Epithelial-mesenchymal transition in oral squamous carcinoma cells

In epithelial-mesenchymal transition (EMT), epithelial cells acquire traits typical for mesenchymal cells, dissociate their cell-cell junctions and gain the ability to migrate. EMT is essential during embryogenesis, but may also mediate cancer progression. Basement membranes are sheets of extracellular matrix that support epithelial cells. They have a major role in maintaining the epithelial phenotype and, in cancer, preventing cell migration, invasion and metastasis. Laminins are the main components of basement membranes and may actively contribute to malignancy. We first evaluated the differences between cell lines obtained from oral squamous cell carcinoma and its recurrence. As the results indicated a change from epithelial to fibroblastoid morphology, E-cadherin to N-cadherin switch, and change in expression of cytokeratins to vimentin intermediate filaments, we concluded that these cells had undergone EMT. We further induced EMT in primary tumour cells to gain knowledge of the effects of transcription factor Snail in this cell model. The E-cadherin repressors responsible for the EMT in these cells were ZEB-1, ZEB-2 and Snail, and ectopic expression of Snail was able to augment the levels of ZEB-1 and ZEB-2. We produced and characterized two monoclonal antibodies that specifically recognized Snail in cell lines and patient samples. By immunohistochemistry, Snail protein was found in mesenchymal tissues during mouse embryonal development, in fibroblastoid cells of healing skin wounds and in fibromatosis and sarcoma specimens. Furthermore, Snail localized to the stroma and borders of tumour cell islands in colon adenocarcinoma, and in laryngeal and cervical squamous cell carcinomas. Immunofluorescence labellings, immunoprecipitations and Northern and Western blots showed that EMT induced a progressive downregulation of laminin-332 and laminin-511 and, on the other hand, an induction of mesenchymal laminin-411. Chromatin immunoprecipitation revealed that Snail could directly bind upstream to the transcription start sites of both laminin α5 and α4 chain genes, thus regulating their expression. The levels of integrin α6β4, a receptor for laminin-332, as well as the hemidesmosomal complex proteins HD1/plectin and BP180 were downregulated in EMT-experienced cells. The expression of Lutheran glycoprotein, a specific receptor for laminin-511, was diminished, whereas the levels of integrins α6β1 and α1β1 and integrin-linked kinase were increased. In quantitative cell adhesion assays, the cells adhered potently to laminin-511 and fibronectin, but only marginally to laminin-411. Western blots and immunoprecipitations indicated that laminin-411 bound to fibronectin and could compromise cell adhesion to fibronectin in a dose-dependent manner. EMT induced a highly migratory and invasive tendency in oral squamous carcinoma cells. Actin-based adhesion and invasion structures, podosomes and invadopodia, were detected in the basal cell membranes of primary tumour and spontaneously transformed cancer cells, respectively. Immunofluorescence labellings showed marked differences in their morphology, as podosomes organized a ring structure with HD1/plectin, αII-spectrin, talin, focal adhesion kinase and pacsin 2 around the core filled with actin, cortactin, vinculin and filamin A. Invadopodia had no division between ring and core and failed to organize the ring proteins, but instead assembled tail-like, narrow actin cables that showed a talin-tensin switch. Time-lapse live-cell imaging indicated that both podosomes and invadopodia were long-lived entities, but the tails of invadopodia vigorously propelled in the cytoplasm and were occasionally released from the cell membrane. Invadopodia could also be externalized outside the cytoplasm, where they still retained the ability to degrade matrix. In 3D confocal imaging combined with in situ gelatin zymography, the podosomes of primary tumour cells were large, cylindrical structures that increased in time, whereas the invadopodia in EMT-driven cells were smaller, but more numerous and degraded the underlying matrix in significantly larger amounts. Fluorescence recovery after photobleaching revealed that the substructures of podosomes were replenished more rapidly with new molecules than those of invadopodia. Overall, our results indicate that EMT has a major effect on the transcription and synthesis of both intra- and extracellular proteins, including laminins and their receptors, and on the structure and dynamics of oral squamous carcinoma cells.

