Glycoprotein non-metastatic melanoma B interacts with epidermal growth factor receptor to regulate neural stem cell survival and differentiation

Abstract The functional recovery following spinal cord injury (SCI) remains a challenge clinically. Among the proteins interacted with the glycoprotein non-metastatic melanoma B (GPNMB), epidermal growth factor receptor (EGFR) during activation is able to promote the proliferation of neural stem cells (NSCs) in the spinal cord. We investigated the roles of GPNMB and EGFR in regulating the survival and differentiation of the NSCs. By overexpression and short-hairpin RNA-mediated knockdown of GPNMB in the NSCs, GPNMB promoted cell viability and differentiation by increasing the expressions of βIII tubulin and CNPase (2′,3′-cyclic nucleotide 3-phosphodiesterase). Using co-immunoprecipitation, we found that EGFR interacted with GPNMB. Furthermore, EGFR had a similar effect as GPNMB on promoting cell viability and differentiation. Overexpression of EGFR reversed the decrease in viability and differentiation caused by the knockdown of GPNMB, and vice versa. Last but not least, we tested the effect of GPNMB and EGFR on several intracellular pathways and found that GPNMB/EGFR modulated the phosphorylated (p)-c-Jun N-terminal kinase (JNK)1/2/JNK1/2 ratio and the p-nuclear factor κB (NF-κB p65)/NF-κB p65 ratio. In sum, our findings demonstrate the interaction between GPNMB and EGFR that regulates cell bioprocesses, with the hope to provide a new strategy of SCI therapy.


Introduction
Spinal cord injury (SCI) has been emerged as a serious and irreversible disease in the central nervous system (CNS) [1], bringing about permanent or temporary loss of function in the motor or sensory capability and leading to destructive neurological and functional deficiency inclusive of paraplegia or quadriplegia [2]. Due to the lack of regeneration capacity, the recovery of the body function as impaired by SCI remains a significant challenge clinically [3]. In this case, the efforts devoted into exploring the functions of endogenous spinal cord neural stem cells (NSCs) shed a light on the treatment of SCI [4,5].
NSCs, primarily indwelling in the CNS including the spinal cord and the brain, are competent to self-renew for the maintenance of the stem cell bank size and to differentiate into neurons for the repairmen of the tissues [4,6], which is likely to be distinguished in the functional recovery of SCI. Given the inclination of NSCs to differentiate into microenvironment-dependent glia lineages, many researchers have committed to facilitating the differentiation into functional neurons from the spinal cord neural progenitor cells (NPCs) [7,8].
The differentiation inducement attempts involve the regulation of the proteins in the spinal cord to trigger the endogenous repair [9]. We started our study by analyzing transcriptome changes via the aberrant gene expressions either in the young or aged patients perplexed by SCI based on the data set GSE93561 and captured 90 genes expressing aberrantly after crossover analysis. Glycoprotein non-metastatic melanoma B (GPNMB) has aroused our interest by dint of its multiple functions, encompassing tissue repairment facilitation, kinase signaling stimulation, cell-cell adhesion and migration acceleration, tumorigenesis promotion, modulation of the cell growth and differentiation, etc. [10]. GPNMB is a type I transmembrane glycoprotein [11], whose expression level is found upregulated in SCI according to the analyses of the above-mentioned data set. Apart from that, GPNMB also exhibits dysregulated expression in the spinal cord transcriptome after peripheral nerve injury [12] and in a high-fat diet-fed male rat model of thoracic spinal contusion [13]. However, the regulatory mechanism of GPNMB in SCI remains uncharacterized.
To figure out the regulatory mechanism of GPNMB in SCI, we adopted bioinformatics analysis to predict the proteins interacting with GPNMB. Notably, epidermal growth factor receptor (EGFR), a transmembrane glycoprotein of ErbB family, was finalized, given that the activation of EGFR contributed to the proliferation of NSCs in SCI [14]. EGFR signaling cascade features in the proliferation, division, differentiation, and survival of the cells [15]. It has been elucidated that EGFR can regulate a variety of downstream pathways, including Janus kinase/ signal transducer and activator of transcription, extracellular signal-regulated kinase/mitogen-activated protein kinase, phosphatidylinositol 3-kinase (PI3K)/Akt, and Notch pathways, and promote the NSC proliferation [16][17][18]. EGF (epidermal growth factor), a ligand of EGFR, activates EGFR and enhances the proliferation of local NSCs, playing an active role in SCI [19]. In line with the above findings, we are dedicating to validating whether GPNMB interacts with EGFR to regulate cell survival and differentiation in SCI, so as to provide a novel insight for the further prognosis for SCI patients.

