Cytoplasmic determinants involved in direct lysosomal sorting, endocytosis, and basolateral targeting of rat lgp120 (lamp-I) in MDCK cells.

Rat lysosomal glycoprotein 120 (lgp120; lamp-I) is a transmembrane protein that is directly delivered from the trans-Golgi network (TGN) to the endosomal/lysosomal system without prior appearance on the cell surface. Its short cytosolic domain of 11 residues encodes determinants for direct lysosomal sorting, endocytosis and, in polarized cells, basolateral targeting. We now characterize the structural requirements in the cytosolic domain required for sorting of lgp120 into the different pathways. Our results show that the cytoplasmic tail is sufficient to mediate direct transport from the trans-Golgi network (TGN) to lysosomes and that a G7-Y8-X-X-I11 motif is crucial for this sorting event. While G7 is only critical for direct lysosomal sorting in the TGN, Y8 and I11 are equally important for lysosomal sorting, endocytosis, and basolateral targeting. Thus, a small motif of five amino acids in the cytoplasmic tail of lgp120 can be recognized by the sorting machinery at several cellular locations and direct the protein into a variety of intracellular pathways.

Expression of macrophage-lymphocyte Fc receptors in Madin-Darby canine kidney cells: polarity and transcytosis differ for isoforms with or without coated pit localization domains.

Many cells of the immune system and certain epithelia express receptors for the Fc domain of IgG (FcR). On mouse macrophages and lymphocytes, two distinct receptor isoforms have been identified, designated FcRII-B1 and FcRII-B2. The isoforms are identical except for an in-frame insertion of 47 amino acids in the cytoplasmic tail of FcRII-B1 that blocks its ability to be internalized by clathrin-coated pits. We have recently found that at least one IgG-transporting epithelium, namely placental syncytial trophoblasts, expresses transcripts encoding a receptor similar or identical to macrophage-lymphocyte FcRII. To determine whether FcRII of hematopoietic cells might also function as a transcytotic receptor if expressed in epithelial cells, FcRII-B1 and -B2 were transfected into Madin-Darby canine kidney (MDCK) cells and grown on permeable filter units. The two FcRII isoforms exhibited different patterns of polarized expression: FcRII-B1 was localized mainly to the apical plasma membrane domain, whereas FcRII-B2 was found predominantly on the basolateral surface. As expected for FcR in placenta, FcRII-B2 and to a lesser extent FcRII-B1 mediated transcellular transport of IgG-complexes from the apical to the basolateral plasma membrane. Neither receptor mediated transcytosis in the opposite direction, although FcRII-B2 also delivered ligand to lysosomes when internalized from either the basolateral or apical domains. Furthermore, FcRII-B2 was capable of transporting monovalent antireceptor antibody Fab fragments across the cell, suggesting that transcytosis was not dependent on receptor cross-linking. These findings suggest the possibility that FcRII can mediate transepithelial IgG transport when expressed in placental syncytial trophoblasts in addition to its "classical" endocytic and signaling activities when expressed in macrophages. Because FcRII-B1 and -B2 are expressed with distinct polarities, the results also suggest that interactions with clathrin-coated pits may play a role in generating the polarized distribution of at least some plasma membrane proteins in MDCK cells.

Fc receptor endocytosis is controlled by a cytoplasmic domain determinant that actively prevents coated pit localization.

