| Macrophages within adipose tissue play a key role in mediating inflammatory responses in adipose tissue that are associated with obesity-related metabolic complications. As adipose tissue is an active endocrine organ, we hypothesized that adipose tissues may also secrete proteins that negatively regulate macrophage-mediated inflammation. We studied the differentiation of preadipocytes(3T3-L1 cells) with a combination of gene expression profiling and bioinformatic techniques to search for highly induced genes that encoded proteins with putative signal peptides in mature adipocytes but not preadipocytes. We found that PAMM, a CXXC-type peroxiredoxin-like 2 domain-containing redox regulatory protein, is a novel secreted protein with potent anti-inflammatory properties.PAMM was secreted from mature human adipocytes but not preadipocytes. PAMM was highly expressed in both white and brown adipose tissues and further increased in obesity status. Overexpression of PAMM significantly attenuated lipopolysaccharide(LPS)-induced macrophage inflammation. Incubation of macrophages with adipocyte-conditional medium treated with anti-PAMM antibody significantly enhanced LPS-induced IL-1β,IL-6,IL-12 and iNOS expression in Raw264.7 cells. In addition, incubation of Raw264.7 cells with purified PAMM protein had a similar anti-inflammatory effect. Moreover, forced overexpression of PAMM in Raw264.7 cells resulted in decreased LPS-induced ERK1/2, p38 and JNK phosphorylation, suggesting that PAMM exerted the anti-inflammatory function probably by suppressing the MAPK signaling pathway. A mutant of CXXC motif of PAMM lost its anti-redox activity but maintained its ability to suppress production of inflammatory cytokines in LPS-stimulated macrophages, suggesting that PAMM’s anti-inflammatory properties may be independent of its anti-oxidant properties. Our results suggest that adipocyte-derived PAMM may suppress macrophage activation by inhibiting MAPK signaling pathway.Part I: PAMM is a novel proteins secreted by adipocytesAims: To search for highly induced genes that encoded proteins with putative signal peptides in mature adipocytes but not preadipocytes,and identify that PAMM is a novel proteins secreted by adipocytes.Methods:(1) 2-day post-confluent 3T3-L1 cells were incubated with a cocktail of DMI(dexamethasone, methylxanthine, and insulin) for 15 days. The cells were stained by Oil O and the images were taken by Nikon microscope system. We performed Affymatrix microarray analysis to compare differences in gene expression profiles in differentiated adipocytes vs. undifferentiated 3T3-L1 cells. Northern blot and ELISA analysis of expression of PAMM are performed during DMI-induced 3T3-L1 differentiation.(2) HEK293 cells were transiently transfected with Flag-PAMM or Flag-MCPIP1. Whole cell lysates and culture medium were analyzed for Flag-PAMM and Flag-MCPIP1 by Western blot analysis with anti-Flag antibody.(3) Northern blot analysis of PAMM mRNA expression in mouse tissues. C57/BL6 mice at 1-month age were began to feed with high-fat diet. The white adipose tissues were collected at different time points as indicated for Northern blot analysis of PAMM mRNA.Results:(1)After incubation with a cocktail of DMI(dexamethasone, methylxanthine and insulin), 3T3-L1 cells can be fully differentiated into adipocytes. PAMM m RNA expression was upregulated by 8 folds in differentiated 3T3-L1 adipocytes vs. undifferentiated 3T3-L1 cells. PAMM is a protein with 229 amino acids that contains a CXXC type peroxiredoxin-like 2 domain. PAMM was significantly increased in the cell lysates and cultured medium after 15 days differentiation(P<0.05).(2) Both Flag-PAMM and Flag-MCPIP1 appeared in whole cell lysates, but only Flag-PAMM appeared in concentrated conditional cultured medium.(3) PAMM mRNA was also broadly expressed in virtually all tissues examined from mice, but was most highly expressed in adipose tissues. PAMM mRNA was significantly increased during high-fat diet-induced obesity.Summary:(1) PAMM, a CXXC-type peroxiredoxin-like 2 domain-containing protein, is upregulated during 3T3-L1 differentiation.(2) PAMM is a novel secreted protein from mature adipocytes.