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. 2024 Sep 27;86(11):1136–1144. doi: 10.1292/jvms.24-0276

A peptide against the N-terminus of myristoylated alanine-rich C kinase substrate promotes neuronal differentiation in SH-SY5Y human neuroblastoma cells

Jannatul FERDOUS 1,2, Kiyotada NAITOU 1, Mitsuya SHIRAISHI 1,*
PMCID: PMC11569876  PMID: 39343539

Abstract

Myristoylated alanine-rich protein kinase C substrate (MARCKS) plays crucial roles in neuronal functions and differentiation. However, specific effects of the myristoylated N-terminal sequence (MANS) peptide, a widely used MARCKS modulator comprising the initial 24 amino acids of MARCKS, on neuronal cells remain unclear. Therefore, in this study, we aimed to examine the effects and action mechanisms of the MANS peptide on SH-SY5Y human neuroblastoma cells, which served as the in vitro neuronal cell models. MANS treatment of SH-SY5Y cells resulted in significant neurite outgrowth within 24 hr, which was as prominent as that induced by seven days of treatment with all-trans retinoic acid, the most common agent used to induce SH-SY5Y cell differentiation. Levels of synaptophysin, a neuronal marker protein, were significantly increased in the MANS peptide-treated cells. Additionally, increased MARCKS levels and decreased MARCKS phosphorylation were observed in MANS peptide-treated cells. Notably, neurite outgrowth induced by the MANS peptide was significantly reduced in MARCKS-knocked-down cells. Overall, these results suggest the MANS peptide as a novel agent for SH-SY5Y cell differentiation, particularly for the analysis of MARCKS functions.

Keywords: all-trans retinoic acid, myristoylated alanine-rich protein kinase C substrate, myristoylated N-terminal sequence peptide, neuronal differentiation, SH-SY5Y cells

Myristoylated alanine-rich protein kinase C substrate (MARCKS) is a rod-shaped ubiquitously expressed protein that plays crucial roles in vertebrate development, regeneration, inflammation, and cancer [20, 22]. MARCKS contains highly conserved domains, including an effector domain (ED) and N-terminal domain (ND), for its functions [8]. ED in the center of the protein is abundant in positively charged lysine residues and susceptible to phosphorylation by protein kinase C (PKC) or Rho-associated protein kinase [24]. ND includes the N-terminal myristoylation site [31, 44]. In the quiescent state, unphosphorylated MARCKS is located at the plasma membrane due to the insertion of the myristoylation site of ND into the lipid bilayer and electrostatic interaction of ED with phosphatidylinositol 4,5-bisphosphate (PIP2) [38, 45]. MARCKS shifts to the cytoplasm upon phosphorylation of serine residues within ED, including serine-159 and serine-163, but dephosphorylation of ED results in the reassociation of MARCKS with the plasma membrane [8, 20, 24, 33, 38].

A unique cell-permeable MARCKS-related peptide, myristoylated N-terminal sequence (MANS), has been developed as a modulator of MARCKS, especially at the myristoylated amino-terminal site. The MANS peptide corresponds to the first 24 amino acids of the MARCKS N-terminus and competes with endogenous MARCKS to bind to the plasma membrane of cells [2, 19, 34], leading to the translocation of MARCKS into the cytosol and release of PIP2 into the local environment [19, 25]. MANS peptide has been widely used as an MARCKS modulator in in vitro and in vivo experimental models [12] to explore the roles of MARCKS in the migration of primary human neutrophils [19], NIH-3T3 fibroblasts [34], mucin secretion from human bronchial epithelial cells [29, 41], lung metastasis [12], breast cancer [11], and mouse models of asthma [2].

