Restricted accessResearch articleFirst published online 2009-03
Inhibition of ERK Promotes Collagen Gel Compaction and Fibrillogenesis to Amplify the Osteogenesis of Human Mesenchymal Stem Cells in Three-Dimensional Collagen I Culture
Tissue morphogenesis remains one of the least understood problems in cell and developmental biology. There is a disconnect between the mechanisms that apply to two-dimensional (2D) cultures and those seen in vivo. Three-dimensional (3D) culture presents a complex stimulus triggering cellular responses that are only partially understood. We compared 2D and 3D cultures of human mesenchymal stem cells in the presence of mitogen-activated protein kinase kinase (MEK) inhibitor, PD98059, to determine the role of extracellular signal-related kinase (ERK) in collagen-induced differentiation. 3D collagen I culture enhanced and accelerated the osteogenic differentiation of human mesenchymal stem cells (hMSC). Contrary to 2D results, the addition of PD98059 induced a significant amplification of osteogenic gene expression and matrix mineralization in 3D cultures. The inhibition of ERK altered cell-mediated compaction, proliferation, and resulted in the development of distinct tissue microstructure. Therefore, we suggest that the ability to reorganize collagen in 3D is an important step in ERK-mediated osteogenic differentiation. This work aims to propose a correlation between osteogenic differentiation and hMSC-directed collagen I remodeling. We present a potential mechanistic link (ERK) through which the three dimensionality of an engineered tissue acts to differentially induce and maintain cellular phenotype during tissue development.
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References
1.
JaiswalRK, JaiswalN, BruderSP, MbalavieleG, MarshakDR, PittengerMF. 2000. Adult human mesenchymal stem cell differentiation to the osteogenic or adipogenic lineage is regulated by mitogen-activated protein kinase. J Biol Chem, 275:9645–9652.
MohlerW, MillardAC, CampagnolaPJ. 2003. Second harmonic generation imaging of endogenous structural proteins. Methods, 29:97–109.
8.
TheodossiouT, RaptiGS, HovhannisyanV, GeorgiouE, PolitopoulosK, YovaD. 2002. Thermally induced irreversible conformational changes in collagen probed by optical second harmonic generation and laser-induced fluorescence. Lasers Med Sci, 17:34–41.
9.
KimBM, EichlerJ, ReiserKM, RubenchikAM, Da SilvaLB. 2000. Collagen structure and nonlinear susceptibility: effects of heat, glycation, and enzymatic cleavage on second harmonic signal intensity. Lasers Surg Med, 27:329–335.
10.
Schenke-LaylandK, RiemannI, DamourO, StockUA, KonigK. 2006. Two-photon microscopes and in vivo multi-photon tomographs—powerful diagnostic tools for tissue engineering and drug delivery. Adv Drug Deliv Rev, 58:878–896.
11.
WangF, HansenRK, RadiskyD, YonedaT, Barcellos-HoffMH, PetersenOW, TurleyEA, BissellMJ. 2002. Phenotypic reversion or death of cancer cells by altering signaling pathways in three-dimensional contexts. J Natl Cancer Inst, 94:1494–1503.
12.
WeaverVM, LelievreS, LakinsJN, ChrenekMA, JonesJC, GiancottiF, WerbZ, BissellMJ. 2002. Beta4 integrin-dependent formation of polarized three-dimensional architecture confers resistance to apoptosis in normal and malignant mammary epithelium. Cancer Cell, 2:205–216.
13.
PedersenJA, SwartzMA. 2005. Mechanobiology in the third dimension. Ann Biomed Eng, 33:1469–1490.
BissellMJ, RadiskyDC, RizkiA, WeaverVM, PetersenOW. 2002. The organizing principle: microenvironmental influences in the normal and malignant breast. Differentiation, 70:537–546.
16.
ParryG, LeeEY, FarsonD, KovalM, BissellMJ. 1985. Collagenous substrata regulate the nature and distribution of glycosaminoglycans produced by differentiated cultures of mouse mammary epithelial cells. Exp Cell Res, 156:487–499.
