Expression of the innate defense receptor S5D-SRCRB in the urogenital tract.

S5D-SRCRB is a novel mouse secretory glycoprotein belonging to the ancient and highly conserved scavenger receptor cysteine-rich superfamily of protein receptors. Available evidence indicates that S5D-SRCRB interacts with conserved microbial cell wall components, as well as with some endogenous proteins, and presents a restricted tissue expression pattern. This study further analyzes the expression of S5D-SRCRB along the mouse urogenital tract. Immunohistochemical staining for S5D-SRCRB was observed in spermatocytes from seminiferous tubules and in the epithelial surface from urethra and bladder, as well as in kidney tubules, mainly from medulla and papilla. Double stainings showed that S5D-SRCRB is expressed in both principal (P) and intercalated (IC) cells from renal collecting ducts (CD). By using an in vitro cell model of IC cell differentiation, preferential expression of S5D-SRCRB was observed in the apical border of terminally differentiated IC. Colocalization of S5D-SRCRB with galectin-3 (Gal-3) was also observed in kidney and bladder, but not in testis, supporting concurrent biochemical studies demonstrating the carbohydrate-dependent interaction of Gal-3 and S5D-SRCRB. Furthermore, upregulation of S5D-SRCRB expression was observed in in vitro and in vivo models of bacterial aggression, reinforcing the emerging view that CD, and specially IC, are important players in innate defense of the urinary tract against infection. Taken together, the results indicate that S5D-SRCRB is an integral component of the urogenital tract involved in innate immune functions.

Prostatic expression of hensin, a protein implicated in epithelial terminal differentiation.

OBJECTIVES: Hensin induces terminal differentiation in rabbit kidney collecting tubule cells. Rabbit hensin and human DMBT1 result from alternative splicing of the same gene. The human DMBT1 gene is located on chromosome 10q25-26, a region often deleted in prostate cancer. In this study we examined the potential role of this gene in terminal differentiation of prostate, as well as its role in prostatic carcinogenesis. METHODS: We searched for deletions of this gene in prostatic cells cultured from cancer and benign tissues using PCR and cDNA cloning. The expression of hensin/DMBT1 in cultured cells and during prostate development was characterized by immunochemistry. RESULTS: No deletions of hensin/DMBT1 similar to those found in glioblastomas, lung and esophageal cancers were observed in prostate cancer or BPH cells. Hensin/DMBT1 protein was localized in intracellular vesicles of epithelial cells in neonatal and 6-week-old mouse prostates. By 6 weeks, hensin/DMBT1 began to localize in the basal lamina of the prostate and vas deferens. In matured 6-month-old prostates, there was extensive deposition of hensin/DMBT1 in the basal lamina. CONCLUSIONS: There is no evidence that hensin/DMBT1 is implicated in prostatic carcinogenesis. The localization of hensin/DMBT1 during maturation raises the possibility that hensin/DMBT1 is involved in terminal differentiation of the prostate and vas deferens.

LRP: a new adhesion molecule for endothelial and smooth muscle cells.

We recently generated a monoclonal antibody that disrupted the association of endothelial cells with their target location during kidney development. Here, we purified the antigen of this monoclonal antibody to homogeneity using rat mesangial cell cytosol. Sequence revealed that it is a previously identified protein, termed the "laminin receptor precursor" (LRP). We found that this protein is expressed in most tissues, but immunocytochemistry revealed that it is present largely or entirely in blood vessels where it is located underneath endothelial cells and in between smooth muscle cells of the vascular wall. Vascular smooth muscle cells such as mesangial cells produce and secrete LRP into their extracellular matrix where it is present in several molecular weight forms. Endothelial cells produce very little if any of the protein, but they bind avidly to LRP-coated dishes. Anti-LRP antibodies prevent the binding of smooth muscle cells to uncoated plates, implying that cells that secrete it use it for attachment. In an assay for heterologous cell-to-cell interaction, antibodies to LRP inhibited the binding of smooth muscle cells to endothelial cells. Maturation and differentiation of blood vessels require interaction between endothelial and smooth muscle cells. LRP is a new component of the mesangial matrix, and we propose that it is an adhesion molecule that mediates an interaction between smooth muscle cells and endothelia.

Induction of terminal differentiation in epithelial cells requires polymerization of hensin by galectin 3.

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.

mu-Protocadherin, a novel developmentally regulated protocadherin with mucin-like domains.

Branching morphogenesis is a central event during the development of kidneys, lungs, and other organs. We previously generated a monoclonal antibody, 3D2-E9, that inhibited branching morphogenesis and caused widespread apoptosis. We now report the purification of its antigen and cloning of its full-length cDNA. Its cDNA encodes an integral membrane protein that contains four cadherin-like ectodomains and a thrice tandemly repeated region enriched in threonine, serine, and proline, similar to those of mucins. We thus term this protein mu-protocadherin, reflecting the hybrid nature of its extracellular region. mu-Protocadherin is expressed in two forms that are developmentally regulated, with the shorter isoform lacking the mucin-like repeats. Expression of the long isoform in heterologous cells results in adhesion of the expressing cells, suggesting that it is a new cell adhesion molecule. mu-Protocadherin contains both N and O glycosylations. It is expressed at lateral and basal surfaces of epithelia during kidney and lung development and is located in coated pits. Colocalization of mu-protocadherin with beta-catenin was noted primarily at the junction of the lateral and basal membrane. The cytoplasmic domain contains four proline-rich regions, similar to SH3 binding regions. Thus, it is likely that adhesive interactions mediated by mu-protocadherin induce signaling events that regulate branching morphogenesis.

