Immunolocalization of Substance P and NK-1 Receptor in ADIPOSE Stem Cells

 This article is protected by copyright. All rights reserved 1 Article IMMUNOLOCALIZATION OF SUBSTANCE P AND NK-1 RECEPTOR IN ADIPOSE STEM CELLS† Short title: SP/NK1R system in adipose stem cells Miguel Muñoz1*, Mario F. Muñoz2 and Antonio Ayala2 1 Virgen del Rocío University Hospital, Research Laboratory on Neuropeptides (IBIS), Seville, Spain 2 Department of Biochemistry and Molecular Biology, University of Seville, Spain *Correspondence: Dr. Miguel Muñoz Hospital Infantil Universitario Virgen del Rocío, Unidad de Cuidados Intensivos Pediátricos Av. Manuel Siurot s/n 41013 - Sevilla, Spain E-mail: [email protected] Phone: 34-955012965 Fax: 34-955012921 †This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: [10.1002/jcb.26134] Received 20 March 2017; Revised 12 May 2017; Accepted 12 May 2017 Journal of Cellular Biochemistry This article is protected by copyright. All rights reserved DOI 10.1002/jcb.26134  This article is protected by copyright. All rights reserved 2 ABSTRACT Substance P (SP) is a neuropeptide belonging to the thachykinin peptide family. SP, after binding to its receptor, the neurokinin 1 receptor (NK1R), controls several transcription factors such as NF-κB, hypoxia inducible factor (HIF-1α), c-myc, c-fos, cjun and AP-1. SP and NK1R have a widespread distribution in both the central and peripheral nervous systems. They are also present in cells not belonging to the nervous system (immune cells, placenta, etc.). SP is located in all body fluids, i.e., blood, cerebrospinal fluid, etc., making it ubiquitous throughout the human body. SP and NK1R genes are expressed in the stem cell line TF-1 and in primary stem cells derived from human placental cord blood. However, to our knowledge, the presence of SP and the NK1R receptor in adipose stem cells (ADSC) is unknown. We demonstrated by immunofluorescence the localization of SP and NK1R in human and rat ADSC. SP and NK1R are located in both the cytoplasm and the nucleus of these cells. The NK1R is higher in the nucleus than in the cytoplasm of ADSCs. By Western blot we demonstrated the presence of different isoforms of NK1R that have different subcellular locations in the ADSC. SP induces proliferation and mitogenesis through NK1R in ADSCs. These findings reported here for the first time suggest an important role for a SP/NK1R system, either as genetic and/or epigenetic factor, in both the cytoplasm and nucleus functions of the ADSCs. This article is protected by copyright. All rights reserved Keywords: Substance P; NK1 receptor; adipose tissue stem cells; immunofluorescence. Preamble: “Theories are renewed, while the facts remain” S. Ramon y Cajal (Nobel Prize 1906)  This article is protected by copyright. All rights reserved 3 INTRODUCTION Substance P (SP) is a neuropeptide belonging to the thachykinin peptide family. After binding to their receptor, the neurokinin-1 receptor (NK1R), SP regulates many biological functions such as proliferation of both normal and tumor cells [Munoz and Covenas, 2014] and cell migration [Meshki et al., 2009]. SP and NK1R are widely distributed in the central and peripheral nervous systems, but they are also present in other cells and tissues (immune and cancer cells, placenta, and adipose tissue, for example). SP is located in all body fluids: blood, cerebrospinal fluid, etc., making it ubiquitous in the human body [Munoz and Covenas, 2014]. SP stimulates human peripheral blood monocytes to produce inflammatory cytokines including interleukin-1 (IL-1), IL-6, and tumor necrosis factor alpha (TNF-a) [Ho et al., 1996; Laurenzi et al., 1990; Lee et al., 1994; Lotz et al., 1988], which are involved in the hematopoieticregulating network [Rameshwar and Gascon, 1995]. Bone marrow (BM) cell population (stroma) is partly responsible for in vitro hematopoietic stimulation by SP [Rameshwar et al., 1993]. Fibroblasts, endothelial cells, macrophages, reticular cells, and adipocytes [Dexter et al., 1990] compose the BM stroma and provide the necessary microenvironment for hematopoietic regulation [Dexter et al., 1990]. The mechanisms involved in this regulation are complex and appear to involve