Growth of choroid plexus epithelium vesicles in vitro depends on secretory activity

Although a number of models have been used to study choroid plexus epithelium (CPe) function, analysis in physiological conditions of this polarised epithelium which produces the majority of the cerebrospinal fluid (CSF) and is one of the key barriers between blood and CSF in the brain remains challenging. As CPe cells form polarised CPe vesicles when cultured in Matrigel, we have assessed their behaviour and potential use for pharmacological studies. Like CPe cells in vivo, CPe vesicles express transthyretin, E2f5, Fox-j1 and p73, and contain tight junctions, as indicated by ZO-1 expression and electron microscopy analysis. Time-lapse microscopy shows that CPe cells plated in Matrigel are highly migratory and rapidly form homotypic cell aggregates, which then reorganise to form vesicles whose size increases linearly overtime. Neither aggregate nor vesicle size is affected by AraC treatment, though this inhibitor significantly reduces proliferation in CPe monolayers. Increase in size of vesicles, which have reached a growth plateau is observed following addition of fluorescently-labelled CPe cells, which become incorporated into the vesicle walls. Significantly, treatment with secretion inhibitors blocks vesicle formation and their expansion. These results show that secretion, rather than cell division, controls vesicle growth, consistent with low levels of proliferation and thinning of the CPe observed both in growing vesicles and during CPe development. Therefore, changes in vesicle size can be used to evaluate the effect of putative molecules involved in the regulation of secretion.     

Complexity and developmental changes in the expression pattern of claudins at the blood–CSF barrier

The choroid plexus epithelium controls the movement of solutes between the blood and the cerebrospinal fluid. It has been considered as a functionally more immature interface during brain development than in adult. The anatomical basis of this barrier is the interepithelial choroidal junction whose tightness has been attributed to the presence of claudins. We used quantitative real-time polymerase chain reaction, Western blot and immunohistochemistry to identify different claudins in the choroid plexuses of developing and adult rats. Claudin-1, -2, and -3 were highly and selectively expressed in the choroid plexus as compared to brain or parenchyma microvessels and were localized at epithelial junctions. Claudin-6, -9, -19, and -22 also displayed a previously undescribed choroidal selectivity, while claudin-4, -5, and -16 were enriched in the cerebral microvessels. The choroidal pattern of tight junction protein expression in prenatal brains was already complex and included occludin and zonula occludens proteins. It differed from the adult pattern in that the pore-forming claudin-2, claudin-9, and claudin-22 increased during development, while claudin-3 and claudin-6 decreased. Claudin-2 and claudin-11 presented a mirror image of abundance between lateral ventricle and fourth ventricle choroid plexuses. Imunohistochemical analysis of human fetal and postnatal brains for claudin-1, -2, and -3 demonstrated their early presence and localization at the apico-lateral border of the choroid plexus epithelial cells. Overall, choroidal epithelial tight junctions are already complex in developing brain. The observed differences in claudin expression between developing and adult choroid plexuses may indicate developmental differences in selective blood–cerebrospinal fluid transport functions.     

ErbB2/Neu-induced, cyclin D1-dependent transformation is accelerated in p27-haploinsufficient mammary epithelial cells but impaired in p27-null cells

ErbB2/Neu destabilizes the cyclin-dependent kinase (Cdk) inhibitor p27 and increases expression of cyclin D1. Therefore, we studied the roles of p27 and cyclin D1 in ErbB2-mediated mammary epithelial cell transformation. Overexpression of ErbB2 or cyclin D1 in p27(+/-) primary murine mammary epithelial cells resulted in increased proliferation, cyclin D1 nuclear localization, and colony formation in soft agar compared to those in p27(+/+) cells. In contrast, ErbB2- or cyclin D1-overexpressing p27(-/-) cells displayed reduced proliferation, anchorage-independent growth, Cdk4 activity, cyclin D1 expression, and cyclin D1 nuclear localization compared to wild-type cells. A cyclin D1 mutation in its nuclear export sequence (T286A) partially rescued nuclear localization of cyclin D1 in p27(-/-) cells but did not increase proliferation or Cdk4 kinase activity. Overexpression of E2F1, however, increased proliferation to the same degree in p27(+/+), p27(+/-), and p27(-/-) cells. Mammary glands from MMTV (mouse mammary tumor virus)-neu/p27(+/-) mice exhibited alveolar hyperplasia, enhanced proliferation, decreased apoptosis, and accelerated tumor formation compared to MMTV-neu/p27(+/+) glands. However, MMTV-neu/p27(-/-) glands showed decreased proliferation, cyclin D1 expression, and Cdk4 activity, as well as markedly prolonged tumor latency, compared to MMTV-neu/p27(+/+) glands. These results suggest that p27(+/-) mammary epithelium may be more susceptible to oncogene-induced tumorigenesis, whereas p27-null glands, due to severely impaired cyclin D1/Cdk4 function, are more resistant to transformation.     

