Abstract
When an intercalated epithelial cell line was seeded at low density and allowed to reach confluence, it located the anion exchanger band 3 in the apical membrane and an H+-ATPase in the basolateral membrane. The same clonal cells seeded at high density targeted these proteins to the reverse location. Furthermore, high density cells had vigorous apical endocytosis, and low density cells had none. The extracellular matrix of high density cells was capable of inducing apical endocytosis and relocation of band 3 to the basolateral membrane in low density cells. A 230-kDa extracellular matrix (ECM) protein termed hensin, when purified to near-homogeneity, was able to reverse the phenotype of the low density cells. Antibodies to hensin prevented this effect, indicating that hensin is necessary for conversion of polarity. We show here that hensin was synthesized by both low density and high density cells. Whereas both phenotypes secreted soluble hensin into their media, only high density cells localized it in their ECM. Analysis of soluble hensin by sucrose density gradients showed that low density cells secreted monomeric hensin, and high density cells secreted higher order multimers. When 35S-labeled monomeric hensin was added to high density cells, they induced its aggregation suggesting that the multimerization was catalyzed by surface events in the high density cells. Soluble monomeric or multimeric hensin did not induce apical endocytosis in low density cells, whereas the more polymerized hensin isolated from insoluble ECM readily induced it. These multimers could be disaggregated by sulfhydryl reagents and by dimethylmaleic anhydride, and treatment of high density ECM by these reagents prevented the induction of endocytosis. These results demonstrate that hensin, like several ECM proteins, needs to be precipitated in the ECM to be functional.
Highlights
IntroductionWhen the immortalized cells were seeded on filters at subconfluent density, they eventually formed epithelial monolayers capable of secreting HCO3 into the apical medium
Hensin Is a Secretory Glycoprotein That Is Retained in the extracellular matrix (ECM) of High Density Cells—Pulse-chase experiments showed that the molecular weight of hensin increased after its synthesis, and the intensity of the band eventually decreased with prolonged periods of chase (Fig. 1A)
Cells were labeled with [35S]methionine, and hensin was chased into the cell lysate, ECM, and medium in both low density and high density cells. (ECM is here defined as the guanidine-extractable material remaining on the filter after Triton solubilization.) While hensin accumulated in the ECM of high density cells, very little was seen in low density cells (Fig. 2)
Summary
When the immortalized cells were seeded on filters at subconfluent density, they eventually formed epithelial monolayers capable of secreting HCO3 into the apical medium These cells did not exhibit any apical endocytic activity; they had apical kAE1 and a basolateral Hϩ-ATPase [9]. By using the induction of apical endocytosis as an assay, we purified a protein from high density ECM that was capable of converting the polarity of the low density cells [9] Procedures that disaggregate the ECM type of multimer prevent hensin from inducing apical endocytosis in low density cells These results demonstrate that only multimeric insoluble hensin is capable of inducing endocytosis, reversing the polarity, and inducing terminal differentiation of intercalated cells
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