Neonatal Rat Lung Fibroblasts Research Articles

A novel device to stretch multiple tissue samples with variable patterns: Application for mRNA regulation in tissue-engineered constructs

A broad range of cells are subjected to irregular time varying mechanical stimuli within the body, particularly in the respiratory and circulatory systems. Mechanical stretch is an important factor in determining cell function; however, the effects of variable stretch remain unexplored. In order to investigate the effects of variable stretch, we designed, built and tested a uniaxial stretching device that can stretch three-dimensional tissue constructs while varying the strain amplitude from cycle to cycle. The device is the first to apply variable stretching signals to cells in tissues or three dimensional tissue constructs. Following device validation, we applied 20% uniaxial strain to Gelfoam samples seeded with neonatal rat lung fibroblasts with different levels of variability (0%, 25%, 50% and 75%). RT-PCR was then performed to measure the effects of variable stretch on key molecules involved in cell-matrix interactions including: collagen 1α, lysyl oxidase, α-actin, β1 integrin, β3 integrin, syndecan-4, and vascular endothelial growth factor-A. Adding variability to the stretching signal upregulated, downregulated or had no effect on mRNA production depending on the molecule and the amount of variability. In particular, syndecan-4 showed a statistically significant peak at 25% variability, suggesting that an optimal variability of strain may exist for production of this molecule. We conclude that cycle-by-cycle variability in strain influences the expression of molecules related to cell-matrix interactions and hence may be used to selectively tune the composition of tissue constructs.

Retinoic Acid Inducibility of the Human PDGF-A Gene Is Mediated by 5′-Distal DNA Motifs That Overlap With Basal Enhancer and Vitamin D Response Elements

Retinoic acid (RA) upregulates expression of PDGF ligands and receptors in neonatal rat lung fibroblasts, a process likely to promote maturation of the lung alveolus and possibly microstructures of other organs. A mutational analysis of the gene encoding the PDGF-A ligand has identified a complex retinoic acid response element (RARE) located far upstream of the transcription start site, in a 5'-distal enhanceosome region previously shown to harbor basal and vitamin D-inducible enhancer activity. Maximal RA responsiveness (fourfold) was conferred by nucleotide sequence located between -7064 and -6787, with a variety of deletion and point mutations revealing the importance of at least three nuclear receptor half-sites within the enhancer region (-6851 to -6824), as well as nucleotides located further upstream. Recombinant human retinoic acid receptor/retinoid-X receptor heterodimers bound with high affinity and sequence specificity to multiple regions within the RARE, as demonstrated by electrophoretic mobility shift and DNase I footprinting assays. The addition of RARE activity to previously described functions of the 5'-distal enhanceosome underscores the importance of this region as a key integration point for regulatory control of PDGF-A expression.

Neutrophil elastase-initiated EGFR/MEK/ERK signaling counteracts stabilizing effect of autocrine TGF-β on tropoelastin mRNA in lung fibroblasts

Neutrophil elastase (NE) plays an important role in emphysema, a pulmonary disease associated with excessive elastolysis and ineffective repair of interstitial elastin. Besides its direct elastolytic activity, NE releases soluble epidermal growth factor receptor (EGFR) ligands and initiates EGFR/MEK/ERK signaling to downregulate tropoelastin mRNA in neonatal rat lung fibroblasts (DiCamillo SJ, Carreras I, Panchenko MV, Stone PJ, Nugent MA, Foster JA, and Panchenko MP. J Biol Chem 277: 18938-18946, 2002). We now report that NE downregulates tropoelastin mRNA in the rat fetal lung fibroblast line RFL-6. The tropoelastin mRNA downregulation is preceded by release of EGF-like and TGF-alpha-like polypeptides and requires EGFR/MEK/ERK signaling, because it is prevented by the EGFR inhibitor AG1478 and the MEK/ERK uncoupler U0126. Tropoelastin expression in RFL-6 fibroblasts is governed by autocrine TGF-beta signaling, because TGF-beta type I receptor kinase inhibitor or TGF-beta neutralizing antibody dramatically decreases tropoelastin mRNA and protein levels. Half-life of tropoelastin mRNA in RFL-6 cells is >24 h, but it is decreased to approximately 8 h by addition of TGF-beta neutralizing antibody, EGF, TGF-alpha, or NE. Tropoelastin mRNA destabilization by NE, EGF, or TGF-alpha is abolished by AG1478 or U0126. EGF-dependent tropoelastin mRNA downregulation is reversed upon ligand withdrawal, whereas chronic EGF treatment leads to persistent downregulation of tropoelastin mRNA and protein levels and decreases insoluble elastin deposition. We conclude that NE-initiated EGFR/MEK/ERK signaling cascade overrides the autocrine TGF-beta signaling on tropoelastin mRNA stability and, therefore, decreases the elastogenic response in RFL-6 fibroblasts. We hypothesize that persistent EGFR/MEK/ERK signaling could impede the TGF-beta-induced elastogenesis/elastin repair in the chronically inflamed, elastase/anti-elastase imbalanced lung in emphysema.

