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1.
Biochim Biophys Acta ; 1538(1): 47-58, 2001 Feb 05.
Article in English | MEDLINE | ID: mdl-11341982

ABSTRACT

Oxygen (O(2)) species are involved in a large variety of pulmonary diseases. Among the various cell types that compose the lung, the epithelial cells of the alveolar structure appear to be a major target for oxidant injury. Despite their importance in the repair processes, the mechanisms which regulate the replication of the stem cells of the alveolar epithelium, the type 2 cells, remain poorly understood. Based on the results of several studies which have documented the involvement of the insulin-like growth factor (IGF) system in lung epithelial cell replication, and which have also suggested a role for IGF binding proteins (IGFBPs) in the control of cell proliferation, the aim of the present work was to determine whether IGFBPs could be involved in the modulation of growth of human lung epithelial cells exposed to oxidants. Experiments were performed using a human lung adenocarcinoma cell line (A549) which was exposed for various durations to hyperoxia (95% O(2)). We observed a rapid and reversible growth arrest of the cells after only 24 h of O(2) exposure. When oxidant injury was prolonged, growth arrest was followed by induction of apoptosis with activation of the Fas pathway. These effects were associated with an increased expression of IGFBP-2 and IGFBP-3. In addition, study of localization of these proteins revealed distinct patterns of distribution. IGFBP-3 was mainly present in the extracellular compartment. In comparison, the fraction of IGFBP-2 secreted was less abundant whereas the IGFBP-2 fraction in the intracellular compartment appeared stronger. In addition, analysis of the subcellular localization provided data indicating the presence of IGFBP-2 in the nucleus. Taken together these data support a role for IGFBP-2 and IGFBP-3 in the processes of growth arrest and apoptosis in lung epithelial cells upon oxidant exposure. They also suggest that distinct mechanisms may link IGFBP-2 and IGFBP-3 to the key regulators of the cell cycle.


Subject(s)
Epithelial Cells/drug effects , Insulin-Like Growth Factor Binding Protein 2/metabolism , Insulin-Like Growth Factor Binding Protein 3/metabolism , Lung/drug effects , Oxidants/pharmacology , Acridine Orange , Apoptosis , Blotting, Western , Cell Division/drug effects , Cell Nucleus/metabolism , Epithelial Cells/metabolism , Fluorescent Antibody Technique , Formaldehyde , Humans , Hyperoxia , Insulin-Like Growth Factor Binding Protein 2/analysis , Insulin-Like Growth Factor Binding Protein 3/analysis , Lung/metabolism , Microscopy, Confocal , Polymers , Staining and Labeling , Time Factors , Tumor Cells, Cultured
2.
Am J Physiol Lung Cell Mol Physiol ; 278(1): L42-50, 2000 Jan.
Article in English | MEDLINE | ID: mdl-10645889

ABSTRACT

Retinoids, including retinol and retinoic acid (RA) derivatives, have been shown to be involved in the processes of lung development as well as of lung repair after injury. Recently, we have provided evidence that RA could stimulate proliferation of lung alveolar type 2 epithelial cells (E. Nabeyrat, V. Besnard, S. Corroyer, V. Cazals, and A. Clement. Am. J. Physiol. Lung Cell. Mol. Physiol. 275: L71-L79, 1998). To gain some insight into the mechanisms involved in the mitogenic action of RA, we focused in the present study on the effects of RA on the expression of G(1) phase cyclins and their cell cycle-dependent kinases (Cdks). Experiments were performed with serum-deprived cells cultured in the absence and presence of RA. The results showed no effects of RA on the expression of either cyclins or Cdks. In contrast, RA treatment was found to prevent the decrease in cyclin E-Cdk2 activity observed when cells were growth arrested by serum deprivation. The observation that changes in cyclin E-Cdk2 activity were not associated with modifications in the amount of complexes formed led to the suggestion that the Cdk inhibitory protein (CKI) was involved. Study of the CKI p21(CIP1) revealed marked differences in its expression in the absence and presence of RA, with a dramatic downregulation observed in RA-treated cells. Interestingly, immunoprecipitation experiments provided evidence that the decreased levels of p21(CIP1) were associated with a reduced interaction of this CKI with cyclin E-Cdk2 complexes. These data together with previous results obtained in various situations of type 2 cell growth arrest emphasize the role of p21(CIP1) in the control of lung alveolar epithelial cell proliferation.


