RESUMEN
Early insults associated with cardiac transplantation increase the immunogenicity of donor microvascular endothelial cells (ECs), which interact with recipient alloreactive memory T cells and promote responses leading to allograft rejection. Thus, modulating EC immunogenicity could potentially alter T cell responses. Recent studies have shown modulating mitochondrial fusion/fission alters immune cell phenotype. Here, we assess whether modulating mitochondrial fusion/fission reduces EC immunogenicity and alters EC-T cell interactions. By knocking down DRP1, a mitochondrial fission protein, or by using the small molecules M1, a fusion promoter, and Mdivi1, a fission inhibitor, we demonstrate that promoting mitochondrial fusion reduced EC immunogenicity to allogeneic CD8+ T cells, shown by decreased T cell cytotoxic proteins, decreased EC VCAM-1, MHC-I expression, and increased PD-L1 expression. Co-cultured T cells also displayed decreased memory frequencies and Ki-67 proliferative index. For in vivo significance, we used a novel murine brain-dead donor transplant model. Balb/c hearts pretreated with M1/Mdivi1 after brain-death induction were heterotopically transplanted into C57BL/6 recipients. We demonstrate that, in line with our in vitro studies, M1/Mdivi1 pretreatment protected cardiac allografts from injury, decreased infiltrating T cell production of cytotoxic proteins, and prolonged allograft survival. Collectively, our data show promoting mitochondrial fusion in donor ECs mitigates recipient T cell responses and leads to significantly improved cardiac transplant survival.
Asunto(s)
Trasplante de Corazón , Dinámicas Mitocondriales , Animales , Linfocitos T CD8-positivos , Células Endoteliales , Rechazo de Injerto/etiología , Rechazo de Injerto/prevención & control , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BLRESUMEN
Previously, we demonstrated neuraminidase (NEU) activity or NEU1 expression, specifically, is increased in the kidneys of lupus mice and urine of human patients with nephritis. Additionally, NEU activity mediates IL-6 secretion from lupus-prone MRL/lpr primary mouse mesangial cells (MCs) in response to an IgG mimic. IL-6 mediates glomerular inflammation and promotes tissue damage in patients and mouse strains with lupus nephritis. This study further elucidates the mechanisms by which NEU activity and NEU1 specifically mediates the release of IL-6 and other cytokines from lupus-prone MCs. We demonstrate significantly increased release of multiple cytokines and NEU activity in MRL/lpr MCs in response to serum from MRL/lpr mice (lupus serum). Inhibiting NEU activity significantly reduced secretion of three of those cytokines: IL-6, GM-CSF and MIP1α. Message levels of Il-6 and Gm-csf were also increased in response to lupus serum and reduced when NEU activity was inhibited. Neutralizing antibodies to cell-surface receptors and MAPK inhibitors in lupus serum- or LPS-stimulated MCs indicate TLR4 and p38 or ERK MAP kinase signalling play key roles in the NEU-mediated secretion of IL-6. Significantly reduced IL-6 release was observed in C57BL/6 (B6) Neu1+/+ primary MCs compared with wild-type (Neu1+/+) B6 MCs in response to lupus serum. Additional results show inhibiting NEU activity significantly increases sialic acid-containing N-glycan levels. Together, our novel observations support a role for NEU activity, and specifically NEU1, in mediating release of IL-6 from lupus-prone MCs in response to lupus serum through a TLR4-p38/ERK MAPK signalling pathway that likely includes desialylation of glycoproteins.
Asunto(s)
Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Riñón/metabolismo , Lupus Eritematoso Sistémico/metabolismo , Nefritis Lúpica/metabolismo , Células Mesangiales/fisiología , Neuraminidasa/metabolismo , Receptor Toll-Like 4/metabolismo , Animales , Células Cultivadas , Citocinas/metabolismo , Femenino , Regulación de la Expresión Génica , Humanos , Riñón/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos MRL lpr , Neuraminidasa/genética , Suero/metabolismo , Transducción de SeñalRESUMEN
The development of nephritis is a leading cause of morbidity and mortality in lupus patients. Although the general pathophysiological progression of lupus nephritis is known, the molecular mediators and mechanisms are incompletely understood. Previously, we demonstrated that the glycosphingolipid (GSL) catabolic pathway is elevated in the kidneys of MRL/lpr lupus mice and human lupus patients with nephritis. Specifically, the activity of neuraminidase (NEU) and expression of Neu1, an enzyme in the GSL catabolic pathway is significantly increased. To better understand the role and mechanisms by which this pathway contributes to the progression of LN, we analyzed the expression and effects of NEU activity on the function of MRL/lpr lupus-prone mesangial cells (MCs). We demonstrate that NEU1 and NEU3 promote IL-6 production in MES13 MCs. Neu1 expression, NEU activity, and IL-6 production are significantly increased in stimulated primary MRL/lpr lupus-prone MCs, and blocking NEU activity inhibits IL-6 production. NEU1 and NEU3 expression overlaps IgG deposits in MCs in vitro and in renal sections from nephritic MRL/lpr mice. Together, our results suggest that NEU activity mediates IL-6 production in lupus-prone MCs possibly through an IgG-receptor complex signaling pathway.