Macrophages and B-lymphocytes express two major isoforms of Fc receptor (FcRII-B2 and FcRII-B1) that exhibit distinct capacities for endocytosis. This difference in function reflects the presence of an in-frame insertion of 47 amino acids in the cytoplasmic domain of the lymphocyte isoform (FcRII-B1) due to alternative mRNA splicing. By expressing wild type and mutant FcRII cDNAs in fibroblasts, we have now examined the mechanism by which the insertion acts to prevent coated pit localization and endocytosis. We first identified the region of the FcRII-B2 cytoplasmic domain that is required for rapid internalization. Using a biochemical assay for endocytosis and an immuno-EM assay to determine coated pit localization directly, we found that the distal half of the cytoplasmic domain, particularly a region including residues 18-31, as needed for coated pit-mediated endocytosis. Elimination of the tyrosine residues at position 26 and 43, separately or together, had little effect on coated pit localization and a partial effect on endocytosis of ligand. Since the FcRII-B1 insertion occurs in the membrane-proximal region of the cytoplasmic domain (residue 6) not required for internalization, it is unlikely to act by physically disrupting the coated pit localization determinant. In fact, the insertion was found to prevent endocytosis irrespective of its position in the cytoplasmic tail and appeared to selectively exclude the receptor from coated regions. Moreover, receptors bearing the insertion exhibited a temperature- and ligand-dependent association with a detergent-insoluble fraction and with actin filaments, perhaps in part explaining the inability of FcRII-B1 to enter coated pits.

Intracellular routing of wild-type and mutated polymeric immunoglobulin receptor in hippocampal neurons in culture.

Certain epithelial cells synthesize the polymeric immunoglobulin receptor (pIgR) to transport immunoglobulins (Igs) A and M into external secretions. In polarized epithelia, newly synthesized receptor is first delivered to the basolateral plasma membrane and is then, after binding the Ig, transcytosed to the apical plasma membrane, where the receptor-ligand complex is released by proteolytic cleavage. In a previous work (Ikonen et al., 1993), we implied the existence of a dendro-axonal transcytotic pathway for the rabbit pIgR expressed in hippocampal neurons via the Semliki Forest Virus (SFV) expression system. By labeling surface-exposed pIgR in live neuronal cells, we now show (a) internalization of the receptor from the dendritic plasma membrane to the dendritic early endosomes, (b) redistribution of the receptor from the dendritic to the axonal domain, (c) inhibition of this movement by brefeldin A (BFA) and (d) stimulation by the activation of protein kinase C (PKC) via phorbol myristate acetate (PMA). In addition, we show that a mutant form of the receptor lacking the epithelial basolateral sorting signal is directly delivered to the axonal domain of hippocampal neurons. Although this mutant is internalized into early endosomes, no transcytosis to the dendrites could be observed. In epithelial Madin-Darby Canine Kidney (MDCK) cells, the mutant receptor could also be internalized into basolaterally derived early endosomes. These results suggest the existence of a dendro-axonal transcytotic pathway in neuronal cells which shares similarities with the basolateral to apical transcytosis in epithelial cells and constitute the basis for the future analysis of its physiological role.

Vitamin D--dependent calcium binding protein: immunocytochemical localization in chick kidney.

A vitamin D--dependent calcium binding protein in the chick kidney that was detected by immunocytochemical techniques was localized exclusively in the distal convoluted tubule, the initial collecting tubule, and the early part of the collecting tubule. The intercalated (mitochondria-rich) cells in these tubular segments were negative for the calcium binding protein. Subcellularly, the protein was found in the cytosol and the nucleus of the tubular cells. The results suggest a role for vitamin D--dependent calcium binding protein in intracellular calcium metabolism rather than a direct involvement in membrane-mediated calcium reabsorption in the avian kidney.

Cytoplasmic domain heterogeneity and functions of IgG Fc receptors in B lymphocytes.

B lymphocytes and macrophages express closely related immunoglobulin G (IgG) Fc receptors (Fc gamma RII) that differ only in the structures of their cytoplasmic domains. Because of cell type-specific alternative messenger RNA splicing, B-cell Fc gamma RII contains an insertion of 47 amino acids that participates in determining receptor function in these cells. Transfection of an Fc gamma RII-negative B-cell line with complementary DNA's encoding the two splice products and various receptor mutants indicated that the insertion was responsible for preventing both Fc gamma RII-mediated endocytosis and Fc gamma RII-mediated antigen presentation. The insertion was not required for Fc gamma RII to modulate surface immunoglobulin-triggered B-cell activation. Instead, regulation of activation involved a region of the cytoplasmic domain common to both the lymphocyte and macrophage receptor isoforms. In contrast, the insertion did contribute to the formation of caps in response to receptor cross-linking, consistent with suggestions that the lymphocyte but not macrophage form of the receptor can associate with the detergent-insoluble cytoskeleton.

