Resistin induces expression of proinflammatory cytokines and chemokines in human articular chondrocytes via transcription and messenger RNA stabilization

Abstract

Objective: To elucidate the effects of resistin on human articular chondrocytes and to generate a picture of their regulation at the transcriptional and posttranscriptional levels.

Methods: Human articular chondrocytes were cultured with resistin. Changes in gene expression were analyzed at various doses and times. Cells were also treated with the transcription inhibitor actinomycin D after resistin treatment or with the NF-kappaB inhibitor IKK-NBD before resistin treatment. Gene expression was tested by quantitative real-time polymerase chain reaction. Computational analysis for transcription factor binding motifs was performed on the promoter regions of differentially expressed genes. TC-28 chondrocytes were transfected with CCL3 and CCL4 promoter constructs, pNF-kappaB reporter, and NF-kappaB and CCAAT/enhancer binding protein beta (C/EBPbeta) expression vectors with or without resistin.

Results: Resistin-treated human articular chondrocytes increased the expression of cytokines and chemokines. Levels of messenger RNA (mRNA) for matrix metalloproteinase 1 (MMP-1), MMP-13, and ADAMTS-4 also increased, while type II collagen alpha1 (COL2A1) and aggrecan were down-regulated. The cytokine and chemokine genes could be categorized into 3 groups according to the pattern of mRNA expression over a 24-hour time course. One pattern suggested rapid regulation by mRNA stability. The second and third patterns were consistent with transcriptional regulation. Computational analysis suggested the transcription factors NF-kappaB and C/EBPbeta were involved in the resistin-induced up-regulation. This prediction was confirmed by the cotransfection of NF-kappaB and C/EBPbeta and the IKK-NBD inhibition.

Conclusion: Resistin has diverse effects on gene expression in human chondrocytes, affecting chemokines, cytokines, and matrix genes. Messenger RNA stabilization and transcriptional up-regulation are involved in resistin-induced gene expression in human chondrocytes.

Figure 1

Response of proinflammatory cytokines, chemokines and matrix molecules to resistin. Chondrocytes from normal human knee cartilage (A, B), and chondrocytes from the preserved area of OA cartilage (C, D) were treated with various doses of resistin for 24 h. A, Genes with relative change of mRNA less than 10-fold. B, Genes with relative change of mRNA over 10-fold. Dose response of cytokines and chemokines to resistin: C, transcripts that continue increase over 100 ng/ml; D, transcripts that were stable or decrease after 100 ng/ml. Quantitative real-time PCR was performed with indicated primers (Supplemental Table 1) and the change of mRNA was normalized to GAPDH mRNA, and compared with no resistin treatment (set as 1). Each bar of (A, B) represents the mean ± S.D. from three experiments with cells from the same patient. Each bar of (C, D) represents the mean ± S.D. from three patient pools, and each was repeated three times.

Figure 2

Time course of response of cytokines, chemokines and matrix molecules to resistin. Chondrocytes from the preserved areas of cartilage from knees of OA patients were treated with 100 ng/ml of resistin for 0, 1, 4, 8, and 24 h. Quantitative real-time PCR was performed with indicated primers (Supplemental Table 1) and the change of mRNA was normalized to GAPDH mRNA. Each bar represents the mean ± S.D. of change compared with zero time (set as 1) from three patient pools, and each was repeated three times.

Figure 3

Involvement of transcriptional regulation in the expression of cytokines and chemokines to resistin. A, Resistin stimulates the activity of pNF-κB Luc reporter in TC28 human chondrocytes. Relative luciferase activity indicates the fold expression relative to the activity of zero time (set as 1) in the presence of resistin (100 ng/ml). B, The candidate C/EBPβ and NF-κB binding sites in human CCL3 (-1395) and CCL4 (-1281) constructs. C/EBPβ and IKK2 stimulate the expression of CCL3 (C) and CCL4 (D) promoter in TC28 human chondrocytes. The CCL3 and CCL4 promoter constructs were co-transfected with C/EBPβ and IKK2 expression plasmids into T/C-28a2 cells without or with resistin (100 ng/ml) 8 h (CCL4) or 24 h (CCL3). The luciferase activity of the empty vector in the absence of resistin is set as 1. Each bar represents the mean ± S.D.

Figure 4

IKK-NBD peptide inhibits the expression of cytokines and chemokines in human articular chondrocytes with resistin treatment. Human articular chondrocytes were pretreated with vehicle (DMSO), IKK-NBD peptide 100 μM or 200 μM , or IKK-NBD control peptide (100 μM) for 2 h and then exposed to resistin (100 ng/ml) (A, B, C) or IL-1β (1ng/ml) (D) for 4 h. After resistin or IL-1β treatment, total RNA was isolated, and real-time quantitative PCR was performed with indicated primers (Supplemental Table 1). Each bar represents the mean ± S.D. of change compared with resistin alone or IL-1β alone (set as 1) from three patient pools, and each was repeated three times. The p value of 100 μM IKK-NBD (^o) or 200 μM IKK-NBD (*) was compared with resistin alone based on student's t-test (^o and *, means p < 0.05; ^oo and **, means p < 0.01; ***, means p < 0.001).