Jens Strelau

Involvement of growth differentiation factor-15/macrophage inhibitory cytokine-1 (GDF-15/MIC-1) in oxLDL-induced apoptosis of human macrophages in vitro and in arteriosclerotic lesions.

Growth differentiation factor-15/macrophage inhibitory cytokine-1 (GDF-15/MIC-1) is a new member of the transforming growth factor beta (TGF-β) superfamily, which has most recently been found in activated macrophages (MΦ). We have now investigated GDF-15/MIC-1 in human MΦ after exposure to oxidized low-density lipoproteins (oxLDL) related mediators in vitro and in arteriosclerotic carotid arteries. Using RT-PCR and Western blotting a pronounced induction of GDF-15/MIC-1 expression by oxLDL, C6-ceramide, tumor necrosis factor (TNFα) and hydrogen peroxide (H2O2) was found in cultured human MΦ. In 11 human arteriosclerotic carotid arteries, immunohistochemical analyses supported by computer-assisted morphometry and regression analyses demonstrated a significant colocalization of GDF-15/MIC-1 immunoreactivity (IR) with oxLDL IR and manganese superoxide dismutase (MnSOD) IR in CD68 immunoreactive (ir) MΦ, which were also expressing AIF-IR (apoptosis-inducing factor), caspase-3-IR (CPP32), PARP-IR, c-Jun/AP-1-IR and p53-IR. Our data suggest that GDF-15/MIC-1 is inducible in human MΦ by oxLDL and its mediators in vitro and is supposed to contribute to oxidative stress dependent consequences in arteriosclerotic plaques, e.g. modulating apoptosis and inflammatory processes in activated MΦ.

ERK activation promotes neuronal degeneration predominantly through plasma membrane damage and independently of caspase-3

Our recent studies have shown that extracellular-regulated protein kinase (ERK) promotes cell death in cerebellar granule neurons (CGN) cultured in low potassium. Here we report that the “death” phenotypes of CGN after potassium withdrawal are heterogeneous, allowing the distinction between plasma membrane (PM)–, DNA-, and PM/DNA-damaged populations. These damaged neurons display nuclear condensation that precedes PM or DNA damage. Inhibition of ERK activation either by U0126 or by dominant-negative mitogen-activated protein kinase/ERK kinase (MEK) overexpression results in a dramatic reduction of PM damaged neurons and nuclear condensation. In contrast, overexpression of constitutively active MEK potentiates PM damage and nuclear condensation. ERK-promoted cellular damage is independent of caspase-3. Persistent active ERK translocates to the nucleus, whereas caspase-3 remains in the cytoplasm. Antioxidants that reduced ERK activation and PM damage showed no effect on caspase-3 activation or DNA damage. These data identify ERK as an important executor of neuronal damage involving a caspase-3–independent mechanism.

TGF-β and the regulation of neuron survival and death

Abstract Transforming growth factor-βs (TGF-βs) constitute a superfamily of multifunctional cytokines with important implications in morphogenesis, cell differentiation, and tissue remodeling. In the developing nervous system, TGF-β2 and -β3 occur in radial and astroglial cells as well as in many populations of postmitotic, differentiating neurons. TGF-β1 is restricted to the choroid plexus and meninges. In addition to functions related to glial cell maturation and performances, TGF-β2 and -β3 are important regulators of neuron survival. In contrast to neurotrophic factors, as for example, neurotrophins, TGF-βs are most likely not neurotrophic by themselves. However, they can dramatically increase the potency of select neurotrophins, fibroblast growth factor-2, ciliary neurotrophic factor, and glial cell line-derived neurotrophic factor (GDNF). In the case of GDNF, we have shown that GDNF fails to promote the survival of highly purified neuron populations in vitro unless it is supplemented with TGF-β. This also applies to the in vivo situation, where antibodies to all three TGF-β isoforms fully prevent the trophic effect of GDNF on axotomized, target-deprived neurons. In addition to the TGF-β isoforms -β2 and -β3, other members of the TGF-β superfamily are expressed in the nervous system having important roles in embryonic patterning, cell migration, and neuronal transmitter determination. We have cloned and expressed a novel TGF-β, named growth/differentiation factor-15 (GDF-15). GDF-15 is synthesized in the choroid plexus and released into the CSF, but also occurs in all regions investigated of the developing and adult brain. GDF-15 is a potent trophic factor for developing and 6-OHDA-lesioned midbrain dopaminergic neurons in vitro and in vivo, matching the potency of GDNF.

