Gene-expression profiles of epithelial cells treated with EMD in vitro: analysis using complementary DNA arrays.

BACKGROUND AND OBJECTIVE: During surgical periodontal treatment, EMD is topically applied in order to facilitate regeneration of the periodontal ligament, acellular cementum and alveolar bone. Suppresion of epithelial down-growth is essential for successful periodontal regeneration; however, the underlying mechanisms of how EMD influences epithelial wound healing are poorly understood. In the present study, the effects of EMD on gene-expression profiling in an epithelial cell line (HSC-2) model were investigated. MATERIAL AND METHODS: Gene-expression modifications, determined using a comparative genome-wide expression-profiling strategy, were independently validated by quantitative real-time RT-PCR. Additionally, cell cycle, cell growth and in vitro wound-healing assays were conducted. RESULTS: A set of 43 EMD-regulated genes was defined, which may be responsible for the reduced epithelial down-growth upon EMD application. Gene ontology analysis revealed genes that could be attributed to pathways of locomotion, developmental processes and associated processes such as regulation of cell size and cell growth. Additionally, eight regulated genes have previously been reported to take part in the process of epithelial-to-mesenchymal transition. Several independent experimental assays revealed significant inhibition of cell migration, growth and cell cycle by EMD. CONCLUSION: The set of EMD-regulated genes identified in this study offers the opportunity to clarify mechanisms underlying the effects of EMD on epithelial cells. Reduced epithelial repopulation of the dental root upon periodontal surgery may be the consequence of reduced migration and cell growth, as well as epithelial-to-mesenchymal transition.

Late endosomal traffic of the epidermal growth factor receptor ensures spatial and temporal fidelity of mitogen-activated protein kinase signaling.

Mitogen-activated protein kinase (MAPK) signaling is regulated by assembling distinct scaffold complexes at the plasma membrane and on endosomes. Thus, spatial resolution might be critical to determine signaling specificity. Therefore, we investigated whether epidermal growth factor receptor (EGFR) traffic through the endosomal system provides spatial information for MAPK signaling. To mislocalize late endosomes to the cell periphery we used the dynein subunit p50 dynamitin. The peripheral translocation of late endosomes resulted in a prolonged EGFR activation on late endosomes and a slow down in EGFR degradation. Continuous EGFR signaling from late endosomes caused sustained extracellular signal-regulated kinase and p38 signaling and resulted in hyperactivation of nuclear targets, such as Elk-1. In contrast, clustering late endosomes in the perinuclear region by expression of dominant active Rab7 delayed the entry of the EGFR into late endosomes, which caused a delay in EGFR degradation and a sustained MAPK signaling. Surprisingly, the activation of nuclear targets was reduced. Thus, we conclude that appropriate trafficking of the activated EGFR through endosomes controls the spatial and temporal regulation of MAPK signaling.

Cell biologists sort things out: Analysis and purification of intracellular organelles by flow cytometry.

Flow cytometry was established originally for measuring DNA content and for the analysis of cell-surface markers in combination with cell sorting. During the past two decades, it has added new dimensions to various areas of immunology and medicine. Increased sensitivity and precision of flow cytometers, accompanied by the development of new fluorescent dyes and probes, has led to new applications in molecular cell biology and genetics. This article focuses on applications of flow cytometry in analysis and sorting of intracellular organelles.

Biogenesis of phagolysosomes proceeds through a sequential series of interactions with the endocytic apparatus.

We have examined the modifications occurring during the transformation of phagosomes into phagolysosomes in J-774 macrophages. The use of low density latex beads as markers of phagosomes (latex bead compartments, LBC) allowed the isolation of these organelles by flotation on a simple sucrose gradient. Two-dimensional gel electrophoresis, immunocytochemistry, and biochemical assays have been used to characterize the composition of LBC at different time points after their formation, as well as their interactions with the organelles of the endocytic pathway. Our results show that LBC acquire and lose various markers during their transformation into phagolysosomes. Among these are members of the rab family of small GTPases as well as proteins of the lamp family. The transfer of the LBC of lamp 2, a membrane protein associated with late endocytic structures, was shown to be microtubule dependent. Video-microscopy showed that newly formed phagosomes were involved in rapid multiple contacts with late components of the endocytic pathway. Collectively, these observations suggest that phagolysosome formation is a highly dynamic process that involves the gradual and regulated acquisition of markers from endocytic organelles.