SH-SY5Y cells, derived from human bone marrow metastatic neuroblastoma [5, 30], are widely used as in vitro neuronal models for neurological research of Parkinson’s disease, Alzheimer’s disease, ischemia, and neurotoxicity [37, 47]. SH-SY5Y cells exhibit a unique capacity to differentiate into mature neuron-like cells upon treatment with various chemicals or modification of the culture medium [17, 23]. Therefore, these cells are suitable models for neuroscience research of cell proliferation and differentiation [39]. MARCKS is highly expressed in the nervous system and involved in neuronal cellular functions, such as migration, endo/exocytosis, neurite initiation, neurite outgrowth, differentiation, synaptic plasticity, and dendrite arborization [7, 9, 10, 13, 40, 42, 43, 48]. However, the specific effects of MANS peptide on neuronal cells, including SH-SY5Y cells, and underlying mechanisms remain unknown.

In this study, we aimed to investigate the effects of the MANS peptide on the morphology and MARCKS expression and phosphorylation in SH-SY5Y cells to determine its potential as an MARCKS modulator in neuronal cells. Treatment of SH-SY5Y cells with the MANS peptide induced neurite outgrowth within 24 hr, along with the increase in MARCKS expression and decrease in MARCKS phosphorylation. Furthermore, levels of synaptophysin, a neuronal marker protein, were significantly increased in the MANS peptide-treated SH-SY5Y cells. Neurite outgrowth induced by the MANS peptide was significantly suppressed in cells with reduced MARCKS expression. These results indicate that the MANS peptide is a useful agent for SH-SY5Y cell differentiation, particularly to analyze MARCKS functions in neurons.

MATERIALS AND METHODS

Reagents and antibodies

Dulbecco’s modified Eagle’s medium/Ham’s F-12 (DMEM/F-12; 1:1), Dulbecco’s phosphate-buffered saline, 0.25% trypsin-EDTA, SuperSep 10% gel, and all-trans retinoic acid (ATRA) were purchased from Fujifilm Wako (Osaka, Japan). Fetal bovine serum (FBS) was obtained from Mediatech (Woodland, CA, USA). The Cell Counting Kit-8 was purchased from Dojindo Laboratories (Kumamoto, Japan). Human MARCKS small interfering RNA (siRNA; SHSS180966) and negative control siRNA was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Accutase, radioimmunoprecipitation assay buffer, protease inhibitor cocktail, Blocking One solution, and penicillin–streptomycin was purchased from Nacalai Tesque (Kyoto, Japan). Immobilon Forte Western horseradish peroxidase (HRP) substrate was obtained from Millipore (Burlington, MA, USA). Anti-microtubule-associated protein 2 (MAP2; 17490-1-AP), anti-growth-associated protein 43 (GAP43; 16971-1-AP), anti-β-tubulin III (10094-1-AP), anti-synaptophysin (17785-1-AP), anti-MARCKS (20661-1-AP), and HRP-conjugated anti-rabbit IgG (SA00001-2) antibodies were purchased from Proteintech (Rosemont, IL, USA). Anti-pS159/163 MARCKS (sc-12971-R) and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; FL-335) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). MANS and random N-terminal sequence (RNS) peptides were synthesized by GenScript Inc. (Tokyo, Japan). The sequence of the MANS peptide was myristic acid-GAQFSKTAAKGEAAAERPGEAAVA, and the RNS control peptide sequence was myristic acid-GTAPAAEGAGAEVKRASAEAKQAF.

Cell culture, siRNA transfection, and peptide treatment

SH-SY5Y cells (ATCC, Manassas, VA, USA) were cultured in DMEM/F-12 supplemented with 10% FBS and antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin) in a humidified incubator at 37°C with 5% CO2. The cells were seeded in 35-mm dishes 24 hr before the experiment at a density of 1.4 × 104 cells/cm2 for morphological observations and 6 × 104 cells/cm2 for western blotting. For a cell viability assay, cells were seeded in 96-well plates at a density of 4 × 104 or 6 × 104 cells/cm2. In experiments involving MARCKS knockdown, transfection of human MARCKS siRNA or negative control siRNA was performed using ScreenFect A Plus, as previously described [16]. The cells were seeded 48 hr before the experiments at a density of 4 × 104 cells/cm2. SH-SY5Y cells were treated with 30 µM MANS or RNS peptide in serum-free DMEM/F-12 after 8 hr of serum-starvation. Then, MANS peptide concentration was determined via preliminary experiments using 10–100 µM MANS peptide. High concentrations of the MANS peptide (100 µM) exerted cytotoxic effects on cell morphology, leading to cell shrinkage, round cells floating in the medium, and presence of cell debris after 24 hr of treatment. For ATRA treatment, SH-SY5Y cells were incubated with 10 µM ATRA or ATRA vehicle (0.2% ethanol) for seven days in DMEM/F-12 containing 1% FBS and antibiotics. The medium was renewed every three days.

