Article |
Address correspondence to Dr. Heike Peterziel, Department of Neuroanatomy and Center for Neurosciences (IZN), Im Neuenheimer Feld 307, 2.OG, D-69120 Heidelberg, Germany. Tel.: 49-6221-548314. Fax: 49-6221-545604. E-mail: heike.peterziel{at}urz.uni-heidelberg.de
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: neurotrophic factors; lipid raft; GFR1; tyrosine kinases; MAPK pathway
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
TGFßs are widely distributed cytokines with prominent roles in development and cell cycle control (Roberts and Sporn, 1990; Alexandrow and Moses, 1995; Krieglstein et al., 1995). TGFßs have been implicated in the regulation of neuronal survival of motoneurons (Martinou et al., 1990), sensory (Chalazonitis et al., 1992), and midbrain dopaminergic neurons (Krieglstein and Unsicker, 1994; Poulsen et al., 1994), for example. However, TGFßs show no or marginal effects on highly enriched, serum-free neuron cultures, such as sensory neurons (Krieglstein and Unsicker, 1996), suggesting that TGFß may require cooperating factors for eliciting its trophic effects. Along this line, TGFß actions are always referred to as being contextually dependent, suggesting that other molecules present significantly determine TGFß actions and vice versa (Nathan and Sporn, 1991; Unsicker and Krieglstein, 2000). Putative contextual actions of TGFßs and additional molecules may also underlie the seemingly conflicting effects of TGFß in the regulation of both survival and death of ciliary ganglionic (CG) neurons (Krieglstein et al., 2000). With regard to the regulation of survival, TGFß cooperates with GDNF, whereas TGFß-induced cell death may be the consequence of a lack of the appropriate neurotrophic factor, like GDNF, or may be the result of cooperativity with other yet unidentified cytokines.
A fundamental question regarding GDNF-TGFß cooperativity concerns the underlying molecular mechanism. GDNF signals via binding as a homodimer to a heterotetrameric complex of two glycosyl-phosphatidylinositol (GPI)-anchored GFR1 (Jing et al., 1996; Treanor et al., 1996) and two molecules of the transmembrane tyrosine kinase Ret (Durbec et al., 1996; Trupp et al., 1996). Upon autophosphorylation of Ret, two signaling pathways are activated: one is the Ras/Raf/MAPK pathway, and the other involves activation of PI3 kinase and its downstream target PKB/Akt (Chiariello et al., 1998; Trupp et al., 1999).
The present study identifies the Ras/MAPK pathway as the survival promoting signaling cascade activated by treatment of CG neurons with GDNF and TGFß. We show that TGFß mediates recruitment of the GPI-linked GFR1 to the site of consecutive signaling, probably lipid rafts.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this study we have asked how the cooperation of these two factors is mediated on the molecular level using primary chick embryonic (E8) CG neurons as a model system. Survival of CG neurons is strictly dependent on the presence of neurotrophic support. Fig. 1 A demonstrates that GDNF plus TGFß promotes survival of the cells, whereas the single factors do not. This effect was dose dependent with concentrations of GDNF ranging from 2 to 10 ng/ml. However, TGFß was almost as effective at 0.125 ng/ml as at 2 ng/ml (Fig. 1 B). As a positive control, ciliary neurotrophic factor (CNTF) was used, which is known to promote survival of CG neurons independent of TGFß (Krieglstein et al., 1998).
|
|
|
Corroborating this result survival of CG neurons was also promoted when TGFß, initially added without GDNF, was neutralized after 3 h and GDNF was subsequently added. In contrast, neutralization of TGFß at the beginning of the experiment and administration of GDNF alone or GDNF and TGFß after 3 h did not promote survival. This proved that the TGFß antibody was present in excess and completely blocked TGFß activity throughout the culture period. Please note that a consecutive treatment of first 3 h with TGFß followed by GDNF was sufficient to attain GDNF-induced cell survival. Further along this line pretreatment with GDNF, followed by a change of medium and addition of TGFß did not elicit a survival promoting effect (Fig. 3 D). Together, these data suggest that GDNF responsiveness essentially requires pretreatment with TGFß.
