Poster presentation. Abstract: Although it is known that macrophage content in visceral adipose tissue is significantly increased in obese humans and mice, the crosstalk between macrophages and adipocytes and its impact on the expression of genes involved in inflammation that contributes to insulin resistance have not been well elucidated. In this study, we incubated human monocytes, macrophages, or U937 mononuclear cells with adipocytes in a transwell system and studied how cell interaction regulated the expression of osteopontin, a matrix glycoprotein and proinflammatory cytokine that mediates obesity-induced adipose tissue macrophage infiltration and insulin resistance. Results showed that coculture of human monocytes, macrophages or U937 cells with adipocytes led to a 3- to 5-fold increase in osteopontin production when compared to that released by independent cultures of monocytes, macrophages or U937 cells and adipocytes. Moreover, LPS or palmitic acid, which activates Toll-like receptor (TLR)4, and high glucose augmented the coculture-stimulated osteopontin secretion. Real-time PCR studies showed that coculture of U937 cells and adipocytes led to increased osteopontin mRNA in U937 cells, but not adipocytes, suggesting that adipocyte-derived soluble factors may stimulate osteopontin expression in U937 cells. This notion was supported by the observation that osteopontin expression by U937 cells was increased by 2-fold by treatment with adipocyte-conditioned medium. In our studies to explore the mechanism involved in the coculture-stimulated osteopontin expression, we found that the neutralizing antibodies against IL-6 and IL-1b, but not those against IL-1a and TNFa, effectively blocked coculture-augmented osteopontin expression, indicating that IL-6 and IL-1b may stimulate osteopontin expression in the coculture. In support of this finding, experiments showed that IL-6 and IL-1b stimulated osteopontin expression. In conclusion, this study demonstrated that the crosstalk between adipocytes and U937 cells stimulated osteopontin expression via IL-6 and IL-1b and that TLR4 activation and high glucose further enhanced the adipocyte/U937 cell interaction-stimulated osteopontin expression.
Poster presentation. Abstract:We have reported that high glucose and IFN? synergistically stimulate matrix metalloproteinase (MMP)-1, a proteinase involved in diabetic complications, by U937 mononuclear cells. We also reported that pre-exposure to high glucose and IFN? markedly increased lipopolysaccharide (LPS)-stimulated MMP-1 expression. To understand how high glucose and IFN? augment MMP-1 expression, we analyzed their effect on the expression of IFN? receptor, Toll-like receptor (TLR)4, CD14, and MD-2, a protein associated with TLR4 and conferring LPS responsiveness. Results showed that high glucose and IFN? had no effect on the expression of IFN? receptor, TLR4, and CD14, but stimulated MD-2. IFN? alone stimulated MD-2 expression by 3.4-fold (0.49 vs 0.15, MD-2/GAPDH mRNA) and high glucose alone had no significant stimulation. Interestingly, the combination of high glucose and IFN? further increased MD-2 expression by 1.7-fold as compared to the combination of normal glucose and IFN? (0.85 vs 0.49, p<0.01). These data indicate that MD-2 expression is specifically upregulated by high glucose and IFN?. More interestingly, we found that neutralizing antibody to MD-2 blocked high glucose and IFN?-stimulated MMP-1 expression. The antibody at 2.5 ?g/ml had 70% blocking while control antibody did not have significant effect. Since it is known that transcription factor AP-1 plays a crucial role in MMP-1 expression, we further determined the effect of high glucose and IFN? on the expression of c-Jun and c-Fos, two major subunits for AP-1. Results showed that high glucose and IFN? increased c-Jun expression by 1.8- and 2.1-fold, respectively, but high glucose plus IFN? increased c-Jun expression by 5.2-fold. Similarly, high glucose and IFN?increased c-Fos expression by 2.3- and 1.4-fold, respectively, but high glucose plus IFN? increased c-Fos expression by 3.6-fold. In conclusion, this study demonstrated that high glucose and IFN? stimulate MMP-1 expression in U937 cells by upregulating MD-2 and AP-1 expression.
Poster presentation. Abstract: Matrix metalloproteinases (MMPs) play a crucial role in the destabilization of atherosclerotic plaques that may lead to plaque rupture-triggered acute coronary syndrome. Although it is known that interleukin 6 (IL-6) is associated with atherosclerosis, and diabetes increases acute coronary events, the regulation of MMP expression in mononuclear cells by IL-6 and the impact of high glucose on the regulation have not been well documented. In the present study, we found that IL-6 stimulated MMP-1 expression in a concentration- and time-dependent manner in U937 mononuclear cells and high glucose further increased IL-6-stimulated MMP-1 expression by 2-fold. By comparing the stimulatory effect of IL-6 with that of lipopolysaccharide (LPS) on MMP-1 expression, we found that IL-6 was 3-fold more effective than LPS at 1 and 10 ng/ml, suggesting that IL-6 is a potent stimulator on MMP-1 expression by U937 cells. Furthermore, results showed that high glucose, IL-6 and LPS stimulated MMP-1 expression by 1.4-, 3.9-, 6.1-fold, respectively, after 24 h treatment, but the combination of high glucose, IL-6 and LPS led to a 53.3-fold increase, revealing a remarkable synergistic effect between high glucose, IL-6, and LPS on MMP-1 expression. In the studies to elucidate how high glucose and IL-6 act synergistically for MMP-1 upregulation, we found that while either high glucose or IL-6 increased c-Jun expression by 2.5-fold, the combination of high glucose and IL-6 stimulated c-Jun expression by 5-fold. In contrast, high glucose did not enhance IL-6-stimulated c-Fos expression. These results suggest that high glucose and IL-6 exert synergy on MMP-1 expression by upregulating c-Jun, an immediate early gene and a major subunit for transcription factor AP-1 that is known to play a key role in MMP-1 transcription. Our further studies showed that the extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) pathways, but not Janus kinases /signal transducer and activator of transcription (JAK/STAT)3 cascade, were involved in high glucose and IL-6-stimulated MMP-1 expression. Taken together, this study showed that high glucose and IL-6 synergistically stimulate MMP-1 expression in U937 mononuclear cells through c-Jun and ERK and JNK cascades.