Transforming growth factor-beta does not alter interleukin-1 expression in cultured human macrophages

Transforming growth factor-beta (TGF beta) is a growth modulator that stimulates the growth of fibroblastic cells but inhibits the growth of cells of epithelial origin. TGF beta also influences the production of extracellular matrix proteins, and of proteases and the type 1 plasminogen activator inhibitor (PAI-1) by cultured cells. TGF beta appears also to have various immunoregulatory effects, suppressing both T- and B-cell activities. It has been proposed that it might increase the expression of interleukin-1 (IL-1) mRNA in cultured human monocytes, thus potentiating immune functions. To analyze the role of TGF beta in IL-1 production we have now quantitated the effect of this factor on the production of biologically active IL-1 as well as IL-1 beta mRNA expression. The effect of TGF beta on IL-1 production optimally activated with bacterial lipopolysaccharide (LPS) was also studied. It was found that IL-1 activity and mRNA levels were rapidly elevated by LPS but not by TGF beta. Culture fluids from monocytes treated with TGF beta alone or with TGF beta plus LPS inhibited the proliferation of the test thymocytes. After gel filtration, the media from TGF beta-treated cultures showed no activity in the molecular weight area of IL-1 (approx. 15 kD), while the supernatants from TGF beta plus LPS-induced cells contained IL-1 activity in these fractions, the magnitude of which was, however, at the same level as in the culture fluids derived from cells stimulated with LPS alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inducible overexpression of cingulin in stably transfected MDCK cells does not affect tight junction organization and gene expression

Cingulin is a component of the cytoplasmic domain of vertebrate tight junctions (TJ). Mutation or down-regulation of cingulin in cultured cells results in changes in gene expression. Some of these changes are dependent on RhoA, whose activity is regulated by GEF-H1, which is inactivated by binding to cingulin at junctions. To gain further insights on the function of cingulin through dominant-negative effects, we cloned and sequenced canine cingulin, and developed stable MDCK cell lines where either full-length cingulin, or head or rod+tail domains were inducibly overexpressed. Surprisingly, analysis of these clones by immunoblotting, microarray, immunofluorescence, measurement of transepithelial resistance, and cell density showed that the overexpression of either full-length cingulin or its domains does not significantly affect TJ protein levels, gene expression, RhoA activity, cell density, doubling time, and the organization and function of TJ. These results suggest that compensatory mechanisms prevent dominant-negative effects in this model system, and that modulation of cellular functions by cingulin occurs within physiological protein levels.     

Nerve growth factor expression response to induction agent booster dosing in transfected human embryonic kidney cells

To assess whether nerve growth factor (NGF) expression would respond to booster dosing with the inducing agent ponasterone A, human embryonic kidney cells (HEK-293) were transfected with human NGF cDNA. Cells were cultured for 5 days in media with or without ponasterone A. On day 5, controls received a ponasterone A media replacement, whereas experimental groups received ponasterone A booster media replacement. NGF protein expression bioactivity was assessed using a PC-12 cell bioassay and the concentration of secreted NGF was quantified using NGF enzyme-linked immunosorbent assay. Cells with and without ponasterone A were left for 5 days without changing the medium. On day 5, the supernatants were collected and flash-frozen for enzyme-linked immunosorbent assay. The ponasterone A-positive and -negative booster medium was replaced in the appropriate wells. Supernatants were collected from the wells at 2, 4, and 6 days after the booster dose and removal of original supernatant. The medium was flash-frozen for enzyme-linked immunosorbent assay (1.5 ml), and the remaining 500 mul was transferred to PC-12 cells seeded onto 12-well plates to determine NGF bioactivity. All experiments were performed in quadruplicate. NGF production was measured daily by enzyme-linked immunosorbent assay over a 6-day period after the ponasterone A booster to a maximal release of 1233 +/- 130 pg/ml at day 6 (11 days after original induction). Maximal NGF production per 10(3) cells was 2.5 +/- 0.61 pg at day 6. Bioactivity was determined by percentage differentiation (per 100 cells counted) at 26, 52, and 98 percent for ponasterone A-treated wells on 2, 4, and 6 days after booster dosing (7, 9, and 11 days after induction), respectively. PC-12 cell differentiation was not visualized in the ponasterone A-negative control wells. Human NGF-EcR-293 cells can inducibly secrete bioactive NGF when exposed to the induction agent ponasterone A. Furthermore, repeated bioactive NGF expression peaks beyond that previously demonstrated can be achieved using induction agent booster dosing, indicating the ability to regulate the system over an extended period.     

