Survival-promoting Activities Research Articles

Molecular Targets of Pinocembrin Underlying Its Regenerative Activities in Human Keratinocytes.

Pinocembrin is one of the well-known compounds in the group of flavonoids. The pharmacological activities of pinocembrin in association with wound-healing activities have been reported. However, its effects on the aspect of cellular interaction underlying growth and survival are still unidentified in human keratinocytes. Our previous study reported that Boesenbergia rotunda potently stimulated survival and proliferation of a human keratinocyte cell line (HaCaT). On the basis that pinocembrin is revealed to be one of the major constituents of this plant, we aimed to define the survival- and proliferation-enhancing effects of this compound at the cellular level. Results from the current study confirmed that pinocembrin induced an increase in HaCaT cell number. At the signaling perspective, we identified that pinocembrin significantly triggered ERK1/2 and Akt activation. The stimulating effects of pinocembrin were clearly inhibited by MEK and PI3K inhibitors authenticating that proliferation- and survival-promoting activities of pinocembrin were mainly acted on these two signaling cascades. Altogether, we successfully identified that pinocembrin functions to induce keratinocyte proliferation and survival, at least by provoking MAPK and PI3K pathways. Our study encourages the fact that pinocembrin is one of the interesting natural flavonoid compounds to be developed as a wound closure-promoting agent.

Triterpenoids with neurotrophic activity from Ganoderma lucidum

A new triterpenoid, 4,4,14α-trimethyl-5α-chol-7,9(11)-dien-3-oxo-24-oic acid (1), together with seven known triterpenoids, were isolated from the dried fruiting bodies of Ganoderma lucidum. Their structures were elucidated by extensive spectroscopic analyses. Bioassay results revealed that compounds 1 and methyl ganoderic acid B (5) had nerve growth factor-like neuronal survival-promoting effects, whereas compounds 1, and 4–7 showed brain-derived neurotrophic factor-like neuronal survival-promoting activities.

Vascular endothelial growth factor as a survival factor for human islets: effect of immunosuppressive drugs

Rapamycin, part of the immunosuppressive regimen of the Edmonton protocol, has been shown to inhibit vascular endothelial growth factor (VEGF) production and VEGF-mediated survival signalling in tumour cell lines. This study investigates the survival-promoting activities of VEGF in human islets and the effects of rapamycin on islet viability. Levels of VEGF and its receptors in isolated human islets and whole pancreas was determined by western blotting and immunostaining. Islet viability following VEGF or immunosuppressive drug treatment was determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Islet VEGF release was measured by ELISA. Mouse islets infected with an adenovirus expressing the gene for VEGF were transplanted syngeneically into streptozotocin-induced diabetic mice, with blood glucose levels measured three times per week. Isolated human islets produced multiple isoforms of VEGF and VEGF receptors 1, 2 and 3 and the coreceptor neuropilin 1. Exogenous VEGF (10 ng/ml) prevented human islet death induced by serum starvation, which suggests that VEGF can act as a survival factor for human islets. Transplantation of mouse islets infected with a VEGF-expressing adenovirus in a syngeneic model, improved glycaemic control at day 1 post-transplantation (p < 0.05). Rapamycin at 10 and 100 ng/ml significantly reduced islet VEGF release (by 37 +/- 4% and 43 +/- 6%, respectively; p < 0.05) and at 100 ng/ml reduced islet viability (by 36 +/- 9%) and insulin release (by 47 +/- 7%, all vs vehicle-treated controls; p < 0.05). Tacrolimus had no effect on islet VEGF release or viability. Our data suggest that rapamycin may have deleterious effects on islet survival post-transplantation, both through a direct effect on islet viability and indirectly through blockade of VEGF-mediated revascularisation.

