To develop a cellular model that would enable chemical-genetic characterization of Nrg1−ErbB4 signaling and discovery of new small-molecule probes, we chose the neuroendocrine cell line PC12 (23
), derived from a rat pheochromocytoma, as a model neuronal system for several reasons. First, PC12 cells do not naturally express ErbB4 but are known to express EGFR (ErbB1), ErbB2, and ErbB3, so this would allow us to express exogenous genetically altered ErbB4 receptors. Second, PC12 cells engineered to express human ErbB4 have been reported to differentiate and undergo neuritogenesis upon treatment with recombinant Nrg1 (24
), which we reasoned could provide a phenotype that could be quantified using automated microscopy and image analysis. Finally, NGF-induced differentiation has been extensively studied in PC12 cells, and much is known about the downstream signaling pathways, which we reasoned would assist in comparing the selectivity of any compounds that we identify.
To create a system for chemical-genetic study, we prepared a stable PC12 cell line that coexpresses green fluorescent protein (GFP) and the human ErbB4 isoform JMa-Cyt2 and a control cell line PC12-GFP that stably expresses GFP but lacks ErbB4. The PC12-ErbB4-GFP cells were examined by fluorescence imaging for the effects of NGF and Nrg1 on neurite outgrowth. As expected, both the PC12-ErbB4-GFP and the PC12-GFP cell lines exhibited the characteristic morphological changes indicative of neuronal differentiation involving neurite outgrowth when treated with NGF (Figure A). Under low magnification (<10×), the distribution of coexpressed GFP is uniform throughout the cell body and neurite projections, and the fluorescent image reliably represents the whole cell body and attached processes (Figure B). Nrg1 stimulation of PC12-ErbB4-GFP cells, but not PC12-GFP, for 2 days resulted in significant neurite outgrowth similar to the effects of NGF. This result indicated that although PC12 cells express ErbB3, which can also bind to Nrg1 and heterodimerize with other ErbB family members, the presence of the ErbB4 receptor is necessary for Nrg1 signaling to stimulate neuritogenesis. In addition, EGF treatment failed to stimulate neurite outgrowth in both cell lines (Figure A), suggesting that although EGFR-activation might also activate other ErbB receptors via heterodimerization, it is not capable of triggering neuritogenesis in the presence or absence of ErbB4.
Figure 1 Nrg1 induces neurite outgrowth and Erk1/2 phosphorylation in PC12-ErbB4-GFP cells. (A) PC12-GFP (vector) and PC12-ErbB4-GFP (ErbB4) cells were seeded in a 96-well plate at 1200 cells/well and incubated at 5% CO2, 37 °C, for 12 h and then were (more ...)
ErbB4 is known to activate downstream effectors of neurotrophic factor pathways such as mitogen-activated protein kinase (MAPK). To verify that PC12-ErbB4-GFP cells activate MAPK pathway components in response to Nrg1, we examined the ability of NGF and Nrg1 treatment to activate of the MAPK pathway as measured by phosphorylation of extracellular signal-regulated kinase (Erk1/2). In both the PC12-GFP and PC12-ErbB4-GFP cell lines, NGF induced a rapid phosphorylation of Erk1/2, which peaked as early as 5 min after treatment. In contrast, Nrg1 induced a strong phosphorylation of Erk1/2 only in PC12-ErbB4-GFP cells (Figure C). In addition, it has been reported that Nrg1 increases the release of the intracellular domain of ErbB4, ErbB4-ICD, in neural precursor cells (25
). However, we did not observe this effect in PC12 cells (Figure C). We also found that a γ-secretase inhibitor that is known to inhibit the cleavage of ErbB4 did not inhibit Nrg1-induced neurite outgrowth in PC12-ErbB4 cells (data not shown).