Regulation of elastin gene transcription by interleukin-1 beta-induced C/EBP beta isoforms.

We previously showed that interleukin (IL)-1beta decreases elastin gene transcription through activation of the NF-kappaB subunit p65 in neonatal rat lung fibroblasts. The present study was undertaken to further explore the molecular mechanisms responsible for the inhibitory effect of IL-1beta on elastin gene transcription. We found that cycloheximide blocked IL-1beta-induced downregulation of elastin mRNA but did not inhibit IL-1beta-induced translocation of p65 into the nucleus. IL-1beta treatment increased CCAAT/enhancer-binding protein (C/EBP)beta mRNA and protein levels including liver-enriched activating protein (LAP) and liver-enriched inhibitory protein (LIP), which was cycloheximide sensitive. C/EBPbeta isoforms bound a GCAAT-containing sequence in the proximal elastin promoter as determined by electrophoretic gel shift studies and confirmed by using specific anti-C/EBPbeta antibodies and by competition studies with oligonucleotides. Transient transfection of LIP expression vectors strongly decreased the transcriptional activity of the cotransfected elastin promoter and decreased levels of endogenous elastin mRNA. We demonstrated that IL-1beta-induced downregulation of elastin mRNA is dependent on NF-kappaB activation and C/EBPbeta expression. These results indicate that IL-1beta treatment activates NF-kappaB, which subsequently induces LIP expression and inhibition of elastin gene transcription.

NF-kappaB induced by IL-1beta inhibits elastin transcription and myofibroblast phenotype.

Interleukin (IL)-1beta released after lung injury regulates the production of extracellular matrix components. We found that IL-1beta treatment reduced the rate of elastin gene transcription by 74% in neonatal rat lung fibroblasts. Deletion analysis of the rat elastin promoter detected a cis-acting element located at -118 to -102 bp that strongly bound Sp1 and Sp3 but not nuclear factor (NF)-kappaB. This element mediated IL-1beta-induced inhibition of the elastin promoter. IL-1beta treatment did not affect the level of Sp1 but did induce translocation of the p65 subunit of NF-kappaB. Overexpression of p65 decreased elastin promoter activity and markedly reduced elastin mRNA. Immunoprecipitation studies indicated an interaction between the p65 subunit and Sp1 protein. Microarray analysis of mRNA isolated after overexpression of p65 or treatment with IL-1beta revealed downregulation of alpha-smooth muscle actin and calponin mRNAs. Expression of these genes is associated with the myofibroblast phenotype. These results indicate that IL-1beta activates the nuclear localization of NF-kappaB that subsequently interacts with Sp1 to downregulate elastin transcription and expression of the myofibroblast phenotype.

Peroxisome proliferators alter lipid acquisition and elastin gene expression in neonatal rat lung fibroblasts.

During the alveolar stage of lung development, lipid droplet-laden interstitial cells are present at the base of elongating alveolar septa. These cells that have been named lipid interstitial cells or lipofibroblasts (LFs) may supply lipids for surfactant production, the synthesis of membrane phospholipids, and/or energy metabolism. They also have myofibroblastic characteristics and participate in the generation of the interstitial elastic fiber network, that is, in the pulmonary alveolar septum. To understand how this cell regulates its lipid-storing and elastin-producing properties, we have examined the effects of peroxisome proliferators on the expression of the genes that are associated with an elastin-producing myofibroblastic phenotype or an adipocyte-like phenotype. Two known ligands for peroxisome proliferator-activated receptors, 5,8,11,14-eicosatetraynoic acid (ETYA) and 15-deoxy-delta-12,14-prostaglandin J2 (15-dPGJ2), decrease elastin gene transcription and the steady-state levels of tropoelastin (TE) and alpha-smooth muscle actin mRNAs in cultured LFs. Concurrently, cultured LFs increase the expression of adipocyte lipid binding protein, which is regarded as an adipocyte-specific protein, and accumulate lipid droplets. Their abilities to store lipids and express desmin intermediate filaments, alpha-smooth muscle actin, and smooth muscle myosin heavy chain in contractile filaments in vitro illustrate similarities among the pulmonary LF, the hepatic lipocyte, and the contractile interstitial cell, which contribute to the repair reaction in the lung after pulmonary injury.