Role of Basement Membrane and Extracellular Matrix Proteins in the Adhesion and Spreading of Immortalized Human Corneal Epithelial Cells

The repair of corneal wounds requires both epithelial cell adhesion and migration. Basement membrane (BM) and extracellular matrix (ECM) proteins function in these processes via integrin and non-integrin receptors. We have studied the adhesion, spreading and migration of immortalized human corneal epithelial (HCE) cells and their interactions with the laminins (Lms), fibronectins and tenascins produced. Human corneal BM expresses Lms-332 and -511, while Lm-111 was not found in these experiments. HCE cells produced both processed and unprocessed Lm-332, whereas neither Lm-111 nor Lm-511 was produced. Because HCE cells did not produce Lm-511, although it was present in corneal BM, we suggest that Lm-511 is produced by stromal keratocytes. The adhesion of HCE cells to Lms-111, -332 and -511 was studied first by determining the receptor composition of HCE cells and then by using quantitative cell adhesion assays. Immunofluorescence studies revealed the presence of integrin α2, α3, α6, β1 and β4 subunits. Among the non-integrin receptors, Lutheran (Lu) was found on adhering HCE cells. The cells adhered via integrin α3β1 to both purified human Lms-332 and -511 as well as to endogenous Lm-332. However, only integrin β1 subunit functioned in HCE cell adhesion to mouse Lm-111. The adhesion of HCE cells to Lm-511 was also mediated by Lu. Since Lm-511 did not induce Lu into focal adhesions in HCE cells, we suggest that Lm-511 serves as an ECM ligand enabling cell motility. HCE cells produced extradomain-A fibronectin, oncofetal fibronectin and tenascin-C (Tn-C), which are also found during corneal wound healing. Monoclonal antibodies (MAbs) against integrins α5β1 and αvβ6 as well as the arginine-glycine-aspartic acid (RGD) peptide inhibited the adhesion of HCE cells to fibronectin. Although the cells did not adhere to Tn-C, they adhered to the fibronectin/Tn-C coat and were then more efficiently inhibited by the function-blocking MAbs and RGD peptide. During the early adhesion, HCE cells codeposited Lm-332 and the large subunit of tenascin-C (Tn-CL) beneath the cells via the Golgi apparatus and microtubules. Integrin β4 subunit, which is a hemidesmosomal component, did not mediate the early adhesion of HCE cells to Lm-332 or Lm-332/Tn-C. Based on these results, we suggest that the adhesion of HCE cells is initiated by Lm-332 and modulated by Tn-CL, as it has been reported to prevent the assembly of hemidesmosomes. Thereby, Tn-CL functions in the motility of HCE cells during wound healing. The different distribution of processed and unprocessed Lm-332 in adhering, spreading and migrating HCE cells suggests a distinct role for these isoforms. We conclude that the processed Lm-332 functions in cell adhesion, whereas the unprocessed Lm-332 participates in cell spreading and migration.

Improving oncolytic adenoviral therapies for gastrointestinal cancers and tumor initiating cells

