Hensin remodels the apical cytoskeleton and induces columnarization of intercalated epithelial cells: processes that resemble terminal differentiation

Abstract

Intercalated epithelial cells exist in a spectrum of phenotypes; at one extreme, beta cells secrete HCO3 by an apical Cl/HCO3 exchanger and a basolateral H+ ATPase. When an immortalized beta cell line is seeded at high density it deposits in its extracellular matrix (ECM) a new protein, hensin, which can reverse the polarity of several proteins including the Cl/HCO3 exchanger (an alternately spliced form of band 3) and the proton translocating ATPase. When seeded at low density and allowed to form monolayers these polarized epithelial cells maintain the original distribution of these two proteins. Although these cells synthesize and secrete hensin, it is not retained in the ECM, but rather, hensin is present in a large number of intracellular vesicles. The apical cytoplasm of low density cells is devoid of actin, villin, and cytokeratin19. Scanning electron microscopy shows that these cells have sparse microvilli, whereas high density cells have exuberant apical surface infolding and microvilli. The apical cytoplasm of high density cells contains high levels of actin, cytokeratin19, and villin. The cell shape of these two phenotypes is different with high density cells being tall with a small cross-sectional area, whereas low density cells are low and flat. This columnarization and the remodeling of the apical cytoplasm is hensin-dependent; it can be induced by seeding low density cells on filters conditioned by high density cells and prevented by an antibody to hensin. The changes in cell shape and apical cytoskeleton are reminiscent of the processes that occur in terminal differentiation of the intestine and other epithelia. Hensin is highly expressed in the intestine and prostate (two organs where there is a continuous process of differentiation). The expression of hensin in the less differentiated crypt cells of the intestine and the basal cells of the prostate is similar to that of low density cells; i.e., abundant intracellular vesicles but no localization in the ECM. On the other hand, as in high density cells hensin is located exclusively in the ECM of the terminally differentiated absorptive villus cells and the prostatic luminal cell. These studies suggest that hensin is a critical new molecule in the terminal differentiation of intercalated cell and perhaps other epithelial cells.

Figure 1

Distribution of hensin in intercalated cells seeded at high and low densities. (A) Intercalated cells seeded at low density (left) and high density (right) were either unpermeabilized, or fixed with methanol and permeabilized with saponin, and stained with anti-hensin antibodies and a nuclear stain (Hoechst 33342). As can be seen, hensin is accessible to the anti-hensin antibodies only in the unpermeabilized high density cells, not in the low density cells. The apical cytoplasmic localization of hensin in low density cells and the extracellular localization of hensin in high density cells can be seen in two pictures (bottom) of permeabilized cells. (B) Colocalization of hensin with collagen type IV in high density cells. Unpermeabilized high and low density intercalated cells were stained with guinea pig anti-hensin polyclonal antibodies and mouse anti–collagen type IV mAb along with a nuclear stain as described in Materials and Methods. Low density cells had no extracellular deposits of hensin (red), although collagen IV (green) was visible in the basement membrane. However in high density cells both hensin and collagen IV were extracellular and colocalized (yellow). In these pictures the nuclear staining is shown in blue.

Figure 2

Biochemical characterization of the synthesis and secretion of hensin. (A) Low density and high density cells were pulse-labeled with [³⁵S]methionine for 5 min at the indicated time intervals after seeding and analyzed by immunoprecipitation as described in Materials and Methods. Densitometric analysis of the results is shown below. (B) Intercalated cells seeded at low or high density, cultured for 5 d, and labeled with [³⁵S]methionine for 12 h. Hensin was immunoprecipitated from the apical and basolateral media. Both low and high density cells secreted hensin in a polarized manner to the basolateral medium.

Figure 3

Distribution of villin in low density and high density phenotypes. Low and high density phenotype intercalated cells were stained with anti-villin mAb (red) and the nuclear stain Hoechst 33342 (green).