interactions between the stroma and progenitors/stem cells, mediated by cytokines, neurotrophic factors, and neuropeptide [Rameshwar and Gascon, 1995]. SP mediates production of the stem cell factor and IL-1 in bone marrow stroma [Rameshwar and Gascon, 1995] and has a potent stimulatory effect on hematopoiesis [Hiramoto et al., 1998]. SP and NK1R genes are expressed in the human stem cell line (TF-1) and primary stem cells derived from human placental cord blood (HPCB) [Li et al., 2000]. After binding to NK1R, SP elicits bone marrow stromal cell osteogenic  This article is protected by copyright. All rights reserved 4 activity, osteoclast differentiation, and resorption activity in vitro in a concentration dependent manner [Wang et al., 2009]. It has been reported recently that SP enhances the proliferation and migration potential of BM mesenchymal stem cells (MSCs) [Dubon and Park, 2015]. Additionally, SP stimulates proliferation of spinal neural stem cells in spinal cord injury via the mitogen-activated protein kinase signalling pathway [Kim et al., 2015]. Moreover, SP has a protective effect on BM MSC against apoptosis induced by serum deprivation [Fu et al., 2015]. Adipose tissue contains mesenchymal stem cells called adipose tissue stem cells (ADSC) [Gimble et al., 2007; Lim et al., 2014; Schaffler and Buchler, 2007; Trounson, 2012; Trounson and McDonald, 2015]. The proportion of MSC is 500 times higher in adipose tissue than in bone marrow [Fraser et al., 2006; Fraser et al., 2008], so that a large number of cells can be obtained without a large number of passes, decreasing the risk of chromosomal abnormalities induced senescence in cultures [Tarte et al., 2010]. The ADSC not only have the potential to differentiate into cells of mesodermal origin and organs, but they also have the ability to differentiate into neurons, endocrine cells of the pancreas, hepatocytes, endothelial cells and cardiomyocytes [Schaffler and Buchler, 2007]. ADSCs can repair and regenerate the tissues by several mechanisms [Dimmeler et al., 2014; Feisst et al., 2015; Trounson and McDonald, 2015; Tsuji et al., 2014]. There are no previous data on whether SP and its receptor are present in ADSC. Therefore, the expression, localization and distribution of NK1R and SP were evaluated by immunofluorescence and Western blot in human and non-human ADSC. The possible effect of SP and its receptor on ADSC proliferation and mitogenesis is also evaluated.  This article is protected by copyright. All rights reserved 5 MATERIAL AND METHODS Samples. Human adipose tissue samples were obtained from patients undergoing elective liposuction surgery and maintained at 4ºC. Approval for obtaining human tissue was obtained from a local IRB committee. The patients signed an informed consent form; the result being unanimous approval. In the case of animals, all experiments were carried out according to the guidelines of the European Union Council (Directive 2010/63/UE) and to Spanish regulations (BOE 34/11370, RD 53/2013) and were approved by the Ethic Committee of the University of Seville. Male Wistar rats (300500 g) were kept at a constant temperature of 22 ± 1ºC and a relative humidity of 60%, with a light-dark cycle of 12 h and free access to food and water. Isolation of Stromal Vascular Fraction (SVF). 200-300 ml of lipoaspirate was washed with sterile phosphate buffered saline (PBS) to remove blood. The whole sample was centrifuged at 430 x g at 4°C for 10 min. After centrifugation, the aqueous fraction was removed. The lipid fractions were diluted with an equal volume of collagenase solution (Collagenase NB 4G, Serva) 0.3 U/ml in PBS (Wünsch units). The mixture was incubated at 37°C for 60 min in an orbital shaker. After digestion, an equal volume of Dulbecco's Modified Eagles Medium (DMEM) containing 20% fetal bovine serum (FBS) was added. The mixture was centrifuged at 600 x g for 10 min and the pellet was resuspended in 10 ml of expansion medium for non-hematopoietic stem cells (NHMiltenyi Biotec). Cell suspension was passed through a 100 µm filter (BD Biosciences). The filtrate was centrifuged at 300 x g for 10 min. The pellet was resuspended in 5 ml of NH medium and passed through a 40 µm