Although the treatment of most cancers has improved steadily, only few metastatic solid tumors can be cured. Despite responses, refractory clones often emerge and the disease becomes refractory to available treatment modalities. Furthermore, resistance factors are shared between different treatment regimens and therefore loss of response typically occurs rapidly, and there is a tendency for cross-resistance between agents. Therefore, new agents with novel mechanisms of action and lacking cross-resistance to currently available approaches are needed. Modified oncolytic adenoviruses, featuring cancer-celective cell lysis and spread, constitute an interesting drug platform towards the goals of tumor specificity and the implementation of potent multimodal treatment regimens. In this work, we demonstrate the applicability of capsid-modified, transcriptionally targeted oncolytic adenoviruses in targeting gastric, pancreatic and breast cancer. A variety of capsid modified adenoviruses were tested for transductional specificity first in gastric and pancreatic cancer cells and patient tissues and then in mice. Then, oncolytic viruses featuring the same capsid modifications were tested to confirm that successful transductional targeting translates into enhanced oncolytic potential. Capsid modified oncolytic viruses also prolonged the survival of tumor bearing orthotopic models of gastric and pancreatic cancer. Taken together, oncolytic adenoviral gene therapy could be a potent drug for gastric and pancreatic cancer, and its specificity, potency and safety can be modulated by means of capsid modification. We also characterized a new intraperitoneal virus delivery method in benefit for the persistence of gene delivery to intraperitoneal gastric and pancreatic cancer tumors. With a silica implant a steady and sustained virus release to the vicinity of the tumor improved the survival of the orthotopic tumor bearing mice. Furthermore, silica gel-based virus delivery lowered the toxicity mediating proimflammatory cytokine response and production of total and anti-adenovirus neutralizing antibodies (NAbs). On the other hand, silica shielded the virus against pre-excisting NAbs, resulting in a more favourable biodistribution in the preimmunized mice. The silica implant might therefore be of interest in treating intraperitoneally disseminated disease. Cancer stem cells are thought to be resistant to conventional cancer drugs and might play an important role in cancer relapse and the formation of metastasis. Therefore, we examined if transcriptionally modified oncolytic adenoviruses are able to kill these cells. Complete eradication of CD44+CD24-/low putative breast cancer stem cells was seen in vitro, and significant antitumor activity was detected in CD44+CD24-/low –derived tumor bearing mice. Thus, genetically engineered oncolytic adenoviruses have potential in destroying cancer initiating cells, which may have relevance for the elimination of cancer stem cells in humans.

Vascular growth factors and progenitor cells in cardiac allograft arteriosclerosis

Heart transplantation is the only therapeutic modality for many end-stage heart diseases but poor long-term survival remains a challenging problem. This is mainly due to the development of cardiac allograft arteriosclerosis (TxCAD) that is an accelerated form of coronary artery disease. Both traditional cardiovascular and transplantation-related risk factors for TxCAD have been identified but options for therapy are limited. TxCAD involves dysfunction of cardiac allograft vascular cells. Activated endothelial cells (EC) regulate allograft inflammation and secrete smooth muscle cell (SMC) growth factors. In turn, SMC and their progenitors invade the intima of the injured vessels and occlude the affected coronary arteries. Different vascular growth factors have to be delicately regulated in normal vascular development. In the present study, experimental heterotopic transplantation models were used to study the role of angiogenic and pro-inflammatory vascular endothelial growth factor (VEGF), EC growth factor angiopoietin (Ang), and SMC mitogen platelet-derived growth factor (PDGF) in the development of TxCAD. Pharmacological and gene transfer approaches were used to target these growth factors and to assess their therapeutic potential. This study shows that alloimmune response in heart transplants upregulates VEGF expression, and induces allograft angiogenesis that involves donor-derived primitive EC. Intracoronary adenoviral VEGF gene transfer increased macrophage infiltration, intimal angiogenesis and TxCAD. VEGF inhibition with PTK787 decreased allograft inflammation and TxCAD, and simultaneous PDGF inhibition with imatinib further decreased TxCAD. Specific inhibition of two VEGF-receptors (VEGFR) decreased allograft inflammation and TxCAD, and VEGFR-2 inhibition normalized the density of primitive and mature capillaries in the allografts. Adenovirus-mediated transient Ang1 expression in the allograft had anti-inflammatory and anti-arteriosclerotic effects. Adeno-associated virus (AAV)-mediated prolonged Ang1 or Ang2 expression had similar anti-inflammatory effects. However, AAV-Ang1 activated allograft SMC whereas AAV-Ang2 had no effects on SMC activation and decreased the development of TxCAD. These studies indicate an interplay of inflammation, angiogenesis and arteriosclerosis in cardiac allografts, and show that vascular growth factors are important regulators in the process. Also, VEGF inhibition, PDGF inhibition and angiopoietin therapy with clinically-relevant pharmacological agents or novel gene therapy approaches may counteract vascular dysfunction in cardiac allografts, and have beneficial effects on the survival of heart transplant patients in the future.

