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• W2070673977 abstract "Vasculature is an important component of the neural stem cell niche in brain. It regulates neural stem/progenitor (NS/P) cell self-renewal, differentiation, and migration. In the neurogenic niches of adult brain, NS/P cells lie close to blood vessels, and proliferating NS/P cells frequently contact the vasculature. In the present study we showed that NS/P cells in co-culture with brain endothelial (bEND) cells activated endothelial G proteins and p38 mitogen-activated protein kinase (p38 MAPK) and stimulated cytokine/chemokine expression. These NS/P cell-induced endothelial responses took place during NS/P cell and bEND cell direct contact and were critically dependent on the expression of the type II transmembrane serine protease matriptase (MTP) by NS/P cells, because knocking down of MTP in NS/P cells impaired and re-expression of MTP restored their ability to induce endothelial cytokine/chemokine expression, p38 MAPK, or G protein activation. Cholera toxin blocked NS/P cell-induced endothelial responses, suggesting that the endothelial G protein activated by NS/P MTP is in the Gs subfamily. The addition of p38 MAPK inhibitor impaired NS/P cell-induced endothelial cytokine/chemokine expression. The known G protein-coupled receptor substrate of MTP, protease-activated receptor 2, was not involved in this system. These results revealed a novel signaling pathway in neural stem cell vascular niches that is mediated by neural MTP and endothelial Gs protein signaling at the cell-cell interface. This is the first report of direct cell-cell signaling between NS/P and bEND cells.Background: Vasculature is a key component of the brain neurogenic stem cell niche.Results: Knockdown of matriptase/epithin in neural progenitor cells or blocking of Gs protein activity in brain endothelial cells impairs contact-induced endothelial signaling activation and gene stimulation.Conclusion: Matriptase/epithin and Gs protein signaling guide contact communication in neurovascular niche.Significance: The study advances our understanding of cell-cell communication in the neurogenic vascular niche. Vasculature is an important component of the neural stem cell niche in brain. It regulates neural stem/progenitor (NS/P) cell self-renewal, differentiation, and migration. In the neurogenic niches of adult brain, NS/P cells lie close to blood vessels, and proliferating NS/P cells frequently contact the vasculature. In the present study we showed that NS/P cells in co-culture with brain endothelial (bEND) cells activated endothelial G proteins and p38 mitogen-activated protein kinase (p38 MAPK) and stimulated cytokine/chemokine expression. These NS/P cell-induced endothelial responses took place during NS/P cell and bEND cell direct contact and were critically dependent on the expression of the type II transmembrane serine protease matriptase (MTP) by NS/P cells, because knocking down of MTP in NS/P cells impaired and re-expression of MTP restored their ability to induce endothelial cytokine/chemokine expression, p38 MAPK, or G protein activation. Cholera toxin blocked NS/P cell-induced endothelial responses, suggesting that the endothelial G protein activated by NS/P MTP is in the Gs subfamily. The addition of p38 MAPK inhibitor impaired NS/P cell-induced endothelial cytokine/chemokine expression. The known G protein-coupled receptor substrate of MTP, protease-activated receptor 2, was not involved in this system. These results revealed a novel signaling pathway in neural stem cell vascular niches that is mediated by neural MTP and endothelial Gs protein signaling at the cell-cell interface. This is the first report of direct cell-cell signaling between NS/P and bEND cells. Background: Vasculature