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• W2074080709 abstract "dlk1/FA1 (delta-like 1/fetal antigen-1) is a member of the epidermal growth factor-like homeotic protein family whose expression is known to modulate the differentiation signals of mesenchymal and hematopoietic stem cells in bone marrow. We have demonstrated previously that Dlk1 can maintain the human bone marrow mesenchymal stem cells (hMSC) in an undifferentiated state. To identify the molecular mechanisms underlying these effects, we compared the basal gene expression pattern in Dlk1-overexpressing hMSC cells (hMSC-dlk1) versus control hMSC (negative for Dlk1 expression) by using Affymetrix HG-U133A microarrays. In response to Dlk1 expression, 128 genes were significantly up-regulated (with >2-fold; p < 0.001), and 24% of these genes were annotated as immune response-related factors, including pro-inflammatory cytokines, in addition to factors involved in the complement system, apoptosis, and cell adhesion. Also, addition of purified FA1 to hMSC up-regulated the same factors in a dose-dependent manner. As biological consequences of up-regulating these immune response-related factors, we showed that the inhibitory effects of dlk1 on osteoblast and adipocyte differentiation of hMSC are associated with Dlk1-induced cytokine expression. Furthermore, Dlk1 promoted B cell proliferation, synergized the immune response effects of the bacterial endotoxin lipopolysaccharide on hMSC, and led to marked transactivation of the NF-κB. Our data suggest a new role for Dlk1 in regulating the multiple biological functions of hMSC by influencing the composition of their microenvironment “niche.” Our findings also demonstrate a role for Dlk1 in mediating the immune response. dlk1/FA1 (delta-like 1/fetal antigen-1) is a member of the epidermal growth factor-like homeotic protein family whose expression is known to modulate the differentiation signals of mesenchymal and hematopoietic stem cells in bone marrow. We have demonstrated previously that Dlk1 can maintain the human bone marrow mesenchymal stem cells (hMSC) in an undifferentiated state. To identify the molecular mechanisms underlying these effects, we compared the basal gene expression pattern in Dlk1-overexpressing hMSC cells (hMSC-dlk1) versus control hMSC (negative for Dlk1 expression) by using Affymetrix HG-U133A microarrays. In response to Dlk1 expression, 128 genes were significantly up-regulated (with >2-fold; p < 0.001), and 24% of these genes were annotated as immune response-related factors, including pro-inflammatory cytokines, in addition to factors involved in the complement system, apoptosis, and cell adhesion. Also, addition of purified FA1 to hMSC up-regulated the same factors in a dose-dependent manner. As biological consequences of up-regulating these immune response-related factors, we showed that the inhibitory effects of dlk1 on osteoblast and adipocyte differentiation of hMSC are associated with Dlk1-induced cytokine expression. Furthermore, Dlk1 promoted B cell proliferation, synergized the immune response effects of the bacterial endotoxin lipopolysaccharide on hMSC, and led to marked transactivation of the NF-κB. Our data suggest a new role for Dlk1 in regulating the multiple biological functions of hMSC by influencing the composition of their microenvironment “niche.” Our findings also demonstrate a role for Dlk1 in mediating the immune response. Human bone marrow-derived mesenchymal stem cells (hMSC) 2The abbreviations used are: hMSC, human bone marrow mesenchymal stem cells; MSC, human bone marrow mesenchymal stem cells; FA1, fetal antigen 1; hMSC-TERT, hMSC-telomerized cells; LPS, bacterial endotoxin lipopolysaccharide; NF-κB, nuclear factor-κB; SGM, standard growth medium; MEM, minimal essential medium; FCS, fetal calf serum; CM, conditioned