One possible explanation for this TGFß requirement may be an effect on the expression of Ret and GFR1. Therefore, we investigated the mRNA level of both receptors using semi-quantitative RT-PCR with primers specific for chicken Ret and GFR
1 in the presence and absence of TGFß. As shown in Fig. 3 E, there was no change in the amount of the two receptor mRNAs. Consistent with the unchanged mRNA level, we found no effect of TGFß pretreatment on the GFR
1 protein level in a Western blot analysis using a GFR
1-specific antibody. We next investigated the time dependence for pretreatment with TGFß. We found increased ERK phosphorylation in GDNF-treated neurons after 30 min, which peaked after 3 h and disappeared after 6 h (Fig. 3 F). This kinetics is also indicative of an effect independent of protein synthesis. One possible explanation could be that the availability of the GDNF receptors for interaction with their ligand at the plasma membrane might be regulated by TGFß.
In the presence of a soluble GFR1, TGFß is not required to promote survival and activation of ERK by GDNF
GFR1 is attached to the outer cell membrane by a GPI modification (Jing et al., 1996). We have previously shown that cleavage of this GPI anchor from the membrane by phosphoinositide-specific phospholipase C disrupts the survival promoting effect of GDNF and TGFß, and that this effect can be partly reversed by adding TGFß before cleavage by phosphoinositide-specific phospholipase C (Krieglstein et al., 1998). This observation strongly argued for an effect of TGFß on the recruitment of GFR
1 to or its stabilization on the plasma membrane. Therefore, we asked whether an exogenously added receptor may confer GDNF responsiveness to CG neurons in the absence of TGFß.
To test this hypothesis we added a GFR1-IgG chimeric protein to the supernatant of the primary neuron cultures. Cell counts after 24 h showed that GDNF fully promoted CG neuron survival in the presence of soluble GFR
1, indicating that addition of TGFß is not required under these conditions (Fig. 4 A). Thus, TGFß may function by recruiting the GFR
1 by yet unknown mechanisms. Interestingly, additional treatment with TGFß further increased numbers of surviving cells, suggesting that TGFß may also affect components of the GDNF signaling pathway other than GFR
1. Neutralization of endogenous TGFß resulted in a minor decrease in survival. Along this line, phosphorylation of ERK in the presence of soluble GFR
1 also does not require TGFß. However, Akt phosphorylation was only observed if the cells were treated simultaneously with GDNF and TGFß (Fig. 4 B).
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
TGFß treatment induces clustering of GFR1 in lipid microdomains on the cell surface
TGFß induction of GDNF responsiveness occurred within 30 min, excluding an effect that is dependent on new protein synthesis. Consistently, TGFß-induced GDNF responsiveness did not involve upregulation of expression of the receptors cRet and GFR1. Our data suggest that TGFß does affect the availability of GFR
1 for the ligand GDNF. This is shown by the following: (a) the inhibitory effect of BFA on ERK phosphorylation; (b) the clustering of GFR
1 immunoreactivity and the costaining of GFR
1 and the lipid raft marker GM1; and (c) preliminary observations that TGFß pretreatment leads to a shift of GFR
1 from cytoplasmic to membrane fractions of CG neuron protein extracts (unpublished data).
It has been shown that GFR1 is necessary to recruit Ret to lipid rafts, which attenuates downstream signaling pathways as well as neuronal differentiation and survival (Tansey et al., 2000; Paratcha et al., 2001). Lipid rafts are thought to represent specialized signaling organelles within the plasma membrane because of an enrichment of many adaptor and signaling molecules (Anderson, 1998). Tansey et al. (2000) have analyzed the functional importance of the GPI anchorage of GFR
1 in mediating GFR
1/Ret signalling. They were able to show that both the anchored and the soluble GFR
1 mediate Ret translocation to lipid rafts, and thereby attenuate downstream kinase activity. Paratcha et al. (2001) extended these studies by providing additional evidence, suggesting that Ret-translocation is best mediated by soluble GFR
1, which may be provided by ectopic sources. Most studies addressing GFR
1/Ret interaction have been performed using cell lines which were transfected with either GFR
1 alone or both GFR
1 and Ret. In this context, the overexpressed GPI-linked receptor is constitutively present in the lipid raft fraction on the cellular membrane. So far, it has not been addressed whether the presence of endogenous GFR
1 at the plasma membrane might be subject to regulation. It has been shown that GPI-linked proteins continuously shuttle between the Golgi complex and the plasma membrane without involvement of new synthesis of this proteins (Nichols et al., 2001). Regulation of this cycling might thus play an important role in lipid raft associated signaling cascades.
