Clemens Suter-Crazzolara

Expression of a novel member of the TGF-beta superfamily, growth/differentiation factor-15/macrophage-inhibiting cytokine-1 (GDF-15/MIC-1) in adult rat tissues.

Abstract We have cloned a novel member of the transforming growth factor-β (TGF-β) superfamily from a human placental cDNA library. The sequence is identical to five very recently published sequences, of which only one (macrophage inhibitory cytokine-1, MIC-1) has been characterized in terms of function. In light of the present data demonstrating the wide distribution of the mRNA and putative multifunctionality, we propose to name this molecule growth/differentiation factor-15/MIC-1 (GDF-15/MIC-1). The deduced amino acid sequence reveals typical features of a secreted molecule. The epithelium of the choroid plexus is the only site in the adult brain expressing detectable levels of GDF-15/MIC-1 mRNA. Many epithelia of non-neural tissues including those of the prostate and intestinal mucosa, bronchi and bronchioli, secretory tubuli of the submandibular gland, and lactating mammary gland are prominent sites of GDF-15/MIC-1 synthesis. GDF-15/MIC-1 is also strongly expressed by macrophages in the adrenal gland. Thus, GDF-15/MIC-1, like many other members of the TGF-β superfamily, is widely distributed in adult tissues, being most strongly expressed in epithelial cells and macrophages.

Characterization of the rat, mouse, and human genes of growth/differentiation factor-15/macrophage inhibiting cytokine-1 (GDF-15/MIC-1)

We have isolated the rat, mouse and human genes of a distant member of the TGF-beta superfamily, growth/differentiation factor-15/macrophage inhibiting cytokine-1 (GDF-15/MIC-1) by screening of genomic libraries. All three genes are composed of two exons, and contain one single intron that interrupts the coding sequences at identical positions within the prepro-domain of the corresponding proteins. The predicted proteins contain the structural hallmarks of members of the TGF-beta superfamily, including the seven conserved carboxy-terminal cysteine residues that form the cystine knot. The orthologous molecules show the lowest sequence conservation of all members of the TGF-beta superfamily. RT-PCR reveals an abundant expression of GDF-15/MIC-1 mRNA in numerous embryonic and adult organs and tissues. Promoter analysis of the rat promoter indicates the presence of multiple regulatory elements, including a TATA-like sequence as well as several SP1, AP-1 and AP-2 sites. Deletion analysis suggests that a 350 bp region upstream of the start of the open reading frame appears to be the most important for regulation of transcription.

Gdnf is expressed in two forms in many tissues outside the Cns

A recently cloned neurotrophic factor, Glial Cell Line-Derived Neurotrophic Factor (GDNF), has been implicated in the survival and morphological and functional differentiation of midbrain dopaminergic neurones in vitro. GDNF has therefore been proposed as a factor which may have utility in the treatment of Parkinsons disease. In the present study, we have used RT-PCR to analyse the distribution of GDNF mRNA throughout the newborn rat (P0). We show that GDNF transcripts are present in kidney, lung, bone, heart, liver, spleen, sciatic nerve and blood. Two separate GDNF transcripts are present in different ratios in each tissue investigated. Sequence analysis of both these mRNA species revealed that the shorter transcript (sGDNF) contains a deletion of 78 bp in comparison to the published sequence for GDNF. We speculate that this shorter mRNA arose due to alternative splicing.

Neural functions of the transforming growth factors β

II. The TGF-13 isoforms and their functions in the nervous system 1. TGF-I3s are widely distributed in the developing and adult nervous system 2. TGF-13s control important events in neural development (a) TGF-13s govern early steps in morphogenesis and phenotypic decisions (b) TGF-13s and radial glial cell functions (c) TGF-I3s control proliferation of neural progenitors (d) TGF-13s control neuron survival (e) GF-I3s affect axon growth 3. TGF-I3s orchestrate the responses of the nervous system to lesions (a) TGF-13s co-operate with and determine the actions of other cytokines (b) Expression of TGF-13s is altered in neural lesions and neurodegenerative diseases (c) TGF-13s regulate astroglial cell proliferation and phenotype (d) TGF-13s and the microglial response (e) TGF-13s and the Schwann cell response (f) TGF-13s are potent neuroprotective agents 4. TGF-I3s in the treatment of neurological disorders

