E4 Isoform Research Articles

Age-related macular degeneration and functional promoter and coding variants of the apolipoprotein E gene

Age-related macular degeneration (AMD) is a frequent, multifactorial disease of the central retina and a major cause of irreversible vision loss in industrialized countries. Apolipoprotein E (APOE) has been consistently associated with AMD, particularly its two functional isoforms E2 (predisposing) and E4 (protective). The biological correlate of this association, however, is still unclear. In this study, we have defined an extended haplotype block encompassing the entire APOE gene locus, including known coding as well as cis-regulatory promoter variants. Of the five extended APOE haplotypes common in the general population, two were found to be significantly associated with AMD, namely G-G-G-G-epsilon2 (odds ratio [OR], 1.59; 95% confidence interval [CI], 1.19-2.12) and T-G-A-G-epsilon4 (OR, 0.76; 95% CI, 0.58-0.99). When analyzing common extended haplotype combinations, T-C-G-G-epsilon3/T-G-A-G-epsilon4 exhibited the most prominent effect (OR, 0.32; 95% CI, 0.20-0.51). Intriguingly, we also found one extended epsilon3-haplotype, G-G-G-A-epsilon3, to be protective in the homozygous state (OR, 0.65; 95% CI, 0.49-0.87). Since single nucleotide polymorphism (SNP) rs405509:G>T is a constituent of the extended epsilon-haplotype block and is known to significantly influence APOE promoter activity, we hypothesize that both the relative rate of APOE isoform expression in conjunction with established functional differences of the respective isoforms may be crucial in mediating AMD pathology. This would also imply that genotyping of the core epsilon-haplotypes alone is not sufficient to estimate AMD risk, but that determination of extended haplotype combinations, including the functional promoter SNP rs405509, is required instead.

The first missense mutation causing Rett syndrome specifically affecting the MeCP2_e1 isoform

We report the identification of the first de novo mutation at a highly conserved residue within the polyalanine stretch in the N-terminal region of the brain-dominant protein isoform MeCP2_e1 in a girl with classical Rett syndrome. The missense mutation, p.Ala2Val, leads to severe developmental delay, microcephaly, no language, severe epilepsy, and cognitive impairment. To evaluate the pathogenic potentials of the MECP2 mutation specific to the MeCP2_e1 isoform detected in this patient, full-length wild-type and mutated cDNAs were cloned in eukaryotic expression vectors to generate a fusion protein with c-myc, and constructs were transfected in COS7 cells. In vitro studies demonstrated that, like wild-type MeCP2e_1, the N-terminal mutant is localized in the nucleus. Neither transcriptional nor translational effect on the MeCP2_e2 isoform was observed in fibroblasts from the p.Ala2Val patient, suggesting that MeCP2_e1 is involved in other functional process. These data suggest the important involvement of the N-terminus in the function of MeCP2 protein, and provide further evidence for the major impact of a specific MeCP2e_1 deficiency in the development of intellectual processing.

Defective assembly of a hybrid vacuolar H+-ATPase containing the mouse testis-specific E1 isoform and yeast subunits

Mammalian vacuolar-type proton pumping ATPases (V-ATPases) are diverse multi-subunit proton pumps. They are formed from membrane Vo and catalytic V1 sectors, whose subunits have cell-specific or ubiquitous isoforms. Biochemical study of a unique V-ATPase is difficult because ones with different isoforms are present in the same cell. However, the properties of mouse isoforms can be studied using hybrid V-ATPases formed from the isoforms and other yeast subunits. As shown previously, mouse subunit E isoform E1 (testis-specific) or E2 (ubiquitous) can form active V-ATPases with other subunits of yeast, but E1/yeast hybrid V-ATPase is defective in proton transport at 37 °C (Sun-Wada, G.-H., Imai-Senga, Y., Yamamoto, A., Murata, Y., Hirata, T., Wada, Y., and Futai, M., 2002, J. Biol. Chem. 277, 18098–18105). In this study, we have analyzed the properties of E1/yeast hybrid V-ATPase to understand the role of the E subunit. The proton transport by the defective hybrid ATPase was reversibly recovered when incubation temperature of vacuoles or cells was shifted to 30 °C. Corresponding to the reversible defect of the hybrid V-ATPase, the Vo subunit a epitope was exposed to the corresponding antibody at 37 °C, but became inaccessible at 30 °C. However, the V1 sector was still associated with Vo at 37 °C, as shown immunochemically. The control yeast V-ATPase was active at 37 °C, and its epitope was not accessible to the antibody. Glucose depletion, known to dissociate V1 from Vo in yeast, had only a slight effect on the hybrid at acidic pH. The domain between Lys26 and Val83 of E1, which contains eight residues not conserved between E1 and E2, was responsible for the unique properties of the hybrid. These results suggest that subunit E, especially its amino-terminal domain, plays a pertinent role in the assembly of V-ATPase subunits in vacuolar membranes.