LFA-1 Integrin beta2 Chain Phosphorylation Regulates Protein Interactions and Mediates Signals in T Cells

Integrins are heterodimeric transmembrane adhesion receptors composed of alpha- and beta-subunits and they are vital for the function of multicellular organisms. Integrin-mediated adhesion is a complex process involving both affinity regulation and coupling to the actin cytoskeleton. Integrins also function as bidirectional signaling devices, regulating cell adhesion and migration after inside-out signaling, but also signal into the cell to regulate growth, differentiation and apoptosis after ligand binding. The LFA-1 integrin is exclusively expressed in leukocytes and is of fundamental importance for the function of the immune system. The LFA-1 integrins have short intracellular tails, which are devoid of catalytic activity. These cytoplasmic domains are important for integrin regulation and both the alpha and beta chain become phosphorylated. The alpha chain is constitutively phosphorylated, but the beta chain becomes phosphorylated on serine and functionally important threonine residues only after cell activation. The cytoplasmic tails of LFA-1 bind to many cytoskeletal and signaling proteins regulating numerous cell functions. However, the molecular mechanisms behind these interactions have been poorly understood. Phosphorylation of the cytoplasmic tails of the LFA-1 integrin could provide a mechanism to regulate integrin-mediated cytoskeletal interactions and take part in T cell signaling. In this study, the effects of phosphorylation of LFA-1 integrin cytoplasmic tails on different cellular functions were examined. Site-specific phosphorylation of both the alpha- and beta-chains of the LFA-1 was shown to have a role in the regulation of the LFA-1 integrin.Alpha-chain Ser1140 is needed for integrin conformational changes after chemokine- or integrin ligand-induced activation or after activation induced by active Rap1, whereas beta-chain binds to 14-3-3 proteins through the phosphorylated Thr758 and mediates cytoskeletal reorganization. Thr758 phosphorylation also acts as a molecular switch to inhibit filamin binding and allows 14-3-3 protein binding to integrin cytoplasmic domain, and it was also shown to lead to T cell adhesion, Rac-1/Cdc42 activation and expression of the T cell activation marker CD69, indicating a signaling function for Thr758 phosphorylation in T cells. Thus, phosphorylation of the cytoplasmic tails of LFA-1 plays an important role in different functions of the LFA-1 integrin in T cells. It is of vital importance to study the mechanisms and components of integrin regulation since leukocyte adhesion is involved in many functions of the immune system and defects in the regulation of LFA-1 contributes to auto-immune diseases and fundamental defects in the immune system.

Role of HMGB1 in cells of the circulation

The matrix of blood is a liquid plasma that transports molecules and blood cells within vessels lined by endothelial cells. High-mobility group B1 (HMGB1) is a protein expressed in blood cells. Under normal circumstances, HMGB1 is virtually absent from plasma, but during inflammation or trauma its level in plasma is increased. In resting and quiescent cells, HMGB1 is usually localized in the intracellular compartment, with the exception of motile cells that express HMGB1 on their outer surface to mediate cell migration. During cell transformation or immune cell activation HMGB1 can be actively secreted outside of the cell. Further, when a cell is damaged, HMGB1 can passively leak into extracellular environment. Extracellular HMGB1 can then participate in regulation of the immune response and under some conditions it can mediate lethality in systemic inflammatory response. The aim of this study was to evaluate the expression and functions of HMGB1 in cells of the vascular system and to investigate the prognostic value of circulating HMGB1 in severe sepsis and septic shock. HMGB1 was detected in platelets, leukocytes, and endothelial cells. HMGB1 was released from platelets and leukocytes, and it was found to mediate their adhesive and migratory functions. During severe infections the plasma levels of HMGB1 were elevated; however, no direct correlation with lethality was found. Further, the analysis of proinflammatory mechanisms suggested that HMGB1 forms complexes with other molecules to activate the immune system. In conclusion, HMGB1 is expressed in the cells of the vascular system, and it participates in inflammatory mechanisms by activating platelets and leukocytes and by mediating monocyte migration.

