IMR-90 Lung Fibroblasts Research Articles

The Expression of Heat Shock Protein 70 Decreases with Cellular Senescencein Vitroand in Cells Derived from Young and Old Human Subjects

Because heat shock proteins have been shown to play a critical role in protecting cells from hyperthermia and other types of stresses, it was of interest to determine what effect cellular senescencein vitroand cells culturedin vitrofrom young and old human donors have on the ability of cells to regulate the expression of heat shock protein 70 (hsp70), the most prominent and most evolutionary conserved of the heat shock proteins. The ability of early and late passage IMR-90 lung fibroblasts and epidermal melanocytes and skin fibroblasts obtained from young and old human donors to express hsp70 was determined after a brief heat shock. We found that the levels of hsp70 protein and mRNA were lower in late passage cells and cells from old donors than in early passage cells and cells from young donors. The binding activity of the heat shock transcription factor HSF1, as measured by a gel shift assay, was significantly higher in early passage cells and cells from young donors in comparison to late passage cells and cells from old donors. In addition, the levels of HSF1 decreased significantly in late passage cells and cells from old donors in comparison to early passage cells and cells from young donors. Thus, our study demonstrates that the induction of hsp70 by hyperthermia in fibroblasts is significantly lower in late passage fibroblasts and in fibroblasts from old donors. In addition, our study shows that the decline in hsp70 expression during cellular senescencein vitroand in cells derived from old human subjects is paralleled by a decrease in the levels of HSF1.

Pharmacological characterization of a nonpeptide bradykinin B2 receptor antagonist, FR165649, and agonist, FR190997.

1. The nonpeptide bradykinin (BK) B2 receptor antagonist, FR165649 (8-[2,6-dichloro-3-[N-[(E)-4-(N-methylcarbamoyl)cinnamidoacetyl ]-N-methylamino]benzyloxy]-2-methylquinoline), and agonist, FR190997 (8-[2,6-dichloro-3-[N-[(E)-4-(N-methylcarbamoyl) cinnamidoacetyl]-N-methylamino]benzyloxy]-2-methyl-4-(2-pyridyl methoxy)quinoline) have been identified. These compounds have a common chemical structure, and the 2-pyridylmethoxy group is the only structural difference between them. 2. Both FR165649 and FR190997 displaced [3H]-BK binding to B2 receptors in guinea-pig ileum membranes, with an IC50 of 4.7 x 10(-10) M and 1.5 x 10(-9) M, respectively. They also displaced [3H]-BK binding to B2 receptors in human lung fibroblast IMR-90 cells, with an IC50 of 1.6 x 10(-9) M and 9.8 x 10(-10) M, respectively. 3. In guinea-pig isolated ileum-preparations, FR165649 had no agonistic effect on contraction and caused parallel rightward shifts of the concentration-response curves to BK on contraction. Analysis of the data produced a nominal pA2 value of 9.2+/-0.1 (n=5) and a slope of 1.4+/-0.1 (n=5). On the other hand, FR190997 induced concentration-dependent contraction of guinea-pig ilea with a pD2 of 7.9+/-0.2 and the contraction was inhibited by a specific peptide bradykinin B2 receptor antagonist, Hoe 140 (D-Arg-[Hyp3, Thi5, D-Tic7, Oic8]BK) in a non-competitive manner. 4. In IMR-90 cells, FR165649 had no agonistic effect on phosphatidyl inositol (PI) hydrolysis and caused parallel rightward shifts (approximately 200 fold shift at 10(-7) M) of the concentration-response curves to BK on PI hydrolysis. FR190997 induced concentration-dependent PI hydrolysis in IMR-90 cells with a pD2 of 8.4+/-0.1, and this effect was inhibited by Hoe 140. 5. These results indicate that FR165649 and FR190997 are, respectively, a potent bradykinin B2 receptor antagonist and agonist, and that the agonistic activity depends on the small part of the nonpeptide ligand. FR165649 and FR190997 may be useful tools for studying the relationship between ligands and receptors.

Molecular analysis of H2O2-induced senescent-like growth arrest in normal human fibroblasts: p53 and Rb control G1 arrest but not cell replication.