Localization of organic anion transporting polypeptide 3 (oatp3) in mouse brain parenchymal and capillary endothelial cells

Organic anion transporting polypeptide 3 (oatp3) transports various CNS-acting endogenous compounds, including thyroid hormones and prostaglandin E2, between extra- and intracellular spaces, suggesting a possible role in CNS function. The purpose of this study was to clarify the expression and localization of oatp3 in the mouse brain. RT-PCR analysis revealed that oatp3 mRNA is expressed in brain capillary-rich fraction, conditionally immortalized brain capillary endothelial cells, choroid plexus, brain and lung, but not in liver or kidney, where oatp1, 2 and 5 mRNAs were detected. Immunohistochemical analysis with anti-oatp3 antibody suggests that oatp3 protein is localized at the brush-border membrane of mouse choroid plexus epithelial cells. Furthermore, intense immunoreactivity was detected in neural cells in the border region between hypothalamus and thalamus, and in the olfactory bulb. Immunoreactivity was also detected in brain capillary endothelial cells in the cerebral cortex. These localizations in the mouse brain suggest that oatp3 plays roles in blood-brain and -cerebrospinal fluid barrier transport of organic anions and signal mediators, and in hormone uptake by neural cells.     

E2F1 confers anticancer drug resistance by targeting ABC transporter family members and Bcl-2 via the p73/DNp73-miR-205 circuitry

Resistance to anti-neoplastic agents is the major cause of therapy failure, leading to disease recurrence and metastasis. E2F1 is a strong inducer of apoptosis in response to DNA damage through its capacity to activate p53/p73 death pathways. Recent evidence, however, showed that E2F1, which is aberrantly expressed in advanced malignant melanomas together with antagonistic p73 family members, drives cancer progression. Investigating mechanisms responsible for dysregulated E2F1 losing its apoptotic function, we searched for genomic signatures in primary and late clinical tumor stages to allow the prediction of downstream effectors associated with apoptosis resistance and survival of aggressive melanoma cells. We identified miR-205 as specific target of p73 and found that upon genotoxic stress, its expression is sufficiently abrogated by endogenous DNp73. Significantly, metastatic cells can be rescued from drug resistance by selective knockdown of DNp73 or overexpression of miR-205 in p73-depleted cells, leading to increased apoptosis and the reduction of tumor growth in vivo. Our data delineate an autoregulatory circuit, involving high levels of E2F1 and DNp73 to downregulate miR-205, which, in turn, controls E2F1 accumulation. Finally, drug resistance associated to this genetic signature is mediated by removing the inhibitory effect of miR-205 on the expression of Bcl-2 and the ATP-binding cassette transporters A2 (ABCA2) and A5 (ABCA5) related to multi-drug resistance and malignant progression. These results define the E2F1-p73/DNp73-miR-205 axis as a crucial mechanism for chemoresistance and, thus, as a target for metastasis prevention.     

Active nuclear import and export pathways regulate E2F-5 subcellular localization

Epidermal keratinocyte differentiation is accompanied by differential regulation of E2F genes, including up-regulation of E2F-5 and its concomitant association with the retinoblastoma family protein p130. This complex appears to play a role in irreversible withdrawal from the cell cycle in differentiating keratinocytes. We now report that keratinocyte differentiation is also accompanied by changes in E2F-5 subcellular localization, from the cytoplasm to the nucleus. To define the molecular determinants of E2F-5 nuclear import, we tested its ability to enter the nucleus in import assays in vitro using digitonin-permeabilized cells. We found that E2F-5 enters the nucleus through mediated transport processes that involve formation of nuclear pore complexes. It has been proposed that E2F-4 and E2F-5, which lack defined nuclear localization signal (NLS) consensus sequences, enter the nucleus in association with NLS-containing DP-2 or pRB family proteins. However, we show that nuclear import of E2F-5 only requires the first N-terminal 56 amino acid residues and is not dependent on interaction with DP or pRB family proteins. Because E2F-5 is predominantly cytoplasmic in undifferentiated keratinocytes and in other intact cells, we also examined whether this protein is subjected to active nuclear export. Indeed, E2F-5 is exported from the nucleus through leptomycin B-sensitive, CRM1-mediated transport, through a region corresponding to amino acid residues 130-154. This region excludes the DNA- and the p130-binding domains. Thus, the subcellular distribution of E2F-5 is tightly regulated in intact cells, through multiple functional domains that direct nucleocytoplasmic shuttling of this protein.     

