HEK293FT Cells Research Articles

Microchip amplifier for in vitro, in vivo, and automated whole cell patch-clamp recording.

Patch clamping is a gold-standard electrophysiology technique that has the temporal resolution and signal-to-noise ratio capable of reporting single ion channel currents, as well as electrical activity of excitable single cells. Despite its usefulness and decades of development, the amplifiers required for patch clamping are expensive and bulky. This has limited the scalability and throughput of patch clamping for single-ion channel and single-cell analyses. In this work, we have developed a custom patch-clamp amplifier microchip that can be fabricated using standard commercial silicon processes capable of performing both voltage- and current-clamp measurements. A key innovation is the use of nonlinear feedback elements in the voltage-clamp amplifier circuit to convert measured currents into logarithmically encoded voltages, thereby eliminating the need for large high-valued resistors, a factor that has limited previous attempts at integration. Benchtop characterization of the chip shows low levels of current noise [1.1 pA root mean square (rms) over 5 kHz] during voltage-clamp measurements and low levels of voltage noise (8.2 μV rms over 10 kHz) during current-clamp measurements. We demonstrate the ability of the chip to perform both current- and voltage-clamp measurement in vitro in HEK293FT cells and cultured neurons. We also demonstrate its ability to perform in vivo recordings as part of a robotic patch-clamping system. The performance of the patch-clamp amplifier microchip compares favorably with much larger commercial instrumentation, enabling benchtop commoditization, miniaturization, and scalable patch-clamp instrumentation.

A novel centrosome and microtubules associated subcellular localization of Nogo-A: Implications for neuronal development

Oligodendrocyte-derived neurite-outgrowth inhibitor Nogo-A and its restriction mechanism are well-known. Recently, Nogo-A is reported to be abundantly expressed in neurons, however, the concrete link between neuronal Nogo-A and neuronal development is poorly understood. In the present study, we used Neuro2A and COS7 cell lines to clarify that Nogo-A largely distributed in the centrosome and microtubules-rich regions. When endogenous Nogo-A was down-regulated with RNA interference, the percentage of cell differentiation and the total neurite length of Neuro2A exposed to valproic acid (VPA) decreased sharply. Furthermore, in primary neurons, acetylated α-tubulin decreased at the tips of neurites where endogenous Nogo-A was still highly expressed. In HEK293FT cell lines, Nogo-A overexpression could redistribute acetylated α-tubulin but not change the level of α-tubulin. Together, our data discovered that centrosome- and microtubules-localized Nogo-A positively regulates neuronal differentiation and neurite outgrowth of Neuro2A cell lines, implicating the essential roles of subcellular Nogo-A in neuronal development.

Abstract 480: CDK9 (a novel BRCA1/BARD1 interaction partner) downregulates BRCA1-mediated transcription

