The formation of extracellular matrix during chondrogenic differentiation of mesenchymal stem cells correlates with increased levels of xylosyltransferase I.
In vitro differentiation of mesenchymal stem cells (MSCs) into chondrogenic cells and their transplantation is promising as a technique for the treatment of cartilaginous defects. But the regulation of extracellular matrix (ECM) formation remains elusive. Therefore, the objective of this study was to analyze the regulation of proteoglycan (PG) biosynthesis during the chondrogenic differentiation of MSCs. In different stages of chondrogenic differentiation, we analyzed mRNA and protein expression of key enzymes and PG core proteins involved in ECM development. For xylosyltransferase I (XT-I), we found maximum mRNA levels 48 hours after chondrogenic induction with a 5.04 +/- 0.58 (mean +/- SD)-fold increase. This result correlates with significantly elevated levels of enzymatic XT-I activity (0.49 +/- 0.03 muU/1 x 10(6) cells) at this time point. Immunohistochemical staining of XT-I revealed a predominant upregulation in early chondrogenic stages. The highly homologous protein XT-II showed 4.7-fold (SD 0.6) increased mRNA levels on day 7. To determine the differential expression of heparan sulfate (HS), chondroitin sulfate (CS), and dermatan sulfate (DS) chains, we analyzed the mRNA expression of EXTL2 (alpha-4-N-acetylhexosaminyltransferase), GalNAcT (beta-1,4-N-acetylgalactosaminyltransferase), and GlcAC5E (glucuronyl C5 epimerase). All key enzymes showed a similar regulation with temporarily downregulated mRNA levels (up to -87-fold) after chondrogenic induction. In accordance to previous studies, we observed a similar increase in the expression of PG core proteins. In conclusion, we could show that key enzymes for CS, DS, and HS synthesis, especially XT-I, are useful markers for the developmental stages of chondrogenic differentiation.
Prante
Christian
C
Institut für Laboratoriums und Transfusionsmedizin, Herz und Diabeteszentrum NRW, Universitätsklinik der Ruhr-Universität Bochum, Bad Oeynhausen, Germany.
Bieback
Karen
K
Funke
Carsten
C
Schön
Sylvia
S
Kern
Susanne
S
Kuhn
Joachim
J
Gastens
Martin
M
Kleesiek
Knut
K
Götting
Christian
C
D002454:Cell Differentiation / Q000235:genetics / Q000502:physiology*; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology / Q000378:metabolism*; D020219:Chondrogenesis / Q000235:genetics / Q000502:physiology; D005109:Extracellular Matrix / Q000235:genetics / Q000378:metabolism*; D016326:Extracellular Matrix Proteins / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression / Q000235:genetics; D006801:Humans; D007150:Immunohistochemistry; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism*; D010430:Pentosyltransferases / Q000235:genetics / Q000378:metabolism*; D011509:Proteoglycans / Q000378:metabolism; D000097608:UDP Xylose-Protein Xylosyltransferase
D016326:Extracellular Matrix Proteins; D010430:Pentosyltransferases; D011509:Proteoglycans; D000097608:UDP Xylose-Protein Xylosyltransferase