Telomerase and neuronal marker status of differentiated NT2 and SK‐N‐SH human neuronal cells and primary human neurons

Upon treatment with retinoic acid, NTera-2 (NT2) human teratocarcinoma and SK-N-SH neuroblastoma cells can be induced to terminally differentiate into postmitotic neuronal cells. The neuronal cell yield obtained from the NT-2 cells is partially dependent on the time of differentiation (24-55 days). SK-N-SH cells differentiate into a mixed population of neuronal and epithelium-like cells. Here we report modified protocols that increase the number of differentiated NT-2 and SK-N-SH cells and that establish an enriched neuronal SK-N-SH-derived cell population essentially devoid of nonneuronal cells. Differentiated cells express the cytoskeleton-associated protein tau and other typical neuronal markers, such as Map2, Ngn1, NeuroD, Mash1, and GluR which are also expressed in primary human fetal neurons. Telomerase activity is down-regulated in differentiated cells, which is consistent with the telomerase status of primary fetal human neurons. Thus, differentiated NT2 and SK-N-SH cells may represent an excellent source for studies investigating the role of telomerase or other survival-promoting activities in protecting human neuronal cells from cell death-mediating stresses associated with neurodegenerative diseases.

Functional abnormalities of heparan sulfate in mucopolysaccharidosis-I are associated with defective biologic activity of FGF-2 on human multipotent progenitor cells

AbstractIn mucopolysaccharidosis-I (MPS-I), α-L-iduronidase deficiency leads to progressive heparan sulfate (HS) and dermatan sulfate (DS) glycosaminoglycan (GAG) accumulation. The functional consequences of these accumulated molecules are unknown. HS critically influences tissue morphogenesis by binding to and modulating the activity of several cytokines (eg, fibroblast growth factors [FGFs]) involved in developmental patterning. We recently isolated a multipotent progenitor cell from postnatal human bone marrow, which differentiates into cells of all 3 embryonic lineages. The availability of multipotent progenitor cells from healthy volunteers and patients with MPS-I (Hurler syndrome) provides a unique opportunity to directly examine the functional effects of abnormal HS on cytokine-mediated stem-cell proliferation and survival. We demonstrate here that abnormally sulfated HS in Hurler multipotent progenitor cells perturb critical FGF-2–FGFR1-HS interactions, resulting in defective FGF-2–induced proliferation and survival of Hurler multipotent progenitor cells. Both the mitogenic and survival-promoting activities of FGF-2 were restored by substitution of Hurler HS by normal HS. This perturbation of critical HS–cytokine receptor interactions may represent a mechanism by which accumulated HS contributes to the developmental pathophysiology of Hurler syndrome. Similar mechanisms may operate in the pathogenesis of other diseases where structurally abnormal GAGs accumulate.

Comparative proteomic analysis identifies protein disulfide isomerase and peroxiredoxin 1 as new players involved in embryonic interdigital cell death

In this study, we used comparative proteomics to identify proteins that were involved in the regulation of interdigital cell death. The protein profiles of embryonic day (E) 12.5 and 13.5 mouse hindlimb interdigital tissues were compared to identify proteins that were differentially expressed. The interdigital cells are irreversibly committed to programmed cell death (PCD) at E13.5, whereas they are developmentally plastic at E12.5. We established that protein disulfide isomerase (PDI) expression was up-regulated at E13.5, while peroxiredoxin 1 (Prdx1) expression was down-regulated at this time point. Semiquantitative reverse transcriptase-polymerase chain reaction and Western blot analyses confirmed the data obtained from the two-dimensional electrophoresis gels. Furthermore, we were able to up-regulate PDI expression by manipulating the E12.5 interdigital tissues to die during culture, although this up-regulation was not possible when cell survival was promoted. In addition, we could inhibit interdigital cell death and expression of proapoptotic genes (Bmp-4 and Bambi) by treating interdigital tissues with PDI antibodies and bacitracin (a PDI enzyme inhibitor). These findings suggested that PDI was involved in the activation and maintenance of interdigital cell death. Conversely, we determined that Prdx1 expression was maintained when interdigital cultures were manipulated to survive but down-regulated when the cultures were permitted to die. The result suggested that Prdx1 was involved in maintaining interdigital cell survival. However, we were unable to induce interdigital cell death by means of RNA interference-mediated silencing of Prdx1 expression, indicating that Prdx1 down-regulation is not sufficient for PCD to occur. Proteomic analysis of the Prdx1 knock-down cells revealed that the level of NF-kappaB inhibitor epsilon (IkappaBepsilon) was dramatically reduced. Furthermore, we found an increase in NFkappaB activation and reactive oxygen species (ROS) levels in the cytoplasm as a result of Prdx1 knockdown. We also found that silencing Prdx1 made the interdigital cells more susceptible to ROS-induced cell death. Taken together, our study identifies two new players in interdigital cell death and highlights that PCD is regulated by a delicate balance of proapoptotic and survival-promoting activities.