Abstract BRCA1 gene encodes a 1863 amino-acid protein comprising a RING finger domain in the N-terminus and two tandem BRCT domains (tBRCT) in C-terminal region. When fused to a heterologous DNA-binding domain, BRCA1 C-terminal region is capable of activating transcription. In addition, BRCA1 was found associated with the RNA polymerase II holoenzyme (RNApolII), a number of transcription factors and regulators. BRCA1 cancer-associated mutations located in the C-terminal region of the protein lead to an impaired transcriptional activation phenotype - the ability to trigger transcription seems to be an important feature of BRCA1 tumor suppression function. BARD1 (BRCA1 associated RING domain 1) also encloses a N-terminal RING finger and a C-terminal tBRCT domains. The BRCA1/BARD1 heterodimer was found to ubiquitinate RNApolII leading to a reduced transcription initiation. Recently, our group described a protein interaction network that identified putative interacting partners of seven different proteins enclosing the tBRCT domain. Among other hits, Cyclin-Dependent Kinase 9 (CDK9) was identified as a common interaction partner of BRCA1 and also BARD1 tBRCTs. CDK9 is a component of the positive transcription elongation factor b complex (P-TEFb), which is involved in co-transcriptional histone modification, mRNA processing, mRNA export and transcriptional elongation. The interaction between constitutive CDK9 and BARD1 was confirmed through co-immunoprecipitation (Co-IP) assay using HEK293FT cells nuclear extracts. We also demonstrated that CDK9 isoforms were able to interact both with BARD1 N- and C-terminal regions in a GST pulldown assay. In addition, the BRCA1/CDK9 constitutive interaction was confirmed by Co-IP assay in HeLa cells. We showed that, differently from BARD1, BRCA1 tBRCT interacts only with the 42KDa isoform of CDK9. In order to investigate CDK9 role in BRCA1 mediated transcription, the kinase expression was downregulated (by shRNA methodology) in HeLa cells, resulting in a two-fold increase in transcription (observed in a luciferase reporter assay). On the other hand, the super expression of CDK9 42KDa isoform leads to the suppression of BRCA1 mediated transcription. These data suggest that the CDK9 42KDa isoform modulates BRCA1 mediated transcription through its tBRCT domain. Now, we are extending our approach to investigate which genes are regulated by this complex. We also ought to investigate the role of BARD1 in the CDK9 regulation function. A better understanding of these interactions will help to unravel the BRCA1 mediated transcription and its tumor suppression function. Citation Format: Thales C. Nepomuceno, Vanessa C. Fernandes, Giuliana De Gregoris, Renato S. Carvalho, Álvaro N. Monteiro, Marcelo A. Carvalho. CDK9 (a novel BRCA1/BARD1 interaction partner) downregulates BRCA1-mediated transcription. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 480. doi:10.1158/1538-7445.AM2014-480

Overexpressed NLRC3 Acts as an Anti-Inflammatory Cytosolic Protein

The novel nucleotide oligomerization domain (NOD)-like receptor (NLR) with a caspase activation and recruitment domain (CARD) 3 (NLRC3) protein belongs to the NLR family of cytosolic pathogen recognition receptors. NLRC3 has the characteristic NOD and leucine-rich repeat configuration with a less well defined CARD. T lymphocytes are known to have high NLRC3 expression, which may be involved in suppression of T cell activation. Here, we report that NLRC3 is a cytoplasmic protein that negatively regulates pro-IL-1β maturation. Among well-known inflammasome components, NLRC3 can interact with apoptosis-associated speck-like protein containing a CARD (ASC) and caspases 1 and 5. Transient transfection of NLRC3 into stable EGFP-ASC-expressing HEK293FT cells reduces NLR family, pyrin domain-containing 3 (NLRP3)/cryopyrin-induced formation of ASC specks in a dose- and time-dependent manner. This suggests that NLRC3 can regulate ASC speck formation, caspase-1 activation and IL-1β maturation. We show for the first time that inflammasome-like complexes assemble when caspase-1 and ASC are cotransfected together with NLRC3 in HEK293FT cells. However, overexpression of NLRC3 with NLRP3/cryopyrin inflammasome components suppresses pro-caspase-1 cleavage and IL-1β processing. Our study suggests that NLRC3 negatively regulates inflammatory responses.

Rapid and Efficient Assembly of Transcription Activator-Like Effector Genes by USER Cloning

Transcription activator-like effectors (TALEs) that were related to bacteria immune system have lately been employed in a promising approach of precise gene targeting. Because of the repetitive characteristics of TALEs, existing TALE assembly methods are either very complicated, time-consuming, or too tricky to be handled in common labs. Here, we reported a rapid, efficient and easy method for TALE assembly. This method takes advantage of uracil-specific excision reagent (USER), an enzyme that can cleave DNA constructs and create long, unique single-strand DNA overhangs. Upon USER treatment, the overhangs on each individual TALE repeat unit can be rejoined hierarchically to form pentamers in a ligation-independent manner. Eventually, three pentamers are assembled into a full TALE construct by Golden Gate cloning. TALE nucleases (TALENs) generated with this method exhibit high genome-editing activity in human cells such as HEK293FT cells. Using this method, we have successfully synthesized three TALEN pairs targeting endogenous Tet1 locus, and proved that all can specifically target Tet1 gene, though in various degree. Comparing to other methods of TALEN assembly, this one is much less labor intensive and fairly faster, and positive clones can be obtained at high efficiency within only two days. We thus contribute to an easier approach for effective TALENs synthesis, which may highly facilitate the wide application of TALEN technology in genome editing, especially for human cells that require precise targeting.