medium; PBS, phosphate-buffered saline; MAPK, mitogen-activated protein kinase; HSC, hematopoietic stem cells; EGF, epidermal growth factor; IL, interleukin; XTT, 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide. 2The abbreviations used are: hMSC, human bone marrow mesenchymal stem cells; MSC, human bone marrow mesenchymal stem cells; FA1, fetal antigen 1; hMSC-TERT, hMSC-telomerized cells; LPS, bacterial endotoxin lipopolysaccharide; NF-κB, nuclear factor-κB; SGM, standard growth medium; MEM, minimal essential medium; FCS, fetal calf serum; CM, conditioned medium; PBS, phosphate-buffered saline; MAPK, mitogen-activated protein kinase; HSC, hematopoietic stem cells; EGF, epidermal growth factor; IL, interleukin; XTT, 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide. are a group of clonogenic cells present among the bone marrow stroma and capable of multilineage differentiation into mesoderm-type cells such as osteoblast, adipocyte, and chondrocyte (1Kassem M. Kristiansen M. Abdallah B.M. Basic Clin. Pharmacol. Toxicol. 2004; 95: 209-214Crossref PubMed Scopus (186) Google Scholar) and possibly other non-mesoderm type cells (2Jiang Y. Jahagirdar B.N. Reinhardt R.L. Schwartz R.E. Keene C.D. Ortiz-Gonzalez X.R. Reyes M. Lenvik T. Lund T. Blackstad M. Du J. Aldrich S. Lisberg A. Low W.C. Largaespada D.A. Verfaillie C.M. Nature. 2002; 418: 41-49Crossref PubMed Scopus (5125) Google Scholar). Moreover, hMSC provide supportive stroma for growth and differentiation of hematopoietic stem cells (HSC) and hematopoiesis (3Majumdar M.K. Thiede M.A. Haynesworth S.E. Bruder S.P. Gerson S.L. J. Hematother. Stem Cell Res. 2000; 9: 841-848Crossref PubMed Scopus (401) Google Scholar). Understanding the mechanisms that control the differentiation decisions of hMSC is thus of the utmost importance from a basic bone biology point of view. It is also important for the clinical use of the hMSC in transplantation and regenerative medicine protocols (1Kassem M. Kristiansen M. Abdallah B.M. Basic Clin. Pharmacol. Toxicol. 2004; 95: 209-214Crossref PubMed Scopus (186) Google Scholar).dlk1/Pref-1 (delta-like 1/pre-adipocyte factor-1) is a transmembrane protein of the EGF-like homeotic superfamily. It is expressed from an imprinted gene paternally expressed at 14q32. Its extracellular domain contains six cysteine-rich EGF-like repeats similar to those found in the Delta/Notch/Serrate family of signaling molecules (4Fleming R.J. Semin. Cell Dev. Biol. 1998; 9: 599-607Crossref PubMed Scopus (159) Google Scholar, 5Laborda J. Sausville E.A. Hoffman T. Notario V. J. Biol. Chem. 1993; 268: 3817-3820Abstract Full Text PDF PubMed Google Scholar). Dlk1 plays a critical role in modulating cell fate decisions throughout development (6Laborda J. Histol. Histopathol. 2000; 15: 119-129PubMed Google Scholar). This is illustrated by the presence of high prenatal mortality, growth retardation, obesity, skeletal malformations, and abnormalities of hematopoiesis in mice deficient in Dlk1 (7Moon Y.S. Smas C.M. Lee K. Villena J.A. Kim K.H. Yun E.J. Sul H.S. Mol. Cell. Biol. 2002; 22: 5585-5592Crossref PubMed Scopus (350) Google Scholar, 8Sakajiri S. O'Kelly J. Yin D. Miller C.W. Hofmann W.K. Oshimi K. Shih L.Y. Kim K.H. Sul H.S. Jensen C.H. Teisner B. Kawamata N. Koeffler H.P. Leukemia (Baltimore). 2005; 19: 1404-1410Crossref PubMed Scopus (75) Google Scholar). Also, in the human syndrome of maternal uniparental disomy 14 (where Dlk1 is silent), patients exhibit obesity, hypotonia, premature puberty, macrocephaly, short stature, and small hands (9Berends M.J. Hordijk R. Scheffer H. Oosterwijk J.C. Halley D.J. Sorgedrager N. Am. J. Med. Genet. 