is a key component of the brain neurogenic stem cell niche. Results: Knockdown of matriptase/epithin in neural progenitor cells or blocking of Gs protein activity in brain endothelial cells impairs contact-induced endothelial signaling activation and gene stimulation. Conclusion: Matriptase/epithin and Gs protein signaling guide contact communication in neurovascular niche. Significance: The study advances our understanding of cell-cell communication in the neurogenic vascular niche. Neurogenic stem cell niches in the central nervous system consist of various cell types including neural stem/progenitor (NS/P) 2The abbreviations used are: NS/Pneural stem/progenitorbENDbrain endothelialMTPmatriptaseSVZsubventricular zoneLVlateral ventriclePTXpertussis toxinCTXcholera toxinCXCL10CXC motif chemokine 10PARprotease-activated receptorGPCRG protein-coupled receptorGFAPglial fibrillary acidic protein. cells, blood vessel cells, ependymal cells, astrocytes, microglia, and oligodendrocytes. A number of observations show that the vasculature is an important component of neural stem cell niches. In the developing CNS, nervous and vascular systems are both derived from the neural tube (1Temple S. The development of neural stem cells.Nature. 2001; 414: 112-117Crossref PubMed Scopus (1272) Google Scholar). Endothelial cells and neural stem cells appear at the same stages during development and are co-localized in the neural geminal zones (2Zerlin M. Goldman J.E. Interactions between glial progenitors and blood vessels during early postnatal corticogenesis. Blood vessel contact represents an early stage of astrocyte differentiation.J. Comp. Neurol. 1997; 387: 537-546Crossref PubMed Scopus (87) Google Scholar). Invasion of spouts from the perineural vascular plexus into the ventral region of the neural tube are associated with the proliferation of neuroepithelium. In the adult brain, neural stem cells and their progeny in the subventricular zone (SVZ) of the lateral ventricle (LV) and in the subgranular zone of the dentate gyrus in the hippocampus lie close to the blood vessels (3Alvarez-Buylla A. Lim D.A. For the long run. Maintaining germinal niches in the adult brain.Neuron. 2004; 41: 683-686Abstract Full Text Full Text PDF PubMed Scopus (1146) Google Scholar). It has been shown that neurogenesis and angiogenesis are coupled processes in subgranular zone (4Palmer T.D. Willhoite A.R. Gage F.H. Vascular niche for adult hippocampal neurogenesis.J. Comp. Neurol. 2000; 425: 479-494Crossref PubMed Scopus (1599) Google Scholar). In rostral migratory stream, blood vessels are tightly associated with the newborn SVZ neuroblasts and are involved in their migration (5Tavazoie M. Van der Veken L. Silva-Vargas V. Louissaint M. Colonna L. Zaidi B. Garcia-Verdugo J.M. Doetsch F. A specialized vascular niche for adult neural stem cells.Cell Stem Cell. 2008; 3: 279-288Abstract Full Text Full Text PDF PubMed Scopus (822) Google Scholar). Similarly, in the olfactory bulb, blood vessels serve as scaffolding for radial migration of neural progenitor cells (6Bovetti S. Hsieh Y.C. Bovolin P. Perroteau I. Kazunori T. Puche A.C. Blood vessels form a scaffold for neuroblast migration in the adult olfactory bulb.J. Neurosci. 2007; 27: 5976-5980Crossref PubMed Scopus (172) Google Scholar). After ischemic insult, neuroblasts of the SVZ are also found migrating along the sides of blood vessels toward the damaged sites (7Yamashita T. Ninomiya M. Hernández Acosta P. García-Verdugo J.M. Sunabori T. Sakaguchi M. Adachi K. Kojima T. Hirota Y. Kawase T. Araki N. Abe K. Okano H. Sawamoto K. Subventricular zone-derived neuroblasts migrate and differentiate into mature neurons in the post-stroke adult striatum.J. Neurosci. 