Human diploid fibroblasts lose the capacity to proliferate and enter a state termed replicative senescence after a finite number of cell divisions in culture. When treated with sub-lethal concentrations of H2O2, pre-senescent human fibroblasts enter long-term growth arrest resembling replicative senescence. To understand the molecular basis for the H2O2-induced growth arrest, we determined the cell cycle distribution, levels of p53 tumour suppressor and p21 cyclin-dependent kinase inhibitor proteins, and the status of Rb phosphorylation in H2O2-treated cells. A 2-h pulse of H2O2 arrested the growth of IMR-90 fetal lung fibroblasts for at least 15 days. The arrested cells showed a G1 DNA content. The level of p53 protein increased 2- to 3-fold within 1.5 h after H2O2 exposure but returned to the control level by 48 h. The induction of p53 protein was dose dependent, beginning at 50-75 microM and reaching a maximum at 100-250 microM. The induction of p53 did not appear to correlate with the level of DNA damage as measured by the formation of 8-oxo-2'-deoxyguanosine in DNA. The level of p21 protein increased about 18 h after H2O2 exposure and remained elevated for at least 21 days. During this period, Rb remained underphosphorylated. The induction of p53 by H2O2 was abolished by the iron chelator deferoxamine and the protein synthesis inhibitor cycloheximide. The human papillomavirus protein E6, when introduced into the cells, abolished the induction of p53, reduced the induction of p21 to a minimal level and allowed Rb phosphorylation and entry of the cells into S-phase. The human papillomavirus protein E7 reduced the overall level of Rb and also abolished H2O2-induced G1 arrest. Inactivating G1 arrest by E6, E7 or both did not restore the replicative ability of H2O2-treated cells. Thus H2O2-treated cells show a transient elevation of p53, high level of p21, lack of Rb phosphorylation, G1 arrest and inability to replicate when G1 arrest is inactivated.

Transcription Factor Nuclear Factor κB Regulates the Inducible Expression of the Human B1 Receptor Gene in Inflammation

Expression of the bradykinin B1 receptor gene is up-regulated in vascular smooth muscle cells (VSMCs) in response to a variety of inflammatory stimuli. We isolated the 5'-flanking region of the human bradykinin B1 receptor gene and examined its promoter activity by transient transfection analysis. This region (-2582 to +34) showed promoter activity inducible by lipopolysaccharide (LPS), tumor necrosis factor alpha (TNF-alpha), and interleukin-1beta (IL-1beta) in VSMCs. Further deletion analysis revealed that constructs containing 111 base pairs of 5'-flanking sequence were sufficient for transcriptional induction. Mutagenesis of a nuclear factor kappaB (NF-kappaB)-like site at -64 to -55 abolished most of the LPS, TNF-alpha, and IL-1beta inducibility, whereas a mutation of a cyclic AMP response element at -50 to -43 markedly reduced the basal promoter activity, and a mutation of the activator protein 1 (AP-1) site at -78 to -72 had minimal effects. Nuclear extracts from LPS, TNF-alpha, and IL-1beta-treated VSMCs, IL-1beta-treated human hepatoma HepG2, and human lung fibroblast IMR-90 cells showed strong inducible binding activity to the NF-kappaB-like site by gel shift assays. These results demonstrated that NF-kappaB-like nuclear factor was involved in the inducible expression of the human bradykinin B1 receptor gene during inflammatory processes.

P27Kip1 overexpression causes apoptotic death of mammalian cells.

p27KiP1, a member of the Cip/Kip family of cyclin-dependent kinase (cdk) inhibitors, has been implicated in mediating G1 arrest in response to a variety of growth inhibitory signals. Its importance in regulating cell growth is emphasized by the fact that mice lacking p27Kip1 are abnormally large and display hyperplasia of multiple tissues. However, these mice retain the ability to undergo G1 arrest in response to growth inhibitory signals, suggesting that p27KiP1 may serve other functions important for controlling tissue growth. In the present study, we utilized an adenoviral vector-based expression system to examine the consequences of p27Kip1 overexpression in the human carcinoma cell lines A549, HeLa and RKO, in human melanoma SK-MEL-110 cells, in human lung fibroblasts IMR90 and in the rat fibroblast line Rat1. We demonstrate that overexpression of p27Kip1 leads to apoptotic cell death in all cell types, and further show that ectopic expression of Bcl-2 can protect HeLa cells from apoptosis mediated by p27Kip1 overexpression. To our knowledge, this is the first study demonstrating that p27Kip1 can induce apoptosis. Our findings provide new insight into the possible functions of this growth regulatory protein, and support the potential utility of gene therapeutic approaches aimed at elevating p27Kip1 expression for treatment of human cancers.

Vitamin D receptor in IMR-90 human fibroblasts and antiproliferative effect of 1,25-dihydroxyvitamin D3.

