Publications by authors named "Eleni Aggelidou"

17 Publications

  • Page 1 of 1

Electrospun wound dressings containing bioactive natural products: physico-chemical characterization and biological assessment.

Biomater Res 2021 Jul 16;25(1):23. Epub 2021 Jul 16.

Laboratory of Organic Chemistry, School of Chemical Engineering, Aristotle University of Thessaloniki (AUTh), 54124, Thessaloniki, Greece.

Background: Current research on skin tissue engineering has been focusing on novel therapies for the effective management of chronic wounds. A critical aspect is to develop matrices that promote growth and uniform distribution of cells across the wound area, and at the same time offer protection, as well as deliver drugs that help wound healing and tissue regeneration. In this context, we aimed at developing electrospun scaffolds that could serve as carriers for the bioactive natural products alkannin and shikonin (A/S).

Methods: A series of polymeric nanofibers composed of cellulose acetate (CA) or poly(ε-caprolactone) (PCL) and varying ratios of a mixture of A/S derivatives, has been successfully fabricated and their physico-chemical and biological properties have been explored.

Results: Scanning electron microscopy revealed a uniform and bead-free morphology for CA scaffolds, while for PCL beads along the fibers were observed. The average diameters for all nanofibers ranged between 361 ± 47 and 487 ± 88 nm. During the assessment of physicochemical characteristics, CA fiber mats exhibited a more favored profile, while the assessment of the biological properties of the scaffolds showed that CA samples containing A/S mixture up to 1 wt.% achieved to facilitate attachment, survival and migration of Hs27 fibroblasts. With respect to the antimicrobial properties of the scaffolds, higher drug-loaded (1 and 5 wt.%) samples succeeded in inhibiting the growth of Staphylococcus epidermidis and S. aureus around the edges of the fiber mats. Finally, carrying out a structure-activity relationship study regarding the biological activities (fibroblast toxicity/proliferation and antibacterial activity) of pure A/S compounds - present in the A/S mixture - we concluded that A/S ester derivatives and the dimeric A/S augmented cell proliferation after 3 days, whereas shikonin proved to be toxic at 500 nM and 1 μM and alkannin only at 1 μM. Additionally, alkannin, shikonin and acetyl-shikonin showed more pronounced antibacterial properties than the other esters, the dimeric derivative and the A/S mixture itself.

Conclusions: Taken together, these findings indicate that embedding A/S derivatives into CA nanofibers might be an advantageous drug delivery system that could also serve as a potential candidate for biomedical applications in the field of skin tissue engineering.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s40824-021-00223-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8284004PMC
July 2021

Novel electrospun poly-hydroxybutyrate scaffolds as carriers for the wound healing agents alkannins and shikonins.

Regen Biomater 2021 Jun 10;8(3):rbab011. Epub 2021 Jun 10.

Laboratory of Organic Chemistry, School of Chemical Engineering, Aristotle University of Thessaloniki (AUTh), Thessaloniki 54124, Greece.

The aim of this study was to investigate the potential of novel electrospun fiber mats loaded with alkannin and shikonin (A/S) derivatives, using as carrier a highly biocompatible, bio-derived, eco-friendly polymer such as poly[(R)-3-hydroxybutyric acid] (PHB). PHB fibers containing a mixture of A/S derivatives at different ratios were successfully fabricated via electrospinning. Αs evidenced by scanning electron microscopy, the fibers formed a bead-free mesh with average diameters from 1.25 to 1.47 μm. Spectroscopic measurements suggest that electrospinning marginally increases the amorphous content of the predominantly crystalline PHB in the fibers, while a significant drug amount lies near the fiber surface for samples of high total A/S content. All scaffolds displayed satisfactory characteristics, with the lower concentrations of A/S mixture-loaded PHB fiber mats achieving higher porosity, water uptake ratios, and entrapment efficiencies. The dissolution studies revealed that all samples released more than 70% of the encapsulated drug after 72 h. All PHB scaffolds tested by cell viability assay were proven non-toxic for Hs27 fibroblasts, with the 0.15 wt.% sample favoring cell attachment, spreading onto the scaffold surface, as well as cell proliferation. Finally, the antimicrobial activity of PHB meshes loaded with A/S mixture was documented for and .
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/rb/rbab011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8240617PMC
June 2021

The Marine Polysaccharide Ulvan Confers Potent Osteoinductive Capacity to PCL-Based Scaffolds for Bone Tissue Engineering Applications.

