FLI1 Levels Impact CXCR3 Expression and Renal Infiltration of T Cells and Renal Glycosphingolipid Metabolism in the MRL/lpr Lupus Mouse Strain.

The ETS factor Friend leukemia virus integration 1 (FLI1) is a key modulator of lupus disease expression. Overexpressing FLI1 in healthy mice results in the development of an autoimmune kidney disease similar to that observed in lupus. Lowering the global levels of FLI1 in two lupus strains (Fli1(+/-)) significantly improved kidney disease and prolonged survival. T cells from MRL/lpr Fli1(+/-) lupus mice have reduced activation and IL-4 production, neuraminidase 1 expression, and the levels of the glycosphingolipid lactosylceramide. In this study, we demonstrate that MRL/lpr Fli1(+/-) mice have significantly decreased renal neuraminidase 1 and lactosylceramide levels. This corresponds with a significant decrease in the number of total CD3(+) cells, as well as CD4(+) and CD44(+)CD62L(-) T cell subsets in the kidney of MRL/lpr Fli1(+/-) mice compared with the Fli1(+/+) nephritic mice. We further demonstrate that the percentage of CXCR3(+) T cells and Cxcr3 message levels in T cells are significantly decreased and correspond with a decrease in renal CXCR3(+) cells and in Cxcl9 and Cxcl10 expression in the MRL/lpr Fli1(+/-) compared with the Fli1(+/+) nephritic mice. Our results suggest that reducing the levels of FLI1 in MRL/lpr mice may be protective against development of nephritis in part through downregulation of CXCR3, reducing renal T cell infiltration and glycosphingolipid levels.

Different signaling mechanisms regulating IL-6 expression by LPS between gingival fibroblasts and mononuclear cells: seeking the common target.

To reduce connective tissue IL-6 level stimulated by LPS, it is essential to control IL-6 expression in both mononuclear cells and fibroblasts. However, it is unclear whether the regulatory mechanisms for both cells are similar or not. In this study, we found that signaling pathways mediating LPS-stimulated IL-6 in mononuclear U937 cells and fibroblasts were different. Furthermore, our studies showed that while LPS activated AP-1 and NFκB in U937 cells, it only activated NFκB in fibroblasts. Analysis of nuclear AP-1 subunits showed that LPS stimulated c-Fos, Fra-1 and Jun D activities in U937 cells, but not fibroblasts. The lack of ERK involvement in LPS-stimulated IL-6 in fibroblasts was further supported by the observations that simvastatin, which is known to target ERK-AP-1, failed to inhibit LPS-stimulated IL-6 by fibroblasts. Finally, we showed that targeting NFκB pathway was highly effective in inhibition of LPS-stimulated IL-6 in coculture of U937 cells and fibroblasts.

Adipocyte-mononuclear cell interaction, Toll-like receptor 4 activation, and high glucose synergistically up-regulate osteopontin expression via an interleukin 6-mediated mechanism.

Although it has been reported that osteopontin, a matrix glycoprotein and proinflammatory cytokine, mediates obesity-induced adipose tissue macrophage infiltration and insulin resistance, it remains unclear how osteopontin is up-regulated in adipose tissue in obese humans and animals. In this study, we incubated U937 mononuclear cells with adipocytes in a transwell system and studied how cell interaction regulated osteopontin expression. Results showed that coculture of U937 cells with adipocytes led to a marked increase in osteopontin production when compared with that released by independent cultures of U937 cells. Moreover, lipopolysaccharide or palmitic acid-induced TLR4 activation and high glucose further augmented the coculture-stimulated osteopontin secretion. Similar observations were made in the coculture of human primary monocytes and adipocytes. Real time PCR studies showed that coculture of U937 cells and adipocytes increased osteopontin mRNA in U937 cells, but not adipocytes, suggesting that adipocyte-derived soluble factor may stimulate osteopontin expression by U937 cells. In our studies to explore the underlying mechanism, we found that the neutralizing antibodies against interleukin (IL)-6 or IL-6 small interfering RNA transfection in adipocytes effectively inhibited coculture-stimulated osteopontin expression, suggesting that IL-6 released by adipocytes plays an essential role in the coculture-stimulated osteopontin expression by U937 cells. In conclusion, this study has demonstrated that cell interaction, TLR4 activation, and high glucose up-regulate osteopontin expression, and adipocyte-derived IL-6 played a major role in the up-regulation.