Morphological observations and neurite length measurement

Cell morphology was observed using inverted microscope CKX53 (Olympus Optical, Tokyo, Japan) equipped with a digital camera (WRAYCAM-EL310; WRAYMER, Osaka, Japan) at 10× magnification. Images were captured using the MicroStudio software (WRAYMER), and neurite length was measured using the “Freehand Line” and “Measure” tools in the ImageJ software (NIH, Bethesda, MD, USA). Cellular extensions exceeding 50 µm (twice the cell body diameter), which were measured as the distance between the tip and attachment of the process to the cell body, were considered as neurites and used to calculate the percentage of neurite-bearing cells and average neurite length, as previously described [6, 40]. At least three randomly selected fields, with more than 200 cells per field, were analyzed in each dish.

Cell viability assay

The viability of SH-SY5Y cells was determined using the Cell Counting Kit-8, following the manufacturer’s instructions. Cells were seeded at a density of 6 × 104 cells/cm2 in 96-well plates and treated with 30 µM MANS or RNS for up to 24 hr. Cell viability was assessed by measuring the change in absorbance of WST-8 at 450 nm using a MultiSKAN microplate reader (Thermo Fisher Scientific), at 1 and 24 hr. Untreated SH-SY5Y cells were considered 100% viable. For separate measurement, siRNA-transfected SH-SY5Y cells were seeded in 96-well plates at a density of 4 × 104 cells/cm2 and their viability was accessed after 48 hr transfection, as described above.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting

SH-SY5Y cells in 35-mm dishes were washed with ice-cold phosphate-buffered saline and lysed with the radioimmunoprecipitation assay buffer containing a protease and phosphatase inhibitor cocktail. Protein concentration in the lysate was quantified using the Bradford method. The proteins were separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis using SuperSep Ace 10% polyacrylamide gels and transferred onto polyvinylidene fluoride membranes. After blocking with the Blocking one solution for 1 hr at room temperature, the membranes were incubated with the anti-MAP2 (1:20,000), anti-GAP43 (1:20,000), anti-β-tubulin III (1:20,000), anti-synaptophysin (1:25,000), anti-pS159/163 MARCKS (1:5,000), anti-MARCKS (1:5,000), and anti-GAPDH (1:5,000) antibodies overnight at 4°C. Then, the membranes were incubated with HRP-conjugated anti-rabbit IgG (1:10,000) used as secondary antibodies for 1 hr at room temperature. Immunoreactive proteins were visualized using the Immobilon Forte Western HRP substrate on the Lumicube Imager (Liponics, Tokyo, Japan). ImageJ software was used for the densitometric analysis of protein bands. GAPDH was used to normalize the expression of other proteins, and expression of each protein in control cells (no treatment) was considered as 100%. The amount of phosphorylated MARCKS at serine-159/163 (P-MARCKS) was normalized to the total amount of pan-MARCKS.

Statistical analyses

Results are expressed as the mean ± standard error of the mean (SEM). Statistical analyses were conducted using the Student’s t-test to compare two groups and Tukey’s honest significant difference to compare multiple groups using the R software (R-Project for Statistical Computing; www.r-project.org/). Statistical significance was set at P<0.05.