Optogenetic activation of Gq signalling modulates pacemaker activity of cardiomyocytes

Investigation of Gq signalling with pharmacological agonists of Gq-coupled receptors lacks spatio-temporal precision. The aim of this study was to establish melanopsin, a light-sensitive Gq-coupled receptor, as a new tool for the investigation of spatial and temporal effects of Gq stimulation on pacemaking in cardiomyocytes at an early developmental stage. A vector for ubiquitous expression of melanopsin was tested in HEK293FT cells, which showed light-induced production of inositol-1,4,5-trisphosphate and elevation of intracellular Ca(2+) concentration. Mouse embryonic stem cells were stably transfected with this plasmid and differentiated into spontaneously beating embryoid bodies (EBs). Cardiomyocytes within EBs showed melanopsin expression and illumination (60 s, 308.5 nW/mm(2), 470 nm) of EBs increased beating rate within 10.2 ± 1.7 s to 317.1 ± 16.3% of baseline frequency. Illumination as short as 5 s was sufficient for generating the maximal frequency response. After termination of illumination, baseline frequency was reached with a decay constant of 27.1 ± 2.5 s. The light-induced acceleration of beating frequency showed a sigmoid dependence on light intensity with a half maximal effective light intensity of 41.7 nW/mm(2). Interestingly, EBs showed a high rate of irregular contractions after termination of high-intensity illumination. Local Gq activation by illumination of a small region in a functional syncytium of cardiomyocytes led to pacemaker activity within the illuminated area. Light-induced Gq activation in melanopsin-expressing cardiomyocytes increases beating rate and generates local pacemaker activity. We propose that melanopsin is a powerful optogenetic tool for the investigation of spatial and temporal aspects of Gq signalling in cardiovascular research.

Elevated YAP and Its Downstream Targets CCN1 and CCN2 in Basal Cell Carcinoma: Impact on Keratinocyte Proliferation and Stromal Cell Activation

Elevated YAP and Its Downstream Targets CCN1 and CCN2 in Basal Cell Carcinoma: Impact on Keratinocyte Proliferation and Stromal Cell Activation

Modulation of Inactivation Kinetics of the Bacterial Sodium Channel Nachbac Suggests a Complex Mode of Inhibition by Isoflurane

The bacterial sodium channel NaChBac is a prokaryotic ancestor of eukaryotic voltage-gated sodium channels (Nav), which are critical for action potential generation and propagation in nervous and cardiac tissue. Like all mammalian Nav isoforms examined thus far, NaChBac is inhibited by clinically relevant concentrations of the inhaled volatile anesthetic isoflurane, and inhibition is accompanied by enhancement of slow or C-type inactivation (Ouyang et al., 2007). However, a detailed mechanistic explanation of the interplay between C-type inactivation and isoflurane inhibition is lacking. To shed light on the relationship between inactivation and inhibition, we introduced point mutations known to alter inactivation in NaChBac (G219A, G219P, G229A, and S195E), expressed the channels in HEK293FT cells, and applied isoflurane during whole-cell patch clamp recording. Preliminary data support the idea that isoflurane acts by multiple mechanisms invovling multiple sites on NaChBac. Channel mutations that enhance inactivation show greater current reduction by isoflurane, suggesting that isoflurane binds more favorably to inactivated channels. However, isoflurane exhibits both tonic and use-dependent block of the essentially non-inactivating NaChBac G219P mutant, indicating that binding also occurs in the closed/resting and open/conducting states. A detailed biophysical and pharmacological profile of these NaChBac inactivation variants together with a recent molecular dynamics simulation study showing isoflurane interacting with NaChBac at three distinct binding sites (Carnavale et al, 2013) supports multiple mechanisms of state-dependent inhibtion. Such functional data help clarify the complex pharmacological effects of volatile anesthetics on Nav channels, and contribute to better understanding of C-type inactivation in these proteins. Supported by NIH grant GM 58055 (RS and HH) and German Research Foundation fellowship 5767/1-1 (TM).