1999; 84: 76-79Crossref PubMed Scopus (65) Google Scholar). Dlk1 has been known for several years as a negative regulator of adipocyte differentiation (10Smas C.M. Sul H.S. Cell. 1993; 73: 725-734Abstract Full Text PDF PubMed Scopus (560) Google Scholar). Recent data suggest that it is involved in many differentiation processes, including hematopoiesis (11Moore K.A. Pytowski B. Witte L. Hicklin D. Lemischka I.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4011-4016Crossref PubMed Scopus (175) Google Scholar), pancreatic islet cell differentiation (12Carlsson C. Tornehave D. Lindberg K. Galante P. Billestrup N. Michelsen B. Larsson L.I. Nielsen J.H. Endocrinology. 1997; 138: 3940-3948Crossref PubMed Scopus (83) Google Scholar), Schwann cell differentiation (13Costaglioli P. Come C. Knoll-Gellida A. Salles J. Cassagne C. Garbay B. FEBS Lett. 2001; 509: 413-416Crossref PubMed Scopus (25) Google Scholar), and hepatic cell differentiation (14Tanimizu N. Tsujimura T. Takahide K. Kodama T. Nakamura K. Miyajima A. Gene Expr. Patterns. 2004; 5: 209-218Crossref PubMed Scopus (77) Google Scholar). Recently, we have identified Dlk1 as a novel regulator of hMSC differentiation (15Abdallah B.M. Jensen C.H. Gutierrez G. Leslie G.Q. Jensen T.G. Kassem M. J. Bone Miner. Res. 2004; 19: 841-852Crossref PubMed Google Scholar). Cellular overexpression of Dlk1 or adding it as a soluble protein to hMSC led to inhibition of hMSC differentiation to osteoblasts or adipocytes (15Abdallah B.M. Jensen C.H. Gutierrez G. Leslie G.Q. Jensen T.G. Kassem M. J. Bone Miner. Res. 2004; 19: 841-852Crossref PubMed Google Scholar). The extracellular domain of the dlk1 is cleaved by tumor necrosis factor-α-converting enzyme (16Wang Y. Sul H.S. Mol. Cell. Biol. 2006; 26: 5421-5435Crossref PubMed Scopus (103) Google Scholar) and was first identified in the amniotic fluid (17Fay T.N. Jacobs I. Teisner B. Poulsen O. Chapman M.G. Stabile I. Bohn H. Westergaard J.G. Grudzinskas J.G. Eur. J. Obstet. Gynecol. Reprod. Biol. 1988; 29: 73-85Abstract Full Text PDF PubMed Scopus (76) Google Scholar) and hence named fetal antigen 1 (FA1) (18Jensen C.H. Krogh T.N. Hojrup P. Clausen P.P. Skjodt K. Larsson L.I. Enghild J.J. Teisner B. Eur. J. Biochem. 1994; 225: 83-92Crossref PubMed Scopus (136) Google Scholar). FA1 is the biologically active part of the molecule, and its serum levels change in some pathological conditions, e.g. growth hormone excess (acromegaly) (19Andersen M. Jensen C.H. Stoving R.K. Larsen J.B. Schroder H.D. Teisner B. Hagen C. J. Clin. Endocrinol. Metab. 2001; 86: 5465-5470Crossref PubMed Scopus (11) Google Scholar). In contrast to other members of Delta/Notch/Serrate family, dlk1 protein does not have the DSL (Delta, Serrate, and LAG2) domain mediating Notch-Delta interaction, and thus it is not known whether dlk1 functions as a ligand or as a receptor (6Laborda J. Histol. Histopathol. 2000; 15: 119-129PubMed Google Scholar). However, recent data based on a yeast two-hybrid system suggested that dlk1 interacts with Notch 1 and inhibits Notch signaling (20Baladron V. Ruiz-Hidalgo M.J. Nueda M.L. Diaz-Guerra M.J.M. Garcia-Ramirez J.J. Bonvini E. Gubina E. Laborda J. Exp. Cell Res. 2005; 303: 343-359Crossref PubMed Scopus (164) Google Scholar). Also, other signaling pathways have been demonstrated to be affected by the expression of Dlk1, e.g. mitogen-activated protein kinase (MAPK) (21Ruiz-Hidalgo M.J. Gubina E. Tull L. Baladron V. Laborda J. Exp. Cell Res. 2002; 274: 178-188Crossref PubMed Scopus (44) Google Scholar) and insulin-like growth factor-I receptor-mediated p42/p44 MAPK activation (22Zhang H. Noohr J. Jensen C.H. Petersen R.K. Bachmann E. Teisner B. Larsen L.K. Mandrup S. Kristiansen K. J. Biol. Chem. 2003; 278: 20906-20914Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Despite this pleiotropic function of Dlk1 in many developmental and differentiation processes, little is known about the mechanisms mediating its regulatory function and its target genes.To identify the mechanisms