GDNF mRNA levels are induced by FGF-2 in rat C6 glioblastoma cells

Glia cell line-derived neurotrophic factor (GDNF), a recently cloned member of the transforming growth factor-beta (TGF-beta) superfamily, has been implicated in the survival, morphological and functional differentiation of midbrain dopaminergic neurons and motoneurons in vitro and in vivo. The factor may thus have utility in the treatment of various human neurodegenerative disorders. Mechanisms regulating expression of GDNF in normal and diseased brain as a possible means to increase the local availability of GDNF are only beginning to be explored. We have established and employed a competitive reverse transcriptase-polymerase chain reaction (RT-PCR) to study and compare levels of expression of GDNF mRNA in several cell types and to investigate its regulation. GDNF expression was clearly evident in primary cultured astrocytes, the glioma B49 and C6 cell, but less pronounced in the Schwannoma RN22 cell lines. Little or no signal could be observed in neuroblastoma cell lines (IMR32, LAN-1) or the pheochromocytoma cell line PC12, emphasizing the glial character of this factor. Using the C6 cell line we found that fibroblast growth factor-2 (FGF-2; bFGF) can increase GDNF mRNA levels, whereas FGF-1, platelet-derived growth factor (PDGF), and vasoactive intestinal polypeptide (VIP) are apparently ineffective. Several other factors (forskolin, kainic acid, triiodothyronine dexamethasone, GDNF, TGF-beta 1, and interleukin-6) appear to have slightly negative effects on GDNF mRNA levels at the concentrations tested. To further explore the relationship between FGF-2 and GDNF, we also addressed the question whether GDNF, like FGF-2, may have an effect on C6 cell proliferation. We conclude that (1) glial and glial tumor cells, rather than neuronal cell lines, express GDNF, (2) that FGF-2 has a prominent inductive effect on GDNF expression and (3) that GDNF stimulates C6 cell proliferation. Finally, these data suggest that neurotrophic actions of FGF-2 in mixed glial-neuronal cell cultures might be mediated in part by GDNF.

Fibroblast growth factor-2 requires glial-cell-line-derived neurotrophic factor for exerting its neuroprotective actions on glutamate-lesioned hippocampal neurons.

FGF-2 is a potent neurotrophic factor for several populations of CNS neurons and has been shown to protect hippocampal neurons from glutamate-induced cell death in vitro and in vivo. Mechanisms underlying the neurotrophic and protective actions of FGF-2 have been resolved only in part. Using glutamate-treated cultured hippocampal neurons we show that FGF-2 shares its neuroprotective capacity with GDNF. Hippocampal neurons express glial-cell-line-derived neurotrophic factor (GDNF), its receptors c-Ret and the lipid-anchored GDNF family receptor-alpha1 (GFRalpha-1), and the FGF receptor 1 (FGFR I). Neutralizing antibodies to GDNF abolish the neuroprotective effect of FGF-2. In support of the notion that GDNF is required to permit the protective effects of FGF-2 we find that FGF-2 up-regulates GDNF and GFRalpha-1 in hippocampal neurons. Furthermore, FGF-2-induced GDNF causes enhanced phosphorylation of c-Ret and the signaling components Akt and Erk. A putative downstream target of FGF-2 and GDNF are bcl-2 gene family members, whose mRNAs are differentially up-regulated by the two factors. Together, these data suggest that GDNF is an important protective factor for glutamate-lesioned hippocampal neurons and an essential mediator of the neuroprotective actions of FGF-2.