RESULTS

Morphological changes in SH-SY5Y cells induced by the MANS peptide

The effect of the MANS peptide on SH-SY5Y cell morphology was examined (Fig. 1A). Control (no treatment) cells exhibited large cell bodies with truncated neurites, whereas treatment with 30 µM RNS peptide for 24 hr did not cause any discernible changes in SH-SY5Y cell morphology. In contrast, cells treated with the MANS exhibited short cell bodies and long neurite projections, indicative of neuronal differentiation. To quantitatively assess neurite outgrowth, we calculated the percentages of neurite-bearing cells (Fig. 1B and 1C) and average neurite lengths (Fig. 1D and 1E) of cells treated with MANS peptides or ATRA. The MANS peptide, but not the RNS peptide, increased the percentage of neurite-bearing cells and average neurite length in a time-dependent manner, with a significant increase in the percentage of neurite-bearing cells observed 1 hr after treatment with MANS compared to that in RNS-treated cells (Fig. 1B and 1D). ATRA is commonly used to induce the differentiation of SH-SY5Y cells [17, 23, 47]. Here, we compared the neurite outgrowth induced by 10 µM ATRA for seven days after treatment and that induced by the MANS peptide. Although a significant but slight increase in the percentage of neurite-bearing cells was observed 1 day after treatment with ATRA, increase in the number of neurite-bearing cells and average neurite length comparable to that induced by MANS treatment for 24 hr was observed after seven days of ATRA treatment (Fig. 1A, 1C, and 1E).

Fig. 1.

Fig. 1.

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Induction of neurite outgrowth in SH-SY5Y cells treated with the myristoylated N-terminal sequence (MANS) peptide or all-trans retinoic acid (ATRA). (A) Representative images of the control (no treatment) cells and cells treated with the MANS peptide (30 µM) for 24 hr or ATRA (10 µM) for seven days. Scale bar=100 µm. Percentages of neurite-bearing cells treated with the (B) MANS peptide and (C) ATRA. Average neurite lengths in cells treated with the (D) MANS peptide and (E) ATRA. Results are expressed as the mean ± standard error of the mean (SEM; n=3). *P<0.05 and **P<0.01 compared to the cells treated with the random N-terminal sequence (RNS) or ATRA-vehicle by Student’s t-test.

Given that ATRA is known to induce differentiation as well as growth inhibition in SH-SY5Y cells [21, 27, 35, 36], we examined the effect of the MANS peptide on cell proliferation in a cell viability assay. Treatment of SH-SY5Y cells with 30 µM MANS peptide slightly decreased cell viability at 24 hr versus that of RNS-treated cells, although no significant difference in cell viability was observed after 1 hr treatment. (Supplementary Fig. 1A).

Neuronal marker protein levels in MANS peptide-treated SH-SY5Y cells

Neuronal differentiation of SH-SY5Y cells is accompanied by the increased expression levels of neuronal marker proteins [18]. Therefore, we determined the levels of the neuronal marker proteins, MAP2, GAP43, β-tubulin, and synaptophysin, using western blotting (Fig. 2). Among these marker proteins, synaptophysin levels were commonly elevated in cells. Treatment with the MANS peptide for 24 hr caused an approximately 1.5-fold increase in synaptophysin levels compared to that in RNS-treated cells (Fig. 2B), whereas treatment with ATRA for seven days caused an approximately 2.2-fold increase in synaptophysin levels compared to that in vehicle-treated cells (Fig. 2C).

Fig. 2.

Fig. 2.

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Neuronal marker protein levels in SH-SY5Y cells treated with the myristoylated N-terminal sequence (MANS) peptide or all-trans retinoic acid (ATRA). (A) Representative western blot images of the neuronal markers, microtubule-associated protein 2 (MAP2), growth-associated protein 43 (GAP43), β-tubulin, and synaptophysin, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), in cells treated with the MANS peptide (30 µM) for 24 hr or ATRA (10 µM) for seven days. Relative expression levels of neuronal markers in cells treated with the (B) MANS peptide and (C) ATRA. Protein expression of each neuronal marker in the control (no treatment) cells was considered as 100%. Results are expressed as the mean ± SEM (n=3). *P<0.05 and **P<0.01 compared to the cells treated with the random N-terminal sequence (RNS) or ATRA-vehicle by Student’s t-test.