Efficient Production of Retroviruses Using PLGA/bPEI-DNA Nanoparticles and Application for Reprogramming Somatic Cells

Reprogramming of somatic cells to pluripotent cells requires the introduction of factors driving fate switches. Viral delivery has been the most efficient method for generation of induced pluripotent stem cells. Transfection, which precedes virus production, is a commonly-used process for delivery of nucleic acids into cells. The aim of this study is to evaluate the efficiency of PLGA/ bPEI nanoparticles in transfection and virus production. Using a modified method of producing PLGA nanoparticles, PLGA/bPEI-DNA nanoparticles were examined for transfection efficiency and virus production yield in comparison with PLGA-DNA, bPEI-DNA nanoparticles or liposome-DNA complexes. After testing various ratios of PLGA, bPEI, and DNA, the ratio of 6:3:1 (PLGA:bPEI:DNA, w/w/w) was determined to be optimal, with acceptable cellular toxicity. PLGA/bPEI-DNA (6:3:1) nanoparticles showed superior transfection efficiency, especially in multiple gene transfection, and viral yield when compared with liposome-DNA complexes. The culture supernatants of HEK293FT cells transfected with PLGA/bPEI-DNA of viral constructs containing reprogramming factors (Oct4, Sox2, Klf4, or c-Myc) successfully and more efficiently generated induced pluripotent stem cell colonies from mouse embryonic fibroblasts. These results strongly suggest that PLGA/bPEI-DNA nanoparticles can provide significant advantages in studying the effect of multiple factor delivery such as in reprogramming or direct conversion of cell fate.

Non-specific Adsorption of Crude Cell Lysate on Surface Plasmon Resonance Sensors

Non-specific adsorption of the molecular components of biofluids is ubiquitous in the area of biosensing technologies, severely limiting the use of biosensors in real-world applications. The surface chemistries developed to prevent non-specific adsorption of crude serum are not necessarily suited for sensing in other biosamples. In particular, the diagnostic potential of differential expression of proteins in tissues makes cell lysate attractive for disease diagnostics using solid biopsies. However, crude cell lysate poses a significant challenge for surface chemistries because of a large concentration of highly adherent lipids. Contrary to the non-specific adsorption in crude serum being suppressed by hydrophilic surfaces, the surface plasmon resonance (SPR) analysis of serine-, aspartic-acid-, histidine-, leucine-, and phenylalanine-based peptide monolayers revealed that hydrophobic and positively charged peptides decreased non-specific adsorption when using lysate from HEK 293FT cells. A polyethylene glycol (PEG) monolayer resulted in 2-fold greater fouling than the best peptide [3-MPA-(His)2(Leu)2(Phe)2-OH] under the same conditions. Matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF MS) analysis of the adsorbate from cell lysate confirmed that lipids are the main source of non-specific adsorption. Importantly, the mass spectrometry (MS) study revealed that both the number of lipids identified and their intensity decreased with decreasing non-specific adsorption. A peptide monolayer thus provides an efficient mean to suppress non-specific adsorption from this human cell lysate.

Characterization of Three Vasopressin Receptor 2 Variants: An Apparent Polymorphism (V266A) and Two Loss-of-Function Mutations (R181C and M311V)