Lactate boosts TLR4 signaling and NF-kappaB pathway-mediated gene transcription in macrophages via monocarboxylate transporters and MD-2 up-regulation.

It has been shown that lactate induces insulin resistance. However, the underlying mechanisms have not been well understood. Based on our observation that lactate augments LPS-stimulated inflammatory gene expression, we proposed that lactate may enhance TLR4 signaling in macrophages, which has been shown to play an important role in insulin resistance in adipocytes. In this study, we demonstrated that lactate stimulated MD-2, a coreceptor for TLR4 signaling activation, NF-kappaB transcriptional activity, and the expression of inflammatory genes in human U937 histiocytes (resident macrophages). Similar enhancement of the inflammatory gene expression by lactate was also observed in human monocyte-derived macrophages. The essential role of MD-2 in lactate-augmented TLR4 signaling was confirmed by observation that the suppression of MD-2 expression by small interfering RNA led to significant inhibition of inflammatory gene expression. To further elucidate how lactate treatment enhances TLR4 activation, we showed that the augmentation of inflammatory gene expression by lactate was abrogated by antioxidant treatment, suggesting a critical role of reactive oxygen species in the enhancement of TLR4 activation by lactate. Finally, we showed that alpha-cyano-4-hydroxycinnamic acid, a classic inhibitor for monocarboxylate transporters, blocked lactate-augmented inflammatory gene expression and nuclear NF-kappaB activity, indicating that lactate transport through monocarboxylate transporters is required for lactate-enhanced TLR4 activation. Collectively, this study documents that lactate boosts TLR4 activation and NF-kappaB-dependent inflammatory gene expression via monocarboxylate transporters and MD-2 up-regulation.

IL-6 and high glucose synergistically upregulate MMP-1 expression by U937 mononuclear phagocytes via ERK1/2 and JNK pathways and c-Jun.

Matrix metalloproteinases (MMPs) play a pivotal role in tissue remodeling and destruction in inflammation-associated diseases such as cardiovascular disease and periodontal disease. Although it is known that interleukin (IL)-6 is a key proinflamatory cytokine, it remains unclear how IL-6 regulates MMP expression by mononuclear phagocytes. Furthermore, it remains undetermined how IL-6 in combination with hyperglycemia affects MMP expression. In the present study, we investigated the regulatory effect of IL-6 alone or in combination with high glucose on MMP-1 expression by U937 mononuclear phagocytes. We found that IL-6 is a powerful stimulator for MMP-1 expression and high glucose further augmented IL-6-stimulated MMP-1 expression. We also found that high glucose, IL-6, and lipopolysaccharide act in concert to stimulate MMP-1 expression. In the studies to elucidate underlying mechanisms, the extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) pathways were found to be required for stimulation of MMP-1 by IL-6 and high glucose. We also observed that IL-6 and high glucose stimulated the expression of c-Jun, a key subunit of AP-1 known to be essential for MMP-1 transcription. The role of c-Jun in MMP-1 expression was confirmed by the finding that suppression of c-Jun expression by RNA interference significantly inhibited MMP-1 expression. Finally, we demonstrated that similarly to U937 mononuclear phagocytes, IL-6 and high glucose also stimulated MMP-1 secretion from human primary monocytes. In conclusion, this study demonstrated that IL-6 and high glucose synergistically stimulated MMP-1 expression in mononuclear phagocytes via ERK and JNK cascades and c-Jun upregulation.

