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Regulator of G protein signaling 5 (RGS5) inhibits sonic hedgehog function in mouse cortical neurons
Molecular and Cellular Neuroscience 83 (2017) 65–73
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Regulator of G protein signaling 5 (RGS5) inhibits sonic hedgehog function in mouse cortical neurons Chuanliang Liu a,b,1, Qiongqiong Hu a,1, Jia Jing a, Yun Zhang a, Jing Jin a, Liulei Zhang a, Lili Mu a, Yumei Liu a, Bo Sun a, Tongshuai Zhang a, Qingfei Kong a, Guangyou Wang a, Dandan Wang a, Yao Zhang a, Xijun Liu a, Wei Zhao a, Jinghua Wang a,⁎, Tao Feng c,⁎⁎, Hulun Li a,d a
Department of Neurobiology, Harbin Medical University, Heilongjiang Provincial Key Laboratory of Neurobiology, Harbin, Heilongjiang 150086, China Vocational College Daxing'an Mountains, Jiagedaqi District, Heilongjiang 165000, China c Department of Neurology, The Nangang Branch of Heilongjiang Provincial Hospital, Harbin, Heilongjiang 150001, China d Key Laboratories of Education Ministry for Myocardial Ischemia Mechanism and Treatment, Harbin, Heilongjiang 150086, China b
a r t i c l e
i n f o
Article history: Received 11 September 2016 Revised 21 February 2017 Accepted 20 June 2017 Available online 03 July 2017 Keywords: RGS5 Sonic hedgehog Cortical neurons
a b s t r a c t Regulator of G protein signaling 5 (RGS5) acts as a GTPase-activating protein (GAP) for the Gαi subunit and negatively regulates G protein-coupled receptor signaling. However, its presence and function in postmitotic differentiated primary neurons remains largely uncharacterized. During neural development, sonic hedgehog (Shh) signaling is involved in cell signaling pathways via Gαi activity. In particular, Shh signaling is essential for embryonic neural tube patterning, which has been implicated in neuronal polarization involving neurite outgrowth. Here, we examined whether RGS5 regulates Shh signaling in neurons. RGS5 transcripts were found to be expressed in cortical neurons and their expression gradually declined in a time-dependent manner in culture system. When an adenovirus expressing RGS5 was introduced into an in vitro cell culture model of cortical neurons, RGS5 overexpression significantly reduced neurite outgrowth and FM4-64 uptake, while cAMP-PKA signaling was also affected. These findings suggest that RGS5 inhibits Shh function during neurite outgrowth and the presynaptic terminals of primary cortical neurons mature via modulation of cAMP. © 2017 Elsevier Inc. All rights reserved.
1. Introduction Regulator of G protein signaling (RGS) proteins are signal transduction molecules which contribute to the regulation of heterotrimeric G proteins. RGS proteins have been classified into nine subfamilies based on conserved sequences present in their catalytic RGS domain. These subfamilies (and their members) include: A/RZ (RGS17, RGS19, RGS20, RET-RGS1), B/R4 (RGS1-RGS5, RGS8, RGS13, RGS16, RGS18, RGS21), C/R7 (RGS6, RGS7, RGS9, RGS11), D/R12 (RGS10, RGS12, RGS14), E/RA (axin, conductin), F/GEF (p115-RhoGEF, PDZ-RhoGEF, LARG), G/GRK (GRK1-GRK7), H/SNX (SNX13, SNX14, SNX25), and I/DAKAP2 (D-AKAP2, RGS22) (Jules et al., 2015). These various RGS subfamilies regulate different G proteins. For example, the RGS-R4 subfamily exhibits specificity for Gαi and Gαq, yet does not appear to interact
⁎ Correspondence to: J. Wang, Department of Neurobiology, Harbin Medical University, Harbin, Heilongjiang 150086, China. ⁎⁎ Correspondence to: T. Feng, Department of Neurology, the Nangang Branch of Heilongjiang Provincial Hospital, Harbin, Heilongjiang 150001, China E-mail addresses: [email protected] (J. Wang), [email protected] (T. Feng). 1 Chuanliang Liu and Qiongqiong Hu contributed equally to this work.
http://dx.doi.org/10.1016/j.mcn.2017.06.005 1044-7431/© 2017 Elsevier Inc. All rights reserved.
with Gαs (Bansal et al., 2007). The R4 subfamily member, RGS5, is first identified and isolated from a neuroblastoma cDNA library (Seki et al., 1998) and is a tissue-specific signaling protein that negatively regulates Gαi activity. Conversely, activation of Gαi inhibits the activity of the membrane enzyme, adenylyl cyclase, which converts ATP to cAMP. RGS5 has also been reported involved in many pathophysiological processes including atherosclerosis (Cheng et al., 2015; Li et al., 2004), blood pressure regulation (Cho et al., 2008), cardiac hypertrophy (Deng et al., 2012), lipid metabolism (Xiao et al., 2009), bronchial smooth muscle cell contraction, and asthma (Yang et al., 2012). Elevated expression of RGS5 has also been found in various cancers (Sethakorn and Dulin, 2013). During brain development, cortical neurons undergo neuron polarization, axon outgrowth, dendritogenesis, the formation of the synapse, as well as synaptic function maturation. To further ascertain the subcellular distribution and the expression of mRNAs or proteins on specific developmental stages, and to address fundamental questions regarding neuronal polarity, spine development, and synaptic plasticity, neonatal neuron cultures have been used. As a result, several cell signaling pathways have been reported to mediate neural development, including the Notch signaling pathway, the Wnt/β-catenin signaling pathway, and the hedgehog signaling pathway (Arenas, 2014). Sonic hedgehog
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(Shh) is a secreted molecule that binds patched (Ptch) receptors which are expressed on Shh-responding cells. Upon receptor binding, Smoothened (Smo), a G-protein coupled receptor (GPCR), transduces an intracellular signal which converges to activate the zinc finger protein transcription factor, Gli. Shh signaling has been shown to control cell growth, survival, and differentiation in a wide variety of cells (He and
Lu, 2013; Stewart et al., 2002). In the nervous system, Shh signaling is essential for patterning of the embryonic neural tube, and it also function s in developing neurons. In primary hippocampal neurons, Shh regulates autophagy (Petralia et al., 2013) and presynaptic terminal function (Mitchell et al., 2012). In primary cortical neurons, Shh promotes neurite outgrowth (He et al., 2016). In adult sensory neurons,
Fig. 1. Expression profile of RGS5 mRNA in C57BL/6 adult mouse tissues. A. Fold-expression of RGS5 mRNA detected in various tissues obtained from C57BL/6 adult mice relative to smooth muscle tissue. n = 3. B. Fold-expression of RGS5 mRNA detected in various central nervous system cellular components as indicated relative to neurons. n = 3. C. Fold-expression of the RGS-R4 subfamily members detected in cultured cortical neurons relative to RGS5. n = 3. D. Fold-expression of RGS5 in cortical neurons cultured in vitro at the different time points indicated relative to the levels detected after 4 h. n = 3. E. Fold-expression of RGS5 in cortical neurons cultured from mice with various age. n = 3. F. Fold-expression of RGS5 in primary cortical neurons versus the Neuro 2a, CATH.a, and C17.2 cell lines. n = 3. Values represent the mean ± SD value from three experiments. *P b 0.05, ***P b 0.001 compared with the Neuron group in B. *P b 0.05 compared with the Neuron group in D. *P b 0.05, **P b 0.01, ***P b 0.001 compared with the 1D group in E. ***P b 0.001 compared with the Neuron group in F.
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knockdown of Shh resulted in decreased outgrowth and branching (Martinez et al., 2015). In recent studies, cAMP-protein kinase A (PKA) has been found to inhibit Shh signaling (Makinodan and Marneros, 2012). However, it remains unknown whether RGS5 served as a regulator of Gαi signaling, contributes to Shh signaling through cAMP in developing neurons. Therefore, in this study, RGS5 expression is investigated during cortical neuron development in an in vitro model. A possible role for RGS5 in regulating Shh signaling is also examined.
2. Materials and methods
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2.2. RNA isolation and quantitative PCR analysis Total RNA was extracted from cells or tissues using TRIzol reagent (Thermo Fisher Scientific), according to the manufacturer's instructions. Reverse transcription was performed using random hexaprimers and Moloney murine leukemia virus (M-MLV; Thermo Fisher Scientific) reverse transcriptase. Real-time PCR was performed with a BIO-RAD CFX96 Touch Real-Time PCR detection system, SYBR TransStart Green qPCR SuperMix (TransGen Biotech, China), and gene-specific primers. Primer sequences are listed in Supplemental Table S1. The 2− ΔΔCT method was used to analyze the relative expression levels of the genes of interest, with detection of 18S rRNA used as an internal control.