Asunto(s)
Interleucina-6/metabolismo , Nefritis Lúpica/enzimología , Células Mesangiales/enzimología , Neuraminidasa/metabolismo , Animales , Línea Celular , Proliferación Celular , Modelos Animales de Enfermedad , Femenino , Inhibidores de Glicósido Hidrolasas/farmacología , Inmunoglobulina G/farmacología , Nefritis Lúpica/sangre , Nefritis Lúpica/tratamiento farmacológico , Nefritis Lúpica/patología , Masculino , Células Mesangiales/efectos de los fármacos , Células Mesangiales/patología , Ratones Endogámicos MRL lpr , Neuraminidasa/antagonistas & inhibidores , Neuraminidasa/genética , Receptores de IgG/metabolismo , Transducción de Señal , Regulación hacia ArribaRESUMEN
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.
Asunto(s)
Glicoesfingolípidos/metabolismo , Riñón/fisiología , Nefritis/tratamiento farmacológico , Proteína Proto-Oncogénica c-fli-1/metabolismo , Receptores CXCR3/metabolismo , Subgrupos de Linfocitos T/inmunología , Linfocitos T/inmunología , Animales , Antígenos CD/metabolismo , Movimiento Celular/efectos de los fármacos , Quimiocina CXCL10/genética , Quimiocina CXCL10/metabolismo , Quimiocina CXCL9/genética , Quimiocina CXCL9/metabolismo , Regulación de la Expresión Génica , Humanos , Riñón/efectos de los fármacos , Lactosilceramidos/metabolismo , Ratones , Ratones Endogámicos MRL lpr , Ratones Noqueados , Nefritis/inmunología , Neuraminidasa/metabolismo , Proteína Proto-Oncogénica c-fli-1/genética , Receptores CXCR3/genéticaRESUMEN
Connective tissue growth factor (CTGF) is a fibrogenic cytokine that promotes fibrosis in various organs. In the heart, both cardiomyocytes (CM) and cardiac fibroblasts have been reported as a source of CTGF expression, aiding cardiac fibrosis. Although the mammalian target of rapamycin (mTOR) forms 2 distinct complexes, mTORC1 and mTORC2, and plays a central role in integrating biochemical signals for protein synthesis and cellular homeostasis, we explored its role in CTGF expression in adult feline CM. CM were stimulated with 10 µM phenylephrine (PE), 200 nM angiotensin (Ang), or 100 nM insulin for 24 hours. PE and Ang, but not insulin, caused an increase in CTGF mRNA expression with the highest expression observed with PE. Inhibition of mTOR with torin1 but not rapamycin significantly enhanced PE-stimulated CTGF expression. Furthermore, silencing of raptor and rictor using shRNA adenoviral vectors to suppress mTORC1 and mTORC2, respectively, or blocking phosphatidylinositol 3-kinase (PI3K) signaling with LY294002 (LY) or Akt signaling by dominant-negative Akt expression caused a substantial increase in PE-stimulated CTGF expression as measured by both mRNA and secreted protein levels. However, studies with dominant-negative delta isoform of protein kinase C demonstrate that delta isoform of protein kinase C is required for both agonist-induced CTGF expression and mTORC2/Akt-mediated CTGF suppression. Finally, PE-stimulated CTGF expression was accompanied with a corresponding increase in Smad3 phosphorylation and pretreatment of cells with SIS3, a Smad3 specific inhibitor, partially blocked the PE-stimulated CTGF expression. Therefore, a PI3K/mTOR/Akt axis plays a suppressive role on agonist-stimulated CTGF expression where the loss of this mechanism could be a contributing factor for the onset of cardiac fibrosis in the hypertrophying myocardium.