P3-294: Post-translational modifications of apolipoprotein-e in the human brain

Polymorphism of apolipoprotein-E (apoE) is the greatest genetic risk factor for developing Alzheimer's Disease (AD). We have investigated the influence of polymorphism and disease status on apoE post-translational modification in the human brain. Human brain homogenate samples were prepared from hippocampal, frontal and occipital regions obtained from AD and control donors of known apoE genotype. ApoE was examined by western blot under either reducing or non-reducing conditions. ApoE was also examined in SKNSH neuronal cells and rabbit brain homogenate prepared immediately after death. A greater percentage of apoE was present as fragmentation products in the control E3/E3 brain (n=5) when compared to the diseased E4/E4 brain (n=5). This trend reached statistical significance in all three brain regions examined (p<0.05). Analysis of diseased E3/E3 brains (n=3) has so far revealed a non-significant trend for greater fragmentation than diseased E4/E4 but less than control E3/E3. Examination of the brain samples under non-reducing conditions revealed that a substantial proportion of apoE is present as a homo-dimer in E3/E3 brains regardless of the disease status. This was not observed in E4/E4 brains as E4 lacks a cysteine residue and thus is unable to form disulphide linked bonds. The contribution of post-mortem delay and contamination by serum and cerebro spinal fluid seems an unlikely cause for this phenomenon as the homo-dimer was also observed in fresh rabbit brain and a neuronal cell line. ApoE fragmentation in the brain is most strongly correlated with the E3 isoform and the absence of AD. ApoE E3 exists in the brain as a homo-dimer, thus representing a major difference between E3 and E4 in the conformation, and possibly function, of brain apoE.

Relationship of the ApoE polymorphism to plasma lipid traits among South Asians, Chinese, and Europeans living in Canada

BackgroundThe prevalence of cardiovascular risk factors and atherosclerosis vary between ethnic groups. We examined the apolipoprotein E (ApoE) polymorphism, its association with lipid traits and atherosclerosis, and its influence on ethnic variations on lipid traits. MethodsIn a randomly sampled cross-sectional study of 985 South Asian, Chinese, and European Canadians, three common isoforms of ApoE (E2, E3 and E4), plasma lipid concentrations and atherosclerosis of the carotid artery were measured. ResultsThe E2, E3 and E4 allele frequencies were 5.7%, 84.0%, and 10.2%, respectively, and differed significantly between ethnic groups. There was a strong, stepwise association between ApoE and each plasma lipid trait, except triglycerides. The E4 genotype was associated with higher low-density lipoprotein cholesterol (p for trend<0.001), ApoB (p<0.001), ApoB/ApoA ratio (p<0.001) and lipoprotein (a) (p<0.001), and lower ApoA (p<0.001) and high-density lipoprotein cholesterol (HDL-C) (p=0.005). A similar pattern of effects was observed across all ethnic groups. Ethnicity accounted for modest variation in ApoA, ApoB/ApoA ratio and HDL-C (4.2–5.0%), whereas ApoE isoforms explained only a small proportion of variability (0.3–1.4%). Dietary and lifestyle factors accounted for modest variation in several traits including HDL-C (5.6% and 5.0%, respectively). Carotid atherosclerosis was lower among individuals with E2 isoform in keeping with the effect of E2 on lipid levels. ConclusionsThe ApoE isoform is associated with plasma lipoproteins in all ethnic groups, yet explains only a small proportion of the inter-ethnic variation for most plasma lipoproteins. Additional genetic variants and/or health behaviors likely contribute to ethnic variations in plasma lipid traits.