Hyaluronic acid (HA) binding to CD44 activates Rac1 and induces lamellipodia outgrowth.

Both cell adhesion protein CD44 and its main ligand hyaluronic acid (HA) are thought to be involved in several processes ultimately requiring cytoskeleton rearrangements. Here, we show that the small guanine nucleotide (GTP)-binding protein, Rac1, can be activated upon HA binding to CD44. When applied locally to a passive cell edge, HA promoted the formation of lamellipodial protrusions in the direction of the stimulus. This process was inhibited by the prior injection of cells with dominant-negative N17Rac recombinant protein or by pretreatment of cells with monoclonal anti-CD44 antibodies, interfering with HA binding, implying the direct involvement of CD44 in signaling to Rac1.

Rab8, a small GTPase involved in vesicular traffic between the TGN and the basolateral plasma membrane.

Small GTP-binding proteins of the rab family have been implicated as regulators of membrane traffic along the biosynthetic and endocytic pathways in eukaryotic cells. We have investigated the localization and function of rab8, closely related to the yeast YPT1/SEC4 gene products. Confocal immunofluorescence microscopy and immunoelectron microscopy on filter-grown MDCK cells demonstrated that, rab8 was localized to the Golgi region, vesicular structures, and to the basolateral plasma membrane. Two-dimensional gel electrophoresis showed that rab8p was highly enriched in immuno-isolated basolateral vesicles carrying vesicular stomatitis virus-glycoprotein (VSV-G) but was absent from vesicles transporting the hemagglutinin protein (HA) of influenza virus to the apical cell surface. Using a cytosol dependent in vitro transport assay in permeabilized MDCK cells we studied the functional role of rab8 in biosynthetic membrane traffic. Transport of VSV-G from the TGN to the basolateral plasma membrane was found to be significantly inhibited by a peptide derived from the hypervariable COOH-terminal region of rab8, while transport of the influenza HA from the TGN to the apical surface and ER to Golgi transport were unaffected. We conclude that rab8 plays a role in membrane traffic from the TGN to the basolateral plasma membrane in MDCK cells.

Rab17, a novel small GTPase, is specific for epithelial cells and is induced during cell polarization.

The rab subfamily of small GTPases has been demonstrated to play an important role in the regulation of membrane traffic in eukaryotic cells. Compared with nonpolarized cells, epithelial cells have distinct apical and basolateral transport pathways which need to be separately regulated. This raises the question whether epithelial cells require specific rab proteins. However, all rab proteins identified so far were found to be equally expressed in polarized and nonpolarized cells. Here we report the identification of rab17, the first epithelial cell-specific small GTPase. Northern blot analysis on various mouse organs, revealed that the rab17 mRNA is present in kidney, liver, and intestine but not in organs lacking epithelial cells nor in fibroblasts. To determine whether rab17 is specific for epithelial cells we studied its expression in the developing kidney. We found that rab17 is absent from the mesenchymal precursors but is induced upon their differentiation into epithelial cells. In situ hybridization studies on the embryonic kidney and intestine revealed that rab17 is restricted to epithelial cells. By immunofluorescence and immunoelectron microscopy on kidney sections, rab17 was localized to the basolateral plasma membrane and to apical tubules. Rab proteins associated with two distinct compartments have been found to regulate transport between them. Therefore, our data suggest that rab17 might be involved in transcellular transport.

ADP ribosylation factor and a 14-kD polypeptide are associated with heparan sulfate-carrying post-trans-Golgi network secretory vesicles in rat hepatocytes.

