Synthesis of fibronectin and laminin by type II pulmonary epithelial cells

1996 ◽  
Vol 270 (2) ◽  
pp. L215-L223 ◽  
Author(s):  
S. E. Dunsmore ◽  
Y. C. Lee ◽  
C. Martinez-Williams ◽  
D. E. Rannels
Keyword(s):  
Extracellular Matrix ◽  
Epithelial Cells ◽  
Primary Culture ◽  
Messenger Rna ◽  
Type Ii ◽  
Matrix Deposition ◽  
Pulmonary Epithelial Cells ◽  
The Matrix ◽  
Average Proportion ◽  
Type Ii Cell

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.

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

1997 ◽  
Vol 273 (1) ◽  
pp. L86-L92
Author(s):  
S. E. Dunsmore ◽  
Y. C. Lee ◽  
D. E. Rannels
Keyword(s):  
Extracellular Matrix ◽  
Epithelial Cells ◽  
Cell Phenotype ◽  
Type Ii Cells ◽  
Mrna Levels ◽  
Hormone Regulation ◽  
Type Ii ◽  
Adrenal Hormone ◽  
Pulmonary Epithelial Cells ◽  
Type Ii Cell

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.

Extracellular matrix synthesis and turnover by type II pulmonary epithelial cells

1992 ◽  
Vol 262 (5) ◽  
pp. L582-L589 ◽  
Author(s):  
D. E. Rannels ◽  
S. E. Dunsmore ◽  
R. N. Grove
Keyword(s):  
Extracellular Matrix ◽  
Steady State ◽  
Epithelial Cells ◽  
Primary Cultures ◽  
Calf Serum ◽  
Type I ◽  
Type Ii Cells ◽  
Type Ii ◽  
Pulmonary Epithelial Cells ◽  
Type Ii Cell

Both type I and type II pulmonary epithelial cells contact the extracellular matrix (ECM). Type II cell-ECM interactions are bidirectional; they involve matrix-mediated modulation of type II cell differentiation, as well as cellular synthesis and deposition of ECM components. The present experiments examine the kinetics of accumulation of newly synthesized proteins in cell and matrix fractions from primary cultures of type II pneumocytes. Cycloheximide-sensitive incorporation of [3H]leucine into total protein of both the cell and ECM fractions was linear for 24–30 h, when steady-state labeling was reached and maintained to at least day 8. Over this interval, the cells enlarged but did not divide. Newly synthesized proteins recovered in the matrix fraction averaged 1–2% of those in the cells. Relative rates of radiolabeling of matrix proteins peaked at culture day 2 and increased in the absence of serum. In short-pulse studies, initial rates of protein synthesis were equal on culture days 1 and 3; this suggested that the steady-state labeling kinetics above reflected protein turnover. This was supported by rapid loss of radioactivity from the ECM after fresh type II cells were seeded on a prelabeled, cell-free matrix surface. Fresh or conditioned Dulbecco's modified Eagle's medium containing 10% fetal calf serum had little effect on matrix stability. These results demonstrate regulated deposition and turnover of a complex ECM by type II cells and provide a basis for further investigations of factors that control these processes.

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

1995 ◽  
Vol 269 (6) ◽  
pp. L754-L765 ◽  
Author(s):  
S. E. Dunsmore ◽  
C. Martinez-Williams ◽  
R. A. Goodman ◽  
D. E. Rannels
Keyword(s):  
Extracellular Matrix ◽  
Epithelial Cells ◽  
Primary Culture ◽  
Biological Effects ◽  
Primary Cultures ◽  
Matrix Composition ◽  
Type Ii Cells ◽  
Type Ii ◽  
Pulmonary Epithelial Cells ◽  
Culture Surface

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.

Connexin expression by alveolar epithelial cells is regulated by extracellular matrix

2001 ◽  
Vol 280 (2) ◽  
pp. L191-L202 ◽  
Author(s):  
Yihe Guo ◽  
Cara Martinez-Williams ◽  
Clare E. Yellowley ◽  
Henry J. Donahue ◽  
D. Eugene Rannels
Keyword(s):  
Extracellular Matrix ◽  
Epithelial Cells ◽  
Gap Junction ◽  
Intercellular Communication ◽  
Translational Regulation ◽  
Alveolar Epithelial Cells ◽  
Alveolar Type ◽  
Type Ii ◽  
Gap Junction Intercellular Communication ◽  
Type Ii Cell

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.