Intracellular calcium-binding proteins: more sites than insights.

Calcium ions as biological regulators exert their effects in part via interaction with a wide variety of intracellular calcium-binding proteins. One class of these proteins shares a common calcium-binding motif, the EF-hand. A consensus amino acid sequence for this motif has aided the identification of new members of this family of EF-hand proteins, which now has about 170 members. A few of these proteins are present in all cells, whereas the vast majority are expressed in a tissue-specific fashion. The physiological function of a few of these proteins is known to be achieved via a calcium-dependent interaction with other proteins, thereby regulating their activity. The elucidation of the interactions and functions of the majority of these proteins remains a challenging task for the coming years.

Transcytosis of the polymeric immunoglobulin receptor in cultured hippocampal neurons.

BACKGROUND: A wide variety of proteins are transported across epithelial cells by vesicular carriers. This process, transcytosis, is used to generate cell surface polarity and to transport macromolecules between the luminal and serosal sides of the epithelial layer. The polymeric immunoglobulin receptor is a well-characterized transcytotic molecule in epithelia. It binds to its ligand, polymeric immunoglobulin, at the basolateral surface, and the receptor-ligand complex is transcytosed to the apical surface, where the ligand is released. Our previous studies have shown that hippocampal neurons may employ mechanisms similar to those of epithelial cells to sort proteins to two plasma membrane domains. The machinery used for axonal delivery recognizes proteins that are targeted apically in epithelia, whereas basolaterally destined proteins are delivered to the dendrites. It has not been clear, however, whether transcytosis occurs in neurons. RESULTS: We report expression of the polymeric immunoglobulin receptor in cultured hippocampal neurons, using a Semliki Forest Virus expression system, and show by immunofluorescence microscopy that the newly synthesized receptor is targeted from the Golgi complex predominantly to the dendrites - only about 20% of the infected neurons display axonal immunofluorescence. Addition of ligand leads to significant redistribution of the receptor to the axons, shown by an approximately three-fold increase in axonal immunoreactivity with the anti-receptor antibodies. CONCLUSIONS: Our results suggest that a transcytotic route, analogous to that in epithelia, exists in neurons, where it transports proteins from the somatodendritic to the axonal domain. Cultured neurons expressing the polymeric immunoglobulin receptor offer an experimental system that should be useful for further characterization of this novel neuronal pathway at the molecular and functional level.

Effect of vitamin D status on the equilibrium between occupied and unoccupied 1,25-dihydroxyvitamin D intestinal receptors in the chick.

The dynamic equilibrium between in vivo occupied and unoccupied 1,25-dihydroxyvitamin D(3)[1,25(OH)(2)D(3)] receptors of the chick intestinal mucosa was investigated by the exchange assay previously reported [(1980). J. Biol. Chem.255: 9534-9537]. These parameters and their correlation to biological response, i.e., the levels of intestinal vitamin D-dependent calcium binding protein (CaBP), were assessed under different physiological conditions. After a single 1,25(OH)(2)D(3) injection (3.25 nmol), occupied receptor levels increased sharply to a maximum between 1 and 2 h, followed by a rapid decline. A single dose of 1alpha-hydroxy-vitamin D(3) [1alpha(OH)D(3)], an analog that requires 25-hydroxylation for biological activity, resulted in a protracted, albeit lower, response with maximal receptor occupancy at 6 h and half maximal levels 24 h after injection. The intestinal receptor occupancy patterns mirrored the serum 1,25(OH)(2)D(3) levels after either 1,25(OH)(2)D(3) or 1alpha(OH)D(3) treatment. Additionally, time-course (half-life) of blood disappearance of 1,25(OH)(2)D(3) and occupied receptor levels were similar (1.9 and 2.3 h, respectively), suggesting that the amount of occupied 1,25(OH)(2)D(3) receptor is determined by a simple equilibrium between serum 1,25(OH)(2)D(3) and unoccupied receptors. A dose-response study after intramuscular 1,25(OH)(2)D(3) injection yielded a hyperbolic curve with an apparent plateau at 70% receptor occupancy, corresponding to 5 nmol 1,25(OH)(2)D(3) injected. Half-maximal occupancy was reached after a dose of 1 nmol 1,25(OH)(2)D(3), corresponding to 1.5 ng 1,25(OH)(2)D(3)/ml serum. From this value the apparent K(d) in vivo is 3.7 nM, which is similar to that determined in vitro. A 10-fold increase in the 1alpha(OH)D(3) dose resulted in less than a doubling of the levels of serum 1,25(OH)(2)D(3), occupied 1,25(OH)(2)D(3) receptors, or CaBP. Under all experimental conditions, there was a positive correlation between occupied receptor and CaBP levels; however, the slope of the lines depended on the times chosen for the assays due in part to the lag period for CaBP induction and its accumulation within the cell. Conversely, the correlation between serum 1,25-(OH)(2)D(3) levels and occupied receptor levels yielded a single regression line independent of the observation time. Short and long-term treatment with different vitamin D metabolites, estrogen, progesterone, or cortisol did not affect the levels of total intestinal 1,25(OH)(2)D(3) receptor. Under normal physiological conditions, only 10-15% of the total 1,25(OH)(2)D(3) receptor population was occupied by ligand. These studies provide a basis for further investigations of physiological and biochemical parameters of the vitamin D endocrine system and their clinical applications.