Microbe-induced Innate Immune Responses in Human Dendritic Cells

Two types of antigen-presenting cells (APCs), macrophages and dendritic cells (DCs), function at the interface of innate and adaptive immunity. Through recognition of conserved microbial patterns, they are able to detect the invading pathogens. This leads to activation of signal transduction pathways that in turn induce gene expression of various molecules required for immune responses and eventually pathogen clearance. Cytokines are among the genes induced upon detection of microbes. They play an important role in regulating host immune responses during microbial infection. Chemotactic cytokines, chemokines, are involved in migratory events of immune cells. Cytokines also promote the differentiation of distinct T cell responses. Because of the multiple roles of cytokines in the immune system, the cytokine network needs to be tightly regulated. In this work, the induction of innate immune responses was studied using human primary macrophages or DCs as cell models. Salmonella enterica serovar Typhimurium served as a model for an intracellular bacterium, whereas Sendai virus was used in virus experiments. The starting point of this study was that DCs of mouse origin had recently been characterized as host cells for Salmonella. However, only little was known about the immune responses initiated in Salmonella-infected human DCs. Thus, cellular responses of macrophages and DCs, in particular the pattern of cytokine production, to Salmonella infection were compared. Salmonella-induced macrophages and DCs were found to produce multiple cytokines including interferon (IFN) -gamma, which is conventionally produced by T and natural killer (NK) cells. Both macrophages and DCs also promoted the intracellular survival of the bacterium. Phenotypic maturation of DCs as characterized by upregulation of costimulatory and human leukocyte antigen (HLA) molecules, and production of CCL19 chemokine, were also detected upon infection with Salmonella. Another focus of this PhD work was to unravel the regulatory events controlling the expression of cytokine genes encoding for CCL19 and type III IFNs, which are central to DC biology. We found that the promoters of CCL19 and type III IFNs contain similar regulatory elements that bind nuclear factor kappaB (NF-kappaB) and interferon regulatory factors (IRFs), which could mediate transcriptional activation of the genes. The regulation of type III IFNs in virus infection resembled that of type I IFNs a cytokine class traditionally regarded as antiviral. The induction of type I and type III IFNs was also observed in response to bacterial infection. Taken together, this work identifies new details about the interaction of Salmonella with its phagocytic host cells of human origin. In addition, studies provide information on the regulatory events controlling the expression of CCL19 and the most recently identified IFN family genes, type III IFN genes.