Extracellular matrix fibronectin alters connexin43 expression by alveolar epithelial cells

2001 ◽  
Vol 280 (4) ◽  
pp. L680-L688 ◽  
Author(s):  
Andrea I. Alford ◽  
D. Eugene Rannels
Keyword(s):  
Extracellular Matrix ◽  
Epithelial Cells ◽  
Gap Junction ◽  
Primary Culture ◽  
Alveolar Epithelial Cells ◽  
Dependent Manner ◽  
Type Ii ◽  
Cx43 Expression ◽  
Cx43 Protein ◽  
Alveolar Epithelial

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.

Assembly of exogenous fibronectin into type II cell extracellular matrix

1997 ◽  
Vol 272 (5) ◽  
pp. L908-L915 ◽  
Author(s):  
J. W. Swisher ◽  
D. E. Rannels
Keyword(s):  
Extracellular Matrix ◽  
Calf Serum ◽  
Cell Types ◽  
Biologically Active ◽  
Matrix Proteins ◽  
Type Ii ◽  
Attachment Protein ◽  
Pulmonary Epithelial Cells ◽  
Type Ii Cell ◽  
Exogenous Origin

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.

Spermidine uptake by type II pneumocytes: interactions of amine uptake pathways

1989 ◽  
Vol 257 (6) ◽  
pp. L346-L353 ◽  
Author(s):  
D. E. Rannels ◽  
R. Kameji ◽  
A. E. Pegg ◽  
S. R. Rannels
Keyword(s):  
Epithelial Cells ◽  
Primary Culture ◽  
Gradient Centrifugation ◽  
Maximal Velocity ◽  
Dependent Manner ◽  
Type Ii ◽  
Transport Pathway ◽  
Concentration Dependent Manner ◽  
Pulmonary Epithelial Cells ◽  
Extracellular Sodium

Uptake of exogenous spermidine by type II pulmonary epithelial cells in primary culture is inhibited by several amines. The present studies further detail the interaction of those alternative substrates with the pathway for spermidine (SPD) transport. Transport activity was measured on the first day of primary culture in type II cells isolated by elastase digestion, followed by density gradient centrifugation and differential adherence in vitro. Spermidine uptake was inhibited in a concentration-dependent manner by the polyamines putrescine (PUTR) and spermine (SPM), by the drug methylglyoxal bis-(guanylhydrazone) (MGBG), and by the herbicide paraquat (PQ). The order of effectiveness of these competitors was SPM greater than PUTR approximately MGBG much greater than PQ. The kinetics of inhibition by SPM were mixed, with an increase in the apparent Michaelis constant and a reduction in the maximal velocity of the SPD uptake pathway. Cellular uptake of SPD and PUTR was inhibited by replacement of extracellular sodium with choline or lithium (PUTR greater than SPD), but SPM uptake was unaffected. PQ appeared to interact with the polyamine transport pathway with low affinity. Compared with SPD and SPM, PQ was most effective as an inhibitor of PUTR uptake, and like PUTR, uptake of the herbicide was sharply inhibited as extracellular sodium was reduced. These observations suggest the presence of diverse pathways for uptake of exogenous polyamines by type II pulmonary epithelial cells and indicate that PQ probably enters the cells by the sodium-dependent transport system favored by PUTR.

Turnover of fibronectin and laminin by alveolar epithelial cells

1995 ◽  
Vol 269 (6) ◽  
pp. L766-L775 ◽  
Author(s):  
S. E. Dunsmore ◽  
C. Martinez-Williams ◽  
R. A. Goodman ◽  
D. E. Rannels
Keyword(s):  
Extracellular Matrix ◽  
Epithelial Cells ◽  
Alveolar Epithelial Cells ◽  
Matrix Composition ◽  
Type Ii ◽  
Alveolar Epithelial ◽  
Extracellular Compartment ◽  
Type Ii Cell ◽  
Turn Over

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.

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