Transgene-induced CCWGG methylation does not alter CG methylation patterning in human kidney cells

Several reports suggest that C^mCWGG methylation tends not to co-exist with ^mCG methylation in human cells. We have asked whether or not methylation at CCWGG sites can influence CG methylation. DNA from cells expressing an M.EcoRII–GFP fusion was actively methylated at CCWGG sites. CG methylation as measured by R.HpaII/R.MspI ratios was unchanged in cells expressing the transgene. Cloned representatives of C^mCWGG methylated DNA often contained, or were adjacent to an ALU repeat, suggesting that M.EcoRII-GFP actively methylated gene-rich R-band DNA. The transgenic methyltransferase applied C^mCWGG methylation to a representative human promoter that was heavily methylated at CG dinucleotides (the SERPINB5 promoter) and to a representative promoter that was essentially unmethylated at CG dinucleotides (the APC promoter). In each case, the CG methylation pattern remained in its original state, unchanged by the presence of neighboring C^mCWGG sites. Q-PCR measurements showed that RNA expression from the APC gene was not significantly altered by the presence of C^mCWGG in its promoter. Kinetic studies suggested that an adjacent C^mCWGG methylation site influences neither the maintenance nor the de novo methylation activities of purified human Dnmt1. We conclude that C^mCWGG methylation does not exert a significant effect on CG methylation in human kidney cells.     

Modulation of glucagon receptor expression and response in transfected human embryonic kidney cells

The modulation of glucagonreceptor (GR) expression and biological response was investigated inhuman embryonic kidney cell (HEK-293) clones permanently expressing theGR with different densities. The GR mRNA expression level in theseclones was upregulated by cellular cAMP accumulation and presented agood correlation with both the protein expression level and the maximumnumber of glucagon binding sites. However, the determination ofglucagon-induced cAMP accumulation in these cell lines revealed thatthe enhancement of receptor expression did not lead to a proportionalincrease in cAMP formation. Under these conditions, the maximumcAMP production induced by NaF and forskolin was not significantlydifferent among selected clones, regardless of the receptor expressionlevel. High receptor-expressing clones showed the greatestsusceptibility for agonist-induced desensitization compared with cloneswith lower GR expression levels. The results of the present studysuggest that the GR can recruit non-GR-specific desensitizationmechanism(s). Furthermore, the partial inhibition or alteration of theoverall cAMP synthesis pathway at the receptor level may be a necessary adaptive step for a cell in response to a massive increase in membranereceptor expression level.

     

Posttranscriptional control of human gamma interferon gene expression in transfected mouse fibroblasts.

Human gamma interferon genomic DNA was introduced into NIH 3T3 fibroblasts by calcium phosphate precipitation and was not expressed in these cells at the cytoplasmic mRNA or protein level. Treatment of the transfected cells with cycloheximide (1 microgram/ml) induced the accumulation of cytoplasmic gamma interferon mRNA and biologically active human gamma interferon. Analysis of the nuclear enriched RNA from untreated cells indicated that human gamma interferon mRNA was present, suggesting that cycloheximide may act by inhibiting a specific nuclease or may enhance the processing or transport of the RNA from the nucleus to the cytoplasm.     