Subject(s)
CDC2-CDC28 Kinases , Cyclins/metabolism , Proto-Oncogene Proteins , Pulmonary Alveoli/cytology , Tretinoin/pharmacology , Animals , Cell Division/drug effects , Cells, Cultured , Cyclin E/metabolism , Cyclin-Dependent Kinase 2 , Cyclin-Dependent Kinase 4 , Cyclin-Dependent Kinase Inhibitor p21 , Cyclin-Dependent Kinases/metabolism , Down-Regulation , Epithelial Cells/cytology , Epithelial Cells/metabolism , G1 Phase/physiology , Protein Serine-Threonine Kinases/metabolism , Pulmonary Alveoli/metabolism , Rats
3.
Biochim Biophys Acta ; 1448(3): 349-62, 1999 Jan 11.
Article in English | MEDLINE | ID: mdl-9990287

ABSTRACT

The surface of the pulmonary alveolus is a major target for oxidant injury, and its proper repair following injury is dependent on the proliferative response of the stem cells of the alveolar epithelium, the type 2 cells. In previous studies on the mechanisms controlling this response, we have documented involvement of several components of the IGF system, and mainly of the IGF binding protein-2 (IGFBP-2). We have provided evidence that this binding protein was associated with inhibition of DNA synthesis of type 2 cells exposed to oxidants and that its expression was regulated mostly at the level of transcription. In the present study, we focused on the factors involved in this regulation. From examination of the IGFBP-2 gene promoter sequence which revealed the presence of four potential binding sites for transcription factors of the NF-kappa B/Rel family, we hypothesized that NF-kappa B might be involved in the transcriptional activation of IGFBP-2 in oxidant-exposed cells. Data reported herein demonstrated that NF-kappa B activated IGFBP-2 promoter in transient transfection assays, and that exposure of cells to hyperoxia was associated with accumulation of the active form of NF-kappa B. Using gel shift analysis, we documented in O2-treated cells an increased binding to the four NF-kappa B binding sites. We also showed that accumulation of NF-kappa B was associated with a decrease in the inhibitory molecule I kappa B-alpha. Based on the current knowledge on NF-kappa B regulation, it is likely that in a number of situations associated with injury of lung alveolar epithelial cells signaling events involving accumulation of NF-kappa B converge to activate IGFBP-2 and to block entry into S phase.


Subject(s)
Hyperoxia/metabolism , Insulin-Like Growth Factor Binding Protein 2/genetics , NF-kappa B/metabolism , Promoter Regions, Genetic , Pulmonary Alveoli/metabolism , Animals , Base Sequence , Binding Sites/genetics , Cell Cycle , Cell Division , Cell Line , DNA/genetics , DNA/metabolism , DNA Probes/genetics , Epithelial Cells/metabolism , Pulmonary Alveoli/cytology , Pulmonary Alveoli/injuries , Rats , Signal Transduction , Transcriptional Activation , Transfection
4.
Am J Physiol ; 275(1): L71-9, 1998 07.
Article in English | MEDLINE | ID: mdl-9688937

ABSTRACT

Retinoids, including retinol and retinoic acid (RA) derivatives, are important molecules for lung growth and homeostasis. The presence of RA receptors and of RA-binding proteins in the alveolar epithelium led to suggest a role for RA on alveolar epithelial cell replication. In the present study, we examined the effects of RA on proliferation of the stem cells of the alveolar epithelium, the type 2 cells. We showed that treatment of serum-deprived type 2 cells with RA led to a stimulation of cell proliferation, with an increase in cell number in a dose-dependent manner. To gain some insights into the mechanisms involved, we studied the effects of RA on the expression of several components of the insulin-like growth factor (IGF) system that have been shown to be associated with the growth arrest of type 2 cells, mainly the IGF-binding protein-2 (IGFBP-2), IGF-II, and the type 2 IGF receptor. We documented a marked decrease in the expression of these components upon RA treatment. Using conditioned media from RA-treated cells, we provided evidence that the proliferative response of type 2 cells to RA was mediated through production of growth factor(s) distinct from IGF-I. We also showed that RA was able to reduce the decrease in cell number observed when type 2 cells were treated with transforming growth factor (TGF)-beta1. These results together with the known stimulatory effect of TGF-beta1 on IGFBP-2 expression led to suggest that RA may be associated with type 2 cell proliferation through mechanisms interfering with the TGF-beta1 pathway.