Simvastatin suppresses LPS-induced MMP-1 expression in U937 mononuclear cells by inhibiting protein isoprenylation-mediated ERK activation.

Matrix metalloproteinase (MMP) plays a crucial role in periodontal disease and is up-regulated by oral Gram-negative, pathogen-derived LPS. In this study, we reported that simvastatin, a 3-hydroxyl-3-methylglutaryl-CoA reductase inhibitor, effectively inhibited LPS-stimulated MMP-1 as well as MMP-8 and MMP-9 expression by U937 mononuclear cells. Our studies showed that the geranylgeranyl transferase inhibitor inhibited LPS-stimulated MMP-1 expression, and addition of isoprenoid intermediate geranylgeranyl pyrophosphate (GGPP) reduced the inhibitory effect of simvastatin on LPS-stimulated MMP-1 expression. We also demonstrated that simvastatin inhibited the activation of Ras and Rac, and the inhibition was abolished by addition of GGPP. The above results indicate that protein isoprenylation is involved in the regulation of MMP-1 expression by LPS and simvastatin. Moreover, we showed that simvastatin inhibited LPS-stimulated nuclear AP-1, but not NF-kappaB activity, and the inhibition was reversed by addition of GGPP. Simvastatin also inhibited LPS-stimulated ERK but not p38 MAPK and JNK. Finally, we showed that the inhibition of LPS-stimulated ERK activation by simvastatin was reversed by GGPP. Taken together, this study showed that simvastatin suppresses LPS-induced MMP-1 expression in U937 mononuclear cells by targeting protein isoprenylation-mediated ERK activation.

Dasatinib Attenuates Pressure Overload Induced Cardiac Fibrosis in a Murine Transverse Aortic Constriction Model.

Reactive cardiac fibrosis resulting from chronic pressure overload (PO) compromises ventricular function and contributes to congestive heart failure. We explored whether nonreceptor tyrosine kinases (NTKs) play a key role in fibrosis by activating cardiac fibroblasts (CFb), and could potentially serve as a target to reduce PO-induced cardiac fibrosis. Our studies were carried out in PO mouse myocardium induced by transverse aortic constriction (TAC). Administration of a tyrosine kinase inhibitor, dasatinib, via an intraperitoneally implanted mini-osmotic pump at 0.44 mg/kg/day reduced PO-induced accumulation of extracellular matrix (ECM) proteins and improved left ventricular geometry and function. Furthermore, dasatinib treatment inhibited NTK activation (primarily Pyk2 and Fak) and reduced the level of FSP1 positive cells in the PO myocardium. In vitro studies using cultured mouse CFb showed that dasatinib treatment at 50 nM reduced: (i) extracellular accumulation of both collagen and fibronectin, (ii) both basal and PDGF-stimulated activation of Pyk2, (iii) nuclear accumulation of Ki67, SKP2 and histone-H2B and (iv) PDGF-stimulated CFb proliferation and migration. However, dasatinib did not affect cardiomyocyte morphologies in either the ventricular tissue after in vivo administration or in isolated cells after in vitro treatment. Mass spectrometric quantification of dasatinib in cultured cells indicated that the uptake of dasatinib by CFb was greater that that taken up by cardiomyocytes. Dasatinib treatment primarily suppressed PDGF but not insulin-stimulated signaling (Erk versus Akt activation) in both CFb and cardiomyocytes. These data indicate that dasatinib treatment at lower doses than that used in chemotherapy has the capacity to reduce hypertrophy-associated fibrosis and improve ventricular function.

ß3 integrin in cardiac fibroblast is critical for extracellular matrix accumulation during pressure overload hypertrophy in mouse.