Contributions of the Carboxyl-Terminal Helical Segment to the Self-Association and Lipoprotein Preferences of Human Apolipoprotein E3 and E4 Isoforms

To understand the molecular basis for the different self-association and lipoprotein preferences of apolipoprotein (apo) E isoforms, we compared the effects of progressive truncation of the C-terminal domain in human apoE3 and apoE4 on their lipid-free structure and lipid binding properties. A VLDL/HDL distribution assay demonstrated that apoE3 binds much better than apoE4 to HDL 3, whereas both isoforms bind similarly to VLDL. Removal of the C-terminal helical regions spanning residues 273-299 weakened the ability of both isoforms to bind to lipoproteins; this led to the elimination of the isoform lipoprotein preference, indicating that the C-terminal helices mediate the lipoprotein selectivity of apoE3 and apoE4 isoforms. Gel filtration chromatography experiments demonstrated that the monomer-tetramer distribution is different for the two isoforms with apoE4 being more monomeric than apoE3 and that removal of the C-terminal helices favors the monomeric state in both isoforms. Consistent with this, fluorescence measurements of Trp-264 in single-Trp mutants revealed that the C-terminal domain in apoE4 is less organized and more exposed to the aqueous environment than in apoE3. In addition, the solubilization of dimyristoylphosphatidylcholine multilamellar vesicles is more rapid with apoE4 than with apoE3; removal of the C-terminal helices significantly affected solubilization rates with both isoforms. Taken together, these results indicate that the C-terminal domain is organized differently in apoE3 and apoE4 so that apoE4 self-associates less and binds less than apoE3 to HDL surfaces; these alterations may lead to the pathological sequelae for cardiovascular and neurodegenerative diseases.

Detrimental Effects of Apolipoprotein E4: Potential Therapeutic Targets in Alzheimers Disease

As the major genetic risk factor for Alzheimer's disease, the apolipoprotein (apo) E4 isoform is a promising therapeutic target. ApoE4 likely contributes to Alzheimer's disease pathology by interacting with multiple factors through various pathways. Interactions with the amyloid beta peptide and the amyloid cascade, for example, may lead to cognitive decline and neurodegeneration. Alternatively, apoE4 might act independently of the amyloid beta peptide. Our working hypothesis is that apoE has isoform-specific effects on neuronal repair and remodeling. One or more injurious agents could result in neuronal damage, requiring neuronal repair or remodeling. The injurious agents (or "second hits") may be genetic, metabolic, or environmental. Potential therapeutic strategies include changing the structure of apoE4 to be more apoE3-like, inhibiting the protease that cleaves apoE4 into toxic fragments, and protecting mitochondria from apoE4 toxicity. Structural features that distinguish apoE4 and apoE3 determine their functional differences and hold the key to understanding how apoE4 is involved in Alzheimer's disease.

Structural Basis for the Recognition and Cross-linking of Amyloid Fibrils by Human Apolipoprotein E

Apolipoprotein (apo) E is a well characterized lipid-binding protein in plasma that also exists as a common nonfibrillar component of both cerebral and systemic amyloid deposits. A genetic link between a common isoform of apoE, apoE4, and the incidence of late onset Alzheimer disease has drawn considerable attention to the potential roles of apoE in amyloid-related disease. We examined the interactions of apoE with amyloid fibrils composed of apoC-II and the amyloid-beta (Abeta) peptide. Aggregates of apoE with Abeta and apoC-II are found in Alzheimer and atherosclerotic plaques, respectively. Sedimentation velocity and fibril size distribution analysis showed that apoE3 and E4 isoforms bind and noncovalently cross-link apoC-II fibrils in a similar manner. This ability to cross-link apoC-II fibrils was abolished by the dissociation of the apoE tetramer to monomers or by thrombin cleavage to yield separate N- and C-terminal domains. Preparative ultracentrifuge binding studies indicated that apoE and the isolated N- and C-terminal domains of apoE bind with submicromolar affinities to both apoC-II and Abeta fibrils. Fluorescence quenching and resonance energy transfer experiments confirmed that both domains of apoE interact with apoC-II fibrils and demonstrated that the binding of the isolated N-terminal domain of apoE to apoC-II or Abeta fibrils is accompanied by a significant conformational change with helix three of the domain moving relative to helix one. We propose a model involving the interaction of apoE with patterns of aligned residues that could explain the general ability of apoE to bind to a diverse range of amyloid fibrils.