Int J Mol Sci 2021 Mar 17;22(6). Epub 2021 Mar 17.

Section of Pharmacognosy and Chemistry of Natural Products, Department of Pharmacy, National and Kapodistrian University of Athens, Panepistimiopolis Zografou, 15771 Athens, Greece.

Hybrid composites of synthetic and natural polymers represent materials of choice for bone tissue engineering. Ulvan, a biologically active marine sulfated polysaccharide, is attracting great interest in the development of novel biomedical scaffolds due to recent reports on its osteoinductive properties. Herein, a series of hybrid polycaprolactone scaffolds containing ulvan either alone or in blends with κ-carrageenan and chondroitin sulfate was prepared and characterized. The impact of the preparation methodology and the polysaccharide composition on their morphology, as well as on their mechanical, thermal, water uptake and porosity properties was determined, while their osteoinductive potential was investigated through the evaluation of cell adhesion, viability, and osteogenic differentiation of seeded human adipose-derived mesenchymal stem cells. The results verified the osteoinductive ability of ulvan, showing that its incorporation into the polycaprolactone matrix efficiently promoted cell attachment and viability, thus confirming its potential in the development of biomedical scaffolds for bone tissue regeneration applications.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ijms22063086DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8002638PMC
March 2021

Cartilage Regeneration with Cell-Seeded Natural Biomaterial Scaffold Implants: 15-Year Study.

Tissue Eng Part B Rev 2021 Mar 10. Epub 2021 Mar 10.

Department of Physiology and Pharmacology, Faculty of Health Sciences and cGMP Regenerative Medicine Facility, School of Medicine, Aristotle University of Thessaloniki (A.U.Th), Thessaloniki, Greece.

Articular cartilage can be easily damaged from human's daily activities, leading to inflammation and to osteoarthritis, a situation that can diminish the patients' quality of life. For larger cartilage defects, scaffolds are employed to provide cells the appropriate three-dimensional environment to proliferate and differentiate into healthy cartilage tissue. Natural biomaterials used as scaffolds, attract researchers' interest because of their relative nontoxic nature, their abundance as natural products, their easy combination with other materials, and the relative easiness to establish Marketing Authorization. The last 15 years were chosen to review, document, and elucidate the developments on cell-seeded natural biomaterials for articular cartilage treatment . The parameters of the experimental designs and their results were all documented and presented. Considerations about the newly formed cartilage and the treatment of cartilage defects were discussed, along with difficulties arising when applying natural materials, research limitations, and tissue engineering approaches for hyaline cartilage regeneration.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/ten.TEB.2020.0295DOI Listing
March 2021

An effective device and method for enhanced cell growth in 3D scaffolds: Investigation of cell seeding and proliferation under static and dynamic conditions.

Mater Sci Eng C Mater Biol Appl 2020 Sep 11;114:111060. Epub 2020 May 11.

Department of Physiology and Pharmacology, School of Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki (A.U.Th), Thessaloniki, Greece; cGMP Regenerative Medicine Facility, Department of Physiology and Pharmacology, School of Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki (A.U.Th), Thessaloniki, Greece.

Cell adhesion on 3D-scaffolds is a challenging task to succeed high cell densities and even cell distribution. We aimed to design a 3D-cell Culture Device (3D-CD) for static seeding and cultivation, to be used with any kind of scaffold, limiting cell loss and facilitating nutrient supply. 3D printing technology was used for both scaffold and device fabrication. Apart from testing the device, the purpose of this study was to assess and compare static and dynamic seeding and cultivation methods, of wet and dry scaffolds, under normoxic and hypoxic conditions and their effects on parameters such as cell seeding efficiency, cell distribution and cell proliferation. Human adipose tissue was harvested and cultured in 3D-printed poly(epsilon-caprolactone) scaffolds. Micro-CT scans were performed and projection images were reconstructed into cross section images. We created 3D images to visualize cell distribution and orientation inside the scaffolds. The group of prewetted scaffolds was the most favorable to cell attachment. The 3D-cell Culture Device (3D-CD) enhanced cell seeding efficiency with almost no cell loss. We suggest that the most favorable outcome can be produced with static seeding in the device for 24 h, followed either by static cultivation in the same device or by dynamic cultivation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.msec.2020.111060DOI Listing
September 2020

Hybrid Sponge-Like Scaffolds Based on Ulvan and Gelatin: Design, Characterization and Evaluation of Their Potential Use in Bone Tissue Engineering.

