title: Chondrocyte hypertrophy and apoptosis induced by GROalpha require three-dimensional interaction with the extracellular matrix and a co-receptor role of chondroitin sulfate and are associated with the mitochondrial splicing variant of cathepsin B.
abstract: CXCR2 ligands contribute to chondrocyte hypertrophy and apoptosis, important determinants in cartilage pathophysiology. We unraveled the kinetics of signaling, biochemical, transcriptional, and morphological events triggered by GROalpha in human osteoarthritic chondrocytes kept in three-dimensional culture. p38 MAPK activation was assessed with a highly sensitive ELISA. Effector caspase activation was evaluated by cleavage of a fluorogenic substrate. Gene expression of key markers of hypertrophy (MMP-13, Runx-2) and matrix synthesis (aggrecan), and of cathepsin B isoform CB(-2,3) was evaluated by real time PCR. Occurrence of the morphological markers of apoptosis was investigated by transmission electron microscopy (TEM). GROalpha led to p38 MAPK activation in passaged chondrocytes cultured in micromass but not as a high-density monolayer. This caused the downstream triggering of chondrocyte hypertrophy (MMP-13 and Runx-2 upregulation, and calcium deposition) and apoptosis/anoikis following concurrence of matrix degrading activity, and inhibition of matrix synthesis which also involved the induction of CB(-2,3). These phenomena proved to be dependent on the co-receptor role of sulfated glycosaminoglycans (sGAG) and the activation of p38 MAPK, since they were abrogated either by preincubation with soluble chondroitin-4 sulfate or p38 MAPK inhibitors. The co-receptor role of sGAG was further demonstrated by colocalization experiments of these molecules with GROalpha in the stimulated micromasses. These findings suggest that extracellular matrix exerts a regulatory role in chondrocytes differentiation, and that meaningful investigation of the effects of chemokines on chondrocyte biology requires culture conditions respectful of both the differentiated status of the chondrocytes and of their three-dimensional interaction with the extracellular matrix.
authors.affiliation: Laboratorio di Immunologia e Genetica, Istituti Ortopedici Rizzoli, Bologna, Italy.
authors.forename: Eleonora
authors.initials: E
authors.lastname: Olivotto
authors.forename: Roberta
authors.initials: R
authors.lastname: Vitellozzi
authors.forename: Patricia
authors.initials: P
authors.lastname: Fernandez
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Falcieri
authors.forename: Michela
authors.initials: M
authors.lastname: Battistelli
authors.forename: Sabrina
authors.initials: S
authors.lastname: Burattini
authors.forename: Annalisa
authors.initials: A
authors.lastname: Facchini
authors.forename: Flavio
authors.initials: F
authors.lastname: Flamigni
authors.forename: Spartaco
authors.initials: S
authors.lastname: Santi
authors.forename: Andrea
authors.initials: A
authors.lastname: Facchini
authors.forename: Rosa Maria
authors.initials: RM
authors.lastname: Borzi'
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D017398:Alternative Splicing / Q000235:genetics; D017209:Apoptosis / Q000502:physiology; D002356:Cartilage / Q000166:cytology / Q000378:metabolism; D002401:Cathepsin B / Q000235:genetics / Q000378:metabolism*; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D054360:Chemokine CXCL1; D019743:Chemokines, CXC / Q000378:metabolism* / Q000494:pharmacology; D019902:Chondrocytes / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D002809:Chondroitin Sulfates / Q000378:metabolism*; D050659:Core Binding Factor Alpha 1 Subunit / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D005109:Extracellular Matrix / Q000378:metabolism*; D006025:Glycosaminoglycans / Q000378:metabolism; D006801:Humans; D006984:Hypertrophy / Q000378:metabolism / Q000503:physiopathology; D053509:Matrix Metalloproteinase 13 / Q000378:metabolism; D008875:Middle Aged; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D015854:Up-Regulation / Q000187:drug effects / Q000502:physiology; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
chemical_list: C475829:CXCL1 protein, human; D054360:Chemokine CXCL1; D019743:Chemokines, CXC; D050659:Core Binding Factor Alpha 1 Subunit; D006025:Glycosaminoglycans; D024101:Mitochondrial Proteins; D020033:Protein Isoforms; C493709:RUNX2 protein, human; D002809:Chondroitin Sulfates; D048051:p38 Mitogen-Activated Protein Kinases; D002401:Cathepsin B; D053509:Matrix Metalloproteinase 13