent BALB/c 3T3 murine fibroblasts by platelet-derived growth factor (PDGF) or fibroblast growth factor (FGFs) is accompanied by induction of c-myc gene expression. To study the role of c-myc in cell growth, we transfected BALB/c 3T3 cells with a plasmid construct containing a glucocorticoid-inducible c-myc gene. When these transfected cells were growth arrested in PDGF-FGF-freedefined medium, glucocorticoid treatment induced S-phase DNA synthesis. This induction of DNA synthesis was inefficient, and cell proliferation was not evident, suggesting that growth factors act through stimulation of c-myc expression together with other intracellular events.