2006; 26: 6627-6636Crossref PubMed Scopus (613) Google Scholar). Early studies suggested that brain endothelial cells influenced NS/P cells through the action of their secreted factors. Indeed, in vitro studies have shown that diffusible factors from endothelial cells maintain and promote NS/P cell self-renewal (8Shen Q. Goderie S.K. Jin L. Karanth N. Sun Y. Abramova N. Vincent P. Pumiglia K. Temple S. Endothelial cells stimulate self-renewal and expand neurogenesis of neural stem cells.Science. 2004; 304: 1338-1340Crossref PubMed Scopus (1285) Google Scholar) and migration (9Wang L. Zhang Z.G. Zhang R.L. Gregg S.R. Hozeska-Solgot A. LeTourneau Y. Wang Y. Chopp M. Matrix metalloproteinase 2 (MMP2) and MMP9 secreted by erythropoietin-activated endothelial cells promote neural progenitor cell migration.J. Neurosci. 2006; 26: 5996-6003Crossref PubMed Scopus (262) Google Scholar). neural stem/progenitor brain endothelial matriptase subventricular zone lateral ventricle pertussis toxin cholera toxin CXC motif chemokine 10 protease-activated receptor G protein-coupled receptor glial fibrillary acidic protein. It was recently demonstrated that neural stem cells and transit-amplifying cells in the LV-SVZ directly contact blood vessels at sites devoid of coverage by astrocyte endfeet and pericyte (5Tavazoie M. Van der Veken L. Silva-Vargas V. Louissaint M. Colonna L. Zaidi B. Garcia-Verdugo J.M. Doetsch F. A specialized vascular niche for adult neural stem cells.Cell Stem Cell. 2008; 3: 279-288Abstract Full Text Full Text PDF PubMed Scopus (822) Google Scholar). LV-SVZ neurogenesis and injury-induced regeneration occur at these specialized neurovasculature contact sites (5Tavazoie M. Van der Veken L. Silva-Vargas V. Louissaint M. Colonna L. Zaidi B. Garcia-Verdugo J.M. Doetsch F. A specialized vascular niche for adult neural stem cells.Cell Stem Cell. 2008; 3: 279-288Abstract Full Text Full Text PDF PubMed Scopus (822) Google Scholar, 10Shen Q. Wang Y. Kokovay E. Lin G. Chuang S.M. Goderie S.K. Roysam B. Temple S. Adult SVZ stem cells lie in a vascular niche. A quantitative analysis of niche cell-cell interactions.Cell Stem Cell. 2008; 3: 289-300Abstract Full Text Full Text PDF PubMed Scopus (824) Google Scholar). An important regulatory mechanism for LV-SVZ neurogenesis may lie within the cell contact interface between the blood vessels and the NS/P cells. Communication between endothelial cells and NS/P cells appears to be a two-way street, each cell type regulates the behavior of the other. It was shown that NS/P cell-derived nitric oxide induces the endothelial expression of VEGF and BDNF (11Li Q. Ford M.C. Lavik E.B. Madri J.A. Modeling the neurovascular niche. VEGF- and BDNF-mediated cross-talk between neural stem cells and endothelial cells. An in vitro study.J. Neurosci. Res. 2006; 84: 1656-1668Crossref PubMed Scopus (163) Google Scholar). BDNF and VEGF in turn activate brain endothelial cell angiogenesis. Nitric oxide also stimulates NS/P cell proliferation by activating endothelial NOS (11Li Q. Ford M.C. Lavik E.B. Madri J.A. Modeling the neurovascular niche. VEGF- and BDNF-mediated cross-talk between neural stem cells and endothelial cells. An in vitro study.J. Neurosci. Res. 2006; 84: 1656-1668Crossref PubMed Scopus (163) Google Scholar). This may represent one mechanism for reciprocal regulation between neurogenesis and angiogenesis. The cellular interaction mechanisms at NS/P cell-blood vessel direct contact sites are largely unexplored. A better understanding of the molecular signals that mediate interactions between NS/P cells and brain endothelial (bEND) cells would be important not only for the maintenance and differentiation of NS/P cells but also for blood vessel regulation. In the present studies we explored the interaction mechanisms between NS/P cells and bEND cells during direct cell contact. We