beta1-integrin mediates myelin-associated glycoprotein signaling in neuronal growth cones
- Eyleen LK Goh†1, 2,
- Ju Kim Young†1, 2,
- Kenichiro Kuwako3,
- Marc Tessier-Lavigne4,
- Zhigang He3,
- John W Griffin2, 5 and
- Guo-li Ming1, 2, 5Email author
© Goh et al; licensee BioMed Central Ltd. 2008
Received: 06 October 2008
Accepted: 15 October 2008
Published: 15 October 2008
Several myelin-associated factors that inhibit axon growth of mature neurons, including Nogo66, myelin-associated glycoprotein (MAG) and oligodendrocyte myelin glycoprotein (OMgp), can associate with a common GPI-linked protein Nogo-66 receptor (NgR). Accumulating evidence suggests that myelin inhibitors also signal through unknown NgR-independent mechanisms. Here we show that MAG, a RGD tri-peptide containing protein, forms a complex with β1-integrin to mediate axonal growth cone turning responses of several neuronal types. Mutations that alter the RGD motif in MAG or inhibition of β1-integrin function, but not removal of NgRs, abolish these MAG-dependent events. In contrast, OMgp-induced repulsion is not affected by inhibition of b1-integrin function. We further show that MAG stimulates tyrosine phosphorylation of focal adhesion kinase (FAK), which in turn is required for MAG-induced growth cone turning. These studies identify β1-integrin as a specific mediator for MAG in growth cone turning responses, acting through FAK activation.
Myelin-associated glycoprotein (MAG), a component of myelin in the central and peripheral nervous system, promotes neurite outgrowth during the embryonic development, but inhibits axonal regeneration in the adult nervous system [1–9]. Following damage to the adult CNS, disruption of the myelin sheath leads to the release in abundance of a soluble fragment containing the MAG extracellular domain, which possesses potent inhibitory activity for neurite outgrowth . A receptor complex consisting of NgR, p75/TROY and Lingo-1 has been shown to mediate the inhibitory activities of three major myelin-associated inhibitors: MAG, Nogo66 (an extracellular domain of NogoA) and OMgp [11–19]. While certain classes of neurons from p75 knockout mice exhibit reduced responses to myelin inhibitors, several types of neurons lacking NgRs are still inhibited by these factors [20–23]. In particular, a recent study using NgR germ-line knockout mice and short-hairpin RNA (shRNA) interference suggests that NgR is only partially involved in the acute growth cone collapse induced by MAG and OMgp, but may not be required for the long-term growth inhibitory actions of these two factor . Thus, it is likely that an additional signaling mechanism is critical for transducing the signaling of MAG and possibly other myelin-associated inhibitors.
Integrins, consisting of α and β chains, are heterodimeric receptors for components of the extracellular matrix and for specific ligands . Extensive studies have shown that integrins are important for cytoskeleton dynamics, cell adhesion and migration . Emerging evidence also suggests that integrins regulate neurite extension, axonal guidance and neuronal migration through direct or indirect mechanisms . Many downstream signaling of guidance cues and integrins converges onto common pathways that regulate cytoskeleton rearrangement, thus integrins and guidance cues could also modulate effects of each other [27–30]. In addition, exogenous laminin as a substrate impedes MAG and myelin inhibitory activity on neurite initiation and outgrowth [31, 32]. These results suggest the existence of competitive crosstalk between integrin ligands and inhibitory factors associated with myelin and glia scar.
Here we demonstrated that β1-integrin acts as a receptor for MAG to mediate growth cone responses independent of NgRs in mammalian neurons. Our study identifies a novel signaling mechanism for MAG and may have significant implications for therapeutic modulation of MAG functions in the adult nervous system.
MAG interacts with β1-integrin
We next examined the requirement of the RGD motif in MAG for its association with β1-integrin. Biochemical analysis showed that the association between MAG and β1-integrin was attenuated by the disintegrin echistatin, a viper-venom-derived RGD peptide that specifically inhibitsβ1 and β3 containing integrins , and by Ha2/5, a specific β1-integrin function blocking antibody (Fig. 1E). We also constructed a mutant form of MAG (Fig. 1A), in which the RGD motif was mutated to KGE (MAG-KGE) and is not recognized by integrins . Under the same experimental condition, purified MAG-KGE was unable to interact with β1-integrin whereas purified MAG-RGD (wild-type) could (Fig. 1C). Taken together, these results demonstrated that the association between MAG and β1-integrin is direct and occurs via a classical mode of integrin-ligand interaction[33, 34].
