Daidzein induces neuritogenesis in DRG neuronal cultures

Absract Background Daidzein, a phytoestrogen found in isoflavone, is known to exert neurotrophic and neuroprotective effects on the nervous system. Using primary rat dorsal root ganglion (DRG) neuronal cultures, we have examined the potential neurite outgrowth effect of daidzein. Methods Dissociated dorsal root ganglia (DRG) cultures were used to study the signaling mechanism of daidzein-induced neuritogenesis by immunocytochemistry and Western blotting. Results In response to daidzein treatment, DRG neurons showed a significant increase in total neurite length and in tip number per neuron. The neuritogenic effect of daidzein was significantly hampered by specific blockers for Src, protein kinase C delta (PKCδ) and mitogen-activated protein kinase/extracellular signal-regulated kinase kinases (MEK/ERK), but not by those for estrogen receptor (ER). Moreover, daidzein induced phosphorylation of Src, PKCδ and ERK. The activation of PKCδ by daidzein was attenuated in the presence of a Src kinase inhibitor, and that of ERK by daidzein was diminished in the presence of either a Src or PKCδ inhibitor. Conclusion Daidzein may stimulate neurite outgrowth of DRG neurons depending on Src kinase, PKCδ and ERK signaling pathway.


Background
Daidzein, found mainly in soy food products and herbs like red clover [1,2], is one of the most studied and most potent phytoestrogens. Phytoestrogens are estrogenic compounds of plant origin, and have structures and functions similar to the mammalian endogenous hormone estrogen [3]. Both estrogen and phytoestrogens can bind to intracellular estrogen receptors (ERs) to trigger downstream signal transduction pathways and achieve various biological functions [4]. Phytoestrogen acts mainly as an ER agonist. It may also function as an antagonist, by inhibition of aromatase activity in breast cancer cells, and blockage of estrogen uptake by uterine cells [5]. This mixed ER agonist/antagonist property probably explains the potential benefit of phytoestrogen in breast cancer prevention [6][7][8].
In response to ligand binding, ERs can signal through both genomic (classical) and non-genomic (non-classical) pathways [9,10]. In the genomic pathway, upon binding to estrogen, ERs dimerize and interact with the estrogen responsive element (ERE) in the regulatory regions of estrogen responsive genes, thereby regulating the transcription of E2sensitive genes, e.g. c-fos, TGF-α, and angiotensinogen [9]. The non-genomic pathway involves the activation of other signal transduction pathways that lead to rapid and diverse physiological responses, including calcium and potassium influxes through cell membrane, and activation of second messenger systems such as cAMP/PKA, MAPK, PI3K/Akt, and G protein [10,11]. The precise mechanisms of nongenomic effects of estrogen are not clear and have been suggested to be mediated by membrane-associated ERα, ERβ, or the orphan G-protein-coupled receptor 30 (GPR30) [12,13]. Activation of non-nuclear ERα, for example, can stimulate endothelial cell proliferation via G protein, Src, and eNOS activation [14]. In cultured osteoblastic cells, daidzein has been suggested to activate a non-classical membrane ER-β pathway that involves phospholipase C-β2 (PLC-β2)/PKC and PI3K/cSrc [15].
Emerging evidence, however, indicates that for some of the phytoestrogen effects, ER activation may not be required. For instance, genistein, a rich phytoestrogen in soybeans, was shown to exert growth inhibitory effects in ER-negative breast cancer cells [16][17][18]. Compared to 17-β estradiol, the most biologically active estrogen in mammals, daidzein has a significantly lower affinity for both ER-α and ER-β [19]. Moreover, daidzein could induce anti anti-proliferative effects in both ER-positive and ER-negative pancreatic cells [20]. Together these observations raise the possibility that daidzein may also exert its pharmacological effect via an ER-independent signaling pathway.
Daidzein is known to exert significant neuronal protection and neuritogenic effects for a variety of cultured neuronal cells, e.g. hippocampal neurons, cortical neurons, dorsal root ganglion (DRG) neurons, and PC12 cells [21][22][23][24]. In hippocampal neuron, the neuritogenic mechanism involves ERβ-PKCα-GAP43 signaling. To further understand the diversity of the intracellular signaling mechanisms of daidzein, in the current study we focused on daidzeininduced neurite outgrowth in cultured DRG neurons. DRG culture is a well-characterized system for investigating the mechanism of neuritogenesis [25][26][27], and for screening neuroprotective drugs for peripheral neuropathies [28]. Studies using DRG cultures have shed light on the pathogenic mechanisms of peripheral nervous system diseases and the regeneration of spinal cord injury [29][30][31]. Here we showed that in cultured DRG neurons daidzein induced notable neuritogenesis via an ER-independent signaling pathway. In addition, we presented several lines of evidence suggesting that daidzein-induced neurite outgrowth in DRG neurons may be primarily mediated by the Src kinase, PKCδ and ERK signaling pathway. Cell culture DRG cultures were prepared as described previously [25]. Briefly, P2 rat pups were put on ice and then decapitated to harvest DRG. DRG were then dissected out under microscope and dissociated with 0.25% trypsin and 0.05% collagenase (Sigma) in HBSS solution, for 30 min at 37°C. These ganglia were then dispersed by mechanically trituration with glass pipettes. The pellet from low-speed centrifugation was re-suspended in phenol-red free L-15 Leibovitz media, supplemented with 1.2 g/L of NaHCO 3 , 5% fetal bovine serum, 100 IU/mL of penicillin, and streptomycin (Gibco). Cells were plated on collagen-coated coverslips for immunocytochemistry, and on 35 mm uncoated culture dishes for protein quantification by Western blot. The medium was changed to serum free L-15 for day in vitro (DIV) 2 cultured DRG cells. Cultures were maintained at 37°C in an atmosphere of 95% air and 5% CO 2 .

