Keratin-mediated resistance to stress and apoptosis in simple epithelial cells in relation to health and disease.

Epithelial cells such as hepatocytes exhibit highly polarized properties as a result of the asymmetric distribution of subsets of receptors at unique portions of the surface membrane. While the proper targeting of these surface receptors and maintenance of the resulting polarity depend on microtubules (MTs), the Golgi sorting compartment, and different actin-filament networks, the contribution of keratin intermediate filaments (IFs) has been unclear. Recent data show that the latter cytoskeletal network plays a predominant role in providing resistance to various forms of stress and to apoptosis targeted to the surface membrane. In this context, we first summarize our knowledge of the domain- or assembly-related features of IF proteins and the dynamic properties of IF networks that may explain how the same keratin pair K8/K18 can exert multiple resistance-related functions in simple epithelial cells. We then examine the contribution of linker protein(s) that integrate interactions of keratin IFs with MTs and the actin-cytoskeleton network, polarity-dependent surface receptors and cytoplasmic organelles. We next address likely molecular mechanisms by which K8/K18 can selectively provide resistance to a mechanical or toxic stress, or to Fas-mediated apoptosis. Finally, these issues on keratin structure-function are examined within a context of pathological anomalies emerging in tissue architecture as a result of natural or targeted mutations, or posttranslational modifications at specific amino acid residues. Clearly. the data accumulated in recent years provide new and significant insights on the role of K8/K18, particularly under conditions where polarized cells resist to stressful or apoptotic insults.

An evolutionarily conserved NPC subcomplex, which redistributes in part to kinetochores in mammalian cells.

The nuclear pore complexes (NPCs) are evolutionarily conserved assemblies that allow traffic between the cytoplasm and the nucleus. In this study, we have identified and characterized a novel human nuclear pore protein, hNup133, through its homology with the Saccharomyces cerevisiae nucleoporin scNup133. Two-hybrid screens and immunoprecipitation experiments revealed a direct and evolutionarily conserved interaction between Nup133 and Nup84/Nup107 and indicated that hNup133 and hNup107 are part of a NPC subcomplex that contains two other nucleoporins (the previously characterized hNup96 and a novel nucleoporin designated as hNup120) homologous to constituents of the scNup84 subcomplex. We further demonstrate that hNup133 and hNup107 are localized on both sides of the NPC to which they are stably associated at interphase, remain associated as part of a NPC subcomplex during mitosis, and are targeted at early stages to the reforming nuclear envelope. Throughout mitosis, a fraction of hNup133 and hNup107 localizes to the kinetochores, thus revealing an unexpected connection between structural NPCs constituents and kinetochores. Photobleaching experiments further showed that the mitotic cytoplasm contains kinetochore-binding competent hNup133 molecules and that in contrast to its stable association with the NPCs the interaction of this nucleoporin with kinetochores is dynamic.

Simple epithelium keratins 8 and 18 provide resistance to Fas-mediated apoptosis. The protection occurs through a receptor-targeting modulation.

Keratins 8 and 18 belong to the keratin family of intermediate filament (IF) proteins and constitute a hallmark for all simple epithelia, including the liver. Hepatocyte IFs are made solely of keratins 8 and 18 (K8/K18). In these cells, the loss of one partner via a targeted null mutation in the germline results in hepatocytes lacking K8/K18 IFs, thus providing a model of choice for examining the function(s) of simple epithelium keratins. Here, we report that K8-null mouse hepatocytes in primary culture and in vivo are three- to fourfold more sensitive than wild-type (WT) mouse hepatocytes to Fas-mediated apoptosis after stimulation with Jo2, an agonistic antibody of Fas ligand. This increased sensitivity is associated with a higher and more rapid caspase-3 activation and DNA fragmentation. In contrast, no difference in apoptosis is observed between cultured K8-null and WT hepatocytes after addition of the Fas-related death-factors tumor necrosis factor (TNF) alpha or TNF-related apoptosis-inducing ligand. Analyses of the Fas distribution in K8-null and WT hepatocytes in culture and in situ demonstrate a more prominent targeting of the receptor to the surface membrane of K8-null hepatocytes. Moreover, altering Fas trafficking by disrupting microtubules with colchicine reduces by twofold the protection generated against Jo2-induced lethal action in K8-null versus WT hepatocytes. Together, the results strongly suggest that simple epithelium K8/K18 provide resistance to Fas-mediated apoptosis and that this protection occurs through a modulation of Fas targeting to the cell surface.

Nuclear pore complexes form immobile networks and have a very low turnover in live mammalian cells.

