Imbalance of plastin-3 levels disturbs cartilage mechanotransduction and leads to early stage osteoarthritis

INTRODUCTION: Plastin-3 (PLS3) is an F-actin-binding and -bundling protein and is consequently involved in all processes dependent on F-actin
dynamics [1]. Because the actin cytoskeleton is connected via cell surface integrins to the extracellular matrix (ECM), it is of central importance for
the transmission of both chemical and mechanical stimuli in both directions inside -out and outside-in the cell [2]. This linkage between the actin
cytoskeleton and the ECM plays a crucial role in tissue maintenance and health. In osteoarthritis (OA) an impaired mechanotransduction via the actin
cytoskeleton is regarded as an important factor [3]. The aim of our study was to characterize the impact of both Pls3 knockout (KO) and PLS3
overexpression (OE) on knee joint articular cartilage mechanotransduction in mice.
METHODS: We studied the influence of ubiquitous Pls3 KO [4] and PLS3 OE (homozygous PLS3V5 transgene) [5] on cartilage homeostasis in
comparison to the C57BL/6N (WT) mice. All experimental protocols were performed in agreement with the guidelines of the German animal protection
law and were approved by a licensing committee (Institutional review Board: “Landesamt für Natur, Umwelt und Verbraucherschutz Nordrhein Westfalen”,
#§4.19.002, §4.21.026 and #81-02.04.2019.A479). For phenotypic characterization, the knee joint articular cartilage of PLS3 OE, Pls3 KO and WT (4-
8 animals/genotype) of 6-month-old mice of both sexes were analyzed. Cartilage degeneration was determined using a modified Osteoarthritis
Research Society International (OARSI) score [6]. In addition, we isolated chondrocytes from knee joints of newborn WT, Pls3 KO and PLS3 OE
mice to analyze F-actin structures under unloaded control conditions and mechanical stimulation using the FX4000 Flexcell system. Cyclic tensil e
strain of 6 % was applied for 30 min on three consecutive days. F-actin structures and effects on the ECM formation were evaluated by
immunofluorescence and immunoblotting.
To identify proteins that are regulated depending on PLS3 levels, an unbiased proteome analysis was performed on tibial articular cartilage of 6-
month-old mice.
RESULTS SECTION: 6-month-old PLS3 OE female mice showed a significantly (p<0.05) increased OARSI score in comparison to WT mice.
Furthermore, in both PLS3 OE and Pls3 KO mice we observed a significantly (p<0.05) reduced collagen II staining in tibial articular cartilage as
well as diminished collagen II levels in protein extracts. Cyclic stretching of primary chondrocytes induced an intense PLS3 staining in
chondrocytes of both WT and PLS3 OE mice. In Pls3 KO cells, the F-actin level was reduced (p<0.05) upon mechanical stimulation while no
alterations were observed in PLS3 OE and WT cells. In addition, pro-collagen II levels were reduced (p<0.05) in PLS3 OE and Pls3 KO
chondrocytes following mechanical loading.
In WT and PLS3 OE chondrocytes, staining intensity of the mechanosensor zyxin was enhanced (p<0.05) after mechanical stimulation. In contrast,
zyxin did not show any response to mechanical loading in Pls3 KO cells. Proteome analysis of Pls3 KO and PLS3 OE mice revealed enhanced
levels of candidates involved in autophagy while PLS3 OE mice lack proteins of apoptosis.
DISCUSSION: An imbalance of PLS3 led to reduced collagen II levels in vivo and in vitro. The reduction might contribute to the early stage OA
observed in the corresponding mouse lines. Proteome analysis revealed downregulated apoptosis and upregulated autophagy pathways in PLS3 OE
and Pls3 KO mice, which might contribute to early stage OA. PLS3 levels might influence many essential OA-relevant cellular processes in
addition to mechanotransduction like endocytosis, vesicle trafficking, autophagy and apoptosis.
SIGNIFICANCE/CLINICAL RELEVANCE: PLS3 plays an important role in cartilage mechanotransduction. As altered mechanotransduction is a risk
factor for OA development PLS3 could be a novel target, in particular for mechanically induced OA subtypes.
REFERENCES:
[1] Wolff et al. (2021). Cell Mol Life Sci 78(13),5275-5301.
[2] Martino et al. (2018). Front Physiol 9,824.
[3] Blain (2009). Int J Exp Pathol 90(1),1-15.
[4] Neugebauer et al. (2018). Hum Mol Genet 27(24),4249-62.
[5] Ackermann et al. (2013). Hum Mol Genet 22(7),1328–47.
[6] Glasson et al. (2010). Osteoarthritis Cartilage 18,S17-S23.
ACKNOWLEDGEMENTS: This project was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation, FOR2722 to BW &
AN (project ID 407176282 and 384170921).