Subject(s)
Epithelial Cells/cytology , Insulin-Like Growth Factor Binding Protein 2/biosynthesis , Insulin-Like Growth Factor II/biosynthesis , Pulmonary Alveoli/cytology , Receptor, IGF Type 2/biosynthesis , Stem Cells/cytology , Tretinoin/pharmacology , Animals , Animals, Newborn , Cell Division/drug effects , Cell Line , DNA/biosynthesis , Epithelial Cells/drug effects , Epithelial Cells/physiology , Gene Expression Regulation/drug effects , Kinetics , Pulmonary Alveoli/physiology , Rats , Stem Cells/drug effects , Stem Cells/physiology , Time Factors
5.
Respir Med ; 92(7): 951-60, 1998 Jul.
Article in English | MEDLINE | ID: mdl-10070569

ABSTRACT

In the development of lung damage induced by oxidative stress, it has been proposed that changes in alveolar macrophages (AM) function with modifications in cytokine production may contribute to altered repair processes. To characterize the changes in profiles of cytokine production by macrophages exposed to oxidants, the effects of hyperoxia (95% O2) on interleukin (IL)-1 beta, IL-6, IL-8, and tumour necrosis factor-alpha (TNF-alpha) expression were studied. Experiments were first performed using AM obtained from control subjects and children with interstitial lung disease. Results showed that a 48 h O2 exposure was associated with two distinct patterns of response: a decrease in TNF-alpha, IL-1 beta and IL-6 expression, and an increase in IL-8. To complete these observations we used U937 cells that were exposed for various durations to hyperoxia. We confirmed that a 48 h O2 exposure led to similar changes with a decrease in TNF-alpha, IL-1 beta and IL-6 production and an increase in IL-8. Interestingly, this cytokine response was preceded during the first hours of O2 treatment by induction of TNF-alpha, IL-1 beta and IL-6. These data indicate that hyperoxia induces changes in the expression of macrophages inflammatory cytokines, and that these modifications appear to be influenced by the duration of O2 exposure.


Subject(s)
Cytokines/metabolism , Hyperoxia/metabolism , Lung Diseases, Interstitial/metabolism , Macrophages, Alveolar/metabolism , Bronchoalveolar Lavage Fluid , Cells, Cultured , Child , Gene Amplification , Gene Expression Regulation , Humans , Interleukin-1/metabolism , Interleukin-6/metabolism , Interleukin-8/metabolism , Oxidative Stress , RNA, Messenger , Time Factors , Tumor Necrosis Factor-alpha/metabolism
6.
J Biol Chem ; 271(41): 25117-25, 1996 Oct 11.
Article in English | MEDLINE | ID: mdl-8810266

ABSTRACT

The alveolar surface of the lung is a major target for oxidant injury, and its repair following injury is dependent on the ability of its stem cells, the type 2 cells, to initiate proliferation. From previous studies it is likely that events located before the entry into the S phase of the cell cycle and involving several components of the insulin-like growth factor system as well as of transforming growth factor-beta (TGF-beta) play a key role in growth regulation of oxidant-exposed type 2 epithelial cells. To gain further insights into these mechanisms, we explored the effects of O2 exposure on G1 cyclins and their cyclin-dependent kinases (CDKs). We documented an increased expression of these genes in O2-treated type 2 cells. However, despite this induction, a dramatic decrease in cyclin E-CDK2 activity, but not in cyclin D-CDK4 activity, was found. The concomitant induction of CDK inhibitory proteins (CKIs), mainly p21(CIP1), suggests that accumulation of inactive cyclin E-CDK2 activity is due to CKI binding. We also provided evidence that the mechanisms regulating this process involved TGF-beta as anti-TGF-beta antibody treatment was able to reduce the oxidant-induced inhibition of cyclin E-CDK2 activity. Taken together, these results suggest that oxidants may block entry into S phase by acting on a subset of late G1 events whose alterations are sufficient to impair the activation of cyclin E-CDK2 complexes.


