Abstract
Osteopontin (OPN), expressed by various immune cells, modulates both innate and adaptive immune responses. Different immune cells have shown differential expression of the two isoforms of OPN: secreted form of OPN (sOPN) and intracellular form of OPN (iOPN). However, the molecular mechanisms that control opn gene expression and the OPN isoforms produced by immune cells remain largely unknown. In this study, we demonstrate that OPN mRNA and protein expression are significantly up-regulated upon stimulation with TLR agonists in macrophages. Interestingly, we find that macrophages constitutively express the secreted form of OPN (sOPN), while the intracellular form of OPN (iOPN) is induced following the stimulation with TLR agonists. Phosphoinositide 3-kinase (PI3K), extracellular signal-regulated kinase (ERK), and c-Jun NH(2)-terminal kinase (JNK) that are activated by LPS stimulation were shown to upregulate OPN expression. In addition, chromatin immunoprecipitation (CHIP) assays showed that AP-1 binds to the proximal AP-1 site in the OPN promoter from LPS-stimulated macrophages. Mutation of the AP-1 site in OPN promoter completely ablates LPS-induced OPN promoter activation. Knockdown of c-Jun and c-Fos expression by small interfering RNA (siRNA) significantly decreases LPS-induced OPN expression. Stable cell lines with iOPN overexpression and knockdown showed that TLR-induced iOPN expression is a negative regulator for interferon-beta (IFN-beta) production. Our findings provide new insight into the transcriptional regulation of opn gene and further clarify the isoforms and functions of OPN produced by macrophages.
Highlights
Including immune responses, inflammation, tumor growth, and metastasis, bone formation, and remodeling [1, 2]
The results indicate that TLR stimulation can induce OPN expression through TLR-induced Phosphoinositide 3-kinase (PI3K), Jun NH2-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and AP-1 activation
LPS-induced OPN protein expression at 4 and 8 h after LPS stimulation was greatly decreased by JNK inhibitor SP60012 treatment and ERK inhibitor PD98089 treatment, but not by P38 kinase inhibitor SB203580 treatment (Fig. 5C). These results demonstrate that LPS-induced MAP kinases JNK and ERK activation is associated with significantly increased OPN mRNA and intracellular form of OPN (iOPN) protein expression in macrophages
Summary
Mice and Reagent—C57BL/6J mice were obtained from Joint Ventures Sipper BK Experimental Animal (Shanghai, China). The mouse OPN promoter plasmid with the AP-1 binding site mutation was constructed by two-step PCR and described previously [20]. The expression plasmids for the secreted form of OPN (sOPN) was constructed by RT-PCR with primers: OPN-F, 5Ј-CGCGAATTCCATGAGATTGGCAGTGATTTG-3Ј, and OPN-R, 5Ј-CGCGGATCCTTAGTTGACCTCAGAAGATG-3Ј; the 885-bp fragment was inserted into the mammalian expression vector pcDNA3.1/HisB (Invitrogen). Mouse macrophage cell line RAW 264.7 was obtained from the American Type Culture Collection (Manassas, VA) and cultured at 37 °C under 5% CO2 in DMEM supplemented with 10% heat-inactivated FCS, 100 units/ml penicillin, and 100 g/ml streptomycin. Overexpression and Knockdown of iOPN in Macrophages— To stably overexpress or knockdown iOPN in macrophages, RAW 264.7 cells were transfected with the iOPN expression plasmid or OPN shRNA plasmid and selected with 800 g/ml G418 (Invitrogen) for 2–3 weeks, and the cells were pooled, expanded, and used for the following experiments. The average size of the sonicated DNA fragments subjected to immunoprecipitation was 500 bp as determined by ethidium bromide gel electrophoresis
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
