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Extracellular Matrix Modulates Expression of Connexin Messenger RNA and Protein by Alveolar Epithelial Cells^*

^* From the Departments of Cellular and Molecular Physiology (Drs. Guo and Alford, and Ms. Martinez-Williams), and Anesthesia (Dr. Rannels); The Pennsylvania State University College of Medicine; Hershey, PA.

Correspondence to: D. Eugene Rannels, PhD, Distinguished Professor of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine H166, 500 University Dr, Hershey, PA 17033; e-mail: grannels{at}psu.edu

Key Words: alveolar epithelium • connexin • extracellular matrix • gap junction

Cell-to-cell communication through gap junction channels plays an important role in both the physiologic and pathophysiologic function of many tissues and cell types.¹ Data from several laboratories have documented expression and function of gap junction proteins, connexins (Cx), in alveolar epithelial cells, both in vivo and in vitro.^{2 3 4 5} Nevertheless, little is known concerning the biology of gap junctions in the alveolar region of the lung. Type II alveolar epithelial cells express at least six Cxs in primary culture. Among these, Cx26 and Cx43 appear to be regulated in a reciprocal manner, at both the messenger RNA (mRNA) and the protein levels, as a function of culture time.⁴ Cx26 mRNA expression declines 40% between the day of type II cell isolation (day 0) and day 1 of primary culture. Cx26 message then remains relatively stable through day 6, as the cells progressively acquire characteristics similar to those of type I epithelial cells. Cx26 protein falls to 20% of the day 0 value by day 1 and decreases further thereafter. Conversely, expression of Cx43 mRNA increases significantly between day 0 and day 3, in concert with a rapid increase in Cx43 protein. The present studies begin to examine whether differential regulation of these Cxs may reflect the biological consequences of type II cell interactions with specific components of the extracellular matrix (ECM).⁶

Experimental Procedures

All studies were carried out using primary cultures of type II alveolar epithelial cells isolated from the lungs of male Sprague-Dawley rats, according to established procedures.⁷ On day 0, freshly isolated cells were plated at a density of 2.1 x 10⁵/cm² on surfaces of either tissue culture plastic (PL), a fibronectin-rich ECM assembled by type II cells over 3 days of primary culture (ECM-3),⁷ or the laminin-rich ECM product of the EHS sarcoma, matrigel (MG).⁹ Both cell isolation and culture methods, as well as analytical procedures, have been described elsewhere in detail.^{4 8}

Results and Discussion

Expression of Cx26 and Cx43 mRNA and protein by type II alveolar epithelial cells changes substantially as a function of time in primary culture (Fig 1) . In each panel of Figure 1 , Cx expression on culture day 3 was calculated relative to that in freshly isolated day 0 control (CNT) type II cells. Levels of Cx expression on day 0 were assigned a relative value of 1.0 for each measured parameter (open bars). Compared to day 0, culture of type II cells on laminin-rich MG elevates both Cx26 mRNA and protein more than twofold by day 3 (right bars, panels A and B, respectively). At the same time, MG maintains other features of the type II cell phenotype.^{6 9} In contrast, day 0 Cx26 mRNA and protein expression decline by as much as 85% percent in cells plated on PL or on a preassembled, cell-derived fibronectin-rich matrix (ECM-3). The latter observations are in close agreement with results of previous studies,⁴ documenting the similarity of these culture surfaces. Note that on day 3, MG supports expression of Cx26 protein at levels more than 10-fold above those in cells cultured on fibronectin (compare to PL or ECM-3).

View larger version (37K): [in this window] [in a new window] [Download PPT slide]   Figure 1. Effects of ECM on expression of Cx26 and Cx43. Expression of Cx mRNA and protein in type II cells cultured 3 days on a PL surface, on a fibronectin-rich ECM-3, or on laminin-rich MG was compared to that in freshly isolated day 0 CNT cell preparations. Densitometry values from Northern blot analysis (panels A and C) were normalized against the CNT gene, EFTu.4 10 Resulting mRNA expression data, along with data from Western blot analysis (panels B and D), are expressed relative to day 0 (assigned a value of 1.0). Note that the maximal value on the ordinate is 10 in panels A through C, but is 100 in panel D. Numerical values for each bar are shown to facilitate comparison of results.

Together these results are consistent with the hypothesis that ECM components play a significant role in regulation of the profile of Cxs expressed by alveolar epithelial cells. The data further suggest a role for cell-matrix interactions in long-term regulation of gap junction communication between cells at the alveolar surface.⁴ The latter premise remains to be explored.

At present, the observations discussed above cannot be extrapolated to deduce either the physiologic or pathophysiologic role of gap junction communication in the normal or injured lung. In this context, it is essential to recognize that patterns of Cx expression may not be reflected directly in the structure or function of gap junction channels, which can exhibit heteromeric composition, as well as selectivity in channel conductance. Furthermore, these studies are not inclusive of all Cxs expressed by alveolar epithelial cells. The present observations do, however, provide strong evidence that expression of gap junction components is regulated in alveolar epithelium at both the transcriptional and translational levels. The results thus provide a framework for further investigation of regulation of gap junction structure and function in cells of the alveolar epithelium.

Footnotes

Abbreviations: Cx = connexin; CNT = control; EFTu = elongation factor Tu; ECM = extracellular matrix; ECM-3, fibronectin-rich ECM; MG = matrigel; mRNA = messenger RNA; PL = plastic

Supported by American Heart Association grant 9750145N and National Heart, Lung, and Blood Institute grant HL-31560.

References

1. Nicholson, SM, Bruzzone, R (1997) Gap junctions: getting the message through. Curr Biol 7,R340-R344[CrossRef][ISI][Medline]
2. Abraham, V, Chou, M, DeBolt, MK, et al (1999) Phenotypic control of gap junctional communication by alveolar epithelial cells. Am J Physiol 272,L825-L834
3. Bartels, H, Oestern, H-J, Voss-Wermbter, G (1980) Communicating-occluding junction complexes in the alveolar epithelium. Am Rev Respir Dis 121,1017-1024[ISI][Medline]
4. Lee, Y-C, Yellowley, CE, Li, Z, et al (1997) Expression of functional gap junctions in cultured pulmonary alveolar epithelial cells. Am J Physiol 272,L1105-L1114[Abstract/Free Full Text]
5. Schneeberger, EE, Walters, DV, Oliver, RE (1978) Development of intercellular junctions in the pulmonary epithelium of the foetal lamb. J Cell Sci 32,307-324[Abstract]
6. Dunsmore, SE, Rannels, DE (1996) Extracellular matrix biology in the lung. Am J Physiol 270,L3-L27[Abstract/Free Full Text]
7. Rannels, SR, Fisher, CS, Heuser, LJ, et al (1987) Culture of type II pneumocytes on a type II cell-derived fibronectin-rich matrix. Am J Physiol 53,C759-C765
8. Rannels SR, Rannels DE. Isolation and culture of type II pulmonary epithelial cells. In: JE Celis, ed. Cell biology: a laboratory handbook. Orlando, FL: Academic Press, 1994; 116–123
9. Rannels, SR, Yarnell, JA, Fisher, CS, et al (1987) The role of laminin in the maintenance of type II cell morphology and function. Am J Physiol 53,C835-C845
10. Levine, RA, Serdy, M, Guo, L, et al (1993) Elongation factor Tu as a control gene for mRNA analysis of lung development and other differentiation and growth regulated systems. Nucl Acids Res 21,4426[Free Full Text]

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