Arginine vasopressin (AVP) is released from the posterior pituitary and controls water homeostasis. AVP binding to vasopressin V2 receptors (V2Rs) located on kidney collecting duct epithelial cells triggers activation of Gs proteins, leading to increased cAMP levels, trafficking of aquaporin-2 water channels, and consequent increased water permeability and antidiuresis. Typically, loss-of-function V2R mutations cause nephrogenic diabetes insipidus (NDI), whereas gain-of-function mutations cause nephrogenic syndrome of inappropriate antidiuresis (NSIAD). Here we provide further characterization of two mutant V2Rs, R181C and M311V, reported to cause complete and partial NDI respectively, together with a V266A variant, in a patient diagnosed with NSIAD. Our data in HEK293FT cells revealed that for cAMP accumulation, AVP was about 500- or 30-fold less potent at the R181C and M311V mutants than at the wild-type receptor respectively (and about 4000- and 60-fold in COS7 cells respectively). However, in contrast to wild type V2R, the R181C mutant failed to increase inositol phosphate production, while with the M311V mutant, AVP exhibited only partial agonism in addition to a 37-fold potency decrease. Similar responses were detected in a BRET assay for β-arrestin recruitment, with the R181C receptor unresponsive to AVP, and partial agonism with a 23-fold decrease in potency observed with M311V in both HEK293FT and COS7 cells. Notably, the V266A V2R appeared functionally identical to the wild-type receptor in all assays tested, including cAMP and inositol phosphate accumulation, β-arrestin interaction, and in a BRET assay of receptor ubiquitination. Each receptor was expressed at comparable levels. Hence, the M311V V2R retains greater activity than the R181C mutant, consistent with the milder phenotype of NDI associated with this mutant. Notably, the R181C mutant appears to be a Gs protein-biased receptor incapable of signaling to inositol phosphate or recruiting β-arrestin. The etiology of NSIAD in the patient with V266A V2R remains unknown.

Profiling Epidermal Growth Factor Receptor and Heregulin Receptor 3 Heteromerization Using Receptor Tyrosine Kinase Heteromer Investigation Technology

Heteromerization can play an important role in regulating the activation and/or signal transduction of most forms of receptors, including receptor tyrosine kinases (RTKs). The study of receptor heteromerization has evolved extensively with the emergence of resonance energy transfer based approaches such as bioluminescence resonance energy transfer (BRET). Here, we report an adaptation of our Receptor-Heteromer Investigation Technology (Receptor-HIT) that has recently been published as the G protein-coupled receptor (GPCR) Heteromer Identification Technology (GPCR-HIT). We now demonstrate the utility of this approach for investigating RTK heteromerization by examining the functional interaction between the epidermal growth factor (EGF) receptor (EGFR; also known as erbB1/HER1) and heregulin (HRG) receptor 3 (HER3; also known as erbB3) in live HEK293FT cells using recruitment of growth factor receptor-bound protein 2 (Grb2) to the activated receptors. We found that EGFR and HER3 heteromerize specifically as demonstrated by HRG inducing a BRET signal between EGFR/Rluc8 and Grb2/Venus only when HER3 was co-expressed. Similarly, EGF stimulation promoted a specific BRET signal between HER3/Rluc8 and Grb2/Venus only when EGFR was co-expressed. Both EGF and HRG effects on Grb2 interaction are dose-dependent, and specifically blocked by EGFR inhibitor AG-1478. Furthermore, truncation of HER3 to remove the putative Grb2 binding sites appears to abolish EGF-induced Grb2 recruitment to the EGFR-HER3 heteromer. Our results support the concept that EGFR interacts with Grb2 in both constitutive and EGF-dependent manners and this interaction is independent of HER3 co-expression. In contrast, HER3-Grb2 interaction requires the heteromerization between EGFR and HER3. These findings clearly indicate the importance of EGFR-HER3 heteromerization in HER3-mediated Grb2-dependent signaling pathways and supports the central role of HER3 in the diversity and regulation of HER family functioning.

