Glial cell line-derived neurotrophic factor (GDNF) is expressed in the human kidney and is a growth factor for human mesangial cells.

BACKGROUND: Glial cell line-derived neurotrophic factor (GDNF), a recently cloned member of the transforming growth factor-beta (TGF-beta) superfamily, is a potent neurotrophic factor in vitro and in vivo. GDNF is essential for nephrogenesis and the highest expression of GDNF is found in the developing kidney. Increased plasma GDNF levels have recently been documented in patients with chronic renal failure; the source and role of this increase, however, remain unclear. No data are available about the expression of GDNF in human adult kidney or human adult mesangial cell (HMC) cultures. We hypothesized that GDNF, similar to other members of the TGF-beta superfamily, might play a role as a growth factor in the pathogenesis of glomerulosclerosis. METHODS: To address this hypothesis, we first investigated (by RT-PCR) the expression of GDNF mRNA and the mRNAs of the GDNF receptors Ret and GFRalpha-1 in (i) adult human renal cortex and medulla and (ii) in HMC in culture. The results were compared to the expression of these molecules in different developmental stages of the rat kidney. We found that both GDNF and its receptors were expressed in human adult kidney and HMC. Since this finding implicates a role for GDNF beyond nephrogenesis, i.e. in renal physiology/pathophysiology, we investigated the effect of GDNF on HMC growth, i.e. (i) cellular protein synthesis as an index of hypertrophy ([(3)H]methionine incorporation), (ii) DNA synthesis ([(3)H]thymidine incorporation) and cell proliferation (cell numbers) as indices of hyperplasia, and (iii) extracellular matrix synthesis, i.e. collagenous and non-collagenous extracellular proteins ([(3)H]proline incorporation into the collagenase-sensitive and -insensitive fraction). HMC cultures were used as a surrogate model for the development of glomerulosclerosis. RESULTS: GDNF induced a biphasic growth stimulatory effect in HMC with stimulation at the lowest concentration used (2 ng/ml) but had no effect at higher concentrations (20 and 50 ng/ml). In contrast, cellular protein synthesis and extracellular matrix synthesis were significantly and dose-dependently increased by GDNF. CONCLUSIONS: These results suggest that GDNF, similar to other members of the TGF-beta superfamily, might play a role as a growth factor for mesangial cells and might thus be a player in the pathogenesis of glomerulosclerosis.

Growth/differentiation factor-15/macrophage inhibitory cytokine-1 is a novel trophic factor for midbrain dopaminergic neurons in vivo.

Transforming growth factor-betas (TGF-betas) constitute an expanding family of multifunctional cytokines with prominent roles in development, cell proliferation, differentiation, and repair. We have cloned, expressed, and raised antibodies against a distant member of the TGF-betas, growth/differentiation factor-15 (GDF-15). GDF-15 is identical to macrophage inhibitory cytokine-1 (MIC-1). GDF-15/MIC-1 mRNA and protein are widely distributed in the developing and adult CNS and peripheral nervous systems, including choroid plexus and CSF. GDF-15/MIC-1 is a potent survival promoting and protective factor for cultured and iron-intoxicated dopaminergic (DAergic) neurons cultured from the embryonic rat midbrain floor. The trophic effect of GDF-15/MIC-1 was not accompanied by an increase in cell proliferation and astroglial maturation, suggesting that GDF-15/MIC-1 probably acts directly on neurons. GDF-15/MIC-1 also protects 6-hydroxydopamine (6-OHDA)-lesioned nigrostriatal DAergic neurons in vivo. Unilateral injections of GDF-15/MIC-1 into the medial forebrain bundle just above the substantia nigra (SN) and into the left ventricle (20 microgram each) immediately before a 6-OHDA injection (8 microgram) prevented 6-OHDA-induced rotational behavior and significantly reduced losses of DAergic neurons in the SN. This protection was evident for at least 1 month. Administration of 5 microgram of GDF-15/MIC-1 in the same paradigm also provided significant neuroprotection. GDF-15/MIC-1 also promoted the serotonergic phenotype of cultured raphe neurons but did not support survival of rat motoneurons. Thus, GDF-15/MIC-1 is a novel neurotrophic factor with prominent effects on DAergic and serotonergic neurons. GDF-15/MIC-1 may therefore have a potential for the treatment of Parkinson's disease and disorders of the serotonergic system.