Materials (Basel) 2020 Apr 9;13(7). Epub 2020 Apr 9.

Section of Pharmacognosy and Chemistry of Natural Products, Department of Pharmacy, National and Kapodistrian University of Athens, Panepistimiopolis Zografou, 15771 Athens, Greece.

Ulvan, a bioactive natural sulfated polysaccharide, and gelatin, a collagen-derived biopolymer, have attracted interest for the preparation of biomaterials for different biomedical applications, due to their demonstrated compatibility for cell attachment and proliferation. Both ulvan and gelatin have exhibited osteoinductive potential, either alone or in combination with other materials. In the current work, a series of novel hybrid scaffolds based on crosslinked ulvan and gelatin was designed, prepared and characterized. Their mechanical performance, thermal stability, porosity, water-uptake and degradation ability were assessed, while their morphology was analyzed through scanning electron microscopy. The prepared hybrid ulvan/gelatin scaffolds were characterized by a highly porous and interconnected structure. Human adipose-derived mesenchymal stem cells (hADMSCs) were seeded in selected ulvan/gelatin hybrid scaffolds and their adhesion, survival, proliferation, and osteogenic differentiation efficiency was evaluated. Overall, it was found that the prepared hybrid sponge-like scaffolds could efficiently support mesenchymal stem cells' adhesion and proliferation, suggesting that such scaffolds could have potential uses in bone tissue engineering.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ma13071763DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7178717PMC
April 2020

Assessment of cartilage regeneration on 3D collagen-polycaprolactone scaffolds: Evaluation of growth media in static and in perfusion bioreactor dynamic culture.

Colloids Surf B Biointerfaces 2019 Nov 29;183:110403. Epub 2019 Jul 29.

Department of Physiology and Pharmacology, School of Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki (A.U.Th), Thessaloniki, Greece; cGMP Regenerative Medicine Facility, Department of Physiology and Pharmacology, School of Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki (A.U.Th), Thessaloniki, Greece. Electronic address:

Efforts on bioengineering are directed towards the construction of biocompatible scaffolds and the determination of the most favorable microenvironment, which will better support cell proliferation and differentiation. Perfusion bioreactors are attracting growing attention as an effective, modern tool in tissue engineering. A natural biomaterial extensively used in regenerative medicine with outstanding biocompatibility, biodegradability and non-toxic characteristics, is collagen, a structural protein with undisputed beneficial characteristics. This is a study designed according to the above considerations. 3D printed polycaprolactone (PCL) scaffolds with rectangular pores were coated with collagen either as a coating on the scaffold's trabeculae, or as a gel-cell solution penetrating scaffolds' pores. We employed histological, molecular and imaging techniques to analyze colonization, proliferation and chondrogenic differentiation of Adipose Derived Mesenchymal Stem Cells (ADMSCs). Two different differentiation culture media were employed to test chondrogenic differentiation on gelated and non gelated PCL scaffolds in static and in perfusion bioreactors dynamic culture conditions. In dynamic culture, non gelated scaffolds combined with our in house TGF-β based medium, augmented chondrogenic differentiation performance, which overall was significantly less favorable compared to StemPro™ propriety medium. The beneficial mechanical stimulus of dynamic culture, appears to outgrow the disadvantage of the "weaker" TGF-β medium used for chondrogenic differentiation. Even though cells in static culture grew well on the scaffold, there was limited penetration inside the construct, so the purpose of the 3D culture was not fully served. In contrast dynamic culture achieved better penetration and uniform distribution of the cells within the scaffold.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.colsurfb.2019.110403DOI Listing
November 2019

Hyaline cartilage next generation implants from adipose-tissue-derived mesenchymal stem cells: Comparative study on 3D-printed polycaprolactone scaffold patterns.

J Tissue Eng Regen Med 2019 02;13(2):342-355

Department of Physiology and Pharmacology, School of Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki (A.U.Th), Thessaloniki, Greece.