Mechanisms of TGFß-induced recruitment of GFR1 to its active site at the plasma membrane
There are several possible ways that TGFß may be mediating the availability of GFR1 at lipid rafts. First, it has to be clarified whether TGFß is acting via its receptors, TßRI and TßRII, and whether downstream signaling of TGFß is required. GDNF-TGFß cooperativity might also involve a direct interaction of the corresponding receptors at the plasma membrane. Furthermore, cooperativity could be due to an indirect effect involving a protein for which the receptors compete. One example for such a competition has been shown for binding of caveolin-1 to the NGF receptors TrkA and p75 which determines the NGF response of PC12 cells (Bilderback et al., 1999). Caveolin-1 is a principal component of cholesterol-enriched plasma membrane microdomains, called caveolae, which are involved in vesicular transport and signal transduction (for review see Schlegel and Lisanti, 2001). It has been proposed that caveolin could function as a transmembrane adaptor molecule that couples GPI-linked proteins with signaling molecules during GPI-membrane trafficking or GPI-mediated signal transduction events (Sargiacomo et al., 1993).
It has been shown that TßRI interacts with caveolin-1, and that upon binding of TGFß to its receptors at the membrane, this interaction is increased and leads to downregulation of the TGFß response, possibly by internalization of TßRI (Razani et al., 2001). Whether a similar interaction of GFR1 with caveolin-1 occurs and whether this is changed in the presence of TGFß remains to be established.
Cooperative effects of GDNF with other factors on the survival of different neuronal populations
The requirement of a synergism between GDNF and another factor for the survival of specific neuronal populations has been described for BDNF (sensory neurons of the nodose-petrosal ganglion complex; Erickson et al., 2001), CNTF (mouse photoreceptors; Ogilvie et al., 2000), IGF-I (motoneurons; Bilak and Kuncl, 2001; Bilak et al., 2001), osteogenin-1/bone morphogenetic protein 7, a member of the TGFß superfamily (explanted embryonic chicken ganglia and dissociated ganglionic neuron; Bengtsson et al., 1998), and cardiotrophin-1 (embryonic rat motoneurons; Arce et al., 1998). These findings underline the importance of modulation of the GDNF response by other (growth) factors, but the mechanistic explanation for any of these synergisms has yet to be provided.
Interference between TGFß signaling and signal transduction through receptor tyrosine kinases
In contrast to GDNF synergisms, molecular mechanisms of interaction of TGFß family members with other signaling cascades have been studied in various cellular systems. Interferon- inhibits TGFß-induced phosphorylation of Smad3 and downstream TGFß effects by an effect involving new protein synthesis, namely by induction of the expression of the antagonistic SMAD, Smad7, through Jak1 and Stat1 (Ulloa et al., 1999). Synergistic effects involving direct interaction of Stat proteins and Smads have been reported for Smad3 (activated by TGFß) and STAT3 (activated by Interleukin-6) in the Hep3B hepatoma cell line (Yamamoto et al., 2001) and for Smad1 (activated by bone morphogenetic protein 2) and STAT3 (activated by leukemia inhibitory factor) on primary fetal neural progenitor cells during the induction of astrocyte differentiation (Nakashima et al., 1999). These activities were due to physical interactions between STAT3 and the Smads bridged by the transcriptional coactivator p300.