2.1. Primary cultures of cortical neurons 2.3. Immunostaining and imaging Cerebral cortical neurons were prepared from neonatal C57BL/6 mice within 24 h of birth. Briefly, meninges were stripped and cortical tissue was incubated for 10 min at 37 °C with 0.125% trypsin. The cell suspension was collected and passed through a 40-μm pore filter and the filtrate was centrifuged at 2000 rpm for 5 min. After the cells were resuspended in Dulbecco's Modified Eagle Medium (DMEM; Sigma, USA) containing 2% B27 Supplement (Thermo Fisher Scientific, USA), they were seeded into poly-L-lysine-coated 6-well plates or 25 cm2 cell culture flasks at a concentration of 2 × 106 cells/ml. Seeded cells were acclimated to 10% DMEM for 4–6 h and then were maintained in DMEM containing 2% B27, 1 mM L-glutamine, 0.1 mg/ml streptomycin, and 100 U/ml penicillin.
Cultured cells were fixed with 4% paraformaldehyde (PFA) and then quenched with 100 nM glycine. Samples were blocked in PBS containing 5% goat serum, 2% bovine serum albumin (BSA), 0.2% Triton X-100, and 0.1% sodium azide. Immunostaining was performed as previously described (Wang et al., 2015). The primary antibodies that were used are listed in Supplemental Table S2. In addition, nuclei were stained with DAPI before the stained samples were mounted with Dako fluorescent mounting media (DAKO Corp., USA). Images of the stained cells were acquired with a Zeiss LSM 700 confocal microscope and were analyzed with ZEN 2009 software (Carl Zeiss, Germany).
Fig. 2. Characterization of the adenovirus system used for overexpression of RGS5. A. Expression of RGS5 fused to GFP in Neuro 2a cells as detected by confocal microscopy. Scale bar, 12.5 μm. B. Relative expression of RGS5 to GFP as assessed by real-time PCR. n = 3. ***P b 0.001 compared with the GFP group. C. Immunofluorescent staining of RGS5 in Neuro 2a cells that were transfected with RGS5-GFP. The red signal represents staining for RGS5 and nuclear DNA was stained with DAPI. Scale bar, 16.7 μm. D. Western blot analysis of RGS5 protein levels in cells infected with GFP control or RGS5 overexpression adenoviruses. n = 3. E. Immunofluorescent staining of RGS5 in primary neurons. Cells were fixed and stained with an anti-RGS5 antibody (red) and nuclei were stained with DAPI (blue). Scale bar, 25 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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2.4. Construction of a recombinant adenovirus for RGS5 overexpression Recombinant adenoviruses expressing RGS5 were constructed with the AdEasy adenoviral vector system (Luo et al., 2007). Both the adenoviral plasmid (pAdEasy-1) and shuttle vector (pAdTrack-CMV) for this system were provided by Dr. Shihuan Kuang from Purdue University. This pAdTrack-CMV vector contains two separate cytomegalovirus (CMV) promoters that independently drive expression of the green fluorescent protein (GFP) and RGS5. RGS5 was PCR amplified from mouse brain cDNA and was cloned into the pAdTrack-CMV vector. A FLAG epitope tag (DYKDDDDK) was fused to the N-terminus of RGS5
with standard cloning techniques. It was previously demonstrated that addition of an N-terminal FLAG tag to RGS5 does not affect its function (Anger et al., 2004). Neurons were infected with the constructed adenoviruses at 37 °C for 48–96 h. 2.5. Analysis of FM4-64 dye uptake Neurons were incubated in 50 mM KCl containing 5 μM FM4-64 (Thermo Fisher Scientific) at 37 °C for 2 min, and then were incubated in PBS containing 5 μM FM4-64 at 37 °C for 10 min. Extracellular FM464 dye was subsequently removed with PBS washes. Optical density
Fig. 3. Activation of Shh signaling pathway in cultured cortical neuron stimulated by Shh. A. Relative expression of members of the Shh-signaling cascade in cultured cortical neuron as assessed by real-time PCR. n = 3. B. Shh promotes neurite outgrowth in cultured cortical neurons. Immunostaining of NF200 was performed to show the neurite. Scale bar, 25 μm. Quantification of total neurite lengths is shown. At least 100 cells/culture were analyzed. *P b 0.05, **P b 0.01 compared with the Control group. C. The expression of Smo, Ptch1 and Gli1 in neurons stimulated by 500 ng/ml Shh was determined by real-time PCR. n = 3. *P b 0.05 compared with the Control group. D. Western blot analysis of Smo and Ptch1 protein levels in neurons stimulated by 500 ng/ml Shh.
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values were recorded by an Infinite F200 pro instrument (TECAN, Italy) with excitation and emission wavelengths of 555 nm and 680 nm, respectively. Data were corrected using blank values obtained from samples that were not incubated with the FM4-64 dye.
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of the membranes, bound antibodies were visualized with an Enhanced Chemiluminescence System (ECL) using a C-DiGit Blot Scanner (LICOR Biosciences, USA). 2.7. Measurement of intracellular cAMP levels
2.6. Western blot analysis Total protein concentrations were determined for each extract according to the bicinchoninic acid (BCA) method. Proteins from each sample were then denatured at 100 °C in boiling water for 5 min, loaded into 12% SDS-PAGE gels, and separated by electrophoresis. After the proteins were transferred to polyvinylidene fluoride (PVDF) membranes, the membranes were blocked with 5% skim milk for 2 h at room temperature (RT). The membranes were subsequently incubated with anti-RGS5 (1:500 in blocking buffer), anti-Smo (1:200 in blocking buffer), anti-Ptch1 (1:500 in blocking buffer) and anti-GAPDH (1:1000 in blocking buffer) antibodies overnight at 4 °C. After three washes, the membranes were incubated with horseradish peroxidase-conjugated anti-mouse secondary antibodies (1:1000 in TBS) or anti-rabbit secondary antibodies (1:1000 in TBS) 1 h at RT. After another three washes
Intracellular cAMP levels were measured using an immunoassay kit (BioVision, USA) according to the manufacturer's instructions. Briefly, cells were incubated in 1 ml 0.1 M HCl for 20 min at RT to extract cAMP. After a centrifugation step at 12,000 ×g for 10 min, the supernatants were assayed using an acetylation procedure according to the manufacturer's protocol. Absorption values at 450 nm were determined using an Infinite F200 pro system (TECAN) and cAMP activity was normalized to protein concentration and expressed as picomoles of activity per microgram of protein. 2.8. PKA assay PKA activity was measured using a cAMP-Dependent PKA kit (Promega, USA), according to the manufacturer's instructions. Briefly,
Fig. 4. RGS5 overexpression significantly reduces Shh-mediated function in cortical neurons. A. NeurphologyJ analysis of neurite outgrowth in one single primary cortical neuron following Shh treatment. Neurites are shown in green, ending points in cyan, soma in red, and attachment points in yellow in the merged image. Scale bar, 30 μm. B–F. Quantitative data regarding neurite outgrowth of primary cortical neurons overexpressing RGS5 and treated with Shh as measured by NeurphologyJ. Mean endpoints (B), mean neurite length (C), mean neurite number (D), mean number of attachment points (E), and mean soma area (F) data are presented. Values represent the mean ± SD value from three experiments. *P b 0.05, **P b 0.01 compared with the GFP group. G. RGS5 over-expression inhibited neurite outgrowth in Shh-treated primary cortical neurons. At least 100 cells/culture were analyzed. Scale bar, 200 μm. H. Quantitative measurements of FM dye uptake by Shh-treated primary cortical neurons expressing GFP or overexpressing RGS5. n = 3. **P b 0.01 compared with the GFP group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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cells were lysed with cold PKA extraction buffer (25 mM Tris-HCl (pH 7.4), 0.5 mM EGTA, 0.5 mM EDTA, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 10 mM β-mercaptoethanol, 0.5 mM PMSF), then were assayed for phosphate incorporation into a PepTag A1 peptide (LRRASLG). Reaction products were separated on 0.8% agarose gels and bands of interest were resected and melted in a gel solubilization solution with heating. Absorbance values for these bands at 570 nm were determined using an Infinite F200 pro system (TECAN). These data were compared with the data for the control cells.