Automated Live-Cell Neurite Outgrowth Assay
Having validated that stimulation of PC12-ErbB4-GFP cells with Nrg1 could induce neuritogenesis, we sought next to determine whether the outgrowth phenotype was suitable for use with automated imaging and image analysis. The expression of soluble GFP in PC12 cells allowed the use of automated microscopy to acquire fluorescent images for the quantitative analysis of cell number and morphological changes associated with neuronal differentiation over a developmental time course. To minimize cell clumping and intersection of neurites in 96- or 384-well plates for time periods of up to 4 days, we seeded cells at a low density of ~4000 cells/cm2
. Imaging with an ImageXpress 5000A automated microscope equipped with 4× objective enabled the acquisition of the entire well of a 384-well plate in one image with sufficient resolution to accurately detect and quantify various properties of neurites and cell bodies. A typical pixel map of the segmentation mask generated by the MetaXpress software is shown in Figure A. Cell bodies were identified as pixel blocks with minimum area of 200 μm2
and maximum width of 40 μm, and the neurites were subsequently identified as line objects longer than 10 μm and connected to each cell body. The mean neurite length per cell for each well was quantified as a single parameter (see the Methods
section for a more detailed description of imaging and analysis parameters), represented as “mean outgrowth per cell (μm)”. We accounted for variation in cell density due to seeding or to the effect of antiproliferative compounds by quantifying neurite outgrowth on a per-cell basis. The use of automated image analysis methods enabled the accurate assessment of morphological properties of hundreds of cells per well without human bias of which cells to measure the properties.
Figure 2 Nrg1- and NGF-induced neurite outgrowth is dose-dependent and quantitatively measurable. PC12-ErbB4-GFP cells were seeded in 384-well assay plates at 400 cells/well for 12 h and then left untreated or treated with Nrg1 or NGF at indicated concentrations. (more ...)
We studied the robustness of our automated neurite detection by comparing the dose response of PC12-ErbB4-GFP cells to Nrg1 and NGF as a function of time with image acquisition every 24 h. Both NGF and Nrg1 stimulated a continuous increase in mean neurite length over a four- day time course. NGF-treated cells appeared to differentiate more slowly than Nrg1-treated cells in the first 24 h, but after four days, the mean length of neurites per cell in both treatments was similar (Figure B). This delayed response to NGF, but similar overall effect after four days, might be explained by an up regulation of the expression levels of the tyrosine kinase receptor TrkA (26
) or a secondary receptor of NGF, such as p75NTR27
, which is known to potentiate the activity of TrkA through the formation of a high-affinity NGF receptor. The mean neurite length exhibited a strong correlation to the dose of Nrg1 or NGF added to the cells especially under concentrations of 10 ng/mL at day two (Figure C) and under 20 ng/mL at day four (Figure D). These data suggested that we could use automated microscopy to measure neurite length as a phenotype for screening.
ErbB4 Activation Is Sufficient for Nrg1-Dependent Neuritogenesis
Having established methods for quantitatively measuring Nrg1-induced neuritogenesis in PC12-ErbB4-GFP cells, before embarking on a screen for chemical modulators, we sought to better understand the signaling events associated with Nrg1−ErbB4 signaling to assist in the eventual downstream characterization of any probes that might be identified. Besides activating ErbB4 homodimers, Nrg1 can mediate the formation of ErbB dimers consisting of ErbB3 (21
), as well as EGFR (20
). Thus, although ErbB4 is required for Nrg1 to promote neurite outgrowth, since all ErbB family members are expressed in the PC12-ErbB4-GFP cells, it remained unclear whether the other ErbB receptors were also playing a role. To address specifically the contribution of other ErbB family members to Nrg1−ErbB4 dependent neurite outgrowth, we individually reduced the expression of ErbB1/EGFR, ErbB2, and ErbB3 with pools of small inhibitory RNAs (siRNAs). The efficacy and specificity of each ErbB receptor siRNA pool was verified by Western blotting (Figure A). The Nrg1-driven neurite outgrowth was diminished only when ErbB4 expression was reduced (Figure B,C), suggesting that ErbB1/EGFR, ErbB2, and ErbB3 do not contribute significantly to Nrg1-dependent neuritogenesis in PC12 cells and that ErbB4 is necessary for the observed effects of Nrg1 on neuritogenesis. These results are consistent with the observation that PC12 cells only extended neurites in response to Nrg1 when the ErbB4 receptor was expressed. Surprisingly, we observed that reducing ErbB3 expression enhanced Nrg1-induced neurite outgrowth, suggesting that ErbB3 inhibits some aspect of neuritogenesis in our PC12 cell system. While the molecular basis for this observation is not understood, it suggests that ErbB3, although lacking a functional kinase domain, may contribute to the signaling of kinase-active ErbB receptors as well as other proteins important for neuritogenesis (28
Figure 3 Nrg1-driven neurite outgrowth is ErbB4- and Erk1/2-dependent. PC12-ErbB4-GFP cells were seeded in a 96-well assay plate at 3000 cells/well for 12 h followed by transfection of siRNAs specific for EGFR, ErbB2, ErbB3, ErbB4, and scrambled (SC), as indicated, (more ...)