Phenotypic plasticity and terminal differentiation of the intercalated cell: the hensin pathway.

The intercalated cell of the collecting tubule exists in a spectrum of types. The alpha form secretes acid by an apical H(+) ATPase and a basolateral Cl:HCO(3) exchanger which is an alternatively spliced form of the red cell band 3 (kAE1), while the beta form secretes HCO(3) by having these transporters on the reverse membranes. In a clonal cell line of the beta form we found that seeding density causes this conversion. A new protein, termed hensin, was deposited in the extracellular matrix of high-density cells which on purification reversed the polarity of the transporters. Hensin also induced the expression of the microvillar protein, villin, and caused the appearance of the apical terminal web proteins, cytokeratin 19 and actin, all of which led to the development of an exuberant microvillar structure. In addition, hensin caused the beta cells to assume a columnar shape. All of these studies demonstrate that the conversion of polarity in the intercalated cell, at least in vitro, represents terminal differentiation and that hensin is the first protein in a new pathway that mediates this process. Hensin, DMBT1, CRP-ductin, and ebnerin are alternately spliced products from a single gene located on human chromosome 10q25-26, a region often deleted in several cancers, especially malignant gliomas. Hensin is expressed in many epithelial cell types, and it is possible that it plays a similarly important role in the differentiation of these epithelia as well.

One hundred years of membrane permeability: does Overton still rule?

The Overton Rule states that entry of any molecule into a cell is governed by its lipid solubility. Overton's studies led to the hypothesis that cell membranes are composed of lipid domains, which mediate transport of lipophilic molecules, and protein 'pores', which transport hydrophilic molecules. Recent studies, however, have shown that hydrophobic molecules are also transported by families of transporter proteins.

Terminal differentiation in epithelia: the Hensin pathway in intercalated cells.

The intercalated cell of the collecting tubule exists in a spectrum of types. The alpha form secretes acid by an apical H+-ATPase and a basolateral CI:HCO3 exchanger, which is an alternatively spliced form of the red cell band 3 (kAE1), and the beta form secretes HCO3 by having these transporters on the reverse membranes. In a clonal cell line of the beta form, we found that seeding density causes conversion of beta cells to the alpha form. A new protein, termed hensin, was deposited in the extracellular matrix (ECM) of high-density cells, which, on purification, reversed the polarity of the transporters. Hensin also induced the expression of the microvillar protein villin and caused the appearance of the apical terminal web proteins, cytokeratin 19 and actin; all of which led to the development of an exuberant microvillar structure. In addition, hensin caused the beta cells to assume a columnar shape. All of these studies show that the conversion of polarity in the intercalated cell, at least in vitro, represents terminal differentiation and that hensin is the first protein in a new pathway that mediates this process. Hensin, DMBT1, CRP-ductin, and ebnerin are alternately spliced products from a single gene located in human chromosome 10q25-26, a region often deleted in several cancers, especially malignant gliomas. Hensin is expressed in many epithelial cell types and it is possible that it plays a similarly important role in the differentiation of these epithelia as well.

Hensin, the polarity reversal protein, is encoded by DMBT1, a gene frequently deleted in malignant gliomas.

The band 3 anion exchanger is located in the apical membrane of a beta-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 alpha-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.

Only multimeric hensin located in the extracellular matrix can induce apical endocytosis and reverse the polarity of intercalated cells.

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.

Expression of green fluorescent protein in the ureteric bud of transgenic mice: a new tool for the analysis of ureteric bud morphogenesis.

The growth and branching of the ureteric bud is a complex process that is ultimately responsible for the organization of the collecting duct system as well as the number of nephrons in the metanephric kidney. While the genes involved in the regulation of this process have begun to be elucidated, our understanding of the cellular and molecular basis of ureteric bud morphogenesis remains rudimentary. Furthermore, the timing and sequence of branching and elongation that gives rise to the collecting system of the kidney can only be inferred from retrospective staining or microdissection of fixed preparations. To aid in the investigation of these issues, we developed strains of transgenic mice in which a green fluorescent protein (GFP) is expressed in the ureteric bud under the control of the Hoxb7 promoter. In these mice, GFP is expressed in every branch of the ureteric bud throughout renal development, and in its derivative epithelia in the adult kidney. As GFP fluorescence can be easily visualized in living tissue, this allows the dynamic pattern of ureteric bud growth and branching to be followed over several days when the kidneys are cultured in vitro. Using confocal microscopy, branching of the ureteric bud in all three dimensions can be analyzed. These mice represent an extremely powerful tool to characterize the normal patterns of ureteric bud morphogenesis and to investigate the response of the bud to growth factors, matrix elements, and other agents that regulate its growth and branching.