Evidence for inhibitory effects of receptor tyrosine kinase pathways on signaling of members of the TGFß family concerns the level of signal transduction through phosphorylated Smads. It has been shown that EGF and hepatocyte growth factor exhibit an inhibitory effect on bone morphogenetic proteininduced Smad1 activation by phosphorylation of specific serines in the linker region of Smad1. This phosphorylation is catalysed by the MAPK ERK in mink lung epithelial cells (Kretzschmar et al., 1997). In contrast to our findings of a TGFß-GDNF cooperativity, in these cases the cells receive opposing regulatory inputs through the receptor tyrosine kinases and the serine/threonine kinase. There are also reports about how receptor tyrosine kinases (RTKs) or downstream kinases can positively interfere with the TGFß signaling cascade. For example, in contrast to their above described inhibitory effect, hepatocyte growth factor and EGF have been shown to induce Smad2 phosphorylation and nuclear translocation in epithelial cells in the absence of TGFß and independent of TGFß receptors (de Caestecker et al., 1998). These observations suggest that the quality of interference of RTK- and TGFß-induced pathways is dependent on the context of the analyzed cellular system. In contrast to all these observations in which RTK signaling positively affects TGFß signaling, in our neuronal model, TGFß facilitates RTK signaling, primarily by exerting an effect on the GFR1 receptor. Whether there also exists a physical interaction of downstream effectors of TGFß and GDNF which could explain the differential TGFß requirement for Akt phosphorylation needs to be examined.
Interaction of Smads with other transcription factors on the promoter level
Cooperativity of the TGFß signaling pathway with other pathways has also been described to involve regulation of ligand-induced gene transcription at the promoter level. A nuclear Smad3/Smad4 can activate a TPA responsive gene promoter element directly or through interaction with both c-Jun and c-Fos (Zhang et al., 1998). Smad3 has also been reported to potentiate ligand-induced transactivation of the vitamin D receptor as a coactivator through binding of the steroid receptor coactivator-1 protein in the nucleus (Yanagi et al., 1999; Yanagisawa et al., 1999) or by binding to a Smad binding element adjacent to a vitamin D receptor binding element on the human osteocalcin promoter (Subramaniam et al., 2001). In human neuroblastoma cells, CNTF and TGFß synergistically induce vasoactive intestinal peptide (VIP) mRNA expression and transcription through the cytokine response element in the VIP promoter. This synergy is dependent on binding of Smad, STAT, and AP-1 to distinct sites within the VIP promoter. Whether any physical interaction of the signaling components is necessary has not been analyzed in this report (Pitts et al., 2001).
As discussed above, crosstalks of TGFß family members with other signaling pathways have been described to occur both in the cytosol and the nucleus (for review see Wrana, 2000). Our finding that TGFß cooperativity also involves molecular interactions at the plasma membrane adds a new point of view to signaling interactions of RTKs and TGFß signaling.
In conclusion, our data show for the first time that TGFß signaling facilitates tyrosine kinase signaling by modulating the availability of the GDNF receptor on the cell surface. TGFß-mediated recruitment of the GFR1 is crucial for GDNF-dependent signaling and survival.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The cell line Neuro2a was cultured in DME medium containing 10% bovine calf serum, 100 U/ml penicillin, 0.50 µg/ml streptomycin, 100 µg/ml neomycin (Invitrogen) and 1% nonessential amino acids. The stably transfected Neuro2a-GFR1 clone, provided by M. Saarma (University of Helsinki, Helsinki, Finland) was cultured in RPMI containing 10% bovine calf serum, 100 U/ml penicillin, 0.50 µg/ml streptomycin and 250 µg/ml G418. Total cell lysates were obtained by solubilizing the cells in Laemmli sample buffer (80 mM Tris, pH 6.8, 2% SDS, 10% glycerol, 2% ß-mercaptoethanol, 0.01% bromophenol blue) and sonication.
Evaluation of cell survival
CG neurons were seeded onto PORN/laminine-coated microtiter plates (A/2; Costar) at a density of 3,000 cells per well with or without different recombinant neurotrophic factors CNTF, GDNF, FGF2, and TGFß3. In the experiments where 5 µg/ml insulin was added to the culture medium, 1,000 cells were seeded per well. The recombinant GFR1/Fc chimera (soluble GFR
1) was obtained from R&D and applied in a concentration of 100 nM. The concentrations of the kinase inhibitors were as follows: Wortmannin (Calbiochem) 100 nM; PD98059 (Calbiochem) 50 µM; LY294002 (Calbiochem) 5 µM; and U0126 (Promega) 10 µM. After 24 h, cultures were fixed by addition of 2.5% glutaraldehyde in PBS. Numbers of surviving neurons were determined by direct counting using phase contrast microscopy.