We used additive manufacturing to fabricate 3D-printed polycaprolactone scaffolds of different geometry topologies and porosities. We present a comparative analysis of hyaline cartilage development from adipose-tissue-derived mesenchymal stem cells (ADMSCs) on three different, newly designed scaffold geometry patterns. The first scaffold design (MESO) was based on a rectilinear layer pattern. For the second pattern (RO45), we employed a 45° rotational layer loop. The design for the third scaffold (3DHC) was a three-dimensional honeycomb-like pattern with a hexagonal cellular distribution and small square shapes. We examined cell proliferation, colonization, and differentiation, in relation to the scaffold's structure, as well as to the mechanical properties of the final constructs. We gave emphasis on the scaffolds, both microarchitecture and macroarchitecture, for optimal and enhanced chondrogenic differentiation, as an important parameter, not well studied in the literature. Among the three patterns tested, RO45 was the most favourable for chondrogenic differentiation, whereas 3DHC better supported cell proliferation and scaffold penetration, exhibiting also the highest rate of increase onto the mechanical properties of the final construct. We conclude that by choosing the optimal scaffold architecture, the resulting properties of our cartilaginous constructs can better approximate those of the physiological cartilage.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/term.2798DOI Listing
February 2019

Isolation and prolonged expansion of oral mesenchymal stem cells under clinical-grade, GMP-compliant conditions differentially affects "stemness" properties.

Stem Cell Res Ther 2017 Nov 2;8(1):247. Epub 2017 Nov 2.

Department of Conservative Dentistry, Periodontology and Preventive Dentistry, Hannover Medical School (MHH), Hannover, Germany.

Background: Development of clinical-grade cell preparations is central to meeting the regulatory requirements for cellular therapies under good manufacturing practice-compliant (cGMP) conditions. Since addition of animal serum in culture media may compromise safe and efficient expansion of mesenchymal stem cells (MSCs) for clinical use, this study aimed to investigate the potential of two serum/xeno-free, cGMP culture systems to maintain long-term "stemness" of oral MSCs (dental pulp stem cells (DPSCs) and alveolar bone marrow MSCs (aBMMSCs)), compared to conventional serum-based expansion.

Methods: DPSC and aBMMSC cultures (n = 6/cell type) were established from pulp and alveolar osseous biopsies respectively. Three culture systems were used: StemPro_MSC/SFM_XenoFree (Life Technologies); StemMacs_MSC/XF (Miltenyi Biotek); and α-MEM (Life Technologies) with 15% fetal bovine serum. Growth (population doublings (PDs)), immunophenotypic (flow cytometric analysis of MSC markers) and senescence (β-galactosidase (SA-β-gal) activity; telomere length) characteristics were determined during prolonged expansion. Gene expression patterns of osteogenic (ALP, BMP-2), adipogenic (LPL, PPAR-γ) and chondrogenic (ACAN, SOX-9) markers and maintenance of multilineage differentiation potential were determined by real-time PCR.

Results: Similar isolation efficiency and stable growth dynamics up to passage 10 were observed for DPSCs under all expansion conditions. aBMMSCs showed lower cumulative PDs compared to DPSCs, and when StemMacs was used substantial delays in cell proliferation were noted after passages 6-7. Serum/xeno-free expansion produced cultures with homogeneous spindle-shaped phenotypes, while serum-based expansion preserved differential heterogeneous characteristics of each MSC population. Prolonged expansion of both MSC types but in particular the serum/xeno-free-expanded aBMMSCs was associated with downregulation of CD146, CD105, Stro-1, SSEA-1 and SSEA-4, but not CD90, CD73 and CD49f, in parallel with an increase of SA-gal-positive cells, cell size and granularity and a decrease in telomere length. Expansion under both serum-free systems resulted in "osteogenic pre-disposition", evidenced by upregulation of osteogenic markers and elimination of chondrogenic and adipogenic markers, while serum-based expansion produced only minor changes. DPSCs retained a diminishing (CCM, StemPro) or increasing (StemMacs) mineralization potential with passaging, while aBMMSCs lost this potential after passages 6-7 under all expansion conditions.