underlying the biological effects of Dlk1 in hMSC, we employed the DNA microarray approach to discover specific molecular pathways regulated by Dlk1.We found that expression of Dlk1 increased the production of a large number of inflammatory and immune response-related factors, including cytokines, chemokines, and complement factors by hMSC, and these changes were associated with its effects on hMSC differentiation into adipocytes, osteoblasts, and its hematopoiesis-support function.EXPERIMENTAL PROCEDURESCell Culture—The establishment and the characterization of hMSC-TERT (immortalized hMSC cells, used as a control with no Dlk1 expression) and hMSC-dlk1 (Dlk1-overexpressing cells, derived from hMSC-TERT) cell lines have been described previously (15Abdallah B.M. Jensen C.H. Gutierrez G. Leslie G.Q. Jensen T.G. Kassem M. J. Bone Miner. Res. 2004; 19: 841-852Crossref PubMed Google Scholar, 23Simonsen J.L. Rosada C. Serakinci N. Justesen J. Stenderup K. Rattan S.I. Jensen T.G. Kassem M. Nat. Biotechnol. 2002; 20: 592-596Crossref PubMed Scopus (684) Google Scholar). Normal hMSC cultures were established from bone marrow aspirates obtained from young donors (n = 5, males 25–30 years old) by aspiration from the iliac crest as described previously (24Kassem M. Mosekilde L. Eriksen E.F. J. Bone Miner. Res. 1993; 8: 1453-1458Crossref PubMed Scopus (77) Google Scholar). A written consent was obtained from each participant, and the study was approved by the local Scientific-Ethical committee. All cells were cultured in a standard growth medium (SGM) containing minimal essential medium (MEM; Invitrogen) supplemented with 10% fetal calf serum (FCS) and 1% penicillin/streptomycin (Invitrogen) at 37 °C in a humidified atmosphere containing 5% CO2.Collecting the Conditioned Media and Purification of FA1— hMSC-TERT or hMSC-dlk1 cells were cultured at 3 × 103 cells/cm2 in Petri dishes in standard growth medium. At 80–90% cell confluence, medium was replaced by serum-free MEM, and cells were cultured for 2 additional days. Conditioned media (CM) were then collected either from hMSC-TERT cells (control/CM) or hMSC-dlk1 (dlk1/CM), centrifuged at 1000 × g for 5 min at 4 °C to remove cell debris, and used neat or diluted 1:2 (v/v) or 1:4 (v/v) with serum-free MEM.The full soluble ectodomain, active form of dlk1 protein named FA1, was purified from the collected serum-free CM of cultured hMSC-dlk1 (dlk1/CM) using immunospecific affinity chromatography as described previously (25Jensen C.H. Teisner B. Hojrup P. Rasmussen H.B. Madsen O.D. Nielsen B. Skjodt K. Hum. Reprod. (Oxf.). 1993; 8: 635-641Crossref PubMed Scopus (82) Google Scholar). dlk1/CM depleted of FA1 (FA1-/CM) were also collected from the affinity chromatography eluents.We were careful to avoid any endotoxin contaminations in our culture system or during FA1 preparation. To ensure that, the endotoxin levels were measured in all conditioned media and FA1 preparation as nondiluted research samples using the Endotoxin-Testing Service at Cambrex BioScience (test code 95-101; Cambrex BioScience Verviers S.p.r.l., Belgium). All conditioned media (control/CM, dlk1/CM, and FA1-/CM) and FA1 preparation used in this study were negative for contamination with endotoxins with less than 0.05 endotoxin unit/ml.Study of the Effect of FA1-containing CM and Purified FA1 on the Cytokine Expression Profile of hMSC—Normal hMSC cells established from healthy donors were cultured in 6-well plates at 3 × 104 cells/cm2 in standard growth medium. At 90–100% cell confluence, cells were cultured either in control/CM (obtained from hMSC-TERT), in dlk1/CM supplemented with 10% FCS, or in SGM supplemented with purified FA1 (1 or 5 μg/ml) for 24 h.Cell Differentiation Assays—To induce osteoblast differentiation, cells were cultured in 6-well plates at a density of 3 × 104 cells/cm2 in