Effects of Long-term Vitamin K (Phylloquinone) Intake on Retina Aging

Emerging evidence suggests neuroprotective functions of vitamin K and/or vitamin K-dependent proteins. We investigated the effect of dietary vitamin K on retina aging (thinning). Female Sprague-Dawley rats were maintained from weaning on low (80 μg kg<PRE>-1</PRE> diet), adequate (500 μg kg<PRE>-1</PRE> diet) or high (2000 μg kg<PRE>-1</PRE> diet) levels of vitamin K1 (phylloquinone). Relative concentrations of brain vitamin K associated with these diets were 1: 3.3: 25 (K1) and 1: 2.7: 9.0 (menaquinone-4). Histomorphometry of old (21 month) rats revealed positive associations between vitamin K and thickness of retina layers, especially in the equatorial/peripheral retina. No association of diet and retina thickness was detected among young (6 month) animals. The sparing effect of vitamin K in the retina was most evident in the inner plexiform layer and in the photoreceptor inner and outer segments. Surprisingly, we observed no effect of vitamin K on the age-dependent loss of photoreceptor cells, interneurons or ganglion cells. These data suggest a role for vitamin K in maintaining the aging retina and suggest that the sparing effect of vitamin K does not reflect the survival-promoting (anti-apoptotic) activities of vitamin K-dependent proteins.

Modulation of inflammation by vasoactive intestinal peptide and bombesin: lack of effects on neutrophil apoptosis.

Inhibition of neutrophil apoptosis has been identified as a prominent feature in chronic inflammation, parenchymal damage, and unresolved organ dysfunction. Lung injury animal models suggest that the neuropeptides vasoactive intestinal peptide and bombesin are protective. Therefore, in vitro effects of VIP and bombesin on apoptosis of normal human neutrophils were tested. For measuring effects on cell survival and apoptosis, trypan dye exclusion, colorimetric MTT assay to assess cell survival, and caspase-3 assay and annexin-V binding for analysing apoptosis rates were used. Foetal calf serum, Fas ligand, and tumour necrosis factor-alpha served as modulatory control agents; survival-promoting and apoptosis-inducing activities of the respective agents were confirmed. Vasoactive intestinal peptide and bombesin, however, failed to significantly affect cell death in neutrophils. Data suggest that direct regulation of neutrophil apoptosis is unlikely to be among the mechanisms of lung-protective actions of VIP and bombesin.

??????????????

A large number of compounds have been reported to prevent ischemia-induced neuronal death, whereas there are few described to enhance recovery of brain functions. Since neurotrophins do not only prevent neuronal death but also protect neuronal circuits, they may be potential candidates. However, their poor penetration of the blood-brain-barrier hampers their development as therapeutic agents. In this context, low-molecular-weight compounds that possess neurite outgrowth- and neuronal survival-promoting activities may be alternative candidates. Neurite outgrowth-promoting prostaglandins, which were recently-synthesized based on the chemical structure of anti-tumor cyclopentenone prostaglandin derivatives, have been characterized by their neurotrophic effects on neurons in the central nervous system. In this paper, we present a review of these compounds as therapeutic agents against several neurodegenerative diseases.

Molecular cloning of POEM: a novel adhesion molecule that interacts with alpha8beta1 integrin.