Asunto(s)
Factor de Crecimiento del Tejido Conjuntivo/agonistas , Factor de Crecimiento del Tejido Conjuntivo/biosíntesis , Miocitos Cardíacos/metabolismo , Serina-Treonina Quinasas TOR/biosíntesis , Angiotensinas/farmacología , Animales , Gatos , Células Cultivadas , Miocitos Cardíacos/efectos de los fármacos , Fenilefrina/farmacologíaRESUMEN
The transcription factor Fli-1, a member of the ETS family of transcription factors, is implicated in the pathogenesis of lupus disease. Reduced Fli-1 expression in lupus mice leads to decreased renal Cxcl10 mRNA levels and renal infiltrating CXCR3+ T cells that parallels reduced renal inflammatory cell infiltration and renal damage. Inflammatory chemokine CXCL10 is critical for attracting inflammatory cells expressing the chemokine receptor CXCR3. The CXCL10/CXCR3 axis plays a role in the pathogenesis of various inflammatory diseases including lupus. Our data here demonstrate that renal CXCL10 protein levels are significantly lower in Fli-1 heterozygous MRL/lpr mice compared to wild-type MRL/lpr mice. Knockdown of Fli-1 significantly reduced CXCL10 secretion in mouse and human endothelial cells, and human mesangial cells, upon LPS or TNFα stimulation. The Fli-1 inhibitor, Camptothecin, significantly reduced CXCL10 production in human monocyte cells upon interferon stimulation. Four putative Ets binding sites in the Cxcl10 promoter showed significant enrichment for FLI-1; however, FLI-1 did not directly drive transcription from the human or mouse promoters, suggesting FLI-1 may regulate CXCL10 expression indirectly. Our results also suggest that the DNA binding domain of FLI-1 is necessary for regulation of human hCXCR3 promotor activity in human T cells and interactions with co-activators. Together, these results support a role for FLI-1 in modulating the CXCL10-CXCR3 axis by directly or indirectly regulating the expression of both genes to impact lupus disease development. Signaling pathways or drugs that reduce FLI-1 expression may offer novel approaches to lupus treatment.
Asunto(s)
Células Endoteliales , Proteína Proto-Oncogénica c-fli-1 , Animales , Humanos , Ratones , Quimiocina CXCL10/genética , Quimiocina CXCL10/metabolismo , Células Endoteliales/metabolismo , Riñón/patología , Ratones Endogámicos MRL lpr , Proteína Proto-Oncogénica c-fli-1/genética , Proteína Proto-Oncogénica c-fli-1/metabolismo , Receptores CXCR3/genética , Receptores CXCR3/metabolismoRESUMEN
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.
Asunto(s)
Fibroblastos/efectos de los fármacos , Interleucina-6/inmunología , Lipopolisacáridos/farmacología , Monocitos/efectos de los fármacos , FN-kappa B/inmunología , Células Cultivadas , Fibroblastos/inmunología , Encía/citología , Humanos , Interleucina-6/genética , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/inmunología , Monocitos/inmunología , FN-kappa B/genética , Inhibidores de Proteínas Quinasas/farmacología , ARN Mensajero/inmunología , Células U937RESUMEN
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.
Asunto(s)
Adipocitos/efectos de los fármacos , Glucosa/farmacología , Interleucina-6/metabolismo , Leucocitos Mononucleares/efectos de los fármacos , Osteopontina/genética , Receptor Toll-Like 4/metabolismo , Adipocitos/citología , Adipocitos/metabolismo , Anticuerpos/farmacología , Comunicación Celular , Células Cultivadas , Técnicas de Cocultivo , Relación Dosis-Respuesta a Droga , Ensayo de Cambio de Movilidad Electroforética , Ensayo de Inmunoadsorción Enzimática , Flavonoides/farmacología , Expresión Génica/efectos de los fármacos , Humanos , Imidazoles/farmacología , Interleucina-6/genética , Interleucina-6/inmunología , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/metabolismo , Lipopolisacáridos/farmacología , Osteopontina/metabolismo , Piridinas/farmacología , Interferencia de ARN , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Factor de Transcripción AP-1/metabolismo , Células U937 , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
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.
