Connexin expression by alveolar epithelial cells is regulated by extracellular matrix

Yihe Guo1, Cara Martinez-Williams1, Clare E. Yellowley2, Henry J. Donahue1,2, and D. Eugene Rannels1,3

Departments of 1 Cellular and Molecular Physiology, 2 Orthopaedics and Rehabilitation, and 3 Anesthesia, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

connexin 43; connexin 26; gap junction; lung; cell-extracellular matrix interactions


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

INTERACTIONS BETWEEN alveolar epithelial cells and the underlying extracellular matrix (ECM) are essential to the functional integrity of the lung. For example, biological effects mediated through ECM components appear to be involved in the repair of alveolar epithelium after injury (for a review, see Ref. 18). Cell-ECM interactions have been investigated by culture of isolated cells on ECMs of differing composition and biological activity (37). Alveolar type II cells cultured on plastic surfaces lose typical lamellar bodies, rapidly accumulate cellular protein, and alter expression of genes associated with their differentiated function. With time, the cells assume many type I cell-like characteristics (7, 13, 35). These changes are accelerated in cells plated on a preformed, fibronectin-rich, type II cell-derived ECM (38) or on culture wells coated with purified fibronectin (14). In contrast, a laminin-coated culture surface or a laminin-rich matrix (Matrigel) can prevent or reverse these changes in cellular phenotype (43). These observations suggest that alveolar type II cells, like many epithelial cell populations (5), respond to signals from the ECM by altering their biological characteristics.

Alveolar epithelial cells establish direct communication with neighboring cells via specialized junctional complexes, including both gap junctions and tight junctions (3, 4, 26, 29, 30, 41). Functional gap junctions involve paired hexameric assemblies (connexons) of integral membrane proteins (connexins). These complexes couple cells electrically and metabolically via gated axial pores that offer low resistance to the passage of ions, electrical current, or molecules of low molecular weight and thereby connect the cytoplasmic compartments of adjacent cells. Gap junction channels can restrict movement of specific molecules via regulated gating of channel conductance in a connexon-specific manner that is determined by the gating properties and selectivity of individual connexin subunits (9, 32).

In addition to acute regulation of gap junction channel conductance, the extent of cell-to-cell communication in the distal lung is likely to be determined by cell type- and condition-specific differential patterns of connexin expression modulated at both the transcriptional and translational levels. For example, Carson et al. (12) reported transient expression of connexin (Cx) 26 in airway epithelium of newborn ferrets, with a significant decline as a function of postnatal age (12). Parallel molecular and immunologic studies demonstrated expression of Cx26 mRNA and protein in alveolar epithelial cells of adult ferret and human lungs where levels of connexin expression were more stable. The latter observations are supported by ultrastructural freeze-fracture studies that confirm the presence of gap junction plaques in the alveolar region (4, 11, 41).

Similarly, in alveolar epithelial cells, expression of Cx26 and Cx32 is downregulated in the early stages of primary cell culture (26), whereas expression of Cx43 and Cx46 is elevated at later culture intervals (1, 26). These patterns reflect differing levels of mRNA abundance as well as the balance between synthesis and turnover of connexin proteins as a function of culture time.

Biological and molecular mechanisms that underlie differential expression of alveolar epithelial cell connexins are poorly understood. It is well known, however, that fibronectin promotes cell attachment and differentiation (28, 31, 37, 38). In primary cell culture, type II alveolar epithelial cells assemble a biologically active fibronectin-rich ECM derived both from endogenous fibronectin synthesis (16, 38) and from fibronectin derived from exogenous sources such as serum (46). The molecular composition of the ECM changes as a function of time, particularly during the early stages of primary culture (17, 46). In this context, it is plausible that fibronectin and other matrix components play a role in the regulation of connexin expression and thereby in the extent of cell coupling (25). These regulatory pathways are likely to be of pathophysiological significance under conditions of lung injury where a provisional fibronectin-rich ECM is assembled at the alveolar surface and repopulated by a subsequent proliferative response of the alveolar epithelium (15).

In the present studies, we begin to examine the hypothesis that ECM composition modulates both connexin expression and the extent of gap junction intercellular communication (GJIC) between adjacent alveolar epithelial cells. The results demonstrate that ECMs rich in fibronectin or laminin have contrasting regulatory effects on gap junction expression and thereby suggest that specific cell-matrix interactions and gap junction-mediated cell-to-cell communication are closely linked biological processes in this essential lung cell population.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Culture of alveolar type II epithelial cells. Type II cells were isolated from the lungs of male Sprague-Dawley rats (150-175 g body weight; Charles River Laboratories) according to methods previously described (39). The day of cell isolation was designated as day 0. For immunocytochemical studies, type II cells were plated on Permanox chamber slides (Nunc, Naperville, IL) at a density of 2.4 × 105 cells/cm2. For both Northern and Western blot analyses, the cells were plated at a density of 2.1 × 105 cells/cm2 on 100-mm and 6-well plastic culture dishes, respectively (Falcon, Franklin Lakes, NJ). In functional assays of GJIC, the cells were plated on glass coverslips within six-well plates. All experiments were terminated between day 0 and day 8 of primary culture.

