B Saitta1, J Elphingstone2, S Limfat2, R Shkhyan2, D Evseenko3. 1. Department of Orthopaedic Surgery, Keck School of Medicine of University of Southern California, Los Angeles, CA, 90033, USA; Division of Nephrology and Hypertension, Department of Medicine and USC/UKRO Kidney Research Center, Keck School of Medicine of the University of Southern California, Los Angeles, CA, 90033, USA. 2. Department of Orthopaedic Surgery, Keck School of Medicine of University of Southern California, Los Angeles, CA, 90033, USA. 3. Department of Orthopaedic Surgery, Keck School of Medicine of University of Southern California, Los Angeles, CA, 90033, USA; Stem Cell Research and Regenerative Medicine Keck School of Medicine of University of Southern California, Los Angeles, CA, 90033, USA. Electronic address: evseenko@usc.edu.
Abstract
OBJECTIVE: Upregulation of calcium/calmodulin-dependent kinase II (CaMKII) is implicated in the pathogenesis of osteoarthritis (OA) and reactivation of articular cartilage hypertrophy. However, direct inhibition of CaMKII unexpectedly augmented symptoms of OA in animal models. The role of CaMKII in OA remains unclear and requires further investigation. METHODS: Analysis of CaMKII expression was performed in normal human and OA articular chondrocytes, and signaling mechanisms were assessed in articular, fetal and Pluripotent Stem Cell (PSC)-derived human chondrocytes using pharmacological (KN93), peptide (AC3-I) and small interfering RNA (siRNA) inhibitors of CaMKII. RESULTS: Expression levels of phospho-CaMKII (pCaMKII) were significantly and consistently increased in human OA specimens. BMP2/4 activated expression of pCaMKII as well as COLII and COLX in human adult articular chondrocytes, and also increased the levels and nuclear localization of SMADs1/5/8, while TGFβ1 showed minimal or no activation of the chondrogenic program in adult chondrocytes. Targeted blockade of CaMKII with specific siRNAs decreased levels of pSMADs, COLII, COLX and proteoglycans in normal and OA adult articular chondrocytes in the presence of both BMP4 and TGFβ1. Both human fetal and PSC-derived chondrocytes also demonstrated a decrease of chondrogenic differentiation in the presence of small molecule and peptide inhibitors of CaMKII. Furthermore, immunoprecipitation for SMADs1/5/8 or 2/3 followed by western blotting for pCaMKII showed direct interaction between SMADs and pCaMKII in primary chondrocytes. CONCLUSION: Current study demonstrates a direct role for CaMKII in TGF-β and BMP-mediated responses in primary and PSC-derived chondrocytes. These findings have direct implications for tissue engineering of cartilage tissue from stem cells and therapeutic management of OA.
OBJECTIVE: Upregulation of calcium/calmodulin-dependent kinase II (CaMKII) is implicated in the pathogenesis of osteoarthritis (OA) and reactivation of articular cartilage hypertrophy. However, direct inhibition of CaMKII unexpectedly augmented symptoms of OA in animal models. The role of CaMKII in OA remains unclear and requires further investigation. METHODS: Analysis of CaMKII expression was performed in normal human and OA articular chondrocytes, and signaling mechanisms were assessed in articular, fetal and Pluripotent Stem Cell (PSC)-derived human chondrocytes using pharmacological (KN93), peptide (AC3-I) and small interfering RNA (siRNA) inhibitors of CaMKII. RESULTS: Expression levels of phospho-CaMKII (pCaMKII) were significantly and consistently increased in humanOA specimens. BMP2/4 activated expression of pCaMKII as well as COLII and COLX in human adult articular chondrocytes, and also increased the levels and nuclear localization of SMADs1/5/8, while TGFβ1 showed minimal or no activation of the chondrogenic program in adult chondrocytes. Targeted blockade of CaMKII with specific siRNAs decreased levels of pSMADs, COLII, COLX and proteoglycans in normal and OA adult articular chondrocytes in the presence of both BMP4 and TGFβ1. Both human fetal and PSC-derived chondrocytes also demonstrated a decrease of chondrogenic differentiation in the presence of small molecule and peptide inhibitors of CaMKII. Furthermore, immunoprecipitation for SMADs1/5/8 or 2/3 followed by western blotting for pCaMKII showed direct interaction between SMADs and pCaMKII in primary chondrocytes. CONCLUSION: Current study demonstrates a direct role for CaMKII in TGF-β and BMP-mediated responses in primary and PSC-derived chondrocytes. These findings have direct implications for tissue engineering of cartilage tissue from stem cells and therapeutic management of OA.
Authors: Rick B Vega; Koichi Matsuda; Junyoung Oh; Ana C Barbosa; Xiangli Yang; Eric Meadows; John McAnally; Chris Pomajzl; John M Shelton; James A Richardson; Gerard Karsenty; Eric N Olson Journal: Cell Date: 2004-11-12 Impact factor: 41.582
Authors: Bettina Keller; Tao Yang; Yuqing Chen; Elda Munivez; Terry Bertin; Bernhard Zabel; Brendan Lee Journal: PLoS One Date: 2011-01-28 Impact factor: 3.240
Authors: Dinesh Parate; Alfredo Franco-Obregón; Jürg Fröhlich; Christian Beyer; Azlina A Abbas; Tunku Kamarul; James H P Hui; Zheng Yang Journal: Sci Rep Date: 2017-08-25 Impact factor: 4.379
Authors: Ling Wu; Carolina Bluguermann; Levon Kyupelyan; Brooke Latour; Stephanie Gonzalez; Saumya Shah; Zoran Galic; Sundi Ge; Yuhua Zhu; Frank A Petrigliano; Ali Nsair; Santiago G Miriuka; Xinmin Li; Karen M Lyons; Gay M Crooks; David R McAllister; Ben Van Handel; John S Adams; Denis Evseenko Journal: Stem Cell Reports Date: 2013-12-12 Impact factor: 7.765
Authors: Steven Woods; Paul A Humphreys; Nicola Bates; Sophie Alice Richardson; Shweta Yogesh Kuba; Imogen R Brooks; Stuart A Cain; Susan J Kimber Journal: Cells Date: 2021-03-24 Impact factor: 7.666