3-Aminobenzamide does not alter DNA repair in human fibroblasts through modulation of deoxynucleoside triphosphate pools

3-Aminobenzamide does not deplete cellular purine deoxynucleoside triphosphate pools as do the structurally-related ribonucleotide reductase inhibitors, the hydroxy- and amino-substituted benzohydroxamic acids. Thus, the previously reported ability of 3-aminobenzamide to inhibit de novo synthesis of DNA purines does not appear to be due to a direct effect on pools via inhibition of ribonucleotide reductase. The enhancement rather than inhibition by 3-aminobenzamide of DNA repair in the present studies, however, leaves open the possibility that pool modulation may play a role in cell systems where repair inhibitory effects are seen.     

Targeted Sprouty1 overexpression in cardiac myocytes does not alter myocardial remodeling or function

The mitogen activated protein kinase (MAPK) signaling pathway regulates multiple events leading to heart failure including ventricular remodeling, contractility, hypertrophy, apoptosis, and fibrosis. The regulation of conserved intrinsic inhibitors of this pathway is poorly understood. We recently identified an up-regulation of Sprouty1 (Spry1) in a targeted approach for novel inhibitors of the MAPK signaling pathway in failing human hearts following reverse remodeling. The goal of this study was to test the hypothesis that up-regulated expression of Spry1 in cardiac myocytes would be sufficient to inhibit ERK1/2 activation and tissue remodeling. We established a murine model with up-regulated Spry1 expression in cardiac myocytes using the alpha-myosin heavy chain promoter (α-MHC). Heart weight and cardiac myocyte morphology were unchanged in adult male α-MHC–Spry1 mice compared to control mice. Ventricular function of α-MHC–Spry1 mice was unaltered at 8 weeks or 1 year of age. These findings were consistent with the lack of an effect of Spry1 on ERK1/2 activity. In summary, targeted up-regulation of Spry1 in cardiac myocytes is not sufficient to alter cell or tissue remodeling consistent with the lack of an effect on ERK1/2 activity.     

Modulation of muscarinic-receptor expression in human embryonic lung fibroblasts by platelet-derived growth factor.

Platelet-derived growth factor (PDGF) is known to have regulatory control of a large number of cellular components, including various receptors. We show that muscarinic acetylcholine receptors of the m2 subtype on CCL 137 human fibroblasts in culture are affected by PDGF treatment. A time-dependent down-regulation is observed in steady-state RNA levels, followed by a decrease in ligand-binding capacity. Minimum RNA levels are attained at 11 h; minimum binding capacity is observed after 24 h of treatment. To our knowledge, this is the first example of negative gene control by PDGF.     

The effects of wild-type and mutant HFE expression upon cellular iron uptake in transfected human embryonic kidney cells

In order to measure the effects of HFE (haemochromatosis) upon iron uptake, stable expression of wild-type and C282Y, H63D and S65C mutant HFE cDNA was established in HEK 293 cells. Control cells were transfected with empty vector. Expression of HFE mRNA and protein was detected in the cell lines transfected with HFE cDNA, but not in the control cell line. The ferritin concentration in wild-type cells cultured in 40 microM ferric ammonium citrate was 69% of that in control cells and 81% of that in C282Y cells. The ferritin concentration in H63D cells was intermediate between wild-type and C282Y and the ferritin concentration in S65C cells was similar to wild-type cells. Uptake of transferrin-iron in wild-type, C282Y and control cells was measured over 45 min. The Hill coefficients for transferrin-iron uptake were similar. The V(max) for transferrin-iron uptake in wild-type cells was 59.5% of control cells and 69.5% of C282Y cells. Estimates of K(m) were 232 nM for wild-type cells, 338 nM for C282Y cells and 570 nM for controls. Transferrin receptor levels were lowered, but not significantly, in the HFE transfected cells. The results show that HFE reduces transferrin-iron uptake, probably as an uncompetitive inhibitor.