Asunto(s)
Ácido Láctico/metabolismo , Antígeno 96 de los Linfocitos/metabolismo , Macrófagos/metabolismo , Transportadores de Ácidos Monocarboxílicos/metabolismo , FN-kappa B/metabolismo , Receptor Toll-Like 4/metabolismo , Antioxidantes/farmacología , Ensayo de Inmunoadsorción Enzimática , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/inmunología , Humanos , Ácido Láctico/inmunología , Lipopolisacáridos/inmunología , Antígeno 96 de los Linfocitos/genética , Antígeno 96 de los Linfocitos/inmunología , Macrófagos/inmunología , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/inmunología , FN-kappa B/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Receptor Toll-Like 4/inmunología , Transcripción Genética/efectos de los fármacos , Transcripción Genética/inmunología , Regulación hacia ArribaRESUMEN
Although solid-organ transplantation has evolved steadily with many breakthroughs in the past 110 y, many problems remain to be addressed, and advanced therapeutic strategies need to be considered. T-cell immunometabolism is a rapidly advancing field that has gathered much attention recently, providing ample mechanistic insight from which many novel therapeutic approaches have been developed. Applications from the field include antitumor and antimicrobial therapies, as well as for reversing graft-versus-host disease and autoimmune diseases. However, the immunometabolism of T cells remains underexplored in solid-organ transplantation. In this review, we will highlight key findings from hallmark studies centered around various metabolic modes preferred by different T-cell subtypes (categorized into naive, effector, regulatory, and memory T cells), including glycolysis, glutaminolysis, oxidative phosphorylation, fatty acid synthesis, and oxidation. This review will discuss the underlying cellular signaling components that affect these processes, including the transcription factors myelocytomatosis oncogene, hypoxia-inducible factor 1-alpha, estrogen-related receptor alpha, and sterol regulatory element-binding proteins, along with the mechanistic target of rapamycin and adenosine monophosphate-activated protein kinase signaling. We will also explore potential therapeutic strategies targeting these pathways, as applied to the potential for tolerance induction in solid-organ transplantation.
Asunto(s)
Enfermedades Autoinmunes , Enfermedad Injerto contra Huésped , Trasplante de Órganos , Glucólisis , Humanos , Trasplante de Órganos/efectos adversos , Transducción de SeñalRESUMEN
The importance of altered glycosphingolipid (GSL) metabolism is increasingly gaining attention as a characteristic of multiple chronic kidney diseases. Previously, we reported elevated levels of GSLs and neuraminidase (NEU) enzyme activity/expression in the urine or kidney of lupus patients and lupus-prone mice, and demonstrated NEU activity mediates the production of cytokines by lupus-prone mouse primary mesangial cells. This mediation occurs in part through TLR4 and p38/ERK MAPK signalling in response to lipopolysaccharide (LPS) and lupus serum (LS). However, the precise role of NEU1, the most abundant NEU in the kidney, is incompletely known. In this study, we investigated the effect of genetically reduced Neu1 levels in vitro and in vivo. Mesangial cells from non-autoimmune prone Neu1+/- C57BL/6 mice had significantly reduced NEU activity, cytokine expression and cytokine secretion in response to LS and LPS, thereby suggesting reducing Neu1 expression may reduce the inflammatory response in lupus nephritis. Disease was assessed in female B6.SLE1/2/3 lupus-prone mice with genetically reduced levels (Neu1+/-) or wild-type levels (Neu1+/+) of Neu1 from 28 to 44 weeks of age along with aged-matched C57BL/6 controls. Renal disease was unexpectedly mild in all B6.SLE1/2/3 mice despite evidence of systemic disease. B6.SLE1/2/3 Neu1+/- mice exhibited significantly reduced levels of renal NEU1 expression and changes in renal α-2,6 linked sialylated N-glycans compared to the Neu1+/+ or healthy C57BL/6 mice, but measures of renal and systemic disease were similar between the B6.SLE1/2/3 Neu1+/+ and Neu1+/- mice. We conclude that NEU1 is the NEU largely responsible for mediating cytokine release by mesangial cells, at least in vitro, but may not be involved in modulating renal GSL levels in vivo or impact onset of nephritis in lupus-prone mice. However, the effect of reduced NEU1 levels on disease may not be appreciated in the mild disease expression in our colony of B6.SLE1/2/3 mice. The impact of the altered renal sialylated N-glycan levels and potential role of NEU1 with respect to established nephritis (late disease) in lupus-prone mice bears further investigation.
Asunto(s)
Citocinas/metabolismo , Nefritis Lúpica/metabolismo , Células Mesangiales/metabolismo , Neuraminidasa/metabolismo , Animales , Modelos Animales de Enfermedad , Femenino , Riñón/efectos de los fármacos , Riñón/metabolismo , Lipopolisacáridos/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Células Mesangiales/efectos de los fármacos , Ratones , Ratones Endogámicos C57BLRESUMEN
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.