Preparation of ECM-coated culture surfaces. To obtain a biologically assembled fibronectin-rich ECM, freshly isolated type II cells were cultured for 3 days on six-well plates in DMEM containing 10% fetal bovine serum (38). After a rinse with cold 1 mM EDTA in phosphate-buffered saline (PBS), the culture wells were incubated with 1 ml/well of 0.25 M NH4OH containing 1 mM EDTA and 1 mM phenylmethylsulfonyl fluoride (PMSF) at 4°C for 30 min. The cells were removed from the plate by swirling until the monolayer lifted. The remaining matrix was rinsed twice with cold EDTA-PBS as previously described (36). To remove remnants of the cells, a second incubation at 4°C (15 min) in 1 ml of 1 M NaCl containing 50 mM Tris and EDTA-PMSF was followed by a rinse with cold EDTA-PBS. Type II cell-derived ECM assembled over 3 days of primary culture (ECM-3) was stored in EDTA-PBS containing 1 mM PMSF (4°C).

To prepare a laminin-rich Matrigel culture surface, a frozen solution of Engelbreth-Holm-Swarm (EHS) tumor matrix components (10 mg protein/ml Eagle's modified essential medium; Collaborative Research) was thawed slowly at 4°C and diluted to 5 mg/ml with ice-cold DMEM. The diluted material (0.5 ml) was spread onto six-well plates and placed in the cell incubator at 37°C. Gel formation was complete by 60 min, at which time freshly isolated type II cells were plated on the Matrigel surface (40).

Immunocytochemistry and quantitation of gap junction plaques. Immunocytochemical methods were used to examine the subcellular distribution of the gap junction protein Cx43 and the tight junction protein zonula occludens-1 (ZO-1). Alveolar epithelial cells plated on Permanox chamber slides were fixed with 95% methanol and 5% acetic acid for 90 min followed by rehydration in PBS for 5 min. The cells were blocked with 10% normal goat or donkey serum in PBS for 60 min at room temperature. Polyclonal rabbit anti-Cx43 antiserum (Zymed Laboratories, San Francisco, CA) and monoclonal antibodies against ZO-1 diluted 1:200 in PBS were used in overnight incubations at 4°C. After a subsequent wash with 4% goat or donkey serum for 60 min, the cultures were stained with either Fluorolink Cy3-labeled goat anti-rabbit IgG (1:200 dilution; Amersham) or Cy2-conjugated donkey anti-rat IgG (1:200 dilution; Jackson ImmunoResearch Laboratories, West Grove, PA) for 3 h at room temperature. The cultures were then washed three times (10 min each) with PBS. The slide chambers and gaskets were removed, and the slides were mounted with Fluoromount-G (Fisher, Pittsburgh, PA) before visualization with a Zeiss LSM 10 confocal microscope. Control slides were processed identically, but the primary antibody was replaced by normal goat or donkey serum. Cx43-immunopositive labeled punctate structures, which represent gap junction plaques, were counted with the same confocal microscope. Under each experimental condition, 50 cells were selected at random for counting.

Quantitation of intercellular coupling. Intercellular transfer of the fluorescent dye Lucifer yellow was used to evaluate functional coupling of alveolar epithelial cells via gap junctions as detailed previously (19). Cells cultured on glass coverslips were rinsed with Tyrode solution (140 mM NaCl, 4 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 5 mM HEPES, and 10 mM glucose; pH 7.45) and transferred to an inverted microscope. Single cells were loaded over a 2-min interval with a glass micropipette with 10% Lucifer yellow dissolved in 1 M LiCl2. Spread of the dye between the cells was visualized under epifluorescent illumination by excitation at 450-490 nm; emitted light was filtered at 515 nm. The number of coupled cells was counted 3 min after the micropipette was removed. Under each experimental condition, at least six cells were loaded with dye.

To evaluate coupling of type II cells and type I-like cells, freshly isolated type II cells were labeled with the lipophilic red fluorescent dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI). DiI does not traverse gap junction channels. The cells were loaded for 10 min (37°C) with DiI at a final concentration of 8.75 µg/ml in DMEM containing 10% fetal bovine serum. DiI-labeled cells were plated at a ratio of 1:50 over a confluent day 3 alveolar epithelial cell monolayer and incubated for 4 h to allow formation of gap junction channels. Single DiI-labeled type II cells were then microinjected with Lucifer yellow according to methods described above. The number of adjacent cells containing Lucifer yellow was evaluated after 5 min.

Antibody blocking. In some experiments, antibodies were used to inhibit cell interactions with the underlying ECM. Polyclonal antibodies against bovine fibronectin (diluted 1:50 in DMEM; Calbiochem) were added to ECM-3-coated culture wells in the absence of serum for at least 1 h before the cells were plated.