Ethics statement
All the research studies related to animal use were complied with the relevant national regulations and institutional policies for the care and use of animals. This study was conducted on the premise of authorization from the Ethic Committee of Experimental Animals of Taizhou Central Hospital (Taizhou University Hospital) with approval number SJWK202001010. Every effort was exploited to minimize the pain and discomfort to the animals.

NPC differentiation
NPC differentiation inducement was implemented as previously described [22]. Briefly, cells were digested and resuspended into single-cell suspension, followed by the seeding in culture dishes coated with poly-L-lysine (P4707; Sigma-Aldrich). Thereafter, cells were cultured in DMEM/F-12 medium supplemented with 2% B27 and 1% FBS to induce differentiation. The culture medium was replaced every 2 days. NPCs cultured in DMEM/F-12 medium without any treatment served as the control group.

Quantitative reverse-transcription polymerase chain reaction (qRT-PCR)
Relative GPNMB and EGFR mRNA expression levels were measured by qRT-PCR. Briefly, total RNAs were extracted via TRIzol reagent (15596026; Invitrogen), whose quantities and purities were determined by a spectrophotometer (ND-LITE-PR;

Statistical analysis
All values were presented as mean ± standard deviation (SD). Independent samples t test was applied for the analysis between two variables. One-way analysis of variance was adopted to analyze one categorical independent variable in multiple groups, followed by Bonferroni post hoc analysis. GraphPad Prism 8 software (GraphPad, CA, USA) was utilized for data analysis. For measurements, P < 0.05 was perceived as statistical significance.

GPNMB promoted neuronal viability and differentiation while shGPNMB performed differently
Post bone marrow NSC extraction and differentiation inducement, we utilized Western blot assay to detect the differentiation degree of the cells. Upregulated protein expression of the neural differentiation marker, βIII tubulin, indicated the successful differentiation of NSCs into neurons (Figure 1a, P < 0.001). Then, we measured GPNMB expression changes after cell differentiation via qRT-PCR and Western blot assay. Results from both assays demonstrated that GPNMB expression level was upregulated in the differentiated cells relative to that in control cells (Figure 1b and c, P < 0.001). Next, we transfected the cells with the overexpressed or silenced GPNMB plasmid to figure out the impact of GPNMB upon cell viability and neural differentiations. shGPNMB was used for further experiment due to its more efficiency of GPNMB knock down ( Figure A1a, P < 0.01). Moreover, qRT-PCR and Western blot assays demonstrated that overexpressed GPNMB promoted while shGPNMB reduced GPNMB expression, indicating the success of transfection (Figure 1d-e, P < 0.001). Besides, CCK-8 assay manifested that GPNMB overexpression boosted cell viability; yet, shGPNMB restrained cell viability in the endured test time (Figure 1f, P < 0.05). Finally, neural differentiation was assessed by the immunolabeling profiles of the neuronal marker βIII tubulin and oligodendrocyte marker CNPase [24], followed by the measurement of Western blot assay, the results of which displayed that overexpressed GPNMB upregulated βIII tubulin and CNPase expression levels (Figure 1g-h, P < 0.05), whilst shGPNMB downregulated the expressions of the neural differentiation markers (Figure 1g-h, P < 0.001). (g) Immunofluorescence assay was adopted to assess the status of neuronal differentiation (magnification 200×, scale bar 100 µm). Red part referred to the targeted proteins, and blue meant the nuclei as stained by DAPI. (h) Relative neural differentiation markers' (βIII tubulin and CNPase) expressions were measured by Western blot assay. GAPDH was set as the internal reference. ** P < 0.01 or *** P < 0.001 vs Control; + P < 0.05 or +++ P < 0.001 vs NC; ## P < 0.01, ### P < 0.001 vs shNC. All results represent means ± SD of triplicate determinations. qRT-PCR: quantitative reverse-transcription polymerase chain reaction; GPNMB: glycoprotein non-metastatic melanoma B; shNC: short-hairpin-negative control; CCK-8: cell counting kit-8; CNPase: 2′,3′-cyclic nucleotide 3′ phosphodiesterase.

