J Korean Med Sci.  2006 Feb;21(1):100-106. 10.3346/jkms.2006.21.1.100.

Evidence for Cyclooxygenase-2 Association with Caveolin-3 in Primary Cultured Rat Chondrocytes

• Kwak JO
• Lee WK
• Kim HW
• Jung SM
• Oh KJ
• Jung SY
• Huh YH
• Cha SH

• KMID: 2157791
• DOI: http://doi.org/10.3346/jkms.2006.21.1.100

Abstract

The purpose of this study was to demonstrate the cellular localization of cyclooxygenase-2 (COX-2) and caveolin-3 (Cav-3) in primarily cultured rat chondrocytes. In normal rat chondrocytes, we observed relatively high levels of Cav-3 and a very low level of COX-2 mRNA and protein. Upon treating the chondrocytes with 5 microM of CdCl2 (Cd) for 6 hr, the expressions of COX-2 mRNA and protein were increased with the decreased Cav-3 mRNA and protein expressions. The detergent insoluble caveolae-rich membranous fractions that were isolated from the rat chondrocytes and treated with Cd contained the both proteins of both COX-2 and Cav-3 in a same fraction. The immuno-precipitation experiments showed complex formation between the COX-2 and Cav-3 in the rat chondrocytes. Purified COX-2 with glutathione S-transferase-fused COX-2 also showed complex formation with Cav-3. Confocal and electron microscopy also demonstrated the co-localization of COX-2 and Cav-3 in the plasma membrane. The results from our current study show that COX-2 and Cav-3 are co-localized in the caveolae of the plasma membrane, and they form a protein-protein complex. The co-localization of COX-2 with Cav-3 in the caveolae suggests that the caveolins might play an important role for regulating the function of COX-2.

Keyword

Chondrocytes; Cyclooxygenase 2; Caveolins; Microscopy, Confocal; Microscopy, El

MeSH Terms

Animals
Animals, Newborn
Blotting, Western
Cadmium Chloride/pharmacology
Caveolae/drug effects/metabolism/ultrastructure
Caveolin 3/*genetics/metabolism
Cell Membrane/drug effects/metabolism
Cells, Cultured
Chondrocytes/cytology/drug effects/*metabolism
Cyclooxygenase 2/*genetics/metabolism
Gene Expression
Immunoprecipitation
Microscopy, Confocal
Microscopy, Electron
RNA, Messenger/genetics/metabolism
Rats
Reverse Transcriptase Polymerase Chain Reaction

Figure

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  Fig. 1 Morphology and characterization of primarily cultured rat chondrocytes. (A) Light microscopy of confluently grown chondrocytes. (B) Messenger RNA expression of type II collagen and aggrecan in the cultured rat chondrocytes. Total RNA (500 ng) isolated from rat chondrocytes was reverse transcribed and polymerase chain reaction was performed (35 cycles). PCR products were separated using 1% agarose gel and stained with ethidium bromide. (C) Isolated membrane protein (30 µg/lane) was separated by electrophoresis and presence of each type II collagen and aggrecan was detected with respective type II collagen and aggrecan antibodies. Data was shown typical result from 3 independent experiments. M, size marker; C, type II collagen; A, aggrecan.

• 

  Fig. 2 Expression of COX-2 and Cav-3 mRNA and protein in primarily cultured rat chondrocytes. Cells were changed with medium containing 0.5% FBS in order to exclude serum effect prior to 12 hr of Cd treatment. Then, cells were treated with 5 µM of CdCl2 for 6 hr. After treatment, cells were collected with Tri Reagent for RT-PCR and with lysis buffer for Western blot analysis. The conditions of RT-PCR and Western blotting were same as those of Fig. 1. The COX-2 and Cav-3 antibodies (in Box). Data was shown typical result from 3 independent experiments. ctl; control, Cd; cadmium treated.

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  Fig. 3 Western blot analysis of COX-2 and Cav-3 in caveolae-rich membrane fraction. Fractions numbered 4 and 5 are the detergent resistant caveolae-rich fractions obtained by sucrose density gradient centrifugations and the fractions numbered 9 to 12 are the detergent soluble membrane fractions. Fractions obtained from sucrose density gradient centrifugation of detergent solubilized rat chondrocytes were subjected to Western blot analysis with respective antibodies. The result was revealed from 2 separate experiments.

• 

  Fig. 4 Complex formation between COX-2 and Cav-3 in the rat chondrocytes. Cell lysates and purified GST fusion proteins (incubated for 2 hr at 37℃ were immunoprecipitated initially with antibodies of COX-2 or Cav-3 and the respective immunoprecipitated (IP) proteins (15 µg) were loaded onto each lane of a 10% SDS-polyacrylamide gel. After separation of the proteins by electrophoresis and transferring the separated proteins to a membrane, the membrane was immunoblotted (IB) using the indicated respective antibodies. Cell lysates were immunoprecipitated initially with COX-2 antibody (A) or Cav-3 antibody (B). The immunoprecipitational result for GST fusion proteins was shown in (C).

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  Fig. 5 Confocal and electron microscopy of rat chondrocytes immuno-cytochemically stained with COX-2 and caveolin antibodies. Cells were double stained with fluorescein isothiocyanate- and Texas Red-conjugated secondary antibodies after an initial treatment with either Cav-3 or COX-2 antibodies separately and then examined under a confocal microscope at their respective wavelengths. For each experiment, a least 50 cells were examined and the presented images represent typical staining pattern for the majority of examined cells. a, Cav-3; b, COX-2; c, merged image of a and b; d and e; stained only with secondary antibodies without the prior treatment of either Cav-3 or COX-2 antibodies (A). Cells were double stained with different size of gold particle-conjugated secondary antibodies after an initial treatment with either Cav-3 or COX-2 antibodies separately and then examined under an electron microscope. Arrow showed structure of caveolae. # and * represented COX-2 (15 nm particles) and Cav-3 (10 nm particles), respectively.

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