The microenvironment of neuron cells plays a crucial role in regulating neural development and regeneration. Hyaluronic acid (HA) biomaterial has been applied in a wide range of medical and biological fields and plays important roles in neural regeneration. PC12 cells have been reported to be capable of endogenous NGF synthesis and secretion. The purpose of this research was to assess the effect of HA biomaterial combining with PC12 cells conditioned media (PC12 CM) in neural regeneration. Using SH-SY5Y cells as an experimental model, we found that supporting with PC12 CM enhanced HA function in SH-SY5Y cell proliferation and adhesion. Through RP-nano-UPLC-ESI-MS/MS analyses, we identified increased expression of HSP60 and RanBP2 in SH-SY5Y cells grown on HA-modified surface with cotreatment of PC12 CM. Moreover, we also identified factors that were secreted from PC12 cells and may promote SH-SY5Y cell proliferation and adhesion. Here, we proposed a biomaterial surface enriched with neurotrophic factors for nerve regeneration application.
Nerve injury is an important topic in the world of medicine and there are many nerve injury cases reported every year. These injuries usually have caused a decreased quality of life because of reduction in motor, sensory and autonomic functions [
Hyaluronan (hyaluronic acid, HA), a component of the extracellular matrix, is a glycosaminoglycan applicable to biomaterial. During embryogenesis, the concentration of HA is at the peak in undifferentiated cells and decreases at the beginning of cell differentiation [
Cell to cell interaction is important for cell fate determination, providing the first evidence for short-range regulatory mechanisms of cell differentiation. The conditioned medium (CM), which contains growth factors and differentiation regulation factors that are released from the cultured cells, could be used to promote cell differentiation into specific lineages [
In this study, we examined the effects of HA and PC12 CM in SH-SY5Y cells. SH-SY5Y is one kind of human derived cell line which is used in scientific research. The original cell line, called SK-N-SH, was subcloned and isolated from a bone marrow biopsy, which had been taken from a female with neuroblastoma. This cell line has been widely used as a model of neuron diseases as these cells possess many biochemical and functional properties of neurons. SH-SY5Y cells have been widely used as in vitro models of neurological studies, including analysis of neuronal differentiation, metabolism, and function related to neurodegenerative and neuroadaptive processes, neurotoxicity, and neuroprotection. It can be differentiated to a more mature neuron-like phenotype that is characterized by dopaminergic markers and, as such, has been used to study Parkinson’s disease [
The surface of a 9 MHz QCM gold electrode (ANT Tech, Taiwan) was washed with 1 M HCl, rinsed with DI water, followed by drying at room temperature. The frequency of the electrode measured by the QCM (ADS, ANT Tech, Taiwan) was assigned as
For seeding SH-SY5Y cells, the HA-modified electrodes, decorated by biopolymer layers, were sterilized with 70% (v/v) ethanol and then exposed to ultraviolet light. The 4 × 104 SH-SY5Y cells in serum-free medium were added to each well in the presence of the aforementioned electrodes and incubated at 37°C in 5% CO2 for 12 hours for investigation of the adhesion of the cells on those electrodes. After the incubations, the electrodes were washed with PBS, and then frequency shifts were measured by the QCM to quantify the adhesions of SH-SY5Y cells on electrodes.
The surface characterization of the coverslips decorated with HA was observed using a Fourier transform infrared spectrometer (FT-IR, Spectrum One system, PerkinElmer, USA).
For human neuroblastoma cell line, SH-SY5Y cells were cultured in Dulbecco’s Modified Eagle Medium: nutrient mixture F-12 (DMEM/F12) medium (Gibco, Invitrogen, USA) with 10% FBS plus 1% antibiotics. In the pheochromocytoma cell line of the rat adrenal medulla, PC12 cells were maintained in DMEM medium with 10% horse serum, 5% FBS plus 1% antibiotics. Those cells were incubated in 5% CO2 at 37°C for 48 hours.