found that NS/P cells induce an endothelial signaling pathway and lead to the production of cytokines/chemokines. Interestingly these endothelial responses were critically dependent on the expression of a type II transmembrane serine protease in NS/P cells and involve an endothelial Gs protein signal. NS/P cells were differentiated from the Sox1-GFP knock-in mouse ES cells (46C ES cells, obtained from Dr. Austin Smith at University of Edinburgh, UK (12Ying Q.L. Stavridis M. Griffiths D. Li M. Smith A. Conversion of embryonic stem cells into neuroectodermal precursors in adherent monoculture.Nat. Biotechnol. 2003; 21: 183-186Crossref PubMed Scopus (1163) Google Scholar)). Differentiation of NS/P cells was carried out by placing 46C ES cells on a gelatin-coated surface in neuronal differentiation medium (referred to as N2B27 medium) as described previously (13Fang J.D. Chou H.C. Tung H.H. Huang P.Y. Lee S.L. Endogenous expression of matriptase in neural progenitor cells promotes cell migration and neuron differentiation.J. Biol. Chem. 2011; 286: 5667-5679Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). GFP+ NS/P cells were collected on day 6 using an ARIA fluorescence-activated cell sorter (BD Biosciences) and were used in the co-culture experiments. For neurosphere culture, 46C ES cell-derived NS/P cells were cultured on an uncoated surface for 6 days. The Sox1-GFP-positive NS/P cell spheroids were then collected. The day 14 mouse embryonic neurocortex neurospheres were purchased from STEMCELL Technologies (Vancouver, Canada). Adult NS/P cells were isolated from SVZ of the LV from 8–12-week-old male FVB mouse as described previously (13Fang J.D. Chou H.C. Tung H.H. Huang P.Y. Lee S.L. Endogenous expression of matriptase in neural progenitor cells promotes cell migration and neuron differentiation.J. Biol. Chem. 2011; 286: 5667-5679Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar); the mouse brain endothelial cell line bEnd.3 was purchased from the Bioscience Collection and Research Center (Hsinchu, Taiwan) and was routinely maintained in DMEM supplemented with 10% FBS. For cell-cell contact co-culture, bEnd.3 cells were plated on 100-mm2 cell culture dishes the previous day to allow attachment. The medium was removed, the cells were washed and changed to N2B27 medium, and NS/P cells were then laid on the top of the attached bEnd.3 cells. Over 90% of NS/P cells attached to bEnd.3 cells in 2–3 h. Twenty-four hours later, NS/P cells were detached from bEnd.3 cells by repeated pipetting, which removed almost all the NS/P cells without detaching bEnd.3 cells as monitored microscopically and by GFP fluorescent of NS/P cells. Cell purity was examined further by RT-PCR for expression of endothelial marker FLK1 and the absence of neural stem/progenitor molecule nestin. After removing detached NS/P cells, bEnd.3 cells were detached from the culture dish with trypsin. All of the cells were then collected by centrifugation. For Fig. 2B, NS/P cells and bEnd.3 cells were mixed and seeded on a coverslip in endothelial cultural medium containing low serum (1%). After the cells attached (within 3 h), the medium was replaced with N2B27 medium and co-cultured for 24 h. For noncontact co-culture, bEnd.3 cells were allowed to attach overnight to the 6-well plate; Falcon Transwells with pore sizes of 8 μm (BD Labware, Franklin Lakes, NJ) were placed on each well, and Sox1-GFP+ NS/P cells were then added into the Transwell. Twenty-four hours later, the Transwell was removed, and bEnd.3 cells were collected as described above. Reagents for cell culture were all from Invitrogen. In cultures where toxin or inhibitor was tested, they were added directly to the medium. Pertussis toxin (PTX), cholera toxin (CTX), and SB203580 were from Sigma. The recombinant