EGFR interacted with GPNMB and its expression level was upregulated in the differentiated cells
To figure out the regulatory mechanism of GPNMB, we adopted STRING to analyze the proteins interacting with GPNMB ( Figure 2a). Ultimately, EGFR was selected as the candidate on the basis of literature study. To validate the interaction between GPNMB and EGFR, we performed Co-IP assay with identification that GPNMB coprecipitated with EGFR (Figure 2b and c). Then, we conducted qRT-PCR and Western blot assay again to measure the expression level of EGFR and discovered that EGFR was upregulated in the differentiated cells relative to that in control cells (Figure 2d-e, P < 0.001).

EGFR boosted cell viability and partly reversed the effect of shGPNMB
Subsequently, we further explored the mechanism with the participation of aberrant EGFR expression through transfection. ShEGFR was used for next experiment because of its more efficiency of EGFR knock down ( Figure A1b, P < 0.001). As supported by the measurements from qRT-PCR and Western blot assays, overexpressed EGFR upregulated EGFR expression, whereas shEGFR downregulated EGFR expression, which indicated the success of transfection (Figure 3a and b, P < 0.001). Then, we implemented CCK-8 assay to reveal the effect of EGFR on cell viability, finding that shEGFR suppressed cell viability; yet, GPNMB boosted cell viability and could partly reverse the inhibiting Figure 2: EGFR interacted with GPNMB and its expression level was downregulated after cell differentiation. (a) STRING (https://www.stringdb.org/) was adopted to comprehensively analyze the data for PPI network. GPNMB interacted with EGFR. (b and c) A Co-IP assay was performed to identify if GPNMB coprecipitated with EGFR. (d and e) Relative EGFR expression was measured by qRT-PCR and Western blot assay. GAPDH was set as the internal reference. ** P < 0.01 *** P < 0.001 vs control. All results represent means ± SD of triplicate determinations. STRING: Retrieval of Interacting Genes Database; PPI: protein-protein interaction; EGFR: epidermal growth factor receptor; Co-IP: co-immunoprecipitation.
3.4 EGFR facilitated p-JNK1/2/JNK1/2 ratio and NF-κB p65 expression and partly counteracted the inhibitory effects of shGPNMB on the JNK/NF-κB signaling pathway while shEGFR displayed oppositely Then, we adopted the immunofluorescence assay to explore the status of neuronal differentiation after the second-phase transfection, followed by Western blot assay. The results uncovered that overexpressed GPNMB increased βIII tubulin and CNPase protein expressions, while shEGFR decreased βIII tubulin and CNPase expressions and could partly neutralize the promotive effects of GPNMB on the expressions of these two proteins (Figure 4a, P < 0.001). Moreover, shGPNMB lessened βIII tubulin and CNPase protein expressions, whereas EGFR enhanced βIII tubulin and CNPase expressions and partly offset the restraining function of shGPNMB in the above aspects (Figure 4b, P < 0.001). Finally, we validated our conjecture through detecting JNK/NF-κB signaling pathway-related indicators via Western blot assay. GPNMB boosted the p-JNK1/2/JNK1/2 ratio and the p-NF-κB p65/NF-κB p65 ratio, but shEGFR worked inversely and partly reversed the enhancing effects of GPNMB on JNK/ NF-κB signaling pathway-related markers (Figure 4c, P < 0.05). On the contrary, shGPNMB reduced the p-JNK1/2/ JNK1/2 ratio and the p-NF-κB p65/NF-κB p65 ratio; yet, EGFR facilitated these ratios and partly counteracted the inhibitory effect of shGPNMB on the JNK/NF-κB signaling pathway-associated markers (Figure 4d, P < 0.001).
