Sam68 Promotes NF-κB Activation and Apoptosis Signaling in Articular Chondrocytes during Osteoarthritis.
OBJECTIVE
To investigate the expression of Sam68 in articular cartilage of knee osteoarthritis (OA) and the relationship between Sam68 and NF-κB activation and apoptosis signaling in OA articular chondrocytes.


METHODS
Sam68 expression in normal and osteoarthritic cartilage was assessed by immunohistochemistry and RT-PCR on both meniscal/ligamentous injury (MLI)-induced OA rat model and the clinical human OA cartilage tissues. Sam68 expression in chondrocytes under tumor necrosis factor-alpha (TNF-α) stimuli was also assessed by immunoblot. Inhibiting Sam68 in chondrocytes under TNF-α stimuli was conducted using small interfering RNA (siRNA) and its influence on the expression of apoptotic marker and catabolic genes was examined by immunoblot. The mechanism of how Sam68 stimulates NF-κB activity was determined by co-immunoprecipitation and immunoblot analysis of nuclear and cytoplasmic fractions of TNF-α-treated chondrocytes for p65 and Sam68.


RESULTS
Sam68 expression was increased in OA cartilage tissues and chondrocytes under TNF-α stimuli. Inhibition of Sam68 by siRNA significantly decreased the expression of apoptotic markers (cleaved caspase-3 and cleaved PARP) in chondrocytes following TNF-α-stimulation. Sam68 knockdown suppressed Iκ-B degradation and p65 nuclear transportation in TNF-α-treated chondrocytes, indicating a suppressed NF-κB activation. Upon TNF-α exposure, the nuclear transportation of Sam68 and its interaction with p65 was detected in chondrocytes. Furthermore, Sam68 knockdown also alleviated the TNF-α-induced catabolic marker (MMP13, ADAMTS5, iNOS and IL-6) expression.


CONCLUSIONS
The highly expressed Sam68 promotes NF-κB signaling activation, catabolic gene expression and cellular apoptosis in TNF-α-treated chondrocytes, which may provide better insights into the pathophysiology of OA and a potential target for its treatment.
Department of Orthopaedics, Affiliated Hospital of Nantong University, Nantong, 226001, Jiangsu Province, China.|Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Nantong University, Nantong, 226001, Jiangsu Province, China.
Libin
L
Xu
Department of Orthopaedics, Affiliated Hospital of Nantong University, Nantong, 226001, Jiangsu Province, China.|Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Nantong University, Nantong, 226001, Jiangsu Province, China.
Chi
C
Sun
Department of Orthopaedics, Affiliated Hospital of Nantong University, Nantong, 226001, Jiangsu Province, China.|Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Nantong University, Nantong, 226001, Jiangsu Province, China.
Sihui
S
Zhang
Department of Orthopaedics, Affiliated Hospital of Nantong University, Nantong, 226001, Jiangsu Province, China.|Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Nantong University, Nantong, 226001, Jiangsu Province, China.
Xinbao
X
Xu
Department of Orthopaedics, Affiliated Hospital of Nantong University, Nantong, 226001, Jiangsu Province, China.|Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Nantong University, Nantong, 226001, Jiangsu Province, China.
Leilei
L
Zhai
Department of Orthopaedics, Affiliated Hospital of Nantong University, Nantong, 226001, Jiangsu Province, China.|Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Nantong University, Nantong, 226001, Jiangsu Province, China.
Youhua
Y
Wang
Department of Orthopaedics, Affiliated Hospital of Nantong University, Nantong, 226001, Jiangsu Province, China.|Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Nantong University, Nantong, 226001, Jiangsu Province, China.
Shitao
S
Wang
Department of Orthopaedics, Affiliated Hospital of Nantong University, Nantong, 226001, Jiangsu Province, China.|Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Nantong University, Nantong, 226001, Jiangsu Province, China.
Zhongbing
Z
Liu
Department of Orthopaedics, Traditional Chinese Medical Hospital of Nantong City, Nantong, 226001, Jiangsu Province, China.
Hongbing
H
Cheng
National Glycoengineering Research Center and State Key Laboratory of Microbial Technology, Shandong University, Jinan, 250100, Shandong Province, China.
Min
M
Xiao
Department of Orthopaedics, Affiliated Hospital of Nantong University, Nantong, 226001, Jiangsu Province, China.|Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Nantong University, Nantong, 226001, Jiangsu Province, China.
Ran
R
Tao
Department of Orthopaedics, Affiliated Hospital of Nantong University, Nantong, 226001, Jiangsu Province, China. zdm@ntu.edu.cn.|Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Nantong University, Nantong, 226001, Jiangsu Province, China. zdm@ntu.edu.cn.|Department of Pathogen Biology, Medical College, Nantong University, Nantong, 226001, Jiangsu Province, China. zdm@ntu.edu.cn.
Dongmei
http://orcid.org/0000-0002-7183-1851
D
Zhang
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D000368:Aged; D000818:Animals; D017209:Apoptosis; D002358:Cartilage, Articular / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D019902:Chondrocytes / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007719:Knee Joint / Q000378:metabolism / Q000473:pathology; D008297:Male; D008875:Middle Aged; D016328:NF-kappa B / Q000378:metabolism*; D020370:Osteoarthritis, Knee / Q000378:metabolism* / Q000473:pathology; D034741:RNA, Small Interfering / Q000235:genetics; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D004268:DNA-Binding Proteins; C489559:KHDRBS1 protein, human; D016328:NF-kappa B; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; D014409:Tumor Necrosis Factor-alpha
Apoptosis; NF-κB; Osteoarthritis; Sam68; TNF-α