(3) PAMM expression is associated with obese.Part II: PAMM significantly inhibits the expression of inflammatory cytokines in macrophages, and study its mechanismsAims: To examined the effects of PAMM on the expression of inflammatory cytokines of macrophage, and study the mechanisms by which PAMM suppresses LPS-induced expression of inflammatory cytokines in Raw264.7 cellsMethods:(1) Raw264.7 cells were transiently transfected with Flag-PAMM expression plasmid or control plasmid. Cell lysates from transfected cells were analyzed by western blot. Transfected Raw264.7 cell were stimulated with PBS or 0.1 μg/ml LPS for 8 hours. Relative mRNA levels of IL-1β, IL-6, IL-12 and iNOS were detected by qPCR.(2) 3T3-L1 cells were incubated with a cocktail of DMI for 15 days to induce differentiation into adipocytes. The cell lysates and cultured medium were collected for western blot analysis. The conditional cultured medium from above experiments was pre-incubated with anti-PAMM(1.0 ng/ml) or control IgG for 1 hour, then added the medium into Raw264.7 cells and incubated for 1 hour. Then the cells were stimulated with PBS or 0.1 μg/ml LPS for 8 hours. Relative m RNA level of IL-1β, IL-6, IL-12 and iNOS was detected by qPCR.(3) PAMM was purified from BL21 E. coli by Akata wash. Purified PAMM was examined by Coomassie blue staining and western blot analysis. Raw264.7 cells were incubated with 0, 0.1 or 0.5 μg/ml of purified PAMM protein for 30 min and then stimulated with PBS or 0.1 μg/ml LPS for 8 hours. Relative mRNA levels of IL-1β, IL-6, IL-12 and iNOS were detected by qPCR.(4) Raw264.7 cells were transiently transfected with PAMM expression plasmid or control plasmid. After 24 hours, the transfected cells were stimulated with 100 ng/ml LPS for 0, 15 and 60 minutes. Cell lysates were extracted and Western blot was performed to detect total protein levels and the phosphorylation of ERK1/2, p38,JNK and IKKβ.(5) Raw264.7 cells were transiently transfected with PAMM or PAMMC88 G expression plasmids or control plasmid. After 24 hours, transfected cells were treated with PBS or 400 μM of H2O2 for 2 hours. The GSH/GSSG ratio was measured in the cytosolic fraction from these cells. The transfected cells were stimulated with or without 0.1 μg/ml LPS for 8 hours. Relative mRNA levels of IL-6, IL-8 and iNOS were detected by qPCR.Results:(1) PAMM overexpression in Raw264.7 cells was confirmed by Western blot, and overexpression of PAMM markedly attenuated LPS–induced mRNA expression for IL-1β, IL-6, IL-12, and iNOS(P<0.001).(2) PAMM protein could be detected in the cultured medium from mature 3T3-L1 adipocytes, and neutrolized PAMM in the cultured medium by anti-PAMM significantly increased LPS-induced IL-1β, IL-6, IL-12 and iNOS expression in Raw264.7 cells(P<0.001).(3) Purified PAMM protein dose-dependently suppressed the mRNA expression of IL-1β, IL-6, IL-12 and iNOS in LPS-stimulated Raw264.7 cells(P<0.001).(4) Overexpression of PAMM significantly attenuated LPS-induced phosphorylation of ERK1/2, p38 and JNK,but failed to suppress LPS-induced phosphorylation of IKKβ.(5) Overexpression of PAMM, but not PAMMC88 G, significantly prevented H2O2-caused decreases in the GSH/GSSG ratio. Overexpression of either PAMM,or the PAMMC88 G mutant, inhibited LPS-induced expression of IL-6 and IL-8. In contrast, PAMMC88 G failed to suppress iNOS expression as PAMM.Summary:(1) Overexpression of PAMM inhibited inflammatory genes(IL-1β, IL-6, IL-12 and iNOS)expression in Raw264.7 cells.(2) Secreted PAMM from adipocytes significantly increased LPS-induced IL-1β, IL-6, IL-12 and iNOS expression in Raw264.7 cells.(3) Purified PAMM protein inhibited inflammatory genes expression in Raw264.7 cells.(4) Overexpression of PAMM significantly attenuated LPS-induced phosphorylation of ERK1/2, p38 and JNK, suggesting PAMM suppresses inflammatory responses in LPS-stimulated macrophages by suppressing the MAPK signaling pathways.(5) PAMM has the ability of anti-oxidative stress, suggesting PAMM functions as a redox regulatory protein and that the CXXC motif is essential for its anti-redox activity. PAMM’s ability to suppress inflammatory cytokine production in LPS-stimulated macrophages is unrelated to its antioxidant properties.Conclusion:(1) PAMM, a CXXC-type peroxiredoxin-like 2 domain-containing redox regulatory protein, is a novel secreted protein from mature adipocytes.(2) PAMM’s ability to suppress inflammatory cytokine production in LPS-stimulated macrophages is related to its ability to suppress the activity of MAPK(ERK, P38 MAPK, JNK), and unrelated to its antioxidant properties. |
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