To further investigate the effect of RGS5 overexpression in cultured mouse cortical neurons, a Flag-Tag modified RGS5 adenovirus overexpression system was constructed by using pAD-Track and the AdEasy1 adenovirus packaging system. The resulting Flag-RGS5 adenoviruses were packaged in 293A cells and delivered to DIV8 primary neurons. Intracellular expression of RGS5 was subsequently confirmed by Western blot (Fig. 2D). Immunofluorescence assays further demonstrated that the expression level of RGS5 dramatically increased in the neurons that were infected with the RGS5 adenovirus for five days (Fig. 2E).
2.9. Statistical analysis Data are presented as the mean ± standard deviation (SD) from three or more experiments. Differences between groups were analyzed with a two-tailed Student's t-test for paired and unpaired data and oneway analysis of variance (ANOVA) for multiple groups (GraphPad Software, USA). Neurite outgrowth was analyzed according to established methods using the NeurphologyJ plugin of ImageJ software (Ho et al., 2011). The level of significance was set at P b 0.05. 3. Results 3.1. Expression of RGS5 in the nervous system Initially, the expression profile for RGS5 in various mouse tissues was determined by real-time PCR. RGS5 was found to be abundantly expressed in skeletal muscle, brain, and heart, and was present at low levels in the adrenal gland, adipose tissue, and lung. RGS5 was not detected in spleen and smooth muscle (Fig. 1A). To determine which subsets of cells in the brain express RGS5, cultured cortical neurons, astrocytes, oligodendrocytes, and microglia were assayed. Cortical neurons were found to express the highest levels of RGS5 transcripts, while astrocytes and oligodendrocytes expressed intermediate levels of RGS5 transcripts (Fig. 1B). RGS5 transcripts were barely detectable in microglia (Fig. 1B). Expression of members of the RGS-R4 subfamily (RGS1–5, RGS8, RGS13, RGS16, RGS18, RGS21) was also assayed in the cultured cortical neurons. Only RGS1, RGS2, RGS4, RGS5, RGS8, RGS13, and RGS16 were detected (Fig. 1C). To investigate the dynamics of RGS5 expression in the cultured neuron model, RGS5 transcripts were assayed at different time points. A time-dependent decrease in RGS5 expression was observed with prolonged culturing, and a significant decrease in RGS5 transcripts was detected five days after the initial seeding of the cortical neurons (Fig. 1D). Also, a decrease in RGS5 mRNA expression was found in cultured cortical neurons (Fig. 1E) with increasing age. These results suggest that RGS5 may inhibit the growth of neurons. It has been reported that RGS5 is expressed in multiple cell lines, including human lung cancer cell line H1299 (Xu et al., 2015), human liver cancer cell line SNU-398 (Hu et al., 2013), mouse teratocarcinoma cell line F9 (Feigin and Malbon, 2007), and mouse embryonic mesenchymal cell line C3H10T1/2 (Mahoney et al., 2013). When three mouse neural cell lines were assayed (Neuro 2a, CATH.a, and C17.2), RGS5 mRNA was not detected in any of them (Fig. 1F).
3.3. RGS5 overexpression significantly diminished Shh-mediated function in cortical neurons It has been reported that Shh promotes both the neurite outgrowth (He et al., 2016) and the maturation of presynaptic terminals in primary cultured neurons (Mitchell et al., 2012). The expression of some numbers of the Shh-signaling cascade (Smo, Ptch1, Gli2 and Gli3) was found in cultured cortical neurons (Fig. 3A). To confirm the effect of Shh on neurite outgrowth, primary cortical neurons were incubated with various concentrations of Shh (10/100/500/1000 ng/ml). As shown in Fig. 3B, Shh significantly increased neurite outgrowth compared with untreated control. At all doses of Shh used, no detectable cytotoxicity was observed as determined using lactate dehydrogenase release assays (Fig. S2). When stimulated with 500 ng/ml Shh, the expression of Smo and Ptch1 was significantly increased, indicating that the Shh pathway was activated (Fig. 3C, Fig. 3D). Furthermore, cyclopamine, which is a Smo inhibitor, blocked the Shh mediated neurite outgrowth confirming that the neurite outgrowth was Smo dependent (Fig. S3). In addition, RGS5 has been shown to diminish Shh function in C3H10T1/2 cells (Mahoney et al., 2013). To test whether RGS5 overexpression effect Shh function in primary cortical neurons, DIV8 neurons were infected with a GFP control adenovirus and an adenovirus overexpressing RGS5 for 24 h. These two groups of primary cortical neurons
3.2. An adenovirus system was used to overexpress RGS5 in cultured mouse cortical neurons To observe the cellular distribution and function of RGS5, a eukaryotic expression plasmid (pLVX-AcGFP-N1) was constructed and delivered into mouse Neuro 2a cells with Lipofectamine 2000. An RGS5-GFP signal was observed to be uniformly distributed throughout each cell with confocal microscopy (Fig. 2A). Expression of GFP and RGS5 were also confirmed by real-time PCR (Fig. 2B), while protein levels of RGS5 were verified in immunofluorescence assays (Fig. 2C). Meanwhile, degradation of exogenously produced RGS5 was found to be very rapid compared with GFP (Fig. S1).
Fig. 5. RGS5 overexpression significantly reduces Shh signaling in cortical neurons. A. Relative expression of Smo, Ptch1 and Gli1 in GFP and RGS5 overexpression neurons stimulated by 500 ng/ml Shh as assessed by real-time PCR. n = 3. *P b 0.05 compared with the GFP group. B. Western blot analysis of Smo and Ptch1 protein levels in GFP and RGS5 overexpression neurons stimulated by 500 ng/ml Shh.
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were then stimulated with Shh (500 ng/ml) for an additional 48 h. RGS5 overexpression significantly reduced the number of neurites under single neuron magnification power (Fig. 4A), and corresponded with fewer endpoints, shorter neurite lengths, a reduced number of attachment points, and a decreased soma area (Fig. 4, B-F). Endocytosis by the neurons overexpressing RGS5 was also examined following FM4-64 staining of the presynaptic terminals. Compared with the neurons which were infected with the GFP control adenovirus, infection with the RGS5 overexpressing adenovirus resulted in a significant decrease in the amount of FM4-64 fluorescence (Fig. 4H). As shown in Fig. 5, following overexpression of RGS5, the expression of Smo and Ptch1 was also inhibited, but no Gli1 expression were significant changed which suggests RGS5 acts through the non-canonical Shh signaling pathway (Berretta et al., 2016). Therefore, it appears that an increase in RGS5 expression leads to a reduction in Shh-mediated function in cortical neurons.
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3.4. cAMP-PKA contributes to RGS5-mediated inhibition of Shh signaling in cortical neurons It is hypothesized that cAMP-PKA inhibits Shh signaling in both mouse and Drosophila melanogaster (Makinodan and Marneros, 2012; Noveen et al., 1996), and RGS5 is a molecule that negatively regulates Gαi, a protein which inhibits the production of cAMP from ATP. To test whether the RGS5-mediated inhibition of Shh function observed in the present study involves modulation of cAMP levels, cAMP concentrations in GFP control and RGS5 overexpressing cortical neurons stimulated with Shh were measured. As shown in Fig. 6A, an increase cAMP production was detected in response to overexpression of RGS5. Immunofluorescence staining of cAMP subsequently confirmed these results (Fig. S4). Interestingly, the distribution of cAMP was characterized by large clusters in the GFP control neurons, while the RGS5 overexpressing neurons exhibited smaller clusters of cAMP that were distributed
Fig. 6. cAMP-PKA contributes to RGS5 mediated inhibition of Shh signaling in cortical neurons. Intracellular cAMP levels (A) and PKA activity (B) were measured for Shh-treated neurons expressing GFP or overexpressing RGS5. n = 3. C–H. H89 (5 μM) treatment reversed RGS5 mediated inhibition of Shh function as demonstrated by measuring mean endpoints (C), mean neurite length (D), mean neurite number (E), mean number of attachment points (F), mean soma area (G), and mean optical density (H). At least 100 cells/culture were analyzed. Data are expressed as the mean ± SD from three experiments. *P b 0.05 compared with the GFP group in A and B; *P b 0.05, **P b 0.01 compared with the RGS5 group in C–H.
