Necrotic cell-derived high mobility group box 1 attracts antigen-presenting cells but inhibits hepatocyte growth factor-mediated tropism of mesenchymal stem cells for apoptotic cell death.

Tissue damage due to apoptotic or necrotic cell death typically initiates distinct cellular responses, leading either directly to tissue repair and regeneration or to immunological processes first, to clear the site, for example, of potentially damage-inducing agents. Mesenchymal stem cells (MSC) as well as immature dendritic cells (iDC) and monocytes migrate to injured tissues. MSC have regenerative capacity, whereas monocytes and iDC have a critical role in inflammation and induction of immune responses, including autoimmunity after tissue damage. Here, we investigated the influence of apoptotic and necrotic cell death on recruitment of MSC, monocytes and iDC, and identified hepatocyte growth factor (HGF) and the alarmin high mobility group box 1 (HMGB1) as key factors differentially regulating these migratory responses. MSC, but not monocytes or iDC, were attracted by apoptotic cardiomyocytic and neuronal cells, whereas necrosis induced migration of monocytes and iDC, but not of MSC. Only apoptotic cell death resulted in HGF production and HGF-mediated migration of MSC towards the apoptotic targets. In contrast, HMGB1 was predominantly released by the necrotic cells and mediated recruitment of monocytes and iDC via the receptor of advanced glycation end products. Moreover, necrotic cardiomyocytic and neuronal cells caused an HMGB1/toll-like receptor-4-dependent inhibition of MSC migration towards apoptosis or HGF, while recruitment of monocytes and iDC by necrosis or HMGB1 was not affected by apoptotic cells or HGF. Thus, the type of cell death differentially regulates recruitment of either MSC or monocytes and iDC through HGF and HMGB1, respectively, with a dominant, HMGB1-mediated role of necrosis in determining tropism after tissue injury.

Future of cord blood for non-oncology uses.

For the last 5 years cord blood (CB) has been under intense experimental investigation in in vitro differentiation models and in preclinical animal models ranging from bone to muscle regeneration, cardiovascular diseases including myocardial and peripheral arterial disease, stroke and Parkinson's disease. On the basis of its biological advantages, CB can be an ideal source for tissue regeneration. However, in the hype of the so-called 'plasticity', many cell types have been characterized either on cell surface Ag expression alone or by RNA expression only, and without detailed characterization of genetic pathways; frequently, cells are defined without analysis of cellular function in vitro and in vivo, and the definition of the lineage of origin and cells have not been defined in preclinical studies. Here, we explore not only the most consistent data with regard to differentiation of CB cells in vitro and in vivo, but also show technical limitations, such as why in contrast to cell populations isolated from fresh CB, cryopreserved CB is not the ideal source for tissue regeneration. By taking advantage of numerous CB units discarded due to lack of sufficient hematopoietic cells for clinical transplantation, new concepts to produce off-the-shelf products are presented as well.

In vitro differentiation of human cord blood-derived unrestricted somatic stem cells towards an endodermal pathway.

BACKGROUND: Pluripotent unrestricted somatic stem cells (USSC) from UC blood can differentiate into hepatic cells in the in utero sheep model, resulting in 20% human albumin-producing parenchymal hepatic cells without cell fusion or tumor-formation events. Additionally, we have shown in vitro differentiation of USSC by hepatocyte growth factor and oncostatin M induction, causing changes in the gene expression towards the endodermal lineage. Positive glycogen synthase expression and a positive periodic acid-schiff reaction demonstrated a functional production of polysaccharides in the cells. METHODS: We describe the in vitro differentiation of USSC towards an endodermal pathway using different matrices, growth factors and organic substances. Also, co-cultures of USSC with primary cells of endodermal tissue were prepared to mimic the biologic niche. We investigated the effect of direct co-culture of USSC with primary rat hepatocytes or with sheep tissue of endodermal origin. Direct co-cultures were set up to ensure cell-cell contacts. For co-cultures without cell-cell contacts, transwell inlays with 1-microm membranes were used to separate the cells. Furthermore, the effect of endodermally conditioned medium was investigated. Changes in the gene expression patterns were analyzed by RT-PCR. RESULTS: We have shown that USSC can differentiate in vitro into an endodermal-like cell with a phenotype similar to hepatic cells. Differentiation of USSC with growth factors, retinoic acid, matrigel matrix and different co-cultures led to an increased expression of albumin and also to the detection of GSC, SOX 17, Cyp2B6, Cyp3A4, Gys2, HNF4a, ISL-1 and Nkx6.1. In addition, functional albumin secretion was observed. DISCUSSION: Although the differentiation assays demonstrated here produce only an immature hepatocyte-like cell, endodermaly differentiated USSC might be a useful alternative for cell replacement in the future.