mouse MTP protein (purchased from R & D System) is the catalytic serine protease domain and has specific activity >4,000 pmol/min/μg supplied by the manufacturer. The recombinant MTP protein was either added directly to the medium or coated on the surface of culture dishes prior seeding bEnd.3 cells. Total RNA was prepared using RNeasy mini kit (Qiagen) following the manufacturer's protocol. RNA integrity was assessed by electrophoresis, and RNA concentration was measured with a NanoDrop ND-1000 spectrophotometer (Thermo Scientific, Waltham, MA). First strand cDNA was synthesized from 1 μg of RNA using Superscript III reverse transcriptase and oligo(dT) primers (Invitrogen). The resulting cDNA was then used in PCRs. Gene-specific primers were as follows: S15 (mouse ribosomal protein S15): forward, 5′-TTCCGCAAGTTCACCTACC-3′, and reverse, 5′-TGCTTCACGGGTTTGTAGGT-3′; MTP: forward, 5′-CACTTCCATTATCGGAATGTGCG-3′, and reverse, 5′-GGATGTCGCCGGTCAGTATTGGTTATCA-3′; IL6: forward, 5′-GTTGTGCAATGGCAATTCTG-3′, and reverse, 5′-TGGTCTTGGTCCTTAGCCAC-3′; FLK1 (vascular endothelial growth factor receptor 2): forward, 5′-CC AAAAACCAATATGCCCTGAT-3′, and reverse, 5′-TCTC TCAGTACAATGCCTGAATCTTC-3′. IL24: forward, 5′-GATGACATCACAAGCATCCG-3′, and reverse, 5′-ATTTCTGCATCCAGGTCAGG-3′; CXC motif chemokine 10 (CXCL10): forward, 5′-ATGAACCCAAGTGCTGCC-3′, and reverse, 5′-TTCATCGTGGCAATGATCTC-3′; and nestin: forward, 5′-GGGAGCTCGGAGCCCAAGGA-3′, and reverse, 5′-GGGCATGGCTGCCCTCATGG-3′. Transfection of siRNA or mammalian expression plasmid was performed using Lipofectamine 2000 (Invitrogen) as described previously (13Fang J.D. Chou H.C. Tung H.H. Huang P.Y. Lee S.L. Endogenous expression of matriptase in neural progenitor cells promotes cell migration and neuron differentiation.J. Biol. Chem. 2011; 286: 5667-5679Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). MTP knockdown or overexpression was checked by RT-PCR or Western blot. The siRNA against mouse St14 and a control siRNA with no target (nonsilencing siRNA) were purchased from Qiagen. The MTP overexpression plasmid is a pSPORT6-CMV vector carrying the full-length mouse MTP cDNA. Cell lysates were prepared in buffer containing 50 mm Tris-HCl (pH7.5), 1 mm EDTA, 1% Triton X-100, protease inhibitors, and phosphatase inhibitors. After 10 min of centrifugation at 13,000 × g, the supernatants were collected, and protein concentrations were determined by SuperSignal® West Pico protein assay kit (Pierce) using bovine serum albumin as a standard. An equal amount of protein was subjected to SDS-PAGE, followed by Western blot as described (14Lee S.L. Dickson R.B. Lin C.Y. Activation of hepatocyte growth factor and urokinase/plasminogen activator by matriptase, an epithelial membrane serine protease.J. Biol. Chem. 2000; 275: 36720-36725Abstract Full Text Full Text PDF PubMed Scopus (354) Google Scholar). Antibodies to p38 MAP kinase, phospho-p38 MAP kinase, PKCα, and phospho-PKCα were all from Cell Signaling Technology (Danvers, MA). Cell staining followed the procedure described previously (13Fang J.D. Chou H.C. Tung H.H. Huang P.Y. Lee S.L. Endogenous expression of matriptase in neural progenitor cells promotes cell migration and neuron differentiation.J. Biol. Chem. 2011; 286: 5667-5679Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). In brief, the cells were seeded on coverslips. After culture, they were fixed, permeabilized with 0.5% Triton X-100, and blocked with 4% horse serum. After incubation with primary antibody, the cells were then incubated with green or red fluorescence-conjugated secondary antibodies. The cell nuclei were counterstained with DAPI and analyzed using epifluorescence microscope (Olympus BX51). Cytokines released into the culture medium was assayed by ELISA according to the manufacturer's