In this study, PC12 CM were collected, filtered, and mixed with equal volumes of fresh DMEM/F12 medium for SH-SY5Y cells to be cultivated on HA surface. To collect PC12 CM, the PC12 cells were rinsed with phosphate buffer saline (PBS) and then incubated in serum-free DMEM medium for 12 hours. Then, the supernatants of the medium were collected and filtered with 0.22
SH-SY5Y cell lysates or PC12 CM were transferred into 1.5 mL tubes and reduced with 1 M dithiothreitol (DTT, USB Corporation, USA) in 25 mM NH4HCO3 at 37°C. After 3 hours, protein samples were alkylated with 1 M iodoacetamide (IAA, Amersham Biosciences, USA) in the dark at room temperature for 30 min. After the proteins were digested by sequencing-grade modified porcine trypsin (Promega, USA) overnight at 37°C, 2
RP-nano-UPLC ESI-MS/MS analyses (nanoACQUITY UPLC, Waters, Milford, MA, coupled to an ion trap mass spectrometer, LTQ Orbitrap Discovery Hybrid FTMS, Thermo, San Jose, CA) were conducted according to standard procedures described below. Briefly, a sample of the desired peptide digest was loaded into the reverse phase column (symmetry C18, 5
All MS and MS/MS data were analyzed and processed using the Mascot software (Version 2.2.1, Matrix Science, London, UK) against the Swiss-Prot database. The search parameters were set as follows: 0.5 Da for MS/MS tolerance, 10 ppm for MS tolerance, carbamidomethylation (C) as the fixed modification, deamidated (NQ), oxidation (M), phospho (ST) and phospho (Y) as the variable modification, and 2 for missing cleavage. Proteins were initially annotated by similar search conditions using UniProtKB/Swiss-Prot databases (SIB Swiss Institute of Bioinformatics, Lausanne, Switzerland). The protein-protein interaction pathways were performed by String 9.1 Web software (SIB Swiss Institute of Bioinformatics, Lausanne, Switzerland) [
Confirmation of protein identities was performed by Western blotting. Protein extracts were prepared in lysis buffer and each cell lysate sample (1
The cell viability was determined by BrdU cell proliferation assay kit (Millipore). The assay was performed according to the manufacturer’s instructions. Briefly, 1 × 103 SH-SY5Y cells were seeded in a sterile 96-well tissue culture plate and incubated for 24 to 72 hours. Then, cells were incubated in the culture medium containing BrdU reagent for 2 hours. Fixing solution was added before the absorbance was measured at 450 nm using an ELISA reader (Multiskan EX, Thermo Scientific, Vantaa, Finland).
The SH-SY5Y cells were grown on coverslips in 12-well culture plates. After 24 hours' incubation, the cells were fixed (60% methanol and 40% acetone) at −20°C for 30 min and then permeabilized (0.5% Triton X-100) at room temperature for 5 min. After rinsing with PBS, the cells were blocked (6% bovine serum albumin) and then incubated with primary and secondary antibodies. The nuclei and cytoskeleton of the cells were stained with DAPI (Sigma-Aldrich, USA), vimentin (Vimentin DyLight 488 Antibody, Epitomics, USA), monoclonal mouse anti-HSP60 (Stressgen, USA), and polyclonal rabbit anti-RanBP2 (Abcam, USA), respectively. After rinsing with PBS, the cells were mounted with ProLong® Gold Antifade Reagent (Invitrogen). The images were acquired by a microscope equipped with fluorescence light source (FLoid Cell Fluorescence Imaging Station, Invitrogen).
All calculations used the SigmaStat statistical software (Jandel Science Corp., San Rafael, CA, USA). All statistical significance was evaluated at 95% of confidence level or better. Data are presented as mean ± standard error.
In our previous studies, the QCM system was applicable to the quantitative analysis of adsorption of HA and adhesion of cells on electrodes [
Frequency shifts of QCM and weights of adhered SH-SY5Y cells on the electrodes decorated with HA-modified surface for 24 to 72 hours of cell incubation.
Cell adhesion |
|
|
---|---|---|
DMEM/F12 medium | ||
24 hrs |
|
|
48 hrs |
|
|
72 hrs |
|
|
DMEM/F12 medium-PC12 CM | ||
24 hrs |
|
|
48 hrs |
|
|
72 hrs |
|
|
Data are expressed as mean ± standard error,
HA-modified surfaces of coverslips were also routinely characterized using FT-IR spectra. The FR-IR spectra in the range of 500–4000 cm−1 for HA surfaces were presented in Figure
The assignment of FT-IR bands for HA-modified surface.
Function group | Wavenumber (cm−1) |
---|---|
C-O-C stretching, O-H deformation, C=O deformation | 894.9 |
C-O-C, C-O, C-O-H stretching | 1049.1 |
CH2, CH3 C-O-H deformation, C-O with C=O combination | 1321.0 |
NH deformation | 1406.9 |
C=O carboxyl amide I | 1616.1 |
CH stretching | 2893.8 |
NH with C=O combination | 3261.2 |
NH stretching and OH stretching | 3433.8 |
The FT-IR spectra show the frequency region from 4000 to 500 cm−1 of modified surfaces and (a) nonmodified and (b) HA-modified coverslips.
HA is well known to promote fibroblasts proliferation and enhance cell adhesion [
The relative percentage of cell viability obtained from BrdU cell proliferation assay. Differential treatments of SH-SY5Y cells were seeded in 96-well tissue culture plates and incubated for 24 to 72 h. Cells were treated with BrdU reagent for 2 h and fixed before the absorbance was measured at 450 nm.