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.

An infrastructure for comparative genomics to functionally characterize genes and proteins.

Current genome projects are resulting in a flood of sequence data. The interpretation of these sequences is lagging, and optimized data analysis strategies need to be developed. Much can be learned from comparing different genomes, as genomes of distant organisms may still encode proteins with high sequence similarity. The order of genes (co linearity) in genomes may also be conserved to some extend. We have employed both these observations to create a multi-functional, computational analysis system (genomeSCOUT) which allows for rapid identification and functional characterization of genes and proteins through genome comparison. With a number of independent algorithms, information about different levels of protein homology (concerning e.g. paralogs, orthologs and clusters of orthologous groups, COGs) and gene order is collected and stored in several value added databases. These databases are then used for interactive comparison of genomes and subsequent analysis. The application is based on the well established data integration system SRS. This ensures (1) fast handling of large genomic data sets, (2) straightforward access to a multitude of biological databases, (3) unique linking functions between these databases, (4) highly efficient collection of information on genes and proteins, and 5. fully integrated and user friendly graphical representations of search results. This application can be used for projects as diverse as the correct annotation of genomes, the optimization of (micro) organisms for industrial production, or the identification of drug targets.

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.

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.

We have cloned a novel member of the transforming growth factor-beta (TGF-beta) 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-beta superfamily, is widely distributed in adult tissues, being most strongly expressed in epithelial cells and macrophages.

Developmental expression of GDNF, neurturin and their receptors in rat hippocampus.

GLIAL cell-line derived neurotrophic factor (GDNF) and neurturin (NTN) are members of a new family of factors within the transforming growth factor-beta (TGFbeta) super-family. Signaling by GDNF and NTN depends on the tyrosine kinase receptor Ret and on GFRalpha-1 and -2. We have investigated by competitive RT-PCR the developmental expression of these molecules within the rat hippocampus. All transcripts reach high expression levels between postnatal day 0 and 14 (P0-P14), a critical period of hippocampal differentiation, suggesting roles for these molecules in this process. All mRNAs (with the exception of Ret) can be detected as early as embryonic day 14 (E14). The absence of Ret expression at E14 suggests that both GDNF and NTN may employ an additional, unknown receptor. NTN expression shows a biphasic expression pattern, with maximum expression levels at E14 and P5.

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-beta (TGF-beta) superfamily, is a potent neurotrophic factor for several neuron populations in the central and peripheral nervous system. Members of the neurotrophin, neurokine, and TGF-beta 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-beta 1 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).

The gene encoding bovine brain-derived neurotrophic factor (BDNF).

We present the nucleotide (nt) sequence for the cDNA encoding bovine brain-derived neurotrophic factor (BDNF). The nt and peptide sequences were compared to those of other BDNF genes from other species.

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.

Glial cell line-derived neurotrophic factor (GDNF) is a widely distributed member of the transforming growth factor-beta 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.

Expression of TGF-beta type II receptor mRNA in the CNS.

Members of the TGF-beta family have been described to bind to a heteromeric complex of two types of serine/threonine kinase receptors, named T beta R-I and T beta R-II. These receptor molecules are essential for TGF-beta-specific signalling. Several type I and type II receptors have been identified by a variety of methods. TGF-beta 2 and 3 are widely expressed in the CNS and exert multiple functions on neurones and glia. Although the T beta R-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 T beta R-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 T beta R-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-beta 2 and 3 may signal in the brain by means of the same type-I and -II receptors as found in peripheral organs.

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.

Characterisation of a new allele of pale cress and its role in greening in Arabidopsis thaliana.

A recessive mutant with white leaves was identified in a screen of a population of T-DNA-tagged Arabidopsis thaliana plants. The mutation is lethal, but plants develop almost to maturity under sterile conditions. The white areas in leaves are devoid of developed chloroplasts, but the plants frequently develop green sectors which contain green chloroplasts. Molecular characterisation of the affected gene revealed that the mutant is allelic to pale cress (pac), a recently described mutation, and was therefore named pac-2. Sequencing of cDNAs and the genomic region revealed several noteworthy features of this genetic locus. In pac-2 the T-DNA had inserted in the region of the promoter and abolished transcription of the PAC gene completely. Cytokinin induced greening in mature, white homozygous pac-2 plants, and therefore is likely to be responsible for the greening observed in callus and shoots induced on roots from such plants. However, the PAC transcript was found to be absent in both white leaves and green callus. Thus, since cytokinin induced greening in the absence of PAC RNA this plant hormone appears to be able to bypass PAC function.