filter. Finally, cell numbers were determined in this cell suspension. Magnetic separation of Human ADSC (hADSC). ADSC were magnetically labeled by using CD271 (LNGFR)-APC antibodies and Anti-APC MicroBeads (Miltenyi  This article is protected by copyright. All rights reserved 6 Biotec). CD271 is a well-known marker on mesenchymal stem cells, also known as mesenchymal stromal cells (MSCs), from bone marrow aspirate or lipoaspirate [Quirici et al., 2002]. The separation is performed in columns inserted in an extremely strong magnet, which induces in the column matrix a sufficiently high intense magnetic field for retaining the labeled cells. Unlabeled cells pass freely through the column and are discarded. After removing the column from the magnet, cells are eluted with washing buffer. The procedure is as follows: The cell suspension was centrifuged at 300 x g for 10 min. The pellet was resuspended in 80 μl of rinse buffer (Miltenyi Biotec). Ten μl of CD271-APC antibody were added. After 10 min at 4ºC, cells were centrifuged at 300 x g for 10 min. Cells are resuspended with 70 μl of buffer rinse and 20 μl of Anti-APC Microbeads were added. The samples were incubated for 15 min at 4°C. Cells were washed and centrifuged at 300 x g for 10 min. Finally, the pellet was resuspended in 500 μl of wash buffer and the sample was passed through the column inserted in the magnetic field according to the manufacturer´s instructions. Cell expansion. After magnetic separation, cells were centrifuged at 350 x g and resuspended in 1 ml of NH expansion medium preheated at 37°C. The cells were cultured in a 25 cm2 flask by adding 4 ml of NH Expansion Medium. Passaging of hADSC. Cell cultures were washed twice with PBS. Enough volume of Trypsin / EDTA (0.05% / 0.53 mM) (Gibco) was added to cover the cells and they were then incubated at 37°C for 5 min. Afterwards, DMEM with 20% FBS was added and the sample was centrifuged at 300 x g for 10 min at room temperature. The cells were resuspended in 2 ml of NH expansion medium. Rat Stromal Vascular Fraction. Animals were anaesthetized with ketamine/xylazine (100/12.5 mg/kg). Adipose tissue of the abdominal or lumbar region was obtained and  This article is protected by copyright. All rights reserved 7 immediately washed with 20 ml of PBS to remove blood. Fat was treated as human samples to obtain SVF. Labeling and magnetic separation of rat ADSC (rADSC). Magnetic isolation of rADSC was carried out by the same procedure used for hADSC. In this case, the antibody was against the surface antigen CD90.1 (thymocyte differentiation antigen1.1) of rADSC cells. The mouse monoclonal antibody reacts with rat CD90 (Thy-1), a GPI-anchored conserved glycoprotein membrane. In the rat, the CD90 antigen is expressed on thymocytes, recent thymic emigrants, hematopoietic stem cells, neurons and mesenchymal stromal cells [Davies et al., 2015]. Cell Expansion. The protocol for cell expansion was the same as for human, using DMEM, 20% FBS and 1% antibiotic as culture medium. Phenotyping of hADSC. Phenotyping of ADSC was carried out by flow cytometry and a commercial kit (MSC Phenotyping Kit human, MiltenyiBiotec) containing the necessary antibodies and fluorophores. The fluorophore-conjugated monoclonal antibodies were: CD14-PerCP, CD20-PerCP, CD34-PerCP, CD45-PerCP, CD73-APC, CD90-FITC and CD105-PE. Phenotyping of rADSC. In this case the cells must be positive for CD90.1 and CD29 and negative for CD11b, CD34, and CD45. The monoclonal antibodies were the following: anti-CD34-PE (SantaCruz, USA), anti-CD45-PE (BD Pharmingen), antiCD11b-PE (Bio-Legend), anti-CD29-APC (Miltenyi Biotec) and anti-CD90.1-FITC (Miltenyi Biotec). Assay of hADSC differentiation into adipocytes. The cells were diluted with adipocytes differentiation medium (Miltenyi Biotec). The medium was changed every three days. On the 21stday, adipogenic differentiation was confirmed by the formation of neutral lipid-vacuoles stainable with Oil Red O (Sigma Aldrich, USA).  