Nrg1-Dependent Neuritogenesis Is Inhibited by MEK inhibitors but Not PI3K Inhibitors
To investigate the downstream components of Nrg1−ErbB4 signaling that triggers neuritogenesis, we specifically examined two key kinase cascades coupled to receptor tyrosine kinase signaling, MEK and PI3K pathways. The ERK kinase (MEK) has long been known to mediate NGF-induced PC12 differentiation (29
). In our system, two specific MEK inhibitors, PD098059 and U0126, attenuated both NGF- and Nrg1-induced neurite outgrowth (Figure D,E). These results further validated that both Nrg1- and NGF-induced neuritogenesis are dependent on the Erk1/2 cascade. PI3K is another important component of many neurotrophic factor signal transduction pathways. Although we used the human ErbB4 isoform JMa-Cyt2, which lacks the PI3K binding domain, it has been demonstrated that all ErbB4 isoforms, including Cyt2 isoforms, associate with and activate PI3K (30
). Consistent with this finding, we observed an up-regulation of phosphorylated Akt (Ser473), an indicator of PI3K activation, when cells were exposed to Nrg1 (data not shown). To determine whether both NGF- and NRG1-induced neuritogenesis requires PI3K signaling, PC12-ErbB4-GFP cells were treated with two structurally distinct PI3K inhibitors, LY294002 and wortmannin. Both of these PI3K inhibitors caused an inhibitory effect on NGF-induced neurite outgrowth as expected (31
). In contrast, neither LY294002 nor wortmannin were capable of inhibiting Nrg1-induced neurite outgrowth at doses ranging from 0.1 to 10 μM (Figure B). Collectively, these results suggest that although both Nrg1 and NGF stimulate Erk1/2 and PI3K cascades, activation of PI3K is not required for neuritogenesis induced by Nrg1 in our PC12 cellular system.
Discovery of Small-Molecule Probes of Nrg1-Induced Neuritogenesis
Having shown that our cellular model activated neuritogenesis in an Nrg1−ErbB4-dependent manner and retained the ability to respond to NGF, we next screened for small molecules that could specifically modulate Nrg1−ErbB4 signaling without affecting NGF-induced neuritogenesis. We initially tested 400 known bioactive small molecules at a single dose (~10 μM) for their ability to modulate neurite outgrowth induced by the addition of Nrg1 and NGF (see Supplementary Tables 1 and 2, Supporting Information
, for the complete list of compounds tested and resulting high-content imaging data). A total of three 384-well plates (one DMSO control plate and two compound plates each containing 200 bioactives and 184 DMSO control wells) were screened (Figure A).
Figure 4 Image-based assay of 400 bioactive small molecules. (A) Summary of the overall HTS performed using the PC12-ErbB4-GFP cell line. Cells were seeded in 384-well assay plate at 400 cells/well and incubated for 12 h. Compounds were then pin-transferred into (more ...)