Serine proteinase inhibitors influence the stability of tropoelastin mRNA in neonatal rat lung fibroblast cultures

Elastin, an elastic extracellular structural protein, is a polymer comprised of soluble tropoelastin (TE) monomers that are joined by covalent cross-links and become insoluble. In cultured vascular smooth muscle cells, the steady-state level of TE mRNA is influenced by soluble elastin moieties in the culture medium, either TE or its fragmentation products. We have hypothesized that an enzyme-mediated proteolytic event may modulate the quantities of TE and its fragmentation products in the culture medium of mesenchymal cells, and thereby indirectly regulate the steady-state level of TE mRNA. Neonatal rat lung fibroblasts were cultured in the presence or absence of the serine proteinase inhibitor, aprotinin, and the quantities of soluble elastin and TE mRNA were analyzed. Exposures to aprotinin lasting up to 12 h increased the soluble elastin content of the culture medium. The increase in the soluble elastin content did not reflect an increase in TE mRNA, which diminished after exposures for 12 h or longer. The decrease in TE mRNA resulted from a decrease in its half-life, rather than a decrease in the rate of TE gene transcription. Aprotinin did not reduce TE mRNA in plasminogen-depleted cultures, but the effect of aprotinin was evident when purified plasminogen was added back to the cultures. Therefore, a serine proteinase, possibly plasmin, may participate in a feedback mechanism and modulate the quantity of TE in lung fibroblast cultures. This mechanism may help ensure that intracellular TE synthesis occurs in tandem with extracellular elastin deposition and cross-linking.

Regulation of lysyl oxidase expression in lung fibroblasts by transforming growth factor-beta 1 and prostaglandin E2.

The regulation of lysyl oxidase produced by cultured, lipid-enriched, neonatal rat lung fibroblasts was explored. The presence of 40 pM of transforming growth factor-beta 1 (TGF-beta 1) in overnight cultures increased levels of enzyme secreted into the medium by 1.6-fold while steady-state levels of lysyl oxidase mRNA increased similarly. In contrast, incubation of these cultures with 100 nM of prostaglandin E2 (PGE2) reduced enzyme activity levels by 40 to 50% although steady-state mRNA was not changed. Consistent with the effect of PGE2, the presence of indomethacin stimulated levels of secreted enzyme activity. When present in cultures simultaneously with TGF-beta 1, PGE2 prevented the stimulation beyond control levels seen with TGF-beta 1 alone. Densitometry of protein bands immunoprecipitated by antibody to lysyl oxidase indicated that the degree of conversion of the 50 kD proenzyme to the 29 kD enzyme was not significantly altered by TGF-beta 1 or PGE2. However, the net accumulation of all forms of lysyl oxidase protein was increased by TGF-beta 1 and decreased by PGE2. These results indicate that TGF-beta 1 and specific prostaglandin(s) exert opposing effects on the expression of lysyl oxidase in these lung fibroblasts.

Basic fibroblast growth factor decreases elastin production by neonatal rat lung fibroblasts.

During pulmonary development, there is a burst in elastin synthesis by interstitial fibroblasts coincident with the period of alveolar septal elongation. Little is known about the regulation of elastin synthesis by these cells, although several endocrine and paracrine factors influence lung fibroblast elastin production. Because alveolar septal elongation is accompanied by a decrease in capillary endothelial cell mitosis, we have hypothesized that a reduction in basic fibroblast growth factor (bFGF), an endothelial cell mitogen, may occur concomitantly with an increase in elastin synthesis. This temporal relationship suggests that bFGF may influence elastin production by interstitial fibroblasts. Therefore, we have examined the effects of bFGF on elastin production by postconfluent, serum-free cultures of lipid interstitial fibroblasts (LF). Elastin production was quantitated by analyzing the incorporation of 3H-valine into the soluble elastin precursor tropoelastin (TE). Exogenous bFGF decreased the quantity of newly synthesized TE in the culture media and cell layers of LF. The level of newly synthesized TE in the media was decreased to 36% and 48% of the unexposed control when LF were exposed for 48 h to 10 or 75 ng/ml bFGF, respectively. Northern analysis demonstrated that the decrease in TE was accompanied by a similar decrease in elastin mRNA. Transient transfection experiments using an elastin promoter/CAT gene construct demonstrated that bFGF exposure decreased elastin promoter activity. These results suggest that bFGF decreases elastin transcription. Exposure to an anti-bFGF antibody neutralized endogenous bFGF and increased soluble elastin production by LF. Our studies indicate that exogenous and endogenous bFGF can suppress elastin production by LF. A similar effect may occur in the intact lung during development or chronic inflammation.