This article is protected by copyright. All rights reserved 8 Assay of hADSC differentiation into Osteoblast. Cells were diluted with osteoblast differentiation medium (Miltenyi Biotec). On the 10thday, osteogenesis was assessed by phosphatase alkaline staining (Sigma Aldrich). Differentiation assays in rADSC. For differentiation into adipocytes, cells were cultured in differentiation medium (StemXVivo Media-R&D System) with added adipogenic supplements contained in the kit. Detection was performed as indicated for hADSC. For osteaoblast differentiation assay, cells were cultured in the same medium plus osteogenic supplements. Phosphatase alkaline detection was the same as hADSC. Immunofluorescence determination in culture cell. Cells were cultured in a 24-well plate containing gelatin-coated coverslips. When cells reached 80% confluence, the culture media were removed from each well and washed twice with PBS. One ml of fixative solution (4% paraformaldehyde) was added to each well and the plates were kept for 20 min at 4°C. Wells were washed twice with PBS and incubated with blocking solution (PBS plus 1% fetal bovine serum) for 1 h at room temperature. After removing the blocking solution, each well was washed 3 times with PBS. Cells were incubated with rabbit-derived anti-NK1R (which specifically corresponds to residues KTMTESSSFYSNMLA, corresponding to the C-terminus of NK1R (amino acids 393407)) (Sigma, 1:1000) or rabbit-derived anti-Substance P (Millipore, 1:1000), the presence of 10% serum and 0.25% Triton X-100 at 4°C overnight. The wells were washed 3 times with PBS and incubated with the anti-rabbit secondary antibody conjugated to Alexa Fluor 488 (Thermo Fisher) diluted 1:500 in 0.25% Triton X-100 and Hoechst (Sigma) diluted 1: 2000 in PBS for 1 h at 37°C. After incubation, the samples were washed 3 times with PBS and mounted with mounting fluid for fluorescence (DAKO). Samples were visualized in a confocal microscope (Zeiss LSM7  This article is protected by copyright. All rights reserved 9 DUO). As negative control, the primary antibody was omitted and replaced by nonimmune serum. Subcellular fractionation. Cells were detached by scrapping in cold PBS and were centrifuged at 15,000g for 20 s at 4ºC and resuspended in buffer A (10 mM Hepes, 2 mM MgCl2, 15 mM KCl, 0.1 mM EDTA, 0.1% NP-40, 250 mM Sucrose, 1 mM DTT; pH 7.6) containing protease inhibitors and incubated 7 min on ice. Cells were centrifuged at 1,000 x g for 10 min to separate the cytoplasmic fraction of the cell extract. The nuclei were lysed by incubation on ice for 45 min with RIPA buffer (20 mM Tris–HCl, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, and 1 mM sodium orthovanadate) containing protease inhibitors. The lysate was then centrifuged at 15,000 x g for 20 min. The resulting supernatant was used as the nuclear fraction of the cell extract. The subcellular fractions were separated by SDS–PAGE and analyzed by immunoblot using antibodies against cytoplasmic and nuclear marker proteins. Immunoblot. Protein content of the samples was estimated with a Pierce BCA protein assay kit (Pierce Biotechnology, USA). Protein samples were separated by SDS–PAGE (12% acrylamide) and transferred to a PVDF membrane (GE Healthcare, USA) at 100 V for 1 h. The membranes were incubated with blocking buffer (5% dry milk in 20 mM Tris–HCl, pH 7.5, 500 mM NaCl, 0.05% Tween 20) for 1 h at room temperature. Membranes were then incubated overnight at 4ºC in blocking solution containing the following antibodies: NK1R (1:2000), α-tubulin (1:3000), β-actin (1:3000; Sigma), and hnRNP (1:2000; Santa Cruz Biotechnology, Santa Cruz, CA, USA). After incubation, the membranes were washed in 20 mM Tris–HCl, pH 7.5, 500 mM NaCl, 0.05% Tween 20 and incubated with peroxidase-conjugated anti-  This article is protected by copyright. All rights reserved 16 important in cell spreading and cell migration [Meshki et al., 2009]. Although the above mentioned mechanisms can explain the immunolocalization of SP and NK1R in the cytoplasm of stem cells, the meaning of the location of SP and NK1R inside stem cell nuclei remains unknown. Because SP and NK1R regulate several cytoplasmic signalling pathways extracellularly, the presence of SP and NK1R inside the stem cell nucleus is theoretically unnecessary. It is known that localization of a peptide and its receptor is involved in the function of the cellular compartment where they are