Cell morphological features measured from each image using MetaXpress software included (1) percent of cells with significant neurite growth, (2) number of cells, (3) total neurite outgrowth, (4) mean neurite outgrowth length per cell, (5) normalized mean neurite outgrowth per cell, (6) mean number of processes per cell, (7) mean branches per cell, and (8) mean cell body area. Each feature is described in the Methods
section in more detail, and the complete data set for Nrg1- and NGF-treated wells is provided in Supplementary Tables 1 and 2, respectively, in the Supporting Information
. Supplementary Tables 3−10, Supporting Information
, provide a global statistical analysis of the eight cellular features, an assessment of the degree to which the cellular feature is normally distributed, and the observed relationships of each feature to each other in the form of Pearson correlation coefficient. Based upon these global analyses, as shown in Supplementary Figure 3, Supporting Information
, a graphical representation of the two-dimensional Pearson correlation map between the eight cellular features in the Nrg1 and NGF high-content imaging screens reveals that many of these cellular features are highly correlated across the 400 compound treatments and there existed differences in the global feature profiles between Nrg1 and NGF. However, we also noticed that there were potentially informative relationships among the cellular and neurite features. For instance, in the case of Nrg1-treated cells (see Supplementary Figure 4, Supporting Information
), the mean neurite length feature was correlated with the percentage of cells with significant neurite outgrowth (r
= 0.93) and the number of processes per cell (r
= 0.95) but was more moderately correlated with the number of branches measured per cell (r
= 0.70). In contrast, the mean neurite length per cell correlated less with cell body size (r
= 0.5) suggesting that Nrg1-induced neuritogenesis can be separated from the control of soma size.
Since we found that the mean neurite length per cell feature was strongly positively correlated to the dose and duration of Nrg1 and NGF treatment (Figure ), we chose this feature as a surrogate of Nrg1 and NGF signaling for use in image-based screening while recognizing that analysis of other features may lead to different types of modulators of Nrg1 signaling. With this feature, in our assay, the 752 DMSO control wells for each treatment exhibited consistent background levels of neurite outgrowth and changes in cell number (see Supplementary Tables 3 and 4, Supporting Information
). The mean neurite length values from each set of DMSO control treatments was taken as the baseline value. For subsequent data visualization, the mean neurite length value in each well upon Nrg1 or NGF induction was normalized to the respective baseline value (Figure B). Within the library of 400 known bioactives, 51 compounds led to a significant reduction in cell number in either the Nrg1 or NGF treatment conditions as defined by a threshold of having less than 100 cells after two days of incubation. While these compounds may have additional phenotypes at lower concentrations, they were not considered further in the studies reported here. The remaining 349 compounds were categorized into nine classes based on their relative activities compared with the DMSO controls and their specificities toward Nrg1- and NGF-induced neurite outgrowth (Figure B) using a simple fold-change cutoff of 2-fold for molecules that potentiated neurite outgrowth and 0.5-fold for molecules that inhibited outgrowth. The numbers of bioactives in each category based upon this classification are summarized in Figure C.
Characterization of 4-Anilino-quinazoline-Based Inhibitors of Nrg1 Signaling
To identify specific inhibitors of Nrg1−ErbB4 signaling, we first focused our analysis on those compounds that inhibited Nrg1-induced neurite outgrowth but had no effect on NGF-induced neurite outgrowth. From our original screen, we noted that two 4-anilino-quinazoline-containing compounds, WHI-P180 and CL-387,785, satisfied our selection criteria (Figure C) and had no effect on the cell number, indicating that they did not alter cell proliferation over the two-day time course.
While both WHI-P180 and CL-387,785 were previously shown to inhibit EGFR (32
), little was known about the ability of these two compounds to inhibit Nrg1 signaling. 4-Anilino-quinazoline-based compounds are known to reversibly and competitively bind to the ATP pocket of EGFR (34
). To further explore the ability of 4-anilino-quinazoline-based compounds to inhibit Nrg1-signaling, we tested four commonly used small molecules of this structural class: AG1478, PD158780, Iressa (gefitinib), and Tarceva (erlotinib) (Figure A), along with nine additional 4-anilino-quinazolines also classified as EGFR inhibitors (Supplementary Figure 2, Supporting Information
). Iressa and Tarceva are Food and Drug Administration (FDA)-approved drugs used for the treatment of non-small-cell lung cancer through a mechanism thought to involve the inhibition of the tyrosine kinase activity of EGFR and have been optimized for their pharmacological properties and safety in humans. All four 4-anilino-quinazolines inhibited Nrg1-induced outgrowth in a dose-dependent manner, while PD158780 appeared to have a lower potency (at ~2 μM) against Nrg1 stimulation and decrease the mean length of neurites induced by NGF at the same concentration. On the other hand, AG1478, Iressa, and Tarceva had no significant effect on NGF-induced neurite outgrowth but all inhibited Nrg1-induced neurite outgrowth with EC50
’s of ~500 nM (Figure B). The two irreversible EGFR inhibitors, CL-387,785 and PD168393, were both superior inhibitors of Nrg1-induced neurite outgrowth (EC50
≈ 100 nM), while PD168393 exhibited the strongest inhibition of NGF-induced neurite outgrowth as well (Supplementary Figure 2, Supporting Information
). CP-724,714, reported to be an inhibitor ErbB2 selective over EGFR (35
), was the weakest compound to inhibit Nrg1 (EC50
≈ 4 μM) (Supplementary Figure 2, Supporting Information
). Taken together, most of the 4-anilino-quinazoline-based compounds in this study inhibit Nrg1-induced neurite outgrowth with various efficacies and specificities over NGF-induced neurite outgrowth.