This study examines the effect of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], serum or forskolin on the proliferation of IMR-90 fetal lung fibroblasts and demonstrates, for the first time, the presence of 1,25(OH)2D3 receptor (VDR) in this cell line. In quiescent, subconfluent cultures neither the treatment with 100 nM 1,25(OH)2D3 nor that with 50 microM forskolin influenced proliferation, while a significant increase was observed after incubation of the cells with 10% fetal bovine serum. Either cell number, determined on growing IMR-90 human fibroblasts after 48 or 72 h incubation with 100 nM 1,25(OH)2D3 or [3H]thymidine incorporation (24, 48 or 72 h incubation) significantly decreased, while protein content per cell increased. Northern blot analysis revealed the expression of the VDR gene, the VDR mRNA bands being prominent in 1,25(OH)2D3, serum or forskolin treated fibroblasts. VDR mRNA levels slightly decreased, when growing fibroblasts were exposed to 100 nM 1,25(OH)2D3 for 48 or 72 h.

Pharmacological characterization of a novel, orally active, nonpeptide bradykinin B 2 receptor antagonist, FR167344

To investigate the pathophysiological role of bradykinin and to develop a drug for inflammatory diseases, we discovered an orally active, nonpeptide bradykinin B 2 receptor antagonist, FR167344, N-[ N-[3-[(3-bromo-2-methylimidazo[1,2-a]pyridin-8-yl)oxymethyl]-2,4-dichlorophenyl]- N-methylaminocarbonylmethyl]-4-(dimethylaminocarbonyl) cinnamylamide hydrochloride. This compound competitively displaced [ 3 H ]bradykinin binding to bradykinin B 2 receptors present in guinea pig ileum membrane with an IC 50 value of 6.6×10 −10 M. In isolated guinea pig ileum preparations, it also antagonized bradykinin-induced contraction with a pA 2 value of 9.3. In human lung fibroblast IMR-90 cells, FR167344 displaced [ 3 H ]bradykinin binding to human bradykinin B 2 receptors with an IC 50 value of 1.3×10 −8 M, but not [ 3 H ]des-Arg 10–kallidin binding to human bradykinin B 1 receptors. In vivo, oral administration of FR167344 inhibited bradykinin-induced bronchoconstriction in guinea pigs and the bradykinin-induced hypotensive response for 6 h in rats. These results show that FR167344 is a potent, selective, orally active and long acting bradykinin B 2 receptor antagonist.

Modulation of tissue plasminogen activator biosynthesis by phosphatidylinositol liposomes in human fetal lung fibroblasts

Phosphatidylinositol (PI) liposomes at 40 microM increased tissue plasminogen activator (t-PA) biosynthesis by human fetal lung fibroblasts IMR-90 (FLF), after 5 days of incubation by 7.4 +/- 1.4 times of the control level. Other phospholipid liposomes, such as phosphatidylserine (PS), phosphatidylcholine (PC), and phosphatidylglycerol (PG), had no effect on t-PA biosynthesis by FLF. The induction of t-PA biosynthesis by PI liposomes was inhibited by specific inhibitors of phosphoinositide pathway: gentamycin and lithium chloride. Thus, gentamycin inhibited the effect of PI liposomes on t-PA biosynthesis by 76% (P less than 0.001), while it had no effect on control FLF. Likewise, lithium chloride inhibited t-PA biosynthesis of both PI-treated and control FLF by greater than 84%. The induction of t-PA biosynthesis by PI liposomes was dependent on RNA transcription and independent of DNA biosynthesis.

Sulfate transport in human lung fibroblasts (IMR-90).

Sulfate transport in a fibroblast cell line derived from human lung (IMR-90) occurred mainly via high- and low-affinity, SITS-sensitive pathways and to a lesser extent by an SITS-insensitive mechanism. In low-ionic-strength media (sucrose substituted for salts) the apparent Km of the carrier-mediated sulfate influx was 1 mM. At 0.3 mM, the sulfate concentration normally found in human serum, the contribution of the SITS-insensitive pathway was negligible. In physiological salts solution, an SITS-sensitive, high-affinity (Km 34 +/- 14 microM) sulfate influx system was observed at extracellular sulfate concentrations less than 100 microM. Between 100 and 500 microM sulfate, the range normally found in human serum, sulfate influx occurred via an SITS-sensitive, low-affinity pathway and to a small extent by an SITS-insensitive mechanism. Extracellular chloride inhibited the influx and stimulated the efflux of sulfate. Bicarbonate and thiosulfate inhibited sulfate influx but had no effect on sulfate efflux. Phosphate, arsenate, or Na+ did not affect sulfate uptake. These results indicate that in human lung fibroblast IMR-90 cells sulfate is transported mainly via an SO4(2-)/Cl- exchange system independent of the phosphate or Na+ transport. Since sulfate concentration as high as 50 mM only slightly increased sulfate efflux, SO4(2-)/SO4(2-) exchange is probably a minor component of sulfate uptake.