located. Thus, the presence of SP and its receptor in the nucleus of stem cells suggests that both are involved in the nuclear function. Our results also show a positive correlation between DNA concentration and NK1R expression (Fig. 8D), suggesting the possible involvement of nuclear NK1R in the mitogenesis process. In addition, it is known that SP exerts its effect only once it is bound to its receptor and both are found in the nucleus of stem cells. This means that the nucleus of ADSC contains everything needed for the SP to work and thus SP might regulate gene expression of stem cells. Similarly to that, SP modulates emotional conduct under control of the limbic system, the presence of SP in the cell nucleus could be involved in stem cell behavior. Up to now, the neurotransmitter, neuropeptides and neuromodulators were exclusively considered as cell signaling molecules in multicellular organisms. However, these findings demonstrate that neuropeptides are not exclusive of multicellular organisms. Indeed, the localization of SP and NK1R in stem cell nuclei opens the door to understanding that stem cells possibly have a rudimentary brain cell. Furthermore, it has been reported that SP, calcitonin gene related peptide (CGRP) and NK1R are located in the nucleus of the dorsal root ganglia subpopulation cells in rats. However, the physiological significance remains unknown [Boer and  This article is protected by copyright. All rights reserved 17 Gontijo, 2006]. Also, it has been reported that SP and NK1R are located in both normal and tumor cell nuclei [Brener et al., 2009; Esteban et al., 2009; Munoz et al., 2013; Munoz and Covenas, 2014; Munoz et al., 2012; Munoz et al., 2014; Munoz et al., 2010]. Due to its nuclear localization, SP has been considered a genetic neuromodulator [Munoz et al., 2010]. Here, we have demonstrated that nuclear localization of SP and NK1R is higher than in cytoplasm of adipose stem cells. This suggests that SP has both cytoplasmic and nuclear functions; most likely the nuclear function is the more important since the SP effect is concentration-dependent. Additionally, it has been reported that SP regulates some transcription factors such as NF-κB, c-myc, c-fos, c-jun, AP-1, and hypoxiainducible factor (HIF-1α) that are involved in inflammation (NF-kB), mitogenesis and cellular differentiation (c-fos, cjun and AP-1) and angiogenesis and vasculogenesis (HIF-1α) [Koh et al., 2010; Luo et al., 1996; Munoz et al., 2015; Walczak-Drzewiecka et al., 2008]. The expression of SP in the nuclei of adipose stem cells suggests that SP could play two roles in modifying the gene expression of adipose stem cells: indirectly, by regulating transcription factors and directly, via nuclear NK1R regulation. These findings are in agreement with the high presence of SP and NK1R within the nucleus of normal and tumour cells [Munoz et al., 2015]. This means that the SP peptide through the NK1R could regulate the nuclear activity of stem cells and that SP could act either as genetic or epigenetic nuclear factor to regulate gene expression. In regards to its epigenetic role, it has been described that SP induces the expression of some gene via histone deacetylase activity in colonic epithelial cells [Koon et al., 2007]. Indeed, the increased expression of NK1R in the nucleus of ADSC during mitosis over the levels found in non-dividing cells (Fig. 6) demonstrated that the neurogenic factors (SP) could regulate the genetic cell cycle machinery of stem cells.  This article is protected by copyright. All rights reserved 18 In summary, we demonstrate here for the first time the immunolocalization of SP and NK1R in human and rat ADSCs. Both are located in the cytoplasm and the nucleus of these cells. The immunolocalization of the NK1R is higher in the nucleus than in the cytoplasm of ADSCs. The isoform of 58 kDa NK1R isoform was mainly present in the nucleus, whereas the level of the 33 kDa isoform was higher in the cytoplasm. SP induces proliferation and mitogenesis through NK1R in ADSCs. 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Walczak-Drzewiecka A, Ratajewski M, Wagner W, Dastych J. 2008. HIF-1alpha is upregulated in activated mast cells by a process that involves calcineurin and NFAT. J.Immunol. 