During terminal differentiation, epithelia become columnar and develop specialized apical membrane structures (microvilli) and functions (regulated endocytosis and exocytosis). Using a clonal intercalated epithelial cell line, we found that high seeding density induced these characteristics, whereas low density seeding maintained a protoepithelial state. When cells were plated at low density, but on the extracellular matrix of high density cells, they converted to the more differentiated phenotype. The extracellular matrix (ECM) protein responsible for this activity was purified and found to be a large 230-kD protein, which we termed hensin. High density seeding caused hensin to be polymerized and deposited in the extracellular matrix, and only this form of hensin was able to induce terminal differentiation. Antibodies to hensin blocked the change in phenotype. However, its purification to homogeneity resulted in loss of activity, suggesting that an additional protein might be necessary for induction of terminal differentiation. Here, we found that a 29-kD protein specifically associates with hensin in the ECM. Addition of purified p29 restored the activity of homogenously purified hensin. Mass fingerprinting identified p29 as galectin 3. Purified recombinant galectin 3 was able to bind to hensin and to polymerize it in vitro. Seeding cells at high density induced secretion of galectin 3 into the ECM where it bundled hensin. Hence, the high density state causes a secretion of a protein that acts on another ECM protein to allow the new complex to signal the cell to change its phenotype. This is a new mechanism of inside-out signaling.

During skeletal development, osteoblasts produce large amounts of extracellular matrix proteins and must therefore increase their secretory machinery to handle the deposition. The accumulation of unfolded protein in the endoplasmic reticulum induces an adoptive mechanism called the unfolded protein response (UPR). We show that one of the most crucial UPR mediators, inositolrequiring protein 1a (IRE1a), and its target transcription factor X-box binding protein 1 (XBP1), are essential for bone morphogenic protein 2-induced osteoblast differentiation. Furthermore, we identify Osterix (Osx, a transcription factor that is indispensible for bone formation) as a target gene of XBP1. The promoter region of the Osx gene encodes two potential binding motifs for XBP1, and we show that XBP1 binds to these regions. Thus, the IRE1a-XBP1 pathway is involved in osteoblast differentiation through promoting Osx transcription.

Intercalated epithelial cells exist in a spectrum of phenotypes; at one extreme, β cells secrete HCO3 by an apical Cl/HCO3 exchanger and a basolateral H+ ATPase. When an immortalized β 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.

Two forms of intercalated cells are present in kidney collecting tubules, the ␣ cell has apical endocytosis, apical H ϩ -ATPase and basolateral band 3, while ␤ cells have reversed polarity of these proteins and no apical endocytosis. When a ␤ cell line was seeded at high density, it changed into the ␣ form. We previously showed that a partially purified 230 kD extracellular matrix protein of high density cells was able to retarget band 3 from apical to basolateral domains and stimulated apical endocytosis in vitro (Van Adelsberg, J., J.C. Edwards, J. Takito, B. Kiss, and Q. Al-Awqati. 1994. Cell. 76:1053-1061). We now purify this protein, which was named hensin, to near homogeneity and find that it belongs to the macrophage scavenger receptor cysteine rich (SRCR) family. An antibody, generated against a fusion protein made from a partial cDNA recognized a 230-kD protein in rabbit kidney and in the intercalated cell line.