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throughout the cytoplasm (Fig. S4). We also confirmed the cAMP function in the absence of RGS5 manipulation; neurite outgrowth was supported by cAMP agonist db-cAMP. It is interesting that neurite outgrowth was suppressed when neurons were stimulated by Shh + db-cAMP compared with Shh or db-cAMP stimulated alone (Fig. S5). Considering that elevated cAMP levels promote PKA activation, and that PKA is a negative regulator of Shh signaling, PKA activation is also examined. In the assays performed, an increase in intracellular PKA activity was detected that paralleled the increase in cAMP levels previously observed (Fig. 6B). Moreover, when the RGS5 overexpressing neurons were incubated with Shh in the presence of the PKA-specific inhibitor, H89 (5 μM), RGS5-mediated inhibition of Shh function was prevented (Fig. 6, C–H). Therefore, in combination, these results suggest that cAMP-PKA plays a role in RGS5-mediated inhibition of Shh function. 4. Discussion In this study, the distribution of RGS5 expression in mouse tissues was investigated by using real-time PCR. Consistent with the results of previous studies (Seki et al., 1998), the expression of RGS5 was found at high levels in skeletal muscle, brain, and heart. In contrast, RGS5 was expressed at low levels in the adrenal gland, adipose, and lung tissues, and was not detected in spleen and smooth muscle. In the central nervous system, higher levels of RGS5 mRNA were detected in cortical neurons compared with astrocytes and oligodendrocytes, while the RGS5 transcript was not detected in microglia. RGS-R4 is the largest RGS protein family and it includes RGS proteins 1–5, 8, 13, 16, 18, and 21. RGS-R4 proteins are the smallest RGS proteins in size and they contain short peptide sequences that flank the RGS box with one notable exception (RGS3) (Bansal et al., 2007). Interestingly, when we examined whether members of the RGS-R4 subfamily were expressed in cultured cortical neurons, only RGS 3, 18, and 21 were not detected. For RGS5, its expression declined as the duration of in vitro culturing increased. Thus, RGS5 may mediate an inhibitory effect during the development of cortical neurons. RGS5 structure is highly homologous with two other members of the RGS-R4 family, RGS4 and RGS16. These three RGS proteins contain an Nterminal cysteine residue which can be oxidized in vivo by nitric oxide and oxygen (Park et al., 2015). Meanwhile, the N terminus of RGS5 contains an N-end rule determinant that controls its ubiquitylation and expression (Bodenstein et al., 2007). In the presence of MG-132, ubiquitylation of exogenous RGS5 was significantly inhibited. The ability of RGS to promote the hydrolysis of GTP, thereby reducing the life span of the active form of the GTP α subunit, has been well characterized. Accordingly, RGS proteins are emerging as important negative regulators of GPCR signaling pathways. Moreover, it is hypothesized that functional diversity exists among RGS proteins. Regarding RGS5, this member of the RGS protein superfamily has recently been reported to regulate many biological processes by inhibiting several Gαimediated signaling pathways (Deng et al., 2012; Gunaje et al., 2011; Hamzah et al., 2008; Li et al., 2010; Takata et al., 2008). Cellular signaling cascades that originate from adenylyl cyclase and that are activated during neural development are mostly controlled by Gαs proteins. Furthermore, activation of adenylyl cyclase has been shown to result in a transient accumulation of cAMP (Cameron et al., 1988). Elevated levels of cytoplasmic cAMP activate PKA which acts as a negative regulator of Shh signaling. In a previous study, Shh was shown to activate autophagy in hippocampal neurons (Petralia et al., 2013) and to alter the architecture and function of presynaptic terminals in cultured hippocampal neurons (Mitchell et al., 2012). Very few studies have explored the function of RGS5 in cortical neurons during neurite outgrowth. Here, overexpression of RGS5 was achieved with an adenovirus system that successfully infected primary cortical neurons. The resulting data provide evidence that overexpression
of RGS5 inhibits neurite outgrowth of primary cortical neurons which have undergone Shh stimulation. In a previous study, presynaptic terminals exhibited an increase in size and uptake of FM dye following Shh stimulation (Mitchell et al., 2012). In the present study, RGS5 overexpression reduced the endocytosis capacity of presynaptic terminals in Shh stimulated cortical neurons for FM4-64 dye. The cAMP-PKA pathway has been shown to suppress Shh signaling (Makinodan and Marneros, 2012). The downstream components in the Shh pathway are potential targets for PKA phosphorylation. Therefore, in the present study, it was hypothesized that cAMP-PKA signaling is involved in the regulation of Shh by RGS5 overexpression. Support for this hypothesis was obtained in the present study when RGS5 overexpression resulted in an increased level of intracellular cAMP and an increased activity of PKA in the cultured neuron model examined. Furthermore, H89, a specific inhibitor of PKA, disrupted the inhibition of Shh function by RGS5 overexpression. Together, these results indicate that cAMP-PKA signaling mediates an inhibition of Shh function that is induced by RGS5 overexpression. Therefore, in conclusion, the results of the present study demonstrate that RGS5 antagonizes Shh signaling and inhibits neurite outgrowth, and this is partially attributed to cAMP-PKA signaling. However, in vivo studies of Shh-induced neurite extension are needed, especially studies that will determine whether other pathways are involved in RGS5-mediated antagonism of Shh signaling. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.mcn.2017.06.005. Acknowledgments The authors would like to thank Dr. Hongwei Xu for her helpful technical assistance, Dr. Shihuan Kuang (Purdue University) for the AdEasy system. This study was financially supported by the National Natural Science Foundation of China (81671234, 81000512, 81471227, 81430035, 31671112), Natural Science Foundation of Heilongjiang Province (LC2016031), and Academician Weihan Yu Outstanding Youth Training Foundation Project (20150901). References Anger, T., Zhang, W., Mende, U., 2004. Differential contribution of GTPase activation and effector antagonism to the inhibitory effect of RGS proteins on Gq-mediated signaling in vivo. J. Biol. Chem. 279, 3906–3915. Arenas, E., 2014. Wnt signaling in midbrain dopaminergic neuron development and regenerative medicine for Parkinson's disease. J. Mol. Cell Biol. 6, 42–53. Bansal, G., Druey, K.M., Xie, Z., 2007. R4 RGS proteins: regulation of G-protein signaling and beyond. Pharmacol. Ther. 116, 473–495. Berretta, A., Gowing, E.K., Jasoni, C.L., Clarkson, A.N., 2016. Sonic hedgehog stimulates neurite outgrowth in a mechanical stretch model of reactive-astrogliosis. Sci Rep 6, 21896. Bodenstein, J., Sunahara, R.K., Neubig, R.R., 2007. N-terminal residues control proteasomal degradation of RGS2, RGS4, and RGS5 in human embryonic kidney 293 cells. Mol. Pharmacol. 71, 1040–1050. Cameron, S., Levin, L., Zoller, M., Wigler, M., 1988. cAMP-independent control of sporulation, glycogen metabolism, and heat shock resistance in S. cerevisiae. Cell 53, 555–566. Cheng, W.L., Wang, P.X., Wang, T., Zhang, Y., Du, C., Li, H., Ji, Y., 2015. Regulator of G-protein signalling 5 protects against atherosclerosis in apolipoprotein E-deficient mice. Br. J. Pharmacol. 172, 5676–5689. Cho, H., Park, C., Hwang, I.Y., Han, S.B., Schimel, D., Despres, D., Kehrl, J.H., 2008. Rgs5 targeting leads to chronic low blood pressure and a lean body habitus. Mol. Cell. Biol. 28, 2590–2597. Deng, W., Wang, X., Xiao, J., Chen, K., Zhou, H., Shen, D., Li, H., Tang, Q., 2012. Loss of regulator of G protein signaling 5 exacerbates obesity, hepatic steatosis, inflammation and insulin resistance. PLoS One 7, e30256. Feigin, M.E., Malbon, C.C., 2007. RGS19 regulates Wnt-beta-catenin signaling through inactivation of Galpha(o). J. Cell Sci. 120, 3404–3414. Gunaje, J.J., Bahrami, A.J., Schwartz, S.M., Daum, G., Mahoney Jr., W.M., 2011. PDGF-dependent regulation of regulator of G protein signaling-5 expression and vascular smooth muscle cell functionality. Am. J. Phys. Cell Phys. 301, C478–C489. Hamzah, J., Jugold, M., Kiessling, F., Rigby, P., Manzur, M., Marti, H.H., Rabie, T., Kaden, S., Grone, H.J., Hammerling, G.J., Arnold, B., Ganss, R., 2008. Vascular normalization in Rgs5-deficient tumours promotes immune destruction. Nature 453, 410–414. He, L., Lu, Q.R., 2013. Coordinated control of oligodendrocyte development by extrinsic and intrinsic signaling cues. Neurosci. Bull. 29, 129–143.