Embryonic Stem Cell–Based Modeling of Tau Pathology in Human Neurons

Alterations in the microtubule (MT)-associated protein, tau, have emerged as a pivotal phenomenon in several neurodegenerative disorders, including frontotemporal dementia and Alzheimer's disease. Although compelling lines of evidence from various experimental models suggest that hyperphosphorylation and conformational changes of tau can cause its aggregation into filaments, the actual tau species and effective mechanisms that conspire to trigger the degeneration of human neurons remain obscure. Herein, we explored whether human embryonic stem cell-derived neural stem cells can be exploited to study consequences of an overexpression of 2N4R tau (two normal N-terminal and four MT-binding domains; n-tau) versus pseudohyperphosphorylated tau (p-tau) directly in human neurons. Given the involvement of tau in MT integrity and cellular homeostasis, we focused on the effects of both tau variants on subcellular transport and neuronal survival. By using inducible lentiviral overexpression, we show that p-tau, but not n-tau, readily leads to an MC-1-positive protein conformation and impaired mitochondrial transport. Although these alterations do not induce cell death under standard culture conditions, p-tau-expressing neurons cultured under non-redox-protected conditions undergo degeneration with formation of axonal varicosities sequestering transported proteins and progressive neuronal cell death. Our data support a causative link between the phosphorylation and conformational state of tau, microtubuli-based transport, and the vulnerability of human neurons to oxidative stress. They further depict human embryonic stem cell-derived neurons as a useful experimental model for studying tau-associated cellular alterations in an authentic human system.

BMP Induces Cochlin Expression to Facilitate Self-renewal and Suppress Neural Differentiation of Mouse Embryonic Stem Cells

BMP4 maintains self-renewal of mouse embryonic stem cells (ESCs) in collaboration with LIF. Here, we report the identification of a novel key BMP target gene, cochlin (Coch) in mouse ESCs. Coch can be significantly up-regulated by BMP4 specifically in ESCs but not in somatic differentiated cells, and this up-regulation is dependent on the BMP signaling mediators Smad1/5 and Smad4. Overexpression of Coch can partially substitute BMP4 to promote self-renewal of mouse ESCs together with LIF, whereas knockdown of Coch impairs self-renewal marker gene expression even in the presence of both BMP4 and LIF. Further studies showed that COCH could mimic BMP4 in repressing neural differentiation of mouse ESCs upon LIF withdrawal and the inhibitory effect of BMP4 on neural differentiation is compromised by Coch knockdown. Taken together, our data suggest that COCH is a part of the downstream target network of BMP signaling and serves as another important effector to fine-tune mouse ESC fates.

Interaction of 14-3-3σ with KCMF1 suppresses the proliferation and colony formation of human colon cancer stem cells

To investigate the biological function of 14-3-3σ protein and to look for proteins that interact with 14-3-3σ protein in colon cancer stem cells. Reverse transcription polymerase chain reaction was performed to amplify the 14-3-3σ gene from the mRNA of colon cancer stem cells. The gene was then cloned into the pGEM-T vector. After being sequenced, the target gene 14-3-3σ was cut from the pGEM-T vector and cloned into the pGBKT7 yeast expression plasmid. Then, the bait plasmid pGBKT7-14-3-3σ was transformed into the yeast strain AH109. After the expression of the pGBKT7-14-3-3σ fusion protein in the AH109 yeast strain was accomplished, a yeast two-hybrid screening assay was performed by mating AH109 with Y187 that contained a HeLa cDNA library plasmid. The interaction between the 14-3-3σ protein and the proteins obtained from positive colonies was further confirmed by repeating the yeast two-hybrid screen. After extracting and sequencing the plasmids from the positive colonies, we performed a bioinformatics analysis. A coimmunoprecipitation assay was performed to confirm the interaction between 14-3-3σ and the proteins obtained from the positive colonies. Finally, we constructed 14-3-3σ and potassium channel modulatory factor 1 (KCMF1) siRNA expression plasmids and transfected them into colon cancer stem cells. The bait plasmid pGBKT7-14-3-3σ was constructed successfully, and the 14-3-3σ protein had no toxic or autonomous activation effect on the yeast. Nineteen true-positive colonies were selected and sequenced, and their full-length sequences were obtained. We searched for homologous DNA sequences for these sequences from GenBank. Among the positive colonies, four coding genes with known functions were obtained, including KCMF1, quinone oxidoreductase (NQO2), hydroxyisobutyrate dehydrogenase (HIBADH) and 14-3-3σ. For the subsequent coimmunoprecipitation assay, the plasmids PCDEF-Flag-14-3-3σ, PCDEF-Myc-KCMF1, PCDEF-Myc-NQO2 and PCDEF-Myc-HIBADH were successfully constructed, and the sequences were further confirmed by DNA sequencing. The Fugene 6 reagent was used to transfect the plasmids, and fluorescence-activated cell sorting analysis showed the transfection efficiency was 97.8% after 48 h. The HEK 293FT cells showed the stable expression of the PCDEF-Flag-14-3-3σ, PCDEF-Myc-KCMF1, PCDEF-Myc-NQO2 and PCDEF-Myc-HIBADH plasmids. After anti-Myc antibody immunoprecipitation with Myc-KCMF1, Myc-NQO2 and Myc-HIBADH from cell lysates, the presence of Flag-14-3-3σ protein in the immunoprecipitated complex was determined by western blot analysis. The knock-down expression of the 14-3-3σ and KCMF1 proteins significantly inhibited cell proliferation and colony formation of SW1116csc. Genes of the proteins that interacted with 14-3-3σ were successfully screened from a HeLa cDNA library. KCMF1 and 14-3-3σ protein may affect the proliferation and colony formation of human colon cancer stem cells.