Constitutive secretory vesicles carrying heparan sulfate proteoglycan (HSPG) were identified in isolated rat hepatocytes by pulse-chase experiments with [35S]sulfate and purified by velocity-controlled sucrose gradient centrifugation followed by equilibrium density centrifugation in Nycodenz. Using this procedure, the vesicles were separated from plasma membranes, Golgi, trans-Golgi network (TGN), ER, endosomes, lysosomes, transcytotic vesicles, and mitochondria. The diameter of these vesicles was approximately 100-200 nm as determined by electron microscopy. A typical coat structure as described for intra-Golgi transport vesicles or clathrin-coated vesicles could not be seen, and the vesicles were not associated with the coat protein beta-COP. Furthermore, the vesicles appear to represent a low density compartment (1.05-1.06 g/ml). Other constitutively secreted proteins (rat serum albumin, apolipoprotein E, and fibrinogen) could not be detected in purified HSPG-carrying vesicles, but banded in the denser fractions of the Nycodenz gradient. Moreover, during pulse-chase labeling with [35S]methionine, labeled albumin did not appear in the post-TGN vesicle fraction carrying HSPGs. These findings indicate sorting of HSPGs and albumin into different types of constitutive secretory vesicles in hepatocytes. Two proteins were found to be tightly associated with the membranes of the HSPG carrying vesicles: a member of the ADP ribosylation factor family of small guanine nucleotide-binding proteins and an unknown 14-kD peripheral membrane protein (VAPP14). Concerning the secretory pathway, we conclude from these results that ADP ribosylation factor proteins are not only involved in vesicular transport from the ER via the Golgi to the TGN, but also in vesicular transport from the TGN to the plasma membrane.

A novel 14-kilodalton protein interacts with the mitogen-activated protein kinase scaffold mp1 on a late endosomal/lysosomal compartment.

We have identified a novel, highly conserved protein of 14 kD copurifying with late endosomes/lysosomes on density gradients. The protein, now termed p14, is peripherally associated with the cytoplasmic face of late endosomes/lysosomes in a variety of different cell types. In a two-hybrid screen with p14 as a bait, we identified the mitogen-activated protein kinase (MAPK) scaffolding protein MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK) partner 1 (MP1) as an interacting protein. We confirmed the specificity of this interaction in vitro by glutathione S-transferase pull-down assays and by coimmunoprecipitation, cosedimentation on glycerol gradients, and colocalization. Moreover, expression of a plasma membrane-targeted p14 causes mislocalization of coexpressed MP1. In addition, we could reconstitute protein complexes containing the p14-MP1 complex associated with ERK and MEK in vitro.The interaction between p14 and MP1 suggests a MAPK scaffolding activity localized to the cytoplasmic surface of late endosomes/lysosomes, thereby combining catalytic scaffolding and subcellular compartmentalization as means to modulate MAPK signaling within a cell.

Analysis of CD44-containing lipid rafts: Recruitment of annexin II and stabilization by the actin cytoskeleton.

CD44, the major cell surface receptor for hyaluronic acid (HA), was shown to localize to detergent-resistant cholesterol-rich microdomains, called lipid rafts, in fibroblasts and blood cells. Here, we have investigated the molecular environment of CD44 within the plane of the basolateral membrane of polarized mammary epithelial cells. We show that CD44 partitions into lipid rafts that contain annexin II at their cytoplasmic face. Both CD44 and annexin II were released from these lipid rafts by sequestration of plasma membrane cholesterol. Partition of annexin II and CD44 to the same type of lipid rafts was demonstrated by cross-linking experiments in living cells. First, when CD44 was clustered at the cell surface by anti-CD44 antibodies, annexin II was recruited into the cytoplasmic leaflet of CD44 clusters. Second, the formation of intracellular, submembranous annexin II-p11 aggregates caused by expression of a trans-dominant mutant of annexin II resulted in coclustering of CD44. Moreover, a frequent redirection of actin bundles to these clusters was observed. These basolateral CD44/annexin II-lipid raft complexes were stabilized by addition of GTPgammaS or phalloidin in a semipermeabilized and cholesterol-depleted cell system. The low lateral mobility of CD44 in the plasma membrane, as assessed with fluorescent recovery after photobleaching (FRAP), was dependent on the presence of plasma membrane cholesterol and an intact actin cytoskeleton. Disruption of the actin cytoskeleton dramatically increased the fraction of CD44 which could be recovered from the light detergent-insoluble membrane fraction. Taken together, our data indicate that in mammary epithelial cells the vast majority of CD44 interacts with annexin II in lipid rafts in a cholesterol-dependent manner. These CD44-containing lipid microdomains interact with the underlying actin cytoskeleton.