Retinoic acid increases elastin in neonatal rat lung fibroblast cultures.

The factors that regulate elastin synthesis during pulmonary alveolar septal formation have not been identified. Because maximal alveolar elastin synthesis occurs over a relatively brief period (postnatal days 4-14 in the rat), we hypothesized that changes in the local concentrations of factors that regulate elastin synthesis may precede or accompany this period. Because pulmonary retinoid stores decline just before the fourth postnatal day, we also hypothesized that this decline could be accompanied by the utilization of retinoic acid, one of the most biologically active retinoids, in a regulatory process that increases elastin synthesis. If these hypotheses are correct, then retinoic acid should increase elastin synthesis by pulmonary cells. Therefore, cultures of neonatal rat lung fibroblasts were exposed to retinoic acid, and elastin production was quantitated. Retinoic acid produced a two- to threefold increase in the steady-state level of elastin mRNA, in soluble elastin, and in insoluble elastin. The transcriptional initiation rate of the elastin gene was 1.8-fold higher in nuclei that were isolated from retinoic acid-treated cells than in nuclei that were isolated from control cells. This indicates that the increase in steady-state elastin mRNA results, at least partially, from an increase in elastin transcription. Lung fibroblasts that were isolated from 8-day-old rats, but not cultured, contained retinoic acid. These findings suggest that retinoic acid is a potential regulator of elastin synthesis in developing pulmonary alveoli.

Effects of Heparin and Other Glycosaminoglycans on Elastin Production by Cultured Neonatal Rat Lung Fibroblasts

Proteoglycans are important structural elements of the extracellular matrix, and may contribute to the dynamic architecture of the lung and also influence pulmonary gas and solute exchange. The potential for proteoglycans and glycosaminoglycans to modulate the synthesis and deposition of elastin, another important extracellular matrix component of the lung, has not been established. Therefore the effects of glycosaminoglycans on the steady-state level of elastin mRNA, the incorporation of [ 3H]valine into tropoelastin, the distribution of soluble elastin in the medium and cell layer, and insoluble elastin deposition have been examined using cultured neonatal rat lung fibroblasts. Heparin decreases the soluble elastin content of the culture medium while increasing the soluble elastin content of the cell layer. This altered partitioning of soluble elastin is associated with an increase in steady-state elastin mRNA and an increase in the deposition of insoluble elastin in the extracellular matrix. Some of these effects may result from the binding of heparin to soluble elastin at physiological concentrations of NaCl. The galactosamine-containing glycosaminoglycans, chondroitin sulfate and dermatan sulfate, differ from heparin in that they increase the quantity of soluble elastin in the culture medium and decrease the deposition of insoluble elastin in the extracellular matrix. Proteoglycans, which are present in most elastic tissues, may participate in the regulation of elastin synthesis and deposition during periods of new elastin formation.

Influences of endogenous and exogenous TGF-beta on elastin in rat lung fibroblasts and aortic smooth muscle cells.

The factors that regulate elastin production during neonatal lung development have not been elucidated. Previous investigations suggested that transforming growth factor-beta (TGF-beta) increases elastin production by neonatal rat lung fibroblasts (LF). We examined whether this effect of TGF-beta was unique to these cells or was evident in other neonatal cells, which constitutively produce elastin, or in cells from adults, whose constitutive elastin production is low. We have quantitated soluble elastin, elastin mRNA, and TGF-beta production in primary cultures of smooth muscle cells (SMC) and LF from neonatal and adult rats and have examined the alterations in soluble elastin and elastin mRNA that result from adding 100 pM exogenous TGF-beta 1 to these cultures. Unsupplemented cultures of LF and SMC obtained from neonatal rats exhibited higher steady-state levels of elastin mRNA and contained more soluble elastin in their culture medium than did cells from adult animals. When neonatal LF were supplemented with 100 pM TGF-beta 1, they showed a significant increase in the soluble elastin content of their culture medium and their steady-state elastin mRNA. Neither LF obtained from adults nor SMC obtained from neonatal or adult rats significantly increased their soluble elastin or steady-state elastin mRNA after the addition of exogenous TGF-beta. When neonatal LF were supplemented with an anti-TGF-beta neutralizing antibody, the soluble elastin content of the culture medium decreased significantly. These data suggest that the responsiveness of elastin expression to TGF-beta is limited to neonatal LF and that endogenous TGF-beta influences elastin production by neonatal LF.

