Perinatal mesenchymal stromal cells of the human decidua restore continence in rats with stress urinary incontinence induced by simulated birth trauma and regulate senescence of fibroblasts from women with stress urinary incontinence.

Stress urinary incontinence (SUI) is a condition that causes the involuntary loss of urine when making small efforts, which seriously affects daily life of people who suffer from it. Women are more affected by this form of incontinence than men, since parity is the main risk factor. Weakening of the pelvic floor tissues is the cause of SUI, although a complete understanding of the cellular and molecular mechanisms of the pathology is still lacking. Reconstructive surgery to strengthen tissue in SUI patients is often associated with complications and/or is ineffective. Mesenchymal stromal cells from the maternal side of the placenta, i.e. the decidua, are proposed here as a therapeutic alternative based on the regenerative potential of mesenchymal cells. The animal model of SUI due to vaginal distention simulating labor has been used, and decidual mesenchymal stromal cell (DMSC) transplantation was effective in preventing a drop in pressure at the leak point in treated animals. Histological analysis of the urethras from DMSC-treated animals after VD showed recovery of the muscle fiber integrity, low or no extracellular matrix (ECM) infiltration and larger elastic fibers near the external urethral sphincter, compared to control animals. Cells isolated from the suburethral connective tissue of SUI patients were characterized as myofibroblasts, based on the expression of several specific genes and proteins, and were shown to achieve premature replicative senescence. Co-culture of SUI myofibroblasts with DMSC via transwell revealed a paracrine interaction between the cells through signals that mediated DMSC migration, SUI myofibroblast proliferation, and modulation of the proinflammatory and ECM-degrading milieu that is characteristic of senescence. In conclusion, DMSC could be an alternative therapeutic option for SUI by counteracting the effects of senescence in damaged pelvic tissue.

New Therapeutic Approaches for Allergy: A Review of Cell Therapy and Bio- or Nano-Material-Based Strategies.

Allergy constitutes a major health issue due to its large prevalence. The established therapeutic approaches (allergen avoidance, antihistamines, and corticosteroids) do not address the underlying causes of the pathology, highlighting the need for other long-term treatment options. Antigen-specific immunotherapy enables the long-term control of allergic diseases by promoting immunological tolerance to the allergen. However, efficacious immunotherapies are not available for all possible allergens, and the risk of undesired reactions during therapy remains a concern, especially in patients with severe allergic reactions. In this context, two types of therapeutic strategies appear especially promising for the future in the context of allergy: cell therapy and bio- or nano-material-based therapy. In this review, the main strategies developed this far in these two types of strategies are discussed, with several examples illustrating the different approaches.

Cell therapy for factor V deficiency: An approach based on human decidua mesenchymal stem cells.

Deficiency of factor V is a congenital autosomal recessive coagulopathy associated with mutations in the F5 gene that results in mild-to-severe bleeding episodes. Factor V is a component of the prothrombinase complex responsible for accelerating conversion of prothrombin to thrombin. At the present time there are no therapeutic factor V concentrates available. This study was designed to lay the preliminary foundations for future cell-based therapy for patients with severe factor V deficiency. The study showed that hepatospheres, which produce coagulation factors VIII, IX, and V, synthetize and store intracellular glycogen and express albumin levels up to 8 times higher than those of undifferentiated cells. Factor IX and factor V gene expression increased significantly in hepatospheres as compared to undifferentiated cells, whereas factor VIII gene expression remained constant. The factor V protein was detected in the hepatospheres´ secretome. Considering the enormous potential of mesenchymal stem cells as therapeutic agents, this study proposes a highly reproducible method to induce differentiation of mesenchymal stem cells from human placenta to factor V-producing hepatospheres. This strategy constitutes a preliminary step towards a curative treatment of factor V deficiency through advanced therapies such as cell therapy.

Endostatin Genetically Engineered Placental Mesenchymal Stromal Cells Carrying Doxorubicin-Loaded Mesoporous Silica Nanoparticles for Combined Chemo- and Antiangiogenic Therapy.