Parathyroid hormone responses to catecholamines and to changes of extracellular calcium in cows.

Modifications of the plasma level of immunoreactive parathyroid hormone (PTH) in cattle were induced by changes of the plasma concentrations of epinephrine, isoproterenol, or calcium. During abrupt hypocalcemia, PTH, obtained by infusions with ethylene glycol-bis (beta-aminoethylether) N, N'-tetraacetate (EGTA), increased during the first 4-8 min. After a transient decline, the hormone levels rose again and remained elevated. Infusions of calcium suppressed the hypocalcemia-induced augmentation of PTH levels within a few minutes. Prolonged epinephrine (and isoproterenol) infusions also rapidly increased PTH levels, however, in this case, they returned to basal concentrations after 50-60 min. Additional epinephrine infusions could not further raise PTH values. Moreover, three short-lasting infusions of epinephrine (7 min each), given at 30-min intervals, increased PTH levels to the same extent, whereas additional infusions were much less effective. The PTH response to epinephrine was completely restored, when the interval after a prolonged epinephrine infusion had been prolonged to > 100 min. During moderate hypocalcemia, occurring at the end of EGTA infusions and lasting for 90 min, the PTH response to a short-lasting epinephrine infusion (7 min) was more pronounced than in normocalcemic animals. During severe hypocalcemia, in which superimposed short-lasting infusions of EGTA (7 min) led to an additional abrupt fall of plasma calcium concentrations but not to a corresponding additional rise of the PTH levels, epinephrine rapidly and further increased PTH concentrations. On the other hand, at the end of prolonged infusions of epinephrine, when additional infusions of epinephrine were ineffective in raising PTH levels, EGTA-induced hypocalcemia consistently increased PTH concentrations. The EGTA-induced augmentation of PTH levels was enhanced by epinephrine and isoproterenol but not by propranolol. The present findings indicate, that variations of the extracellular calcium concentrations and beta-adrenergic agonists modify PTH levels by two different and independent mechanisms. On the other hand, it appears that the magnitude of change of the PTH levels to either stimulus is partially modulated by exposure to the other.

Basolateral sorting of furin in MDCK cells requires a phenylalanine-isoleucine motif together with an acidic amino acid cluster.