Apolipoprotein E and Alzheimer's disease: molecular mechanisms and therapeutic opportunities

Multiple genetic and environmental factors are likely to contribute to the development of Alzheimer's disease (AD). The most important known risk factor for AD is presence of the E4 isoform of apolipoprotein E (apoE). Epidemiological studies demonstrated that apoE4 carriers have a higher risk and develop the disease and an early onset. Moreover, apoE4 is the only molecule that has been associated with all the biochemical disturbances characteristic of the disease: amyloid-beta (Aβ) deposition, tangle formation, oxidative stress, lipid homeostasis deregulation, synaptic plasticity loss and cholinergic dysfunction. This large body of evidence suggest that apoE is a key player in the pathogenesis of AD. This short review examines the current facts and hypotheses of the association between apoE4 and AD, as well as the therapeutic possibilities that apoE might offer for the treatment of this disease.

Apolipoprotein E genotype and traumatic brain injury in children—association with neurological outcome

To determine whether the presence of Apolipoprotein E epsilon4 genotype (ApoE epsilon4) is associated with outcomes of traumatic brain injury in children. The ApoE genotype was examined in the group of 70 pediatric patients who suffered from traumatic brain injury. The group consists of 48 boys and 22 girls, and the most frequent was the E3 isoform of ApoE. Polymerase chain reaction/restriction fragment length polymorphism method was used for the ApoE genotype assessment. The severity of trauma was assessed by Glasgow Coma Scale and graded into three categories. The presence of focal neurology signs, comparing the admission and dimission status, and duration of hospital care were observed. The neurological outcome after 1 year was assessed by Glasgow Outcome Scale. Trauma severity was compared with the neurological outcome, according to different ApoE genotypes. For statistical processing, t test, nonparametric Wilcoxon test, Fisher, and chi(2) tests were used. Our results suggest the association between the ApoE genotype and outcome of traumatic brain injury in children. Patients with ApoE epsilon4 genotype were more likely to have severe clinical symptomatology and unfavorable neurological outcome after traumatic brain injury compared to significantly better outcome with other ApoE genotype.

Studies on the glutathione S‐transferase proteome of adult Drosophila melanogaster: Responsiveness to chemical challenge

GSTs from adult Drosophila melanogaster have been partially purified using three different affinity chromatography media and separated by 2-DE. Nine GSTs have been identified by MALDI-TOF MS. In the absence of special treatments, eight GSTs could be positively identified. These were DmGSTs D1 (the dominant Delta isoform which was present in five protein zones of differing pI) and D3 (and possibly also D5); the Epsilon-class GSTs E3, 6, 7 and 9 and a previously uncharacterised, probable member of the class, CG16936. The Sigma-class DmGSTS1 was prominent. DmGSTD2 was detected only after pretreatment of the flies with Phenobarbital (PhB). Treatment with Paraquat (PQ) led to an increase in the total GST activity, as measured with the substrates 1-chloro-2,4-dinitrobenzene (CDNB) and 3,4-dichloro-nitrobenzene (DCNB) and an increase in the relative amounts of the D1, D3, E6 and E7 isoforms. PhB treatment led to increases in the relative amounts of the D1, D2, E3, E6, E7 and E9 isoforms detected with a possible depression in the relative amount of GSTS1. CG16936 was unaffected by either pretreatment.