Combination therapies constitute a powerful tool for cancer treatment. By combining drugs with different mechanisms of action, the limitations of each individual agent can be overcome, while increasing therapeutic benefit. Here, we propose employing tumor-migrating decidua-derived mesenchymal stromal cells as therapeutic agents combining antiangiogenic therapy and chemotherapy. First, a plasmid encoding the antiangiogenic protein endostatin was transfected into these cells by nucleofection, confirming its expression by ELISA and its biological effect in an ex ovo chick embryo model. Second, doxorubicin-loaded mesoporous silica nanoparticles were introduced into the cells, which would act as vehicles for the drug being released. The effect of the drug was evaluated in a coculture in vitro model with mammary cancer cells. Third, the combination of endostatin transfection and doxorubicin-nanoparticle loading was carried out with the decidua mesenchymal stromal cells. This final cell platform was shown to retain its tumor-migration capacity in vitro, and the combined in vitro therapeutic efficacy was confirmed through a 3D spheroid coculture model using both cancer and endothelial cells. The results presented here show great potential for the development of combination therapies based on genetically-engineered cells that can simultaneously act as cellular vehicles for drug-loaded nanoparticles.

Premature senescence of placental decidua cells as a possible cause of miscarriage produced by mycophenolic acid.

BACKGROUND: Successful pregnancy is supported by a healthy maternal-fetal interface (i.e., the decidual tissues) which holds the conceptus and safeguards it against stressors from the beginning of pregnancy. Any disturbance of this interface can presumably lead to the loss of pregnancy. The use of the immunosuppressive drug mycophenolic acid (MPA) should be discontinued in pregnancy given its abortive and embryotoxic effects. Direct teratogenic effects have been observed in mammalian embryos cultured in MPA, but the underlying mechanisms of abortion by MPA are less understood. METHODS: Decidual stromal cells isolated from human placentas are cultured in the presence of clinically relevant doses of MPA. Data regarding the effects of MPA on the proliferation and viability of decidua cultures are first analysed and then, molecular pathways contributing to these effects are unravelled. RESULTS: MPA treatment of decidual stromal cells results in loss of proliferation capacity and a decrease in the viability of decidua cultures. The molecular pathways involved in the effects of MPA on decidual stromal cells are a reduction in pre-rRNA synthesis and subsequent disruption of the nucleolus. The nucleolar stress stabilizes p53, which in turn, leads to a p21-mediated cell cycle arrest in late S and G2 phases, preventing the progression of the decidua cells into the mitosis. Furthermore, MPA does not induce apoptosis but activate mechanisms of autophagy and senescence in decidual stromal cells. CONCLUSION: The irreversible growth arrest of decidua cells, whose role in the maintenance of the pregnancy microenvironment is known, may be one cause of miscarriage in MPA treated pregnant women.

Cell-Based Nanoparticles Delivery Systems for Targeted Cancer Therapy: Lessons from Anti-Angiogenesis Treatments.

The main strategy of cancer treatment has focused on attacking the tumor cells. Some cancers initially responsive to chemotherapy become treatment-resistant. Another strategy is to block the formation of tumor vessels. However, tumors also become resistant to anti-angiogenic treatments, mostly due to other cells and factors present in the tumor microenvironment, and hypoxia in the central part of the tumor. The need for new cancer therapies is significant. The use of nanoparticle-based therapy will improve therapeutic efficacy and targeting, while reducing toxicity. However, due to inefficient accumulation in tumor sites, clearance by reticuloendothelial organs and toxicity, internalization or conjugation of drug-loaded nanoparticles (NPs) into mesenchymal stem cells (MSCs) can increase efficacy by actively delivering them into the tumor microenvironment. Nanoengineering MSCs with drug-loaded NPs can increase the drug payload delivered to tumor sites due to the migratory and homing abilities of MSCs. However, MSCs have some disadvantages, and exosomes and membranes from different cell types can be used to transport drug-loaded NPs actively to tumors. This review gives an overview of different cancer approaches, with a focus on hypoxia and the emergence of NPs as drug-delivery systems and MSCs as cellular vehicles for targeted delivery due to their tumor-homing potential.

Suicide-gene transfection of tumor-tropic placental stem cells employing ultrasound-responsive nanoparticles.