Northern blot analysis. Total RNA was isolated from type II cells cultured on 100-mm plates with the TRI Reagent protocol (Molecular Research Center, Cincinnati, OH). RNA concentration and purity were estimated spectrophotometrically. Northern blot analysis was performed as previously described (26). Twenty micrograms of total RNA were subjected to electrophoresis on 1.2% agarose-0.4 M formaldehyde surface tension gels and then transferred to a nylon membrane. After capillary blotting, the filters were hybridized overnight in a rolling incubator at 65°C with a prehybridization and hybridization sodium phosphate-based solution consisting of 7% sodium dodecyl sulfate (SDS), 0.5 M Na2HPO4, 1% nonfat dry milk, and 1 mM EDTA. Washes were performed in 2× saline-sodium phosphate-EDTA (0.3 M NaCl, 20 mM NaH2PO4, pH 7.4, and 20 mM EDTA, pH 7.4) containing 0.5% SDS at 65°C for 30 min. For autoradiography, the blots were exposed to Fuji RX film with intensifying screens at -70°C. Northern blots were subsequently quantified with a PhosphorImager SI with ImageQuant software (Molecular Dynamics, Sunnyvale, CA). Radioactivity associated with bands representing connexin mRNA was compared with that of a control probe against elongation factor Tu (EFTu) to normalize for minor differences in loading (27).

Western blot analysis of cell and membrane proteins. To obtain membrane-enriched and cellular fractions (21), the cells were lysed in cold hypotonic buffer, 10 mM Tris (pH 7.8), containing a standard protease inhibitor cocktail (0.1 mg/ml of aprotinin, 0.1 mg/ml of leupeptin, 0.1 mg/ml of antipain, and 0.01 mg/ml of pepstatin in buffer containing 1 mM EDTA-Na2 and 0.2 mM PMSF). Crude cell and membrane fractions were recovered from the supernatant and pellet, respectively, after centrifugation of the above extracts at 15,000 g (10 min). The resulting pellets were dissolved in 1% SDS containing the above-mentioned protease inhibitors.

Proteins in these fractions were subjected to Western blot analysis to assay expression of Cx26, Cx43, and/or fibronectin. Equal amounts of protein (8) were separated by electrophoresis on 10% SDS-polyacrylamide gels and then transferred electrophoretically to a nitrocellulose membrane with a Transblot apparatus (Bio-Rad, Hercules, CA). For immunoblotting, nonspecific binding sites were blocked with 5% nonfat dry milk and 0.5% Tween 20 in PBS for 15 min. The blots were then incubated for 2 h with polyclonal antibodies against Cx43 (1:3,000 dilution; Zymed) or Cx26 (1:1,000; Zymed) and/or polyclonal antibodies against fibronectin (1:2,500; Calbiochem), washed extensively, and then incubated with the appropriate biotinylated secondary antibodies (Vector Laboratories, Burlingame, CA). Specific proteins were visualized with enhanced chemiluminescence detection reagents (Amersham, Arlington Heights, IL). Control segments of Western blots were processed identically, with the antibodies replaced by antibody diluent. A short exposure to X-ray film was made for densitometric analysis.

Data analysis. Results are expressed as means ± SE of the number of observations indicated. Data were analyzed with a two-tailed Student's t-test. Values of P < 0.05 were considered to be significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Expression of cellular fibronectin and Cx43 are closely associated early in alveolar epithelial cell culture. During the initial phases of primary type II cell culture, day 0 to day 3, expression of Cx43 mRNA increased 10-fold (1, 26). Over the same interval, the abundance of Cx43 protein increased nearly 30-fold (26). To examine whether this increment in Cx43 expression may reflect cell interactions with fibronectin in the ECM, total type II cell protein was sampled over a 68-h culture interval and subjected to Western blot analysis with antibodies specific either to Cx43 or to fibronectin. Expression of the proteins increased in parallel with time in primary culture (Fig. 1, inset). Comparison of the data derived from the time course demonstrates a linear relationship between relative expression of fibronectin and Cx43 over an extensive range of abundance of both proteins (Fig. 1). Maximal expression of cellular fibronectin reached ~1.0 µg/culture well at the 68-h interval. An independent regression analysis showed that cellular fibronectin levels correlated closely with those of both the nonphosphorylated and the two phosphorylated forms of Cx43 (22), which increased in parallel as a function of culture time (numerical values not shown).


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Fig. 1.   Parallel changes in expression of cellular fibronectin and connexin (Cx) 43. Total cell protein was collected at 10 intervals over 68 h of primary cell culture for quantitation of fibronectin and Cx43 expression by Western blot analysis. Primary antibodies against fibronectin and Cx43 did not cross-react (data not shown). Densities of immunopositive fibronectin and Cx43 bands were quantitated and normalized relative to maximal expression of the respective protein at the 68-h interval, which in both cases is designated as 100. Values for nonphosphorylated and 2 forms of phosphorylated Cx43 were combined; expression of these Cx43 forms increased in parallel (data not shown; Ref. 22). Data are means from triplicate observations. The curve was fitted by eye. Inset: relative expression of fibronectin () and Cx43 () as a function of culture time.

