Divergent Innate and Epithelial Functions of the RNA-Binding Protein HuR in Intestinal Inflammation

HuR is an abundant RNA-binding protein acting as a post-transcriptional regulator of many RNAs including mRNAs encoding inflammatory mediators, cytokines, death signalers and cell cycle regulators. In the context of intestinal pathologies, elevated HuR is considered to enhance the stability and the translation of pro-tumorigenic mRNAs providing the rationale for its pharmacological targeting. However, HuR also possesses specific regulatory functions for innate immunity and cytokine mRNA control which can oppose intestinal inflammation and tumor promotion. Here, we aim to identify contexts of intestinal inflammation where the innate immune and the epithelial functions of HuR converge or diverge. To address this, we use a disease-oriented phenotypic approach using mice lacking HuR either in intestinal epithelia or myeloid-derived immune compartments. These mice were compared for their responses to (a) Chemically induced Colitis; (b) Colitis- associated Cancer (CAC); (c) T-cell mediated enterotoxicity; (d) Citrobacter rodentium-induced colitis; and (e) TNF-driven inflammatory bowel disease. Convergent functions of epithelial and myeloid HuR included their requirement for suppressing inflammation in chemically induced colitis and their redundancies in chronic TNF-driven IBD and microbiota control. In the other contexts however, their functions diversified. Epithelial HuR was required to protect the epithelial barrier from acute inflammatory or infectious degeneration but also to promote tumor growth. In contrast, myeloid HuR was required to suppress the beneficial inflammation for pathogen clearance and tumor suppression. This cellular dichotomy in HuR's functions was validated further in mice engineered to express ubiquitously higher levels of HuR which displayed diminished pathologic and beneficial inflammatory responses, resistance to epithelial damage yet a heightened susceptibility to CAC. Our study demonstrates that epithelial and myeloid HuR affect different cellular dynamics in the intestine that need to be carefully considered for its pharmacological exploitation and points toward potential windows for harnessing HuR functions in intestinal inflammation

Activation of Wnt/beta-catenin signaling increases insulin sensitivity through a reciprocal regulation of Wnt10b and SREBP-1c in skeletal muscle cells.

Intramyocellular lipid accumulation is strongly related to insulin resistance in humans, and we have shown that high glucose concentration induced de novo lipogenesis and insulin resistance in murin muscle cells. Alterations in Wnt signaling impact the balance between myogenic and adipogenic programs in myoblasts, partly due to the decrease of Wnt10b protein. As recent studies point towards a role for Wnt signaling in the pathogenesis of type 2 diabetes, we hypothesized that activation of Wnt signaling could play a crucial role in muscle insulin sensitivity.Here we demonstrate that SREBP-1c and Wnt10b display inverse expression patterns during muscle ontogenesis and regeneration, as well as during satellite cells differentiation. The Wnt/beta-catenin pathway was reactivated in contracting myotubes using siRNA mediated SREBP-1 knockdown, Wnt10b over-expression or inhibition of GSK-3beta, whereas Wnt signaling was inhibited in myoblasts through silencing of Wnt10b. SREBP-1 knockdown was sufficient to induce Wnt10b protein expression in contracting myotubes and to activate the Wnt/beta-catenin pathway. Conversely, silencing Wnt10b in myoblasts induced SREBP-1c protein expression, suggesting a reciprocal regulation. Stimulation of the Wnt/beta-catenin pathway i) drastically decreased SREBP-1c protein and intramyocellular lipid deposition in myotubes; ii) increased basal glucose transport in both insulin-sensitive and insulin-resistant myotubes through a differential activation of Akt and AMPK pathways; iii) restored insulin sensitivity in insulin-resistant myotubes.We conclude that activation of Wnt/beta-catenin signaling in skeletal muscle cells improved insulin sensitivity by i) decreasing intramyocellular lipid deposition through downregulation of SREBP-1c; ii) increasing insulin effects through a differential activation of the Akt/PKB and AMPK pathways; iii) inhibiting the MAPK pathway. A crosstalk between these pathways and Wnt/beta-catenin signaling in skeletal muscle opens the exciting possibility that organ-selective modulation of Wnt signaling might become an attractive therapeutic target in regenerative medicine and to treat obese and diabetic populations

Combination of subtherapeutic anti-TNF dose with dasatinib restores clinical and molecular arthritogenic profiles better than standard anti-TNF treatment.