SGM for 24 h. At 70–80% confluence, the medium was replaced by osteogenic medium, consisting of an SGM supplemented with 10 nm 1,25-dihydroxycholecalciferol (vitamin D3) (kindly provided by Leo Pharma, Denmark). The medium was replaced every 3 days.To induce adipocyte differentiation, cells were cultured in 6-well plates at a density of 3 × 104 cells/cm2. At 100% confluence, the medium was replaced by adipogenic induction medium, consisting of low glucose Dulbecco's modified Eagle's medium (Invitrogen) containing 10% FCS supplemented with 100 nm human recombinant insulin (Sigma), 10 nm dexamethasone, 0.25 mm 1-methyl-3-isobutylxanthine (Sigma), and 1 μm rosiglitazone (BRL49653) (kindly provided by Novo Nordisk, Bagsvaerd, Denmark) for 48 h and then shifted into adipogenic induction medium without dexamethasone for another 10 days. Medium was renewed every 3 days.Proliferation Assay of Cultured Human B Cells—Peripheral blood mononuclear cells isolated from buffy coats of healthy blood donors were depleted of CD19+ cells using Dynabeads CD19 Pan B (Dynal, Norway) according to the manufacturer's instructions. Cell-attached magnetic beads were removed by magnet after overnight incubation with DETACHBEAD CD19 (Dynal, Norway). Isolated CD19+ cells were washed twice at 1,200 rpm for 10 min at room temperature in modified Eagle's medium (without phenol red) supplemented with 200 international units/ml penicillin and counted in trypan blue. Cells were then resuspended at 0.5 × 106/ml in RPMI (as a positive control), serum-free conditioned medium from hMSC-TERT (control/CM), or hMSC-dlk1 (dlk1/CM) supplemented with 10% FCS and stimulated with 20 ng/ml recombinant human IL-4 (R&D Systems, Abingdon, UK) and the indicated concentrations of recombinant human CD40L (R&D Systems) or left untreated (nonstimulated). Cells were seeded in flat-bottom 96-well microtiter plates (50,000 cells per well in 100 μl of medium) and incubated at 37 °C. At day 3 of culture, 100 μl of fresh media supplemented with IL-4 and CD40L were added. At day 6 of cell culture, metabolic activity of the cells was measured with an XTT assay (Biological Industries, Israel). Data were represented as fold induction of the proliferation of B cells over control CD19+ cells cultured in nonstimulated CM.Microarrays—Both control (hMSC-TERT) and Dlk1-overexpressing cells (hMSC-dlk1) were cultured in triplicate at 3 × 104 cells/cm2 in Petri dishes in standard growth medium. At 90–100% confluence, highly purified total cellular RNA was isolated from each of three independent cultures per cell line using an RNeasy kit (Qiagen Nordic, West Sussex, UK) according to the manufacturer's instructions. First- and second-strand cDNA syntheses were performed from 8 μg of total RNA using the SuperScript Choice System (Invitrogen) according to the manufacturer's instructions. cRNA was synthesized from cDNA and biotinylated using the BioArray High Yield RNA transcript labeling kit (Enzo® kit, Enzo Diagnostics, Farmingdale, NY), according to the protocol provided by the manufacturer, and hybridized for 16–18 h at 45 °C in a rotisserie to the Affymetrix® Human Genome U133A 2.0 arrays. The arrays were scanned using the GeneArray scanner (Affymetrix).Data analysis was performed using the dChip software (26Li C. Wong W.H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 31-36Crossref PubMed Scopus (2701) Google Scholar). We first normalized our probe level data using the invariant set normalization procedure (26Li C. Wong W.H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 31-36Crossref PubMed Scopus (2701) Google Scholar). The normalized probe level data were converted to model-based gene expression indexes (MBEI, log with base 2) for use as the values of gene expression for a total of 22,000 genes on the Affymetrix 133A 2.0 arrays. The paired t statistic was used to assess the genes that are differentially regulated in the two cell lines by assigning the hMSC-TERT cell line as reference and the hMSC-dlk1 cell line as the experiment group. The false discovery rate (27Tusher V.G. Tibshirani R. Chu G. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5116-5121Crossref PubMed Scopus (9705) Google Scholar) was calculated for each of the genes to account for multiple testing. Significant genes were identified as those with a mean expression ratio between the experiment and the reference groups above 2 and below 2 and at the same time a false discovery rate of <0.05. Hierarchical clustering analysis was applied to cluster the genes as well as the samples using the average linkage method. Gene ontology classifications for all differentially expressed genes by Dlk1 in hMSC were performed using DAVID 2.0 software (28Dennis Jr., G. Sherman B.T. Hosack D.A. Yang J. Gao W. Lane H.C. Lempicki R.A. Genome Biol. 2003; 4: P3Crossref PubMed Google Scholar) and EASE software (29Hosack D.A. Dennis Jr., G. Sherman B.T. Lane H.C. Lempicki R.A. Genome Biol. 2003; 4: R70Crossref PubMed Google Scholar). Genes were classified according to their molecular function and relevant biological process. The microarray data were deposited in the Array Express public data base with assigned accession number (E-MEXP-560).Total RNA Extraction and Real Time PCR Analysis—Total cellular RNA was isolated using a single step method with TRIzol (Invitrogen), according to the manufacturer's instructions. First strand complementary cDNA was synthesized from 4 μg of total RNA using a revertAid H minus first strand cDNA synthesis kit (Fermentas, Copenhagen, Denmark) according to the manual instructions.Real time PCR was performed using the iCycler IQ detection system (Bio-Rad) and SYBR® Green I as a double strand DNA-specific binding dye. Thermocycling was performed in a final volume of 20 μl containing 3 μl of cDNA sample (diluted 1:20), 20 pmol of each primer, and 2× iQ™ SYBR® Green Supermix (Bio-Rad). The quantification of gene expression for each target gene and reference gene was performed in separate tubes using primers as shown in supplemental Table 1. We used a denaturing step at 95 °C for 3 min and 40 cycles of 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 1 min. Each reaction was run in duplicate, and fluorescence data were collected at the end of the extension step in every cycle.To ensure specific amplification, a melting curve was calculated for each PCR by increasing the temperature from 60 to 95 °C with a temperature increment rate of 0.5 °C/10 s. Fold induction and expression level for each target gene were calculated using the comparative CT method as follows: 1/(2ΔCT), where ΔCT is the difference between CT target and CT reference. After normalization to β-actin mRNA (User Bulletin No. 2, PerkinElmer Life Sciences), data were analyzed using optical system software version 3.1 (Bio-Rad) and Microsoft Excel 2000 to generate relative expression values.In Situ Detection of NF-κB Nuclear Translocation—Control cells (hMSC-TERT) were seeded at 3,000 cells/well in 96-well plates and cultured for 2 days in normal medium. Cells were exposed for 15, 30, 60, and 120 min at 37 °C to nondiluted CM prepared either from control or hMSC-dlk1 cells. As a positive control for NF-κB activation, some wells were treated with 1 μg/ml LPS. In situ detection of NF-κB nuclear translocation was carried out as described previously (30Boissy P. Andersen T.L. Abdallah B.M. Kassem M. Plesner T. Delaisse J.M. Cancer Res. 2005; 65: 9943-9952Crossref PubMed Scopus (166) Google Scholar). Briefly, cells were fixed first in 4% paraformaldehyde for 10 min at room temperature and then with cold methanol for 15 min at -20 °C. Cells were incubated with a mouse monoclonal anti-p65 antibody (Santa