Cell adhesion molecules are involved in a number of biological functions, such as cell survival, cell differentiation, tissue repair, and development. A novel molecule, POEM (preosteoblast epidermal growth factor-like repeat protein with meprin, A5 protein, and receptor protein-tyrosine phosphatase mu domain), was isolated by reverse transcription-polymerase chain reaction using a set of degenerate primers designed after other known epidermal growth factor (EGF)-like motifs. From its structure, POEM was suggested to be a novel adhesion molecule with five EGF-like domains, an Arg-Gly-Asp (RGD) cell binding motif, and a meprin, A5 protein, and receptor protein-tyrosine phosphatase mu (MAM) domain. By in situ hybridization using embryonic day 16.5 (E16.5) mouse embryos, strong expression of POEM mRNA was observed in developing kidney renal tubules, parathyroid and thyroid glands, developing bone, tooth germ, and endocrine organs of the brain. The inner ear, skeletal muscle, smooth muscle (except for the vascular system), and skin were also positive for POEM expression. Bacterial recombinant POEM protein containing the RGD sequence and MAM domain showed strong cell adhesion, spreading, and survival-promoting activities. By mutating the RGD sequence to RGE, the cell spreading and survival activities were significantly decreased, but the MAM domain was shown to contribute only to cell adhesion and not to cell spreading and survival-promoting activities. The distribution of POEM in several tissues was close to that of alpha(8)beta(1) integrin. Therefore, we conducted cell adhesion assays using KA8 cells, a K562 leukemia clone stably expressing alpha(8) integrin. Parental K562 cells, which expressed alpha(5)beta(1) integrin, bound to fibronectin but not to POEM. On the other hand, KA8 cells showed strong binding and spreading on both fibronectin and POEM. These results suggest that POEM is a novel ligand for alpha(8)beta(1) integrin and that POEM may be involved in the development and function of various tissues, such as kidney, bone, muscles, and endocrine organs.

Effects of β-Casomorphins on Neuronal Survival in Culture of Embryonic Chick Dorsal Root Ganglion Neurons

We studied the effects of beta-casomorphins (beta-CMs, mu-acting opioid peptides from milk protein beta-casein) on survival of primary-cultured chick dorsal root ganglion neurons in the presence of nerve growth factor. Beta-CM-5 and beta-CM-7 had potent neuronal survival-promoting activities. Beta-CM-4 amide (morphiceptin) and des-Tyr'-beta-CM-7 also exhibited the similar promoting effects, although their effects were very weak. The promoting effect of beta-CM-5 was prevented by co-administration of naloxone, or pretreatment with pertussis toxin. These results suggest that the neuronal survival-promoting effects of beta-CMs might be mediated through opioid receptors coupled to G proteins.

LDL Receptor-Related Protein as a Component of the Midkine Receptor

Midkine (MK) is a heparin-binding growth factor with migration-promoting and survival-promoting activities. To identify signaling receptor(s) of MK, membrane glycoproteins with MK-binding activity were isolated from day 13 mouse embryos by lectin- and MK-affinity chromatography. SDS-PAGE followed by protein sequence analysis revealed the presence of LDL receptor-related protein (LRP) and NCAM in the fraction. The dissociation constant of binding between LRP and MK was 3.5 nM. Receptor-associated protein (RAP), which interfered with the binding, inhibited MK-dependent survival of embryonic neurons. Brushin/megalin, which is also a high molecular weight protein belonging to the LDL receptor family, bound to MK less strongly than LRP. These findings suggest that LRP is a component of the receptor complex for MK.

Coupling of osteopontin and its cell surface receptor CD44 to the cell survival response elicited by interleukin-3 or granulocyte-macrophage colony-stimulating factor.

The receptors for interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) share a common beta subunit, the distal cytoplasmic domain of which is essential for the promotion of cell survival by these two cytokines. Genes whose expression is specifically induced by signaling through the distal cytoplasmic domain of this receptor beta subunit were screened by a subtraction cloning approach in derivatives of a mouse pro-B-cell line. One gene thus identified was shown to encode a protein highly homologous (with only 7 amino acid substitutions) to murine osteopontin (OPN), a secreted adhesion protein. Conditioned medium from cells expressing wild-type OPN, but not that from cells expressing a deletion mutant lacking residues 79 to 140, increased the viability of a non-OPN-producing cell line in the presence of human GM-CSF. Antibody blocking experiments revealed that OPN produced as a result of IL-3 or GM-CSF signaling was secreted into the medium and, through binding to its cell surface receptor, CD44, contributed to the survival-promoting activities of these two cytokines. Furthermore, coupling of the OPN-CD44 pathway to the survival response to IL-3 was also demonstrated in primary IL-3-dependent mouse bone marrow cells. These results thus show that induction of an extracellular adhesion protein and consequent activation of its cell surface receptor are important for the antiapoptotic activities of IL-3 and GM-CSF.