Cell survival assay
The MTT assay, a colorimetric assay for measuring the activity of mitochondrial enzymes, was used to examine whether cell viability was affected by treatmen [32]. In each well of 24-well culture plates, 2 × 10 4 cells were plated and were treated with 0.1% DMSO, different concentration of daidzein or different kinase inhibitors for 24 h. After treatments, cells were washed with phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 1.5 mM KH 2 PO 4 , 8 mM Na 2 HPO 4 , pH 7.4), and incubated in 0.5 mg/ml of 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) solution for 4 h to allow the conversion of (See figure on previous page.) Figure 2 Quantitative analysis of total neurite length and tip number of neurite branches per neuron following various treatments with DMSO, daidzein, or NGF. Significant neurite lengthening and branching were observed in both large (A and B) and small DRG neurons (C) treated with 30 μM daidzein or 100 nM NGF. Three independent experiments were performed. Ten neurons were chosen from each group in one representative experiment for analysis. *, p < 0.05 vs DMSO controls. **, p ≤ 0.01 vs DMSO controls. MTT into the purple formazan product by mitochondrial dehydrogenases. The reaction medium was then removed and the cells were lysed with DMSO for 5 min. The absorbance was read at 590 nm with a spectrophotometer (Beckman Coulter Inc., Fullerton, CA).

Drug treatment
DIV 3 cultured DRG cells received either daidzein at a concentration of 10 μM, 30 μM, 50 μM, or 100 μM, vehicle solution DMSO (final concentration of 0.1%), or NGF of 100 ng/mL, in order to study the effect of daidzein on neurite outgrowth.

Immunocytochemistry
After 24 h of DMSO or daidzein treatment, DRG neurons on cover glasses were fixed for 10 min with 10% formalin in PBS. After washed with PBS, cells were then permeabilized and blocked with 0.15% Triton X-100 and 5% non-fat milk in PBS for 1 h. DRG neurons were then incubated in mouse anti-NF-L antibody overnight at 4°C. After PBS wash, cells were incubated in biotin-conjugated goat antimouse IgG (Vector, Burlingame, CA, USA) at 1:50 dilution for 1 h at room temperature, washed with PBS, then reacted with avidin-biotinylated enzyme complex (Vector) for one hour at room temperature. Following PBS wash, staining was done with peroxidase-chromogen reaction (SG substrate kit, Vector), which was stopped by Trisbuffered saline (TBS: 50 mM Tris-Base, 150 mM NaCl, pH 8.2). Coverslips were then dehydrated by ethanol and xylene, and mounted with Permount (Fisher Scientific, NH, USA). Images were taken on a light microscope, equipped with a Nikon DIX digital camera (Nikon, Tokyo, Japan).