Expression of TGF-β type II receptor mRIMA in the CNS

MEMBERS of the TGF-β family have been described to bind to a heteromeric complex of two types of serine/threonine kinase receptors, named TβR-I and TβR-II. These receptor molecules are essential for TGF-β-specific signalling. Several type I and type II receptors have been identified by a variety of methods. TGF-β 2 and 3 are widely expressed in the CNS and exert multiple functions on neurones and glia. Although the TβR-I molecule is abundant in the CNS, it was unclear whether the type II receptor found in peripheral organs is also expressed in the CNS. Previous, negative findings, seemed to suggest that a novel, as yet undescribed type II receptor may be expressed in the CNS. We used competitive RT-PCR to detect TβR-II mRNA in rats at different stages of development (E18, P6, adult) and in different tissues. We detected this mRNA in the lung, liver, heart, gut, kidney, and pituitary of adult rats. Surprisingly, and in contrast to previous studies, similar levels of the TβR-II mRNA were also detected in several regions of the CNS, namely cortex, midbrain, cerebellum, brain stem and hippocampus. We therefore tentatively conclude that TGF-β2 and 3 may signal in the brain by means of the same type-I and -II receptors as found in peripheral organs.

Expression of neurotrophins and their receptors in the developing and adult rat adrenal gland

We have studied the postnatal expression of neurotrophins, their cognate high-affinity trk receptors and the low-affinity NGF receptor (p75LNGFR) in the rat adrenal gland using RT-PCR. Neurotrophin mRNAs were detectable during the whole postnatal period. Strongest signals were obtained for BDNF and NT4/5. Expression of trkA, trkB, trkC and p75LNGFR was found at all ages studied. Signals for trkA were highest in the adult adrenal medulla, whereas signals for p75LNGFR were highest in the adult adrenal cortex. Cur data suggest still largely enigmatic roles for neurotrophins in functions of the adrenal medulla and possibly also the cortex.

Expression and localization of GDNF in developing and adult adrenal chromaffin cells

Abstract.Glial cell line-derived neurotrophic factor (GDNF) is a widely distributed member of the transforming growth factor-β superfamily and a potent neurotrophic molecule for several neuron populations in the peripheral and central nervous system. We show here that adrenal medullary chromaffin cells synthesize GDNF mRNA and contain immunoreactive GDNF protein. GDNF immunoreactivity can be found as early as embryonic day 16 in chromaffin progenitor cells of the rat adrenal gland and becomes more prominent with age. Most of the chromaffin cells within the adult rat adrenal medulla are GDNF immunoreactive, including both the noradrenergic and adrenergic subpopulations. The functions of adrenal medullary GDNF are still enigmatic but may include both auto/paracrine roles and retrograde trophic support of preganglionic neurons in the spinal cord or of sensory neurons that innervate chromaffin cells.

GDNF INDUCES THE CALRETININ PHENOTYPE IN CULTURES OF EMBRYONIC STRIATAL NEURONS

Glial cell line‐derived neurotrophic factor (GDNF), a member of the transforming growth factor‐β (TGF‐β) superfamily, is a potent neurotrophic factor for several neuron populations in the central and peripheral nervous system. Members of the neurotrophin, neurokine, and TGF‐β families of growth factors can affect neurons beyond their capacity to promote survival. They can play instructive roles including the determination of a particular transmitter phenotype. Here, we show that GDNF enhances the number of calretinin (CaR)‐positive neurons in serum‐free cultures of striatal cells isolated from embryonic rats. The effect is dose‐dependent, can be elicited with concentrations as low as 0.1 ng/ml, and is not accompanied by increased incorporation of 5‐bromo‐2′‐desoxyuridine and appearance of glial fibrillary acidic protein–positive cells. Similar, but weaker effects can be elicited by brain‐derived neurotrophic factor, neurotrophin‐3 and ‐4, fibroblast growth factor‐2. Ciliary neurotrophic factor, nerve growth factor, and TGF‐β1 do not affect striatal CaR expression. GDNF can augment CaR‐positive cells at any time point and with a minimal exposure of 18 hr, suggesting induction of the phenotype rather than increased survival. By reverse transcription polymerase chain reaction (RT‐PCR), we show that GDNF is expressed in the E16 striatum and in cultures derived from this tissue. GDNF also protected striatal CaR‐positive neurons against glutamate toxicity. We conclude that striatal GDNF, in addition to its retrograde trophic role for nigrostriatal dopaminergic neurons, may also act locally within the striatum (e.g., by inducing the CaR phenotype and protecting these cells against toxic insult). J. Neurosci. Res. 50:361–372, 1997.