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CaMKII inhibition in human primary and pluripotent stem cell-derived chondrocytes modulates effects of TGF? and BMP through SMAD signaling.

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 TGF?1 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 TGF?1. 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-? 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.

SUBMITTER: Saitta B

PROVIDER: S-EPMC6309757 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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CaMKII inhibition in human primary and pluripotent stem cell-derived chondrocytes modulates effects of TGFβ and BMP through SMAD signaling.

Saitta B B Elphingstone J J Limfat S S Shkhyan R R Evseenko D D

Osteoarthritis and cartilage 20180908 1

<h4>Objective</h4>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.<h4>Methods</h4>Analysis of CaMKII expression was performed in normal human and OA articular chondrocytes, and signaling mechanisms were as ...[more]

PMID: 30205161

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Project description:The present study evaluated the effects of strontium (Sr) on proliferation and differentiation of chondrocytes isolated from dairy cows, and whether Sr exerts its effects via transforming growth factor β (TGFβ) signaling. The chondrocytes were isolated from patellar cartilage from newborn Holstein bull calves (n = 3, 1 day old, 38.0 ± 2.8 kg, fasting) within 15 min after euthanasia, and treated with different concentrations of Sr (0, 0.1, 1, and 10 μg/ml, as SrCl2·6H2O). After pretreatment with or without activin receptor-like kinase 5 (ALK5) inhibitor (10 μM SB-505124) for 4 h, chondrocytes were incubated with Sr for another 4 h. Overall effects of Sr were evaluated relative to NaCl as the control. In contrast, the 1 μg/ml Sr-treated group served as the control to determine effects of preincubating with SB-505124. Western blot and qRT-PCR were used for measuring expression of proliferation-, differentiation-, and TGFβ1-responsive factors. Data were analyzed using one-way ANOVA in GraphPad Prism 7.0. Incubation with all doses of Sr increased TGFβ1/ALK5-induced SMAD3 phosphorylation, and at 10 μg/ml it inhibited ALK1-induced SMAD1/5/9 phosphorylation. Expression of mRNA and protein of the proliferation-responsive factors type Ⅱ Collagen α1 (COL2A1) and aggrecan (ACAN) was induced by Sr at 1 μg/ml. In contrast, Sr at 10 μg/ml inhibited the expression of differentiation-responsive factors type Ⅹ Collagen α1 (COL10A1) and secreted phosphoprotein 1 (SPP1), and at 1 μg/ml it had the same effect on alkaline phosphatase (ALPL) mRNA and protein levels. Cells were stained with PI/RNase Staining buffer to assess cell cycle activity using flow-cytometry. Incubation with Sr at 1 and 10 μg/ml induced an increase in the number of cells in the S-phase, leading to an increase in the proliferation index. Incubation with SB-505124 inhibited phosphorylation of SMAD3. Abundance of ACAN and COL2A1 mRNA and protein was lower when cells were pre-incubated with SB-505124. Overall, data indicated that Sr promotes proliferation and inhibits differentiation of primary chondrocytes by directing TGFβ1 signaling towards SMAD3 phosphorylation rather than SMAD1/5/9 phosphorylation. Whether these effects occur in vivo remains to be determined and could impact future application of Sr as an experimental tool in livestock.

Dataset Information

CaMKII inhibition in human primary and pluripotent stem cell-derived chondrocytes modulates effects of TGF? and BMP through SMAD signaling.

Publications

CaMKII inhibition in human primary and pluripotent stem cell-derived chondrocytes modulates effects of TGFβ and BMP through SMAD signaling.

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