17.
EmermanJT, PitelkaDR. 1977. Maintenance and induction of morphological differentiation in dissociated mammary epithelium on floating collagen membranes. In Vitro, 13:316–328.
SalasznykRM, KleesRF, HughlockMK, PlopperGE. 2004. ERK signaling pathways regulate the osteogenic differentiation of human mesenchymal stem cells on collagen I and vitronectin. Cell Commun Adhes, 11:137–153.
20.
FierroF, IllmerT, JingD, SchleyerE, EhningerG, BoxbergerS, BornhauserM. 2007. Inhibition of platelet-derived growth factor receptor beta by imatinib mesylate suppresses proliferation and alters differentiation of human mesenchymal stem cells in vitro. Cell Prolif, 40:355–366.
21.
KunathT, Saba-El-LeilMK, AlmousailleakhM, WrayJ, MelocheS, SmithA. 2007. FGF stimulation of the Erk1/2 signalling cascade triggers transition of pluripotent embryonic stem cells from self-renewal to lineage commitment. Development, 134:2895–2902.
22.
SimmonsCA, MatlisS, ThorntonAJ, ChenS, WangCY, MooneyDJ. 2003. Cyclic strain enhances matrix mineralization by adult human mesenchymal stem cells via the extracellular signal-regulated kinase (ERK1/2) signaling pathway. J Biomech, 36:1087–1096.
23.
KookSH, SonYO, ChoiKC, LeeHJ, ChungWT, HwangIH, LeeJC. 2008. Cyclic mechanical stress suppresses myogenic differentiation of adult bovine satellite cells through activation of extracellular signal-regulated kinase. Mol Cell Biochem, 309:133–141.
24.
LivakKJ, SchmittgenTD. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods, 25:402–408.
25.
SalasznykRM, WilliamsWA, BoskeyA, BatorskyA, PlopperGE. 2004. Adhesion to vitronectin and collagen I promotes osteogenic differentiation of human mesenchymal stem cells. J Biomed Biotechnol, 2004:24–34.
26.
GardnerHA. 1999. Integrin signaling in fibrosis and scleroderma. Curr Rheumatol Rep, 1:28–33.
27.
SalasznykRM, KleesRF, WestcottAM, VandenbergS, BennettK, PlopperGE. 2005. Focusing of gene expression as the basis of stem cell differentiation. Stem Cells Dev, 14:608–620.
28.
BruderSP, JaiswalN, RicaltonNS, MoscaJD, KrausKH, KadiyalaS. 1998. Mesenchymal stem cells in osteobiology and applied bone regeneration. Clin Orthop Relat Res, 355,Suppl.:S247–S256.
29.
NgPC, LamCW, WongGW, LeeCH, ChengPS, FokTF, ChanIH, WongE, CheungK, LeeSY. 2002. Changes in markers of bone metabolism during dexamethasone treatment for chronic lung disease in preterm infants. Arch Dis Child Fetal Neonatal Ed, 86:F49–F54.
30.
HuangS, ChenCS, IngberDE. 1998. Control of cyclin D1, p27(Kip1), and cell cycle progression in human capillary endothelial cells by cell shape and cytoskeletal tension. Mol Biol Cell, 9:3179–3193.
31.
McBeathR, PironeDM, NelsonCM, BhadrirajuK, ChenCS. 2004. Cell shape, cytoskeletal tension, and RhoA regulate stem cell lineage commitment. Dev Cell, 6:483–495.
WozniakMA, DesaiR, SolskiPA, DerCJ, KeelyPJ. 2003. ROCK-generated contractility regulates breast epithelial cell differentiation in response to the physical properties of a three-dimensional collagen matrix. J Cell Biol, 163:583–595.
37.
SalasznykRM, KleesRF, WilliamsWA, BoskeyA, PlopperGE. 2007. Focal adhesion kinase signaling pathways regulate the osteogenic differentiation of human mesenchymal stem cells. Exp Cell Res, 313:22–37.