The adhesion receptor ß3 integrin regulates diverse cellular functions in various tissues. As ß3 integrin has been implicated in extracellular matrix (ECM) remodeling, we sought to explore the role of ß3 integrin in cardiac fibrosis by using wild type (WT) and ß3 integrin null (ß3-/-) mice for in vivo pressure overload (PO) and in vitro primary cardiac fibroblast phenotypic studies. Compared to WT mice, ß3-/- mice upon pressure overload hypertrophy for 4 wk by transverse aortic constriction (TAC) showed a substantially reduced accumulation of interstitial fibronectin and collagen. Moreover, pressure overloaded LV from ß3-/- mice exhibited reduced levels of both fibroblast proliferation and fibroblast-specific protein-1 (FSP1) expression in early time points of PO. To test if the observed impairment of ECM accumulation in ß3-/- mice was due to compromised cardiac fibroblast function, we analyzed primary cardiac fibroblasts from WT and ß3-/- mice for adhesion to ECM proteins, cell spreading, proliferation, and migration in response to platelet derived growth factor-BB (PDGF, a growth factor known to promote fibrosis) stimulation. Our results showed that ß3-/- cardiac fibroblasts exhibited a significant reduction in cell-matrix adhesion, cell spreading, proliferation and migration. In addition, the activation of PDGF receptor associated tyrosine kinase and non-receptor tyrosine kinase Pyk2, upon PDGF stimulation were impaired in ß3-/- cells. Adenoviral expression of a dominant negative form of Pyk2 (Y402F) resulted in reduced accumulation of fibronectin. These results indicate that ß3 integrin-mediated Pyk2 signaling in cardiac fibroblasts plays a critical role in PO-induced cardiac fibrosis.

TLR4 activation and IL-6-mediated cross talk between adipocytes and mononuclear cells synergistically stimulate MMP-1 expression.

Obesity is associated with increased monocyte infiltration into adipose tissue and hence increased interaction between adipocytes and monocytes. Although it has been shown that matrix metalloproteinases (MMP) play a critical role in adipose tissue development, the effect of adipocyte and monocyte interaction on MMP production remains largely unknown. Furthermore, although it has been shown that Toll-like receptor 4 (TLR4), a receptor mediating innate immune response, plays an important role in the obesity-associated inflammation and insulin resistance, the effect of TLR4 activation in coculture of adipocytes and monocytes on MMP production has not been investigated. In this study, we cocultured adipocytes with U937 mononuclear cells in a Transwell coculture system and activated TLR4 with lipopolysaccharide or palmitic acid. We found that TLR4 activation and the coculture had a synergistic effect on MMP-1 production. In our further investigation on the underlying mechanisms, it was indicated that adipocyte-derived IL-6 and TLR4 activation acted in concert to synergistically stimulate MMP-1 expression by U937 cells. Taken together, this study has uncovered a novel mechanism potentially involved in MMP-1 up-regulation in adipose tissue, which may facilitate adipose tissue development and obesity.

High glucose and interferon gamma synergistically stimulate MMP-1 expression in U937 macrophages by increasing transcription factor STAT1 activity.

Recent diabetes control and complications trial and epidemiology of diabetes interventions and complications (DCCT/EDIC) and other clinical studies have reported that glucose control in patients with diabetes leads to a significant reduction of cardiovascular events and atherosclerosis, indicating that hyperglycemia plays an essential role in cardiovascular disease in diabetic patients. Although several mechanisms by which hyperglycemia promotes atherosclerosis have been proposed, it remains unclear how hyperglycemia promotes atherosclerosis by interaction with inflammatory cytokines. To test our hypothesis that hyperglycemia interplays with interferon gamma (IFN gamma), a key factor involved in atherosclerosis, to up-regulate the expression of genes such as matrix metalloproteinases (MMPs) and cytokines that are involved in plaque destabilization, U937 macrophages cultured in medium containing either normal or high glucose were challenged with IFN gamma and the expression of MMPs and cytokines were then quantified by real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). Results showed that high glucose and IFN gamma had a synergistic effect on the expression of MMP-1, MMP-9 and IL-1 beta. High glucose also enhanced IFN gamma-induced priming effect on lipopolysaccharide (LPS)-stimulated MMP-1 secretion. Furthermore, high glucose and IFN gamma exert the synergistic effect on MMP-1 expression by enhancing STAT1 phosphorylation and STAT1 transcriptional activity. In summary, this study revealed a novel mechanism potentially involved in diabetes-promoted cardiovascular disease.

