Bhavani S. Kowtharapu

Expression of 3‐hydroxyisobutyrate dehydrogenase in cultured neural cells

The branched‐chain amino acids (BCAAs) – isoleucine, leucine, and valine – belong to the limited group of substances transported through the blood–brain barrier. One of the functions they are thought to have in brain is to serve as substrates for meeting parenchymal energy demands. Previous studies have shown the ubiquitous expression of a branched‐chain alpha‐keto acid dehydrogenase among neural cells. This enzyme catalyzes the initial and rate‐limiting step in the irreversible degradative pathway for the carbon skeleton of valine and the other two branched‐chain amino acids. Unlike the acyl‐CoA derivates in the irreversible part of valine catabolism, 3‐hydroxyisobutyrate could be expected to be released from cells by transport across the mitochondrial and plasma membranes. This could indeed be demonstrated for cultured astroglial cells. Therefore, to assess the ability of neural cells to make use of this valine‐derived carbon skeleton as a metabolic substrate for the generation of energy, we investigated the expression in cultured neural cells of the enzyme processing this hydroxy acid, 3‐hydroxyisobutyrate dehydrogenase (HIBDH). To achieve this, HIBDH was purified from bovine liver to serve as antigen for the production of an antiserum. Affinity‐purified antibodies against HIBDH specifically recognized the enzyme in liver and brain homogenates. Immunocytochemistry demonstrated the ubiquitous expression of HIBDH among cultured glial (astroglial, oligodendroglial, microglial, and ependymal cells) and neuronal cells. Using an RT‐PCR technique, these findings were corroborated by the detection of HIBDH mRNA in these cells. Furthermore, immunofluorescence double‐labeling of astroglial cells with antisera against HIBDH and the mitochondrial marker pyruvate dehydrogenase localized HIBDH to mitochondria. The expression of HIBDH in neural cells demonstrates their potential to utilize valine imported into the brain for the generation of energy.

Expression of pyruvate carboxylase in cultured oligodendroglial, microglial and ependymal cells.

The mitochondrial enzyme, pyruvate carboxylase (PC; EC 6.4.1.1) is considered to play a significant role in the intermediary metabolism of neural tissue. PC-catalyzed carboxylation of pyruvate to oxaloacetate is a major anaplerotic reaction in brain. Anaplerosis is essential for homeostasis of the members of the tricarboxylic acid (TCA) cycle. Several biochemical pathways rely on withdrawing TCA cycle members. Prominent among these are biosynthesis of fatty acids and of non-essential amino acids such as aspartate, asparagine, glutamate and glutamine, gluconeogenesis, glycogen synthesis, and regeneration of NADPH. The expression of PC in brain has already been described and assigned to astrocytes. Since pyruvate carboxylase deficiency is associated with malformations of the brain, e.g., inadequate development of the corpus callosum and the lack of myelination, one can hypothesize that PC may be expressed also in glial cells other than astrocytes. Therefore, the expression of PC was investigated in cultured oligodendroglial, microglial, and ependymal cells. As assessed by RT-PCR, all these cultures contain PC mRNA. This mRNA is generated in a transcription process that is regulated by the “distal class” of promoters of the PC gene. The expression of PC among cultured glial cells was studied with a rabbit antiserum by immunoblotting and immunocytochemistry. The results indicate that PC is not only expressed in cultured astroglial cells but also in cultured oligodendrocytes, microglial cells, and ependymocytes. It appears that the intermediary metabolism of these cells includes the anaplerotic action of PC as well as possibly also functions of the enzyme in biosynthetic pathways and the provision of NADPH for defense against reactive oxygen species.