β1-integrin function is required for MAG-induced growth cone response
To determine whether the function of MAG-integrin interactions are limited to hippocampal neurons, we examined growth cone responses of postnatal rat cerebellar granule cells . Axonal growth cones of these neurons exhibited significant repulsive responses in the MAG gradient (150 μg/ml in the pipette; Fig. 4B). Importantly, MAG-induced repulsion of these neurons was also abolished in the presence of Ha2/5 (1.0 μg/ml; Fig. 4B). These results show that β1-integrin function is required for MAG-induced growth cone responses in different types of mammalian CNS neurons.
β1-integrin function is not required for OMgp-induced growth cone turning
β1-integrin mediates MAG-induced growth cone turning independent of NgR
To directly assess the specific role of NgR in MAG-induced growth cone responses, we examined neurons from NgR null mice . Mouse hippocampal neurons lacking NgR still exhibited significant repulsive responses to the MAG gradient (Fig. 6B), suggesting that NgR is dispensible for MAG-induced growth cone repulsion. More importantly, MAG-induced repulsion of neurons lacking NgR was also abolished by Ha2/5 (Fig. 6B). In addition, we were unable to detect interactions between β1-integrin and any member of the known NgR signaling complex, including NgR, p75, TROY and Lingo-1, either in the presence or absence of MAG [see Additional file 4]. Taken together, these findings are consistent with the notion that β1-integrin mediates MAG-induced growth cone responses independent of the known NgR receptor complex.
FAK mediates MAG-induced growth cone turning downstream of β1-integrin
We further examined specific tyrosine residues of FAK that are phosphorylated upon MAG stimulation in hippocampal neurons. As shown with site-specific phospho-tyrosine FAK antibodies, MAG induced a significant increase in the phosphorylation of FAK at tyrosine residues 397 and 861 (Fig. 7D). Similar β1-integrin-dependent phosphorylation of FAK these tyrosine residues were also found in embryonic cortical neurons treated with MAG (data not shown).
To determine the functional role of FAK and its tyrosine phosphorylation in MAG-induced growth cone turning, we transfected P5 rat hippocampal neurons with shRNA constructs to knockdown the expression of endogenous FAK [see Additional file 5]. Expression of shRNA-FAK, but not control shRNA, abolished MAG-induced repulsion (Fig. 7G). We also transfected neurons with expression constructs for either wild-type FAK (WT-FAK) or a mutant FAK (FAK-Y397/861F) that cannot be phosphorylated on tyrosine residues 397 and 861. Expression of mutant FAK-Y397/861F, but not WT-FAK, also abolished MAG-induced repulsion (Fig. 7E–G). Together, these findings demonstrated that MAG-induced phosphorylation of FAK is essential for growth cone turning responses to MAG.
We provided biochemical and functional evidence that β1-integrin acts as a direct receptor to mediate MAG-induced growth cone responses of mammalian CNS neurons from both embryonic and postnatal stages. We further showed that β1-integrin signaling mediates MAG effects through FAK phosphorylation and is independent of NgR. Taken together, these results demonstarted a common role of β1-integrin in mediating MAG signaling for diverse functions in different neuronal types.
Previous studies led to the finding that Nogo66, OMgp and MAG, three major inhibitors associated with myelin, all bind to NgR and appear to signal at axonal growth cones through a common receptor complex containing NgR, p75/TROY and Lingo-1 [11–19]. Two additional human homologs of NgR (NgR2 and NgR3) are found to be expressed in CNS neurons [52, 53]. While neither binds to Nogo66 , NgR2 appears to bind to MAG . Accumulating evidence suggests that inhibitors associated with myelin may signal independent of NgRs [20–22]. Our growth cone turning results using NgR null neurons and PI-PLC treatment are in agreement with these findings (Fig. 6). MAG has also been reported to inhibit neurite outgrowth through sialoglycoproteins [49, 56] and gangliosides [23, 57] in postnatal DRG neurons and cerebellar granule neurons. Our results with the RAD mutant that has an intact arginine residue to mediate the binding of MAG to sialic acids  (Fig. 2) but failed to induce growth cone responses suggest a specific requirement of β1-integrin in MAG signaling. Whether sialic acids of sialoglycoproteins and gangliosides serve as a co-receptor together with β1-integrin to mediate MAG signaling remains to be determined .