Statins potentiate caspase-3 activity in immortalized murine neurons.

Statins are lipid-lowering drugs that have been shown to reduce atherosclerotic cardiovascular morbidity and mortality. However, there is growing evidence from epidemiological studies that long-term treatment with statins has unwanted effects on extrahepatic tissue and increases the risk for neuropathy. To investigate underlying molecular mechanisms we analyzed whether statins influence the activity of caspase-3 in immortalized neurons. Lovastatin and mevastatin are not able to activate caspase-3 but they strongly potentiate its activity when apoptotic signal transduction is initiated by staurosporine. The increase in caspase-3 activity after coincubation with statins and staurosporine was paralleled by an increase in the protein level of the pro-apoptotic GTPase RhoB. Our data provide evidence that statins enhance neuronal apoptosis and therefore give reasons for a careful evaluation when patients with neurological diseases are treated with these drugs.

GTPase RhoB: an early predictor of neuronal death after transient focal ischemia in mice.

Applying the recently developed DNA array technique to a murine stroke model, we found that the gene coding for RhoB, a member of the family of GTPases that regulate a variety of signal transduction pathways, is upregulated in ischemia-damaged neurons. RhoB immunoreactivity precedes DNA single-strand breaks and heralds the evolving infarct, making it an early predictor of neuronal death. Expression of RhoB colocalized with drastic rearrangement of the actin cytoarchitecture indicates a role for Rho in postischemic morphological changes. Apoptosis in a murine hippocampal cell line was also associated with an early increase in RhoB protein. Activation of caspase-3, a crucial step in apoptosis, could be inhibited by cytochalasin D, a substance that counteracts the actin-modulating activity of Rho GTPases, indicating that Rho proteins may have impact on injury-initiated neuronal signal transduction. Our findings make Rho GTPases potential targets for the development of drugs aimed at limiting neuronal death following brain damage.

Protection against oxidative stress-induced neuronal cell death--a novel role for RU486.

Free radicals and oxidative stress-induced neuronal cell death have been implicated in a variety of neurological disorders. Therefore, neuroprotection is of primary interest in basic and preclinical neuroscience. Here it is shown that RU486 (mifepristone), a potent antagonist of progesterone and glucocorticoid receptors, protects rat primary hippocampal neurons, clonal mouse hippocampal cells and organotypic hippocampal slice cultures against oxidative stress-induced neuronal cell death. 10(-5) M RU486 prevents intracellular peroxide accumulation and cell death induced by amyloid beta protein, hydrogen peroxide and glutamate, neurotoxins that have been implicated in certain neurodegenerative disorders, including Alzheimer's disease. RU486 has a significant protective effect that is independent of the presence and activation of glucocorticoid or progesterone receptors. The neuroprotective activity of this well-studied drug may have an impact on therapeutic interventions for neurodegenerative conditions which involve peroxidation processes, such as stroke and Alzheimer's disease.

A classic cAMP responsive element in the promoter region of the alpha 1-GABAA receptor subunit has non-classic properties.