Subject(s)
Cell Cycle/drug effects , Cyclins/biosynthesis , Oxidants/pharmacology , Oxygen/pharmacology , Pulmonary Alveoli/cytology , Pulmonary Alveoli/metabolism , Animals , Animals, Newborn , Antibodies/pharmacology , Cell Division/drug effects , Cells, Cultured , Cyclin D1 , Cyclin D2 , Cyclin D3 , Cyclin-Dependent Kinase Inhibitor p21 , DNA Primers , Enzyme Inhibitors , Epithelial Cells , Epithelium/drug effects , Epithelium/metabolism , G1 Phase , Molecular Sequence Data , Oncogene Proteins/biosynthesis , Polymerase Chain Reaction , Rats , Receptors, Transforming Growth Factor beta/physiology , Transforming Growth Factor beta/immunology , Transforming Growth Factor beta/pharmacology
7.
Endocrinology ; 137(1): 287-95, 1996 Jan.
Article in English | MEDLINE | ID: mdl-8536625

ABSTRACT

Glucocorticoids have been shown to impair lung growth by altering development of the alveolar structure. To characterize the mechanisms involved in this process, we examined the effects of dexamethasone on proliferation of the stem cells of the alveolar epithelium, the type 2 cells. Treatment of type 2 cells with dexamethasone rapidly decreased DNA synthesis, and this effect was observed for concentrations less than 10(-8)M. Inhibition of cell proliferation by glucocorticoids was associated with a marked accumulation of insulin-like growth factor (IGF)-binding protein-2 (IGFBP-2) in the culture medium. Studies of the mechanisms involved in this accumulation indicated that it was associated with an enhanced production of IGFBP-2 and with a similar increase in the level of IGFBP-2 messenger RNA expression without any changes in its stability, as evaluated by actinomycin D experiments. Furthermore, transfection studies using plasmids conveying expression of luciferase gene transcribed from the fragment of rat IBFBP-2 promoter extending from +12 bp relative to the start of transcription plus 1.4 kilobases of the 5'-flanking sequence showed a stimulation of luciferase activity in cells treated with dexamethasone that was similar to the increase in IGFBP-2 messenger RNA and protein. Study of the other components of the IGF system also revealed induction of IGF-II expression upon treatment with dexamethasone. Together with other previously reported results using various modulators of type 2 cell proliferation, the present study strongly suggests that IGFBP-2 is likely to play an important role in the control of alveolar epithelial cell proliferation.


Subject(s)
Dexamethasone/pharmacology , Insulin-Like Growth Factor Binding Protein 2/biosynthesis , Pulmonary Alveoli/cytology , Pulmonary Alveoli/metabolism , Animals , Cell Cycle/drug effects , Dexamethasone/antagonists & inhibitors , Epithelial Cells , Epithelium/metabolism , Fibroblasts/metabolism , Insulin-Like Growth Factor Binding Protein 2/genetics , Insulin-Like Growth Factor II/metabolism , Mifepristone/pharmacology , RNA, Messenger/metabolism , Rats , Receptors, Somatomedin/metabolism , Thymidine/pharmacokinetics , Transcription, Genetic/drug effects
8.
Endocrinology ; 135(1): 83-91, 1994 Jul.
Article in English | MEDLINE | ID: mdl-7516870