Protein transport to the dendritic plasma membrane of cultured neurons is regulated by rab8p.

In the companion paper (Huber, L. A., S. W. Pimplikar, R. G. Parton, H. Virta, M. Zerial, and K. Simons. J. Cell Biol. 123:35-45) we reported that the small GTPase rab8p is involved in transport from the TGN to the basolateral plasma membrane in epithelia. In the present work we investigated the localization and function of rab8p in polarized hippocampal neurons. By immunofluorescence microscopy we found that rab8p localized preferentially in the somatodendritic domain, and was excluded from the axon. Double-labeling immunofluorescence showed that some of the rab8p co-localized in the dendrites with the Semliki Forest Virus glycoprotein E2 (SFV-E2). An antisense oligonucleotide approach was used to investigate the role of rab8p in dendritic transport of newly synthesized viral glycoproteins. Antisense oligonucleotides corresponding to the initiation region of the rab8 coding sequence were added to the cultured neurons for four days. This treatment resulted in a significant decrease in cellular levels of rab8p and transport of SFV-E2 from the cell body to the dendrites was significantly reduced. However, no effect was observed on axonal transport of influenza HA. From these results we conclude that rab8p is involved in transport of proteins to the dendritic surface in neurons.

The involvement of the small GTP-binding protein Rab5a in neuronal endocytosis.

Rab5a is a small GTPase that regulates fusion of endocytic vesicles to early endosomes. We investigated whether Rab5a is involved in early endocytic traffic in both the axonal and the somatodendritic domains of polarized neurons. Using immunofluorescence, endogenous Rab5a was detected in axons and dendrites. Its localization in axons strongly overlapped that of the synaptic vesicle protein synaptophysin. Indeed, Rab5a co-immunoisolated with synaptophysin-containing vesicles, and antibodies against Rab5a labeled synaptic vesicle-like structures in nerve terminals. The functional association of Rab5a with dendritic and axonal early endosomes was assayed by electron microscopy after overexpression of wild-type and mutant Rab5a in cultured hippocampal neurons. This induced the formation of abnormal endosomes in both the somatodendritic and the axonal domains. These results show a role for Rab5a in axonal and dendritic endocytosis, and the presence of Rab5a on synaptic vesicles indicates that the axonal endosomes participate in the biogenesis of these vesicles.

A deficiency of the small GTPase rab8 inhibits membrane traffic in developing neurons.

One of the major activities of developing neurons is the transport of new membrane to the growing axon. Candidates for playing a key role in the regulation of this intense traffic are the small GTP-binding proteins of the rab family. We have used hippocampal neurons in culture and analyzed membrane traffic activity after suppressing the expression of the small GTP-binding protein rab8. Inhibition of protein expression was accomplished by using sequence-specific antisense oligonucleotides. While rab8 depletion resulted in the blockage of morphological maturation in 95% of the neurons, suppression of expression of another rab protein, rab3a, had no effect, and all neurons developed normal axons and dendrites. The impairment of neuronal maturation by rab8 antisense treatment was due to inhibition of membrane traffic. Thus, by using video-enhanced differential interference contrast microscopy, we observed in the rab8-depleted cells a dramatic reduction in the number of vesicles undergoing anterograde transport. Moreover, by incubating antisense-treated neurons with Bodipy-labeled ceramide, a fluorescent marker for newly formed exocytic vesicles, we observed fluorescence labeling restricted to the Golgi apparatus, whereas in control cells labeling was found also in the neurites. These results show the role of the small GTPase rab8 in membrane traffic during neuronal process outgrowth.

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.

In search of differentially expressed genes and proteins.

A great challenge for modern cell biology is the successful examination of the co-expression of thousands of genes under physiological or pathological conditions and how the expression patterns define the different states of a single cell, tissue or a microorganism. Gene expression can be analyzed today on a large scale by advanced technical approaches for differential screening of proteins and mRNAs. The identification of differentially expressed mRNAs has been successfully applied to understand gene function and the underlying molecular mechanism(-s) of differentiation, development and disease state. Analysis of gene expression by the systematic mapping of thousands of proteins present in a cell or tissue can be achieved by the use of two-dimensional (2D) gel electrophoresis, quantitative computer image analysis, and protein identification techniques. In this article, we comment on some of these techniques and try to stress their advantages and drawbacks. We show how data from RNA/DNA mapping, sequence information from genome projects and protein pattern profiling can be linked with each other and annotated. These comprehensive approaches permit the study of differential gene and protein expressions in cells or tissues.