The cAMP responsive element binding protein (CREB) and the glucocorticoid receptor (GR) have been reported to bind to a 60 bp promoter fragment of the alpha 1-GABAA receptor gene containing a classic cAMP-responsive element (CRE). We inserted this fragment into a hormone responsive element-deleted mouse mammary tumor virus promoter controlling the expression of luciferase. Activation of GR showed no significant change in luciferase expression, but hormone induction by forskolin revealed a reduction in neuronal cell lines. Furthermore, we demonstrate that cellular factors from neuronal cells can bind to the CRE-containing promoter fragment, although competition by unlabeled CRE and GRE oligo-nucleotides is not present. Mutation of the CRE site and deletion of neighboring DNA sequences indicate that the promoter is probably associated with a complex of different regulatory factors.

Glucocorticoids enhance oxidative stress-induced cell death in hippocampal neurons in vitro.

In patients with Alzheimer's disease, hippocampal cells are among the first neuronal cells of the brain to degenerate. Both rat primary hippocampal neurons and cells of the clonal mouse hippocampal cell line HT22 express endogenous functional glucocorticoid receptors (GRs), as shown by transient transfection of cells with a luciferase reporter plasmid containing GR-responsive elements. The influence of activated GRs on oxidative stress-induced neuronal cell death in vitro was investigated employing these hippocampal model systems. Two oxidative stressors were investigated, the free radical-inducing Alzheimer's disease-associated amyloid beta-protein, which is toxic to hippocampal neurons, and the excitatory amino acid glutamate, which induces oxidative cell death in HT22 cells via an increase in intracellular peroxides. Cellular viability was assessed with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide test and trypan exclusion staining, followed by microscopical cell counting. Glucocorticoids strongly increased the vulnerability of the hippocampal cells to amyloid beta-protein and glutamate. This increase could be blocked by the specific GR antagonist RU486. Our data suggest that changes in hippocampal GR homeostasis and regulation may render hippocampal neurons more vulnerable to oxidative stress-induced neuronal degeneration.

Molecular and functional evidence for in vitro cytokine enhancement of human and murine target cell sensitivity to glucocorticoids. TNF-alpha priming increases glucocorticoid inhibition of TNF-alpha-induced cytotoxicity/apoptosis.

Cytokine-induced glucocorticoid secretion and glucocorticoid inhibition of cytokine synthesis and pleiotropic actions act as important safeguards in preventing cytokine overreaction. We found that TNF-alpha increased glucocorticoid-induced transcriptional activity of the glucocorticoid receptor (GR) via the glucocorticoid response elements (GRE) in L-929 mouse fibroblasts transfected with a glucocorticoid-inducible reporter plasmid. In addition, TNF-alpha also enhanced GR number. The TNF-alpha effect on transcriptional activity was absent in other cell lines that express TNF-alpha receptors but not GRs, and became manifest when a GR expression vector was cotransfected, indicating that TNF-alpha, independent of any effect it may have on GR number, has a stimulatory effect on the glucocorticoid-induced transcriptional activity of the GR. Moreover, TNF-alpha increased GR binding to GRE. As a functional biological correlate of this mechanism, priming of L-929 cells with a low (noncytotoxic) dose of TNF-alpha significantly increased the sensitivity to glucocorticoid inhibition of TNF-alpha-induced cytotoxicity/apoptosis. TNF-alpha and IL-1 beta had the same stimulatory action on glucocorticoid-induced transcriptional activity of the GR via the GRE, in different types of cytokine/glucocorticoid target cells (glioma, pituitary, epithelioid). The phenomenon may therefore reflect a general molecular mechanism whereby cytokines modulate the transcriptional activity of the GR, thus potentiating the counterregulation by glucocorticoids at the level of their target cells.

Severe spontaneous carotid artery dissection and multiple aneurysmal dilatations. A case report.