Suppression of TGF-β pathway by pirfenidone decreases extracellular matrix deposition in ocular fibroblasts in vitro

In glaucoma surgery, fibrotic processes occur, leading to impairment of liquid outflow. Activated fibroblasts are responsible for postoperative scarring. The transforming growth factor-β (TGF-β) pathway plays a key role in fibroblast function, differentiation and proliferation. The aim of this study was the characterization of the fibrotic potential of two subtypes of primary human ocular fibroblasts and the attempt to inhibit fibrotic processes specifically, without impairing cell viability. For fibrosis inhibition we focused on the small molecule pirfenidone, which has been shown to prevent pulmonary fibrosis by the decrease of the expression of TGF-β1, TGF-β2 and TGF-β3 cytokines. For in vitro examinations, isolated human primary fibroblasts from Tenon capsule and human intraconal orbital fat tissues were used. These fibroblast subpopulations were analyzed in terms of the expression of matrix components responsible for postoperative scarring. We concentrated on the expression of collagen I, III, VI and fibronectin. Additionally, we analyzed the expression of α-smooth muscle actin, which serves as a marker for fibrosis and indicates transformation of fibroblasts into myofibroblasts. Gene expression was analyzed by rtPCR and synthesized proteins were examined by immunofluorescence and Western blot methods. Proliferation of fibroblasts under different culture conditions was assessed using BrdU assay. TGF-β1 induced a significant increase of cell proliferation in both cell types. Also the expression of some fibrotic markers was elevated. In contrast, pirfenidone decreased cell proliferation and matrix synthesis in both fibroblast subpopulations. Pirfenidone slightly attenuated TGF-β1 induced expression of fibronectin and α-smooth muscle actin in fibroblast cultures, without impairing cell viability. To summarize, manipulation of the TGF-β signaling pathway by pirfenidone represents a specific antifibrotic approach with no toxic side effects in two human orbital fibroblast subtypes. We presume that pirfenidone is a promising candidate for the treatment of fibrosis following glaucoma surgery.

Metabolism of [U-13C]Aspartate by Astroglial Cultures: Nuclear Magnetic Resonance Analysis of the Culture Media

In brain the amino acid L-aspartate serves roles as: (1) putative transmitter, (2) protein precursor, (3) donor of atoms for the biosynthesis of pyrimidine and purine bases, and (4) fuel for energy metabolism. Astrocytes dominate aspartate clearance in brain, and in culture they take up aspartate and quickly metabolize it. In brain, only astrocytes were shown to express the enzymes for de novo pyrimidine biosynthesis. To gain more details about the spectrum of metabolites generated from aspartate and subsequently released by cultured astrocytes a 13C-nuclear magnetic resonance analysis was performed of [U-13C]aspartate supplemented incubation media exposed to astroglial cultures. The results show that astrocytes readily metabolize aspartate and release into their culture media 13C-isotopomers of lactate, glutamine, citrate and alanine. Despite the presence in astroglial cells of two tandem enzymes of pyrimidine biosynthesis and their mRNAs, pyrimidine nucleotide-related heterocyclic compounds such as dihydroorotate and orotate could not be detected in the culture media.

Adenylate kinase 7 is a differentiation marker of kinocilia-bearing cells

Ependymal cells form a mostly single-layered epithelium which covers the surfaces of the cerebral ventricles. Considerable evidence has accumulated for the ependyma to be involved in the pathogenesis of hydrocephalus, a dilation of the ventricles at the expense of brain parenchyma. The underlying mechanism may include gene defects in ependymal cells, which prevent their proper differentiation and render them unable to fulfil their physiological functions. The most prominent marker of ependymal differentiation is the presence of kinocilia at the apical cell surface. In order to identify molecular players involved in the differentiation process, a subtractive cDNA library of ependyma minus brain was screened for ependyma-specific transcripts. One of the resulting candidates is the message for putative adenylate kinase 7 (pAK7), which is exclusively produced in rat tissues known for the presence of kinocilia, namely testis, lung and ependyma, as determined by real-time RT-PCR.

