The interstitial collagenase matrix metalloproteinase-1 (MMP-1) is up-regulated in the lung during pulmonary emphysema. The mechanisms underlying this aberrant expression are poorly understood. Although cigarette smoking is the predominant cause of emphysema, only 15-20% of smokers develop the disease. To define the signaling pathways activated by smoke and to identify molecules responsible for emphysema-associated MMP-1 expression, we performed several in vitro and in vivo experiments. In this study, we showed that cigarette smoke directly induced MMP-1 mRNA and protein expression and increased the collagenolytic activity of human airway cells. Treatment with various chemical kinase inhibitors revealed that this response was dependent on the extracellular regulated kinase-1/2 (ERK) mitogen activated protein kinase pathway. Cigarette smoke increased phosphorylation of residues Thr-202 and Tyr-204 of ERK in airway lining cells and alveolar macrophages in mice at 10 days and 6 months of exposure. Moreover, analysis of lung tissues from emphysema patients revealed significantly increased ERK activity compared with lungs of control subjects. This ERK activity was evident in airway lining and alveolar cells. The identification of active ERK in the lungs of emphysema patients and the finding that induction of MMP-1 by cigarette smoke in pulmonary epithelial cells is ERKdependent reveal a molecular mechanism and potential therapeutic target for excessive matrix remodeling in smokers who develop emphysema.

Pulmonary emphysema is associated with alterations in matrix proteins and protease activity. These alterations may be linked to programmed cell death by apoptosis, potentially influencing lung architecture and lung function.To evaluate apoptosis in emphysema, lung tissue was analysed from 10 emphysema patients and six individuals without emphysema (normal). Morphological analysis revealed alveolar cells in emphysematous lungs with convoluted nuclei characteristic of apoptosis. DNA fragmentation was detected using terminal deoxynucleotide transferase-mediated dUTP nick-end labelling (TUNEL) and gel electrophoresis. TUNEL revealed higher apoptosis in emphysematous than normal lungs. Markers of apoptosis, including active caspase-3, proteolytic fragment of poly (ADP-ribose) polymerase, Bax and Bad, were detected in emphysematous lungs. Linear regression showed that apoptosis was inversely correlated with surface area. Emphysematous lungs demonstrated lower surface areas and increased cell proliferation. There was no correlation between apoptosis and proliferation, suggesting that, although both events increase during emphysema, they are not in equilibrium, potentially contributing to reduced lung surface area.In summary, cell-based mechanisms associated with emphysematous parenchymal damage include increased apoptosis and cell proliferation. Apoptosis correlated with airspace enlargement, supporting epidemiological evidence of the progressive nature of emphysema. These data extend the understanding of cell dynamics and structural changes within the lung during emphysema pathogenesis.

Signal transduction pathways often use a transcriptional component to mediate adaptive cellular responses. Coactivator proteins function prominently in these pathways as the conduit to the basic transcriptional machinery. Here we present a high-throughput cell-based screening strategy, termed the ''coactivator trap,'' to study the functional interactions of coactivators with transcription factors. We applied this strategy to the cAMP signaling pathway, which utilizes two families of coactivators, the cAMP response element binding protein (CREB) binding protein (CBP)/p300 family and the recently identified transducers of regulated CREB activity family (TORCs1-3). In addition to identifying numerous known interactions of these coactivators, this analysis identified NONO (p54 nrb ) as a TORC-interacting protein. RNA interference experiments demonstrate that NONO is necessary for cAMP-dependent activation of CREB target genes in vivo. Furthermore, TORC2 and NONO complex on cAMP-responsive promoters, and NONO acts as a bridge between the CREB/TORC complex and RNA polymerase II. These data demonstrate the utility of the coactivator trap by identification of a component of cAMP-mediated transcription.transcription ͉ signal transduction ͉ cell-based screen ͉ RNA polymerase II ͉ transducer of regulated cAMP response element-binding protein T ranscription is regulated by large multisubunit complexes that can be grouped into three general categories; DNA binding proteins, coregulators (coactivators and corepressors), and basal transcriptional components. DNA binding proteins recognize discrete sequences or response elements within promoters and in general function as scaffolds that direct the recruitment of coregulatory proteins. Coregulators in turn function as a conduit to the basic transcriptional machinery. Coregulators may also influence gene expression via intrinsic enzymatic activity or by recruitment of other enzyme activities (e.g., acetylation, methylation, poly ADPribosylation, ubiquitination, sumoylation, or ATP-dependent remodeling complexes) capable of modifying both transcriptional proteins and chromatin (1). Thus, determining the interaction networks of coregulators recruited by transcription factors and the accompanying enzymatic activities is necessary for understanding the complexities of gene expression.The cAMP signal-transduction pathway activates transcription by stimulating interactions between cAMP response element binding protein (CREB) and two coactivator families, CREBbinding protein (CBP)/p300 and transducers of regulated CREB (TORCs) (2-4). CREB-CBP/p300 interaction occurs when elevations in intracellular cAMP liberate protein kinase A (PKA) catalytic subunits (PKA c ) from PKA regulatory subunits. PKA c directly phosphorylates serine 133 in the kinase-inducible domain of CREB, increasing the affinity of CBP/p300 for CREB (5, 6). CBP/p300 interacts with components of the RNA polymerase II (RNA pol II) complex to facilitate transcription and contains intrinsic acetyltransferase activity specula...

The murine smoke-induced model produces histologic emphysema. The authors sought to assess whether the structural emphysema that occurred correlated with the development of compliance changes. The study exposed 2 strains of mice (CBA/J/J x C57BL/6J and A/J) to chronic cigarette smoke. Lung compliance and morphometry were measured. The smoking model generated significant emphysema in A/J mice in the absence of changes in compliance, lung matrix, or apoptosis. Importantly, there was no correlation between the emphysema measured by lung morphometry and pulmonary compliance. This lack of correlation suggests that the mechanisms involved in anatomic emphysema may be distinct from those that cause the loss of elastic recoil.

Tobacco-related diseases are leading causes of death worldwide, and many are associated with expression of matrix metalloproteinase-1 (MMP-1). We have reported extracellular signal-regulated kinase (ERK)1/2-dependent induction of MMP-1 by cigarette smoke in lung epithelial cells. Our objectives were to define regions of the human MMP-1 promoter required for activation by smoke, to identify differences in responses of the 1G/2G -1607 polymorphic promoters to smoke, and to identify relevant transcription factors whose activity in airway epithelial cells is increased by smoke. The responses of deletion and mutant promoter constructs were measured in transfected cells during exposure to cigarette smoke extract (CSE). DNA oligonucleotide arrays were used to identify transcription factors activated after smoke exposure. CSE activated the MMP-1 promoter, and this induction was prevented by PD98059 blockade of ERK1/2 phosphorylation. Deletion studies revealed the distal 1kb promoter region (-4438 to -3280 upstream of the transcription start site) is essential for CSE induction of MMP-1, and confers activation of a minimal promoter. Studies of 1G and 2G MMP-1 polymorphic promoter variants revealed higher 2G allele basal and CSE-responsive activities than the 1G allele. Cotransfection, mithramycin, and electrophoretic mobility shift assay studies identified activating and repressive roles for Sp1 and PEA3 transcription factors, respectively. Oligonucleotide DNA arrays confirmed activation of Sp1 and PEA3 by CSE. These data demonstrate that the MMP-1 promoter is a direct target of cigarette smoke in lung epithelial cells. This characterization of a smoke response region in the distal MMP-1 promoter has implications for smoking-related diseases such as cancer, heart disease, and emphysema.