Biochemical and Biophysical Research Communications
Potential role of WISP3 (CCN6) in regulating the accumulation of reactive oxygen species
Section snippets
Materials and methods
Cell cultures. The C28I2 chondrocyte line [10] was maintained in DMEM (Invitrogen, CA) supplemented with 10% FBS in a 5% CO2 incubator at 37 °C and passaged at subconfluency. Human umbilical vein endothelial cells (HUVEC) were obtained from Cambrex (MD), maintained in culture medium provided by Cambrex, and split at subconfluency for ∼6 passages.
Expression constructs and transfection. C28I2 cells were transfected with either wild-type/mutant WISP3 expression vectors (WISP3, trp331stop, and
Reduction in endogenous WISP3 protein expression increases the levels of cellular reactive oxygen species (ROS)
Previous studies on the functional significance of WISP3 suggest that WISP3 contributes to connective tissue homeostasis. Thus we investigated the role of WISP3 in the regulation of oxidative stress, a central feature of tissue maintenance/homeostasis [13], [14], [15], [16], [17], [20], [21], [22]. Accordingly, we determined whether inhibition of endogenous WISP3 expression had any effect on cellular ROS levels. Expression of WISP3 was inhibited in the chondrocyte line C28I2 by siRNA
Discussion
Earlier observations have suggested that WISP3 regulates connective tissue specific gene expression and promotes SOD activity, among other functions [10], [11]. Such interpretations of WISP3 function led us to further explore if WISP3 has any role in the regulation of ROS levels, which modulate various cellular events during growth and metabolism, and have often been presented as a central feature in connective tissue disorders and inflammation [12], [13], [14], [15], [16], [19], [20], [21],
Acknowledgments
This work was accomplished by funding from the Arthritis Foundation to M.S. The authors thank Dr. Gourisankar Ghosh for support in protein purification and critical comments, and Dr. M Goldring for chondrocyte lines. The authors also thank Dr. Dennis Carson, the Department of Medicine and SIRA, UCSD, for providing research space. D.M. is an undergraduate student in the Department of Chemistry and Biochemistry, UCSD.
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