Figure 4

Distribution of F-actin, cytokeratin19, and villin in intercalated cells. Intercalated cells seeded at low (left) or high densities (middle) and at low density on a high density ECM (right). They were analyzed by immunocytochemistry 1 wk after seeding as described in Materials and Methods. Actin was visualized by staining with rhodamine-phalloidin, whereas villin and cytokeratin19 were stained with mAb. In the xz sections, the bottom of each image represents the level of the filter. Bar, 25 μm.

Figure 5

Immunoblot analysis of cytokeratin19 in the microfilament fraction of low and high density intercalated cells. Intermediate filaments were extracted from equal number of cells of both low and high density phenotype intercalated cells as described in Materials and Methods and probed with a mAb to cytokeratin19.

Figure 6

Distribution of E-cadherin, fodrin, ZO-1, and tubulin in intercalated cells seeded at low and high seeding densities. Intercalated cells were seeded at the indicated densities and cultured for 1 wk. The cells were fixed and stained with mAb to E-cadherin, fodrin, ZO-1, and tubulin. The xz sections of both phenotypes reveal the characteristic basolateral localizations for E-cadherin and fodrin and the apical tight junction localization for ZO-1. The tubulin network is similar in both phenotypes.

Figure 7

Scanning electron micrographs of the apical surface of intercalated cells seeded at low (LD) and high (HD) densities. Low density intercalated cell images are shown after 1 wk in culture. When cultured for an additional week they showed no change. Bars: (left and middle) 7 μm; (right) 5 μm.

Figure 8

Induction of apical cytoskeleton in low density cells grown on filters conditioned by hensin or other matrices. Intercalated cells were seeded at low density on Transwell filters conditioned with the ECM component of the high density cells or on Transwell filters treated with laminin, fibronectin, or matrigel as described in Materials and Methods. The cells were stained with Hoechst 33322 (green) and rhodamine-phalloidin (red). The top panels represent the projected image of all (8–10 sections of 1 μm each) and the bottom panels represent the xz sections.

Figure 9

Anti-hensin antibodies prevent the reorganization of the actin cytoskeleton in high density cells. High density intercalated cells were seeded in the presence or absence of polyclonal anti-hensin serum (1:100), anti–laminin mAb (30 μg/ml), and anti–collagen IV mAb (60 μg/ml). The culture media with and without the antibodies were replaced daily. Staining with rhodamine-phalloidin was performed 5 d after seeding. A 2-μm thick apical section (left) is shown. A basolateral optical section (right) of the same thickness is shown. Bar, 25 μm.

Figure 10

Distribution of hensin in small intestine and prostate gland. (Top) Rabbit small intestine was fixed, sectioned, and stained with anti-hensin antibodies. Confocal images of intestinal crypts (left) and villi (right) are shown. Note the staining pattern in crypts shows diffuse intracellular staining located mostly in the apical half of the cell. In villi, hensin is present in a basolateral pattern that surrounds the cell. (Bottom) Prostate glands: simultaneous staining with hensin (rhodamine) and cytokeratin7 (fluorescein). (Left) Red indicates the staining pattern of hensin in a typical prostate gland section. (Middle) shows the staining pattern of cytokeratin7, an exclusive marker of basal epithelia in rodent prostate in the same section. The right panel is the combined image.

Figure 10

Distribution of hensin in small intestine and prostate gland. (Top) Rabbit small intestine was fixed, sectioned, and stained with anti-hensin antibodies. Confocal images of intestinal crypts (left) and villi (right) are shown. Note the staining pattern in crypts shows diffuse intracellular staining located mostly in the apical half of the cell. In villi, hensin is present in a basolateral pattern that surrounds the cell. (Bottom) Prostate glands: simultaneous staining with hensin (rhodamine) and cytokeratin7 (fluorescein). (Left) Red indicates the staining pattern of hensin in a typical prostate gland section. (Middle) shows the staining pattern of cytokeratin7, an exclusive marker of basal epithelia in rodent prostate in the same section. The right panel is the combined image.