The nuclear pore complex (NPC) and its relationship to the nuclear envelope (NE) was characterized in living cells using POM121-green fluorescent protein (GFP) and GFP-Nup153, and GFP-lamin B1. No independent movement of single pore complexes was found within the plane of the NE in interphase. Only large arrays of NPCs moved slowly and synchronously during global changes in nuclear shape, strongly suggesting mechanical connections which form an NPC network. The nuclear lamina exhibited identical movements. NPC turnover measured by fluorescence recovery after photobleaching of POM121 was less than once per cell cycle. Nup153 association with NPCs was dynamic and turnover of this nucleoporin was three orders of magnitude faster. Overexpression of both nucleoporins induced the formation of annulate lamellae (AL) in the endoplasmic reticulum (ER). Turnover of AL pore complexes was much higher than in the NE (once every 2.5 min). During mitosis, POM121 and Nup153 were completely dispersed and mobile in the ER (POM121) or cytosol (Nup153) in metaphase, and rapidly redistributed to an immobilized pool around chromatin in late anaphase. Assembly and immobilization of both nucleoporins occurred before detectable recruitment of lamin B1, which is thus unlikely to mediate initiation of NPC assembly at the end of mitosis.

Hepatocyte nuclear factor 3beta (Foxa2) is dispensable for maintaining the differentiated state of the adult hepatocyte.

Liver-specific gene expression is controlled by a heterogeneous group of hepatocyte-enriched transcription factors. One of these, the winged helix transcription factor hepatocyte nuclear factor 3beta (HNF3beta or Foxa2) is essential for multiple stages of embryonic development. Recently, HNF3beta has been shown to be an important regulator of other hepatocyte-enriched transcription factors as well as the expression of liver-specific structural genes. We have addressed the role of HNF3beta in maintenance of the hepatocyte phenotype by inactivation of HNF3beta in the liver. Remarkably, adult mice lacking HNF3beta expression specifically in hepatocytes are viable, with histologically normal livers and normal liver function. Moreover, analysis of >8,000 mRNAs by array hybridization revealed that lack of HNF3beta affects the expression of only very few genes. Based on earlier work it appears that HNF3beta plays a critical role in early liver development; however, our studies demonstrate that HNF3beta is not required for maintenance of the adult hepatocyte or for normal liver function. This is the first example of such functional dichotomy for a tissue specification transcription factor.

Localization of the mixed-lineage kinase DLK/MUK/ZPK to the Golgi apparatus in NIH 3T3 cells.

DLK/MUK/ZPK is a serine/threonine kinase that belongs to the mixed-lineage (MLK) subfamily of protein kinases. As is the case for most members of this family, relatively little is known about the physiological role of DLK/MUK/ZPK in mammalian cells. Because analysis of subcellular distribution may provide important clues concerning the potential in vivo function of a protein, an antiserum was generated against the amino terminal region of murine DLK/MUK/ZPK and used for localization studies in wild-type NIH 3T3 cells. Light microscopic immunocytochemistry experiments performed with the antiserum revealed that DLK/MUK/ZPK was specifically localized in a juxtanuclear structure characteristic of the Golgi complex. In support of this, treatment of cells with brefeldin A, a drug known to disintegrate the Golgi apparatus, caused disruption of DLK/MUK/ZPK perinuclear staining. Ultrastructural observation of NIH 3T3 cells also confirmed this localization, showing that most of the immunoreactivity was detected on membranes of the stacked Golgi cisternae. Consistent with localization studies, biochemical analyses revealed that DLK/MUK/ZPK was predominantly associated with Golgi membranes on fractionation of cellular extracts and was entirely partitioned into the aqueous phase when membranes were subjected to Triton X-114 extraction. On the basis of these findings, we suggest that DLK/MUK/ZPK is a peripheral membrane protein tightly associated with the cytoplasmic face of the Golgi apparatus. (J Histochem Cytochem 47:1287-1296, 1999)

Simplified generation of targeting constructs using ET recombination.

ET recombination is a way to engineer DNA in Escherichia coli using homologous recombination. Here we develop the potential of ET recombination in two ways relevant to complex engineering exercises such as building gene targeting constructs. First, a targeting construct was made in a single step. Second, ET recombination was used to place two unique restriction sites at precise positions in a large genomic clone. Subsequently a complex targeting construct was created by ligation with a multifunctional cassette.

Establishment and immunocharacterization of an immortalized pancreatic cell line derived from the H-2Kb-tsA58 transgenic mouse.

This study describes the establishment and characterization of an immortalized cell line derived from the pancreas of an adult H-2Kb-tsA58 transgenic mouse. These cells, designated IMPAN for IMmortalized PANcreatic cells, displayed a cobblestone appearance typical of confluent epithelial cells and a distinct polarity in the organization of their cytoplasmic organelles. Immunocytochemical studies revealed that all IMPAN cells stained positively for a wide range of markers characteristic of pancreatic acinar cells, namely the secretory products alpha-amylase, chymotrypsinogen, DNAse, the lectinlike secretory protein PAP (pancreatitis associated protein), and the zymogen granule membrane proteins GP-2 and gp300. They also stained positively for carbonic anhydrase II and cytokeratin 19, two proteins characteristic of pancreatic duct cells, as well as for rab3A, a small GTP-binding protein specifically localized in pancreatic islet cells. No reactivity was ever obtained with insulin antibodies. Taken together, these results show that the IMPAN cells exhibit a phenotype comparable to exocrine pancreatic acinar cells. However the expression of some proteins more specific to duct and islet cells make them similar to in vivo or in vitro growing acinar cells. The cell line should be a valuable model to study the mechanisms of growth, differentiation, and transformation of the exocrine pancreatic acinar cell.