Intrinsic Disorder and Autonomous Domain Function in the Multifunctional Nuclear Protein, MeCP2

To probe the tertiary structure and domain organization of native methyl CpG-binding protein 2 (MeCP2), the recombinant human e2 isoform was purified to homogeneity and characterized by analytical ultracentrifugation, CD, and protease digestion. The location of intrinsic disorder in the MeCP2 sequence was predicted using the FoldIndex algorithm. MeCP2 was found to be monomeric in low and high salt and over a nearly 1000-fold concentration range. CD indicated that the MeCP2 monomer was nearly 60% unstructured under conditions where it could preferentially recognize CpG dinucleotides and condense chromatin. Protease digestion experiments demonstrate that MeCP2 is composed of at least six structurally distinct domains, two of which correspond to the well characterized methyl DNA binding domain and transcriptional repression domain. These domains collectively are organized into a tertiary structure with coil-like hydrodynamic properties, reflecting the extensive disorder in the MeCP2 sequence. When expressed as individual fragments, the methyl DNA binding domain and transcriptional repression domain both could function as nonspecific DNA binding domains. The unusual structural features of MeCP2 provide a basis for understanding MeCP2 multifunctionality in vitro and in vivo. These studies also establish an experimental paradigm for characterizing the tertiary structures of other highly disordered proteins.

An apolipoprotein E variant may protect against age-related macular degeneration through cytokine regulation

In this study, the authors investigated the association between apolipoprotein E (ApoE C112R/R158C) single nucleotide polymorphisms (which determine the E2, E3, and E4 isoforms) and age-related macular degeneration (AMD), and the mechanism underlying the association. Genomic DNA was extracted from 133 clinically screened controls, 94 volunteers with a younger mean age, 120 patients with advanced AMD, and 40 archived ocular AMD slides for single nucleotide polymorphism typing. The effects of recombinant ApoE isoforms on CCL2 (a chemokine), CX3CR1 (a chemokine receptor), and VEGF (a cytokine) expression in cultured human retinal pigment epithelium (RPE) cells were tested and serum cholesterol profiles of the clinically screened subjects were analyzed.

Apolipoprotein E deficiency increased microglial activation/CCR3 expression and hippocampal damage in kainic acid exposed mice

Apolipoprotein E (apoE) down-regulates microglial activation and the secretion of inflammatory molecules in an isoform specific fashion (E2 > E3 > E4); the E4 isoform is over-represented in Alzheimer cases while E2 is under-represented. To better define the role of apoE in neurodegeneration, we contrasted apoE knockout ( n = 38) and wild-type mice ( n = 41) with respect to seizure activity, mortality, locomotion, hippocampal microglial activation/chemokine receptor expression, and damage to the hippocampus after nasal administration of kainic acid (KA) (water as controls). Mice lacking apoE demonstrated more hunching and less rearing, more damage to neurons in the CA3 region (mean histopathologic score: 3.7 vs. 1.6, p < 0.05), greater microglial activation confirmed by high levels of CD11b and CD86 expression in hippocampus (CD11b p < 0.01, CD86 p < 0.05), and a greater percentage of activated microglia expressing CC chemokine receptors 3 (CCR3) ( p < 0.05). Taken together, these findings imply that apoE modulates hippocampal damage induced by KA and found early in the sequence of human Alzheimer's brain changes, by modulating microglial activation.

An apolipoprotein E variant may protect against age‐related macular degeneration through cytokine regulation