Cruz Biotechnology) in bovine serum albumin/PBS overnight at 4 °C, washed several times in PBS, and subsequently incubated with a secondary AlexaFluor 568 goat anti-mouse IgG antibody (Invitrogen) in bovine serum albumin/PBS for 1 h at room temperature. After several washes, the nucleus of cells was counterstained with Hoechst 33258 (Sigma). Pictures of Hoechst and anti-p65 immunofluorescence were taken randomly in different areas of the wells with a coolsnap camera (Roper Scientific, Brock & Michelsen, Birkerød, Denmark) plugged to a stage-motorized inverted Axiovert 200 microscope (Zeiss, Brock & Michelsen). Cells showing a bright immunofluorescence for p65 in the nucleus were scored using MetaVue® image analyzing software (Universal Imaging Corp., Brock & Michelsen). The results were presented as the number of cells with NF-κB nuclear translocation in percentage of the total number of nuclei scrutinized.Statistical Analysis—All values are expressed as means ± S.D. Statistical analysis was performed by Student's t test using unpaired t test (two-tailed). p < 0.05 was considered significant.RESULTSTo identify the genetic pathways regulated by the expression of Dlk1, we studied the basal gene expression pattern in Dlk1-overexpressing hMSC cells versus control hMSC-TERT cells using Affymetrix DNA microarrays. A total of 315 probe sets corresponding to 277 genes/expressed sequence tags were found to be differentially expressed in response to Dlk1 expression. Among these, 157 probes corresponding to 128 genes were up-regulated (>2-fold, p < 0.001), whereas158 probe sets corresponding to 149 genes were down-regulated (<2-fold, p < 0.001) in hMSC-dlk1 compared with control cells. Gene annotation based on biological processes and molecular functions revealed the presence of 24% immune response-related genes, whereas the other 76% were assigned to different functional categories, such as signal transduction, cell adhesion, metabolism, apoptosis, and others (Fig. 1, A and B). On the other hand, Dlk1 down-regulated genes were mainly annotated as DNA replication-related genes and cell growth and/or maintenance genes (see the supplemental Table 2).As shown in Table 1, the largest category of the up-regulated genes was annotated as immune regulatory factors, which include several potent pro-inflammatory cytokines, a large number of chemokines, the complement components C3 and C1 that are known as key factors for the activation of the complement pathway (31Mastellos D. Lambris J.D. Trends Immunol. 2002; 23: 485-491Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar), prostaglandin E synthase, and prostaglandin-endoperoxide synthase 2 (COX-2) that are up-regulated in response to IL-1β (32Samad T.A. Moore K.A. Sapirstein A. Billet S. Allchorne A. Poole S. Bonventre J.V. Woolf C.J. Nature. 2001; 410: 471-475Crossref PubMed Scopus (1140) Google Scholar) and KLRC1, a molecule expressed primarily by Natural Killer cells during infection and involved in the recognition of major histocompatibility complex class I HLA-E molecules (33Braud V.M. Allan D.S. O'Callaghan C.A. Soderstrom K. D'Andrea A. Ogg G.S. Lazetic S. Young N.T. Bell J.I. Phillips J.H. Lanier L.L. McMichael A.J. Nature. 1998; 391: 795-799Crossref PubMed Scopus (1716) Google Scholar). In addition, the apoptosis-annotated gene group included numerous genes such as CASP1, CASP8, TNFRSF9, TNFRSF21, and TNFAIP3 that function cooperatively with some cytokines to induce cell death and activate NF-κB signaling (34Heyninck K. Beyaert R. Mol. Cell. Biol. Res. Commun. 