Immunoblotting
The cells were allowed to attach to the PORN/laminine-coated dishes for at least 2 h and treated as indicated. Total cell lysates were subjected to a 810% SDS polyacrylamide gel electrophoresis and electroblotted onto nitrocellulose membranes. For detection of proteins the membranes were incubated with the specific antibodies, prior to the addition of the corresponding secondary antibody conjugated to horseradish peroxidase and detection by the enhanced chemoluminescence method (ECL; Amersham Biosciences). The antibodies were obtained from different sources as follows: antiphospho-ERK and anti-ERK2 from Santa Cruz Biotechnology; antiphospho-Akt (Ser-437) from New England Biolabs; biotinylated anti-GFR1 antibody from R&D Systems; and streptavidine-coupled horseradish peroxidase from Amersham Biosciences. Quantification of immunoreactive bands was performed with Scion Image software and statistics performed with Excel.
Measurement of GFR1 and cRet mRNA levels
Total RNA was extracted from neurons that had been cultured in the presence and absence of TGFß using Total RNA reagent (Biomol) according to the manufacturer's protocol. Three micrograms of RNA were reverse transcribed with MMLV-RT (Promega) in a 50 µl reaction containing the manufacturer's buffer supplemented with 0.8 mM dNTPs and 0.02 µg/µl random hexanucleotides. Aliquots of 4 µl of the reverse transcription reaction were used for amplification in 30 µl PCR reactions with the following specific forward and reverse primers: GFR1 forward: 5'-TTGACAAAGTTCCCCCAAAG-3'; GFR
1 reverse: 5'-GTTCGGTGTCATCACTGTGC-3'; Ret forward: 5'-GATGCTGTCGTGGAGTTCAA-3'; Ret reverse: 5'-TCGTTCACCAAAACATCCAA-3'; ß-actin forward: 5'-CCAGCCATCTTTCTTGGGTA-3'; and ß-actin reverse: 5'-GCGCATTTATGGGTTTTGTT-3'.
Immunocytochemistry
Staining for GFR1 was done with a biotinylated anti-rat GFR
1 antibody (R&D) for either 2 h on ice prior to fixation of the cells with 4% PFA or after extraction with Triton X-100. In the latter case cells were washed in PBS, incubated on ice in buffer (2 mM MgCl2, 10 mM EGTA, 60 mM Pipes, pH 7) and extracted for 8 to 9 min on ice with 1% Triton X-100 diluted in MSB buffer (Ledesma et al., 1998). The cells were washed with cold MSB and fixed with 4% PFA. After washing a secondary biotinylated antibody was added and immunoreactivity was detected by adding streptavidin-Cy3. For visualization of the lipid raft marker GM1 the cells were then labeled for 45 min with 0.1 µg/ml of fluorescein-conjugated cholera toxin B fragment (Sigma-Aldrich) in PBS with 0.1% BSA, washed with PBS, and mounted in mounting solution.
![]() |
Footnotes |
---|
![]() |
Acknowledgments |
---|
This work was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 530, C8).
Submitted: 25 March 2002
Revised: 29 August 2002
Accepted: 3 September 2002
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Anderson, R.G. 1998. The caveolae membrane system. Annu. Rev. Biochem. 67:199225.[CrossRef][Medline]
Arce, V., R.A. Pollock, J.M. Philippe, D. Pennica, C.E. Henderson, and O. deLapeyriere. 1998. J. Neurosci. 18:14401448.