Age-related macular degeneration (AMD) is the leading cause of visual impairment and blindness among the elderly in Western countries. Genetic factors, age, cigarette smoking, nutrition, and exposure to light have been identified as AMD risk factors. In this study, we investigated the association between ApoE C112R/R158C single nucleotide polymorphisms (which determine the E2, E3, and E4 isoforms) and age-related macular degeneration (AMD), and the mechanism underlying the association. Genomic DNA was extracted from 133 clinically screened controls, 94 volunteers with a younger mean age, 120 patients with advanced AMD, and 40 archived ocular AMD slides for single nucleotide polymorphism typing. The effects of recombinant ApoE isoforms on CCL2 (a chemokine), CX3CR1 (a chemokine receptor), and VEGF (a cytokine) expression in cultured human retinal pigment epithelium (RPE) cells were tested and serum cholesterol profiles of the clinically screened subjects were analyzed. ApoE112R (E4) distribution differed significantly between AMD patients and controls. ApoE112R allele frequency was 10.9% in the AMD group when compared with 16.5% in the younger controls and 18.8% in the clinically screened controls. The pathologically diagnosed archived AMD cases had the lowest allele frequency of 5%. No significant differences in ApoE158C (E2) distribution were observed among the groups. A meta-analysis of 8 cohorts including 4,289 subjects showed a strong association between AMD and 112R, but not 158C. In vitro studies found that recombinant ApoE suppresses CCL2 and VEGF expression in RPE cells. However, the E4 isoform showed more suppression than E3 in both cases. These results further confirm the association between ApoE112R and a decreased risk of AMD development. The underlying mechanisms may involve differential regulation of both CCL2 and VEGF by the ApoE isoforms.

Apolipoprotein E-derived peptides reduce CNS inflammation: implications for therapy of neurological disease

The apolipoprotein E4 isoform (apoE4) was initially identified as a susceptibility gene for the development of Alzheimer's disease, and has also recently been associated with poor outcome after acute traumatic and ischemic brain injury. One mechanism by which apoE may influence outcome in acute and chronic neurological disease is by downregulating glial activation and the neuroinflammatory response. Because it does not readily cross the blood-brain barrier (BBB), the apoE holoprotein has limited therapeutic potential. However, smaller peptides derived from the receptor binding region of apoE have been developed that mimic the functional anti-inflammatory and neuroprotective effects of the intact apoE protein. These apoE-derived therapeutic peptides cross the BBB and have been demonstrated to improve functional and histological outcomes in murine models of brain injury. Thus, the development of apoE-derived peptides represent a novel therapeutic strategy for the treatment of acute and chronic neurological disease.

Dynamics of the actin-binding protein drebrin in motile cells and definition of a juxtanuclear drebrin-enriched zone

The actin-binding protein (ABP) drebrin, isoform E2, is involved in remodelling of the actin cytoskeleton and in formation of cell processes, but its role in cell migration has not yet been investigated. Therefore, we have studied the organization of drebrin in motile cultured cells such as murine B16F1 melanoma and human SV80 fibroblast cells, using live cell confocal microscopy. In cells overexpressing DNA constructs encoding drebrin linked to EGFP, numerous long, branched cell processes were formed which slowly retracted and extended, whereas forward movement was halted. In contrast, stably transfected B16F1 cells containing drebrin-EGFP at physiological levels displayed lamellipodia and were able to migrate on laminin. Surprisingly, in such cells, drebrin was absent from anterior lamellipodia but was enriched in a specific juxtanuclear zone, the “drebrin-enriched zone” (DZ), and in the tail. In leading edges of SV80 cells, characterized by pronounced actin microspikes, drebrin was specifically enriched along posterior portions of the microspikes, together with tropomyosin. Drebrin knock-down by small interfering RNAs did not impair movements of SV80 cells. Our results confirm the role of drebrin E2 in the formation of branching processes and further indicate that during cell migration, the protein contributes to retraction of the cell body and the tail but not to lamellipodia formation. In particular, the novel, sizable juxtanuclear DZ structure will have to be characterized in future experiments with respect to its molecular assembly and cell biological functions.

A Novel Rat Homolog of the Saccharomyces cerevisiae Ubiquitin-Conjugating Enzymes UBC4 and UBC5 with Distinct Biochemical Features Is Induced during Spermatogenesis