Furin is a subtilisin-related endoprotease which processes a wide range of bioactive proteins. Furin is concentrated in the trans-Golgi network (TGN), where proteolytic activation of many precursor proteins takes place. A significant fraction of furin, however, cycles among the TGN, the plasma membrane, and endosomes, indicating that the accumulation in the TGN reflects a dynamic localization process. The cytosolic domain of furin is necessary and sufficient for TGN localization, and two signals are responsible for retrieval of furin to the TGN. A tyrosine-based (YKGL) motif mediates internalization of furin from the cell surface into endosomes. An acidic cluster that is part of two casein kinase II phosphorylation sites (SDSEEDE) is then responsible for retrieval of furin from endosomes to the TGN. In addition, the acidic EEDE sequence also mediates endocytic activity. Here, we analyzed the sorting of furin in polarized epithelial cells. We show that furin is delivered to the basolateral surface of MDCK cells, from where a significant fraction of the protein can return to the TGN. A phenylalanine-isoleucine motif together with the acidic EEDE cluster is required for basolateral sorting and constitutes a novel signal regulating intracellular traffic of furin.

The recycling endosome of Madin-Darby canine kidney cells is a mildly acidic compartment rich in raft components.

We present a biochemical and morphological characterization of recycling endosomes containing the transferrin receptor in the epithelial Madin-Darby canine kidney cell line. We find that recycling endosomes are enriched in molecules known to regulate transferrin recycling but lack proteins involved in early endosome membrane dynamics, indicating that recycling endosomes are distinct from conventional early endosomes. We also find that recycling endosomes are less acidic than early endosomes because they lack a functional vacuolar ATPase. Furthermore, we show that recycling endosomes can be reached by apically internalized tracers, confirming that the apical endocytic pathway intersects the transferrin pathway. Strikingly, recycling endosomes are enriched in the raft lipids sphingomyelin and cholesterol as well as in the raft-associated proteins caveolin-1 and flotillin-1. These observations may suggest that a lipid-based sorting mechanism operates along the Madin-Darby canine kidney recycling pathway, contributing to the maintenance of cell polarity. Altogether, our data indicate that recycling endosomes and early endosomes differ functionally and biochemically and thus that different molecular mechanisms regulate protein sorting and membrane traffic at each step of the receptor recycling pathway.

Overexpression of the human neonatal Fc-receptor alpha-chain in trophoblast-derived BeWo cells increases cellular retention of beta2-microglobulin.

Major histocompatibility complex (MHC) class I and MHC class I-type molecules such as the neonatal Fcgamma-receptor, FcRn, are heterodimers consisting of a transmembrane alpha-chain non-covalently associated with beta2-microglobulin (beta2m). Human placental villous syncytiotrophoblast (STB) lacks MHC class I molecules, but express hFcRn that mediates materno-fetal transmission of immunoglobulin G (IgG). Trophoblast-derived BeWo cells that are used to study placental IgG transport likewise express beta2m and low levels of hFcRn alpha-chain. The contribution of FcRn alpha-chain in retention and subcellular distribution of beta2m in STB and BeWo cells is unclear. To investigate this issue, we increased expression of hFcRn alpha-chain in BeWo cells (BeWo/hFcRn) by cDNA transfection. Overexpressed hFcRn protein exhibited the characteristic pH-dependent IgG binding and association with beta2m. In comparison to parental BeWo cells, beta2m mRNA levels in BeWo/hFcRn cells were not significantly altered, but total cell-associated beta2m protein was increased by 120%. Treatment of BeWo and BeWo/hFcRn cells with brefeldin A, an inhibitor of the secretory pathway, abrogated this effect, demonstrating that hFcRn alpha-chain expression retained otherwise secreted beta2m. Flow cytometry revealed that beta2m plasma membrane expression was unaffected by alpha-chain overexpression whereas by fluorescence microscopy a preferential staining of beta2m in peripheral endosomes was observed.

Rat brain calbindin D28: six domain structure and extensive amino acid homology with chicken calbindin D28.