Trophic and protective effects of growth/differentiation factor 5, a member of the transforming growth factor-beta superfamily, on midbrain dopaminergic neurons.

Growth/differentiation factor 5 (GDF5) is a novel member of the transforming growth factor-beta (TGF-beta) superfamily of multifunctional cytokines. We show here that GDF5 is expresed in the developing CNS including the mesencephalon and acts as a neurotrophic, survival promoting molecule for rat dopaminergic midbrain neurons, which degenerate in Parkinson's disease. Recombinant human GDF5 supports dopaminergic neurons, dissected at embryonic day (E) 14 and cultured for 8 days under serum-free conditions, to almost the same extent as TGF-beta 3, and is as effective as glial cell line-derived neurotrophic factor (GDNF), two established trophic factors for midbrain dopaminergic neurons. In contrast to TGF-beta and GDNF, GDF5 augments numbers of astroglial cells in the cultures, suggesting that it may act indirectly and through pathways different from those triggered by TGF-beta and GDNF. GDF5 also protects dopaminergic neurons against the toxicity of N-methylpyridinium ion (MPP+), which selectively damages dopaminergic neurons through mechanisms currently debated in the etiology of Parkinson's disease (PD). GDF5 may therefore now be tested in animal models of PD and might become useful in the treatment of PD.

T-DNA integrations in a new family of repetitive elements of Nicotiana tabacum.

A number of T-DNA insertions in the genome of Nicotiana tabacum were characterized. One class of integrations was found to have occurred in a new family of highly repetitive sequences. Three genomic regions (ecoA, ecoB, and ecoC) were isolated, all of which contain basic units of 180 bp, organized in direct tandem repeats. Several of the 180-bp elements contain an EcoRI recognition site within the repeating unit and are therefore named "eco repeats." All members of this family are weakly homologous in sequence to a previously described class of repeat elements which contained a BamHI site (HRS60 repeat family), which suggests that both groups of sequences are of common evolutionary origin. The allotetraploid genome of N. tabacum is presumed to originate from the hybridization of two diploid genomes. The HRS60 elements previously described have been found exclusively in the genome of one of the ancestors, N. sylvestris, and in N. tabacum itself. Our DNA hybridization data suggest that the eco elements originate from the genome of the other ancestor, N. tomentosiformis. Whereas the HRS60 elements are transcriptionally silent, at least some eco elements appear to be transcribed.

TGF-beta superfamily members promote survival of midbrain dopaminergic neurons and protect them against MPP+ toxicity.

The superfamily of transforming growth factors-beta (TGF-beta) comprises an expanding list of multifunctional proteins serving as regulators of cell proliferation and differentiation. Prominent members of this family include the TGF-beta s 1-5, activins, bone morphogenetic proteins and a recently discovered glial cell line-derived neurotrophic factor (GDNF). In the present study we demonstrate and compare the survival promoting and neuroprotective effects of TGF-beta 1, -2 and -3, activin A and GDNF for midbrain dopaminergic neurons in vitro. All proteins increase the survival of tyrosine hydroxylase-immunoreactive dopaminergic neurons isolated from the embryonic day (E) 14 rat mesencephalon floor to varying extents (TGF-beta s 2.5-fold, activin A and GDNF 1.6-fold). TGF-beta s, activin A and GDNF did not augment numbers of very rarely observed astroglial cells visualized by using antibodies to glial fibrillary acidic protein and had no effect on cell proliferation monitored by incorporation of BrdU. TGF-beta 1 and activin A protected dopaminergic neurons against N-methyl-4-phenylpiridinium ion toxicity. Reverse transcription-polymerase chain reaction (RT-PCR) analysis indicated that TGF-beta 2 mRNA, but not GDNF mRNA, is expressed in the E14 rat midbrain floor and in mesencephalic cultures. We conclude that TGF-beta s 1-3, activin A and GDNF share a neurotrophic capacity for developing dopaminergic neurons, which is not mediated by astroglial cells and not accompanied by an increase in cell proliferation.