Beck, K.D., J. Valverde, T. Alexi, K. Poulsen, B. Moffat, R.A. Vandlen, A. Rosenthal, and F. Hefti. 1995. Mesencephalic dopaminergic neurons protected by GDNF from axotomy-induced degeneration in the adult brain. Nature. 373:339341.[CrossRef][Medline]
Besser, D., M. Presta, and Y. Nagamine. 1995. Elucidation of a signaling pathway induced by FGF-2 leading to uPA gene expression in NIH 3T3 fibroblasts. Cell Growth Differ. 6:10091017.[Abstract]
Bilak, M.M., A.M. Corse, and R.W. Kuncl. 2001. Additivity and potentiation of IGF-I and GDNF in the complete rescue of postnatal motor neurons. Amyotroph. Lateral Scler. Other Motor Neuron Disord. 2:8391.
Bilderback, T.R., V.R. Gazula, M.P. Lisanti, and R.T. Dobrowsky. 1999. Caveolin interacts with Trk A and p75(NTR) and regulates neurotrophin signaling pathways. J. Biol. Chem. 274:257263.
Boulton, T.G., N. Stahl, and G.D. Yancopoulos. 1994. Ciliary neurotrophic factor/leukemia inhibitory factor/interleukin 6/oncostatin M family of cytokines induces tyrosine phosphorylation of a common set of proteins overlapping those induced by other cytokines and growth factors. J. Biol. Chem. 269:1164811655.
Cacalano, G., I. Farinas, L.C. Wang, K. Hagler, A. Forgie, M. Moore, M. Armanini, H. Phillips, A.M. Ryan, L.F. Reichardt, et al. 1998. GFRalpha1 is an essential receptor component for GDNF in the developing nervous system and kidney. Neuron. 21:5362.[Medline]
Chiariello, M., R. Visconti, F. Carlomagno, R.M. Melillo, C. Bucci, V. de Franciscis, G.M. Fox, S. Jing, O.A. Coso, J.S. Gutkind, et al. 1998. Signaling of the Ret receptor tyrosine kinase through the c-Jun NH2-terminal protein kinases (JNKS): evidence for a divergence of the ERKs and JNKs pathways induced by Ret. Oncogene. 16:24352445.[CrossRef][Medline]
de Caestecker, M.P., W.T. Parks, C.J. Frank, P. Castagnino, D.P. Bottaro, A.B. Roberts, and R.J. Lechleider. 1998. Smad2 transduces common signals from receptor serine-threonine and tyrosine kinases. Genes Dev. 12:15871592.
Dudley, D.T., L. Pang, S.J. Decker, S.J. Bridges, and A.R. Saltiel. 1995. A synthetic inhibitor of the mitogen-activated protein kinase cascade. Proc. Natl. Acad. Sci. USA. 92:76867689.[Abstract]
Erickson, J.T., T.A. Brosenitsch, and D.M. Katz. 2001. Brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor are required simultaneously for survival of dopaminergic primary sensory neurons in vivo. J. Neurosci. 21:581589.
Enomoto, H., P.A. Crawford, A. Gorodinsky, R.O. Heuckeroth, E.M. Johnson, Jr., and J. Milbrandt. 2001. RET signaling is essential for migration, axonal growth and axon guidance of developing sympathetic neurons. Development 128:39633974.
Favata, M.F., K.Y. Horiuchi, E.J. Manos, A.J. Daulerio, D.A. Stradley, W.S. Feeser, D.E. Van Dyk, W.J. Pitts, R.A. Earl, F. Hobbs, et al. 1998. Identification of a novel inhibitor of mitogen-activated protein kinase kinase. J. Biol. Chem. 273:1862318632.