181:1665-1672. Wang L, Zhao R, Shi X, Wei T, Halloran BP, Clark DJ, Jacobs CR, Kingery WS. 2009. Substance P stimulates bone marrow stromal cell osteogenic activity, osteoclast differentiation, and resorption activity in vitro. Bone 45:309-320. Zuk PA, Zhu M, Mizuno H, Huang J, Futrell JW, Katz AJ, Benhaim P, Lorenz HP, Hedrick MH. 2001. Multilineage cells from human adipose tissue: implications for cellbased therapies. Tissue Eng 7:211-228.  This article is protected by copyright. All rights reserved 22 FIGURE LEGEND. Figure 1. Characterization of ADSCs. Microscope image showing the adherence of ADSC (A, E). Multipotential differentiation to adipocytes is shown by Oil-O-Red staining (B, F). Osteoblast differentiation is detected by Alkaline Phosphatase staining (C, G). Flow Cytometry analysis show that almost all hADSCcultures express CD73/CD90 (99.6%) and CD90/CD105 (99.7%), while a small portion of hADSC express negative markers (0.11%) (D). rADSC express CD29/CD90.1 (99.1%) and negative markers (10.3%) (H). Scale bar = 50 µm. Figure 2. Immunofluorescence localization of SP in ADSCs. Localization of SP(green) in hADSC and rADSC. Nuclei (Blue) were stained by Hoeschst. Scale bar = 50 µm. Figure 3. Immunofluorescence localization of NK1R in ADSCs. Localization of NK1R (green) in hADSC and rADSC. Nuclei (Blue) are stained by Hoeschst. Scale bar = 50 µm. Figure 4. Intensity profiles of SP in ADSCs. Immunofluorescence intensity profile in hADSC and rADSC of SP (green) was measured using Zen Application 2011 software. The lines were placed across cells and the simultaneously occurring intensity peaks were evaluated. Line scan graphs show the immunofluorescence intensity along the positioned white arrow after subtraction of the background. Scale bar = 50 µm. Figure 5. Intensity profiles of NK1R in ADSCs. Immunofluorescence intensity profile in hADSC and rADSCs of NK-1R (green) was measured using Zen Application 2011 software. The lines were placed across cells and the simultaneously occurring intensity  This article is protected by copyright. All rights reserved 23 peaks were evaluated. Line scan graphs show the immunofluorescence intensity along the positioned white arrow after subtraction of the background. Scale bar = 50 µm. Figure 6. Intensity profiles of NK1R in rADSCs during the process of mitosis. Immunofluorescence intensity profile in rADSC and of NK-1R (green) was measured using Zen Application 2011 software. Line scan graphs show the immunofluorescence intensity along the positioned white arrow. Scale bar = 50 µm. Figure 7. Evidence of subcellular localization of NK1R by WB. a-Tub (50 kDa) and hnRNP (37 kDa) were used as cytoplasmic and nuclear markers while β-Actin (40 kDa) was used as loading control. In addition, two bands of NK1R were observed with different molecular weight (33 kDa and 58 kDa). The optical densities of NK1R bands were divided by the optical densities of β-actin. The results are the mean ± SEM of three independent subcellular location experiments. W: Whole cell fraction, C: Cytoplasmic fraction and N: Nuclear fraction. a: statistically different from whole cells. b: statistically different from cytoplasm. Figure 8. Proliferative effect of SP and mitogenic involvement of NKR1 in ADSCs. MTS viability assay was performed in octuplicate technical replicates per group in three independent experiments. Results are expressed as percentage with respect to the amount found in control (N=3, *p=0.001) (A). rADSCs population labeled with NK1RFITC was gated as R1 in FSC against SSC dot plot (doublets discrimination was removed using an AUX channel). The R1 population was represented in histograms to PI lin and NK1R FITC Log (grey color represents the fluorescence intensity of secondary antibody control) (B). PI lin histogram was gated in to regions R2 (less PI intensity) and R3 (more PI intensity) and both were gated in NK1R FITC Log histograms (C). Mean fluorescence intensity of NK1R-FITC was obtained from three independent experiments (N=3, *p<0.001) (D).  This article is protected by copyright. All rights reserved 24 Figure 1  This article is protected by copyright. All rights reserved 25 Figure 2