instructions. ELISA for mouse IL6, mouse IL24, and mouse CXCL10 were from Invitrogen, UCSN Life Science Inc. (Wuhan, PRC), and R & D Systems, respectively. Purified recombinant protein of each cytokine was used to obtain a standard curve for each assay to determine the concentration of each cytokine in the cell conditioned medium. GTPase activity of cell lysates was measured using a colorimetric assay kit (Innova Biosciences, Cambridge, UK) following the manufacturer's protocol. In brief, cell lysate or buffer was mixed with GTP substrate: the PiColorLock Gold reagent mix. The stabilizer was then added and incubated for 30 min. The absorbance was measured at 650-nm wavelength. A phosphate standard included in the package was used to calculate the specific GTPase activity in each sample of cell lysate. The data are presented as means ± S.D. derived from three to five independent experiments. Two-group t test was used for comparison between groups. For multiple-group comparisons, data were tested by one-way analysis of variance, and Bonferroni adjustment was used for within group comparisons. NS/P cells were seeded on PDL/laminin-coated culture dishes and cultured in N2B27 medium in the absence or presence of recombinant IL6 protein or the conditioned medium before or after IL6 depletion. The medium was changed every other day and cells were harvested after 7 days of culture. The conditioned medium of NS/P cell-bEND cell contact co-culture were harvested after 24-hours and concentrated by Ultra-4 molecular cutoff at 5 kDa (Millipore-Amicon). For IL6 depletion, the concentrated conditioned medium was incubated with IL6 antibody, and the antibody-bond fraction was then removed by protein-A agarose beads. Depletion of IL6 was confirmed by ELISA. NS/P cells cultured directly on bEND cells attached to bEND cells after short incubation. Fig. 1A shows that expression of IL6, IL24, and CXCL10 in bEND cells was induced by the attached NS/P cells. Expression of endothelial molecule VEGF receptor 2 (FLK1) in bEND cells collected after contact co-culture with NS/P cells was similar to that in bEND cells without co-culture. The NS/P marker nestin was not detected in bEND cells after contact co-culture with NS/P cells. The expression of FLK1 and nestin indicated that the endothelial cells collected after contact co-culture were substantially free of NS/P cells. IL6, IL24, or CXCL10 were undetectable in NS/P cells (Fig. 1A, NPC) whether they were cultured without (Fig. 1A, No Co-Cult) or with (Fig. 1A, Co-Cult) bEND cells. Brain endothelial cells (Fig. 1A, bEND) cultured alone (Fig. 1, A, No Co-Cult, and B, No Co-Cult) expressed low or undetectable levels of IL6, IL24, or CXCL10. After co-culture (Fig. 1A, Co-Cult) with NS/P cells, IL6, IL24, and CXCL10 in bEND cells were significantly increased. In contrast to contact co-culture (Fig. 1, A and B, Cont, Co-Cult), IL6, IL24, and CXCL10 in bEND cells co-cultured with NS/P cells in a Transwell (Fig. 1B, N-Cont, Co-Cult), a noncontact co-culture condition, were not induced. Brain endothelial cells cultured with the conditioned medium of NS/P cell culture did not exhibit increased expression of these genes either. 3H.-H. Tung and S.-L. Lee, unpublished observation. Fig. 1C shows that the secretion of IL6, IL24, and CXCL10 proteins after contact co-culture with NS/P cells (Fig. 1C, Cont, Co-Cult), but not in Transwell co-culture (Fig. 1C, No-Cont, Co-Cult), was three to four times greater than bEND cells without co-culture. Evidently, endothelial IL6, IL24, and CXCL10 induction occurs only in cells in direct contact with NS/P cells. The NS/P cells used in these cultures were differentiated from mouse embryonic stem cells. Identical results were observed in bEND cells in contact co-culture with NS/P cells