Spontaneous cervical artery dissections or arterial aneurysms associated with deficiencies of alpha(1)-antitrypsin (alpha(1)-AT) or other inhibitors of proteolytic enzymes have occasionally been reported. However, a coexistence of severe spontaneous internal carotid artery dissection and multiple aneurysmal dilatations associated with alpha(1)-AT phenotype M1S have not yet been presented; herein the authors describe such a patient. In order to avoid the risks associated with intraarterial angiography in a patient in whom an underlying arteriopathy is suspected, only noninvasive techniques were employed. This case demonstrates that magnetic resonance imaging combined with magnetic resonance angiography is a valuable noninvasive method for use in diagnosis and follow-up of carotid artery dissection.

Nuclear orphan receptor as a repressor of glucocorticoid receptor transcriptional activity.

Nuclear orphan receptors belong to the superfamily of ligand-activated transcription factors that show a close structural relationship and sequence homology. Ligands and functions of most of the orphan receptors have not yet been identified. The first nuclear orphan receptors that were cloned displayed a high degree of amino acid identity with the human estrogen receptor and were termed estrogen receptor-related (ERR) 1 and 2. In the present study, we show that ERR2 functions as a potent repressor of transcriptional activity mediated by the glucocorticoid receptor (GR). Transient transfection of different cell lines with a steroid-responsive reporter plasmid and receptor expression plasmids revealed that transcriptional activity mediated by GR in response to agonists was strongly suppressed by coexpression of ERR2. The orphan receptor displayed no promoter activity when expressed without GR. The inhibitory activity of ERR2 is cell-specific and also receptor-specific because transactivation mediated by the progesterone receptor is unaffected by ERR2. Our observations provide evidence that the nuclear orphan receptor ERR2 acts as an endogenous modulator of GR transcriptional activity.

Heterodimerization between mineralocorticoid and glucocorticoid receptors increases the functional diversity of corticosteroid action.

Gene regulation by steroids is mediated by the binding of the endogenous or pharmacological ligand to the corresponding nuclear receptor. Ligand-activated steroid receptors usually regulate the expression of responsive genes by binding to common response elements on DNA as homodimers. However, recent findings indicate that mineralocorticoid and glucocorticoid receptors are able to interact by forming heterodimers. In tissues coexpressing both of these corticosteroid receptors, heterodimerization between them may be a hitherto unrecognized modality for the transcriptional regulation of corticosteroid-responsive genes. In this review, Thorsten Trapp and Florian Holsboer discuss the potential impact of this heterodimerization on corticosteriod physiology and pharmacology.

Neurosteroids: molecular mechanisms of action and psychopharmacological significance.

In addition to the well-known genomic effects of steroid molecules via intracellular steroid receptors, certain steroids rapidly alter neuronal excitability through binding sites on neurotransmitter-gated ion channels. Several of these steroids accumulate in the brain after local synthesis or after metabolization of adrenal steroids. The 3 alpha-hydroxy ring A-reduced pregnane steroids allopregnanolone and tetrahydrodeoxycorticosterone have been thought not to interact with intracellular receptors but enhance gamma-aminobutyric acid (GABA)-medicated chloride currents. When administered systematically in the rat, these neurosteroids display anxiolytic and hypnotic activities that suggest pronounced systemic effects as well as neuropsychopharmacological potential for modulation of sleep and anxiety. We demonstrated that these neurosteroids can regulate gene expression via the progesterone receptor. The induction of DNA-binding and transcriptional activation of the progesterone receptor requires intracellular oxidation of the neurosteroids into progesterone receptor-active 5 alpha-pregnane steroids. Thus, in physiological concentrations these neurosteroids regulate neuronal function through their concurrent influence on transmitter-gated ion channels and gene expression. These findings extend the concept of a "cross-talk" between membrane and nuclear hormone effects and provide a new role for the therapeutic application of these steroids in neurology and psychiatry.

17-beta estradiol protects neurons from oxidative stress-induced cell death in vitro.