Expression of Growth Differentiation Factor-15/ Macrophage Inhibitory Cytokine-1 (GDF-15/MIC-1) in the Perinatal, Adult, and Injured Rat Brain

We and others have recently cloned a new member of the transforming growth factor‐β superfamily, growth differentiation factor‐15/ macrophage inhibitory cytokine‐1 (GDF‐15/MIC‐1). Using in situ hybridization and immunohistochemistry, we determined the distribution of GDF‐15/MIC‐1 mRNA and protein in the perinatal and cryolesioned adult rat brain. The choroid plexus epithelium of all ventricles represents the site of strongest and almost exclusive mRNA expression in the normal perinatal and adult brain. The newborn rat brain reveals GDF‐15/MIC‐1 immunoreactivity (ir) in ependymal cells lining the ventricles, in the striatal subventricular zone, and in populations of nonneural cells of the thalamic/hippocampal lamina affixa, in addition to that in the choroid plexus. Unilateral cryogenic cortical lesioning induced a significant increase of GDF‐15/MIC‐1 mRNA expression and ir at the lesion site and expression in presumed neurons within the dorsal thalamic area. At the lesion site, GDF‐15/MIC‐1‐producing cells showed immuncytochemical features of neurons, macrophages, and activated microglial cells. Flourescent microscopy revealed both intra‐ and extracellular GDF‐15/MIC‐1 ir. Up‐regulation of GDF‐15/MIC‐1 in activated macrophages (Mϕ) is also supported by RT‐PCR, ICC, and Western blot experiments showing pronounced induction of GDF‐15/MIC‐1 expression (mRNA and protein) in retinoic acid/phorbol ester‐stimulated human Mϕ. Our data suggest that 1) GDF‐15/MIC‐1 is secreted into the cerebrospinal fluid and 2) in the newborn brain may penetrate through the ependymal lining and act on developing neurons and/or glial cells. As a constituent of cells in the lamina affixa, the protein might be involved in the regulation of mesenchyme–epithelial interactions. Finally, GDF‐15/MIC‐1 may also act within the antiinflammatory cytokine network activated in CNS lesions. J. Comp. Neurol. 439:32–45, 2001.

Insulin-Like Growth Factor 1 Inhibits Extracellular Signal-Regulated Kinase to Promote Neuronal Survival via the Phosphatidylinositol 3-Kinase/Protein Kinase A/c-Raf Pathway

Extracellular signal-regulated kinase (ERK) activation has been shown to promote neuronal death in various paradigms. We demonstrated previously that the late and sustained ERK activation in cerebellar granule neurons (CGNs) cultured in low potassium predominantly promotes plasma membrane (PM) damage. Here, we examined the effects of a well established neuronal survival factor, insulin-like growth factor 1 (IGF-1), on the ERK cell death pathway. Stimulation of CGNs with IGF-1 induced an early and transient ERK activation but abrogated the appearance of late and sustained ERK. Withdrawal or readdition of IGF-1 after 4 h in low potassium failed to prevent sustained ERK activation and cell death. IGF-1 activated the protein kinase A (PKA) to mediate ERK inhibition via c-Raf phosphorylation at an inhibitory site (Ser259). Phosphatidylinositol 3-kinase (PI3K) or PKA inhibitors, but not a specific Akt inhibitor, abrogated PKA signaling. This suggests that the PI3K/PKA/c-Raf-Ser259 pathway mediates ERK inhibition by IGF-1 independent of Akt. In addition, adenoviral-mediated expression of constitutively active MEK (mitogen-activated protein kinase kinase) or Sindbis viral-mediated expression of mutant Raf Ser259Ala both attenuated IGF-1-mediated prevention of PM damage. Activation of caspase-3 promoted DNA damage. Its inhibition by IGF-1 was both PI3K and Akt dependent but PKA independent. 8-Br-cAMP, an activator of PKA, induced phosphorylation of c-Raf-Ser259 and inhibited ERK activation without affecting caspase-3. This indicates a selective role for PKA in ERK inhibition through c-Raf-Ser259 phosphorylation. Together, these data demonstrate that IGF-1 can positively and negatively regulate the ERK pathway in the same neuronal cell, and provide new insights into the PI3K/Akt/PKA signaling pathways in IGF-1-mediated neuronal survival.

The murine growth differentiation factor 15 is not essential for systemic iron homeostasis in phlebotomized mice

In conditions of increased erythropoiesis, expression of hepcidin, the master regulator of systemic iron homeostasis, is decreased to allow for the release of iron into the blood stream from duodenal enterocytes and macrophages. It has been suggested that hepcidin suppression is controlled by growth differentiation factor 15 (GDF15), a member of the transforming growth factor-β superfamily of cytokines that is secreted from developing erythroblasts. In this study, we analyzed iron-related parameters in mice deficient for GDF15 under steady-state conditions and in response to increased erythropoietic activity induced by blood loss. We demonstrate that GDF15 suppresses the hepatic mRNA expression of some BMP/TGFβ target genes but not of hepcidin, and show that GDF15 is not required to balance iron homeostasis in response to blood loss.

Progressive Postnatal Motoneuron Loss in Mice Lacking GDF-15

Growth/differentiation factor-15 (GDF-15) is a widely expressed distant member of the TGF-β superfamily with prominent neurotrophic effects on midbrain dopaminergic neurons. We show here that GDF-15-deficient mice exhibit progressive postnatal losses of spinal, facial, and trigeminal motoneurons. This deficit reaches a ∼20% maximum at 6 months and is accompanied by losses of motor axons and significant impairment of rotarod skills. Similarly, sensory neurons in dorsal root ganglia (L4, L5) are reduced by 20%, whereas sympathetic neurons are not affected. GDF-15 is expressed and secreted by Schwann cells, retrogradely transported along adult sciatic nerve axons, and promotes survival of axotomized facial neurons as well as cultured motor, sensory, and sympathetic neurons. Despite striking similarities in the GDF-15 and CNTF knock-out phenotypes, expression levels of CNTF and other neurotrophic factors in the sciatic nerve were unaltered suggesting that GDF-15 is a genuine novel trophic factor for motor and sensory neurons.