The PC12 CM was collected and added to the culture medium of the SH-SY5Y cell. After 48 hours' incubation, the results showed that PC12 CM induced SH-SY5Y cell growth and proliferation especially with the HA-modified surface. The HA-modified coverslip combined with treatment of PC12 CM promoted SH-SY5Y cell proliferation after 72 hours' incubation as indicated in Figure
To understand the mechanism and consequence of the increasing of the SH-SY5Y cell proliferation and growth, the proteins in PC12 CM and SH-SY5Y cell lysate were identified by proteomic approaches.
To identify the PC12 secreted proteins related to SH-SY5Y cell proliferation and cell adhesion, the original PC12 CM were collected and the proteins were identified by RP-nano-HPLC-ESI-MS/MS. One hundred seventy-three HA-modified surface proteins were identified and then narrowed down to the number 62, using a threshold of a minimum of three peptides identified in a protein. We found that several proteins (described below) are involved in cell differentiation functions. Table
Proteins identified by the higher confidence level (at least three unique peptide sequences matched) in the PC12 CM which were involved in neuron generation function.
Accession numbers | Protein name | Biological process | Molecular function |
---|---|---|---|
Q66HC8 | Gametogenetin | Cell differentiation |
|
|
|
Double-strand break repair |
|
|
|
Embryo implantation |
|
|
|
Spermatogenesis |
|
P43145 | ADM | Aging | Adrenomedullin receptor binding |
|
|
Androgen metabolic process |
|
|
|
Calcium ion homeostasis |
|
|
|
cAMP-mediated signaling |
|
|
|
Hormone secretion |
|
|
|
Vasculogenesis |
|
|
|
Cell proliferation |
|
|
|
Apoptotic process |
|
Q9JKU6 | Spermatid perinuclear RNA-binding protein | Cell differentiation | DNA binding |
|
|
Multicellular organismal development | RNA binding |
|
|
Spermatogenesis |
|
O35569 | Pro-neuregulin-2, membrane-bound isoform | Epidermal growth factor receptor signaling pathway | Epidermal growth factor receptor binding |
|
|
Intracellular signal transduction | ErbB-3 class receptor binding |
|
|
Organ development |
|
Q62956 | Receptor tyrosine-protein kinase erbB-4 | Cardiac muscle tissue regeneration | ATP binding |
|
|
Cell migration | Receptor signaling protein tyrosine kinase activity |
|
|
Nervous system development | Transmembrane receptor protein tyrosine kinase activity |
|
|
Apoptotic process |
|
|
|
Cell proliferation |
|
|
|
Glucose import |
|
|
|
Odontogenesis |
|
|
|
Protein tyrosine kinase Signaling pathway |
|
P01026 | Complement C3 | Blood coagulation | C5L2 anaphylatoxin chemotactic receptor binding |
|
|
Chemotaxis | Cofactor binding |
|
|
Fatty acid metabolic process | Endopeptidase inhibitor activity |
|
|
Inflammatory response | Lipid binding |
|
|
Glucose transport |
|
|
|
Triglyceride biosynthetic process |
|
|
|
Response to progesterone and estrogen |
|
Q9R172 | Neurogenic locus notch homolog protein 3 | Cell differentiation | Calcium ion binding |
|
|
Multicellular organismal development |
|
|
|
Notch signaling pathway |
|
|
|
Regulation of transcription, DNA-templated |
|
|
|
Tissue regeneration |
|
The expression of Gametogenetin (GGN) was confined to late pachytene spermatocytes and round spermatids, a time window concomitant with the occurrence of meiosis. It was expressed with highest level in diplotene spermatocytes and meiotic germ cells, especially when the nuclear membrane breaks down and the nucleolus is disorganized. In addition to functioning in proliferation of primordial germ cells, POG also involved in spermatogenesis [
Adrenomedullin (ADM) is a member of the calcitonin gene-related peptide (CGRP) family, which has shown neuroprotective functions [
Spermatid perinuclear RNA-binding protein (SPNR) is a microtubule-associated RNA-binding protein [
The Pro-neuregulin-2 (Nrg2) has played a critical role in the growth and development of multiple organ systems, which was also involved in neural and organ development. In the embryo, the Nrg2 was expressed in the brain where it was found in the telencephalon, but not in the hindbrain. The Nrg2 was direct ligand for ErbB 3 and ErbB 4 tyrosine kinase receptors. Concomitantly recruiting ErbB 1 and ErbB 2 coreceptors, the Nrg2 may result in ligand-stimulated tyrosine phosphorylation and activation of the ErbB receptors, which may also promote the heterodimerization with the EGF receptor [
NRG1/ErbB signaling pathways are important in CNS development and may be neuroprotective in brain injury [
The complement system plays an important role in inflammatory diseases and neurodegenerative processes of the CNS [
The interaction of Notch, with its established intercellular signaling pathway, plays a key role in neural development. The Notch-3 activation induces the increase of the progenitor cell number in the central nervous system (CNS) and affects CNS development [
Also, in this study, more than one hundred proteins were identified in SH-SY5Y cell lysate and most of these were identified at the minimal confidence level, which was only one unique peptide sequence matched. Experimental results reported a total of six protein identifications with higher confidence levels (at least three unique peptide sequences matched). The protein-protein interaction pathways were performed by String 9.1 Web software, and proteins identified in this study were marked by arrows (red: SH-SY5Y; green: PC12; Figure
The protein-protein interaction pathways are illustrated. (a) Proteins identified in this study are marked by arrows (red: SH-SY5Y; green: PC12). (b) Two proteins, HSP60 and RanBP2, may turn on the ubiquitin (UBC) pathway, which is responsible for the proliferation and is required for survival of the majority of cells.