Figure 5 Characterization of 4-anilino-quinazolines that inhibit Nrg1-induced neurite outgrowth. (A) Structures of four representative 4-anilino-quinazolines, AG1478, PD158780, Iressa, and Tarceva. (B) PC12-ErbB4-GFP cells were pretreated with serially diluted (more ...)
Inhibition of ErbB4 Activation by Nrg1 Signaling
While Iressa was originally developed to selectively target EGFR (36
), our results described here suggest that Iressa also inhibits ErbB4-dependent neuritogenesis. To characterize the interaction between Iressa and ErbB4 in greater detail and to test whether Iressa inhibits the activation of ErbB4 by Nrg1, ErbB4 was immunoprecipitated from PC12-ErbB4-GFP cells after a short exposure to Nrg1 with or without Iressa treatment. The phosphorylation status of ErbB4, a measure of receptor activity, was examined using a phosphotyrosine specific antibody. Indeed, the phosphorylation of ErbB4 receptors induced by Nrg1 was inhibited when cells were treated with Iressa (2 μM), and the subsequent phosphorylation of the downstream Erk1/2 was also diminished. In contrast, Iressa did not affect NGF-induced activation of Erk1/2, thereby confirming the selectivity observed for the 4-anilino-quinazolines in the initial small-molecule screen (Figure C). In addition, when cells were treated with Nrg1 (20 ng/mL), the phosphorylation levels of ErbB4 and Erk1/2 were diminished by Iressa (0.2−5 μM) in a dose-dependent manner as determined by Western blotting with phospho-ErbB4 and phospho-Erk1/2 antibodies (Figure D,E), respectively. These results indicate that Iressa treatment inhibits ErbB4 receptor activation and its downstream signaling.
Cellular and Biochemical Characterization of Targets of Iressa Involved in Neuritogenesis
Although we demonstrated that ErbB4 activation is necessary and sufficient for Nrg1-induced neuritogenesis (Figure A) and that the activation of ErbB4 is inhibited by Iressa (Figure D,E), it remained possible that the inhibition is indirect through inhibition of trans-phosphorylation by other ErbB family members or other targets. Since ErbB3 lacks kinase activity, we ruled this ErbB receptor out as a direct target. It is also known that Iressa inhibits EGFR (IC50
≈ 30 nM) more potently than ErbB2 (IC50
> 3.7 μM) (36
). Furthermore, the selective ErbB2-inhibitor, CP-724,714, poorly inhibited Nrg1-induced neurite outgrowth (Supplementary Figure 2, Supporting Information
), suggesting that inhibiting ErbB2 is not critical. Thus, we focused our efforts on determining whether the inhibition of ErbB4 is due to an indirect inhibition of EGFR.
To determine whether Iressa acts through ErbB4 to inhibit neurite outgrowth, to complement the chemical treatments described above, we used RNAi-mediated silencing to reduce the levels of ErbB family members and then treated with Iressa. Iressa was found to still effectively diminish neurite outgrowth in cells where expression of EGFR, ErbB2, or ErbB3 was reduced by siRNAs (Figure F), suggesting that none of these three ErbB family members are required for Iressa’s effects on Nrg1 signaling and that their loss-of-function does not potentiate the effect of Iressa. Of note, even though ErbB3 silencing potentiated the effects of Nrg1, the enhanced neurite outgrowth observed upon ErbB3 knock down was still blocked by Iressa treatment. This result suggests either that the neuritogenesis signaling caused by ErbB3 depletion is transduced through ErbB4 signaling itself or that Iressa causes a dominant inhibition of an alternative signaling pathway that mediates the alterations of neuritogenesis caused by loss of ErbB3.