A Trojan-horse strategy for cancer therapy employing tumor-tropic mesenchymal stem cells transfected with a non-viral nanovector is here presented. In this sense, ultrasound-responsive mesoporous silica nanoparticles were coated with a polycation (using two different molecular weights), providing them with gene transfection capabilities that were evaluated using two different plasmids. First, the expression of Green Fluorescent Protein was analyzed in Decidua-derived Mesenchymal Stem Cells after incubation with the silica nanoparticles. The most successful nanoparticle was then employed to induce the expression of two suicide genes: cytosine deaminase and uracil phosphoribosyl transferase, which allow the cells to convert a non-toxic pro-drug (5-fluorocytosine) into a toxic drug (5-Fluorouridine monophosphate). The effect of the production of the toxic final product was also evaluated in a cancer cell line (NMU cells) co-cultured with the transfected vehicle cells, Decidua-derived Mesenchymal Stem Cells. STATEMENT OF SIGNIFICANCE: Cell-mediated cancer therapy has recently attracted great interest. Tumor-homing cells can exert anticancer effects through innate capacities, via transfection with a therapeutic gene or acting as vehicles of therapeutic nanoparticles. In this work, an ultrasound-responsive mesoporous silica nanoparticle (capable of carrying an anticancer drug) is engineered to act as a non-viral transfection agent for tumor-tropic human placental mesenchymal stem cells. The successful transfection of the vehicle cells is evaluated employing different expression plasmids. After transfection with two suicide genes, the vehicle cells are capable of converting a non-toxic pro-drug into a highly toxic molecule, which can also kill surrounding cancer cells in an in vitro co-culture model. This work opens the gate for a plethora of strategies in which both genes and drug-loaded nanoparticles can be transported towards tumor tissues by easily available human mesenchymal stem cells.

Searching for a Cell-Based Therapeutic Tool for Haemophilia A within the Embryonic/Foetal Liver and the Aorta-Gonads-Mesonephros Region.

The development of new strategies based on cell therapy approaches to correct haemophilia A (HA) requires further insights into new cell populations capable of producing coagulation factor VIII (FVIII) and presenting stable engraftment potential. The major producers of FVIII in the adult are liver sinusoidal endothelial cells (LSECs) and in a lesser degree bone marrow-derived cells, both of which have been shown to ameliorate the bleeding phenotype in adult HA mice after transplantation. We have previously shown that cells from the foetal liver (FL) and the aorta-gonads-mesonephros (AGM) haematopoietic locations possess higher LSEC engraftment potential in newborn mice compared with adult-derived LSECs, constituting likely therapeutic targets for the treatment of HA in neonates. However, less is known about the production of FVIII in embryonic locations. Quantitative polymerase chain reaction and Western blot analysis were performed to assess the relative level of FVIII production in different embryonic tissues and at various developmental stages, identifying the FL and AGM region from day 12 (E12) as prominent sources of FVIII. Furthermore, FL-derived VE-cad+CD45-Lyve1+/- endothelial/endothelial progenitor cells, presenting vascular engraftment potential, produced high levels of F8 ribonucleic acid compared with CD45+ blood progenitors or Dlk1+ hepatoblasts. In addition, we show that the E11 AGM explant cultures expanded cells with LSEC repopulation activity, instrumental to further understand signals for in vitro generation of LSECs. Taking into account the capacity for FVIII expression, culture expansion and newborn engraftment potential, these results support the use of cells with foetal characteristics for correction of FVIII deficiency in young individuals.

Vectorization of ultrasound-responsive nanoparticles in placental mesenchymal stem cells for cancer therapy.

A new platform constituted by engineered responsive nanoparticles transported by human mesenchymal stem cells is here presented as a proof of concept. Ultrasound-responsive mesoporous silica nanoparticles are coated with polyethylenimine to favor their effective uptake by decidua-derived mesenchymal stem cells. The responsive-release ability of the designed nanoparticles is confirmed, both in vial and in vivo. In addition, this capability is maintained inside the cells used as carriers. The migration capacity of the nanoparticle-cell platform towards mammary tumors is assessed in vitro. The efficacy of this platform for anticancer therapy is shown against mammary tumor cells by inducing the release of doxorubicin only when the cell vehicles are exposed to ultrasound.