Changes in MARCKS expression and phosphorylation in MANS peptide-treated SH-SY5Y cells

MANS peptide is widely used as an MARCKS modulator. MARCKS is involved in neuronal maturation, including neurite outgrowth [8, 40, 49]. Therefore, changes in MARCKS expression and phosphorylation of cells treated with MANS peptides (Fig. 3B and 3D) or ATRA (Fig. 3C and 3E) were examined via western blotting. MARCKS levels were significantly increased in cells treated with the MANS peptide for 24 hr compared to those in RNS-treated cells (Fig. 3B), whereas P-MARCKS levels were significantly decreased in cells treated with the MANS peptide for 12 or 24 hr (Fig. 3D). Moreover, MARCKS levels were significantly decreased and P-MARCKS levels were slightly decreased in cells treated with ATRA for seven days (Fig. 3C and 3E).

Fig. 3.

Fig. 3.

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Levels of myristoylated alanine-rich protein kinase C substrate (MARCKS) and phosphorylated MARCKS (P-MARCKS) in SH-SY5Y cells treated with the myristoylated N-terminal sequence (MANS) peptide or all-trans retinoic acid (ATRA). (A) Representative western blot images of MARCKS, P-MARCKS, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in cells treated with the MANS peptide (30 µM) for 24 hr or ATRA (10 µM) for seven days. Relative expression levels of MARCKS in cells treated with the (B) MANS peptide and (C) ATRA. Relative expression levels of P-MARCKS in cells treated with the (D) MANS peptide and (E) ATRA. Relative expression levels of MARCKS and P-MARCKS in the control (no treatment) SH-SY5Y cells were considered as 100%. Results are expressed as the mean ± SEM (n=5). *P<0.05 and **P<0.01 compared to the cells treated with the random N-terminal sequence (RNS) or ATRA-vehicle by Student’s t-test.

Effect of MARCKS knockdown on neurite elongation in MANS peptide-treated SH-SY5Y cells

To examine the role of MARCKS in neurite outgrowth induced by the MANS peptide, we observed changes in MARCKS expression and neurite outgrowth in MARCKS-knocked-down cells treated with the MANS peptide for up to 24 hr (Fig. 4). First, we investigated the changes in MARCKS levels after treatment with the MANS and RNS peptides in control siRNA-transfected and MARCKS siRNA-transfected SH-SY5Y cells. In the control (no treatment with MANS and RNS) cells, transfection with MARCKS siRNA decreased the MARCKS levels by 54.0 ± 3.3% (n=4) 48 hr after treatment compared to that in cells treated with the control siRNA (Fig. 4A). In addition, we determined that transfection with MARCKS siRNA did not affect the viability of SH-SY5Y cells compared to that in control siRNA-transfected cells (Supplementary Fig. 1B). Although MARCKS expression was increased after treatment with the MANS peptide in cells transfected with the negative control or MARCKS siRNA, MARCKS levels 24 hr after treatment with the MANS peptide in MARCKS-knocked-down cells were significantly decreased compared to those in the control siRNA-transfected cells (Fig. 4B). Moreover, neurite outgrowth was suppressed in MARCKS-knocked-down cells after 24-hr treatment with the MANS peptide compared to that in the control siRNA-transfected cells (Fig. 4C). The percentage of neurite-bearing cells was significantly increased by the MANS peptide in cells transfected with the control or MARCKS siRNA. However, the percentage of neurite-bearing cells was significantly decreased in MARCKS siRNA-transfected cells 12 and 24 hr after treatment with the MANS peptide compared to that in the control siRNA-transfected cells (Fig. 4D). However, the increase in the average neurite length after MANS peptide treatment was not significantly different between the MARCKS siRNA-transfected and control siRNA-transfected cells (Fig. 4E).