Henderson, C.E., H.S. Phillips, R.A. Pollock, A.M. Davies, C. Lemeulle, M. Armanini, L. Simmons, B. Moffet, R.A. Vandlen, and L.C. Simpson. 1994. GDNF: a potent survival factor for motoneurons present in peripheral nerve and muscle. Science. 266:10621064.[Medline]
Huang, E.J., and L.F. Reichardt. 2001. Neurotrophins: roles in neuronal development and function. Annu. Rev. Neurosci. 24:677736.[CrossRef][Medline]
Iwamori, M., J. Shimomura, and Y. Nagai. 1985. Specific binding of cholera toxin to rat erythrocytes revealed by analysis with a fluorescence-activated cell sorter. J. Biochem. 97:729735.[Abstract]
Kingsley, D.M. 1994. The TGF-beta superfamily: new members, new receptors, and new genetic tests of function in different organisms. Genes Dev. 8:133146.[CrossRef][Medline]
Kretzschmar, M., J. Doody, and J. Massague. 1997. Opposing BMP and EGF signalling pathways converge on the TGF-beta family mediator Smad1. Nature. 389:618622.[CrossRef][Medline]
Krieglstein, K., and K. Unsicker. 1996. Distinct modulatory actions of TGF-beta and LIF on neurotrophin-mediated survival of developing sensory neurons. Neurochem. Res. 21:843850.[Medline]
Krieglstein, K., P. Henheik, L. Farkas, J. Jaszai, D. Galter, K. Krohn, and K. Unsicker. 1998. Glial cell line-derived neurotrophic factor requires transforming growth factor-ß for exerting its full neurotrophic potential on peripheral and CNS neurons. J. Neurosci. 18:98229834.
Lachmund, A., D. Gehrke, K. Krieglstein, and K. Unsicker. 1994. Trophic factors from chromaffin granules promote survival of peripheral and central nervous system neurons. Neuroscience. 62:361370.[CrossRef][Medline]
Ledesma, M.D., K. Simons, and C.G. Dotti. 1998. Neuronal polarity: essential role of protein-lipid complexes in axonal sorting. Proc. Natl. Acad. Sci. USA. 95:39663971.
Martinou, J.C., A. Le Van Thai, A. Valette, and M.J. Weber. 1990. Transforming growth factor beta 1 is a potent survival factor for rat embryo motoneurons in culture. Brain Res. Dev. Brain Res. 52:175181.[Medline]
Nakashima, K., M. Yanagisawa, H. Arakawa, N. Kimura, T. Hisatsune, M. Kawabata, K. Miyazono, and T. Taga. 1999. Synergistic signaling in fetal brain by STAT3-Smad1 complex bridged by p300. Science. 284:479482.
Neet, K.E., and R.B. Campenot. 2001. Receptor binding, internalization, and retrograde transport of neurotrophic factors. Cell. Mol. Life Sci. 58:10211035.[Medline]
Nichols, B.J., A.K. Kenworthy, R.S. Polishchuk, R. Lodge, T.H. Roberts, K. Hirschberg, R.D. Phair, and J. Lippincott-Schwartz. 2001. Rapid cycling of lipid raft markers between the cell surface and Golgi complex. J. Cell Biol. 153:529541.
Oda, K., T. Fujiwara, and Y. Ikehara. 1990. Brefeldin A arrests the intracellular transport of viral envelope proteins in primary cultured rat hepatocytes and HepG2 cells. Biochem. J. 265:161167.[Medline]
Paratcha, G., F. Ledda, L. Baars, M. Coulpier, V. Besset, J. Anders, R. Scott, and C.F. Ibanez. 2001. Released GFRalpha1 potentiates downstream signaling, neuronal survival, and differentiation via a novel mechanism of recruitment of c-Ret to lipid rafts. Neuron. 29:171184.[Medline]
Piiper, A., D. Stryjek-Kaminska, R. Gebhardt, and S. Zeuzem. 1996. Pertussis toxin-sensitive G-proteins inhibit fibroblast growth factor-induced signaling in pancreatic acini. J. Cell. Physiol. 167:5259.[CrossRef][Medline]
Pitts, R.L., S. Wang, E.A. Jones, and A.J. Symes. 2001. Transforming growth factor-beta and ciliary neurotrophic factor synergistically induce vasoactive intestinal peptide gene expression through the cooperation of Smad, STAT, and AP-1 sites. J. Biol. Chem. 276:1996619973.
Poulsen, K.T., M.P. Armanini, R.D. Klein, M.A. Hynes, H.S. Phillips, and A. Rosenthal. 1994. TGF beta 2 and TGF beta 3 are potent survival factors for midbrain dopaminergic neurons. Neuron. 13:12451252.[Medline]
Razani, B., X.L. Zhang, M. Bitzer, G. von Gersdorff, E.P. Bottinger, and M.P. Lisanti. 2001. Caveolin-1 regulates transforming growth factor (TGF)-beta/SMAD signaling through an interaction with the TGF-beta type I receptor. J. Biol. Chem. 276:67276738.