Restrained Th17 response and myeloid cell infiltration into the central nervous system by human decidua-derived mesenchymal stem cells during experimental autoimmune encephalomyelitis.

BACKGROUND: Multiple sclerosis is a widespread inflammatory demyelinating disease. Several immunomodulatory therapies are available, including interferon-ß, glatiramer acetate, natalizumab, fingolimod, and mitoxantrone. Although useful to delay disease progression, they do not provide a definitive cure and are associated with some undesirable side-effects. Accordingly, the search for new therapeutic methods constitutes an active investigation field. The use of mesenchymal stem cells (MSCs) to modify the disease course is currently the subject of intense interest. Decidua-derived MSCs (DMSCs) are a cell population obtained from human placental extraembryonic membranes able to differentiate into the three germ layers. This study explores the therapeutic potential of DMSCs. METHODS: We used the experimental autoimmune encephalomyelitis (EAE) animal model to evaluate the effect of DMSCs on clinical signs of the disease and on the presence of inflammatory infiltrates in the central nervous system. We also compared the inflammatory profile of spleen T cells from DMSC-treated mice with that of EAE control animals, and the influence of DMSCs on the in vitro definition of the Th17 phenotype. Furthermore, we analyzed the effects on the presence of some critical cell types in central nervous system infiltrates. RESULTS: Preventive intraperitoneal injection of DMSCs resulted in a significant delay of external signs of EAE. In addition, treatment of animals already presenting with moderate symptoms resulted in mild EAE with reduced disease scores. Besides decreased inflammatory infiltration, diminished percentages of CD4(+)IL17(+), CD11b(+)Ly6G(+) and CD11b(+)Ly6C(+) cells were found in infiltrates of treated animals. Early immune response was mitigated, with spleen cells of DMSC-treated mice displaying low proliferative response to antigen, decreased production of interleukin (IL)-17, and increased production of the anti-inflammatory cytokines IL-4 and IL-10. Moreover, lower RORγT and higher GATA-3 expression levels were detected in DMSC-treated mice. DMSCs also showed a detrimental influence on the in vitro definition of the Th17 phenotype. CONCLUSIONS: DMSCs modulated the clinical course of EAE, modified the frequency and cell composition of the central nervous system infiltrates during the disease, and mediated an impairment of Th17 phenotype establishment in favor of the Th2 subtype. These results suggest that DMSCs might provide a new cell-based therapy for the control of multiple sclerosis.

Decidua-derived mesenchymal stem cells as carriers of mesoporous silica nanoparticles. In vitro and in vivo evaluation on mammary tumors.

The potential use of human Decidua-derived mesenchymal stem cells (DMSCs) as a platform to carry mesoporous silica nanoparticles in cancer therapy has been investigated. Two types of nanoparticles were evaluated. The nanoparticles showed negligible toxicity to the cells, a fast uptake and a long retention inside them. Nanoparticle location in the cell was studied by colocalization with the lysosomes. Moreover, the in vitro and in vivo migration of DMSCs towards tumors was not modified by the evaluated nanoparticles. Finally, DMSCs transporting doxorubicin-loaded nanoparticles were capable of inducing cancer cell death in vitro. STATEMENT OF SIGNIFICANCE: The use of nanotechnology for anticancer drug delivery has recently attracted great interest. Nanoparticles such as mesoporous silica nanoparticles (MSNs) can reach tumors, either by passive targeting, through the enhanced permeability and retention (EPR) effect, or active targeting, through the functionalization of nanoparticle surface. However, nanotechnology has not yet achieved the expected results in improving drug targeting, highlighting the need for a better localization of the nanoparticles in the tumors. Human mesenchymal stem cells from the decidua of the human placenta (DMSCs) have been observed to migrate towards tumors in a preclinical model of breast cancer. Moreover, they have been shown to inhibit growth of primary tumors and development of new tumors. In this work, combining MSNs and DMSCs, we have studied for the first time whether placental stem cells could be employed as a platform to load nanoparticles and carry them towards tumors for future anticancer therapies.