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

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.

Architectural patterns in branching morphogenesis in the kidney.

During kidney development, several discrete steps generate its three-dimensional pattern including specific branch types, regional differential growth of stems, the specific axes of growth and temporal progression of the pattern. The ureteric bud undergoes three different types of branching. In the first, terminal bifid type, a lateral branch arises and immediately bifurcates to form two terminal branches whose tips induce the formation of nephrons. After 15 such divisions (in humans) of this specifically renal type of branching, several nephrons are induced whose connecting tubules fuse and elongate to form the arcades. Finally, the last generations undergo strictly lateral branching to form the cortical system. The stems of these branches elongate in a highly regulated pattern. The molecular basis of these processes is unknown and we briefly review their potential mediators. Differential growth in three different axes of the kidney (cortico-medullary, dorsoventral and rostro-caudal) generate the characteristic shape of the kidney. Rapid advances in molecular genetics highlight the need for development of specific assays for each of these discrete steps, a prerequisite for identification of the involved pathways. The identification of molecules that control branching (the ultimate determinant of the number of nephrons) has acquired new urgency with the recent suggestion that a reduced nephron number predisposes humans to hypertension and to progression of renal failure.

An endothelial growth factor involved in rat renal development.

In the kidney, there is a close and intricate association between epithelial and endothelial cells, suggesting that a complex reciprocal interaction may exist between these two cell types during renal ontogeny. Thus, we examined whether metanephrogenic mesenchymal cells secrete endothelial mitogens. With an endothelial mitogenic assay and sequential chromatography of the proteins in the media conditioned by a cell line of rat metanephrogenic mesenchymal cells (7.1.1 cells), we isolated a protein whose amino acid analysis identified it as hepatoma-derived growth factor (HDGF). Media conditioned with Cos-7 cell transfected with HDGF cDNA stimulated endothelial DNA synthesis. With immunoaffinity purified antipeptide antibodies, we found that HDGF was widely distributed in the renal anlage at early stages of development but soon concentrated at sites of active morphogenesis and, except for some renal tubules, disappeared from the adult kidney. From a 7.1.1 cells cDNA library, a clone of most of the translatable region of HDGF was obtained and used to synthesize digoxigenin-labeled riboprobes. In situ hybridization showed that during kidney development mRNA for HDGF was most abundant at sites of nephron morphogenesis and in ureteric bud cells while in the adult kidney transcripts disappeared except for a small population of distal tubules. Thus, HDGF is an endothelial mitogen that is present in embryonic kidney, and its expression is synchronous with nephrogenesis.

Phenotypic plasticity in the intercalated cell: the hensin pathway.

The collecting duct of the renal tubule contains two cell types, one of which, the intercalated cell, is responsible for acidification and alkalinization of urine. These cells exist in a multiplicity of morphological forms, with two extreme types, alpha and beta. The former acidifies the urine by an apical proton-translocating ATPase and a basolateral Cl/HCO3 exchanger, which is an alternately spliced form of band 3. This kidney form of band 3, kAE1, is present in the apical membrane of the beta-cell, which has the H+-ATPase on the basolateral membrane. We had suggested previously that metabolic acidosis leads to conversion of beta-types to alpha-types. To study the biochemical basis of this plasticity, we used an immortalized cell line of the beta-cell and showed that these cells convert to the alpha-phenotype when plated at superconfluent density. At high density these cells localize a new protein, which we term "hensin," to the extracellular matrix, and hensin acts as a molecular switch capable of changing the phenotype of these cells in vitro. Hensin induces new cytoskeletal proteins, makes the cells assume a more columnar shape and retargets kAE1 and the H+-ATPase. These recent studies suggest that the conversion of beta- to alpha-cells, at least in vitro, bears many of the hallmarks of terminal differentiation.

Kanadaptin is a protein that interacts with the kidney but not the erythroid form of band 3.

Although epithelial membrane proteins are separately targeted to apical or basolateral domains, some are apically located in one cell type but are basolateral in others. More dramatically, the anion exchanger of a clonal cell line of intercalated cells derived from the kidney can be retargeted from the apical to basolateral domain. This Cl:HCO3 exchanger, kAE1, is an alternately spliced form of the erythroid anion exchanger (AE1, band 3), but unlike band 3 it does not bind ankyrin. Here we identify a new protein (kanadaptin) that binds to the cytoplasmic domain of kAE1 in vitro and in vivo but not to the erythroid AE1 or to ankyrin. No significant homologous proteins have been reported so far. Kanadaptin is widely expressed in epithelial (kidney, lung, and liver) and non-epithelial cells (brain and skeletal and cardiac muscle). In kidney, we found by immunocytochemistry that kanadaptin was only expressed in the collecting tubule. In the intercalated cells of this segment, it colocalized with kAE1 in cytoplasmic vesicles but not when the exchanger was in the basolateral membrane. These results raised the possibility that this protein is involved in the targeting of kAE1 vesicles to their final destination.