Kamala P. Sundararaj

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 C6-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.

Lactate Boosts TLR4 Signaling and NF-κB 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-κB 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 α-cyano-4-hydroxycinnamic acid, a classic inhibitor for monocarboxylate transporters, blocked lactate-augmented inflammatory gene expression and nuclear NF-κB 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-κB-dependent inflammatory gene expression via monocarboxylate transporters and MD-2 up-regulation.

Potent Antitumor Activity of a Novel Cationic Pyridinium-Ceramide Alone or in Combination with Gemcitabine against Human Head and Neck Squamous Cell Carcinomas in Vitro and in Vivo

In this study, a cationic water-soluble ceramide analog l-threo-C6-pyridinium-ceramide-bromide (l-t-C6-Pyr-Cer), which exhibits high solubility and bioavailability, inhibited the growth of various human head and neck squamous cell carcinoma (HNSCC) cell lines at low IC50 concentrations, independent of their p53 status. Consistent with its design to target negatively charged intracellular compartments, l-t-C6-Pyr-Cer accumulated mainly in mitochondria-, and nuclei-enriched fractions upon treatment of human UM-SCC-22A cells [human squamous cell carcinoma (SCC) of the hypopharynx] at 1 to 6 h. In addition to its growth-inhibitory function as a single agent, the supra-additive interaction of l-t-C6-Pyr-Cer with gemcitabine (GMZ), a chemotherapeutic agent used in HNSCC, was determined using isobologram studies. Then, the effects of this ceramide, alone or in combination with GMZ, on the growth of UM-SCC-22A xenografts in SCID mice was assessed following the determination of preclinical parameters, such as maximum tolerated dose, clearance from the blood, and bioaccumulation. Results demonstrated that treatment with l-t-C6-Pyr-Cer in combination with GMZ significantly prevented the growth of HNSCC tumors in vivo. The therapeutic efficacy of l-t-C6-Pyr-Cer/GMZ combination against HNSCC tumors was approximately 2.5-fold better than that of the combination of 5-fluorouracil/cis-platin. In addition, liquid chromatography/mass spectroscopy analysis showed that the levels of l-t-C6-Pyr-Cer in HNSCC tumors weresignificantly higher than its levels in the liver and intestines; interestingly, the combination with GMZ increased the sustained accumulation of this ceramide by approximately 40%. Moreover, treatment with l-t-C6-Pyr-Cer/GMZ combination resulted in a significant inhibition of telomerase activity and decrease in telomere length in vivo, which are among downstream targets of ceramide.

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.

Inhibition of growth and telomerase activity by novel cationic ceramide analogs with high solubility in human head and neck squamous cell carcinoma cells.

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.

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‐κB 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.

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. J. Cell. Biochem. 110: 248–259, 2010.

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.

β3 Integrin in Cardiac Fibroblast Is Critical for Extracellular Matrix Accumulation during Pressure Overload Hypertrophy in Mouse

The adhesion receptor β3 integrin regulates diverse cellular functions in various tissues. As β3 integrin has been implicated in extracellular matrix (ECM) remodeling, we sought to explore the role of β3 integrin in cardiac fibrosis by using wild type (WT) and β3 integrin null (β3−/−) mice for in vivo pressure overload (PO) and in vitro primary cardiac fibroblast phenotypic studies. Compared to WT mice, β3−/− 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 β3−/− 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 β3−/− mice was due to compromised cardiac fibroblast function, we analyzed primary cardiac fibroblasts from WT and β3−/− 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 β3−/− 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 β3−/− cells. Adenoviral expression of a dominant negative form of Pyk2 (Y402F) resulted in reduced accumulation of fibronectin. These results indicate that β3 integrin-mediated Pyk2 signaling in cardiac fibroblasts plays a critical role in PO-induced cardiac fibrosis.

A trend of increase in periodontal interleukin‐6 expression across patients with neither diabetes nor periodontal disease, patients with periodontal disease alone, and patients with both diseases

BACKGROUND AND OBJECTIVE Epidemiological studies have established that patients with diabetes have increased prevalence and severity of periodontal disease. However, the periodontal expression of inflammatory cytokines and matrix metalloproteinases (MMPs) in diabetic patients has not been well characterized. The objective of this study was to determine the difference in the periodontal expression of MMP-1, MMP-8, interleukin-6, tumor necrosis factor-alpha and interleukin-1beta between diabetic and nondiabetic patients. MATERIAL AND METHODS Periodontal tissue specimens were collected from nine nondiabetic patients without periodontal disease (group 1), from 11 nondiabetic patients with periodontal disease (group 2) and from seven diabetic patients with periodontal disease (group 3). The expression of MMP-1, MMP-8, interleukin-6, tumor necrosis factor-alpha and interleukin-1beta was quantified using real-time polymerase chain reaction. RESULTS The nonparametric Kruskal-Wallis test showed that the difference in interleukin-6 expression among the groups was statistically significant (p = 0.04). Furthermore, the generalized Kruskal-Wallis nonparametric linear-by-linear association test showed a statistically significant trend of increase in the expression of interleukin-6 from group 1 to group 2 to group 3 (p = 0.02) and a suggestion of such a trend for MMP-1 (p = 0.05). No increase in MMP-8 expression was observed in patients in group 3 compared to patients in groups 1 and 2. Although the average expression levels of MMP-1, interleukin-1beta and tumor necrosis factor-alpha were increased from group 1 to group 3, the differences were not statistically significant. CONCLUSION A trend of increased interleukin-6 expression in periodontal tissues was observed across patients with neither diabetes nor periodontal disease, patients with periodontal disease alone, and patients with both diseases.