Human decidua-derived mesenchymal stem cells differentiate into functional alveolar type II-like cells that synthesize and secrete pulmonary surfactant complexes.

Lung alveolar type II (ATII) cells are specialized in the synthesis and secretion of pulmonary surfactant, a lipid-protein complex that reduces surface tension to minimize the work of breathing. Surfactant synthesis, assembly and secretion are closely regulated and its impairment is associated with severe respiratory disorders. At present, well-established ATII cell culture models are not available. In this work, Decidua-derived Mesenchymal Stem Cells (DMSCs) have been differentiated into Alveolar Type II- Like Cells (ATII-LCs), which display membranous cytoplasmic organelles resembling lamellar bodies, the organelles involved in surfactant storage and secretion by native ATII cells, and accumulate disaturated phospholipid species, a surfactant hallmark. Expression of characteristic ATII cells markers was demonstrated in ATII-LCs at gene and protein level. Mimicking the response of ATII cells to secretagogues, ATII-LCs were able to exocytose lipid-rich assemblies, which displayed highly surface active capabilities, including faster interfacial adsorption kinetics than standard native surfactant, even in the presence of inhibitory agents. ATII-LCs could constitute a highly useful ex vivo model for the study of surfactant biogenesis and the mechanisms involved in protein processing and lipid trafficking, as well as the packing and storage of surfactant complexes.

[Referrals from primary care to a dedicated headache clinic: analysis of the first 1,000 patients]. / Derivaciones de atencion primaria a una consulta monografica de cefaleas: analisis de los 1.000 primeros pacientes.

INTRODUCTION: It is commonplace practice in dedicated clinics or headache units to deal with complex patients referred from general neurology clinics. In our centre, part of the schedule of the dedicated headache clinic (DHC) is reserved for patients referred from primary care (PC) in the form of one-stop clinics. AIMS: To analyse both the characteristics of the patients referred by PC to DHC and the suitability of the agreed referral criteria, and to compare them with the first visits due to headache in a general neurology clinic. PATIENTS AND METHODS: The study was conducted on a sample of 1,000 patients (741 females and 259 males) in the DHC and 290 (203 females and 87 males) in the general clinic. Data were collected retrospectively and included demographic variables, previous use of symptomatic or preventive treatments, need for complementary examinations and adjustment to referral criteria. Headaches were coded in accordance with the second edition of the International Headache Classification (IHC-2). RESULTS: A total of 1,562 headaches were coded in the DHC group and 444 in the general clinic group; their distribution over the groups of the IHC-2 was similar, and most of them fell into group 1 (migraine). The percentage of patients who had received preventive treatment was higher in the DHC. The percentage of appropriate referrals was high and a little higher still in the general clinic. CONCLUSIONS: The characteristics of the patients referred from PC to a DHC in our milieu were similar to those reported in previous studies. These patients have received treatment more frequently than those who were seen in a general neurology clinic.

TITLE: Derivaciones de atencion primaria a una consulta monografica de cefaleas: analisis de los 1.000 primeros pacientes.Introduccion. Es habitual en las consultas monograficas o unidades de cefaleas que atiendan a pacientes complejos derivados desde consultas generales de neurologia. En nuestro centro, parte de la agenda de la consulta monografica de cefaleas (CMC) se reserva a pacientes remitidos desde atencion primaria (AP) con formato de alta resolucion. Objetivos. Analizar las caracteristicas de los pacientes derivados por AP a la CMC y la adecuacion a los criterios de derivacion consensuados, y compararlos con las primeras visitas por cefalea en una consulta general de neurologia. Pacientes y metodos. Muestra de 1.000 pacientes (741 mujeres y 259 varones) en la CMC y 290 (203 mujeres y 87 varones) en la consulta general. Recogemos prospectivamente variables demograficas, uso previo de tratamientos sintomaticos o preventivos, necesidad de exploraciones complementarias y adecuacion a los criterios de derivacion. Codificamos las cefaleas de acuerdo con la segunda edicion de la Clasificacion Internacional de Cefaleas (CIC-2). Resultados. En el grupo de la CMC se codificaron 1.562 cefaleas, y en la consulta general, 444; su distribucion en los grupos de la CIC-2 era similar, y la mayoria se encuadraba en el grupo 1 (migraña). El porcentaje de pacientes que habian recibido tratamiento preventivo era mayor en la CMC. El porcentaje de derivaciones adecuadas fue alto y algo mayor en la consulta general. Conclusiones. Las caracteristicas de los pacientes derivados desde AP a una CMC en nuestro medio son similares a las previamente descritas. Estos pacientes han recibido tratamiento con mas frecuencia que los atendidos en una consulta general de neurologia.