Human Glial Cell-Line-Derived Neurotrophic Factor: A Structure–Function Analysis

Glial cell-line-derived neurotrophic factor (GDNF) is a protein known to enhance the survival of dopaminergic and motor neurons. It has been shown to have therapeutic potential in the treatment of Parkinson's disease and other neurodegenerative diseases. GDNF gene was modified by deletion and insertion mutagenesis using PCR methods. The various mutants were all highly expressed in Escherichia coli. The recombinant proteins were purified and their survival-promoting activities were determined by motor neurons. The result showed that the C-terminus was critical for structure stability of GDNF, and the α-helix, finger1 and finger2 regions were involved in receptor binding, while the N-terminus was not essential for the biological functions of GDNF.

The Cell Death-promoting Gene DP5, Which Interacts with the BCL2 Family, Is Induced during Neuronal Apoptosis Following Exposure to Amyloid β Protein

DP5, which contains a BH3 domain, was cloned as a neuronal apoptosis-inducing gene. To confirm that DP5 interacts with members of the Bcl-2 family, 293T cells were transiently co-transfected with DP5 and Bcl-xl cDNA constructs, and immunoprecipitation was carried out. The 30-kDa Bcl-xl was co-immunoprecipitated with Myc-tagged DP5, suggesting that DP5 physically interacts with Bcl-xl in mammalian cells. Previously, we reported that DP5 is induced during neuronal apoptosis in cultured sympathetic neurons. Here, we analyzed DP5 gene expression and the specific interaction of DP5 with Bcl-xl during neuronal death induced by amyloid-beta protein (A beta). DP5 mRNA was induced 6 h after treatment with A beta in cultured rat cortical neurons. The protein encoded by DP5 mRNA showed a specific interaction with Bcl-xl. Induction of DP5 gene expression was blocked by nifedipine, an inhibitor of L-type voltage-dependent calcium channels, and dantrolene, an inhibitor of calcium release from the endoplasmic reticulum. These results suggested that the induction of DP5 mRNA occurs downstream of the increase in cytosolic calcium concentration caused by A beta. Moreover, DP5 specifically interacts with Bcl-xl during neuronal apoptosis following exposure to A beta, and its binding could impair the survival-promoting activities of Bcl-xl. Thus, the induction of DP5 mRNA and the interaction of DP5 and Bcl-xl could play significant roles in neuronal degeneration following exposure to A beta.

Determinants of BDNF-induced hippocampal synaptic plasticity: role of the Trk B receptor and the kinetics of neurotrophin delivery.

The neurotrophins are a class of signaling molecules known for their growth and survival-promoting activities during neuronal development. Recent studies suggest that the neurotrophins, including brain-derived neurotrophic factor (BDNF), can also dramatically influence synaptic transmission in the adult hippocampus. The experiments described in this paper indicate that ability of BDNF to potentiate synaptic transmission in the hippocampus relies on functional Trk B receptors. Moreover, the rate at which BDNF is applied to hippocampal synapses is also a potent determinant of whether synaptic potentiation will result. Hippocampal slices perfused with BDNF at a very slow flow rate (e.g., < or = 25 ml/hr) did not show synaptic potentiation. Increasing the rate of BDNF application resulted in synaptic potentiation in which the magnitude and onset kinetics of the potentiation were determined by the rate of BDNF delivery. Immunocytochemical analysis of BDNF detected with confocal microscopy confirmed these electrophysiological observations, indicating that the penetration of BDNF into hippocampal slices is influenced dramatically by the perfusion rate.

Human CNTF and related cytokines: effects on DRG neurone survival

Ciliary neurotrophic factor (CNTF), leukaemia inhibitory factor (LIF), oncostatin M (OSM), interleukin-6 (IL-6), and interleukin-11 (IL-11) are structurally and functionally related cytokines. We compared their survival-promoting activities on embryonic chick and newborn rat dorsal root ganglion (DRG) neurones. Human CNTF showed the well known trophic effect on both chick and rat DRG neurones. Human and murine LIF and, at unphysiologically high doses, human OSM were trophic for rat neurones, but failed to promote chick DRG cell survival. Human IL-11, murine IL-6 and human IL-6 did not improve chick or rat DRG neurone survival; soluble human IL-6 receptor α did not increase sensitivity to human IL-6. Thus, human CNTF as well as murine and human LIF had special neurotrophic properties compared with other related cytokines.