The TP53 pathway has been famously recognized to be connected to the UBC/PI3K/AKT1/mTOR pathway, which is responsible for the proliferation and is required for survival of the majority of cells. The hypothesis of the mTOR pathway is that it acts as a master switch of cellular catabolism and anabolism, thereby determining whether cells grow and proliferate. In particular, the UBC/PI3K/AKT1/mTOR pathway regulates the import and retention of glucose. It provides substrates for glycolysis and the biosynthetic pathways which rely on the supply of glycolytic intermediates. The mTOR pathway, downstream of AKT signaling, regulates the protein translation rate and accelerates the supply of amino acid biosynthesis to generate the charged tRNAs [
To confirm this hypothesis, the proteins in SH-SY5Y cell lysate need to be validated. In addition, there were two proteins, 60 kDa heat shock protein (HSP60, known as HSPD1) and E3 SUMO-protein ligase RanBP2 (RanBP2), identified in SH-SY5Y cell lysate samples, which were involved in cell proliferation, differentiation, development, and cycle regulation. Those two proteins were also involved in the UBC/PI3K/AKT1/mTOR pathway (Figure
Heat shock proteins (HSPs) are overexpressed in a wide range of cells and are implicated in cell proliferation, differentiation, and recognition by the immune system. These proteins have molecular chaperone activity, which can be induced by various environmental stresses. Some HSPs were found to be localized in the synapse [
The detection of HSP60 protein expression on SH-SY5Y cells. (a) Western blotting of HSP60 and
RanBP2, located at the nuclear pore complexes (NPCs) [
The detection of RanBP2 protein expression on SH-SY5Y cells. (a) Western blotting of RanBP2 and
In the result, it showed that HA and PC12 CM may regulate protein expression, such as HSP60 and RanBP2 to promote SH-SY5Y cell proliferation and adhesion. These results were similar to Yamada’s study, which reported that the combination treatment of SHya and FGF-2 increased NHA proliferation [
In this study, SH-SYSY cells were used as a model to examine the effects of HA and PC12 CM in neuron regeneration. We found that stimulation of a HA-modified surface with PC12 CM can promote SH-SYSY cell proliferation and adhesion; the combination of both showed synergy effects on SH-SYSY cell regeneration. Our evidences supported that neurotrophic factor proteins enhance HA function in neurogenesis. Biomaterial surface supported with neurotrophic factor proteins may be utilized in nerve autograft application. We used proteomic analysis to analyze the molecular mechanisms of HA-modified surface and PC12 CM stimuli. Among these proteins, HSP60 and RanBP2 were upregulated in SH-SY5Y cells. The UBC/PI3K/AKT1/mTOR pathway was related to the cell growth and proliferation. Future study will be of the molecular regulations and interaction networks governed by biomaterials and combined with neurotrophic factors.
The authors declare no conflict of interests.
Ming-Hui Yang and Ko-Chin Chen contributed equally to this work.
The authors thank S. Sheldon (Medical Technologist, American Society of Clinical Pathology, retired, MT, ASCP) of Oklahoma University Medical Center Edmond for fruitful discussions and editorial assistance. The authors thank the Center of Excellence for Environmental Medicine, Kaohsiung Medical University, for the assistance in protein identification. This work was supported by Research Grants MOST 103-2320-B-037-025 and MOST 105-0210-01-12-01 (Taiwan Protein Project) from the Ministry of Science and Technology, KMU-TP104E12 and KMU-O104003 (Aim for the Top 500 Universities Grant) from Kaohsiung Medical University, 105-CCH-KMU-005 from CCH-KMU Research Project, and NSYSUKMU104-P032 from NSYSU-KMU Research Project, Taiwan.