Integrin signaling has been shown to be critical for axon guidance and cell migration, either as a direct receptor or as a modulator of guidance signaling . Laminins, when presented as substrates for integrins, are known to promote neurite outgrowth  and have been shown to override inhibitory activities of MAG and myelin-associated factors [31, 32]. It is possible that, in addition to the growth promoting activity of laminin, competitions at the receptor levels by laminins and MAG may also contribute to the enhancement of neurite initiation and outgrowth [60, 61]. Our results also support the notion that integrin signaling plays an instructive, rather than permissive role, in MAG-induced growth cone turning (Fig. 3). Activation of the integrin/FAK pathway is normally associated with enhanced nerve growth/growth cone attraction [26, 62]. Interestingly, β1-integrin signaling is required for both MAG-induced repulsion and attraction of CNS neurons at different developmental stages and under different cellular status (Fig. 4; [see Additional file 2]). A recent study also showed that inhibition of neurite outgrowth by fibrinogen requires β3-integrin function . Taken together, these findings suggest a bi-functional role of integrin/FAK signaling in regulating the dynamics of cytoskeletal proteins. Our results show that β1-integrin serves as a specific receptor for MAG, but not for OMgp. Consistent with the selective involvement of β1-integrin in mediating MAG effects, human and rodent MAG contain a RGD-tri-peptide motif characteristic of integrin binding proteins [33, 34], whereas OMgp and Nogo do not. Interestingly, MAG homologs in fugu and Zebrafish, species with the capacity for axonal regeneration, do not contain an intact RGD motif (Fig. 1). The extent to which different receptors mediate distinct effects of MAG in various species remains to be determined. Our results further demonstrate that integrin/FAK signaling mediates MAG effects independent of the NgR receptor complex. These findings suggest that a diversity of signaling mechansims is likely to be employed to limit axon regeneration in the adult CNS. Given the general role of β1-integrin in mediating diverse functions of MAG in the adult central nervous system, our findings may have implications for novel strategies for therapeutic modulation of MAG functions in the adult nervous system.
Our results show that β1-integrin serves as a specific receptor for MAG, but not for OMgp. Consistent with the selective involvement of β1-integrin in mediating MAG effects, human and rodent MAG contain a RGD-tri-peptide motif characteristic of integrin binding proteins [33, 34], whereas OMgp and Nogo do not. Interestingly, MAG homologs in fugu and Zebrafish, species with the capacity for axonal regeneration, do not contain an intact RGD motif (Fig. 1). The extent to which different receptors mediate distinct effects of MAG in various species remains to be determined. Our results further demonstrate that integrin/FAK signaling mediates MAG effects independent of the NgR receptor complex. These findings suggest that a diversity of signaling mechansims is likely to be employed to limit axon regeneration in the adult CNS. Given the general role of β1-integrin in mediating diverse functions of MAG in the adult central nervous system, our findings may have implications for novel strategies for therapeutic modulation of MAG functions in the adult nervous system.
Primary neuronal cultures
Hippocampal neurons were isolated from the hippocampi embryonic and postnatal rats, or wild-type and NgR knockout mice  as previously described . Similarly, cerebellar neurons were isolated from P5 rat cerebellum . Dissociated neurons were cultured on poly-L-lysine coated plates or coverslips without laminin as previously described . For biochemical analysis, E18 neurons were treated with AraC to eliminate dividing astrocytes and used at 5 days after plating as previously described . For growth cone turning assay, neurons were used between 2–3 days after plating. PI-PLC (1 or 2 units/ml), Ha2/5 (1 μg/ml) or echistatin (100 nM)  were added 30 mins prior to and were present during the growth cone turning assay.
Expression constructs and neuronal transfection
Mutation of MAG-Fc was generated by site directed mutagenesis and confirmed by DNA sequencing. Expression plasmids of wild-type MAG (RGD) or mutant forms of MAG (KGE, RAD) were transfected into 293 Ebna cells and proteins were collected from the media and affinity purified using protein A sepharose. MAG-Fc from R & D systems was also used. The pUEG vector was used to co-express GFP (under the control of the EF1α promoter) and a specific shRNA (under the control of the human U6 promoter in the same vector)[65, 66]. Several shRNAs against different regions of β1-integrin or FAK, and control shRNA against DsRed  were generated. The following short-hairpin sequences were cloned into pUEG vector using a PCR SHAGing strategy : shRNA-control: AGTTCCAGTACGGCTCCAA; shRNA-β1-integrin-3: TGCCTACTTCTGCACGATG; shRNA-FAK1: GCACGTGGCCTGCTATGGA; shRNA-FAK2: GCCTTAACAATGCGTCAGT; and shRNA-FAK3: TCCAGAAGACAGGCTACCG. To validate the specificity and efficiency of shRNAs, pUEG vectors with different shRNAs were transfected into 3T3 cells and cell lysates were prepared for western blot analysis of β1-integrin or FAK expression with specific antibodies, respectively.