Fig. 4.

Fig. 4.

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Effect of MARCKS knockdown on neurite outgrowth in SH-SY5Y cells induced by the myristoylated N-terminal sequence (MANS) peptide. (A) Representative western blot images of myristoylated alanine-rich protein kinase C substrate (MARCKS) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in the control and MARCKS small interfering RNA (siRNA)-transfected cells treated with the MANS peptide (30 µM) for 24 hr. (B) Relative expression levels of MARCKS in the control and MARCKS siRNA-transfected cells treated with the MANS peptide. Relative expression of MARCKS in the control (no random N-terminal sequence (RNS) or MANS treatment) cells was considered as 100%. Representative images of (C), percentages of neurite-bearing cells among (D), and average neurite lengths in (E) the control and MARCKS siRNA-transfected SH-SY5Y cells treated with the MANS peptide (30 µM) for 24 hr. Results are expressed as the mean ± SEM (n=4 for B; n=3 for D and E). *P<0.05 and **P<0.01 compared to the control and MARCKS siRNA-transfected SH-SY5Y cells treated with the RNS by Tukey’s test (B, D) or Student’s t-test (E).

DISCUSSION

Although MARCKS is abundantly expressed in the nervous system and plays important roles in neuronal functions, effects of the MANS peptide, an MARCKS modulator, on neurons remain unclear. SH-SY5Y is a human neuroblastoma cell line commonly used as an in vitro neuronal model for neuroscience research due to its ability to differentiate into neuron-like cells with neurite outgrowth and neuronal marker expression [1, 26, 27, 30]. In this study, treatment of SH-SY5Y cells with the MANS peptide induced neurite outgrowth, increased synaptophysin levels, and changed the expression and phosphorylation status of MARCKS. Neurite outgrowth and MARCKS expression are differentially regulated in cells treated with the MANS peptide or ATRA, which is the most common differentiating agent for SH-SY5Y cells [17, 23, 47], indicating that the MANS peptide induces differentiation of SH-SY5Y cells with characteristics distinct from those induced by ATRA. These findings highlight the potential use of the MANS peptide as an inducer of SH-SY5Y cell differentiation, particularly to explore the neural functions of MARCKS.

Neurite outgrowth is a typical morphological feature of neuronal differentiation in SH-SY5Y cells [27]. Here, treatment of SH-SY5Y cells with 30 µM MANS induced neurite outgrowth within 24 hr of treatment, whereas RNS, a randomly scrambled control peptide, did not cause any obvious morphological changes in cells (Fig. 1A). Notably, MANS peptide induced neurite outgrowth in SH-SY5Y cells in a shorter period than ATRA. Neurite outgrowth was observed 1 hr after treatment with the MANS peptide, and percentage of neurite-bearing cells and average neurite length in cells treated with the MANS peptide for 24 hr (Fig. 1B and 1D) were comparable to those in cells treated with ATRA for seven days (Fig. 1C and 1E). Furthermore, levels of synaptophysin, a neuronal marker protein that promotes neurotransmission [17], were significantly increased in the cells treated with the MANS peptide for 24 hr compared to those in cells treated with RNS (Fig. 2B). Synaptophysin levels were also elevated in the ATRA-treated SH-SY5Y cells after seven days (Fig. 2C), consistent with a previous report [14]. These findings suggest that the MANS peptide induces neuronal differentiation in SH-SY5Y cells in a shorter period than ATRA. Since cell viability was slightly decreased after 24 hr treatment with MANS peptide, we cannot exclude the possibility that differences in cell density affected neurite outgrowth and expression of differentiation markers. However, after 1 hr treatment with MANS peptide, cells showed significant neurite outgrowth without any change in viability, suggesting that alterations in cell growth or density were not involved in the initiation of neurite outgrowth, at least in the 1 hr treatment (Supplementary Fig. 1A).