BACKGROUND New medications for Rheumatoid Arthritis (RA) have emerged in the last decades, including Disease Modifying Antirheumatic Drugs (DMARDs) and biologics. However, there is no known cure, since a significant proportion of patients remain or become non-responders to current therapies. The development of new mode-of-action treatment schemes involving combination therapies could prove successful for the treatment of a greater number of RA patients. METHODS We investigated the effect of the Tyrosine Kinase inhibitors (TKIs) dasatinib and bosutinib, on the human TNF-dependent Tg197 arthritis mouse model. The inhibitors were administered either as a monotherapy or in combination with a subtherapeutic dose of anti-hTNF biologics and their therapeutic effect was assessed clinically, histopathologically as well as via gene expression analysis and was compared to that of an efficient TNF monotherapy. RESULTS Dasatinib and, to a lesser extent, bosutinib inhibited the production of TNF and proinflammatory chemokines from arthritogenic synovial fibroblasts. Dasatinib, but not bosutinib, also ameliorated significantly and in a dose-dependent manner both the clinical and histopathological signs of Tg197 arthritis. Combination of dasatinib with a subtherapeutic dose of anti-hTNF biologic agents, resulted in a synergistic inhibitory effect abolishing all arthritis symptoms. Gene expression analysis of whole joint tissue of Tg197 mice revealed that the combination of dasatinib with a low subtherapeutic dose of Infliximab most efficiently restores the pathogenic gene expression profile to that of the healthy state compared to either treatment administered as a monotherapy. CONCLUSION Our findings show that dasatinib exhibits a therapeutic effect in TNF-driven arthritis and can act in synergy with a subtherapeutic anti-hTNF dose to effectively treat the clinical and histopathological signs of the pathology. The combination of dasatinib and anti-hTNF exhibits a distinct mode of action in restoring the arthritogenic gene signature to that of a healthy profile. Potential clinical applications of combination therapies with kinase inhibitors and anti-TNF agents may provide an interesting alternative to high-dose anti-hTNF monotherapy and increase the number of patients responding to treatment

Activation of Wnt signaling in contracting myotubes.

<p>(<b>A</b>) Over-expression of Wnt10b cDNA down-regulated SREBP-1c protein. Myotubes were transfected with a plasmid encoding the mouse Wnt10b cDNA, then treated or not with 10 nM insulin for 24 hours. Western blot analysis of cytoplasmic (left panel) and nuclear (right panel) protein extracts showing the down-regulation of precursor and mature forms of SREBP-1c following the activation of the Wnt/β-catenin pathway. Wnt10b over-expression induced the nuclear accumulation of active β-catenin and MyoD. Blots were normalized using antibodies raised against the cytoplasmic protein GAPDH or the nuclear protein Lamin A/C. (<b>B</b>) Myotubes were submitted to a 48 hour-treatment with 1 µM BIO, then 10 nM insulin was added for 24 hours. BIO-mediated activation of the Wnt/β-catenin pathway induced SREBP-1c down-regulation, even in the presence of insulin.</p

Effect of BIO on intramyocellular lipid accumulation.

<p>Oil red O staining of intramyocellular lipids in myotubes cultured in 5 mM glucose (<b>A</b>) 25 mM glucose (<b>B</b>) or 25 mM glucose in the presence of 1 µM BIO for 5 days (<b>C</b>). Phase-contrast microphotographs of the same myotubes cultured in 5 mM glucose (<b>D</b>), or 25 mM glucose in the absence (<b>E</b>) or presence (<b>F</b>) of BIO. BIO totally abolished intramyocellular lipid deposition. Scale bar 20 µm.</p

Comparison between insulin and BIO effects on intracellular signaling.

