Implications of post-pneumonectomy compensatory lung growth in pulmonary physiology and disease.

In a number of species, partial pneumonectomy initiates hormonally regulated compensatory growth of the remaining lung lobes that restores normal mass, structure and function. Compensation is qualitatively similar across species, but differs with gender, age and hormonal status. Although the biology of response is best characterized in rats, dogs have proven valuable in defining post-operative physiological adaptations. Most recently, mice were recognized to offer unique opportunities to explore the genetic basis of the response, as well as to evaluate associated detrimental effects of pathophysiological significance in animals exposed to carcinogens. The pneumonectomy model thus offers powerful insight concerning adaptive organ growth.

Extracellular matrix fibronectin alters connexin43 expression by alveolar epithelial cells.

Alveolar type II epithelial cells undergo phenotypic changes and establish gap junction intercellular communication as they reach confluence in primary culture. The pattern of gap junction protein (connexin) expression changes in parallel. Although connexin (Cx)43 mRNA and protein increase significantly by culture day 2, Cx26 and Cx32 expression decline. Along with increasing Cx43 expression, the cells assemble fibronectin derived both from serum in the culture medium and from de novo synthesis into the extracellular matrix (ECM). The present studies indicate that this ECM regulates Cx43 expression. Culture of type II cells in DMEM containing 8-10% fetal bovine serum (FBS) promotes assembly of a fibronectin-rich ECM that stimulates expression of both Cx43 mRNA and protein. Although Cx43 protein expression increased in response to FBS in a dose-dependent manner, fibronectin also elevated Cx43 protein in the absence of FBS. Anti-fibronectin antibody significantly reduced the serum-dependent increase in Cx43 expression. These results support the premise that fibronectin in the ECM contributes to the regulation of Cx43 expression by alveolar epithelial cells in primary culture.

Connexin expression by alveolar epithelial cells is regulated by extracellular matrix.

Extracellular matrix (ECM) proteins promote attachment, spreading, and differentiation of cultured alveolar type II epithelial cells. The present studies address the hypothesis that the ECM also regulates expression and function of gap junction proteins, connexins, in this cell population. Expression of cellular fibronectin and connexin (Cx) 43 increase in parallel during early type II cell culture as Cx26 expression declines. Gap junction intercellular communication is established over the same interval. Cells plated on a preformed, type II cell-derived, fibronectin-rich ECM demonstrate accelerated formation of gap junction plaques and elevated gap junction intercellular communication. These effects are blocked by antibodies against fibronectin, which cause redistribution of Cx43 protein from the plasma membrane to the cytoplasm. Conversely, cells cultured on a laminin-rich ECM, Matrigel, express low levels of Cx43 but high levels of Cx26, reflecting both transcriptional and translational regulation. Cx26 and Cx43 thus demonstrate reciprocal regulation by ECM constituents.

Compensatory lung growth after partial pneumonectomy enhances lung tumorigenesis induced by 3-methylcholanthrene.

In small mammals, partial pneumonectomy (PNX) elicits rapid hyperplastic compensatory growth of the remaining lung parenchyma to restore normal lung mass, structure, and function. In BALB mice subjected to PNX, compensatory lung growth is complete within 10 days. Because cellular hyperplasia contributes to the mechanism of tumor promotion by butylated hydroxytoluene (BHT), we hypothesized that hyperplastic compensatory lung growth would promote tumor formation in carcinogen-treated animals in a manner similar to that observed after BHT. In mice subjected to PNX, within 1 week of treatment with the carcinogen 3-methylcholanthrene (MCA; 10 microg/g body weight), lung tumor multiplicity was 3-7-fold higher in animals subjected to PNX than in mice subjected to a sham operation. The increase in tumor multiplicity occurred when PNX was performed 1, 3, and 6 days before or 1 day after MCA treatment. In the absence of PNX, lung tumor multiplicity in MCA-treated mice given one injection of BHT (200 mg/kg body weight) increased significantly (P < 0.01) as compared to that in mice given MCA alone. Tumor multiplicity continued to increase linearly (R2 = 0.99) with each subsequent BHT injection. Lung tumor multiplicity and tumor size in mice given one or two injections of BHT were comparable to those in animals subjected to PNX. These data demonstrate that post-PNX compensatory lung growth stimulates tumorigenesis in MCA-treated mice and provides a novel model for investigating tumor formation.

