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Induction of CEMIP in Chondrocytes by Inflammatory Cytokines: Underlying Mechanisms and Potential Involvement in Osteoarthritis

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Induction of CEMIP in Chondrocytes by Inflammatory Cytokines: Underlying Mechanisms and Potential Involvement in Osteoarthritis International Journal of Molecular Sciences Article Induction of CEMIP in Chondrocytes by Inflammatory Cytokines: Underlying Mechanisms and Potential Involvement in Osteoarthritis Takashi Ohtsuki 1 , Omer F. Hatipoglu 1, Keiichi Asano 2, Junko Inagaki 3, Keiichiro Nishida 4 and Satoshi Hirohata 1,* 1 Department of Medical Technology, Graduate School of Health Sciences, Okayama University, Okayama 700-8558, Japan; [email protected] (T.O.); [email protected] (O.F.H.) 2 Department of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama 700-8558, Japan; [email protected] 3 Department of Cell Chemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama 700-8558, Japan; [email protected] 4 Department of Orthopaediac Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1,Shikata-cho, Kita-ku, Okayama 700-8558, Japan; [email protected] * Correspondence: [email protected]; Tel.: +81-86-235-6897 Received: 30 March 2020; Accepted: 27 April 2020; Published: 29 April 2020 Abstract: In patients with osteoarthritis (OA), there is a decrease in both the concentration and molecular size of hyaluronan (HA) in the synovial fluid and cartilage. Cell migration-inducing hyaluronidase 1 (CEMIP), also known as hyaluronan (HA)-binding protein involved in HA depolymerization (HYBID), was recently reported as an HA depolymerization-related molecule expressed in the cartilage of patients with OA. However, the underlying mechanism of CEMIP regulation is not well understood. We found that CEMIP expression was transiently increased by interleukine-1β (IL-1β) stimulation in chondrocytic cells. We also observed that ERK activation and NF-κB nuclear translocation were involved in the induction of CEMIP by IL-1β. In addition, both administration of HA and mechanical strain attenuated the CEMIP induction in IL-1β-stimulated chondrocytes. In conclusion, we clarified the regulatory mechanism of CEMIP in chondrocytes by inflammatory cytokines and suggested the potential involvement in osteoarthritis development. Keywords: cell migration-inducing hyaluronidase 1 (CEMIP); chondrocyte; hyaluronan; mechanical strain; nuclear factor kappa B (NF-κB); osteoarthritis 1. Introduction The extracellular matrix of cartilage is composed mainly of hyaluronan (HA)–aggrecan (a major cartilage proteoglycan) network and type II collagens [1–3]. HA is a nonsulfated linear polysaccharide composed of N-acetylglucosamine and glucuronic acid [3]. HA is a major component of the synovial fluid (SF) and provides viscoelasticity retention, lubrication of joints, and cushioning against mechanical stress on articular cartilage [4]. In healthy subjects, hyaluronic acid in articular cartilage has a high molecular weight (HMW; 1200 kDa) and density (~2.5–4 mg/mL) [5]. HA assumes a helical configuration in a solution which can be attributed to hydrogen bonding between hydroxy groups along the chain [6]. As a result, HA can bind to and absorb water approximately 1000 times its own weight [7]. HMW HA-containing SF thus performs various functions such as cushioning, synovitis suppression, and elimination of reactive oxygen species [8]. On the other hand, due to degradation and/or depolymerization, the SF of osteoarthritis (OA) patients contains HA at a lower concentration Int. J. Mol. Sci. 2020, 21, 3140; doi:10.3390/ijms21093140 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 3140 2 of 15 (1–2 mg/mL) with a lower molecular weight (2–2.5 kDa) than the SF in healthy subjects [9]. Accordingly, HA degradation and/or depolymerization are closely related with the onset of OA. In a rat model of OA,Int. injection J. Mol. Sci. of2018 HA, 19, x into the joint cavity protected the cartilage, suggesting an important2 of 16 role of HA in protecting[9]. Accordingly, the joint HA degradation [10]. HA homeostasis and/or depolymeri is maintainedzation are closely through related its synthesis with the onset and of degradation OA. and/orIn depolymerization a rat model of OA, [ 11injection]. However, of HA theinto mechanismthe joint cavity of HAprotected depolymerization the cartilage, suggesting in OA is an not fully understood.important Although role of HA hyaluronidases in protecting the (HYALs), joint [10]. suchHA homeostasis as HYAL2 is and maintain HYAL1,ed through were long its synthesis thought to be key enzymesand degradation for HA depolymerizationand/or depolymerization [12], [11] short. However, interfering the mechanism RNA (siRNA)-mediated of HA depolymerization knockdown of theirin gene OA is expression not fully understood. failed