title: Collagen-induced expression of collagenase-3 by primary chondrocytes is mediated by integrin &alpha;1 and discoidin domain receptor 2: a protein kinase C-dependent pathway.
abstract: OBJECTIVE
  To investigate whether maintaining the chondrocyte's native pericellular matrix prevents collagen-induced up-regulation of collagenase-3 (MMP-13) and whether integrin α1 (ITGα1) and/or discoidin domain receptor 2 (DDR2) modulate MMP-13 expression and which signalling pathway plays a role in collagen-stimulated MMP-13 expression.
  
  
  METHODS
  Goat articular chondrocytes and chondrons were cultured on collagen coatings. Small interfering RNA (siRNA) oligonucleotides targeted against ITGα1 and DDR2 were transfected into primary chondrocytes. Chemical inhibitors for mitogen-activated protein kinase kinase (MEK1) (PD98059), focal adhesion kinase (FAK) (FAK inhibitor 14), mitogen-activated protein kinase 8 (JNK) (SP600125) and protein kinase C (PKC) (PKC412), and a calcium chelator (BAPTA-AM) were used in cell cultures. Real-time PCR was performed to examine gene expression levels of MMP-13, ITGα1 and DDR2 and collagenolytic activity was determined by measuring the amount of hydroxyproline released in the culture medium.
  
  
  RESULTS
  Maintaining the chondrocyte's native pericellular matrix prevented MMP-13 up-regulation and collagenolytic activity when the cells were cultured on a collagen coating. Silencing of ITGα1 and DDR2 reduced MMP-13 gene expression and collagenolytic activity by primary chondrocytes cultured on collagen. Incubation with the PKC inhibitor strongly reduced MMP-13 gene expression levels. Gene expression levels of MMP-13 were also decreased by chondrocytes incubated with the MEK, FAK or JNK inhibitor.
  
  
  CONCLUSIONS
  Maintaining the native pericellular matrix of chondrocytes prevents collagen-induced up-regulation of MMP-13. Both ITGα1 and DDR2 modulate MMP-13 expression after direct contact between chondrocytes and collagen. PKC, FAK, MEK and JNK are involved in collagen-stimulated expression of MMP-13.
authors.affiliation: Department of Oral Cell Biology, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam, The Netherlands.
authors.forename: Lucienne A
authors.initials: LA
authors.lastname: Vonk
authors.forename: Behrouz Z
authors.initials: BZ
authors.lastname: Doulabi
authors.forename: ChunLing
authors.initials: C
authors.lastname: Huang
authors.forename: Marco N
authors.initials: MN
authors.lastname: Helder
authors.forename: Vincent
authors.initials: V
authors.lastname: Everts
authors.forename: Ruud A
authors.initials: RA
authors.lastname: Bank
mesh_terms: D000818:Animals; D019902:Chondrocytes / Q000378:metabolism*; D000070818:Discoidin Domain Receptors; D005260:Female; D006041:Goats; D039423:Integrin alpha1 / Q000235:genetics / Q000378:metabolism; D053509:Matrix Metalloproteinase 13 / Q000096:biosynthesis / Q000235:genetics*; D011493:Protein Kinase C / Q000378:metabolism*; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics* / Q000378:metabolism; D011975:Receptors, Mitogen / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction; D015854:Up-Regulation
chemical_list: D039423:Integrin alpha1; D011975:Receptors, Mitogen; D000070818:Discoidin Domain Receptors; D020794:Receptor Protein-Tyrosine Kinases; D011493:Protein Kinase C; D053509:Matrix Metalloproteinase 13