Role of Corneal Stromal Cells on Epithelial Cell Function during Wound Healing

Following injury, corneal stromal keratocytes transform into repair-phenotype of activated stromal fibroblasts (SFs) and participate in wound repair. Simultaneously, ongoing bi-directional communications between corneal stromal-epithelial cells also play a vital role in mediating the process of wound healing. Factors produced by stromal cells are known to induce proliferation, differentiation, and motility of corneal epithelial cells, which are also subsequently the main processes that occur during wound healing. In this context, the present study aims to investigate the effect of SFs conditioned medium (SFCM) on corneal epithelial cell function along with substance P (SP). Antibody microarrays were employed to profile differentially expressed cell surface markers and cytokines in the presence of SFCM and SP. Antibody microarray data revealed enhanced expression of the ITGB1 in corneal epithelial cells following stimulation with SP whereas SFCM induced abundant expression of IL-8, ITGB1, PD1L1, PECA1, IL-15, BDNF, ICAM1, CD8A, CD44 and NTF4. All these proteins have either direct or indirect roles in epithelial cell growth, movement and adhesion related signaling cascades during tissue regeneration. We also observed activation of MAPK signaling pathway along with increased expression of focal adhesion kinase (FAK), paxillin, vimentin, β-catenin and vasodilator-stimulated phosphoprotein (VASP) phosphorylation. Additionally, epithelial-to-mesenchymal transition (EMT) regulating transcription factors Slug and ZEB1 expression were enhanced in the presence of SFCM. SP enriched the expression of integrin subunits α4, α5, αV, β1 and β3 whereas SFCM increased α4, α5, αV, β1 and β5 integrin subunits. We also observed increased expression of Serpin E1 following SP and SFCM treatment. Wound healing scratch assay revealed enhanced migration of epithelial cells following the addition of SFCM. Taken together, we conclude that SFCM-mediated sustained activation of ZEB1, Slug in combination with upregulated migration-associated integrins and ERK (Extracellular signal-regulated kinase)-FAK-paxillin axis, may lead to induce type 2 EMT-like changes during corneal epithelial wound healing.

Lentiviral Transfection of Ependymal Primary Cultures Facilitates the Characterisation of Kinocilia-specific Promoters

Ependymal primary cultures (EPCs) are an established model for studying ependymal cell biochemistry and the biology of kinocilia-bearing cells. However, the difficulty in causing them to express transgenes at high efficiency has been an important drawback of the system. Indeed plasmid-based transfection attempts remain at an efficiency below 1% and fail to elicit reporter gene expression, namely green fluorescent protein (GFP) synthesis, in any of the kinocilia-bearing cells of the cultures. Human immunodeficiency virus pseudotyped with the vesicular stomatitis virus envelope glycoprotein (HIV/VSV-G) and encoding GFP under the control of the ubiquitously recognised promoter of elongation factor 1 alpha (EF1alpha) also does not cause transgene expression in the kinocilia-bearing cells of an EPC when applied at multiplicities of infection (MOIs) of up to 40 and destroys the culture when the MOI is increased further. In contrast, HIV/VSV-G encoding GFP under the control of a promoter specifically active in kinocilia-bearing cells leads to transgene expression in up to 79% of the kinociliated cells of an EPC when applied at an MOI of 20. This has permitted the initial characterisation of the promoter for the gene specifically transcribed in kinocilia-bearing cells, wdr16. The results have identified two regions of 100 nucleotides length each, which are critical for promoter activity and contain putative binding sites for the transcription factors Foxd1, Sox17 and Spz1. It appears that wdr16 is controlled by a bidirectional promoter also responsible for regulating the syntaxin 8 gene.

Role of Bone Morphogenetic Protein 7 (BMP7) in the Modulation of Corneal Stromal and Epithelial Cell Functions

In the cornea, healing of the wounded avascular surface is an intricate process comprising the involvement of epithelial, stromal and neuronal cell interactions. These interactions result to the release of various growth factors that play prominent roles during corneal wound healing response. Bone morphogenetic proteins (BMPs) are unique multi-functional potent growth factors of the transforming growth factor-beta (TGF-β) superfamily. Treatment of corneal epithelial cells with substance P and nerve growth factor resulted to an increase in the expression of BMP7 mRNA. Since BMP7 is known to modulate the process of corneal wound healing, in this present study, we investigated the influence of exogenous rhBMP7 on human corneal epithelial cell and stromal cell (SFs) function. To obtain a high-fidelity expression profiling of activated biomarkers and pathways, transcriptome-wide gene-level expression profiling of epithelial cells in the presence of BMP7 was performed. Gene ontology analysis shows BMP7 stimulation activated TGF-β signaling and cell cycle pathways, whereas biological processes related to cell cycle, microtubule and intermediate filament cytoskeleton organization were significantly impacted in corneal epithelial cells. Scratch wound healing assay showed increased motility and migration of BMP7 treated epithelial cells. BMP7 stimulation studies show activation of MAPK cascade proteins in epithelial cells and SFs. Similarly, a difference in the expression of claudin, Zink finger E-box-binding homeobox 1 was observed along with phosphorylation levels of cofilin in epithelial cells. Stimulation of SFs with BMP7 activated them with increased expression of α-smooth muscle actin. In addition, an elevated phosphorylation of epidermal growth factor receptor following BMP7 stimulation was also observed both in corneal epithelial cells and SFs. Based on our transcriptome analysis data on epithelial cells and the results obtained in SFs, we conclude that BMP7 contributes to epithelial-to-mesenchymal transition-like responses and plays a role equivalent to TGF-β in the course of corneal wound healing.