isolated either from SVZ of the adult mouse brain LV (Fig. 1D, SVZ) or from neural cortex of 14-day mouse embryo (Fig. 1D, E14), clearly demonstrating that induction of endothelial cytokine/chemokine is a general feature of NS/P cells. These data demonstrate that endothelial expression of cytokines/chemokines is induced by cell contact between NS/P and bEND cells. Our data suggest that signaling between NS/P and bEND cells takes place during cell-cell contact interaction and results in induction of endothelial cytokine/chemokine expression. We investigated whether p38 MAPK or PKCα was activated in bEND cells by contact with NS/P cells. We found that p38 MAPK protein (Fig. 2A, p38) was expressed in both NS/P (Fig. 2A, NPC) and bEND cells (Fig. 2A, bEND). In NS/P cells, p38 MAPK was present in the active, phosphorylated state (Fig. 2A, *p38), and it was not altered by co-culture with bEND cells (Fig. 2A, *p38 in NPC under Co-Cult). p38 MAPK proteins in bEND cells, on the other hand, were not phosphorylated until after contact co-culture with NS/P cells (Fig. 2A, bEND, Co-Cult). PKCα was not detected in NS/P cells; nor was its expression induced by co-culture with bEND cells (Fig. 2A, PKC in NPC). PKCα was present in bEND cells, but it was in a phosphorylated state; neither its expression nor its phosphorylation was affected by co-culture with NS/P cells (Fig. 2A, *PKC in bEND). These observations show that endothelial p38 MAPK, not PKCα, is activated by NS/P cells during cell contact. Cell contact induction of endothelial p38 MAPK phosphorylation was checked by immunofluorescence cell staining. Fig. 2B shows that bEND cells in contact with GFP-positive NS/P cells (Fig. 2B, green fluorescence) were highly stained by antibody to the phosphorylated p38 MAPK (Fig. 2B, red fluorescence, arrowheads). Cells that are not in contact with NS/P cells, on the other hand, were either negative or weakly stained (Fig. 2B, arrows). Weak staining of anti-phosphorylated p38 MAPK was observed occasionally in bEND cells without co-culture (Fig. 2B, left panel in the box) or in bEND cells co-cultured with NS/P cell in Transwell (Fig. 2B, right panel in the box), suggesting basal staining of phosphorylate p38MAP in uninduced bEND cells. Same as that shown in Fig. 2A, p38 MAPK are phosphorylated in NS/P cells whether or not they were in contact with bEND cells (Fig. 2B co-stain of GFP and p38*). Cell staining further confirmed that p38 MAPK activation in bEND cells is promoted by contact with NS/P cells. To investigate whether p38 MAPK signaling is responsible for NS/P cell-induced endothelial IL6, IL24 or CXCL10 expression, co-cultured bEND-NS/P cells were treated with p38 MAPK inhibitor SB203580. In the presence of SB203580, NS/P cells failed to induce endothelial IL6 and CXCL10 expression, showing that induction of these endothelial molecules is mediated by p38 MAPK activation induced during co-culture. IL24 expression induced by NS/P cells, however, was not affected much by SB203580 (Fig. 2C, +p38I, Co-Cult), suggesting that p38 MAPK signaling is not the main responsive pathway for IL24 induction. These data demonstrate that NS/P cells in contact with bEND cells activate endothelial p38 MAPK, which can lead to overproduction of some (if not all) endothelial cytokines/chemokines. The observations described above suggested that cell to cell signaling systems exist at the cell contact interface between NS/P cells and bEND cells. We recently showed that the type II transmembrane serine protease matriptase (MTP) is expressed in NS/P cells (13Fang J.D. Chou H.C. Tung H.H. Huang P.Y. Lee S.L. Endogenous expression of matriptase in neural progenitor cells promotes cell migration and neuron differentiation.J. Biol. Chem. 2011; 286: 5667-5679Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Considering