GDNF family members and their receptors : Expression and functions in two oligodendroglial cell lines representing distinct stages of oligodendroglial development

Glial cell line‐derived neurotrophic factor (GDNF), neurturin (NTN), and persephin (PSP) constitute a subfamily of transforming growth factor‐βs (TGF‐βs) with prominent roles in the regulation of neuron survival and differentiation. Although numerous members of the TGF‐β superfamily are important regulators of glial cell functions in health and disease, it is unknown whether any member of the GDNF subfamily may have functions in normal or pathological glial cell performances. To begin to address this issue, we have studied expression and putative functions of GDNF, NTN, PSP, and their receptors in two cell lines representing models for oligodendrocyte progenitor cells (OLI‐neu) and immature oligodendrocytes (OLN‐93), respectively. RT‐PCR analysis revealed expression of all three growth factor mRNAs in OLI‐neu and OLN‐93 cells. Expression was weak in OLI‐neu cells, while both NTN and PSP mRNAs were strongly expressed in OLN‐93 cells. Furthermore, OLI‐neu and OLN‐93 cells expressed transcripts encoding the GDNF receptors Ret and GFRα‐1. The two splice variants for GFRα‐2 were exclusively synthesized in OLI‐neu cells. Similarly, primary O‐2A progenitor cells and enriched mature oligodendrocytes expressed Ret, GFRα‐1 and GFRα‐2 mRNAs. Both GDNF and NTN stimulated DNA synthesis monitored by BrdU incorporation of OLI‐neu cells in a dose‐dependent fashion. Co‐administration of TGF‐β significantly reduced this effect. Similarly, PDGF co‐applied with GDNF or NTN down‐regulated proliferation in OLI‐neu cells. In contrast, OLN‐93 cells did not respond to GDNF or NTN with increased incorporation of BrdU. Expression of GDNF, NTN, and their receptors and distinct effects in two model cell lines of oligodendrocyte development suggest that functions of members of the GDNF family and their receptors may not be restricted to neurons and may be implicated in oligodendrocyte development. GLIA 26:291–301, 1999.

GDF-15/MIC-1 a novel member of the TGF-beta superfamily.

We have cloned, expressed, and raised antibodies against a novel member of the TGF-beta superfamily, growth/differentiation factor-15 (GDF-15). The predicted protein is identical to macrophage inhibitory cytokine-1 (MIC-1), which was discovered simultaneously. GDF-15 is a more distant member of the TGF-beta superfamily and does not belong to one of the known TGF-beta subfamilies. In the CNS, GDF-15/MIC-1 mRNA is abundantly expressed by the choroid plexus. In addition we have preliminary evidence that GDF-15/MIC-1 is a potent trophic factor for selected classes of neurons in vitro and in vivo. Thus, GDF-15 is a novel neurotrophic factor with prospects for the treatment of disorders of the CNS.

Growth differentiation factor-15 deficiency inhibits atherosclerosis progression by regulating interleukin-6-dependent inflammatory response to vascular injury.

Background Growth differentiation factor (GDF)‐15 is a distant and divergent member of the transforming growth factor‐β superfamily (TGF‐β) . There is growing evidence indicating the involvement of GDF‐15 in various pathologies. Expression of GDF‐15 is induced under conditions of inflammation and increased GDF‐15 serum levels are suggested as a risk factor for cardiovascular diseases. Methods and Results We show here that GDF‐15 and proinflammatory cytokine interleukin (IL)‐6 levels are highly increased (5‐fold) in cultured oxidized low‐density lipoproteins–stimulated peritoneal macrophages derived from GDF‐15+/+/apolipoprotein (apo) E−/−, mice. Notably, IL‐6 induction on oxidized low‐density lipoproteins stimulation is completely abolished in the absence of GDF‐15. Consistent with our in vitro data GDF‐15 mRNA expression and protein levels are upregulated (2.5‐ to 6‐fold) in the atherosclerotic vessel wall of GDF‐15+/+/apoE−/− mice after a cholesterol‐enriched diet. GDF‐15 deficiency inhibits lumen stenosis (52%) and 18FDG uptake (34%) in the aortic arch despite increased serum triglyceride/cholesterol levels and elevated body weight. Immunohistomorphometric investigations of atherosclerotic lesions reveal a decreased percentage of inflammatory CD11b+ (57%) or IL‐6+, leukocytes, and apoptotic cells (74%) after 20 weeks. However, the total number of macrophages and cell density in atherosclerotic lesions of the innominate artery are increased in GDF‐15−/−/apoE−/− mice. Conclusions Our data suggest that GDF‐15 is involved in orchestrating atherosclerotic lesion progression by regulating apoptotic cell death and IL‐6–dependent inflammatory responses to vascular injury.