Actin Dynamics in Muscle Cells

In every cell, actin is a key component involved in migration, cytokinesis, endocytosis and generation of contraction. In non-muscle cells, actin filaments are very dynamic and regulated by an array of proteins that interact with actin filaments and/or monomeric actin. Interestingly, in non-muscle cells the barbed ends of the filaments are the predominant assembly place, whereas in muscle cells actin dynamics was reported to predominate at the pointed ends of thin filaments. The actin-based thin filament pointed (slow growing) ends extend towards the middle of the sarcomere's M-line where they interact with the thick filaments to generate contraction. The actin filaments in muscle cells are organized into a nearly crystalline array and are believed to be significantly less dynamic than the ones in other cell types. However, the exact mechanisms of the sarcomere assembly and turnover are largely unknown. Interestingly, although sarcomeric actin structures are believed to be relatively non-dynamic, many proteins promoting actin dynamics are expressed also in muscle cells (e.g ADF/cofilin, cyclase-associated protein and twinfilin). Thus, it is possible that the muscle-specific isoforms of these proteins promote actin dynamics differently from their non-muscle counterparts, or that actin filaments in muscle cells are more dynamic than previously thought. To study protein dynamics in live muscle cells, I used primary cell cultures of rat cardiomyocytes. My studies revealed that a subset of actin filaments in cardiomyocyte sarcomeres displays rapid turnover. Importantly, I discovered that the turnover of actin filaments depends on contractility of the cardiomyocytes and that the contractility-induced actin dynamics plays an important role in sarcomere maturation. Together with previous studies those findings suggest that sarcomeres undergo two types of actin dynamics: (1) contractility-dependent turnover of whole filaments and (2) regulatory pointed end monomer exchange to maintain correct thin filament length. Studies involving an actin polymerization inhibitor suggest that the dynamic actin filament pool identified here is composed of filaments that do not contribute to contractility. Additionally, I provided evidence that ADF/cofilins, together with myosin-induced contractility, are required to disassemble non-productive filaments in developing cardiomyocytes. In addition, during these studies we learned that isoforms of actin monomer binding protein twinfilin, Twf-1 and Twf-2a localise to myofibrils in cardiomyocytes and may thus contribute to actin dynamics in myofibrils. Finally, in collaboration with Roberto Dominguez s laboratory we characterized a new actin nucleator in muscle cells - leiomodin (Lmod). Lmod localises towards actin filament pointed ends and its depletion by siRNA leads to severe sarcomere abnormalities in cardiomyocytes. The actin filament nucleation activity of Lmod is enhanced by interactions with tropomyosin. We also revealed that Lmod expression correlates with the maturation of myofibrils, and that it associates with sarcomeres only at relatively late stages of myofibrillogenesis. Thus, Lmod is unlikely to play an important role in myofibril formation, but rather might be involved in the second step of the filament arrangement and/or maintenance through its ability to promote tropomyosin-induced actin filament nucleation occurring at the filament pointed ends. The results of these studies provide valuable new information about the molecular mechanisms underlying muscle sarcomere assembly and turnover. These data offer important clues to understanding certain physiological and pathological behaviours of muscle cells. Better understanding of the processes occurring in muscles might help to find strategies for determining, diagnosis, prognosis and therapy in heart and skeletal muscles diseases.

Differentiation of neural stem cells in fragile X syndrome

Multipotent stem cells can self-renew and give rise to multiple cell types. One type of mammalian multipotent stem cells are neural stem cells (NSC)s, which can generate neurons, astrocytes and oligodendrocytes. NSCs are likely involved in learning and memory, but their exact role in cognitive function in the developing and adult brain is unclear. We have studied properties of NSCs in fragile X syndrome (FXS), which is the most common form of inherited mental retardation. FXS is caused by the lack of functional fragile X mental retardation protein (FMRP). FMRP is involved in the regulation of postsynaptic protein synthesis in a group I metabotropic glutamate receptor 5 (mGluR5)-dependent manner. In the absence of functional FMRP, the formation of functional synapses is impaired in the forebrain which results in alterations in synaptic plasticity. In our studies, we found that FMRP-deficient NSCs generated more neurons and less glia than control NSCs. The newborn neurons derived from FMRP-deficient NSCs showed an abnormally immature morphology. Furthermore, FMRP-deficient NSCs exhibited aberrant oscillatory Ca2+ responses to glutamate, which were specifically abolished by an antagonist of the mGluR5 receptor. The data suggested alterations in glutamatergic differentiation of FMRP-deficient NSCs and were further supported by an accumulation of cells committed to glutamatergic lineage in the subventricular zone of the embryonic Fmr1-knockout (Fmr1-KO) neocortex. Postnatally, the aberrant cells likely contributed to abnormal formation of the neocortex. The findings suggested a defect in the differentiation of distinct glutamatergic mGluR5 responsive cells in the absence of functional FMRP. Furthermore, we found that in the early postnatal Fmr1-KO mouse brain, the expression of mRNA for regulator of G-protein signalling-4 (RGS4) was decreased which was in line with disturbed G-protein signalling in NSCs lacking FMRP. Brain derived neurotrophic factor (BDNF) promotes neuronal differentiation of NSCs as the absence of FMRP was shown to do. This led us to study the effect of impaired BDNF/TrkB receptor signaling on NSCs by overexpression of TrkB.T1 receptor isoform. We showed that changes in the relative expression levels of the full-length and truncated TrkB isoforms influenced the replication capacity of NSCs. After the differentiation, the overexpression of TrkB.T1 increased neuronal turnover. To summarize, FMRP and TrkB signaling are involved in normal differentiation of NSCs in the developing brain. Since NSCs might have potential for therapeutic interventions in a variety of neurological disorders, our findings may be useful in the design of pharmacological interventions in neurological disorders of learning and memory.