38.
SalasznykRM, KleesRF, BoskeyA, PlopperGE. 2007. Activation of FAK is necessary for the osteogenic differentiation of human mesenchymal stem cells on laminin-5. J Cell Biochem, 100:499–514.
39.
BrownRA, PrajapatiR, McGroutherDA, YannasIV, EastwoodM. 1998. Tensional homeostasis in dermal fibroblasts: mechanical responses to mechanical loading in three-dimensional substrates. J Cell Physiol, 175:323–332.
HongH, McCulloughCM, StegemannJP. 2007. The role of ERK signaling in protein hydrogel remodeling by vascular smooth muscle cells. Biomaterials, 28:3824–3833.
42.
StegemannJP, HongH, NeremRM. 2005. Mechanical, biochemical, and extracellular matrix effects on vascular smooth muscle cell phenotype. J Appl Physiol, 98:2321–2327.
43.
BatorskyA, LiaoJ, LundAW, PlopperGE, StegemannJP. 2005. Encapsulation of adult human mesenchymal stem cells within collagen-agarose microenvironments. Biotechnol Bioeng, 92:492–500.
44.
XiaH, NhoRS, KahmJ, KleidonJ, HenkeCA. 2004. Focal adhesion kinase is upstream of phosphatidylinositol 3-kinase/Akt in regulating fibroblast survival in response to contraction of type I collagen matrices via a beta 1 integrin viability signaling pathway. J Biol Chem, 279:33024–33034.
45.
TianX, RusanescuG, HouW, SchaffhausenB, FeigLA. 2002. PDK1 mediates growth factor-induced Ral-GEF activation by a kinase-independent mechanism. EMBO J, 21:1327–1338.
46.
DaleyWP, PetersSB, LarsenM. 2008. Extracellular matrix dynamics in development and regenerative medicine. J Cell Sci, 121:255–264.
47.
WardDFJr, SalasznykRM, KleesRF, BackielJ, AgiusP, BennettK, BoskeyA, PlopperGE. 2007. Mechanical strain enhances extracellular matrix-induced gene focusing and promotes osteogenic differentiation of human mesenchymal stem cells through an extracellular-related kinase-dependent pathway. Stem Cells Dev, 16:467–480.
48.
ConstantVA, GagnonA, YarmoM, SoriskyA. 2008. The antiadipogenic effect of macrophage-conditioned medium depends on ERK1/2 activation. Metabolism, 57:465–472.
49.
LiaoQC, LiYL, QinYF, QuarlesLD, XuKK, LiR, ZhouHH, XiaoZS. 2008. Inhibition of adipocyte differentiation by phytoestrogen genistein through a potential downregulation of extracellular signal-regulated kinases 1/2 activity. J Cell Biochem(Epub ahead of print April10.1002/jcb.21753).
50.
ChenTH, ChenWM, HsuKH, KuoCD, HungSC. 2007. Sodium butyrate activates ERK to regulate differentiation of mesenchymal stem cells. Biochem Biophys Res Commun, 355:913–918.
51.
YangB, CaoDJ, SainzI, ColmanRW, GuoYL. 2004. Different roles of ERK and p38 MAP kinases during tube formation from endothelial cells cultured in 3-dimensional collagen matrices. J Cell Physiol, 200:360–369.
52.
KadiyalaS, YoungRG, ThiedeMA, BruderSP. 1997. Culture expanded canine mesenchymal stem cells possess osteochondrogenic potential in vivo and in vitro. Cell Transplant, 6:125–134.
53.
XuL, AndersonAL, LuQ, WangJ. 2007. Role of fibrillar structure of collagenous carrier in bone sialoprotein-mediated matrix mineralization and osteoblast differentiation. Biomaterials, 28:750–761.
54.
CukiermanE, PankovR, StevensDR, YamadaKM. 2001. Taking cell-matrix adhesions to the third dimension. Science, 294:1708–1712.