Epigenetic Regulation of Galectin-3 Expression by β1 Integrins Promotes Cell Adhesion and Migration

Introduction of the integrin β1- but not the β3-subunit in GE11 cells induces an epithelial-to-mesenchymal-transition (EMT)-like phenomenon that is characterized by the loss of cell-cell contacts, cell scattering, increased cell migration and RhoA activity, and fibronectin fibrillogenesis. Because galactose-binding lectins (galectins) have been implicated in these phenomena, we investigated whether galectins are involved in the β1-induced phenotype. We examined 9 galectins and, intriguingly, found that the expression of galectin-3 (Gal-3) is specifically induced by β1 but not by β3. Using β1-β3 chimeric integrins, we show that the induction of Gal-3 expression requires the hypervariable region in the extracellular domain of β1, but not its cytoplasmic tail. Furthermore, Gal-3 expression does not depend on RhoA signaling, serum factors, or any of the major signal transduction pathways involving protein kinase C (PKC), p38 mitogen-activated protein kinase (p38MAPK), extracellular signal-regulated kinase-1/-2 (ERK-1/2), phosphatidylinositol-3-OH kinase (PI3-K), or Src kinases. Instead, Gal-3 expression is controlled in an epigenetic manner. Whereas DNA methylation of the Lgals3 promoter maintains Gal-3 silencing in GE11 cells, expression of β1 causes its demethylation, leading to transcriptional activation of the Lgals3 gene. In turn, Gal-3 expression enhances β1 integrin-mediated cell adhesion to fibronectin (FN) and laminin (LN), as well as cell migration. Gal-3 also promotes β1-mediated cell adhesion to LN and Collagen-1 (Col)-1 in cells that endogenously express Gal-3 and β1 integrins. In conclusion, we identify a functional feedback-loop between β1 integrins and Gal-3 that involves the epigenetic induction of Gal-3 expression during integrin-induced EMT and cell scattering.

Protein Kinase Cα (PKCα) Regulates p53 Localization and Melanoma Cell Survival Downstream of Integrin αv in Three-dimensional Collagen and in Vivo

Protein kinase C α (PKCα) is overexpressed in numerous types of cancer. Importantly, PKCα has been linked to metastasis of malignant melanoma in patients. However, it has been unclear how PKCα may be regulated and how it exerts its role in melanoma. Here, we identified a role for PKCα in melanoma cell survival in a three-dimensional collagen model mimicking the in vivo pathophysiology of the dermis. A pathway was identified that involved integrin αv-mediated up-regulation of PKCα and PKCα-dependent regulation of p53 localization, which was connected to melanoma cell survival. Melanoma survival and growth in three-dimensional microenvironments requires the expression of integrin αv, which acts to suppress p53 activity. Interestingly, microarray analysis revealed that PKCα was up-regulated by integrin αv in a three-dimensional microenvironment-dependent manner. Integrin αv was observed to promote a relocalization of endogenous p53 from the nucleus to the cytoplasm upon growth in three-dimensional collagen as well as in vivo, whereas stable knockdown of PKCα inhibited the integrin αv-mediated relocalization of p53. Importantly, knockdown of PKCα also promoted apoptosis in three-dimensional collagen and in vivo, resulting in reduced tumor growth. This indicates that PKCα constitutes a crucial component of the integrin αv-mediated pathway(s) that promote p53 relocalization and melanoma survival.