Quantification
Immunostained neurons were photographed at 20× or 40× (for morphological demonstration) magnification, and the images transformed into 256 gray scale images. The fields were chosen to locate individual neuron with discernible neurites from nearby neurons. Generally, an average of 2-4 neurons could be seen in a micrograph taken at 20× magnification. The total neurite length was then measured from the somata using a PC-based image analyzer software Image Pro 3.0 Plus (Media Cybernetics, Silver Spring, MD, USA). The signal intensity of bands stained on immunoblot was quantified with Gel pro 3.1 (Media Cybernetics). Student's t-test was used for evaluating statistical differences between the means of different groups, with p value of less than 0.05 considered significant.

Daidzein enhances neuritogenesis in cultured DRG neurons
We first studied the effect of daidzein on neurite outgrowth in primary rat DRG neuronal cultures. DRG neurons were classified into large (diameter ≥ 40 μm) and small (diameter ≤ 40 μm) according to the criteria described by Gavazzi [33]. Based on cell diameter, smallsized unmyelinated neurons that are responsible for pain sensation, and large-sized myelinated that are for proprioception. DIV 3 neurons were incubated with different concentrations of daidzein, DMSO (negative control), or NGF (positive control) for 24 h. Compared to DMSO-treated control cultures, daidzen treatment significantly enhanced neurite extension and branching of large DRG neurons (Figures 1, 2A-B). Quantitative analysis of total neurite length and tip numbers per neuron indicated that the minimal effective concentration of daidzein in promoting neurite outgrowth was 30 μM (length, 4180 ± 246 μm; tip number, 15.0 ± 1.4; DMSO, 2323 ± 128 μm; tip number, 7.8 ± 0.2; p ≤ 0.01, n = 10), which had an effect similar to that of 100 ng/ml NGF ( Figure 2) and was thus used for further study of the compound. The effect of daidzeininduced neuritogenesis was observed similarly in both small-sized and large-sized DRG neurons ( Figures 1G-H,  2C). Furthermore, to examine whether daidzen was toxic to DRG neurons, double staining of cells with DAPI and PI was performed to identify apoptotic and necrotic cells, respectively [34]. No apparent cell death was observed at 30 μM by MTT assay (Figure 3A).

Daidzein treatment activates Src, PKCδ, and ERK
We next examined whether daidzein could activate the Src, PKCδ, or the ERK signaling pathway. By using Western  blotting with antibodies directed against phosphotyrosine-416 of active Src (pSrc416), we found that the pSrc416 level was significantly increased by daidzein treatment ( Figure 6A). Interestingly, pPKCδ levels were also upregulated in the presence of daidzein treatment, and the Src kinase inhibitor PP2 significantly inhibited this activation of pPKCδ by daidzein ( Figure 6B). Moreover, ERK1 and ERK2 were activated by daidzein, and this effect was suppressed by both the Src kinase inhibitor PP2 and the PKCδ inhibitor rottlerin (Figure 7). Interestingly, a dosedependent study of daidzein showed that a maximal activation of Src kinase and ERK was found at 30 μM daidzein ( Figure 8). This data correlates well with the previous observation that the optimal concentration of daidzein to achieve effective neurotigenic activity is 30 μM.