On the basis of a previous research, we further conducted our study on the perspective of functional recovery of SCI, which was impacted by the neuron loss in most occasions [3]. Generally, the main obstacle for SCI treatment would be the effective usage of the stimulated endogenous NPCs. A previous study stated GPNMB as a novel neuroprotective factor in cerebral ischemia-reperfusion injury [26]. Also, a recent study demonstrated that GPNMB could be a novel strategy for peripheral nerve regeneration after transection by promoting the proliferation of Schwann cells as well as expression and secretion of neurotrophic factors and neural adhesion molecules in vitro [27]. In our study, we uncovered that overexpression of GPNMB was beneficial to cell viability and neuronal differentiations as supported by the expression changes of the neural differentiation-associated markers (βIII tubulin and CNPase) in vitro which might avail the restricted recovery after SCI. We took over the studies against GPNMB from Spann et al. and Weng et al. and ulteriorly proved the possible role of GPNMB in SCI from the perspective of probing into the underlying regulation of cell differentiation rather than lingering on the surface.
As for the detailed mechanism, the interaction between GPNMB and EGFR in SCI, on the basis of a research reported by Han et al. who had already demonstrated GPNMB as an activator in cell migration, and its upregulation might be related to the oncogenic property of EGFR in lung cancer [28]. Despite the different study field and research direction, we substantiated the synergistic effect between GPNMB and EGFR in the non-cancer field and raised that the interaction between the two functioned in the biological processes and cell differentiation in NSCs. Liu et al. conducted a study on the individual role of RGFR in SCI and validated the promotive role of EGFR in NSC activation after SCI [14]. Similar to Liu et al., we also highlighted the significance of EGFR in NSC activation for functional recovery after SCI, although two of us focused on a different signaling pathway.
Fang et al. had confirmed the participation of JNK/ NF-κB pathway in SCI through the inhibitory regulation of miR-132-3p on attenuating the injury [29]. It has been evidenced that NF-κB signaling pathway functions prominently in immune response and neuroinflammation; with a great detail, the neuroinflammation could be triggered by the NF-κB signaling pathway after SCI [30]. In addition, inactivation of this pathway can ameliorate the SCI via modulating the inflammatory reaction [31,32]. As for the JNK pathway, its significance has been validated in the contribution to the neuronal apoptosis after neuron injury [33,34]. In our study, we further confirmed the involvement of JNK/NF-κB signaling pathway in SCI and uncovered that GPNMB interacted with EGFR to modulate JNK phosphorylation and NF-κB p65 phosphorylation, thereby making an impact upon neuronal differentiation. This finding is a further extension of Fang et al. in SCI treatment and provides a novel cue in the regulatory mechanism of stimulating endogenous NPCs for SCI therapy. Typically, PI3K/protein kinase B (Akt) and RAS/RAF pathways are the main downstream pathways that EGFR regulates survival and differentiation. These two pathways are not detected in this study, which is the shortcoming of this study. Moreover, whether GPNMB regulates EGFR protein abundance and PI3K/Akt and whether RAS/RAF pathway affects the survival and differentiation of NSCs need further investigation. At present, the study of GPNMB is still in its infancy and the role of GPNMB in the multiple tumors or non-tumor cases requires to be further elucidated. Further studies toward GPNMB in SCI are welcomed and recommended to validate the performances and properties in the actual practice. Besides, the interaction between GPNMB and EGFR or other proteins is worth pursuing due to the extensive scope captured from the bioinformatics analysis. Furthermore, ulterior studies are expected to exploit the actual value of this interaction as therapeutic entity to induce NPCs in terms of feasibility and practicability.
In conclusion, we prove that the interaction between GPNMB and EGFR regulates neuronal survival and differentiation through the modulation of JNK/NF-κB signaling pathway.