Analysis of the intracellular localization of p73 N-terminal protein isoforms TAp73 and ∆Np73 in medulloblastoma cell lines

The protein homologous to the tumor suppressor p53, p73, has essential roles in development and tumorigenesis. This protein exists in a wide range of isoforms with different, even antagonistic, functions. However, there are virtually no detailed morphological studies analyzing the endogenous expression of p73 isoforms at the cellular level in cancer cells. In this study, we investigated the expression and subcellular distribution of two N-terminal isoforms, TAp73 and ΔNp73, in medulloblastoma cells using immunofluorescence microscopy. Both proteins were observed in all cell lines examined, but differences were noted in their intracellular localization between the reference Daoy cell line and four newly established medulloblastoma cell lines (MBL-03, MBL-06, MBL-07 and MBL-10). In the new cell lines, TAp73 and ΔNp73 were located predominantly in cell nuclei. However, there was heterogeneity in TAp73 distribution in the cells of all MBL cell lines, with the protein located in the nucleus and also in a limited non-random area in the cytoplasm. In a small percentage of cells, we detected cytoplasmic localization of TAp73 only, i.e., nuclear exclusion was observed. Our results provide a basis for future studies on the causes and function of distinct intracellular localization of p73 protein isoforms with respect to different protein–protein interactions in medulloblastoma cells.     

Expression of nuclear transport importins beta 1 and beta 3 is regulated during rodent spermatogenesis

Spermatogenic differentiation requires progressive gene expression changes, and proteins required for this must be transported into the nucleus. Many of these contain a nuclear localization signal and are likely to be transported by importin protein family members, each of which recognizes and transports distinct cargo proteins. We hypothesized that importins, as modulators of protein nuclear access, would display distinct expression profiles during spermatogenesis, indicating their potential to regulate key steps in cellular differentiation. This was tested throughout testicular development in rodents. Real-time PCR analysis of postnatal mouse testes revealed changing expression levels of Knpb1 (encoding importin beta 1) and Ranbp5 (encoding beta 3) mRNAs, with Knpb1 highest at 26 days postpartum and Ranbp5 highest in Day 26 and adult testis. Their distinctive cellular expression patterns visualized using in situ hybridization and immunohistochemistry were identical in mouse and rat testes where examined. Within the seminiferous epithelium, Knpb1 mRNA and importin beta1 protein were detected within mitotic Sertoli and germ cells during fetal and early postnatal development, becoming restricted to spermatogonia and spermatocytes in adulthood. Importin beta 3 protein in fetal germ cells displayed a striking difference in intracellular localization between male and female gonads. In adult testes, Ranbp5 mRNA was detected in round spermatids and importin beta 3 protein in elongating spermatids. This is the first comprehensive in situ demonstration of developmentally regulated synthesis of nuclear transport components. The contrasting expression patterns of importins beta 1 and 3 identify them as candidates for regulating nuclear access of factors required for developmental switches.     

Uptake and transport of immunoglobulin by the chick choroid plexus

Summary The choroid plexus from the lateral ventricles of 18-day chick embryos was cultivated as an organ in medium 199 until the degeneration of the stroma. Selected plexuses forming an empty epithelial sac were then incubated with enzyme-treated human immunoglobulin (5S-antibody) and with native human immunoglobulin (7S-antibody). Uptake of the 7S-antibody was observed after 30 min, whereas the 5S-antibody was taken up by the choroid plexus within 1 min, as demonstrated by means of the peroxidase-anti-peroxidase (PAP) technique (Sternberger 1974). The antibodies were located in conspicuous, large vacuoles of the choroid epithelium. Further experiments were performed using only 5S-antibody. In addition to the demonstration of the protein structure of this immunoglobulin, it was also shown that its binding capacity for tetanus toxoid as an antigen remains intact in the intracellular location. It was not possible to observe lysosomal degradation. Moreover, 5S-antibody was detectable in cultures first incubated with 5S-antibody for 30 min and subsequently in antibody-free medium for a further period of 7 to 11 days.The biological significance of the uptake of material from the cerebrospinal fluid and the possibility of the existence of a receptor for 5S-antibody are discussed.     