Based on the cellular results described above, we used three other lines of investigation to further test the hypothesis that the 4-anilino-quinazolines identified here act as direct inhibitors of ErbB4 kinase activity. First, to demonstrate that Iressa interacts with full-length ErbB4 receptor in a physiologically relevant setting, we created a new chemical tool, “iTrap”, consisting of Iressa immobilized on an agarose solid support and performed affinity chromatography (Figure A). iTrap was able to affinity-capture full-length ErbB4 and ErbB4-ICD (intracellular domain containing the kinase domain) in PC12-ErbB4-GFP but not the parental PC12-GFP cells lacking ErbB4 expression (Figure B). Most importantly, the levels of ErbB4 captured by iTrap were diminished by addition of Iressa (50 μM) as a soluble competitor revealing the specificity of the iTrap reagent.
Figure 6 Characterization of Iressa’s effect on Nrg1-induced signaling. (A) Schematic presentation of the agarose bead-conjugated Iressa (iTrap). (B) Whole cell extracts of PC12-GFP and PC12-ErbB4-GFP were premixed with or without 50 μM Iressa (more ...)
To determine whether Iressa directly bound to ErbB4, we used surface plasmon resonance (SPR) binding assays. We carried out SPR binding assays with the kinase domains of ErbB4 and EGFR using Iressa at concentrations ranging from 0 to 20 μM (Figure C,D). We found that Iressa bound both EGFR and ErbB4 with different affinities (Figure E,F), while the specific GSK-3β inhibitor, CHIR-99021, showed no interaction (Figure G,H). Kd's were determined from equilibrium binding measurements and by fitting these equilibrium measurements with a 1:1 interaction model using global parameters. Kd's for Iressa were determined to be approximately 30 and 150 nM for EGFR and ErbB4, respectively.
Finally, the effects of Iressa on the in vitro kinase activity of recombinant ErbB4 and EGFR were measured. Iressa was found to inhibit ErbB4 kinase domain activity in vitro with an IC50 ≈ 1 μM (compared with 50 nM against EGFR), consistent with its EC50 for inhibition of Nrg1-induced neurite outgrowth (Figure I). Thus, in agreement with the iTrap affinity reagent studies and SPR binding assays, these biochemical findings provide support for the potential of direct interaction between Iressa and ErbB4 leading to a block of Nrg1-induced neuritogenesis.
Overall, our screen revealed that among the negative regulators of Nrg1−ErbB4 signaling, anilino-quinazolines are a rich source of inhibitors with diverse levels of efficacy and intra-ErbB family class specificity. Over the past decade, tremendous effort has been invested in ErbB receptor inhibition, especially targeting EGFR and ErbB2, because of their long-recognized role in cancer (42
). As a result, a growing number of ErbB inhibitors have been identified. However, the specificity of these inhibitors has mostly been annotated by comparing EGFR and ErbB2, and no small molecules that are selective inhibitors of ErbB4 are currently available. Based on the close homology among ErbB family members in their kinase domain, several EGFR inhibitors, such as AG1478 and PD158780, have been considered as pan-ErbB inhibitors and used against ErbB4. Previously, these two inhibitors were shown to inhibit Nrg1-signaling and downstream biological consequences such as neurite outgrowth in hippocampal neurons (43
), inhibition of NMDA receptor currents in pyramidal neurons from rodent prefrontal cortex (44
), inhibition of long-term potentiation at Schaffer collateral-CA1 synapses in the hippocampus (45
) and glutamatergic synapse maturation and plasticity (46
). The identification of some of these compounds in our screen suggests that the cell-based imaging assay we developed may provide a surrogate system for identifying compounds that modulate Nrg1−ErbB4 regulated synaptic plasticity. However, dissecting ErbB4-specific inhibition from pan-ErbB inhibition poses a new challenge. We also noticed that, unlike Iressa or Traceva, PD158780 has an inhibitory effect on NGF-induced neurite outgrowth, which confounds the interpretation of results when this compound is used in physiological conditions where other neurotrophic factors might interfere. Thus, caution must be taken when these compounds are used because of potential off-target or indirect effects that might be attributed to inhibition of other hererodimerizing ErbB receptors instead of ErbB4 itself.