Conclusions: These findings indicate there is still a vacant role for development of qualified protocols for clinical-grade expansion of oral MSCs; a key milestone achievement for translation of research from the bench to clinics.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13287-017-0705-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5667471PMC
November 2017

Low Dose Administration of Glutamate Triggers a Non-Apoptotic, Autophagic Response in PC12 Cells.

Cell Physiol Biochem 2015 13;37(5):1750-8. Epub 2015 Nov 13.

Background/aims: Increasing amounts of the neurotransmitter glutamate are associated with excitotoxicity, a phenomenon related both to homeostatic processes and neurodegenerative diseases such as multiple sclerosis.

Methods: PC12 cells (rat pheochromocytoma) were treated with various concentrations of the non-essential amino acid glutamate for 0.5-24 hours. The effect of glutamate on cell morphology was monitored with electron microscopy and haematoxylin-eosin staining. Cell survival was calculated with the MTT assay. Expression analysis of chaperones associated with the observed phenotype was performed using either Western Blotting at the protein level or qRT-PCR at the mRNA level.

Results: Administration of glutamate in PC12 cells in doses as low as 10 μM causes an up-regulation of GRP78, GRP94 and HSC70 protein levels, while their mRNA levels show the opposite kinetics. At the same time, GAPDH and GRP75 show reduced protein levels, irrespective of their transcriptional rate. On a cellular level, low concentrations of glutamate induce an autophagy-mediated pro-survival phenotype, which is further supported by induction of the autophagic marker LC3.

Conclusion: The findings in the present study underline a discrete effect of glutamate on neuronal cell fate depending on its concentration. It was also shown that a low dose of glutamate orchestrates a unique expression signature of various chaperones and induces cell autophagy, which acts in a neuroprotective fashion.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1159/000430250DOI Listing
August 2016

Oxygen-Glucose Deprivation (OGD) Modulates the Unfolded Protein Response (UPR) and Inflicts Autophagy in a PC12 Hypoxia Cell Line Model.

Cell Mol Neurobiol 2016 Jul 4;36(5):701-12. Epub 2015 Aug 4.

Laboratory of Physiology, School of Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki, 54124, Thessaloniki, Greece.

Hypoxia is the lack of sufficient oxygenation of tissue, imposing severe stress upon cells. It is a major feature of many pathological conditions such as stroke, traumatic brain injury, cerebral hemorrhage, perinatal asphyxia and can lead to cell death due to energy depletion and increased free radical generation. The present study investigates the effect of hypoxia on the unfolded protein response of the cell (UPR), utilizing a 16-h oxygen-glucose deprivation protocol (OGD) in a PC12 cell line model. Expression of glucose-regulated protein 78 (GRP78) and glucose-regulated protein 94 (GRP94), key players of the UPR, was studied along with the expression of glucose-regulated protein 75 (GRP75), heat shock cognate 70 (HSC70), and glyceraldehyde 3-phosphate dehydrogenase, all with respect to the cell death mechanism(s). Cells subjected to OGD displayed upregulation of GRP78 and GRP94 and concurrent downregulation of GRP75. These findings were accompanied with minimal apoptotic cell death and induction of autophagy. The above observation warrants further investigation to elucidate whether autophagy acts as a pro-survival mechanism that upon severe and prolonged hypoxia acts as a concerted cell response leading to cell death. In our OGD model, hypoxia modulates UPR and induces autophagy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s10571-015-0250-2DOI Listing
July 2016

An HNF4α-miRNA inflammatory feedback circuit regulates hepatocellular oncogenesis.

Cell 2011 Dec;147(6):1233-47

Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA.

Hepatocyte nuclear factor 4α (HNF4α) is essential for liver development and hepatocyte function. Here, we show that transient inhibition of HNF4α initiates hepatocellular transformation through a microRNA-inflammatory feedback loop circuit consisting of miR-124, IL6R, STAT3, miR-24, and miR-629. Moreover, we show that, once this circuit is activated, it maintains suppression of HNF4α and sustains oncogenesis. Systemic administration of miR-124, which modulates inflammatory signaling, prevents and suppresses hepatocellular carcinogenesis by inducing tumor-specific apoptosis without toxic side effects. As we also show that this HNF4α circuit is perturbed in human hepatocellular carcinomas, our data raise the possibility that manipulation of this microRNA feedback-inflammatory loop has therapeutic potential for treating liver cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cell.2011.10.043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3251960PMC
December 2011

SAFB1 interacts with and suppresses the transcriptional activity of p53.