Transforming Growth Factor-β Increases Elastin Production by Neonatal Rat Lung Fibroblasts

While elastin degradation is a hallmark of pulmonary emphysema, it is likely that elastin synthesis also occurs. However, the supramolecular structure and function of the newly synthesized elastin are abnormal. Very little is known about the regulation of elastin synthesis during the development of emphysema when prominent collections of mononuclear phagocytes are found in and near the alveolar interstitium. Transforming growth factor-beta (TGF-beta) is an important regulator of collagen and fibronectin production in wound healing, which is also accompanied by an influx of mononuclear phagocytes. We hypothesized that TGF-beta may influence elastin production by fibroblasts in the pulmonary interstitium. Therefore, we examined the influence of TGF-beta on the production of elastin by postconfluent cultures of neonatal rat lung fibroblasts. Elastin production was quantitated by analyzing the incorporation of [3H]valine into the soluble elastin precursor tropoelastin (TE). The incorporation of [3H]valine into TE was approximately 2-fold greater in the presence of 40 or 100 pM TGF-beta than in its absence. The intracellular, free [3H]valine pool was increased by 18% in the presence of TGF-beta. Therefore, TGF-beta-related differences in the precursor pool size were not solely responsible for the observed increase in [3H]valine incorporation. Northern analysis demonstrated that the increase in TE was accompanied by a smaller but significant increase in the steady-state level of elastin mRNA. Thus, the observed increase in TE production can be at least partially attributed to a pretranslational effect of TGF-beta.

Mechanisms of Extracellular Matrix Proteoglycan Degradation by Human Neutrophils

Alterations in proteoglycans (PG) located in the pulmonary interstitium may influence extracellular matrix (ECM) structure and assembly during the development of diseases in which increased numbers of neutrophils enter the lung. To evaluate potential mechanisms of PG degradation, neutrophils or purified neutrophil products were incubated with ECM that had been produced by cultured neonatal rat vascular smooth muscle cells (SMC) or lung fibroblasts (LF) and metabolically labeled with 35SO4. Matrix PG solubilization was expressed as a percentage of the spontaneous [35SO4]PG solubilization that occurred in the presence of buffer alone. Solubilization by unstimulated neutrophils was 105.8 +/- 3.1% (mean +/- SEM, n = 6) and 101.7 +/- 3.05 (n = 8) using ECM that had been produced by LF and SMC, respectively. Solubilization by neutrophils that had been stimulated with formyl-methionine-leucine-phenylalanine (FMLP) in the presence of cytochalasin B (CB) was 189.7 +/- 5.8% and 298.2 +/- 26.2% using ECM produced by LF and SMC, respectively. Matrix that had been produced by SMC was used to evaluate which neutrophil products were responsible for the degradation of PG. Addition of a specific inhibitor of neutrophil elastase (NE) to stimulated polymorphonuclear leukocytes (PMN) reduced PG solubilization by 88.3 +/- 4.8% (n = 8). Addition of an inhibitor of cathepsin G (CG), as well, did not further reduce PG degradation. Purified CG and myeloperoxidase solubilized significantly more PG, 125.8 +/- 6.2% (n = 9) and 143.2 +/- 8.1% (n = 6), respectively (P less than 0.01), than was solubilized spontaneously.(ABSTRACT TRUNCATED AT 250 WORDS)

An ozone-generating system and chamber for testing injury to cultured cells.

A system was constructed which alternately exposed cultured cells to specific concentrations of ozone in the gas phase and then to cell culture medium. It was designed to produce a constant mass transfer between the gas phase and the exposed cell phase. Monolayers of neonatal rat lung fibroblasts cultured in miniature glass dishes were used to test for ozone toxicity. The cells were alternately exposed to ozone (0.05, 0.15, 0.45, 1.35, and 4.05 ppm) for 30 sec and then to culture medium for 30 sec over a 1-hr period. Toxicity was measured as inhibition of cell growth 4 days after exposure to ozone. Growth was significantly inhibited at all concentrations with a 50% inhibitory concentration of 0.8 ppm. The system described was effective for quantifying ozone toxicity for cultured lung cells.