Formation of gap junction plaques is time dependent and associated with ECM assembly. Anatomic data cited above provide evidence that both tight junctions and gap junctions are established between adjacent alveolar epithelial cells both in vivo and in vitro. To expand these observations, immunofluorescent confocal microscopy was employed to evaluate the assembly of both tight and gap junction proteins into the plasma membranes of primary alveolar epithelial cell cultures. On day 1, alveolar type II cells attached to the culture surface express the tight junction protein ZO-1 and appear to have begun to form junctional complexes. ZO-1 immunostaining is present, although in some cases interrupted, along the common borders of many adjacent cells (Fig. 2A). Qualitative comparison of Fig. 2, A-C, suggests that ZO-1 expression increases further with time in culture because the pattern of ZO-1 staining in the plane of the plasma membrane becomes more continuous and robust.


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Fig. 2.   Immunohistochemical distribution of tight junction and gap junction proteins in primary type II cell cultures. Type II cells (106) were cultured on Permanox chamber slides on untreated plastic (A-F) or on a surface of cell-derived extracellular matrix (ECM) after 3 days of culture (ECM-3; G-I). After 1 (A, D, and G), 2 (B, E, and H) or 3 (C, F, and I) days of primary cell culture, monolayers were fixed in 95% methanol and 5% acetic acid for immunostaining of zonula occludens-1 (ZO-1; A-C) or Cx43 (D-I). Arrowheads, selected ZO-1-positive tight junction complexes; arrows, selected Cx43-positive gap junction plaques. Bar, 13.6 µm. Related data in text are expressed as means ± SE of counts from 50 cells chosen at random.

In contrast, expression of the gap junction protein Cx43 is more variable at the day 1 culture interval when the borders of individual cells appear to stain in an "all-or-none" pattern (Fig. 2D). An average of 4 ± 1 Cx43-positive gap junction plaques/cell were observed on day 1. On culture days 2 and 3, the punctate structures of gap junction plaques were present at higher levels, averaging 31 ± 1/cell at each interval (P < 0.001; Fig. 2, E and F). Independent observations showed that increasing numbers of gap junction plaques paralleled increased expression of Cx43 protein and elevated cell coupling (26).

The above results reflect previous observations (16, 17) that cultured alveolar epithelial cells assemble a fibronectin-rich ECM with biological activity, again suggesting a role for fibronectin in the regulation of connexin expression. Freshly isolated type II cells were thus plated on a cell-free, fibronectin-rich ECM that had been assembled over 3 days of primary culture (ECM-3). This matrix is comparable to that underlying the cells shown in Fig. 2, C and F. Under these conditions, the number of immunostained Cx43-positive gap junction plaques on day 1 increased threefold (Fig. 2), from 4 ± 1 to 12 ± 1/cell (P < 0.01; Fig. 2, G compared with D). On day 2, there was no significant difference in the number of gap junction plaques per cell on ECM-3 (32 ± 1/cell) compared with those on plastic (31 ± 1/cell; Fig. 2, H and E, respectively). On day 3, the average number of gap junction plaques on ECM-3 (25 ± 1/cell; Fig. 2I) was below the control value (31 ± 1/cell; P < 0.01; Fig. 2F). In parallel, the number of internalized Cx43-immunopositive inclusions increased in ECM cultures on day 3 (Fig. 2I).

Preformed ECM-3 enhances type II cell coupling. To examine functional consequences of the above effects of ECM-3 on the density of membrane-associated Cx43-positive gap junction plaques, the level of gap junction coupling between adjacent epithelial cells was evaluated based on intercellular transfer of the fluorescent dye Lucifer yellow. In day 2 cell cultures (Fig. 3, left bars), Lucifer yellow spread from a single dye-loaded cell to 9 ± 3 adjacent cells within 5 min. In contrast, the number of coupled cells on day 2 doubled to 19 ± 4 in cultures plated on ECM-3 (P < 0.05). In day 3 cultures (Fig. 3, right bars), the number of coupled cells on ECM-3 increased to 53 ± 4 compared with 21 ± 3 in the corresponding day 3 control cells (P < 0.01). These data provide a functional correlate to the results shown in Fig. 2.


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Fig. 3.   Effect of ECM on gap junction communication. Alveolar type II cells were plated on untreated tissue culture wells (control) or on ECM-3. The extent of gap junction communication between adjacent cells was determined on both day 2 and day 3 based on intercellular transfer of Lucifer yellow as described in EXPERIMENTAL PROCEDURES. Data are means ± SE of observations from 32 and 15 dye-loaded cells on day 2 and day 3, respectively.