Monoubiquitination Promotes Calpain Cleavage of the Protein Phosphatase 2A (PP2A) Regulatory Subunit α4, Altering PP2A Stability and Microtubule-associated Protein Phosphorylation

Multiple neurodegenerative disorders are linked to aberrant phosphorylation of microtubule-associated proteins (MAPs). Protein phosphatase 2A (PP2A) is the major MAP phosphatase; however, little is known about its regulation at microtubules. α4 binds the PP2A catalytic subunit (PP2Ac) and the microtubule-associated E3 ubiquitin ligase MID1, and through unknown mechanisms can both reduce and enhance PP2Ac stability. We show MID1-dependent monoubiquitination of α4 triggers calpain-mediated cleavage and switches α4's activity from protective to destructive, resulting in increased Tau phosphorylation. This regulatory mechanism appears important in MAP-dependent pathologies as levels of cleaved α4 are decreased in Opitz syndrome and increased in Alzheimer disease, disorders characterized by MAP hypophosphorylation and hyperphosphorylation, respectively. These findings indicate that regulated inter-domain cleavage controls the dual functions of α4, and dysregulation of α4 cleavage may contribute to Opitz syndrome and Alzheimer disease.

A Unique TGFBI Protein in Granular Corneal Dystrophy Types 1 and 2

Purpose: Types 1 and 2 granular corneal dystrophies (GCD) are primarily associated with accumulation of the R555W and R124H mutant transforming growth factor β-inducible proteins (TGFBIp) in corneal stroma, respectively. However, specific components of TGFBIp responsible for granular deposits have not been delineated. This study was undertaken to identify the mutant TGFBIp components potentially responsible for GCD.Methods: Recombinant TGFBIp of wild-type (WT) and three mutants, R124C, R124H, and R555W, were generated in HEK293FT cells. WT and TGFBIp mutants were collected from cell lysates. Immunoblot analyses were performed with five different antibodies directed against various regions of WT TGFBIp.Results: WT and TGFBIp mutants showed differential reactivities with these antibodies. In contrast to our prior observation in purified WT and TGFBIp mutants, TGFBIp from cell lysates were less prone to polymerize. A unique 35 kD fragment was detected in cell lysates of R555W and R124H, but not in those of WT or R124C, by a commercial antibody raised against amino acids (a.a.) 199–406 of TGFBIp.Conclusions: Monomeric and polymeric WT and TGFBIp mutants were observed in vitro. The 35 kD fragment found only in R555W and R124H, but not in WT and R124C cell lysates, is likely a degraded TGFBIp derived from the central domain of these mutants and this fragment may be contributory to the nonamyloid granular deposits observed in GCD 1 and 2.

Diverse regulation of AKT and GSK-3β by O-GlcNAcylation in various types of cells

Protein kinase B (AKT) and glycogen synthase kinase-3β (GSK-3β) are major components of insulin–AKT signaling that plays crucial roles in various types of tissue. Recent studies found that these two kinases are modified posttranslationally by O-GlcNAcylation. Here, we demonstrate that O-GlcNAcylation regulated phosphorylation/activation of AKT and GSK-3β in different manners in kidney HEK-293FT cells, but did not affect these two kinases in hepatic HepG2 cells. In neuronal cells, O-GlcNAcylation regulated phosphorylation of AKT negatively, but had no effect on GSK-3β. These results suggest protein-specific and cell type-specific regulation of AKT and GSK-3β by O-GlcNAcylation. Therefore, studies on the roles of AKT and GSK-3β O-GlcNAcylation should be done in a tissue- and cell type-specific manner.