Asunto(s)
Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Glucosa/farmacología , Interleucina-6/farmacología , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Metaloproteinasa 1 de la Matriz/genética , Fagocitos/enzimología , Proteínas Proto-Oncogénicas c-jun/metabolismo , Línea Celular , Curcumina/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Lipopolisacáridos/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Metaloproteinasa 1 de la Matriz/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Monocitos/efectos de los fármacos , Monocitos/enzimología , Fagocitos/efectos de los fármacos , Simvastatina/farmacología , Inhibidores Tisulares de Metaloproteinasas/genética , Inhibidores Tisulares de Metaloproteinasas/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Glycosphingolipids (GSLs) hexosylceramides and lactosylceramides are elevated in lupus mice and human patients with nephritis. Whereas other renal diseases characterized by increased GSL levels are thought to be a result of upregulated GSL synthesis, our results suggest elevated hexosylceramides and lactosylceramides in lupus nephritis is a result of increased catabolism of ganglioside GM3 due to significantly increased neuraminidase (NEU) activity. Thus, we hypothesized GM3 would be decreased in lupus nephritis kidneys and blocking NEU activity would reduce GSLs and improve disease in lupus mice. Female MRL/lpr lupus mice were treated with water or the NEU inhibitor oseltamivir phosphate at the onset of proteinuria to block GSL catabolism. Age-matched (non-nephritic) female MRL/MpJ lupus mice served as controls. Renal GM3 levels were significantly higher in the nephritic MRL/lpr water-treated mice compared to non-nephritic MRL/MpJ mice, despite significantly increased renal NEU activity. Blocking GSL catabolism increased, rather than decreased, renal and urine GSL levels and disease was not significantly impacted. A pilot study treating MRL/lpr females with GlcCer synthase inhibitor Genz-667161 to block GSL synthesis resulted in a strong significant negative correlation between Genz-667161 dose and renal GSL hexosylceramide and GM3 levels. Splenomegaly was negatively correlated and serum IgG levels were marginally correlated with increasing Genz-667161 dose. These results suggest accumulation of renal GM3 may be due to dysregulation of one or more of the GSL ganglioside pathways and inhibiting GSL synthesis, but not catabolism, may be a therapeutic approach for treating lupus nephritis.
Asunto(s)
Glicoesfingolípidos/metabolismo , Nefritis Lúpica/tratamiento farmacológico , Nefritis Lúpica/metabolismo , Animales , Ceramidas/metabolismo , Femenino , Gangliósido G(M3)/metabolismo , Riñón/efectos de los fármacos , Riñón/metabolismo , Lactosilceramidos/metabolismo , Ratones , Ratones Endogámicos MRL lpr , Neuraminidasa/metabolismo , Oseltamivir/análogos & derivados , Oseltamivir/uso terapéutico , Ácidos Fosforosos/uso terapéutico , Proyectos Piloto , Proteinuria/tratamiento farmacológico , Proteinuria/metabolismoRESUMEN
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.
Asunto(s)
Activación Enzimática/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Lipopolisacáridos/farmacología , Metaloproteinasa 1 de la Matriz/genética , Simvastatina/farmacología , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Lipopolisacáridos/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Reacción en Cadena de la Polimerasa , Factores de Transcripción/metabolismo , Células U937RESUMEN
OBJECTIVES: Head and neck squamous cell carcinoma (HNSCC) is notoriously resistant to chemotherapy. The sphingolipid ceramide and its analogs have been demonstrated to exert antitumor activity in many cell types; however, the effectiveness of these analogs has been limited by potency and solubility. This study focuses on the effects of novel highly soluble cationic pyridinium-ceramides, alone and in combination with various chemotherapeutic agents, on cell survival, telomerase activity, and cell cycle arrest in HNSCC cell lines in vitro. METHODS: The concentration of pyridinium-ceramides and chemotherapeutic agents that inhibited cell growth by 50% (IC50) was determined by MTT cell survival assays. The cell cycle profiles were determined by flow cytometry. Telomerase activity was determined by telomerase repeat amplification protocol (TRAP) assay. RESULTS: Treatment of the human UM-SCC-22A (SCC of the hypopharynx) cells, as well as various other HNSCC cell lines, with C6-Pyr-Cer resulted in the inhibition of cell survival with an IC50 concentration of approximately 250 to 300 nM at 96 hours, whereas its IC50 was greater than 1000 nM in noncancerous Wi-38 human lung fibroblasts, and adult human epidermal keratinocytes. Moreover, treatment with C6-Pyr-Cer also resulted in cell cycle arrest in G0/G1, which correlated with a significant inhibition of telomerase activity in UM-SCC-22A cells. Additional results demonstrated that the combination of C6-Pyr-Cer with gemcitabine (GMZ) or doxorubicin (DOX), which have the lowest IC50 concentrations among various chemotherapeutic drugs in these cells, enhances the effects of these drugs in the inhibition of telomerase and cell growth. CONCLUSIONS: These data suggest that the novel C6-Pyr-Cer with high solubility and bioavailability may lead to the development of new therapeutic strategies that target telomerase for the treatment of HNSCC.