2001; 4: 259-265Crossref PubMed Scopus (76) Google Scholar). Given the gene expression pattern found, we decided to focus on studying Dlk1-up-regulated genes, especially the pro-inflammatory cytokines.TABLE 1Genes differentially up-regulated by Dlk1 in hMSCGene accession no.Gene nameGene symbolFold changeImmune response NM_000575Interleukin 1αIL1A10.6 NM_000576Interleukin 1βIL1B7.1 NM_000600Interleukin 6 (interferon β2)IL68.5 NM_000584Interleukin 8IL88.5 NM_001511Chemokine (CXC motif) ligand 1 (melanoma growth-stimulating activity, α)CXCL12.4 NM_002089Chemokine (CXC motif) ligand 2CXCL25.2 NM_002090Chemokine (CXC motif) ligand 3CXCL35.4 NM_002993Chemokine (CXC motif) ligand 6 (granulocyte chemotactic protein 2)CXCL618.5 NM_005409Chemokine (CXC motif) ligand 11CXCL115.9 NM_004591Chemokine (CC motif) ligand 20CCL208.1 NM_000064Complement component 3C315.2 NM_001734Complement component 1. s subcomponentC1S3.6 NM_001733Homo sapiens cDNA FLJ36690 fis. clone UTERU2008707. highly similar to COMPLEMENT C1R COMPONENT PRECURSOR (EC 3.4.21.41).C1R3.2 NM_004878Prostaglandin E synthasePTGES3.6 NM_000963Prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase and cyclooxygenase)PTGS25.5 NM_000636Superoxide dismutase 2. MitochondrialSOD26.6 NM_004079Cathepsin SCTSS14.0 NM_002164Indoleamine-pyrrole 2,3-dioxygenaseINDO6.4 NM_002309Leukemia inhibitory factor (cholinergic differentiation factor)LIF2.7 NM_213602CD33 antigen-like 3CD33L325.7 NM_014096Likely ortholog of mouse embryonic epithelial gene 1SLC43A34.6 NM_002607Platelet-derived growth factor-α polypeptidePDGFA2.7 NM_002259Killer cell lectin-like receptor subfamily C. Member 2KLRC28.1 NM_014707Histone deacetylase 9HDAC93.4 NM_006398Ubiquitin DUBD3.4 NM_002084Glutathione peroxidase 3 (plasma)GPX33.4 NM_001005176SP140 nuclear body proteinSP1403.3 NM_005746Pre-B cell colony-enhancing factorPBEF15.6 NM_001432EpiregulinEREG3.1 NM_002123Major histocompatibility complex. class II. DQ β1HLA-DQB12.6 NM_021628Arachidonate lipoxygenase 3ALOXE32.5 NM_014734KIAA0247KIAA02472.5Signal transduction NM_002847Protein-tyrosine phosphatase. Receptor type. N polypeptide 2PTPRN214.6 NM_002849Protein-tyrosine phosphatase. Receptor type. RPTPRR2.5 NM_005012Receptor tyrosine kinase-like orphan receptor 1ROR13.3 NM_005424Tyrosine kinase with immunoglobulin and epidermal growth factor homology domainsTIE13.8 NM_005026Phosphoinositide 3-kinase. Catalytic. δ polypeptidePIK3CD2.6 NM_002222Inositol 1,4,5-triphosphate receptor. Type 1ITPR12.6 NM_001394Dual specificity phosphatase 4DUSP43.5 NM_007207Dual specificity phosphatase 10DUSP102.6 NM_001957Endothelin receptor type AEDNRA3.0 NM_000115Endothelin receptor type BEDNRB3.3 NM_004442EphB2EPHB22.6 NM_006868RAB31. Member RAS oncogene familyRAB314.2 NM_015234G protein-coupled receptor 116GPR1166.3 NM_003236Transforming growth factor-αTGFA8.4 NM_002037FYN oncogene related to SRC. FGR. YESFYN3.6 NM_022748Tensin-like SH2aSH indicates Src homology domain-containing 1TENS13.0 NM_005335Hematopoietic cell-specific Lyn substrate 1HCLS12.8 NM_004163RAB27B. Member RAS oncogene familyRAB27B2.7 NM_003155Stanniocalcin 1STC12.6 NM_002820Parathyroid hormone-like hormonePTHLH4.4 NM_000275Oculocutaneous albinism II (pink-eye dilution homolog. Mouse)OCA23.5 NM_025195Phosphoprotein regulated by mitogenic pathwaysC8FW2.5 NM_001025081Myeli" @default.
• W2074080709 created "2016-06-24" @default.
• W2074080709 creator A5009208757 @default.
• W2074080709 creator A5014389043 @default.
• W2074080709 creator A5019213890 @default.
• W2074080709 creator A5035262769 @default.
• W2074080709 creator A5049717703 @default.
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• W2074080709 creator A5058688311 @default.
• W2074080709 creator A5074757056 @default.
• W2074080709 creator A5004434287 @default.
• W2074080709 date "2007-03-01" @default.
• W2074080709 modified "2023-10-07" @default.
• W2074080709 title "dlk1/FA1 Regulates the Function of Human Bone Marrow Mesenchymal Stem Cells by Modulating Gene Expression of Pro-inflammatory Cytokines and Immune Response-related Factors" @default.
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