Hydrogen peroxide induces oxidative DNA damage in rat type II pulmonary epithelial cells.

Type II epithelial cells, which line the alveolar surface of the lung, are exposed to a variety of potentially mutagenic and carcinogenic insults. The purpose of this study was to determine if type II cells are susceptible to oxidative DNA damage in vitro. Treatment of cultured rat type II lung epithelial cells with hydrogen peroxide led to increased concentrations (nmol/mg DNA) of 12 of 14 monitored DNA base modifications, suggesting oxidative damage by the hydroxyl radical. These base modifications are typically associated with oxidative stress, and elevated levels have been correlated with mutagenesis and carcinogenesis. These data demonstrate that type II cells are indeed vulnerable to oxidative DNA damage.

Inhibition of gap junction communication in alveolar epithelial cells by 18alpha-glycyrrhetinic acid.

Cultured alveolar epithelial cells exhibit gap junction intercellular communication (GJIC) and express regulated levels of connexin (Cx) 43 mRNA and protein. Newly synthesized radiolabeled Cx43 protein equilibrates with phosphorylated Cx43 isoforms; these species assemble to form both connexons and functional gap junction plaques. The saponin 18alpha-glycyrrhetinic acid (GA) rapidly and reversibly blocks GJIC at low concentrations (5 microM). Extended exposure to 18alpha-GA at higher concentrations causes inhibition of GJIC and time- and dose-dependent reductions in both Cx43 protein and mRNA expression. The latter toxic effects are paralleled by disassembly of gap junction plaques and are reversed less readily than acute effects on GJIC. These observations demonstrate 18alpha-GA-sensitive regulation of intercellular communication in epithelial cells from the mammalian lung and suggest a role for Cx43 expression and phosphorylation in acute and chronic regulation of GJIC between alveolar epithelial cells.

Isolation, cloning, and characterization of a novel rat lung zinc finger gene, RLZF-Y.

Zinc-finger (ZF) proteins are widely distributed. The current study reports isolation, cloning and characterization of a novel ZF gene, RLZF-Y. Total RNA from rat lung was reverse transcribed. The 5' and 3' ends were isolated by rapid amplification of cDNA ends (RACE) using primers derived from a previously isolated partial clone. RACE products of 1.5 and 1.1 kb were cloned and sequenced. Identical overlapping sequence of 70 base pairs confirmed representation of the same cDNA approximately 2.5 kb in length. Probes derived from both 5' RACE and 3' RACE products independently hybridized to a 2.5 kb mRNA from rat lung. RLZF-Y mRNA is expressed in lung, brain, heart and kidney; expression is low in liver. Predicted amino acid sequence analysis defined three regions of similarity to known C2H2 ZF proteins: a region containing seven ZF structures characteristic of the Krüppel-like subfamily of ZF genes; a region with sequence similarity to the Krüppel-associated box A (KRAB-A) domain at the amino end; an amino-terminal leucine-rich region (LeR) adjacent to KRAB-A. The presence of KRAB-A and the adjacent LeR implies RLZF-Y protein may function as a transcriptional repressor.

Regulation of extracellular matrix synthesis by TNF-alpha and TGF-beta1 in type II cells exposed to coal dust.

Type II pulmonary epithelial cells respond to anthracite coal dust PSOC 867 with increased synthesis of extracellular matrix (ECM) components. Alveolar macrophages modulate this response by pathways that may involve soluble mediators, including tumor necrosis factor-alpha (TNF-alpha) or transforming growth factor-beta1 (TGF-beta1). The effects of TNF-alpha (10 ng/ml) and/or TGF-beta1 (2 ng/ml) were thus investigated in dust-exposed primary type II cell cultures. In control day 1 or day 3 cultures, TNF-alpha and/or TGF-beta1 had little or no effect on the synthesis of type II cellular proteins, independent of whether the cells were exposed to dust. With PSOC 867 exposure, where ECM protein synthesis is elevated, TNF-alpha and TGF-beta1 further increased both the absolute and relative rates of ECM synthesis on day 3 but had little effect on day 1. Each mediator increased expression of fibronectin mRNA, as well as of ECM fibronectin content, in a manner qualitatively similar to their effects on synthesis. Thus TNF-alpha and TGF-beta1 modulate both ECM synthesis and fibronectin content in coal dust-exposed type II cell cultures.