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Molecular and Cellular Neuroscience journal homepage: www.elsevier.com/locate/ymcne
Regulator of G protein signaling 5 (RGS5) inhibits sonic hedgehog function in mouse cortical neurons Chuanliang Liu a,b,1, Qiongqiong Hu a,1, Jia Jing a, Yun Zhang a, Jing Jin a, Liulei Zhang a, Lili Mu a, Yumei Liu a, Bo Sun a, Tongshuai Zhang a, Qingfei Kong a, Guangyou Wang a, Dandan Wang a, Yao Zhang a, Xijun Liu a, Wei Zhao a, Jinghua Wang a,⁎, Tao Feng c,⁎⁎, Hulun Li a,d a
Department of Neurobiology, Harbin Medical University, Heilongjiang Provincial Key Laboratory of Neurobiology, Harbin, Heilongjiang 150086, China Vocational College Daxing'an Mountains, Jiagedaqi District, Heilongjiang 165000, China c Department of Neurology, The Nangang Branch of Heilongjiang Provincial Hospital, Harbin, Heilongjiang 150001, China d Key Laboratories of Education Ministry for Myocardial Ischemia Mechanism and Treatment, Harbin, Heilongjiang 150086, China b
a r t i c l e
i n f o
Article history: Received 11 September 2016 Revised 21 February 2017 Accepted 20 June 2017 Available online 03 July 2017 Keywords: RGS5 Sonic hedgehog Cortical neurons
a b s t r a c t Regulator of G protein signaling 5 (RGS5) acts as a GTPase-activating protein (GAP) for the Gαi subunit and negatively regulates G protein-coupled receptor signaling. However, its presence and function in postmitotic differentiated primary neurons remains largely uncharacterized. During neural development, sonic hedgehog (Shh) signaling is involved in cell signaling pathways via Gαi activity. In particular, Shh signaling is essential for embryonic neural tube patterning, which has been implicated in neuronal polarization involving neurite outgrowth. Here, we examined whether RGS5 regulates Shh signaling in neurons. RGS5 transcripts were found to be expressed in cortical neurons and their expression gradually declined in a time-dependent manner in culture system. When an adenovirus expressing RGS5 was introduced into an in vitro cell culture model of cortical neurons, RGS5 overexpression significantly reduced neurite outgrowth and FM4-64 uptake, while cAMP-PKA signaling was also affected. These findings suggest that RGS5 inhibits Shh function during neurite outgrowth and the presynaptic terminals of primary cortical neurons mature via modulation of cAMP. © 2017 Elsevier Inc. All rights reserved.
1. Introduction Regulator of G protein signaling (RGS) proteins are signal transduction molecules which contribute to the regulation of heterotrimeric G proteins. RGS proteins have been classified into nine subfamilies based on conserved sequences present in their catalytic RGS domain. These subfamilies (and their members) include: A/RZ (RGS17, RGS19, RGS20, RET-RGS1), B/R4 (RGS1-RGS5, RGS8, RGS13, RGS16, RGS18, RGS21), C/R7 (RGS6, RGS7, RGS9, RGS11), D/R12 (RGS10, RGS12, RGS14), E/RA (axin, conductin), F/GEF (p115-RhoGEF, PDZ-RhoGEF, LARG), G/GRK (GRK1-GRK7), H/SNX (SNX13, SNX14, SNX25), and I/DAKAP2 (D-AKAP2, RGS22) (Jules et al., 2015). These various RGS subfamilies regulate different G proteins. For example, the RGS-R4 subfamily exhibits specificity for Gαi and Gαq, yet does not appear to interact
⁎ Correspondence to: J. Wang, Department of Neurobiology, Harbin Medical University, Harbin, Heilongjiang 150086, China. ⁎⁎ Correspondence to: T. Feng, Department of Neurology, the Nangang Branch of Heilongjiang Provincial Hospital, Harbin, Heilongjiang 150001, China E-mail addresses: [email protected] (J. Wang), [email protected] (T. Feng). 1 Chuanliang Liu and Qiongqiong Hu contributed equally to this work.
http://dx.doi.org/10.1016/j.mcn.2017.06.005 1044-7431/© 2017 Elsevier Inc. All rights reserved.
with Gαs (Bansal et al., 2007). The R4 subfamily member, RGS5, is first identified and isolated from a neuroblastoma cDNA library (Seki et al., 1998) and is a tissue-specific signaling protein that negatively regulates Gαi activity. Conversely, activation of Gαi inhibits the activity of the membrane enzyme, adenylyl cyclase, which converts ATP to cAMP. RGS5 has also been reported involved in many pathophysiological processes including atherosclerosis (Cheng et al., 2015; Li et al., 2004), blood pressure regulation (Cho et al., 2008), cardiac hypertrophy (Deng et al., 2012), lipid metabolism (Xiao et al., 2009), bronchial smooth muscle cell contraction, and asthma (Yang et al., 2012). Elevated expression of RGS5 has also been found in various cancers (Sethakorn and Dulin, 2013). During brain development, cortical neurons undergo neuron polarization, axon outgrowth, dendritogenesis, the formation of the synapse, as well as synaptic function maturation. To further ascertain the subcellular distribution and the expression of mRNAs or proteins on specific developmental stages, and to address fundamental questions regarding neuronal polarity, spine development, and synaptic plasticity, neonatal neuron cultures have been used. As a result, several cell signaling pathways have been reported to mediate neural development, including the Notch signaling pathway, the Wnt/β-catenin signaling pathway, and the hedgehog signaling pathway (Arenas, 2014). Sonic hedgehog
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(Shh) is a secreted molecule that binds patched (Ptch) receptors which are expressed on Shh-responding cells. Upon receptor binding, Smoothened (Smo), a G-protein coupled receptor (GPCR), transduces an intracellular signal which converges to activate the zinc finger protein transcription factor, Gli. Shh signaling has been shown to control cell growth, survival, and differentiation in a wide variety of cells (He and
Lu, 2013; Stewart et al., 2002). In the nervous system, Shh signaling is essential for patterning of the embryonic neural tube, and it also function s in developing neurons. In primary hippocampal neurons, Shh regulates autophagy (Petralia et al., 2013) and presynaptic terminal function (Mitchell et al., 2012). In primary cortical neurons, Shh promotes neurite outgrowth (He et al., 2016). In adult sensory neurons,
Fig. 1. Expression profile of RGS5 mRNA in C57BL/6 adult mouse tissues. A. Fold-expression of RGS5 mRNA detected in various tissues obtained from C57BL/6 adult mice relative to smooth muscle tissue. n = 3. B. Fold-expression of RGS5 mRNA detected in various central nervous system cellular components as indicated relative to neurons. n = 3. C. Fold-expression of the RGS-R4 subfamily members detected in cultured cortical neurons relative to RGS5. n = 3. D. Fold-expression of RGS5 in cortical neurons cultured in vitro at the different time points indicated relative to the levels detected after 4 h. n = 3. E. Fold-expression of RGS5 in cortical neurons cultured from mice with various age. n = 3. F. Fold-expression of RGS5 in primary cortical neurons versus the Neuro 2a, CATH.a, and C17.2 cell lines. n = 3. Values represent the mean ± SD value from three experiments. *P b 0.05, ***P b 0.001 compared with the Neuron group in B. *P b 0.05 compared with the Neuron group in D. *P b 0.05, **P b 0.01, ***P b 0.001 compared with the 1D group in E. ***P b 0.001 compared with the Neuron group in F.
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knockdown of Shh resulted in decreased outgrowth and branching (Martinez et al., 2015). In recent studies, cAMP-protein kinase A (PKA) has been found to inhibit Shh signaling (Makinodan and Marneros, 2012). However, it remains unknown whether RGS5 served as a regulator of Gαi signaling, contributes to Shh signaling through cAMP in developing neurons. Therefore, in this study, RGS5 expression is investigated during cortical neuron development in an in vitro model. A possible role for RGS5 in regulating Shh signaling is also examined.
2. Materials and methods
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2.2. RNA isolation and quantitative PCR analysis Total RNA was extracted from cells or tissues using TRIzol reagent (Thermo Fisher Scientific), according to the manufacturer's instructions. Reverse transcription was performed using random hexaprimers and Moloney murine leukemia virus (M-MLV; Thermo Fisher Scientific) reverse transcriptase. Real-time PCR was performed with a BIO-RAD CFX96 Touch Real-Time PCR detection system, SYBR TransStart Green qPCR SuperMix (TransGen Biotech, China), and gene-specific primers. Primer sequences are listed in Supplemental Table S1. The 2− ΔΔCT method was used to analyze the relative expression levels of the genes of interest, with detection of 18S rRNA used as an internal control.
