Molecular analysis of chondrocytes cultured in agarose in response to dynamic compression.
BACKGROUND
Articular cartilage is exposed to high mechanical loads under normal physiological conditions and articular chondrocytes regulate the composition of cartilaginous matrix, in response to mechanical signals. However, the intracellular pathways involved in mechanotransduction are still being defined. Using the well-characterized chondrocyte/agarose model system and dynamic compression, we report protocols for preparing and characterizing constructs of murine chondrocytes and agarose, and analyzing the effect of compression on steady-state level of mRNA by RT-PCR, gene transcription by gene reporter assay, and phosphorylation state of signalling molecules by Western-blotting. The mouse model is of particular interest because of the availability of a large choice of bio-molecular tools suitable to study it, as well as genetically modified mice.


RESULTS
Chondrocytes cultured in agarose for one week were surrounded by a newly synthesized pericellular matrix, as revealed by immunohistochemistry prior to compression experiments. This observation indicates that this model system is suitable to study the role of matrix molecules and trans-membrane receptors in cellular responsiveness to mechanical stress. The chondrocyte/agarose constructs were then submitted to dynamic compression with FX-4000C Flexercell Compression Plus System (Flexcell). After clearing proteins off agarose, Western-blotting analysis showed transient activation of Mitogen-activated protein kinases (MAPK) in response to dynamic compression. After assessment by capillary electrophoresis of the quality of RNA extracted from agarose, steady-state levels of mRNA expression was measured by real time PCR. We observed an up-regulation of cFos and cJun mRNA levels as a response to compression, in accordance with the mechanosensitive character observed for these two genes in other studies using cartilage explants submitted to compression. To explore further the biological response of mouse chondrocytes to the dynamic compression at the transcriptional level, we also developed an approach for monitoring changes in gene transcription in agarose culture by using reporter promoter constructs. A decrease in promoter activity of the gene coding for type II procollagen, the most abundant protein in cartilage, was observed in response to dynamic loading.


CONCLUSIONS
The protocols developed here offer the possibility to perform an integrated analysis of the molecular mechanisms of mechanotransduction in chondrocytes, at the gene and protein level.
Bougault
Carole
C
UMR 5086, CNRS, Université de Lyon, IFR 128, IBCP, Institut de Biologie et Chimie des Protéines, 7 passage du Vercors F-69367 Lyon FRANCE. c.bougault@ibcp.fr
Paumier
Anne
A
Aubert-Foucher
Elisabeth
E
Mallein-Gerin
Frédéric
F
D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D002478:Cells, Cultured; D019902:Chondrocytes / Q000502:physiology*; D024043:Collagen Type II / Q000502:physiology*; D019245:Compressive Strength / Q000502:physiology; D004548:Elasticity; D005786:Gene Expression Regulation / Q000502:physiology*; D040542:Mechanotransduction, Cellular / Q000502:physiology*; D051379:Mice; D010770:Phosphotransferases / Q000502:physiology*; D011518:Proto-Oncogene Proteins / Q000502:physiology*; D012685:Sepharose / Q000737:chemistry; D013314:Stress, Mechanical
C447413:Col2a1 protein, mouse; D024043:Collagen Type II; D011518:Proto-Oncogene Proteins; D012685:Sepharose; D010770:Phosphotransferases
J Biol Chem. 2000 Sep 1;275(35):27421-38
10856292
J Orthop Res. 1992 Sep;10(5):610-20
1500975
Cell. 1982 Aug;30(1):215-24
7127471
J Cell Biol. 1990 Jul;111(1):257-60
2365733
Arch Biochem Biophys. 2003 Sep 1;417(1):105-11
12921786
J Biol Chem. 2003 Dec 19;278(51):50940-8
12952976
Biochim Biophys Acta. 1998 Oct 21;1405(1-3):67-77
9784610
J Rheumatol. 2007 Mar;34(3):556-62
17309123
J Orthop Res. 1989;7(5):619-36
2760736
Arthritis Rheum. 2005 Aug;52(8):2386-95
16052587
Arthritis Rheum. 2003 Mar;48(3):689-99
12632422
Osteoarthritis Cartilage. 2003 May;11(5):343-50
12744940
J Biomech. 2006;39(8):1489-97
15990101
J Orthop Res. 2001 Jul;19(4):729-37
11518285
Matrix Biol. 1994 Aug;14(4):329-35
7827756
J Cell Sci. 1998 Mar;111 ( Pt 5):573-83
9454731
J Biol Chem. 2002 Sep 13;277(37):33545-58
12082094
Matrix Biol. 1999 Aug;18(4):391-9
10517186
Connect Tissue Res. 1994;31(1):11-21
15609618
J Cell Sci. 1995 Apr;108 ( Pt 4):1497-508
7615670
Biomech Model Mechanobiol. 2007 Jan;6(1-2):113-25
16691412
Biochem Biophys Res Commun. 2006 Jun 16;344(4):1192-9
16650379
J Biol Chem. 2006 Aug 25;281(34):24095-103
16782710
Arthritis Res Ther. 2006;8(4):R135
16872525
Matrix Biol. 2006 Aug;25(6):323-31
16697175
J Orthop Res. 2008 Jun;26(6):847-53
18186131
Arthritis Rheum. 2003 Oct;48(10):2865-72
14558092
Tissue Eng. 2003 Aug;9(4):597-611
13678439
J Biol Chem. 2004 May 7;279(19):19502-11
14960571