The Saccharomyces cerevisiae ubiquitin-conjugating enzymes (E2s) UBC4 and UBC5 are essential for degradation of short-lived and abnormal proteins. We previously identified rat cDNAs encoding two E2s with strong sequence similarity to UBC4 and UBC5. These E2 isoforms are widely expressed in rat tissues, consistent with a fundamental cellular function for these E2s. We now report a new isoform, 8A, which despite having >91% amino acid identity with the other isoforms, shows several novel features. Expression of the 8A isoform appears restricted to the testis, is absent in early life, but is induced during puberty. Hypophysectomy reduced expression of the 8A isoform. In situ hybridization studies indicated that 8A mRNA is expressed mainly in round spermatids. Immunoblot analyses showed that 8A protein is found not only in subfractions of germ cells enriched in round spermatids but also in subfractions containing residual bodies extruded from more mature elongated spermatids, indicating that the protein possesses a longer half-life than the mRNA. Unlike all previously identified mammalian and plant homologs of S. cerevisiae UBC4, which possess a basic pI, the 8A isoform is unique in possessing an acidic pI. The small differences in sequence between the 8A isoform and other rat isoforms conferred differences in biochemical function. The 8A isoform was less effective than an isoform with a basic pI or ineffective in conjugating ubiquitin to certain fractions of testis proteins. Thus, although multiple isoforms of a specific E2 may exist to ensure performance of a critical cellular function, our data demonstrate, for the first time, that multiple genes also permit highly specialized regulation of expression of specific isoforms and that subtle differences in E2 primary structure can dictate conjugation of ubiquitin to different subsets of cellular proteins.

Drebrin, an Actin-Binding, Cell-Type Characteristic Protein: Induction and Localization in Epithelial Skin Tumors and Cultured Keratinocytes

Isoform E2 of drebrin, an actin-binding protein originally identified in neuronal cells, has recently been identified in diverse non-neuronal cells, mostly in association with cell processes and intercellular junctions. Here, we report on the presence of drebrin in normal human skin, epithelial skin cancers, and cultured keratinocytes. Keratinocytes of normal epidermis contain almost no drebrin but the protein is readily seen in hair follicles. By immunohistochemistry and immunoblot, basal cell carcinomas (BCC) are rich in drebrin, and confocal laser scanning and immunoelectron microscopy show accumulation at adhering junctions, in co-localization with actin and partially with plaque proteins. In squamous cell carcinomas, keratoacanthomas, and in epidermal precancers, drebrin is heterogeneously distributed, appearing as mosaics. Primary keratinocyte cultures contain significant amounts of drebrin enriched at adhering junctions. When epithelium-derived cells devoid of drebrin are transfected with drebrin-enhanced green fluorescent protein, constructs accumulate in the cell periphery, and immunoprecipitation shows complexes with actin. During epidermal growth factor induced formation of cell processes, drebrin retains this junction association, as observed by live cell microscopy. Our results suggest novel functions of drebrin such as an involvement in cell-cell adhesion and tumorigenesis and a potential value in diagnosis of BCC.

Apolipoprotein E Regulates Primary Cultured Human Mesangial Cell Proliferation

Background: The role of apolipoprotein (apo) E in kidney disease is still unclear. Animal studies have been performed, but it is doubtful if the conclusions are applicable to human beings. The objective of this study was to determine how apo E acts on human kidneys using primary cultured normal human mesangial cells (NHMCs) rather than animals used in previous studies. Methods: apo E and its isoforms E2, E3 and E4, or combinations with apo B were cocultured with primary NHMCs in serum-free medium. Premix WST-1 Cell Proliferation Assay System and DNA-Prep Reagent System were used to measure the proliferation and apoptosis of NHMCs, respectively. Results: (1) apo E itself increased NHMC proliferation at 24 h of culture, while it inhibited this proliferation after 48 h. (2) At 72 h of culture, apo E alone inhibited NHMC proliferation at concentrations higher than 0.78 µg/ml in concentration-dependent manner. (3) When co-cultured with both apo E and apo B, NHMC proliferation was higher than that with apo E alone and lower than that with apo B alone. (4) At 72 h of culture, apo E2, E3 and E4 inhibited NHMC proliferation at different intensities, with no proliferative effect observed. (5) Neither apo E nor apo B caused NHMC apoptosis. Conclusion: apo E regulates primary NHMC proliferation by (1) inhibiting NHMC proliferation or reducing NHMC proliferation induced by apo B, which implies that apo E has a protective effect on the kidney, and (2) increasing the proliferation under certain conditions.