2.1. Primary cultures of cortical neurons 2.3. Immunostaining and imaging Cerebral cortical neurons were prepared from neonatal C57BL/6 mice within 24 h of birth. Briefly, meninges were stripped and cortical tissue was incubated for 10 min at 37 °C with 0.125% trypsin. The cell suspension was collected and passed through a 40-μm pore filter and the filtrate was centrifuged at 2000 rpm for 5 min. After the cells were resuspended in Dulbecco's Modified Eagle Medium (DMEM; Sigma, USA) containing 2% B27 Supplement (Thermo Fisher Scientific, USA), they were seeded into poly-L-lysine-coated 6-well plates or 25 cm2 cell culture flasks at a concentration of 2 × 106 cells/ml. Seeded cells were acclimated to 10% DMEM for 4–6 h and then were maintained in DMEM containing 2% B27, 1 mM L-glutamine, 0.1 mg/ml streptomycin, and 100 U/ml penicillin.
Cultured cells were fixed with 4% paraformaldehyde (PFA) and then quenched with 100 nM glycine. Samples were blocked in PBS containing 5% goat serum, 2% bovine serum albumin (BSA), 0.2% Triton X-100, and 0.1% sodium azide. Immunostaining was performed as previously described (Wang et al., 2015). The primary antibodies that were used are listed in Supplemental Table S2. In addition, nuclei were stained with DAPI before the stained samples were mounted with Dako fluorescent mounting media (DAKO Corp., USA). Images of the stained cells were acquired with a Zeiss LSM 700 confocal microscope and were analyzed with ZEN 2009 software (Carl Zeiss, Germany).
Fig. 2. Characterization of the adenovirus system used for overexpression of RGS5. A. Expression of RGS5 fused to GFP in Neuro 2a cells as detected by confocal microscopy. Scale bar, 12.5 μm. B. Relative expression of RGS5 to GFP as assessed by real-time PCR. n = 3. ***P b 0.001 compared with the GFP group. C. Immunofluorescent staining of RGS5 in Neuro 2a cells that were transfected with RGS5-GFP. The red signal represents staining for RGS5 and nuclear DNA was stained with DAPI. Scale bar, 16.7 μm. D. Western blot analysis of RGS5 protein levels in cells infected with GFP control or RGS5 overexpression adenoviruses. n = 3. E. Immunofluorescent staining of RGS5 in primary neurons. Cells were fixed and stained with an anti-RGS5 antibody (red) and nuclei were stained with DAPI (blue). Scale bar, 25 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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2.4. Construction of a recombinant adenovirus for RGS5 overexpression Recombinant adenoviruses expressing RGS5 were constructed with the AdEasy adenoviral vector system (Luo et al., 2007). Both the adenoviral plasmid (pAdEasy-1) and shuttle vector (pAdTrack-CMV) for this system were provided by Dr. Shihuan Kuang from Purdue University. This pAdTrack-CMV vector contains two separate cytomegalovirus (CMV) promoters that independently drive expression of the green fluorescent protein (GFP) and RGS5. RGS5 was PCR amplified from mouse brain cDNA and was cloned into the pAdTrack-CMV vector. A FLAG epitope tag (DYKDDDDK) was fused to the N-terminus of RGS5
with standard cloning techniques. It was previously demonstrated that addition of an N-terminal FLAG tag to RGS5 does not affect its function (Anger et al., 2004). Neurons were infected with the constructed adenoviruses at 37 °C for 48–96 h. 2.5. Analysis of FM4-64 dye uptake Neurons were incubated in 50 mM KCl containing 5 μM FM4-64 (Thermo Fisher Scientific) at 37 °C for 2 min, and then were incubated in PBS containing 5 μM FM4-64 at 37 °C for 10 min. Extracellular FM464 dye was subsequently removed with PBS washes. Optical density
Fig. 3. Activation of Shh signaling pathway in cultured cortical neuron stimulated by Shh. A. Relative expression of members of the Shh-signaling cascade in cultured cortical neuron as assessed by real-time PCR. n = 3. B. Shh promotes neurite outgrowth in cultured cortical neurons. Immunostaining of NF200 was performed to show the neurite. Scale bar, 25 μm. Quantification of total neurite lengths is shown. At least 100 cells/culture were analyzed. *P b 0.05, **P b 0.01 compared with the Control group. C. The expression of Smo, Ptch1 and Gli1 in neurons stimulated by 500 ng/ml Shh was determined by real-time PCR. n = 3. *P b 0.05 compared with the Control group. D. Western blot analysis of Smo and Ptch1 protein levels in neurons stimulated by 500 ng/ml Shh.
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values were recorded by an Infinite F200 pro instrument (TECAN, Italy) with excitation and emission wavelengths of 555 nm and 680 nm, respectively. Data were corrected using blank values obtained from samples that were not incubated with the FM4-64 dye.
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of the membranes, bound antibodies were visualized with an Enhanced Chemiluminescence System (ECL) using a C-DiGit Blot Scanner (LICOR Biosciences, USA). 2.7. Measurement of intracellular cAMP levels
2.6. Western blot analysis Total protein concentrations were determined for each extract according to the bicinchoninic acid (BCA) method. Proteins from each sample were then denatured at 100 °C in boiling water for 5 min, loaded into 12% SDS-PAGE gels, and separated by electrophoresis. After the proteins were transferred to polyvinylidene fluoride (PVDF) membranes, the membranes were blocked with 5% skim milk for 2 h at room temperature (RT). The membranes were subsequently incubated with anti-RGS5 (1:500 in blocking buffer), anti-Smo (1:200 in blocking buffer), anti-Ptch1 (1:500 in blocking buffer) and anti-GAPDH (1:1000 in blocking buffer) antibodies overnight at 4 °C. After three washes, the membranes were incubated with horseradish peroxidase-conjugated anti-mouse secondary antibodies (1:1000 in TBS) or anti-rabbit secondary antibodies (1:1000 in TBS) 1 h at RT. After another three washes
Intracellular cAMP levels were measured using an immunoassay kit (BioVision, USA) according to the manufacturer's instructions. Briefly, cells were incubated in 1 ml 0.1 M HCl for 20 min at RT to extract cAMP. After a centrifugation step at 12,000 ×g for 10 min, the supernatants were assayed using an acetylation procedure according to the manufacturer's protocol. Absorption values at 450 nm were determined using an Infinite F200 pro system (TECAN) and cAMP activity was normalized to protein concentration and expressed as picomoles of activity per microgram of protein. 2.8. PKA assay PKA activity was measured using a cAMP-Dependent PKA kit (Promega, USA), according to the manufacturer's instructions. Briefly,
Fig. 4. RGS5 overexpression significantly reduces Shh-mediated function in cortical neurons. A. NeurphologyJ analysis of neurite outgrowth in one single primary cortical neuron following Shh treatment. Neurites are shown in green, ending points in cyan, soma in red, and attachment points in yellow in the merged image. Scale bar, 30 μm. B–F. Quantitative data regarding neurite outgrowth of primary cortical neurons overexpressing RGS5 and treated with Shh as measured by NeurphologyJ. Mean endpoints (B), mean neurite length (C), mean neurite number (D), mean number of attachment points (E), and mean soma area (F) data are presented. Values represent the mean ± SD value from three experiments. *P b 0.05, **P b 0.01 compared with the GFP group. G. RGS5 over-expression inhibited neurite outgrowth in Shh-treated primary cortical neurons. At least 100 cells/culture were analyzed. Scale bar, 200 μm. H. Quantitative measurements of FM dye uptake by Shh-treated primary cortical neurons expressing GFP or overexpressing RGS5. n = 3. **P b 0.01 compared with the GFP group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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cells were lysed with cold PKA extraction buffer (25 mM Tris-HCl (pH 7.4), 0.5 mM EGTA, 0.5 mM EDTA, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 10 mM β-mercaptoethanol, 0.5 mM PMSF), then were assayed for phosphate incorporation into a PepTag A1 peptide (LRRASLG). Reaction products were separated on 0.8% agarose gels and bands of interest were resected and melted in a gel solubilization solution with heating. Absorbance values for these bands at 570 nm were determined using an Infinite F200 pro system (TECAN). These data were compared with the data for the control cells.