Increased lung inflation induces gene expression after pneumonectomy.

Rapid hyperplastic growth of the remaining lung is initiated by partial pneumonectomy in many mammalian species. The response restores normal tissue structure and function. Although physiological control of compensatory lung growth is documented, little is known about the molecular mechanisms that underlie the process. The aim of this study was to investigate the role of mechanical signals in the induction of immediate-early gene (IEG) expression after pneumonectomy. Expression of c-fos and junB increased nine- and fourfold, respectively, in the right lung within 30 min after left pneumonectomy in rats. In contrast, changes in expression of c-jun and c-myc were not observed. When isolated lungs were subjected to elevated airway pressures in vitro, expression of c-fos and junB was induced in a time- and dose-dependent manner similar to that observed in vivo. Similarly, in vitro lung perfusion induced c-fos and junB expression in the absence of increasing lung inflation. These results support the premise that rapid changes in IEG expression after pneumonectomy are initiated by mechanical signaling in the remaining lung. Elevated IEG expression may contribute to initiation of compensatory lung growth.

Targeted identification of zinc finger genes expressed in rat lungs.

Control of alveolar cell growth and differentiation after pneumonectomy likely involves changes in expression of regulatory genes, including those encoding zinc finger (ZF) proteins. To explore this premise, total RNA from the lungs of control and pneumonectomized rats was reverse transcribed; PCRs were performed with degenerate primers corresponding to amino acid sequences HTGEKP and CPECGK(N), which are evolutionarily conserved among ZF genes. Reaction products corresponding to three and four ZF units were isolated and cloned. Sixteen clones were sequenced and found to represent rat lung ZF genes: six clones were highly similar or identical to known ZF genes and ten clones showed lower homology to known ZF genes and thus appear to represent new members of the ZF family. Northern analysis demonstrated differential expression of some ZF genes after pneumonectomy. Thus a PCR-based strategy with primers derived from evolutionarily conserved ZF protein sequences efficiently identifies ZF genes expressed in lung, some of which may play a role in cellular growth and differentiation.

Adrenal hormone regulation of fibronectin synthesis by type II pulmonary epithelial cells.

Previous work suggested an association between changes in the alveolar extracellular matrix and epithelial cell growth in lungs of adrenalectomized rats in vivo. Other studies demonstrated that adrenal hormones modulate extracellular matrix synthesis by type II pulmonary epithelial cells in vitro. Adrenal hormone regulation of type II cell fibronectin synthesis was thus examined. Fibronectin synthesis was quantitated by immunoprecipitation of the metabolically labeled molecule from cells, extracellular matrix, and culture medium. On day 1 of primary culture, synthesis of matrix fibronectin by type II cells isolated from the lungs of adrenalectomized animals was more than twice that by cells isolated from control rats. Adrenalectomy elevated steady-state fibronectin mRNA levels in primary isolates of type II cells cultured for 1 or 3 days. These results suggest that altered fibronectin synthesis and deposition into the extracellular matrix accompany changes in type II cell phenotype that occur after adrenalectomy.

Expression of functional gap junctions in cultured pulmonary alveolar epithelial cells.