Antibodies against fibronectin cause redistribution of Cx43 into the cytoplasm. Antibodies were used to test whether fibronectin present in ECM-3 (17) plays a role in the regulation of Cx43 trafficking and gap junction function (Fig. 4). Cell-free ECM-3 was incubated for 60 min in the absence (Fig. 4, A-C) or presence (Fig. 4, D-F) of anti-fibronectin antibodies (1:50) before freshly isolated type II cells were plated and cultured for 3 days under the same conditions. Confocal microscopy revealed that in the absence of antibody, the cells attached to the plates and flattened as a function of time in culture (Fig. 4, B and C compared with A). The effect of fibronectin on cell shape appeared to be reduced in the presence of the antibodies (Fig. 4, E and F compared with B and C), reflecting inhibition of cell interactions with fibronectin-rich ECM-3 (17). Furthermore, on days 2 and 3, immunopositive Cx43 appeared to be confined mainly to the cytoplasmic compartment in antibody-treated cells (Fig. 4, D-F, arrowheads), whereas Cx43-immunopositive gap junction plaques were more prevalent in the membranes of cells cultured without antibody (Fig. 4, A-C, arrows).


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Fig. 4.   Effect of anti-fibronectin antibody on cellular distribution of Cx43. Alveolar type II cells were plated on ECM-3 in the absence of serum under standard culture conditions (A-C) and in the presence of anti-fibronectin antibody (D-F). On days 1 (A and D), 2 (B and E), and 3 (C and F), cultures were immunostained with antibodies against Cx43. Arrows, typical Cx43-immunopositive gap junction plaques in the plasma membrane; arrowheads, immunopositive Cx43 in the intracellular compartment. In D-F, note the qualitative reduction of Cx43-immunopositive material in the plasma membrane.

These observations were extended with Western blot analysis to evaluate the distribution of immunoreactive Cx43 between the cellular and membrane fractions (Fig. 5). Cx43 expression increased more than sevenfold between culture day 1 and day 3. Under control conditions (Fig. 5A), the portion of total immunopositive Cx43 recovered in the membrane fraction averaged 71%; this value was constant as a function of culture time. In contrast, when cells were cultured in the presence of anti-fibronectin antibody (Fig. 5B), the portion of Cx43 in the membrane fraction averaged 24%, although the overall increase in Cx43 expression was preserved. These observations support the qualitative results shown in Fig. 4, confirming redistribution of Cx43 from the membrane to the cytosol in cells exposed to anti-Cx43 antibody, suggesting a link between phosphorylation and compartmentation of Cx43. The latter possibility remains to be explored in the present model system.


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Fig. 5.   Effect of anti-fibronectin antibody on cellular partitioning of Cx43 protein. Type II cells were plated without serum on ECM-3 in the absence (A) and presence (B) of anti-fibronectin antibody. Antibody (1:50) was added 1 h before the cells were plated (B) and on each subsequent day. Cell and membrane fractions were prepared on culture days 1, 2, or 3 as described in EXPERIMENTAL PROCEDURES. Relative abundance of Cx43 in each fraction was evaluated by Western blot analysis. Total height of the bars represents total Cx43 protein. Day 1 and 2 values are shown in proportion to those on day 3, which are set at 100%. Data are means of 3 independent observations.

Parallel functional assays showed that transfer of Lucifer yellow between adjacent cells was abolished in the presence of anti-fibronectin antibody (Fig. 6). Under control conditions, Lucifer yellow loaded into a single alveolar epithelial cell on day 3 spread rapidly to an average of 17 ± 3 adjacent cells (5 observations) via gap junction channels (Fig. 6A). In contrast, dye moved into 1 ± 2 adjacent cells in the cultures exposed to anti-fibronectin antibody (Fig. 6B).


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Fig. 6.   Anti-fibronectin antibody inhibits gap junction communication. Primary type II cells were plated without serum and cultured for 3 days on ECM-3 in the absence (A) and presence (B) of anti-fibronectin antibody as described in Fig. 5. On day 3, intercellular coupling was evaluated over a 5-min interval based on transfer of Lucifer yellow between adjacent cells (26). Arrows, dye-injected cells.

Laminin-rich Matrigel alters connexin expression in cultured type II cells. The results above suggest a significant role for fibronectin in the regulation of Cx43 expression and distribution as well as in cell coupling. Other matrix components such as laminin may also play a role in regulating cell-to-cell communication. To address this possibility, type II cells were plated on Matrigel, a laminin-rich basement membrane prepared from the mouse EHS sarcoma (23). In initial studies, Western blot analysis confirmed rapid downregulation of Cx26 protein as early as day 1 in cells plated on tissue culture plastic or on ECM-3 (Fig. 7). Compared with day 0, Cx26 was nearly undetectable through 8 subsequent days of cell culture. In contrast, Cx26 expression increased and was sustained in cells cultured on Matrigel, although the abundance of Cx26 protein was somewhat variable with time. Over the same time course, Cx43 expression increased substantially on the plastic culture surface as well as on ECM-3. Conversely, Cx43 expression was nearly abolished in cells cultured on Matrigel.