Faculty advisor program for family medicine residents.

PROBLEM BEING ADDRESSED: In response to the accreditation guidelines of the College of Family Physicians of Canada's (CFPC) Task Force on Intraining Evaluation, the Department of Family Medicine at McGill University implemented a faculty advisor program on July 1, 1993. OBJECTIVE OF PROGRAM: In addition to meeting the requirements of the CFPC, the faculty advisor program was developed to foster communication between residents and faculty, increase opportunities for feedback, promote self-directed learning, and personalize the educational experience of trainees. MAIN COMPONENTS OF PROGRAM: Residents were assigned an advisor. They were expected to meet their advisors monthly to discuss educational objectives, performance, career planning, and any problems. Educational plans were to be completed at each meeting. CONCLUSIONS: Feedback from advisors and residents has been positive, with both groups expressing overall satisfaction with the program. The faculty advisor program will continue but will be modified to address problems identified and better meet the needs of faculty and residents.

Inhibition of cell growth by overexpression of the ZPK gene.

ZPK is a recently described serine/threonine protein kinase that is thought to be involved in the regulation of cell proliferation and differentiation. To directly determine whether ZPK exhibits any effect on cell growth, NIH 3T3 fibroblasts were transfected with an expression vector harboring the murine ZPK cDNA. Stable expression of this construct led to a dramatic reduction in the proliferative capacity of these cells as measured by a colony formation assay in monolayer culture. By contrast, overexpression of a ZPK cDNA with a mutation in the ATP-binding domain did not affect clonal expansion of the transfected cells. These findings suggest that the ZPK gene may act as a negative regulator of cell growth and that this function may be mediated in part by the intrinsic kinase activity of the ZPK protein.

A targeted mouse Otx2 mutation leads to severe defects in gastrulation and formation of axial mesoderm and to deletion of rostral brain.

Mouse Otx2 is a bicoid-class homeobox gene, related to the Drosophila orthodenticle (otd) gene. Expression of this gene is initially widespread in the epiblast at embryonic day 5.5 but becomes progressively restricted to the anterior end of the embryo at the headfold stage. In flies, loss of function mutations in otd result in deletion of pre-antennal and antennal segments; which leads to the absence of head structures derived from these segments. To study the function of Otx2 in mice, we have generated a homeobox deletion mutation in this gene. Mice homozygous for this mutation show severe defects in gastrulation and in formation of axial mesoderm and loss of anterior neural tissues. These results demonstrate that Otx2 is required for proper development of the epiblast and patterning of the anterior brain in mice, and supports the idea of evolutionary conservation of the function of Otd/Otx genes in head development in flies and mice.

The legumin boxes and the 3' part of a soybean beta-conglycinin promoter are involved in seed gene expression in transgenic tobacco plants.

beta-conglycinin is one of the major seed storage proteins in soybean. It is composed of three subunits, namely alpha, alpha' and beta. The expression of beta-conglycinin is highly regulated, being restricted to the embryo during the mid-maturation phase of embryogeny. Two series of constructs were made with the alpha' subunit promoter and the GUS reporter gene to investigate the cis-acting elements involved in the regulated expression of this promoter. The activity of each construct was tested in transgenic tobacco plants. In the first series of constructs, we checked if the 'legumin box', a sequence found in most legume seed storage protein genes as well as in other seed-specific genes, is involved in the regulated expression of the alpha' subunit of the beta-conglycinin gene in tobacco. To this end, both copies of the alpha' subunit promoter legumin boxes were mutagenized in vitro. The transcriptional activity of the single mutants and the double mutant were compared with that of the wild-type promoter. Our results show that the legumin boxes act together to increase transcription of the beta-conglycinin alpha' subunit gene by about a factor of ten. This is the first demonstration of a function for the legumin box in transcriptional regulation. In the second series of experiments, we wished to determine if the 3' part of the promoter (the CCAAT and TATAA region) contains important regulatory elements. We found that this small fragment (-82 to +13 bp) can confer by itself a low level of seed-specific gene expression. Chimaeric promoters constructed from parts of the alpha' subunit promoter and of the constitutive CaMV 35S promoter were also analysed. These constructs also revealed the importance of the CCAAT and TATAA region of the alpha' subunit promoter in seed-specific gene expression.