The potential antioxidant activity of 17-beta estradiol and other steroid hormones in neuronal cells was investigated by studying oxidative stress-induced cell death caused by the neurotoxins amyloid beta protein, hydrogen peroxide and glutamate in the clonal mouse hippocampal cell line HT22. Preincubation of the cells with 10(-5) M 17-beta estradiol prior to addition of the neurotoxins prevented oxidative stress-induced cell damage and ultimately cell death, as detected with cell viability (MTT) and cell lysis (trypan blue exclusion/cell counting; propidium iodide staining) assays. At the DNA level, 17-beta estradiol blocked the DNA degradation caused by glutamate. Other steroid hormones, such as progesterone, aldosterone, corticosterone and the steroid precursor cholesterol, did not protect the cells. The neuronal protection afforded by 17-beta estradiol was estrogen receptor-independent. These data demonstrate a potent neuroprotective activity of the antioxidant 17-beta estradiol, which may have implications for the prevention and treatment of Alzheimer's disease.

Ligand-induced conformational changes in the mineralocorticoid receptor analyzed by protease mapping.

The human mineralocorticoid receptor (MR) binds the agonists aldosterone and cortisol and the antagonist progesterone with a comparably high affinity. We used limited proteolysis of human MR synthesized by in vitro translation to detect structural alterations induced by these different endogenous ligands. Steroid binding induces a conformational change within the receptor protein. This structural alteration renders a fragment of MR resistant to proteolysis. Agonists and antagonist vary in how well they protect the MR fragment against proteolysis. But the two agonists also differ in their ability to protect, indicating that agonists and antagonists, but also different agonists, may induce distinct conformational changes. Ligand-independent removal of MR-associated heat-shock proteins induces no detectable structural change but completely prevents ligand binding of MR.

Regulation of rat mineralocorticoid receptor expression in neurons by progesterone.

We have studied the effects of progesterone on the transcription of the mineralocorticoid receptor (MR) gene in neurons in vitro and in vivo. Progesterone treatment caused a 2.5-fold increase in activity of the MR promoter in transiently transfected N2A neuroblastoma cells. Similarly, MR promoter activity in GH3 pituitary cells was increased 2-fold after treatment with the specific progesterone receptor agonist R5020, with an even greater induction after priming with 17 beta-estradiol. Progesterone treatment also produced a dose-dependent increase in MR messenger RNA (mRNA) levels in primary hippocampal neuron cultures. In vivo, chronic administration of progesterone to estrogen-primed adrenalectomized/ovariectomized rats significantly increased MR mRNA levels in all hippocampal subfields, as determined by semiquantitative in situ hybridization histochemistry. Whereas chronic estradiol treatment decreased MR mRNA levels in the hippocampus, progesterone administration in the absence of estradiol priming was without any effect. These results indicate that 1) progesterone increases MR mRNA levels in vitro and in vivo; 2) the stimulatory effects of progesterone are at least partially mediated by induction of MR promoter activity; and 3) estrogen priming is essential for the effect of progesterone upon MR mRNA in vivo. Further, they suggest the possibility of heterologous regulation of corticosteroid receptors in the brain, whereby the responsiveness of the limbic-hypothalamo-pituitary-adrenal system to corticosteroids may be modulated.

Transcriptional induction of rat mineralocorticoid receptor gene in neurones by corticosteroids.

We investigated the mechanisms by which corticosteroids regulate the expression of the mineralocorticoid receptor (MR) in neurones. Aldosterone and dexamethasone produced a dose-dependent increase of MR and mRNA levels in cultured primary hippocampal neurones. Transient transfection of neuroblastoma cells showed that corticosteroids directly activate the rat MR promoter, indicating that the steroid-induced increase in the MR mRNA concentration is at least partially transcriptional. Progressive 5' deletions of the MR promoter sequence revealed that the promoter induction cannot be assigned to a single element. An oligonucleotide comprising a consensus half-glucocorticoid responsive element located at -319 bp in the MR promoter stimulated the corticosteroid-induced activation of the heterologous promoter. Cloning three of these enhancers in tandem greatly potentiated the responses to glucocorticoids and mineralocorticoids, suggesting that although this element is a weak enhancer it can, in combination with other enhancer elements, induce MR gene expression by both types of corticosteroid receptors.