Interleukin-6 released from fibroblasts is essential for up-regulation of matrix metalloproteinase-1 expression by U937 macrophages in coculture: cross-talking between fibroblasts and U937 macrophages exposed to high glucose.

Matrix metalloproteinases (MMPs) play a key role in periodontal disease. Although it is known that macrophages and fibroblasts are co-localized and express MMPs in the diseased periodontal tissue, the effect of interaction between these two cell types on MMP expression has not been well elucidated. Furthermore although it is known that diabetes is associated with accelerated periodontal tissue destruction, it remains unknown whether hyperglycemia, a major metabolic abnormality in diabetes, regulates MMP expression by affecting the cross-talking between fibroblasts and macrophages. In this study, human gingival fibroblasts and U937 macrophages were cocultured in a two-compartment transwell culture system, and the cells were treated with normal or high glucose. We found that coculture of fibroblasts and U937 macrophages led to an augmentation of MMP-1 expression by U937 macrophages, and high glucose further enhanced this augmentation. Similar observations were also made in the coculture of fibroblasts and human primary monocytes. We also found that interleukin 6 (IL-6) released by fibroblasts was essential for the augmentation of MMP-1 expression by U937 macrophages. Furthermore our results showed that high glucose, IL-6, and lipopolysaccharide had a synergistic effect on MMP-1 expression. Finally our study indicated that MAPK pathways and activator protein-1 transcription factor were involved in the coculture- and high glucose-augmented MMP-1 expression. In conclusion, this study demonstrates that IL-6 derived from fibroblasts is essential for MMP-1 up-regulation by cross-talking between fibroblasts and U937 macrophages exposed to high glucose, revealing an IL-6-dependent mechanism in MMP-1 up-regulation.

Rapid shortening of telomere length in response to ceramide involves the inhibition of telomere binding activity of nuclear glyceraldehyde-3-phosphate dehydrogenase.

Ceramide has been demonstrated as one of the upstream regulators of telomerase activity. However, the role for ceramide in the control of telomere length remains unknown. It is shown here that treatment of the A549 human lung adenocarcinoma cells with C(6)-ceramide results in rapid shortening of telomere length. During the examination of ceramide-regulated telomere-binding proteins, nuclear glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified to associate with both single- and double-stranded telomeric DNA with high specificity in vitro. The association of nuclear GAPDH with telomeres in interphase nuclei was also demonstrated by co-fluorescence in situ hybridization and chromatin immunoprecipitation analysis. Further data demonstrated that the nuclear localization of GAPDH is regulated by ceramide in a cell cycle-dependent manner parallel with the inhibition of its telomere binding activity in response to ceramide. In addition, the results revealed that nuclear GAPDH is distinct from its cytoplasmic isoform and that telomere binding function of nuclear GAPDH is strikingly higher than the cytoplasmic isoform. More importantly, the functional role for nuclear GAPDH in the maintenance and/or protection of telomeric DNA was identified by partial inhibition of the expression of GAPDH using small interfering RNA, which resulted in rapid shortening of telomeres. In contrast, overexpression of nuclear GAPDH resulted in the protection of telomeric DNA in response to exogenous ceramide as well as in response to anticancer drugs, which have been shown to induce endogenous ceramide levels. Therefore, these results demonstrate a novel function for nuclear GAPDH in the maintenance and/or protection of telomeres and also show that mechanisms of the rapid degradation of telomeres in response to ceramide involve the inhibition of the telomere binding activity of nuclear GAPDH.