<p>Myotubes cultured in 5 mM glucose (G5) or 25 mM glucose (G25) concentration were treated with 10 nM insulin for 30 minutes, or with 1 µM BIO for 30 and 60 minutes. (<b>A</b>) Insulin increased GSK-3β^S9 phosphorylation in myotubes cultured in G5 or G25, whereas BIO had no effect. In contrast, BIO decreased GSK-3β^Y216 phosphorylation, whereas insulin had no effect. (<b>B</b>) Insulin increased Akt2^S473 phosphorylation in myotubes cultured in G5, whereas myotubes cultured in G25 were resistant to insulin. BIO increased Akt1^T308 phosphorylation in myotubes cultured in G5, and had a biphasic effect in myotubes cultured in G25. (<b>C</b>) BIO stimulated AMPK-α1^S485 but not AMPK-α2^T172 phosphorylation whatever the glucose concentration, whereas insulin had no effect on AMPK phosphorylation. (<b>D</b>) In contrast to BIO, insulin failed to increase AS160^S588 phosphorylation. BIO showed a biphasic effect in myotubes cultured in G25. (<b>E</b>) Insulin increased Erk1/2 phosphorylation only in myotubes cultured in G5, whereas BIO diminished Erk1/2 phosphorylation in myotubes whatever the glucose concentration. Lower panels show quantifications of 3 independent experiments. Data are expressed as mean±SE. Significant difference between BIO and insulin, (###) p<0.001; (##) p<0.01. Significant difference between control and insulin (***) p<0.001; (**) p<0.01. NS: non significant.</p

Effect of Wnt signaling on glucose transport in contracting myotubes.

<p>(<b>A</b>) Myotubes cultured in 5 mM glucose (G5) or in 25 mM glucose (G25) for 48 hours were transfected with a mouse Wnt10b cDNA or treated with 1 µM BIO. 2-deoxyglucose (2-DG) uptake was then measured in the presence or absence of 10 nM insulin for 30 minutes as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008509#s4" target="_blank">Material and Methods</a>. Data are expressed as mean±SE from 5 independent experiments performed in triplicate. Significant difference from G5, (***) p<0.0001; (**) p<0.02; Significant difference from G25, (###) p<0.0001; (##) p<0.001. (<b>B</b>) BIO induced GLUT4 translocation to the plasma membrane. Myotubes were cultured in 5 mM (G5) or 25 mM glucose (G25) for 48 hours in the presence or absence of 1 µM BIO. Myotubes were treated or not with 10 nM insulin for 30 minutes, then plasma membranes were isolated. Western blot analysis showed that insulin and BIO induced GLUT4 translocation to the plasma membrane, whereas GLUT1 was unaffected. (<b>C</b>) Quantification of GLUT4 and GLUT1 translocation. Data are expressed as mean±SE from 4 independent experiments. Significant difference from G5, (***) p<0.0001; (**) p<0.01; (*) p<0.05.</p

Wnt10b and SREBP-1c are also inversely expressed in cultured satellite cells.