Calbindin D28 cDNA clones were isolated from a rat brain library using a chicken intestinal Calbindin D28 cDNA probe. Nucleotide sequence analysis of these clones shows an open reading frame of 78 nucleotide coding for a 261 amino acid 29,994 dalton protein. The predicted amino acid sequence contains six repeats of a domain with the feature of an EF-hand calcium binding site. In domains II and VI, two of the five oxygen-containing amino acids important for the coordination of calcium are absent, suggesting that these two sites have lost their calcium-binding capability. Comparing the amino acid sequence to that recently reported for the chicken Calbindin D28 there is 79% homology. Tolerating conservative differences, the homology increases to 93%. Interestingly, domains II and VI which have presumably lost their calcium binding ability are very conserved among the two species (81% and 78%, respectively). Since an EF hand calcium binding site requires only certain types of amino acids at certain positions, rather than a specific amino acid sequence, maintaining a calcium binding site is a weak conservation pressure. To explain the high degree of homology of rat and chicken Calbindin D28, and in particular the conservation of the two degenerated domains over the 300 million years since divergence of birds and mammals, additional function(s) of the Calbindin D28 are postulated.

Anaerobic digestion of slaughterhouse waste with UF-membrane separation and recycling of permeate after free ammonia stripping.

Anaerobic digestion can adapt to free ammonia to a certain extent. During the anaerobic digestion of slaughterhouse waste, however, an ammonia concentration of up to 15 g Nl(-1) can be reached in the sludge liquid and this will even inhibit adapted sludge. To lower this concentration, a fraction of the digester liquid must therefore be continuously separated from the digested sludge and the free ammonia stripped before the liquid is recycled to the digester. A mesophilic laboratory digester was successfully operated with an ammonium concentration of 4-5g l(-1) and a pH of 8.0-8.4. After free ammonia stripping, the excess liquid was treated in a laboratory SBR for nitrogen and phosphorus removal before being added to the receiving water. The effluent had no toxic effect on daphnia and algae.

Dimeric recombinant IgA directed against carcino-embryonic antigen, a novel tool for carcinoma localization.

Carcinoembryonic antigen (CEA) has been shown to be one of the best markers for in vivo tumor targeting of radiolabeled antibodies, despite the fact that it is localized predominantly at the apical side of human colon carcinoma cells within the fairly closed pseudolumen structures formed by these tumors. Due to this particular histological localization, a large proportion of the CEA molecules may remain inaccessible to the intravenously injected radiolabeled anti-CEA antibodies of IgG isotype, which are widely used in the clinic. In order to improve targeting, we made a recombinant dimeric IgA, which should have the capacity to translocate from the basolateral to the apical side of the pseudolumen formed by colon carcinoma cells after binding to the polyIg receptor (pIgR). A genomic chimeric mouse-human IgA2 construct was made using one of our most specific anti-CEA hybridomas, CE-25. The chimeric IgA (chIgA) was expressed in the Sp2/0 myeloma cell line. The secreted recombinant antibody was found to consist mostly of a dimeric form of IgA with a molecular weight of about 350 kDa. The dimeric chIgA was shown to translocate efficiently in vitro across a monolayer of epithelial cells expressing the pIgR and to retain full CEA binding activity.

Absence of calbindin-D28 expression in nonclassical 1,25-dihydroxyvitamin D targets: analysis by polymerase chain reaction.

CaBP-D28 mRNA expression in rat heart, testis, and lung was assessed by polymerase chain reaction (PCR). The animal model used was the hyperinduced vitamin D-treated rat (100 ng 1,25-dihydroxyvitamin D subcutaneously, daily for 7 days). For the PCR studies, two pairs of 20 mer oligonucleotide primers (designated 1-4 according to their position on the coding strand, but with primers 3 and 4 in reverse orientation) derived from the rat CaBP-D28 cDNA sequence were tested in various combinations. Optimal conditions were established using a 1:100 dilution of cDNA from normal rat kidney. Bands of the predicted sizes of 869 (1, 3), 994 (1, 4), 725 (2, 3), and 850 (2, 4) nucleotide base pairs resulted, but with varying intensities: 2,4 approximately 1,3 > 1,4 > 2,3. Repeat PCR (recycling after 1:100 dilution and readdition of reagents and primers with at least one different primer) provided strong additional amplification, particularly with the 1,4/2,4 combination. Under these conditions, mixing experiments showed that CaBP-D28 transcripts were detectable at 10(-7)- to 10(-9)-fold lower levels of expression than in D+ kidney. When RNA was isolated and cDNA generated from test tissues from 4 individual vitamin D-stimulated (D+) and vitamin D-deficient (D-) rats, repeat PCR (1,4/2,4 primer combination) provided no evidence of significant CaBP-D28 mRNA expression in the nonclassic target tissues, in contrast to strong bands in both the D- kidney (undiluted) and D+ kidney (1:100 dilution) preparations.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of a polyclonal antiserum against the purified human recombinant calcium binding protein calretinin.