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The band 3 anion exchanger is located in the apical membrane of a β-intercalated clonal cell line, whereas the vacuolar H+-ATPase is present in the basolateral membrane. When these cells were seeded at confluent density, they converted to an α-phenotype, localizing each of these proteins to the opposite cell membrane domain. The reversal of polarity is induced by hensin, a 230-kDa extracellular matrix protein. Rabbit kidney hensin is a multidomain protein composed of eight SRCR (“scavenger receptor, cysteine rich”), two CUB (“C1r/C1s Uegf Bmp1”), and one ZP (“zona pellucida”) domain. Other proteins known to have these domains include CRP-ductin, a cDNA expressed at high levels in mouse intestine (8 SRCR, 5 CUB, 1 ZP), ebnerin, a protein cloned from a rat taste bud library (4 SRCR, 3 CUB, 1 ZP), and DMBT1, a sequence in human chromosome 10q25–26 frequently deleted in malignant gliomas (9 SRCR, 2 CUB, 1 ZP). Rabbit and mouse hensin genomic clones contained a new SRCR that was not found in hensin cDNA but was homologous to the first SRCR domain in DMBT1. Furthermore, the 3′-untranslated regions and the signal peptide of hensin were homologous to those of DMBT1. Mouse genomic hensin was localized to chromosome 7 band F4, which is syntenic to human 10q25–26. These data suggest that hensin and DMBT1 are alternatively spliced forms of the same gene. The analysis of mouse hensin bacterial artificial chromosome (BAC) genomic clone by sequencing and Southern hybridization revealed that the gene also likely encodes CRP-ductin. A new antibody against the mouse SRCR1 domain recognized a protein in the mouse and rabbit brain but not in the immortalized cell line or kidney, whereas an antibody to SRCR6 and SRCR7 domains which are present in all the transcripts, recognized proteins in intestine, kidney, and brain from several species. The most likely interpretation of these data is that one gene produces at least three transcripts, namely, hensin, DMBT1, and CRP-ductin. Hensin may participate in determining the polarized phenotype of other epithelia and brain cells.

SummaryMultinucleated osteoclasts are responsible for bone resorption. Hypermultinucleated osteoclasts are often observed in some bone-related diseases such as Paget's disease and cherubism. The cellular mechanics controlling the size of osteoclasts is poorly understood. We introduced EGFP-actin into RAW 264.7 cells to monitor actin dynamics during osteoclast differentiation. Before their terminal differentiation into osteoclasts, syncytia displayed two main types of actin assembly, podosome clusters and clusters of zipper-like structures. The zipper-like structures morphologically resembled the adhesion zippers found at the initial stage of cell-cell adhesion in keratinocytes. In the zipper-like structure, Arp3 and cortactin overlapped with the distribution of dense F-actin, whereas integrin b3, paxillin and vinculin were localized to the periphery of the structure. The structure was negative for WGA-lectin staining and biotin labeling. The zipper-like structure broke down and transformed into a large actin ring, called a podosome belt. Syncytia containing clusters of zipper-like structures had more nuclei than those with podosome clusters. Differentiated osteoclasts with a podosome belt also formed the zipper-like structure at the cell contact site during cell fusion. The breakdown of the cell contact site resulted in the fusion of the podosome belts following plasma membrane fusion. Additionally, osteoclasts in mouse calvariae formed the zipper-like structure in the sealing zone. Therefore, we propose that the zipper-like actin superstructures might be involved in cell-cell interaction to achieve efficient multinucleation of osteoclasts. Understanding of the zipper-like structure might lead to selective therapeutics for bone diseases caused by hypermultinucleated osteoclasts.

Osteoclasts form a specialized cell–matrix adhesion structure, known as the “sealing zone”, during bone resorption. The sealing zone is a dynamic actin-rich structure that defines the resorption area of the bone. The detailed dynamics and fine structure of the sealing zone have been elusive. Osteoclasts plated on glass do not form a sealing zone, but generate a separate supra-molecular structure called the “podosome belt”. Podosomes are integrin-based adhesion complexes involved in matrix adhesion, cell migration, matrix degradation, and mechanosensing. Invadopodia, podosome-like protrusions in cancer cells, are involved in cell invasion into other tissues by promoting matrix degradation. Both podosomes and invadopodia exhibit actin pattern transitions during maturation. We previously found that Arp2/3-dependent actin flow occurs in all observed assembly patterns of podosomes in osteoclasts on glass. It is known that the actin wave in Dictyostelium cells exhibits a similar pattern transition in its evolution. Because of significant advances in our understanding regarding the mechanism of podosomes/invadopodia formation over the last decade, we revisited the structure and function of the sealing zone in this review, highlighting the possible involvement of self-organized actin waves in the organogenesis of the sealing zone.