Heterodimerization between mineralocorticoid and glucocorticoid receptor: a new principle of glucocorticoid action in the CNS.

In the mammalian central nervous system, responsiveness to glucocorticoids is mediated by both the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR). These pharmacologically distinct receptors are believed to bind to common response elements as homodimers. We provide evidence that MR and GR can form a heterodimeric complex with DNA-binding and transactivation properties different from those of the respective homodimers. There was a high degree of cooperativity of MR and GR in binding to a glucocorticoid response element. Transient transfection of a neuroblastoma cell line revealed a transcriptional response pattern of coexpressed MR and GR distinct from that obtained by MR or GR alone. Our findings demonstrate that heterodimerization of MR and GR is a hitherto unrecognized principle for the transcriptional regulation of glucocorticoid-responsive genes in tissue coexpressing these receptors.

The oestrogen receptor modulates growth of pituitary tumour cells in the absence of exogenous oestrogen.

The GH3 pituitary cell line has been used to investigate the role of the oestrogen receptor (ER) as a modulator of mitogenic signals in tumour cells in the absence of exogenous oestrogen. Using a chemically defined, serum- and oestrogen-free medium, we have demonstrated that the pure steroidal anti-oestrogens ICI 182780 and ICI 164384 are capable of blocking growth by more than 50% after 5 days of culture. Studies with conditioned medium have indicated that the basal growth is due to the secretion of autocrine growth stimulatory substances. Under serum- and oestrogen-free conditions, insulin and IGF-I increased the growth rate of these cells by twofold over a 5-day treatment period, and this effect was also blocked by the anti-oestrogens ICI 182780 and ICI 164384 (50% of maximum inhibition at 0.6 and 6 nM respectively). To explore the potential mechanism by which the ER apparently facilitates the growth factor effects under oestrogen-free conditions, GH3 cells were transiently transfected with a plasmid reporter containing the vitellogenin oestrogen response element (delta MTV-ERE-LUC). We have shown that as well as oestradiol (OE2), insulin and IGF-I induce luciferase activity by between two- and sevenfold (four experiments), and these effects were completely blocked by ICI 182780. In contrast, growth factors and OE2 were unable to induce luciferase expression when transfections were performed with a plasmid reporter lacking the oestrogen response element. The studies presented here strongly suggest that, in the absence of oestrogen, the ER in these pituitary tumour cells has a role in growth, as peptide factors are able to induce its conversion to a state which is capable of up-regulating the transcription of key growth-promoting genes.

The unliganded estrogen receptor (ER) transduces growth factor signals.

In the absence of serum and estrogen, we show that the growth of the prolactin secreting pituitary tumour cell line, GH3 is stimulated by insulin and insulin-like growth factor-1 (IGF-1) and this response is blocked by the steroidal antiestrogens, ICI 164384 and ICI 182780. From conditioned medium (CM) experiments, growth of low density cells (10k/cm2) is increased by the addition of CM from high density cells (100k/cm2) and this growth effect is also blocked by antiestrogen. Transfection studies with a delta MTV-ERE-LUC reporter plasmid show that in the absence of estrogen and serum, both insulin and IGF-1 induce luciferase expression and this is blocked by the pure antiestrogens. No effect of these treatments was apparent when parallel experiments were conducted with a plasmid construct lacking the vitellogenin estrogen response element. From these and other data discussed in this report, we conclude that for GH3 cells, in the absence of estrogen and serum, the ER is transcriptionally activated by intracellular peptide factor pathways and by this means, acts as the key nuclear factor inducing mitogenesis in response to autocrine and exogenously added growth factors.