While this manuscript was in preparation, elegant studies by Krivosheya et al. (41
) demonstrated that treatment of rat hippocampal neurons with soluble Nrg1 resulted in enhanced dendritic arborization through activation of the tyrosine kinase domain of ErbB4 and that RNAi-mediated silencing of ErbB4 decreased the number of primary neurites. These findings are consistent with our findings using RNAi toward ErbB4 in PC12 cells engineered to express this receptor and again provide evidence supporting the role of the kinase activity of ErbB4 in mediating neuritogenesis. However, our results differ in some aspects, as treatment of neurons with the PI3 kinase inhibitor LY294002, but not the MAPK inhibitor PD980059, blocked neurite remodeling upon Nrg1 treatment. We speculate that these differences are due to differences in cell type and culture conditions.
Discovery of Small-Molecule Potentiators of Nrg1-ErbB4 Signaling
In addition to identifying inhibitors such as Iressa, our small-molecule screen also identified small molecules that had no effect on NGF-induced neurite outgrowth but potentiated Nrg1-induced neurite outgrowth. One compound, the indolocarbazole, K-252c (Figure A), satisfied our selection criteria and furthermore had no effect on cell death or proliferation in the concentration range tested. Since K-252c is structurally similar to K-252a, a potent TrkA inhibitor that is widely used for inhibition of NGF-induced processes (e.g., refs (37
), we speculated that K-252a may also have effects on Nrg1−ErbB4 signaling. To test this hypothesis, we first treated the PC12-ErbB4-GFP cells with K-252a and NGF or Nrg1. As expected, K-252a completely inhibited NGF-induced neurite outgrowth at concentrations as low as 50 nM. In contrast, however, similar to K-252c, K-252a significantly potentiated Nrg1-induced neurite outgrowth at the same concentration that inhibited NGF-induced neurite outgrowth. Furthermore, both NGF inhibition and Nrg1 potentiation are dose-dependently modulated by K-252a (Figure B,C). Though we have yet to identify the specific target of K-252a that is responsible for mediating its effect on Nrg1−ErbB4 signaling, we and others have found that small modifications to the scaffold can afford remarkable selectivity (47
). Functionally, the early Erk1/2 phosphorylation in response to Nrg1 is not dramatically affected by K-252a treatment. On the other hand, NGF-induced Erk1/2 phosphorylation was diminished by K-252a (Figure D). These findings, and the potentiation of neuritogenesis phenotype, suggest that K-252a affects Nrg1 signaling in a manner distinct from its effects on Trk receptor mediated signaling.
Figure 7 Characterization of indolocarbazoles that potentiate Nrg1-induced signaling. (A) Structures of K-252a and K-252c. (B) Cells were left untreated or were treated with K-252a (50 nM) for 30 min followed by treatment with 20 ng/mL of Nrg1 or NGF. Images were (more ...)
Overall, the finding that Nrg1-induced neuritogenesis can be potentiated by both K-252a and NGF suggests that ErbB4 signaling in the brain can be enhanced by removing an inhibitory signal or by activating potentially intersecting or parallel signaling networks. It is possible that K-252a acts as a potent modulator of a downstream component shared by all the neurotrophic factors; however in the case of NGF signaling, its inhibitory effect on the TrkA receptor is dominant. K-252a has been shown previously to have a neuroprotective effect in several cell types through a mechanism reportedly due to inhibition of Trk family receptors (49
). The detailed mechanism for K-252a’s ability to potentiate Nrg1-induced signaling as observed here for the first time remains a challenge for future studies to address. While we speculate that the relevant target is a kinase, additional potential targets include other ATP-binding proteins such as ATPases involved in chromatin remodeling (e.g, SWI/SNF family) and cytoskeletal dynamics (e.g., myosin).