Asunto(s)
Antineoplásicos/farmacología , Carcinoma de Células Escamosas/enzimología , Carcinoma de Células Escamosas/patología , Ciclo Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Ceramidas/farmacología , Neoplasias de Cabeza y Cuello/enzimología , Neoplasias de Cabeza y Cuello/patología , Compuestos de Piridinio/farmacología , Telomerasa/efectos de los fármacos , Telomerasa/fisiología , Antineoplásicos/uso terapéutico , Carcinoma de Células Escamosas/tratamiento farmacológico , Ceramidas/uso terapéutico , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Humanos , Compuestos de Piridinio/uso terapéutico , Células Tumorales CultivadasRESUMEN
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.
Asunto(s)
Aorta , Dasatinib/farmacología , Corazón/efectos de los fármacos , Corazón/fisiología , Miocardio/patología , Presión/efectos adversos , Transporte Activo de Núcleo Celular/efectos de los fármacos , Animales , Biomarcadores/metabolismo , Movimiento Celular/efectos de los fármacos , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Constricción , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/metabolismo , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/patología , Fibrosis , Quinasa 1 de Adhesión Focal/antagonistas & inhibidores , Quinasa 1 de Adhesión Focal/metabolismo , Quinasa 2 de Adhesión Focal/antagonistas & inhibidores , Quinasa 2 de Adhesión Focal/metabolismo , Masculino , Ratones , Miocardio/metabolismo , Función Ventricular Izquierda/efectos de los fármacosRESUMEN
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.
Asunto(s)
Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Integrina beta3/metabolismo , Miocardio/metabolismo , Animales , Aorta/fisiopatología , Aorta/cirugía , Becaplermina , Western Blotting , Cardiomegalia/genética , Cardiomegalia/metabolismo , Cardiomegalia/patología , Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Colágeno/metabolismo , Constricción Patológica/fisiopatología , Fibroblastos/efectos de los fármacos , Fibroblastos/patología , Fibronectinas/metabolismo , Quinasa 2 de Adhesión Focal/metabolismo , Hipertrofia , Inmunohistoquímica , Integrina beta3/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Miocardio/patología , Presión , Proteínas Proto-Oncogénicas c-sis/farmacología , Proteína de Unión al Calcio S100A4 , Proteínas S100/metabolismoRESUMEN
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.
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Adipocitos/metabolismo , Interleucina-6/metabolismo , Leucocitos Mononucleares/metabolismo , Metaloproteinasa 1 de la Matriz/biosíntesis , Comunicación Paracrina , Receptor Toll-Like 4/metabolismo , Línea Celular , Técnicas de Cocultivo , Humanos , Obesidad/etiología , Obesidad/metabolismo , Receptor Cross-TalkRESUMEN
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.
Asunto(s)
Glucosa/farmacología , Interferón gamma/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/fisiología , Metaloproteinasa 1 de la Matriz/genética , Factor de Transcripción STAT1/metabolismo , Aterosclerosis/inmunología , Aterosclerosis/metabolismo , Aterosclerosis/fisiopatología , Angiopatías Diabéticas/inmunología , Angiopatías Diabéticas/metabolismo , Angiopatías Diabéticas/fisiopatología , Sinergismo Farmacológico , Humanos , Hiperglucemia/inmunología , Hiperglucemia/metabolismo , Hiperglucemia/fisiopatología , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Macrófagos/metabolismo , Metaloproteinasa 1 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Inhibidor Tisular de Metaloproteinasa-2/metabolismo , Células U937RESUMEN
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.