FEBS Lett 2011 Jan 3;585(1):78-84. Epub 2010 Dec 3.

Department of Chemistry, Aristotle University of Thessaloniki, Thessaloniki, Greece.

A significant amount of nuclear p53 is found associated with the nuclear matrix in cells that were exposed to genotoxic stress. In this study we identified Scaffold attachment factor B1 (SAFB1), a nuclear matrix-associated protein that binds the scaffold or matrix attachment regions (S/MARs) of genomic DNA, as a novel p53-interacting protein. SAFB1 was able to associate with p53 through its C-terminal domain, while significant co-localization of the two proteins was observed in cells treated with 5-fluorouracil or mithramycin. Binding of p53 to SAFB1 had a significant functional outcome, since SAFB1 was shown to suppress p53-mediated reporter gene expression. These data suggest that nuclear matrix-associated proteins may play a critical role in regulating p53 localization and activity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.febslet.2010.11.054DOI Listing
January 2011

Functional characterization of hepatocyte nuclear factor-4 alpha dimerization interface mutants.

FEBS J 2006 May;273(9):1948-58

Department of Genetics, Development and Molecular Biology, Laboratory of Developmental Biology, School of Biology, Aristotle University of Thessaloniki, Greece.

Hepatocyte nuclear factor-4 (HNF-4alpha), a member of the nuclear receptor superfamily, binds DNA exclusively as a homodimer. Dimerization controls important aspects of receptor function, such as DNA binding, protein stability, ligand binding and interaction with coactivators. Crystallographic data of the HNF-4alpha ligand-binding domain (LBD) demonstrated that the homodimer interface is composed of residues in helices 7, 9 and 10 with intermolecular salt bridges, hydrogen bonds and hydrophobic interactions contributing to the stability of the interface. To investigate the importance of the proposed ionic interactions for HNF-4alpha dimerization, interactions critical for formation of the LBD homodimer interface were disrupted by introducing point mutations in residues D261N (H7), E269Q (H7), Q307L (H9), D312N (H9) and Q336L (H10). Mutants were analysed for transactivation, coactivator interaction, DNA binding and dimerization. EMSA analysis showed that the mutants are able to bind DNA as dimers and coimmunoprecipitation assays confirmed dimerization in solution. Furthermore, the mutations do not compromise HNF-4alpha activity and are responsive to PPAR-gamma coactivator-1 (PGC-1). Finally, residue R324, located in the H9/H10 loop, which was suspected to be involved in dimer stabilization via an ionic interaction with residue E276, was studied. In contrast to the conservative substitution R324H the mutation R324L abolishes HNF-4alpha transcriptional activity and coactivator recruitment, revealing that the nature of substitution may play an important role in HNF-4alpha function.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/j.1742-4658.2006.05208.xDOI Listing
May 2006

Distinct amino acid residues may be involved in coactivator and ligand interactions in hepatocyte nuclear factor-4alpha.

J Biol Chem 2005 Jun 12;280(23):21810-9. Epub 2005 Apr 12.

Department of Genetics, Development and Molecular Biology, Laboratory of Developmental Biology, Aristotle University of Thessaloniki, Thessaloniki, Greece 54124.

Hepatocyte nuclear factor-4 (HNF-4) is a transcription factor of the nuclear hormone receptor superfamily that is constitutively active without the addition of exogenous ligand. Crystallographic analysis of the HNF-4alpha and HNF-4gamma ligand binding domains (LBDs) demonstrated the presence of endogenous ligands that may act as structural cofactors for HNF-4. It was also proposed by crystallographic studies that a combination of ligand and coactivator might be required to lock the receptor in its active state. We previously showed that mutations in amino acid residues Ser-181 and Met-182 in H3, Leu-219 and Leu-220 and Arg-226 in H5, Ileu-338 in H10, and Ileu-346 in H11, which line the LBD pocket in HNF-4alpha and come in contact with the ligand, impair its transactivation potential. In the present study, physical and functional interaction assays were utilized with two different coactivators, PGC-1 and SRC-3, to address the role of coactivators in HNF-4 function. We show that the integrity of the hinge (D) domain of HNF-4alpha and the activation function (AF)-2 activation domain region are critical for coactivation. Surprisingly, a different mode of coactivation is observed among the LBD point mutants that lack transcriptional activity. In particular, coactivation is maintained in mutants Ser-181, Arg-226, and Ile-346 but is abolished in mutants Met-182, Leu-219, and Ile-338. Physical interactions confirm this pattern of activation, implying that distinct amino acid residues may be involved in coactivator and ligand interactions, although some residues may be critical for both functions. Our results provide evidence and expand predictions based on the crystallographic data as to the role of coactivators in HNF-4alpha constitutive transcriptional activity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M501221200DOI Listing
June 2005