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Fig. 7.   Differential effects of ECM on connexin expression. Freshly isolated (day 0) alveolar type II epithelial cells were plated and cultured for up to 8 days on plastic tissue culture wells (PL), on ECM-3 (17), or on wells coated with Matrigel (MG) (40). Relative abundance of both Cx26 and Cx43 was determined by Western blot analysis as described in EXPERIMENTAL PROCEDURES.

Regulation of connexin expression by ECM. To determine whether transcriptional and/or translational regulation of connexin gene expression contributed to the above observations, both the relative abundance of mRNAs encoding Cx26 and Cx43 and the expression of the respective proteins were measured daily over an 8-day course of primary cell culture. Cells were plated under three conditions: on tissue culture plastic, on a preformed, fibronectin-rich ECM recovered from day 3 type II cell cultures (ECM-3), or on Matrigel.

Data in Fig. 8 expand the qualitative observations in Fig. 7 by showing quantitative effects of ECM components on the expression of Cx26 mRNA and protein in primary culture. As suggested by previous observations (26), Northern blot analysis revealed that Cx26 mRNA decreased substantially with culture time (Fig. 8). In cells plated on either plastic or a fibronectin-rich ECM-3 surface (Fig. 8, A and B, respectively), Cx26 mRNA declined 65% during the first 2 culture days (P < 0.01) and 80% by day 4 (P < 0.01). Cx26 mRNA remained at low abundance through day 8. These changes are reflected in qualitatively similar decreases in Cx26 protein, which dropped to between 25 and 50% of its initial abundance by day 4 (P < 0.05; Fig. 8, D and E, respectively), where it remained through day 8. In contrast, Cx26 expression was increased or maintained in cells cultured on laminin-rich Matrigel. Cx26 mRNA increased 34% in these cells by day 2 (P < 0.01; Fig. 8C), although Cx26 mRNA abundance declined to 60% of initial levels by day 4 (P < 0.01 vs. day 0). On the same culture surface, Cx26 protein increased threefold by day 2 (P < 0.02) but declined somewhat thereafter, stabilizing at about twice the initial level (Fig. 8F). Expression of both Cx26 mRNA and protein was more variable on Matrigel than on the plastic or ECM-3 surfaces.


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Fig. 8.   Regulation of Cx26 expression by ECM. Primary alveolar epithelial cells were cultured as described in Fig. 7. Relative expression of Cx26 mRNA (A-C) and protein (D-F) was determined as a function of culture time by Northern and Western blot analyses, respectively. Densitometric data from each gel were normalized against peak expression on the respective gel to account for differences in exposure. mRNA data were normalized against the control gene, elongation factor Tu (EFTu) (27). n, No. of experimental observations/data point.

Effects of ECM composition on expression of Cx43 mRNA and protein contrast with those on Cx26 (Fig. 9). On plastic or ECM-3 (Fig. 9, A and B, respectively), Cx43 mRNA increased fivefold by day 2 (P < 0.01) and remained stable through day 8. Cx43 protein showed a qualitatively similar pattern of expression, although in contrast to Cx43 mRNA, abundance of the protein increased 30-fold by day 2 on either plastic or ECM-3 (P < 0.01; Fig. 9, D and E, respectively) and was sustained thereafter (P < 0.01). In contrast, both Cx43 mRNA and protein remained relatively stable as a function of culture time on Matrigel (Fig. 9, C and F, respectively). Together, these results suggest that the pattern of connexin expression by alveolar epithelial cells in vitro is governed, at least in part, through changes in cell phenotype brought about by specific components of the ECM.


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Fig. 9.   Regulation of Cx43 expression by ECM. Primary alveolar epithelial cells were cultured as described in Fig. 7. Relative expression Cx43 mRNA (A-C) and protein (D-F) was determined as described in Fig. 8. n, No. of experimental observations/data point.

GJIC between alveolar epithelial cells of differing phenotypes. The results above raise the question of whether alveolar epithelial cell populations with differing profiles of connexin expression can establish GJIC. To address this issue, experiments were designed to test whether freshly isolated type II cells, which exhibit a relatively high expression of Cx26 and low expression of Cx43, could establish GJIC with cultured alveolar cells that express the converse distribution of these connexins.

To test this possibility, alveolar epithelial cells were cultured on plastic to confer the "type I cell-like" phenotype, with associated high Cx43 and low Cx26 expression. On day 3, a second group of day 0 cells was isolated (relatively high Cx26 but low Cx43; refer to Fig. 7, day 0 lanes). The latter cells, which exhibit the typical rounded "type II cell-like" shape and express type II cell markers, were incubated with the red fluorescent gap junction-impermeable dye DiI and then plated over the day 3 cell monolayer at a ratio of 1:50 (type II to type I). This approach facilitated identification of solitary red DiI-stained type II cells on the underlying unstained monolayer (Fig. 10A). Three DiI-stained cells are marked for orientation (Fig. 10, arrow and arrowheads). At specified intervals, a single rounded type II cell containing DiI (Fig. 10A, arrow) was loaded with the gap junction-permeable dye Lucifer yellow. The cells were tested immediately and after 2 and 4 h for transfer of Lucifer yellow from the DiI-stained cell to its type I cell-like neighbors (Fig. 10B). No Lucifer yellow transfer was evident until 4 h of culture when an average of 18 surrounding day 3 cells contained Lucifer yellow within 5 min. Similar data were obtained after 8 h of coculture. These observations provide evidence that alveolar epithelial cells with type II cell-like and type I cell-like phenotypes can establish GJIC in vitro as can adjacent type I-like cells (26).