ABSTRACT

Pulmonary alveolar type 2 cells act as a reservoir of stem cells which can be induced to proliferate during periods of lung growth and repair after lung injury. Despite the importance of this process, the mechanisms that regulate type 2 cell proliferation have not been well characterized. We show in this study that insulin-like growth factor (IGF)-binding protein-2 (IGFBP-2) accumulates to high levels in culture medium of growth-arrested type 2 cells. This is associated with an increased expression of IGFBP-2 messenger RNA (mRNA). Study of the other components of the IGF system also reveals induction of IGF-II and type 2 IGF receptor mRNA during the process of type 2 cell block of proliferation. When growth-arrested cells are allowed to resume proliferation by the addition of serum, the level of expression of IGFBP-2, type 2 IGF receptor, and IGF-II rapidly decreased. Despite the similarities in the timing of induction, it is likely that these components are not necessarily linked to mediate effects through a single pathway. Indeed, we show that the addition of conditioned medium from growth-arrested cells on proliferative cells results in down-regulation of IGFBP-2 and increased expression of IGF-II and type 2 IGF receptor mRNA. Treatment of the cells with various concentrations of IGF-II affects only the level of expression of type 2 IGF receptor, whereas IGF-I and insulin appear to influence only the expression of IGFBP-2. From the results presented in this study, it can be suggested that IGFBP-2, IGF-II, and type 2 IGF receptor play an important role in the transition of lung alveolar epithelial cells in and out of the cell cycle.


Subject(s)
Carrier Proteins/genetics , Gene Expression , Insulin-Like Growth Factor II/genetics , Pulmonary Alveoli/physiology , Receptor, IGF Type 2/genetics , Animals , Base Sequence , Carrier Proteins/metabolism , Cell Division , Epithelial Cells , Epithelium/metabolism , Epithelium/physiology , Insulin-Like Growth Factor Binding Protein 2 , Molecular Probes/genetics , Molecular Sequence Data , Pulmonary Alveoli/cytology , Pulmonary Alveoli/metabolism , Rats , Receptor, IGF Type 1/metabolism , Receptor, IGF Type 2/metabolism , Somatomedins/genetics , Somatomedins/metabolism
9.
J Biol Chem ; 269(19): 14111-7, 1994 May 13.
Article in English | MEDLINE | ID: mdl-7514600

ABSTRACT

The epithelium of the pulmonary alveolus is a major target for oxidant injury, and its proper repair following injury is dependent on the proliferative response of its stem cells, the type 2 cells. We have recently shown that hyperoxia arrests proliferation of an immortalized type 2 cell line (SV40T-T2) and that expression of several growth-related genes, normally induced near the G1/S and boundary was altered with a block of translation of their mRNA. In the present study we examined the possible role of the insulin-like growth factor (IGF) system and of transforming growth factor-beta 1 (TGF-beta 1) in the arrest of proliferation induced by hyperoxia. We show that IGF-binding protein 2 (IGFBP-2) accumulates to higher levels in culture medium of SV40T-T2 cells whose proliferation has been arrested by hyperoxia. This proliferation arrest is associated with increased expression of IGFBP-2 mRNA and with induction of type 2 IGF receptor and IGF-II mRNAs. When O2-arrested cells were allowed to resume proliferation in normoxia, the level of expression of these genes rapidly decreased to control levels. We also, found that TGF-beta 1 was induced by O2 exposure, that TGF-beta 1 inhibited SV40T-T2 proliferation, and that TGF-beta 1 itself was a potent stimulator of IGFBP-2 expression. These studies suggest a regulatory link between components of the IGF system and TGF-beta 1 in hyperoxic control of cell proliferation of alveolar epithelial cells.


Subject(s)
Carrier Proteins/metabolism , Oxidants/pharmacology , Pulmonary Alveoli/metabolism , Somatomedins/metabolism , Transforming Growth Factor beta/metabolism , Animals , Base Sequence , Carrier Proteins/genetics , Cell Division/drug effects , Cells, Cultured , DNA Primers , Epithelial Cells , Epithelium/drug effects , Epithelium/metabolism , Insulin-Like Growth Factor Binding Protein 2 , Molecular Sequence Data , Oxygen/pharmacology , Pulmonary Alveoli/cytology , Pulmonary Alveoli/drug effects , RNA, Messenger/metabolism , Rats , Somatomedins/genetics , Transforming Growth Factor beta/biosynthesis
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