To further investigate the effect of RGS5 overexpression in cultured mouse cortical neurons, a Flag-Tag modified RGS5 adenovirus overexpression system was constructed by using pAD-Track and the AdEasy1 adenovirus packaging system. The resulting Flag-RGS5 adenoviruses were packaged in 293A cells and delivered to DIV8 primary neurons. Intracellular expression of RGS5 was subsequently confirmed by Western blot (Fig. 2D). Immunofluorescence assays further demonstrated that the expression level of RGS5 dramatically increased in the neurons that were infected with the RGS5 adenovirus for five days (Fig. 2E).
2.9. Statistical analysis Data are presented as the mean ± standard deviation (SD) from three or more experiments. Differences between groups were analyzed with a two-tailed Student's t-test for paired and unpaired data and oneway analysis of variance (ANOVA) for multiple groups (GraphPad Software, USA). Neurite outgrowth was analyzed according to established methods using the NeurphologyJ plugin of ImageJ software (Ho et al., 2011). The level of significance was set at P b 0.05. 3. Results 3.1. Expression of RGS5 in the nervous system Initially, the expression profile for RGS5 in various mouse tissues was determined by real-time PCR. RGS5 was found to be abundantly expressed in skeletal muscle, brain, and heart, and was present at low levels in the adrenal gland, adipose tissue, and lung. RGS5 was not detected in spleen and smooth muscle (Fig. 1A). To determine which subsets of cells in the brain express RGS5, cultured cortical neurons, astrocytes, oligodendrocytes, and microglia were assayed. Cortical neurons were found to express the highest levels of RGS5 transcripts, while astrocytes and oligodendrocytes expressed intermediate levels of RGS5 transcripts (Fig. 1B). RGS5 transcripts were barely detectable in microglia (Fig. 1B). Expression of members of the RGS-R4 subfamily (RGS1–5, RGS8, RGS13, RGS16, RGS18, RGS21) was also assayed in the cultured cortical neurons. Only RGS1, RGS2, RGS4, RGS5, RGS8, RGS13, and RGS16 were detected (Fig. 1C). To investigate the dynamics of RGS5 expression in the cultured neuron model, RGS5 transcripts were assayed at different time points. A time-dependent decrease in RGS5 expression was observed with prolonged culturing, and a significant decrease in RGS5 transcripts was detected five days after the initial seeding of the cortical neurons (Fig. 1D). Also, a decrease in RGS5 mRNA expression was found in cultured cortical neurons (Fig. 1E) with increasing age. These results suggest that RGS5 may inhibit the growth of neurons. It has been reported that RGS5 is expressed in multiple cell lines, including human lung cancer cell line H1299 (Xu et al., 2015), human liver cancer cell line SNU-398 (Hu et al., 2013), mouse teratocarcinoma cell line F9 (Feigin and Malbon, 2007), and mouse embryonic mesenchymal cell line C3H10T1/2 (Mahoney et al., 2013). When three mouse neural cell lines were assayed (Neuro 2a, CATH.a, and C17.2), RGS5 mRNA was not detected in any of them (Fig. 1F).
3.3. RGS5 overexpression significantly diminished Shh-mediated function in cortical neurons It has been reported that Shh promotes both the neurite outgrowth (He et al., 2016) and the maturation of presynaptic terminals in primary cultured neurons (Mitchell et al., 2012). The expression of some numbers of the Shh-signaling cascade (Smo, Ptch1, Gli2 and Gli3) was found in cultured cortical neurons (Fig. 3A). To confirm the effect of Shh on neurite outgrowth, primary cortical neurons were incubated with various concentrations of Shh (10/100/500/1000 ng/ml). As shown in Fig. 3B, Shh significantly increased neurite outgrowth compared with untreated control. At all doses of Shh used, no detectable cytotoxicity was observed as determined using lactate dehydrogenase release assays (Fig. S2). When stimulated with 500 ng/ml Shh, the expression of Smo and Ptch1 was significantly increased, indicating that the Shh pathway was activated (Fig. 3C, Fig. 3D). Furthermore, cyclopamine, which is a Smo inhibitor, blocked the Shh mediated neurite outgrowth confirming that the neurite outgrowth was Smo dependent (Fig. S3). In addition, RGS5 has been shown to diminish Shh function in C3H10T1/2 cells (Mahoney et al., 2013). To test whether RGS5 overexpression effect Shh function in primary cortical neurons, DIV8 neurons were infected with a GFP control adenovirus and an adenovirus overexpressing RGS5 for 24 h. These two groups of primary cortical neurons
3.2. An adenovirus system was used to overexpress RGS5 in cultured mouse cortical neurons To observe the cellular distribution and function of RGS5, a eukaryotic expression plasmid (pLVX-AcGFP-N1) was constructed and delivered into mouse Neuro 2a cells with Lipofectamine 2000. An RGS5-GFP signal was observed to be uniformly distributed throughout each cell with confocal microscopy (Fig. 2A). Expression of GFP and RGS5 were also confirmed by real-time PCR (Fig. 2B), while protein levels of RGS5 were verified in immunofluorescence assays (Fig. 2C). Meanwhile, degradation of exogenously produced RGS5 was found to be very rapid compared with GFP (Fig. S1).
Fig. 5. RGS5 overexpression significantly reduces Shh signaling in cortical neurons. A. Relative expression of Smo, Ptch1 and Gli1 in GFP and RGS5 overexpression neurons stimulated by 500 ng/ml Shh as assessed by real-time PCR. n = 3. *P b 0.05 compared with the GFP group. B. Western blot analysis of Smo and Ptch1 protein levels in GFP and RGS5 overexpression neurons stimulated by 500 ng/ml Shh.
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were then stimulated with Shh (500 ng/ml) for an additional 48 h. RGS5 overexpression significantly reduced the number of neurites under single neuron magnification power (Fig. 4A), and corresponded with fewer endpoints, shorter neurite lengths, a reduced number of attachment points, and a decreased soma area (Fig. 4, B-F). Endocytosis by the neurons overexpressing RGS5 was also examined following FM4-64 staining of the presynaptic terminals. Compared with the neurons which were infected with the GFP control adenovirus, infection with the RGS5 overexpressing adenovirus resulted in a significant decrease in the amount of FM4-64 fluorescence (Fig. 4H). As shown in Fig. 5, following overexpression of RGS5, the expression of Smo and Ptch1 was also inhibited, but no Gli1 expression were significant changed which suggests RGS5 acts through the non-canonical Shh signaling pathway (Berretta et al., 2016). Therefore, it appears that an increase in RGS5 expression leads to a reduction in Shh-mediated function in cortical neurons.
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3.4. cAMP-PKA contributes to RGS5-mediated inhibition of Shh signaling in cortical neurons It is hypothesized that cAMP-PKA inhibits Shh signaling in both mouse and Drosophila melanogaster (Makinodan and Marneros, 2012; Noveen et al., 1996), and RGS5 is a molecule that negatively regulates Gαi, a protein which inhibits the production of cAMP from ATP. To test whether the RGS5-mediated inhibition of Shh function observed in the present study involves modulation of cAMP levels, cAMP concentrations in GFP control and RGS5 overexpressing cortical neurons stimulated with Shh were measured. As shown in Fig. 6A, an increase cAMP production was detected in response to overexpression of RGS5. Immunofluorescence staining of cAMP subsequently confirmed these results (Fig. S4). Interestingly, the distribution of cAMP was characterized by large clusters in the GFP control neurons, while the RGS5 overexpressing neurons exhibited smaller clusters of cAMP that were distributed
Fig. 6. cAMP-PKA contributes to RGS5 mediated inhibition of Shh signaling in cortical neurons. Intracellular cAMP levels (A) and PKA activity (B) were measured for Shh-treated neurons expressing GFP or overexpressing RGS5. n = 3. C–H. H89 (5 μM) treatment reversed RGS5 mediated inhibition of Shh function as demonstrated by measuring mean endpoints (C), mean neurite length (D), mean neurite number (E), mean number of attachment points (F), mean soma area (G), and mean optical density (H). At least 100 cells/culture were analyzed. Data are expressed as the mean ± SD from three experiments. *P b 0.05 compared with the GFP group in A and B; *P b 0.05, **P b 0.01 compared with the RGS5 group in C–H.