Rab and rho GTPases are involved in specific response of periodontal ligament fibroblasts to mechanical stretching.

To investigate the contribution of ras-related signalling molecules to the mechanotransduction process, stretch-sensing human periodontal ligament (PDL) fibroblasts were isolated and cultured in dishes with a flexible bottom. The cells were stimulated by stretching the bottom of the dishes and membrane fractions were prepared and analysed at the level of mapping small GTP-binding proteins by high resolution two-dimensional gel electrophoresis followed by renaturing transfer and an [alpha-32P]GTP-overlay procedure. This analysis revealed that mechanically-stretched PDL fibroblasts exhibit complete down-regulation of rhoA and induction of rab6, rab17 and a putative member of the rab3 subfamily in a cell type-specific manner.

Mechanical loading triggers specific biochemical responses in mandibular condylar chondrocytes.

The effect of mechanical loading on the phosphorylation state of condylar cartilage proteins was investigated by high resolution electrophoretic analysis of 32P-labelled proteins from mechanically stimulated rat mandibular condylar chondrocytes. Specific dephosphorylation (and/or loss) of an acidic, 35-36 kDa protein(s) and of proteins overlapping with members of the ras superfamily of small GTP-binding proteins was observed. These responses may constitute part of a mechanically induced transduction system which establishes the differentiated phenotype.

Inhibitory effect of TIS7 on Sp1-C/EBPalpha transcription factor module activity.

The transcription factors C/EBPalpha and Sp1 functionally interact to induce expression of specific genes during myeloid and epithelial cell differentiation. The C/EBPalpha-Sp1 transcription factor "module" binds to enhancer elements within the upstream regulatory sequences of target genes. In our previous study we identified mouse TPA inducible sequence 7 (TIS7) as a novel co-repressor in epithelial cells undergoing loss of polarity. Increased levels of TIS7 down-regulate the transcription of a specific set of genes. Using bioinformatic analysis we identified a common binding site for the C/EBPalpha-Spl transcription factor module within the upstream regulatory regions of TIS7-regulated genes. The inhibitory effect of TIS7 on C/EBPalpha-Sp1-mediated transcription was confirmed by reporter assays. Our data showed that the TIS7 effect was mediated through specific interference with Sp1 transcriptional activity. Furthermore, TIS7 prevented formation of a complex between Sp1 protein and its consensus DNA binding site. Data presented here further specify the mechanism of action of the transcriptional co-repressor TIS7 as well as document the strength of a bioinformatic approach for the prediction and analysis of transcription factor modules.

Mapping cells and sub-cellular organelles on 2-D gels: 'new tricks for an old horse'.

Nowadays, investigators in all fields are faced with the identification of unknown, up- or down-regulated, modified proteins that they are trying to identify. Two-dimensional (2-D) gel electrophoresis, with its ability to resolve several thousand proteins, is an extremely powerful technique. The current resolution and reproducibility of 2-D gel technology and the establishment of computer assisted 2-D gel protein databases have paved new ways for the identification of proteins.

Myelin membrane biogenesis by oligodendrocytes. Developmental regulation of low molecular weight GTP-binding proteins.

Oligodendrocytes synthesize dramatic amounts of myelin membrane. We hypothesized that this requires unique aspects of vesicular trafficking. Specific stages of the oligodendrocyte lineage were assayed for low molecular weight GTP-binding proteins implicated in the regulation of vesicular transport pathway (two dimensional gel electrophoresis, [alpha-32P]GTP overlay). Consistent with the hypothesis, as oligodendrocytes differentiate from early progenitors to mature myelin-producing cells, > or = 12 small GTP-binding proteins become up-regulated. Myelin membrane also has a complex pattern of GTP-binding proteins. Several of these proteins may be specific to oligodendrocytes, suggesting that oligodendrocytes may utilize cell-type specific GTP-binding proteins for biogenesis and maintenance of the myelin membrane.