Esophageal cancer related gene-4 is a choroid plexus-derived injury response gene: evidence for a biphasic response in early and late brain injury

By virtue of its ability to regulate the composition of cerebrospinal fluid (CSF), the choroid plexus (CP) is ideally suited to instigate a rapid response to traumatic brain injury (TBI) by producing growth regulatory proteins. For example, Esophageal Cancer Related Gene-4 (Ecrg4) is a tumor suppressor gene that encodes a hormone-like peptide called augurin that is present in large concentrations in CP epithelia (CPe). Because augurin is thought to regulate senescence, neuroprogenitor cell growth and differentiation in the CNS, we evaluated the kinetics of Ecrg4 expression and augurin immunoreactivity in CPe after CNS injury. Adult rats were injured with a penetrating cortical lesion and alterations in augurin immunoreactivity were examined by immunohistochemistry. Ecrg4 gene expression was characterized by in situ hybridization. Cell surface augurin was identified histologically by confocal microscopy and biochemically by sub-cellular fractionation. Both Ecrg4 gene expression and augurin protein levels were decreased 24-72 hrs post-injury but restored to uninjured levels by day 7 post-injury. Protein staining in the supraoptic nucleus of the hypothalamus, used as a control brain region, did not show a decrease of auguin immunoreactivity. Ecrg4 gene expression localized to CPe cells, and augurin protein to the CPe ventricular face. Extracellular cell surface tethering of 14 kDa augurin was confirmed by cell surface fractionation of primary human CPe cells in vitro while a 6-8 kDa fragment of augurin was detected in conditioned media, indicating release from the cell surface by proteolytic processing. In rat CSF however, 14 kDa augurin was detected. We hypothesize the initial release and proteolytic processing of augurin participates in the activation phase of injury while sustained Ecrg4 down-regulation is dysinhibitory during the proliferative phase. Accordingly, augurin would play a constitutive inhibitory function in normal CNS while down regulation of Ecrg4 gene expression in injury, like in cancer, dysinhibits proliferation.     

C/EBPalpha regulates formation of S-phase-specific E2F-p107 complexes in livers of newborn mice

We previously showed that the rate of hepatocyte proliferation in livers from newborn C/EBPalpha knockout mice was increased. An examination of cell cycle-related proteins showed that the cyclin-dependent kinase (CDK) inhibitor p21 level was reduced in the knockout animals compared to that in wild-type littermates. Here we show additional cell cycle-associated proteins that are affected by C/EBPalpha. We have observed that C/EBPalpha controls the composition of E2F complexes through interaction with the retinoblastoma (Rb)-like protein, p107, during prenatal liver development. S-phase-specific E2F complexes containing E2F, DP, cdk2, cyclin A, and p107 are observed in the developing liver. In wild-type animals these complexes disappear by day 18 of gestation and are no longer present in the newborn animals. In the C/EBPalpha mutant, the S-phase-specific complexes do not diminish and persist to birth. The elevation of levels of the S-phase-specific E2F-p107 complexes in C/EBPalpha knockout mice correlates with the increased expression of several E2F-dependent genes such as those that encode cyclin A, proliferating cell nuclear antigen, and p107. The C/EBPalpha-mediated regulation of E2F binding is specific, since the deletion of another C/EBP family member, C/EBPbeta, does not change the pattern of E2F binding during prenatal liver development. The addition of bacterially expressed, purified His-C/EBPalpha to the E2F binding reaction resulted in the disruption of E2F complexes containing p107 in nuclear extracts from C/EBPalpha knockout mouse livers. Ectopic expression of C/EBPalpha in cultured cells also leads to a reduction of E2F complexes containing Rb family proteins. Coimmunoprecipitation analyses revealed an interaction of C/EBPalpha with p107 but none with cdk2, E2F1, or cyclin A. A region of C/EBPalpha that has sequence similarity to E2F is sufficient for the disruption of the E2F-p107 complexes. Despite its role as a DNA binding protein, C/EBPalpha brings about a change in E2F complex composition through a protein-protein interaction. The disruption of E2F-p107 complexes correlates with C/EBPalpha-mediated growth arrest of hepatocytes in newborn animals.