We have purified recombinant human calretinin (CR) from Escherichia coli lysates and have produced a polyclonal antiserum against it. The antiserum recognizes determinants conserved in fish, chicken, rat, monkey and human CR. We show its use in the qualitative detection of CR by different methods of immunohistochemistry as well as in the detection of CR on immunoblots.

The calcium-binding protein calretinin-22k, an alternative splicing product of the calretinin gene is expressed in several colon adeno carcinoma cell lines.

An alternatively spliced mRNA for the calcium-binding protein calretinin (CR) is present in the colon adenocarcinoma cell line WiDr. As a consequence of a frame shift, the resulting protein, calretinin-22k (CR-22k), consists of the first 178 amino acids of calretinin followed by a carboxy-terminal peptide of 14 amino acids that is not present in full-length calretinin. Antibodies specific for this C-terminal region have been generated by 2 different methods. A peptide corresponding to the specific C-terminal region of CR-22k was either chemically synthesized and coupled to a carrier protein or was expressed in Escherichia coli as a carboxyterminal fusion to a carrier protein applying recombinant techniques. Both antisera produced in rabbits were tested in Western blots and immuno-histochemical experiments. The antisera recognized human recombinant CR-22k overexpressed in E. coli, but not fulllength calretinin and stained fixed WiDr cells. The presence of CR-22k was also confirmed in the colon cell lines CO115/3 in which mRNA coding for CR-22k mRNA coding for CR-22k mRNA is present as well as in the lines COLO205 and LS-180, all of which also express full-length calretinin. Although the intracellular distribution of CR-22k and CR are similar as evidenced by immunohistochemical stainings, CR-22k is preferentially localized in the nucleus in the cell lines LS-180 and Co115/3 suggesting potentially different roles for the two proteins.

The phytoestrogen genistein enhances osteogenesis and represses adipogenic differentiation of human primary bone marrow stromal cells.

In the present study, we investigated the role of the phytoestrogen genistein and 17beta-estradiol in human bone marrow stromal cells, undergoing induced osteogenic or adipogenic differentiation. Profiling of estrogen receptors (ERs)-alpha, -beta1, -beta2, -beta3, -beta4, -beta5, and aromatase mRNAs revealed lineage-dependent expression patterns. During osteogenic differentiation, the osteoblast-determining core binding factor-alpha1 showed a progressive increase, whereas the adipogenic regulator peroxisome proliferator-activated receptor gamma (PPARgamma) was sequentially decreased. This temporal regulation of lineage-determining marker genes was strongly enhanced by genistein during the early osteogenic phase. Moreover, genistein increased alkaline phosphatase mRNA levels and activity, the osteoprotegerin:receptor activator of nuclear factor-kappaB ligand gene expression ratio, and the expression of TGFbeta1. During adipogenic differentiation, down-regulation in the mRNA levels of PPARgamma and CCAAT/enhancer-binding protein-alpha at d 3 and decreased lipoprotein lipase and adipsin mRNA levels at d 21 were observed after genistein treatment. This led to a lower number of adipocytes and a reduction in the size of their lipid droplets. At d 3 of adipogenesis, TGFbeta1 was strongly up-regulated by genistein in an ER-dependent manner. Blocking the TGFbeta1 pathway abolished the effects of genistein on PPARgamma protein levels and led to a reduction in the proliferation rate of precursor cells. Overall, genistein enhanced the commitment and differentiation of bone marrow stromal cells to the osteoblast lineage but did not influence the late osteogenic maturation markers. Adipogenic differentiation and maturation, on the other hand, were reduced by genistein (and 17beta-estradiol) via an ER-dependent mechanism involving autocrine or paracrine TGFbeta1 signaling.