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Fig. 10.   Gap junction communication between alveolar epithelial cells of type II cell and type I cell-like phenotypes. Experiment is described in text. A: fluorescence microscopic image of freshly isolated type II alveolar epithelial cells labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) and plated (1:50) over day 3 cultures of alveolar epithelial cells. DiI does not traverse gap junction channels. Arrow and arrowheads, specific cells for orientation. B: image of same field shown in A. After 4 h, a single DiI-loaded cell (arrow) was loaded with Lucifer yellow with a glass micropipette. Lucifer yellow diffused to 18 surrounding cells within 5 min. The experiment was performed twice with similar results.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Intercellular communication via gap junctions is of broad biological significance. Based on both anatomic and functional data, it has been known for some time that gap junction channels are present in the gas-exchange region of the lung (3, 4) as well as in airway epithelium (6, 41). There is, however, only limited information concerning differential expression, functional characteristics, or regulation of gap junctions in alveolar epithelial cells.

Specific characteristics of gap junction intercellular signaling pathways that involve calcium and other mediators are well established in nonpulmonary systems (for reviews, see Refs. 10, 20, 24, 34). These encompass diverse areas including, but not limited to, mechanochemical signal transduction, transepithelial ion transport, wound repair, regulation of normal and abnormal cell growth, and the cellular response to injury. Ample evidence implicates these processes as being functionally critical to cells of the peripheral lung. It is thus anticipated that extended investigations centered on basic characteristics of intercellular communication between alveolar cells will yield information relevant to cellular function in both normal and abnormal pulmonary tissues.

At least eight connexins (Cx26, Cx30.3, Cx32, Cx37, Cx40, Cx43, Cx45, and Cx46) expressed in lung tissue appear to be distributed differentially among the major resident cell populations (1, 26). Cx43 was selected for emphasis in the present studies based on observations that it is highly expressed in lung tissue and in primary cultures of alveolar epithelial cells (1, 26). Although Cx43 is subject to both chronic and acute regulation in alveolar epithelium, the mechanisms and significance of this regulation are not well understood. The present studies extend the observation that the expression of Cx43 mRNA and protein increase rapidly during the early phase of alveolar epithelial cell culture. Over the same interval, the cells synthesize and assemble a multicomponent (17) fibronectin-rich ECM with both newly synthesized and exogenous serum-derived fibronectin (16, 46). Both the biological activity and structure of this matrix suggest that its components may regulate Cx43 expression as well as gap junction-mediated cell-to-cell communication in cultured alveolar epithelium. Several lines of evidence support this premise. The linear correlation between fibronectin and Cx43 expression in the early culture interval is consistent with a role for fibronectin in establishing intercellular communication. Over the same time course, fibronectin promotes cell growth, flattening, and subsequent formation of both tight junction complexes (41) and gap junction channels (26) between adjacent cells.

These observations parallel data from nonpulmonary tissues that establish a significant role for ECM constituents in the regulation of gap junction expression and function. For example, an early investigation (45) provided evidence that hepatocytes cultured in the presence of single ECM proteoglycans and glycosaminoglycans (including dermatan sulfate, chondroitin sulfates, heparins, and hyaluronic acid) alter cell morphology, cell coupling, and (where tested) connexin expression in vitro.

The question of whether parallel increases in alveolar epithelial cell expression of fibronectin and Cx43 represent cause and effect or are simply coincidental remains to be resolved. Similar effects are evident when cells are plated on culture surfaces coated with fibronectin (Alford AI and Rannels DE, unpublished observations) independent of whether the source of fibronectin is endogenous or exogenous. These effects are blocked by anti-fibronectin antibodies. Furthermore, in a keratinocyte model of epidermal wound healing, cellular interaction with laminin 5 via the alpha 3beta 1-integrin promoted both assembly of gap junction plaques and GJIC (25). Based on those observations, it appears that integrin-mediated cell-basement membrane interactions do play an important role in promoting cell coupling and subsequent coordination of cell growth to affect wound healing. These observations are of specific interest in the context of injury to the alveolar epithelium, where GJIC between adjacent alveolar epithelial cells may be essential to coordinated repair and reepithelialization of the alveolar surface (42, 44).