Recent observations suggest that cell-cell interactions may modulate the response of the alveolar epithelium to injury. Expression and function of gap junctions were thus evaluated in isolated alveolar type II cells. Freshly isolated (day 0) type II cells expressed mRNAs for gap junctional connexins 26, 32, and 43. Whereas connexin 26 mRNA declined approximately 40% in cultured cells, connexin 32 message decreased rapidly and was not detectable on day 1. In contrast, connexin 43 expression increased 10-fold by day 3 compared with day 0. Western blot confirmed a 30-fold elevation in connexin 43 protein. Connexin 45 mRNA was not detected. Functional gap junction-mediated calcium signal propagation was monitored using fura 2, a calcium-sensitive dye. Membrane deformation in a single type II cell increased intracellular calcium; the signal spread rapidly to neighboring cells by octanol-sensitive pathways. Transfer of Lucifer yellow between cells also was inhibited by octanol. These observations demonstrate functional gap junctions between cultured alveolar epithelial cells and suggest that gap junctional expression and gap junction-mediated cell-cell coupling are regulated with time in culture.

Assembly of exogenous fibronectin into type II cell extracellular matrix.

Type II pulmonary epithelial cells (T2P) in primary culture assemble a biologically active extracellular matrix (ECM) from endogenously synthesized components, including fibronectin. Fibronectin is a well-recognized attachment protein that mediates cell adhesion, migration, and cytodifferentiation. In some cell types, exogenous fibronectin also is incorporated into ECM. The latter pathway of ECM assembly was thus investigated in T2P. Cells were cultured for 3-days in Dulbecco's modified Eagle's medium (DMEM) with or without 10% fetal calf serum (FCS), a source of exogenous fibronectin. Cell and matrix fractions were harvested on culture days 1, 2, and 3 to determine synthesis of cell and matrix proteins and matrix fibronectin content. During 3 days in DMEM containing 10% FCS, T2P flattened and spread to confluence more rapidly than cells in DMEM; they also produced ECM with higher fibronectin content than did cells in DMEM alone. On culture days 2 and 3, 10% FCS doubled (on average) synthesis of ECM fibronectin; in contrast, ECM fibronectin content increased nearly 10-fold. These observations suggest that cultured type II cells incorporate exogenous fibronectin into newly assembled ECM to a greater extent than the newly synthesized glycoprotein. Components of both endogenous and exogenous origin may therefore contribute to T2P assembly of a biologically active ECM.

Regulated stimulation of epithelial cell DNA synthesis by fibroblast-derived mediators.

Interactions of interstitial fibroblasts with nearby epithelial cells are thought to play a role in lung growth and development. The present studies support this premise. Medium conditioned by second-passage lung fibroblasts (FCM) stimulated both DNA synthesis and accumulation in low-density (2 x 10(4)/cm2) cultures of type II alveolar epithelial cells. FCM effects did not require serum; they were time- and dose dependent, with half-maximal FCM activity at 1:8 dilution. A maximal response to FCM required 30 h of exposure. FCM activity was reduced in medium from fibroblasts treated with dexamethasone, suggesting physiological regulation. Type II cells subjected to cyclic mechanical stress demonstrated an increased response to FCM compared with static cultures. FCM activity did not appear to be accounted for by hepatocyte growth factor, keratinocyte growth factor, acidic fibroblast growth factor, or fibronectin. These results suggest that early passage lung fibroblasts release, by regulated pathways, one or more factors that stimulate DNA synthesis by type II cells. Sensitivity to FCM appears to be elevated in type II cell cultures subjected to cyclic mechanical stress.

Synthesis of fibronectin and laminin by type II pulmonary epithelial cells.

Previous investigations demonstrated that type II pulmonary epithelial cells regulate extracellular matrix deposition as a function of time in primary culture. In those studies, the matrix fraction was analyzed as a whole. The present work focused on two components of the type II cell matrix, fibronectin and laminin. These glycoproteins have differing effects on differentiation of type II cells in primary culture. Fibronectin synthesis was quantitated between day 1 and day 6 in the cells, matrix, and medium; laminin synthesis was quantitated only in the cells. Although total fibronectin synthesis was regulated as a function of time in culture, reaching its greatest value on day 2, the average proportion of newly synthesized fibronectin in the cells (35%), medium (50%), and matrix (15%) remained constant over a 6-day interval. Between day 2 and day 6, the relative abundance of fibronectin messenger RNA increased 6.5-fold. Rates of cellular laminin synthesis did not vary with time in culture. These results support a role for differential regulation of fibronectin and laminin synthesis to determine the composition of the type II cell extracellular matrix.