Resistin Stimulates Expression of Chemokine Genes in Chondrocytes via Combinatorial Regulation of C/EBPβ and NF-κB
Abstract
:1. Introduction
2. Results and Discussion
2.1. Resistin Independently Stimulates the Expression of Chemokine Genes in Normal Chondrocytes
2.2. Resistin Stimulates the Expression of C/EBPβ in Chondrocytes
2.3. NF-κB Is Involved in the Up-Regulation of Chemokine Genes by Chondrocytes in Response to Resistin
2.4. C/EBPβ and NF-κB Co-Enhance the Expression of CCL3 and CCL4 in Human Chondrocytes in Response to Resistin
2.5. Immunohistochemistry of C/EBPβ and NF-κB
2.6. Discussion
3. Experimental Section
3.1. Materials
3.2. Cell Culture
3.3. RNA Isolation and Real Time Quantitative PCR
Gene | Primer Sequence |
---|---|
GAPDH | F(5'-ACCCAGAAGACTGTGGATGG-3'), R(5'-GAGGCAGGGATGATGTTCTG-3') |
COL2A1 | F(5'-CCCAGAGGTGACAAAGGAGA-3'), R(5'-CACCTTGGTCTCCAGAAGGA-3') |
Aggrecan | F(5'-GGCACTAGTCAACCCTTTGG-3'), R(5'-CTGAACCCTGGTAACCCTGA-3') |
C/EBPβ | F(5'-CTCGCAGGTCAAGAGCAAGG-3'), R(5'-TCGTCGCTGTGCTTGTCC-3') |
CCL3 (MIP-1α) | F(5'-GCAACCAGTTCTCTGCATCA-3'), R(5'-TGGCTGCTCGTCTCAAAGTA-3') |
CCL3L1 (LD78β) | F(5'-GTCCTCTCTGCACCACTTGC-3'), R(5'-GGAAGATGACACTGGGCTTG-3') |
CCL4 (MIP-1β) | F(5'-GCTTTTCTTACACTGCGAGGA-3'), R(5'-CCAGGATTCACTGGGATCAG-3') |
CCL20 (MIP-3α) | F(5'-GCTTTTCTTACACTGCGAGGA-3'), R(5'-CCAGGATTCACTGGGATCAG-3') |
CXCL1 (GRO-α) | F(5'-GGGAATTCACCCCAAGAAC-3'), R(5'-GATGCAGGATTGAGGCAAG-3') |
CXCL2 (GRO-β) | F(5'-TGCAGGGAATTCACCTCAAG-3'), R(5'-TCTTAACCATGGGCGATGC-3') |
CXCL3 (GRO-γ) | F(5'-ACCGAAGTCATAGCCACACTC-3'), R(5'-GGTGCTCCCCTTGTTCAGTA-3') |
CXCL6 (GCP-2) | F(5'-GTTTACGCGTTACGCTGAGAG-3'), R(5'-ACTTCCACCTTGGAGCACTG-3') |
IL-8 | F(5'-GAAGGTGCAGTTTTGCCAAG-3'), R(5'-TGTGGTCCACTCTCAATCACTC-3') |
IL-1α | F(5'-TGCCTGAGATACCCAAAACC-3'), R(5'-AACAAGTTTGGATGGGCAAC-3') |
IL-1β | F(5'-TCCAGGAGAATGACCTGAGC-3'), R(5'-GTGATCGTACAGGTGCATCG-3') |
NF-κB1 (p50) | F(5'-CCTGGATGACTCTTGGGAAA-3'), R(5'-TCAGCCAGCTGTTTCATGTC-3') |
NF-κB2 (p52) | F(5'-GAACAGCCTTGCATCTAGCC-3'), R(5'-TTTTCAGCAT GGATGTCAGC-3') |
p65 (RelA) | F(5'-TCTGCTTCCAGGTGACAGTG-3'), R(5'-GCCAGAGTTTCGGTTCACTC-3') |
c-Rel | F(5'-CGAACCCAATTTATGACAAC-3'), R(5'-TTTTGTTTCTTTGCTTTATTGC-3') |
RelB | F(5'-CTGCTTCCAGGCCTCATATC-3'), R(5'-CGCAGCTCTGATGTGTTTGT-3') |
IκBα | F(5'-GATCCGCCAGGTGAAGGG-3'), R(5'-GCAATTTCTGGCTGGTTGG-3') |
3.4. Plasmid Constructs
3.5. Transient Transfection and Luciferase Assay
3.6. Immunofluorescence
3.7. Statistical Analysis
4. Conclusions
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Zhang, Z.; Zhang, Z.; Kang, Y.; Hou, C.; Duan, X.; Sheng, P.; Sandell, L.J.; Liao, W. Resistin Stimulates Expression of Chemokine Genes in Chondrocytes via Combinatorial Regulation of C/EBPβ and NF-κB. Int. J. Mol. Sci. 2014, 15, 17242-17255. https://doi.org/10.3390/ijms151017242
Zhang Z, Zhang Z, Kang Y, Hou C, Duan X, Sheng P, Sandell LJ, Liao W. Resistin Stimulates Expression of Chemokine Genes in Chondrocytes via Combinatorial Regulation of C/EBPβ and NF-κB. International Journal of Molecular Sciences. 2014; 15(10):17242-17255. https://doi.org/10.3390/ijms151017242
Chicago/Turabian StyleZhang, Ziji, Zhiqi Zhang, Yan Kang, Changhe Hou, Xin Duan, Puyi Sheng, Linda J. Sandell, and Weiming Liao. 2014. "Resistin Stimulates Expression of Chemokine Genes in Chondrocytes via Combinatorial Regulation of C/EBPβ and NF-κB" International Journal of Molecular Sciences 15, no. 10: 17242-17255. https://doi.org/10.3390/ijms151017242