Human CNTF and related cytokines

Ciliary neurotrophic factor (CNTF), leukaemia inhibitory factor (LIF), oncostatin M (OSM), interleukin-6 (IL-6), and interleukin-11 (IL-11) are structurally and functionally related cytokines. We compared their survival-promoting activities on embryonic chick and newborn rat dorsal root ganglion (DRG) neurones. Human CNTF showed the well known trophic effect on both chick and rat DRG neurones. Human and murine LIF and, at unphysiologically high doses, human OSM were trophic for rat neurones, but failed to promote chick DRG cell survival. Human IL-11, murine IL-6 and human IL-6 did not improve chick or rat DRG neurone survival; soluble human IL-6 receptor alpha did not increase sensitivity to human IL-6. Thus, human CNTF as well as murine and human LIF had special neurotrophic properties compared with other related cytokines.

Effects of vitamin B 6 and its related compounds on survival of cultured brain neurons

The effects of pyridoxine and its derived cofacter, pyridoxal phosphate (PLP) on the survival of primary cultured neurons from fetal rat brain were investigated. Pyridoxine and PLP significantly promoted the neuronal survival of various brain regions in high cell density culture (10 5 cells/cm 2), but showed no positive effects on hippocampal neurons in low cell density culture (5 × 10 3 cells/cm 2). This neurotrophic effect of PLP was remarkably suppressed by picrotoxin and ifenprodil. Aminooxyacetic acid (AOAA), an inhibitor of PLP dependent enzymes, caused significant neuronal loss by itself, and largely counteracted the neurotrophic effect of PLP. Taken together, we presume that vitamin B 6 afforded the survival-promoting activities of cultured neurons by virtue of its crucial coenzymatic actions in the biosynthesis of putative neurotransmitters.

Photoreceptor survival-promoting activity in interphotoreceptor matrix preparations: Characterization and partial purification

It is well established that many types of cells are dependent on trophic factors for their survival. We are investigating whether photoreceptor survival may also be dependent upon such a factor, as well as the possibility that the interphotoreceptor matrix (IPM), which lies between the outer retina and the RPE, may be a source of photoreceptor survival-promoting activities. Well-characterized cultures of embryonic chick retinal neurons and photoreceptors, in which the photoreceptors spontaneously degenerate between 7 and 10 days in culture, were used to test this hypothesis. Crude extracts of IPM were found to support three- to fourfold increases in photoreceptor survival in 10-day cultures. This response was dose-dependent, saturable, and specific for photoreceptors, since there was no difference in the number of non-photoreceptor neurons between treated and control cultures. This photoreceptor survival-promoting activity (PSPA) may function when present either as a substratum-bound molecule or as a medium supplement. PSPA is heat labile, sensitive to freeze-thawing, and stable only within a very narrow pH range. The activity binds to heparin affinity columns but, in contrast to the behavior of growth factors such as FGF, it can be eluted from the columns at low salt concentrations (0·5 m NaCl). PSPA is eluted from a Sephacryl S-200 gel filtration column in two regions with M r = 400–450 kDa and 33 kDa, respectively. SDS-gel electrophoresis suggests that the high molecular weight activity is composed of aggregates of lower molecular weight molecules. While this factor has not been purified to homogeneity, the combination of heparin-agarose affinity chromatography and hydrophobic interaction chromatography on phenyl-Sepharose columns results in 720-fold purification of the activity, with one unit of PSPA activity corresponding to 40 ng protein. This partial purification is accompanied by substantial reduction in the complexity of protein patterns in silver-stained SDS-gels. These findings are potentially important since a macromolecular factor promoting photoreceptor survival could play a role in the normal development of the retina, and could also further our understanding of retinal degenerations such as retinitis pigmentosa.