MARCKS is involved in neurite initiation and elongation in in vitro neuronal cell models, including primary mouse cortical neurons, PC12 cells, and SH-SY5Y cells [4, 7, 8, 28, 40]. As the MANS peptide competes with endogenous MARCKS [15] to modulate its function, we examined the effects of the MANS peptide on MARCKS expression and phosphorylation to determine the role of MARCKS in the MANS-induced differentiation of SH-SY5Y cells. Treatment of cells with the MANS peptide for 24 hr significantly increased the MARCKS levels in SH-SY5Y cells, whereas MARCKS phosphorylation was significantly decreased 12 and 24 hr after treatment with the MANS peptide (Fig. 3B and 3D), which is consistent with a previous report of decreased MARCKS phosphorylation at serine-159/163 after treatment with the MANS peptide in lung cancer and melanoma cells [12, 32]. Both the expression and phosphorylation status of MARCKS affect neuronal development and functions [7, 20]. As dephosphorylation of MARCKS is a crucial step in insulin growth factor-I-induced neurite outgrowth in SH-SY5Y cells [40, 48], decreased MARCKS phosphorylation caused by MANS possibly plays a role in the induction of neurite outgrowth. In contrast, treatment of cells with ATRA for seven days decreased MARCKS levels (Fig. 3C), along with a slight decrease in MARCKS phosphorylation (Fig. 3E). Interestingly, the MANS peptide has been shown to suppress MARCKS phosphorylation induced by phorbol ester via PKC activation [12], suggesting that this inhibitory action may reflect their role as a competitive inhibitor of PKC. However, further studies are necessary to elucidate the underlying mechanism linking the inhibition of MARCKS phosphorylation and the induction of differentiation by the MANS peptide. ATRA decreases the levels of MARCKS, particularly in the membrane fractions, in immortalized mouse hippocampal cell lines [46]. As the MANS peptide leads to MARCKS translocation from the membrane to the cytosol [19, 25], relative decrease in the amount of membrane-binding MARCKS is possibly a common factor in cell differentiation induced by MANS and ATRA. Differential regulation of MARCKS expression and phosphorylation by MANS and ATRA, in addition to the disparate temporal patterns of neurite outgrowth, suggest that distinct types of neuronal differentiation are induced by MANS and ATRA in SH-SY5Y cells.

To assess the role of MARCKS in the induction of neurite outgrowth in SH-SY5Y cells by the MANS peptide, we monitored the effect of MARCKS knockdown on neurite outgrowth. In MARCKS-knocked-down cells, number of neurite-bearing cells and MARCKS levels increased by the MANS peptide were significantly reduced compared to those in cells treated with the control siRNA (Fig. 4B and 4D), suggesting that MARCKS plays a role in MANS-induced neurite outgrowth in SH-SY5Y cells. Many studies have indicated that MARCKS is involved in neurite outgrowth [4, 7, 8, 28, 40]; however, the downstream signaling mechanisms associated with changes in MARCKS expression and phosphorylation remain unclear. In this study, MARCKS knockdown had no effect on average neurite length (Fig. 4E), suggesting that MARCKS plays a crucial role in neurite outgrowth induced by the MANS peptide. In addition to the observation that transfection of MARCKS siRNA did not affect the cell viability (Supplementary Fig. 1B), no change in the percentage of neurite-bearing cells by transfection of SH-SY5Y cells with MARCKS siRNA has been reported [48], suggesting that transfection of MARCKS siRNA did not induce SH-SY5Y cell differentiation. As a limitation of this study, we did not determine the specific mechanisms underlying the differentiation of SH-SY5Y cells induced by the MANS peptide and we focused on examining short- term effects of MANS peptide, warranting further investigation. Moreover, future studies should examine the involvement of PIP2, polysialic acid, and cdc42, which are associated with MARCKS and play important roles in neuronal differentiation [3, 7, 10, 43], in MANS peptide-induced neurite outgrowth.

CONFLICT OF INTEREST

The author declares no conflicts of interest.

Supplementary

Supplement Figure
jvms-86-1136-s001.pdf (16.1KB, pdf)

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