Roberts, A.B., and M.B. Sporn. 1990. The transforming growth factor-ßs. Handbook of Experimental Pharmacology. M.B. Sporn and A.B. Roberts, editors. Springer/Germany, Heidelberg. 419172.
Sargiacomo, M., M. Sudol, Z. Tang, and M.P. Lisanti. 1993. Signal transducing molecules and glycosyl-phosphatidylinositol-linked proteins form a caveolin-rich insoluble complex in MDCK cells. J. Cell Biol. 122:789807.[Abstract]
Sauer, H., C. Rosenblad, and A. Bjorklund. 1995. Glial cell line-derived neurotrophic factor but not transforming growth factor beta 3 prevents delayed degeneration of nigral dopaminergic neurons following striatal 6-hydroxydopamine lesion. Proc. Natl. Acad. Sci. USA. 92:89358939.[Abstract]
Schober, A., R. Hertel, U. Arumae, L. Farkas, J. Jaszai, K. Krieglstein, M. Saarma, and K. Unsicker. 1999. Glial cell line-derived neurotrophic factor rescues target-deprived sympathetic spinal cord neurons but requires transforming growth factor-beta as cofactor in vivo. J. Neurosci. 19:20082015.
Segal, R.A., and M.E. Greenberg. 1996. Intracellular signaling pathways activated by neurotrophic factors. Annu. Rev. Neurosci. 19:463489.[CrossRef][Medline]
Subramaniam, N., G.M. Leong, T.A. Cock, J.L. Flanagan, C. Fong, J.A. Eisman, and A.P. Kouzmenko. 2001. Cross-talk between 1,25-dihydroxyvitamin D3 and transforming growth factor-beta signaling requires binding of VDR and Smad3 proteins to their cognate DNA recognition elements. J. Biol. Chem. 276:1574115746.
Tomac, A., E. Lindqvist, L.F. Lin, S.O. Ogren, D. Young, B.J. Hoffer, and L. Olson. 1995. Protection and repair of the nigrostriatal dopaminergic system by GDNF in vivo. Nature. 373:335339.[CrossRef][Medline]
Trupp, M., M. Ryden, H. Jornvall, H. Funakoshi, T. Timmusk, E. Arenas, and C.F. Ibanez. 1995. Peripheral expression and biological activities of GDNF, a new neurotrophic factor for avian and mammalian peripheral neurons. J. Cell Biol. 130:137148.[Abstract]
Trupp, M., R. Scott, S.R. Whittemore, and C.F. Ibanez. 1999. Ret-dependent and -independent mechanisms of glial cell line-derived neurotrophic factor signaling in neuronal cells. J. Biol. Chem. 274:2088520894.
Unsicker, K., and K. Krieglstein. 2000. Co-activation of TGF-ßs and cytokine signaling pathways are required for neurotrophic functions. Cytokine Growth Factor Rev. 11:97102.[CrossRef][Medline]
Vlahos, C.J., W.F. Matter, K.Y. Hui, and R.F. Brown. 1994. A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). J. Biol. Chem. 269:52415248.
Wrana, J. 2000. Crossing smads. Sci. STKE. 2000:RE1.
Yanagi, Y., M. Suzawa, M. Kawabata, K. Miyazono, J. Yanagisawa, and S. Kato. 1999. Positive and negative modulation of vitamin D receptor function by transforming growth factor-beta signaling through smad proteins. J. Biol. Chem. 274:1297112974.
Yanagisawa, J., Y. Yanagi, Y. Masuhiro, M. Suzawa, M. Watanabe, K. Kashiwagi, T. Toriyabe, M. Kawabata, K. Miyazono, and S. Kato. 1999. Convergence of transforming growth factor-beta and vitamin D signaling pathways on SMAD transcriptional coactivators. Science. 283:13171321.