<p>(<b>A</b>) Western blot analysis showing an inverse expression pattern between Wnt10b and SREBP-1c proteins according to the differentiation stage. In contrast, Wnt3 remained almost unchanged throughout differentiation. SREBP-1c induced the up-regulation of the lipogenic enzyme FAS in myotubes. (<b>B</b>) Wnt10b knockdown was sufficient to up-regulate SREBP-1c and PPARγ mRNAs, whereas Wnt10b over-expression down-regulated their expression. RT-PCR was performed on myoblasts transfected with a scrambled siRNA (lane 1), a pool of 3 Wnt10b siRNAs (lane 2), or a plasmid encoding the mouse Wnt10 cDNA (lane 3) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008509#s4" target="_blank">Material and Methods</a>. (<b>C</b>) SREBP-1 knockdown stimulated Wnt signaling in contracting myotubes. Myotubes were transfected with SREBP-1 siRNAs or a scrambled siRNA, then treated or not with 10 nM insulin for 24 hours. SREBP-1 knockdown was sufficient to induce Wnt10b protein expression in myotubes, particularly in the presence of insulin, and to activate the Wnt/β-catenin pathway, as shown by GSK-3β and β-catenin activities. (<b>D</b>) Wnt10b knockdown induced SREBP-1c protein expression in myoblasts. Myoblasts were transfected with 30 pmoles or 60 pmoles of a pool of 3 Wnt10b siRNAs, or with a scrambled siRNA as a control. Silencing Wnt10b was sufficient to induce SREBP-1c protein expression in myoblasts through the inhibition of Wnt/β-catenin signaling. The blots are representative of 3 independent experiments.</p

Differential expression of SREBP-1c and Wnt proteins during skeletal muscle ontogenesis and regeneration.

<p>(<b>A</b>) Western blot analysis showing inverse expression patterns between Wnt10b and SREBP-1c during ontogenesis. The developmental stages are underlined using antibodies against developmental (MyHC-Dev) and fast (MyHC-2) myosin heavy chains. (<b>B</b>) Western blot analysis of Wnt10b and SREBP-1c protein levels in regenerating (R) adult EDL muscles at 2, 8, and 30 days after crush injury as compared to contralateral control (C) EDL. The down-regulation of SREBP-1c was concomitant with the up-regulation of Wnt10b throughout regeneration. The blots are representative of 3 independent experiments.</p

Combination of subtherapeutic anti-TNF dose with dasatinib restores clinical and molecular arthritogenic profiles better than standard anti-TNF treatment

Background New medications for Rheumatoid Arthritis (RA) have emerged in the last decades, including Disease Modifying Antirheumatic Drugs (DMARDs) and biologics. However, there is no known cure, since a significant proportion of patients remain or become non-responders to current therapies. The development of new mode-of-action treatment schemes involving combination therapies could prove successful for the treatment of a greater number of RA patients. Methods We investigated the effect of the Tyrosine Kinase inhibitors (TKIs) dasatinib and bosutinib, on the human TNF-dependent Tg197 arthritis mouse model. The inhibitors were administered either as a monotherapy or in combination with a subtherapeutic dose of anti-hTNF biologics and their therapeutic effect was assessed clinically, histopathologically as well as via gene expression analysis and was compared to that of an efficient TNF monotherapy. Results Dasatinib and, to a lesser extent, bosutinib inhibited the production of TNF and proinflammatory chemokines from arthritogenic synovial fibroblasts. Dasatinib, but not bosutinib, also ameliorated significantly and in a dose-dependent manner both the clinical and histopathological signs of Tg197 arthritis. Combination of dasatinib with a subtherapeutic dose of anti-hTNF biologic agents, resulted in a synergistic inhibitory effect abolishing all arthritis symptoms. Gene expression analysis of whole joint tissue of Tg197 mice revealed that the combination of dasatinib with a low subtherapeutic dose of Infliximab most efficiently restores the pathogenic gene expression profile to that of the healthy state compared to either treatment administered as a monotherapy. Conclusion Our findings show that dasatinib exhibits a therapeutic effect in TNF-driven arthritis and can act in synergy with a subtherapeutic anti-hTNF dose to effectively treat the clinical and histopathological signs of the pathology. The combination of dasatinib and anti-hTNF exhibits a distinct mode of action in restoring the arthritogenic gene signature to that of a healthy profile. Potential clinical applications of combination therapies with kinase inhibitors and anti-TNF agents may provide an interesting alternative to high-dose anti-hTNF monotherapy and increase the number of patients responding to treatment