Human decidua-derived mesenchymal stromal cells differentiate into hepatic-like cells and form functional three-dimensional structures.

BACKGROUND AIMS: Previously, we have shown that human decidua-derived mesenchymal stromal cells (DMSC) are mesenchymal stromal cells (MSC) with a clonal differentiation capacity for the three embryonic layers. The endodermal capacity of DMSC was revealed by differentiation into pulmonary cells. In this study, we examined the hepatic differentiation of DMSC. METHODS: DMSC were cultured in hepatic differentiation media or co-cultured with murine liver homogenate and analyzed with phenotypic, molecular and functional tests. RESULTS AND CONCLUSIONS: DMSC in hepatic differentiation media changed their fibroblast morphology to a hepatocyte-like morphology and later formed a 3-dimensional (3-D) structure or hepatosphere. Moreover, the hepatocyte-like cells and the hepatospheres expressed liver-specific markers such as synthesis of albumin (ALB), hepatocyte growth factor receptor (HGFR), α-fetoprotein (AFP) and cytokeratin-18 (CK-18), and exhibited hepatic functions including glycogen storage capacity and indocyanine green (ICG) uptake/secretion. Human DMSC co-cultured with murine liver tissue homogenate in a non-contact in vitro system showed hepatic differentiation, as evidenced by expression of AFP and ALB genes. The switch in the expression of these two genes resembled liver development. Indeed, the decrease in AFP and increase in ALB expression throughout the co-culture were consistent with the expression pattern observed during normal liver organogenesis in the embryo. Interestingly, AFP and ALB expression was significantly higher when DMSC were co-cultured with injured liver tissue, indicating that DMSC respond differently under normal and pathologic micro-environmental conditions. In conclusion, DMSC-derived hepatospheres and DMSC co-cultured with liver homogenate could be suitable in vitro models for toxicologic, developmental and pre-clinical hepatic regeneration studies.

Fibronectin increases survival of rat hepatic stellate cells--a novel profibrogenic mechanism of fibronectin.

UNLABELLED: The aims of this study were to determine whether fibronectin increases survival of hepatic stellate cells (HSCs) in starving conditions, and to identify the signal transduction pathways involved in this effect. METHODS: Primary culture of rat HSCs were plated on fibronectin-uncoated or coated culture wells, and grown in the presence of 0.2% or 20% fetal calf serum. Cell apoptosis was measured by an ELISA procedure. Signal transduction pathways were analyzed by inhibiting major intracellular transduction pathways with appropriated inhibitors and by detecting phosphorylated proteins. RESULTS: Fibronectin increased survival of serum deprived HSCs. This effect was abrogated by the presence of the RGD peptide, by silencing FAK expression, and by inhibiting PI3K with LY294002 or wortmannin. Growth of HSCs on fibronectin induced integrin alpha5beta1 expression, tyr397, ser473, and ser136 phosphorylation of FAK, Akt, and Bad, respectively, and the binding of phosphorylated Bad to 14-3-3 proteins. Likewise, fibronectin increased Bcl2/Bax ratio and reduced release of mitochondrial cytochrome c into the cytoplasm, formation of apoptosome, and caspase 9 and 3 activity. These effects were avoided by treatment of cells with PI3K inhibitors. CONCLUSION: Fibronectin increases survival of HSCs via a pathway involving integrin alpha5beta1 receptors, FAK, PI3K, Akt and proteins of Bcl2 family.