Role of S-adenosylmethionine cycle in carcinogenesis

Alterations in enzymatic activities underlying the cellular capacity to maintain functional S-adenosylmethionine (SAM) cycle are associated with modified levels of its constituents. Since SAM is the most prominent donor of methyl group for sustaining the methylation pattern of macromolecules by methyltransferases, its availability is an essential prerequisite for sustaining the methylation pattern of nucleic acids and proteins. In addition, increased intracellular concentrations of S-adenosylhomocysteine and homocysteine, another two constituents of SAM cycle, exerts an inhibitory effect on the enzymatic activity of methyltranferases. While methylation pattern of DNA and histones is considered as an important regulatory hallmark in epigenetically regulated gene expression, amended methylation of several cellular proteins, including transcription factors, affects their activity and stability. Indeed, varied DNA methylome is a common consequence of disturbed SAM cycle and is linked with molecular changes underlying the transformation of the cells that may underlay the carcinogenesis. Here we summarize the recent evidences about the impact of disturbed SAM cycle on carcinogenesis.

Lentiviral vectors as a tool for studying ependyma-specific proteins

The ependyma is a single-layered, ciliated epithelium at the interface between the parenchyma of the central nervous system and the ventricular space. Recently, it has been implicated in the pathogenesis of hydrocephalus, a debilitating dilation of the cerebral ventricles at the expense of the brain parenchyma. We have previously identified marker proteins for ependymal differentiation and kinocilia generation, namely SCO-C3 and putative adenylate kinase 7 (pAK7). In order to functionally characterize these proteins, it is desirable to introduce corresponding expression and RNAi constructs into our ependymal primary culture model system. However, polyciliated ependymal cells in the primary cultures were found to be completely resistant to transfection with naked plasmid DNA, necessitating more efficient means of nucleic acid transfer. Therefore, a lentiviral vector system based on pseudotyped human immunodeficiency virus (HIV) was established. HIV pseudotyped with the vesicular stomatitis virus envelope protein (VSV-G) and bearing constructs encoding either SCO-C3 or pAK7 were generated and verified for infectivity by successful production of stable HEK293T cell lines expressing the respective proteins. As reported by others, the ependyma is easily transfected by HIV/VSV-G in vivo. However, treatment of ependymal primary cultures with this vector resulted in the transfection of only a few polyciliated ependymal cells. While the current transfection efficiency may be insufficient for the proper analysis of target proteins, the principal feasibility of lentiviral transfection of ependymal primary cultures has nevertheless been demonstrated. In order to improve the transfection efficiency, other HIV pseudotypes including HIV/Mokola virus envelope protein, HIV/Rabies virus envelope protein and HIV/Ebola virus envelope protein are being tested. As a byproduct of vector testing, we obtained HEK293T cell lines coexpressing pAK7 fragments and putative pAK7 interaction partners that had previously been identified by yeast two-hybrid screens. These cell lines are now being used to yield additional evidence for the corresponding protein-protein interactions. Cell lines expressing SCO-C3 and pAK7 were further used to test the efficacy of RNAi constructs introduced by other lentiviral vectors. An RNAi sequence yielding almost complete knockdown of pAK7 expression was identified. The successful anti-pAK7 lentivirus is now ready for use in pAK7 knockdown studies both in vitro and in vivo. from Annual Meeting of the Study Group Neurochemistry. International Conference of the Gesellschaft für Biochemie und Molekularbiologie 2006 (GBM 2006): Molecular pathways in health and disease of the nervous system Witten, Germany. 28–30 September 2006