Rat primary hippocampal neurons were transfected with the Amaxa transfection system following protocols from the manufacturer. Briefly, hippocampal neurons were isolated and 100 μl of nucleofector solution was added to resuspend the cell pellet. Different expression constructs (1–5 μg) for GFP, WT-FAK-GFP, FAK-Y397/861F-GFP, WT-Rho-GFP, DN-Rho-GFP, or pUEG vectors for shRNAs [65, 66], were added to the cell suspension and the cell-DNA mix was then transferred to cuvettes for electroporation. The cells were cultured in DMEM with 10% fetal bovine serum for 24 hrs before changing to the serum-free neurobasal medium . GFP+ neurons were identified for the turning assay.
Neurons at 5 days after plating were treated with 2 units/ml PI-PLC, 100 nM Echistatin or 0.5–2.0 μg/ml Ha2/5, and then stimulated with 2.0 μg/ml MAG or 0.5 μg/ml OMgp for the indicated time periods. Cells were then lysed in immunoprecipitation buffer (1% Triton X-100; 150 mM NaCl; 10 mM Tris, pH 7.4; 1 mM EDTA; 1 mM EGTA; 1% Nonidet P-40; 0.2 mM Na3VO4; 1 μg/ml protease inhibitor cocktail; and 0.1 mM PMSF). Samples were immunoprecipitated with polyclonal antibody against FAK (Santa Cruz Biotechnology, Inc.), human Fc (Sigma) or β1-integrin (Chemicon), and then subjected to western blot analysis. The following antibodies were used: monoclonal antibody against tyrosine phosphorylated proteins (pY20, Transduction Laboratories; 1:1000), rabbit polyclonal antibodies against β1-integrin (1:1000), FAK (1:1000), FAK-pY397 (Biosource; 1:1000), FAK-pY861 (Biosource; 1:1000), or human Fc (1:1000). Blots were stripped and reblotted with the same antibodies used for their immunoprecipitation to ensure equal loading of the immunoprecipitated proteins.
For GST pull-down experiments, the extracellular domain (ECD) of β1-integrin was amplified from mouse brain cDNA and cloned into the GST-fusion expression vector (pGEX-4T-1; Amersham-Pharmacia Biotech) to express GST-β1 (ECD) fusion protein. The fusion protein was purified using glutathione beads according to the manufacturer's manual (Amersham-Pharmacia Biotech). Native Fc fragment (2 μg/ml) or MAG-Fc (2 μg/ml) was then added to the purified GST-β1 (ECD) overnight at 4°C. The samples were further processed according to the standard immunoprecipitation protocol as described.
For experiments testing potential interactions between β1-integrin and the NgR receptor complex, HEK293 cells were transfected with expression constructs for NgR, p75, TROY, or Lingo-1, respectively, as previously described . Transfected cells were stimulated with MAG (5 μg/ml) or medium and then were immunoprecipitated with anti-β1-integrin antibodies and immunoblotted for respective components of the NgR receptor complex. Total cell lysates were also examined to show the expression of endogenous β1-integrin and proteins from transfection.
Growth cone turning assay
Microscopic gradients of recombinant MAG (150 μg/ml in the pipette; 1.8 μM) and OMgp (5 μg/ml in the pipette; 0.1 μM) were produced as previously described to induce growth cone turning responses [40, 41, 45, 69]. In some experiments, MAG (150 ng/ml) was added to the bath solution and microscopic gradients were produced with saline or Ha2/5 (0.5 μg/μl) in the pipette. As another control, a gradient of Ha2/5 (0.5 μg/μl in the pipette) was applied in the absence of MAG in the bath. Previous analysis [69, 70] have shown that, under standard pulsing conditions, the average concentration of the factor at the growth cone at a distance of 100 μm from the pipette tip is about 103 fold lower than that in the pipette and the concentration gradient across the growth cone is about 5–10%. Axons were identified as the longest neurite in these cultures at stage 2–3 of hippocampal neurons as previously described . Growth cone assays were carried out for 30 min at room temperature. The turning angle was defined by the angle between the original direction of neurite extension and a line connecting the position of the center of the growth cone at the onset and the end of the 30 min period. To assure accurate measurement of turning angles, only neurons with axonal extension > 5 μm over the 30 min period were included for analysis.
We thank David Ginty, Alex Kolodkin and Ronald Schnaar for critical reading of the manuscript, Lihong Liu and Kurt Sailor for technical help. Supported by grants from the Adelson Medical Research Foundation, the Culpeper Scholarship in Medical Science, March of Dimes, Klingenstein Fellowship Award in the Neuroscience, and National Institute of Health (NS048271) to G-l. M.
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