Critical role of residues defining the ligand binding pocket in hepatocyte nuclear factor-4alpha.

J Biol Chem 2004 Jul 30;279(29):30680-8. Epub 2004 Apr 30.

Aristotle University of Thessaloniki, Faculty of Sciences, School of Biology, Department of Genetics, Development and Molecular Biology, GR-54124, Thessaloniki, Greece.

Hepatocyte nuclear factor-4alpha (HNF-4alpha), a member of the nuclear receptor superfamily, is a crucial regulator of a large number of genes involved in glucose, cholesterol, and fatty acid metabolism. Unlike other members of the superfamily, HNF-4alpha activates transcription in the absence of exogenously added ligand. Recently published crystallographic data show that fatty acids are endogenous ligands for HNF-4. Transcriptional analysis of point mutations of the residues that are located in helices H3, H5, H10, and H11, which have been shown to come in contact with the ligand, resulted in a dramatic decrease in activity, without affecting DNA binding and dimerization. Our results show the importance of residues Ser-181, Met-182 in H3, Leu-219, Leu-220 and Arg-226 in H5, Ile-338 in H10, and Ile-346 in H11 that line the ligand-binding domain pocket in HNF-4alpha and impair its transactivation potential. Structural modeling reveals that the mutations do not cause any large scale structural alterations, and the observed loss in transactivation can be attributed to local changes, demonstrating that these residues play a significant role in maintaining the structural integrity of the HNF-4alpha ligand binding pocket.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M401120200DOI Listing
July 2004

Up-regulation of nitric oxide synthase and modulation of the guanylate cyclase activity by corticotropin-releasing hormone but not urocortin II or urocortin III in cultured human pregnant myometrial cells.

Proc Natl Acad Sci U S A 2002 Mar 19;99(5):3300-5. Epub 2002 Feb 19.

Sir Quinton Hazell Molecular Medicine Research Centre, Department of Biological Sciences, University of Warwick, Gibbet Hill Road, Coventry CV4 7AL, United Kingdom.

The biological actions of corticotropin-releasing hormone (CRH) in the human myometrium during pregnancy and labor are unknown. We hypothesized that CRH may modulate the nitric oxide system, and influence myometrial relaxation/contractility. Incubation of myometrial cells with CRH, but not urocortin II or urocortin III, for 8-16 h significantly induced mRNA and protein expression of endothelial and brain but not inducible nitric oxide synthase (NOS) isoforms. This action resulted in increased activity of soluble guanylate cyclase (GC(s)), demonstrated by the enhanced cGMP-producing capacity of the NO donor, sodium nitroprusside. CRH also caused acute activation of the membrane-bound GC, shown by increased basal or atrial natriuretic peptide (ANP)-stimulated cGMP production. These effects appeared to be mediated via the R1 receptors because the CRH receptor antagonists, astressin and antalarmin but not anti-sauvagine 30, could block them. The acute effects of CRH were significantly reduced by inhibition of protein kinase A (PKA) activity, suggesting it is partially PKA dependent. Activation of protein kinase C (PKC) resulted in significant inhibition of both ANP-and CRH-stimulated cGMP production, suggesting a direct effect of PKC on membrane-bound GC. In conclusion, CRH appears to have a dual effect on myometrial NOS/GC pathway, a short term effect predominantly mediated by PKA, and a long-term effect increasing constitutive NOS expression, mediated by a PKA-independent mechanism. This mechanism could potentially be active during human pregnancy, and, because cGMP stimulates myometrial relaxation, these findings further suggest that during pregnancy CRH primarily activates intracellular signals that contribute to the maintenance of myometrial quiescence.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.052296399DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC122513PMC
March 2002
-->