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throughout the cytoplasm (Fig. S4). We also confirmed the cAMP function in the absence of RGS5 manipulation; neurite outgrowth was supported by cAMP agonist db-cAMP. It is interesting that neurite outgrowth was suppressed when neurons were stimulated by Shh + db-cAMP compared with Shh or db-cAMP stimulated alone (Fig. S5). Considering that elevated cAMP levels promote PKA activation, and that PKA is a negative regulator of Shh signaling, PKA activation is also examined. In the assays performed, an increase in intracellular PKA activity was detected that paralleled the increase in cAMP levels previously observed (Fig. 6B). Moreover, when the RGS5 overexpressing neurons were incubated with Shh in the presence of the PKA-specific inhibitor, H89 (5 μM), RGS5-mediated inhibition of Shh function was prevented (Fig. 6, C–H). Therefore, in combination, these results suggest that cAMP-PKA plays a role in RGS5-mediated inhibition of Shh function. 4. Discussion In this study, the distribution of RGS5 expression in mouse tissues was investigated by using real-time PCR. Consistent with the results of previous studies (Seki et al., 1998), the expression of RGS5 was found at high levels in skeletal muscle, brain, and heart. In contrast, RGS5 was expressed at low levels in the adrenal gland, adipose, and lung tissues, and was not detected in spleen and smooth muscle. In the central nervous system, higher levels of RGS5 mRNA were detected in cortical neurons compared with astrocytes and oligodendrocytes, while the RGS5 transcript was not detected in microglia. RGS-R4 is the largest RGS protein family and it includes RGS proteins 1–5, 8, 13, 16, 18, and 21. RGS-R4 proteins are the smallest RGS proteins in size and they contain short peptide sequences that flank the RGS box with one notable exception (RGS3) (Bansal et al., 2007). Interestingly, when we examined whether members of the RGS-R4 subfamily were expressed in cultured cortical neurons, only RGS 3, 18, and 21 were not detected. For RGS5, its expression declined as the duration of in vitro culturing increased. Thus, RGS5 may mediate an inhibitory effect during the development of cortical neurons. RGS5 structure is highly homologous with two other members of the RGS-R4 family, RGS4 and RGS16. These three RGS proteins contain an Nterminal cysteine residue which can be oxidized in vivo by nitric oxide and oxygen (Park et al., 2015). Meanwhile, the N terminus of RGS5 contains an N-end rule determinant that controls its ubiquitylation and expression (Bodenstein et al., 2007). In the presence of MG-132, ubiquitylation of exogenous RGS5 was significantly inhibited. The ability of RGS to promote the hydrolysis of GTP, thereby reducing the life span of the active form of the GTP α subunit, has been well characterized. Accordingly, RGS proteins are emerging as important negative regulators of GPCR signaling pathways. Moreover, it is hypothesized that functional diversity exists among RGS proteins. Regarding RGS5, this member of the RGS protein superfamily has recently been reported to regulate many biological processes by inhibiting several Gαimediated signaling pathways (Deng et al., 2012; Gunaje et al., 2011; Hamzah et al., 2008; Li et al., 2010; Takata et al., 2008). Cellular signaling cascades that originate from adenylyl cyclase and that are activated during neural development are mostly controlled by Gαs proteins. Furthermore, activation of adenylyl cyclase has been shown to result in a transient accumulation of cAMP (Cameron et al., 1988). Elevated levels of cytoplasmic cAMP activate PKA which acts as a negative regulator of Shh signaling. In a previous study, Shh was shown to activate autophagy in hippocampal neurons (Petralia et al., 2013) and to alter the architecture and function of presynaptic terminals in cultured hippocampal neurons (Mitchell et al., 2012). Very few studies have explored the function of RGS5 in cortical neurons during neurite outgrowth. Here, overexpression of RGS5 was achieved with an adenovirus system that successfully infected primary cortical neurons. The resulting data provide evidence that overexpression
of RGS5 inhibits neurite outgrowth of primary cortical neurons which have undergone Shh stimulation. In a previous study, presynaptic terminals exhibited an increase in size and uptake of FM dye following Shh stimulation (Mitchell et al., 2012). In the present study, RGS5 overexpression reduced the endocytosis capacity of presynaptic terminals in Shh stimulated cortical neurons for FM4-64 dye. The cAMP-PKA pathway has been shown to suppress Shh signaling (Makinodan and Marneros, 2012). The downstream components in the Shh pathway are potential targets for PKA phosphorylation. Therefore, in the present study, it was hypothesized that cAMP-PKA signaling is involved in the regulation of Shh by RGS5 overexpression. Support for this hypothesis was obtained in the present study when RGS5 overexpression resulted in an increased level of intracellular cAMP and an increased activity of PKA in the cultured neuron model examined. Furthermore, H89, a specific inhibitor of PKA, disrupted the inhibition of Shh function by RGS5 overexpression. Together, these results indicate that cAMP-PKA signaling mediates an inhibition of Shh function that is induced by RGS5 overexpression. Therefore, in conclusion, the results of the present study demonstrate that RGS5 antagonizes Shh signaling and inhibits neurite outgrowth, and this is partially attributed to cAMP-PKA signaling. However, in vivo studies of Shh-induced neurite extension are needed, especially studies that will determine whether other pathways are involved in RGS5-mediated antagonism of Shh signaling. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.mcn.2017.06.005. Acknowledgments The authors would like to thank Dr. Hongwei Xu for her helpful technical assistance, Dr. Shihuan Kuang (Purdue University) for the AdEasy system. This study was financially supported by the National Natural Science Foundation of China (81671234, 81000512, 81471227, 81430035, 31671112), Natural Science Foundation of Heilongjiang Province (LC2016031), and Academician Weihan Yu Outstanding Youth Training Foundation Project (20150901). References Anger, T., Zhang, W., Mende, U., 2004. Differential contribution of GTPase activation and effector antagonism to the inhibitory effect of RGS proteins on Gq-mediated signaling in vivo. J. Biol. Chem. 279, 3906–3915. Arenas, E., 2014. Wnt signaling in midbrain dopaminergic neuron development and regenerative medicine for Parkinson's disease. J. Mol. Cell Biol. 6, 42–53. Bansal, G., Druey, K.M., Xie, Z., 2007. R4 RGS proteins: regulation of G-protein signaling and beyond. Pharmacol. Ther. 116, 473–495. Berretta, A., Gowing, E.K., Jasoni, C.L., Clarkson, A.N., 2016. Sonic hedgehog stimulates neurite outgrowth in a mechanical stretch model of reactive-astrogliosis. Sci Rep 6, 21896. Bodenstein, J., Sunahara, R.K., Neubig, R.R., 2007. N-terminal residues control proteasomal degradation of RGS2, RGS4, and RGS5 in human embryonic kidney 293 cells. Mol. Pharmacol. 71, 1040–1050. Cameron, S., Levin, L., Zoller, M., Wigler, M., 1988. cAMP-independent control of sporulation, glycogen metabolism, and heat shock resistance in S. cerevisiae. Cell 53, 555–566. Cheng, W.L., Wang, P.X., Wang, T., Zhang, Y., Du, C., Li, H., Ji, Y., 2015. Regulator of G-protein signalling 5 protects against atherosclerosis in apolipoprotein E-deficient mice. Br. J. Pharmacol. 172, 5676–5689. Cho, H., Park, C., Hwang, I.Y., Han, S.B., Schimel, D., Despres, D., Kehrl, J.H., 2008. Rgs5 targeting leads to chronic low blood pressure and a lean body habitus. Mol. Cell. Biol. 28, 2590–2597. Deng, W., Wang, X., Xiao, J., Chen, K., Zhou, H., Shen, D., Li, H., Tang, Q., 2012. Loss of regulator of G protein signaling 5 exacerbates obesity, hepatic steatosis, inflammation and insulin resistance. PLoS One 7, e30256. Feigin, M.E., Malbon, C.C., 2007. RGS19 regulates Wnt-beta-catenin signaling through inactivation of Galpha(o). J. Cell Sci. 120, 3404–3414. Gunaje, J.J., Bahrami, A.J., Schwartz, S.M., Daum, G., Mahoney Jr., W.M., 2011. PDGF-dependent regulation of regulator of G protein signaling-5 expression and vascular smooth muscle cell functionality. Am. J. Phys. Cell Phys. 301, C478–C489. Hamzah, J., Jugold, M., Kiessling, F., Rigby, P., Manzur, M., Marti, H.H., Rabie, T., Kaden, S., Grone, H.J., Hammerling, G.J., Arnold, B., Ganss, R., 2008. Vascular normalization in Rgs5-deficient tumours promotes immune destruction. Nature 453, 410–414. He, L., Lu, Q.R., 2013. Coordinated control of oligodendrocyte development by extrinsic and intrinsic signaling cues. Neurosci. Bull. 29, 129–143.
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