Discussion
Previously, in cultured osteoblastic cells, daidzein was shown to bind to cell membrane ER-β to activate the phospholipase C β2 (PLC-β2)/PKC and PI3K/cSrc pathways, leading to the expression of several groups of genes for cell differentiation, proliferation, and migration [15]. Similarly, in macrophage, daidzein inhibited the activation of STAT-1 and NF-kB proteins, thereby decreased the expression of iNOS and the production of NO [35]. Moreover, in hippocampal neurons, daidzein was demonstrated to promote neurite outgrowth via ER-β, which in turn may increase the phosphorylation of PKCα and growth associated protein 43 (GAP-43) [22]. In contrast to these ER-dependent signaling systems, here our results revealed that the neuritogenic effects of daidzein in DRG neurons did not mediate through ER α/β or GPR-30 (the third kind of ER receptor), as pretreatment of DRG neurons with specific estrogen receptor antagonists, ICI 182780, tamoxifen, or G-15, did not block the daidzeininduced neuritogenesis.
Most importantly, our studies showed that daidzein treatment increased the phosphorylation of Src kinase and ERK1/ERK2 in cultured DRG neurons. Src kinase, which is activated by various molecules like NGF, laminin, artemin, and anti-Thy-1 antibody, has been shown to be an important signaling pathway involved in the process of DRG neurite outgrowth [27,36,37]. Downstream signaling of Src includes MEK/ERK and PI3K/Akt pathways, which can be activated by NGF to induce neurite extension and branching of DRG neurons [38]. While inhibition of Src kinase by PP2 and suppression of ERK1/2 by U0126 abolished the neuritogenic effect of daidzein, inhibition of Akt by LY294002 had no effect (unpublished observation). Thus, the current study did not support the role of PI3K/Akt pathway in daidzeininduced neuritogenesis. The increased phosphorylation of ERK and daidzein-induced neuritogenesis was blocked by the Src kinase inhibitor PP2, indicating that promotion of neurite outgrowth by daidzein required Src kinase and ERK. As phosphorylated ERK could activate CREB, Cdk5, GAP-43 and other neuritogenesis-related genes [39][40][41], it is possible that daidzein-induced neurite outgrowth is mediated by the Src-ERK pathway. Interestingly, PKCδ has been shown to be phosphorylated and activated by Src kinases in salivary and PC12 cells [42]. Consistent with this notion, we also found that daidzein increased the phosphorylation of PKCδ, and that inhibition of PKCδ by a selective PKCδ inhibitor rottlerin resulted in the suppression of neurite outgrowth, suggesting that PKCδ may also play a role in the signaling cascade induced by daidzein. Different PKC isozymes, including PKCδ, have been found to be activated by neurotrophic agents. For examples, in PC12 cells the activation of ERK by neuritogenic agents, fibroblast growth factor (FGF) and NGF, was dependent on the activation of PKCδ [43]. In diabetic rats, over-expression of PKCδ could ameliorate the retarded neurite outgrowth of DRG neurons [44]. Additional studies are therefore required to elucidate the mechanistic link between daidzein-induced activation of the Src-PKCδ-ERK pathway and the downstream signaling pathways that eventually lead to neuritogenesis.
As mentioned above, we have shown that daidzeininduced neuritogenesis in hippocampal and DRG neurons are mediated by ER-dependent [22] and ER-independent mechanisms, respectively. The precise reasons underlying this striking difference remain unclear. Intrinsic signaling pathways regulating neurite outgrowth could drastically vary among different types of neuronal cells [45,46]. For example, activation and inhibition of the small GTPase Rac1 promotes neurite outgrowths in hippocampal and DRG neurons, respectively [47,48]. Further experiments will be required to determine if the neuritogenic effect of daidzein is also differentially regulated in various regions of the nervous system.
In cultured rat hippocampal neurons, a low concentration of daidzein had neuroprotective action (at 3.9 μM), but it could not promote neuritogenesis or enhance neuronal survival [24]. Using relative affinity binding assay of cellular extracts, the affinity of daidzein for estrogen receptor was estimated to be several hundred times lower than estrogen [19]. Therefore, adequate amount of daidzein is required to achieve biological activities through the estrogen receptor pathways. Studies using daidzein at much higher concentrations (30 to 40 μM) in cultured hippocampal neurons indicate that daidzein can promote neurite extension and protect neurons from glutamate-induced cell death [22,24]. We demonstrated that daidzein at 30 μM increased neurite lengthening and branching for DRG neurons, which was in accordance with the results of previously published reports. In addition, we found 30 μM daidzein had a neuritogenic effect similar to that of NGF, indicating that daidzein had a robust neuritogenic property. Meanwhile, daidzein did not affect DRG neuronal survival at 30 μM. When daidzein was used at a concentration higher than 30 μM, the neuritogenesis was decreased. This finding is in agreement with the observed fact that both Src and ERK achieve the highest levels of phosphorylation at 30 μM daidzein.
Our finding that daidzein facilitates neurite outgrowth of DRG and hippocampal neurons has important implications for the potential facilitation of neural regeneration [49,50]. Preliminary study using daidzein for the treatment of optic nerve injury in rats have shown promising results [23]. Neurite outgrowth is a fundamental step in the establishment of neural connections during development and following injury. It would be interesting to see if daidzein could improve sensory and cognitive function in various animal disease models, e.g. injuries of peripheral nerves, brachial plexus, and spinal cord, as well as Alzheimer's disease. On the other hands, the signaling mechanism of daidzein warrants further investigation. It has been shown that daidzein did not activate Src kinase via ERs, so other upstream regulators of Src could be the potential targets of daidzein. Many downstream effectors of ERK are associated with neurite outgrowth, and daidzein may preferentially activate some of those. Future research will be directed to tackle these questions.

Conclusion
Daidzein enhances neurite outgrowth of cultured rat DRG neurons which is mediated by cooperative action of Src kinase, PKCδ, and MEK.