The ERK5 and ERK1/2 signaling pathways play opposing regulatory roles during chondrogenesis of adult human bone marrow-derived multipotent progenitor cells.
Adult human bone marrow-derived multipotent progenitor cells (MPCs) are able to differentiate into a variety of specialized cell types, including chondrocytes, and are considered a promising candidate cell source for use in cartilage tissue engineering. In this study, we examined the regulation of MPC chondrogenesis by mitogen-activated protein kinases in an attempt to better understand how to generate hyaline cartilage in the laboratory that more closely resembles native tissue. Specifically, we employed the high-density pellet culture model system to assess the roles of ERK5 and ERK1/2 pathway signaling in MPC chondrogenesis. Western blotting revealed that high levels of ERK5 phosphorylation correlate with low levels of MPC chondrogenesis and that as TGF-beta 3-enhanced MPC chondrogenesis proceeds, phospho-ERK5 levels steadily decline. Conversely, levels of phospho-ERK1/2 paralleled the progression of MPC chondrogenesis. siRNA-mediated knockdown of ERK5 pathway components MEK5 and ERK5 resulted in increased MPC pellet mRNA transcript levels of the cartilage-characteristic marker genes SOX9, COL2A1, AGC, L-SOX5, and SOX6, as well as enhanced accumulation of SOX9 protein, collagen type II protein, and Alcian blue-stainable proteoglycan. In contrast, knockdown of ERK1/2 pathway members MEK1 and ERK1 decreased expression of all chondrogenic markers tested. Finally, overexpression of MEK5 and ERK5 also depressed MPC chondrogenesis, as indicated by diminished activity of a co-transfected collagen II promoter-luciferase reporter construct. In conclusion, our results suggest a novel role for the ERK5 pathway as an important negative regulator of adult human MPC chondrogenesis and illustrate that the ERK5 and ERK1/2 kinase cascades play opposing roles regulating MPC cartilage formation.
Bobick
Brent E
BE
Cartilage Biology and Orthopaedics Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland 20892, USA.
Matsche
Alexander I
AI
Chen
Faye H
FH
Tuan
Rocky S
RS
D053687:Adult Stem Cells / Q000201:enzymology*; D000368:Aged; D000369:Aged, 80 and over; D001854:Bone Marrow Cells / Q000201:enzymology*; D002356:Cartilage / Q000378:metabolism; D002454:Cell Differentiation; D002478:Cells, Cultured; D020219:Chondrogenesis / Q000235:genetics; D005260:Female; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D048369:MAP Kinase Kinase 1 / Q000378:metabolism; D048671:MAP Kinase Kinase 5 / Q000378:metabolism; D020935:MAP Kinase Signaling System; D008297:Male; D008875:Middle Aged; D019950:Mitogen-Activated Protein Kinase 1 / Q000235:genetics / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000235:genetics / Q000378:metabolism*; D048054:Mitogen-Activated Protein Kinase 7 / Q000235:genetics / Q000378:metabolism*; D039902:Multipotent Stem Cells / Q000201:enzymology*; D010766:Phosphorylation; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D013234:Stem Cells / Q000201:enzymology*; D013997:Time Factors; D014162:Transfection; D053782:Transforming Growth Factor beta3 / Q000378:metabolism
D012333:RNA, Messenger; D053782:Transforming Growth Factor beta3; D016222:Fibroblast Growth Factor 2; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D048054:Mitogen-Activated Protein Kinase 7; D048369:MAP Kinase Kinase 1; D048671:MAP Kinase Kinase 5; C482563:MAP2K5 protein, human