Culture of alveolar epithelial cells on fibronectin also promotes formation of gap junction plaques as evidenced by increased immunostaining along the common borders of adjacent cells and, in parallel, increased cell coupling. In cells plated on plastic, the density of Cx43-immunopositive plaques appeared to decline between days 2 and 3 (Fig. 2, E compared with F), whereas the efficiency of Lucifer yellow transfer between adjacent cells doubled over the same interval (Fig. 3). Similarly, between day 0 and day 3, the antibody against fibronectin abolished GJIC (Fig. 6). Although this observation is supported by parallel Western blot analysis and by data showing that Cx43-immunopositive gap junction plaques redistribute from the membrane to the cytoplasm in antibody-treated cells, nearly 25% of Cx43 remains membrane-associated under these conditions (Fig. 5).

This apparent discrepancy may reflect recovery of a portion of cytosolic Cx43 in the membrane fraction (33) and/or the degree of uncertainty in quantifying gap junction plaques. Moreover, it is essential to recognize that gap junction channels containing specific connexins demonstrate differential selectivity for charged molecules such as anionic and cationic dyes. Alveolar epithelial cells also express additional connexins, including Cx46 and Cx26, that may contribute to junctional conductance in the present studies (1). The extent to which anion-preferring Cx46 channels contribute to intercellular transfer of negatively charged Lucifer yellow has not been investigated in the current model. Nevertheless, it appears that channels containing Cx26 may offer greater resistance to transfer of the dye than those containing Cx43 (19).

Type II cells plated on laminin-rich Matrigel retain a rounded phenotype for up to 5 days in primary culture. Under these conditions, Cx43 mRNA and protein remain at low abundance, whereas expression of Cx26 mRNA and protein increase. These data suggest that the level of Cx43 expression does not reflect time in culture but rather is a matrix-dependent phenomenon. The effectiveness of Matrigel, particularly in suppressing expression of Cx43 protein, declined with time in culture. It is likely that the small but progressive increase in Cx43 expression between days 0 and 8 (Fig. 9F) reflects proteolytic digestion of the Matrigel surface in vitro (40) in conjunction with the parallel deposition of matrix fibronectin. Similar considerations, along with low levels of Cx26 expression, may account for the relatively high variability in Cx26 expression on Matrigel (Fig. 8, C and F).

The results shown in Fig. 10 reveal that GJIC can be established between alveolar epithelial cells that express type II cell and type I cell-like phenotypes in vitro, suggesting that these cell populations may be coupled in vivo. The latter premise is consistent with the recent observations of Ashino et al. (2) that provide strong functional evidence for exocytosis-linked intercellular propagation of intracellular calcium concentration oscillations between type I and type II cells after lung expansion in situ. Inhibitors of gap junction conductance blocked the above effects on both calcium oscillations and exocytosis, suggesting a role for GJIC in linking these cell populations.

The biological significance of these results must be interpreted conservatively given the large number of connexins expressed in lung tissue and the fact that knowledge of their distribution is incomplete. Type II cells express several connexins simultaneously. Among these, Cx26 and Cx32 are more prevalent in freshly isolated cells than in culture (26). Both the profile and proportions of connexins expressed by the pulmonary epithelium change as a function of culture time as the cells assume a more type I cell-like phenotype (1, 26). These observations raise the possibility that heterotypic pathways of gap junction coupling may be established in the alveolar region. Further efforts to identify specific connexin(s) that account for cell coupling and to confirm that those connexins are expressed in relevant cell populations in vivo are needed to draw conclusions concerning the significance of these observations in the intact organ.

In summary, phenotypic alterations in alveolar epithelial cells with time in primary culture resemble those observed after lung injury in vivo. These changes involve elevated expression and assembly of fibronectin into the ECM and consequent increases in cell size, flattening, and contact. Manipulation of cell phenotype by altering or blocking components of the underlying ECM is associated with differential expression of specific connexin mRNAs and proteins along with significant changes in GJIC. These coordinated events offer strong suggestive evidence that the levels of connexin expression and gap junction coupling may be regulated in response to the altered expression, assembly, and/or composition of specific ECM components, particularly fibronectin and laminin.


    ACKNOWLEDGEMENTS

We thank Dr. Kirk Gilbert for productive discussions and support. We also thank Dr. David Antonetti for the monoclonal antibodies against zonula occludens-1 (ZO-1) and the following investigators for providing cDNA probes: Dr. R. A. Levine for elongation factor Tu (EFTu), Dr. B. J. Nicholson for connexins 26 and 32, and Dr. S. Lye for connexin 43.


    FOOTNOTES

This research was supported by American Heart Association Grant 9750145N and National Heart, Lung and Blood Institute Grant R01-HL-31560.

Address for reprint requests and other correspondence: D. E. Rannels, Dept. of Cellular and Molecular Physiology C4723, The Pennsylvania State Univ. College of Medicine H166, 500 University Dr., Hershey, PA 17033 (E-mail: grannels{at}psu.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 14 June 2000; accepted in final form 6 September 2000.


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