Interleukin-6 increases rat metalloproteinase-13 gene expression through Janus kinase-2-mediated inhibition of serine/threonine phosphatase-2A.

Interleukin-6 (IL-6) increases metalloproteinase-13 (MMP-13) gene expression by increasing phosphorylated c-Jun and by inhibiting serine/threonine phosphatase-2A (PP2A) activity. We investigated the mechanisms by which IL-6 induces c-Jun phosphorylation and PP2A inactivation in Rat-1 fibroblasts. We show that IL-6 increased MMP-13 mRNA, phosphorylated c-Jun, and activator protein 1 (AP1) binding activity without increasing c-Jun-N-terminal kinase (JNK) activity. These effects did not seem to be mediated by ERK, p38 MAP kinase, phosphatidylinositol-3-kinase, calmoduline-dependent protein kinase, protein kinase C (PKC) or protein kinase A since inhibition with specific inhibitors did not abrogate these effects. IL-6 increases PP2A catalytic subunit tyrosine phosphorylation. Inhibition of the tyrosine kinase Jak2, with the specific inhibitor AG490, abrogated this effect. Likewise, this Jak2 inhibitor blocked the effects of IL-6 on c-Jun phosphorylation, AP1 binding activity and metalloproteinase-13 gene expression. We conclude that IL-6 increases MMP-13 gene expression by activation of Jak2, resulting in tyrosine phosphorylation of the catalytic subunit of PP2A, which in turn decreases PP2A activity and prolongs c-Jun phosphorylation.

Reactive oxygen species mediate the down-regulation of mitochondrial transcripts and proteins by tumour necrosis factor-alpha in L929 cells.

In this study, we show that reactive oxygen species production induced by tumour necrosis factor alpha (TNF-alpha) in L929 cells was associated with a decrease in the steady-state mRNA levels of the mitochondrial transcript ATPase 6-8. Simultaneously, the transcript levels of two nuclear-encoded glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphofructokinase, were increased. These changes were associated with decreased protein levels of the ATPase subunit a (encoded by the mitochondrial ATPase 6 gene) and cytochrome c oxidase subunit II, and increased protein levels of phosphofructokinase. Since TNF-alpha had no effect on the amount of mitochondrial DNA, the results suggested that TNF-alpha acted at the transcriptional and/or post-transcriptional level. Reactive oxygen species scavengers, such as butylated hydroxianisole and butylated hydroxytoluene, blocked the production of free radicals, prevented the down-regulation of ATPase 6-8 transcripts, preserved the protein levels of ATPase subunit a and cytochrome c oxidase subunit II, and attenuated the cytotoxic response to TNF-alpha, indicating a direct link between these two phenomena.

Sp1 and Sp3 transcription factors mediate malondialdehyde-induced collagen alpha 1(I) gene expression in cultured hepatic stellate cells.

Malondialdehyde, the end product of lipid peroxidation, has been shown to stimulate collagen alpha1(I) (Col1a1) gene expression. However, mechanisms of this effect are unclear. The purpose of this study was to clarify these mechanisms. Rat hepatic stellate cells were cultured in the presence of 200 microm malondialdehyde, and the effects on collagen gene expression and the binding of nuclear proteins to the col1a1 promoter were analyzed. Malondialdehyde treatment induced an increase in the cellular levels of col1a1 mRNA that was abrogated by pretreating cells with cycloheximide, p-hydroxymercuribenzoate, pyridoxal 5'-phosphate, and mithramycin. Transient transfections showed that malondialdehyde exerted its effect through regulatory elements located between -220 and -110 bp of the col1a1 promoter. Gel retardation assays demonstrated that malondialdehyde increased the binding of nuclear proteins to two elements located between -161 and -110 bp of the col1a1 promoter. These bindings were supershifted with Sp1 and Sp3 antibodies. Finally, malondialdehyde increased cellular levels of the Sp1 and Sp3 proteins and Sp1 mRNA. Our data indicated that treatment of hepatic stellate cells with malondialdehyde stimulated col1a1 gene expression by inducing the synthesis of Sp1 and Sp3 and their binding to two regulatory elements located between -161 and -110 bp of the col1a1 promoter.