its cell surface locality and its substrate diversity, MTP represents a candidate NS/P cell membrane activator protein for inducing endothelial p38 MAPK signaling and the subsequent cytokine/chemokine expression. We tested this possibility by investigating the effect of knocking down the MTP expression in NS/P cells. MTP is highly expressed in NS/P cells; only a small amount of expression was detected in bEND cells (Fig. 3A, No Co-Cult) (13Fang J.D. Chou H.C. Tung H.H. Huang P.Y. Lee S.L. Endogenous expression of matriptase in neural progenitor cells promotes cell migration and neuron differentiation.J. Biol. Chem. 2011; 286: 5667-5679Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Consistent with the results in Fig. 1, contact co-culture with NS/P cells induced endothelial IL6 mRNA expression (Fig. 3A, CT Co-Cult). Contact of NS/P cells and bEND cells did not change the expression of MTP in either cell type (Fig. 3A, CT Co-Cult). MTP mRNA expression in NS/P cells was reduced by specific siRNA (Fig. 3A, M-KD NPC), and it remained knocked down during the course of co-culture with bEND cells (Fig. 3A, KD Co-Cult). These MTP knockdown cells were unable to induce endothelial IL6 mRNA during contact co-culture (Fig. 3A, KD Co-Cult). The ability of NS/P cells to induce endothelial CXCL10 in contact was also impaired by MTP knockdown (Fig. 3B). Interestingly, although IL24 induction was not affected by p38 MAPK inhibitor (Fig. 2), it was impaired by MTP knockdown (Fig. 3B), suggesting that MTP can activate additional endothelial signaling pathways. Semiquantitative RT-PCR is shown in Fig. 3C. Exponential amplification for IL6, IL24, and CXCL10 in bEND cells without co-culture (Fig. 3C, No Co-Cult) or co-cultured with MTP knockdown NS/P cells (Fig. 3C, M-KD Co-Cult) occurred between PCR cycles 25–27. After co-culture with NS/P cells, however, expression of these three molecules reached plateau before or around PCR cycle number 26 (Fig. 3C, Ctrl Co-Cult), confirming that induction of these transcripts in bEND cells by the NS/P cells they directly contact to is dependent on MTP. At the protein level, secretion of all three proteins was induced three or four times after contact with NS/P cells (Fig. 3D, +CTRL NPC). Cells in contact with MTP knockdown NS/P cells (Fig. 3D, +M-KD NPC), on the other hand, expressed all three proteins approximately the same levels as that in cells without co-culture (Fig. 3D, No NPC). Consistent with their mRNA expression, induction of endothelial IL6, IL24, and CXCL10 proteins was lost in MTP knockdown NS/P cell. MTP knockdown NS/P cells also lost their ability to induce endothelial p38 MAPK activation in co-culture (Fig. 3E, +M-KD NPC). Transfection of the MTP expression plasmid into MTP knockdown NS/P cells (Fig. 3F, M-KD/pM) restored MTP expression (Fig. 3F, gel image). These cells regained their ability to induce endothelial IL6, IL24, and CXCL10 in contact co-culture (Fig. 3F, graph, M-KD/pM). These data confirmed that NS/P cells induction of endothelial IL6, IL24, and CXCL10 is a specific effect of MTP. Transfection of MTP expression vector into control NS/P cells induced approximately the same amount of IL6, IL24, or CXCL10 (Fig. 3F, graph, pM). It is obvious that NS/P cell induction of endothelial I" @default.
• W2070673977 created "2016-06-24" @default.
• W2070673977 creator A5040061870 @default.
• W2070673977 creator A5050043195 @default.
• W2070673977 date "2012-06-01" @default.
• W2070673977 modified "2023-09-27" @default.
• W2070673977 title "Neural Transmembrane Protease and Endothelial Gs Protein Activation in Cell Contact-dependent Signaling between Neural Stem/Progenitor Cells and Brain Endothelial Cells" @default.
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