Two levels of MCT1 and CD147 expression in the equine red blood cells and muscle

Monocarboxylate transporters (MCTs), especially the isoforms MCT1 - MCT4, cotransport lactate and protons across the cell membranes. They are thus essential for pH regulation and homeostasis in glycolytic cells such as red blood cells (RBCs), and skeletal muscle cells during intense exercise. In 70% of the Standardbred horses the lactate transport activity (TA) in RBCs is high and transport is mediated mainly by MCTs. In the rest 30% of the Standardbreds MCT mediated transport route is not active and the TA is low. MCTs need an ancillary protein for their proper localization and functioning in the plasma membrane. The ancillary protein for MCT1 and MCT4 is a member of immunoglobulin superfamily, CD147. Here we determined the expression of MCT isoforms and CD147 in equine RBCs and gluteal muscle. We sequenced the cDNA of horse MCT1 and CD147 to achieve horse-specific antibodies and to reveal sequence variations that may affect the TA of RBCs. The amount of MCT1 and CD147 mRNA in muscle were also studied. ---- In all, 73 horses representing different breeds were used. Blood samples were drawn from the jugular vein and muscle samples were taken either from gluteal muscle using biopsy needle or during castration from expendable cremaster muscle. The TA of RBCs was studied using radiolabeled lactate and the amount of MCT isoforms and CD147 in the plasma membranes using Western blotting. The level of mRNA in muscle cells was determined using qPCR. Isoforms MCT1 and MCT2 were found in the RBCs and isoforms MCT1 and MCT4 in the muscle cells of horses. The TA of RBCs was dependent on the expression of CD147 and MCT1 in the plasma membrane. Sequence variations were found in the cDNA of both MCT1 and CD147, but they did not explain the inactivity of MCT1 mediated transport route. The single nucleotide polymorphism (SNP) Met125Val in CD147 that existed parallel with an SNP in 3´-untranslated region explained, however, attenuation in CD147 expression in Standardbreds. A single mutation Ile51Val also decreased the expression of CD147 in one Warmblood. The MCT1 and CD147 mRNA concentrations in the gluteal muscle were higher in horses with higher MCT1 and CD147 expression in RBCs and lower in horses with minor expression of CD147 and MCT1. This suggests that the bimodal distribution of TA is due to differences in transcriptional regulation that is functioning in parallel in MCT1 and CD147 gene.

Metabolism and Translocation of Aminophospholipids in Mammalian Cells

In this study we investigated the metabolism, i.e. remodeling and translocation, of the aminophospholipids phosphatidylserine (PS) and phosphatidylethanolamine (PE). A new method for introduction of exogenous PS and PE molecular species to cultured cells was developed, and combined with mass spectrometry it enabled more detailed follow-up of the metabolism of single molecular species than previously. We found that I) exogenous PS and PE molecular species can be efficiently introduced to cultured cells without compromising cell integrity, II) PS and PE molecular species are remodeled by several phospholipases displaying selectivity based on phopholipid head group and acyl chain composition, III) PS decarboxylase (PSD) and Kennedy pathways provide a different PE molecular species composition to the cellular PE pool. In addition, PE species produced by these pathways are translocated from the site of synthesis to other cell compartments depending on their acyl chain composition. The data obtained in the present study helps to understand cellular phospholipid metabolism in more depth. The data show that effective labeling of cultured cells by exogenous phospholipids does not compromise cell viability and may be used to disturb cellular phospholipid composition to study lipid homeostasis. Remodeling and translocation of PS and PE molecular species is highly selective. The developed method and mass- spectrometric techniques may be used in future studies to understand disturbances in lipid homeostasis for example in diabetes mellitus, thus opening doors to optional scientific approaches to study mechanisms behind pathologies related to lipid disturbances.