to Although decrease hyaluronidases HA-depolymerizing (HYALs), such activity as HYAL2 in skin and fibroblastsHYAL1, were and OA long thought to be key enzymes for HA depolymerization [12], short interfering RNA (siRNA)- chondrocytes [13]. The cell migration-inducing hyaluronidase 1 (CEMIP), also known as hyaluronan mediated knockdown of their gene expression failed to decrease HA-depolymerizing activity in skin (HA)-bindingfibroblasts protein and OA involved chondrocytes in HA [13]. depolymerization The cell migration-inducing (HYBID; alias hyaluronidase KIAA1199), 1 (CEMIP), is another also molecule implicatedknown in theas degradationhyaluronan (HA)-binding of HA in the protein skin and involved the OA in cartilage HA depolymerization [14]. Treatment (HYBID; of these alias cells with siRNAKIAA1199), for CEMIP is dramatically another molecule abolished implicated HA in degradation the degradation [13 of]. HA However, in the skin the and mechanism the OA cartilage underlying the regulation[14]. Treatment of CEMIP of these expression cells with in siRNA arthritic for CEMIP cartilage dramatically remains unclear.abolished HA degradation [13]. InHowever, this study, the wemechanism examined underlying the induction the regulation of CEMIP of CEMIP expression expression by in inflammatory arthritic cartilage cytokines, remains unclear. regulation of CEMIP by HA and mechanical strain, and the underlying intracellular signaling In this study, we examined the induction of CEMIP expression by inflammatory cytokines, mechanismsregulation in chondrocytic of CEMIP by cells.HA and mechanical strain, and the underlying intracellular signaling mechanisms in chondrocytic cells. 2. Results 2. Results 2.1. Immunolocalization of CEMIP in OA Articular Cartilage 2.1. Immunolocalization of CEMIP in OA Articular Cartilage Serial sections of cartilage samples derived from patients with severe OA were subjected to histology withSerial Safraninsections of O cartilage staining samples and immunostaining derived from patients with with specific severe antibodiesOA were subjected for CEMIP to and histology with Safranin O staining and immunostaining with specific antibodies for CEMIP and NITEGE (aggrecan fragment cleaved by ADAMTS). In OA patients’ cartilage, the CEMIP expression NITEGE (aggrecan fragment cleaved by ADAMTS). In OA patients’ cartilage, the CEMIP expression was observedwas observed in transitional in transitional and and radial radial zones zones (Figure(Figure 11).). CEMIP-positive CEMIP-positive chondrocytes chondrocytes (Figure (Figure 1c) 1c) were locatedwere located in the in NITEGE-positive the NITEGE-positive areas areas (dark (dark brown,brown, Figure Figure 1b)1b) of of OA OA cartilage. cartilage. (a) (b) (c) FigureFigure 1. CEMIP 1. CEMIP expression expression was was examined examined inin OAOA cartilage. Serial Serial sections sections of OA of cartilage OA cartilage samples samples were subjectedwere subjected to Safranin to Safranin O staining O staining (a (),a), immunostaining immunostaining with with antibodies antibodies to NITEGE to NITEGE (b), and (b CEMIP), and CEMIP (c). Areas with severe OA show weak staining with Safranin O (a). Strong NITEGE-immunostaining (c). Areas with severe OA show weak staining with Safranin O (a). Strong NITEGE-immunostaining was shown in damaged areas in OA cartilage (b). CEMIP-positive chondrocytes were located in the was shown in damaged areas in OA cartilage (b). CEMIP-positive chondrocytes were located in the c NITEGE-positive area of OA cartilage ( ). The boxed areas in the top are shown at higher magnification at the bottom. Int. J. Mol. Sci. 2018, 19, x 3 of 16 Int. J. Mol.NITEGE-positive Sci. 2020, 21, 3140 area of OA cartilage (c). The boxed areas in the top are shown at higher3 of 15 magnification at the bottom. 2.2.2.2. E ffEffectsects of of Inflammatory Inflammatory Cytokines Cytokines on on CEMIP CEMIP mRNA mRNA Expression Expression in in Chondrocytic Chondrocytic Cells Cells WeWe examined examined the the e ffeffectsects of of various various inflammatory inflammatory cytokines cytokines on on CEMIP CEMIP mRNA mRNA expression expression in in OUMS-27OUMS-27 cells cells using using qRT-PCR. qRT-PCR. IL-1 IL-1β βsignificantly significantly induced induced CEMIP CEMIP mRNA mRNA expression expression starting starting at at 6 h6 h andand reached reached its its peak peak at at 12 12 h h (Figure (Figure2a). 2a). CEMIP CEMIP mRNA mRNA expression expression also also increased increased in in OUMS-27 OUMS-27 cells cells stimulatedstimulated by by the the combination combination of of IL-1 IL-1ββand and tumor tumor necrosis necrosis factor factor (TNF) (TNF)αα(Figure (Figure2b), 2b), compared compared to to thatthat with with IL-1 IL-1βalone.βalone. IL-6 IL-6 together together with with soluble soluble IL-6 IL-6 receptor
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