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.

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.

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.

Composition of extracellular matrix of type II pulmonary epithelial cells in primary culture.

Type II pulmonary epithelial cells in primary culture synthesize and deposit an extracellular matrix which has reciprocal biological effects on cellular differentiation. The present work establishes conditions for metabolic labeling of matrix constituents and for separation of cells from the associated matrix; it also defines matrix composition, which does not appear to change qualitatively between days 2 and 6 of primary culture. Type II cells synthesize and deposit a spectrum of radiolabeled components on the culture surface. These include fibronectin, laminin, type IV collagen, and plasminogen activator inhibitor-1, along with additional unidentified proteins. Few radiolabeled proteins in medium conditioned by type II cells bind nonspecifically to the culture surface in the absence of cells. Fibroblasts and macrophages, which may contaminate the primary cultures, do not appear to contribute substantially to the type II cell matrix. These results demonstrate that type II cells synthesize and deposit a complex multicomponent extracellular matrix. The work provides a basis for further investigations of bidirectional interactions between type II cells and the extracellular matrix.

Turnover of fibronectin and laminin by alveolar epithelial cells.

Type II pulmonary epithelial cells in primary culture synthesize and assemble a multicomponent extracellular matrix which exhibits biological activity in vitro. Simultaneously, the pneumocytes degrade components of the underlying matrix, such that matrix composition may be determined by the balance of synthesis and turnover. The present work defines turnover of the specific matrix glycoproteins, fibronectin and laminin, both in the type II cell and in its extracellular matrix. Pulse-chase experiments demonstrate that both fibronectin and laminin, identified by immunoprecipitation, turn over rapidly in the cell and extracellular matrix compartments, with half-lives < 10 h. In the cell compartment, initial rates of laminin turnover are more rapid than those of fibronectin on culture day 2, but these rates are similar on day 6. Matrix fibronectin also turns over rapidly, with similar rates on day 2 and day 6. During the chase interval, small but increasing amounts of immunoprecipitable fibronectin are detected in the medium, suggesting that a portion of the glycoprotein may be released to the extracellular compartment, rather than degraded. Alternatively, release of immunoreactive glycoprotein may involve ongoing processing and secretion of residual radiolabeled fibronectin by the cells. The results suggest that matrix composition may be determined by turnover, as well as synthesis, of its components.