Vance B. Matthews

Cellular Cholesterol Depletion Triggers Shedding of the Human Interleukin-6 Receptor by ADAM10 and ADAM17 (TACE)

Interleukin-6 (IL-6) activates cells by binding to the membrane-bound IL-6 receptor (IL-6R) and subsequent formation of a glycoprotein 130 homodimer. Cells that express glycoprotein 130, but not the IL-6R, can be activated by IL-6 and the soluble IL-6R which is generated by shedding from the cell surface or by alternative splicing. Here we show that cholesterol depletion of cells with methyl-β-cyclodextrin increases IL-6R shedding independent of protein kinase C activation and thus differs from phorbol ester-induced shedding. Contrary to cholesterol depletion, cholesterol enrichment did not increase IL-6R shedding. Shedding of the IL-6R because of cholesterol depletion is highly dependent on the metalloproteinase ADAM17 (tumor necrosis factor-α-converting enzyme), and the related ADAM10, which is identified here for the first time as an enzyme involved in constitutive and induced shedding of the human IL-6R. When combined with protein kinase C inhibition by staurosporine or rottlerin, breakdown of plasma membrane sphingomyelin or enrichment of the plasma membrane with ceramide also increased IL-6R shedding. The effect of cholesterol depletion was confirmed in human THP-1 and Hep3B cells and in primary human peripheral blood monocytes, which naturally express the IL-6R. For decades, high cholesterol levels have been considered harmful. This study indicates that low cholesterol levels may play a role in shedding of the membrane-bound IL-6R and thereby in the immunopathogenesis of human diseases.

Brain-derived neurotrophic factor is produced by skeletal muscle cells in response to contraction and enhances fat oxidation via activation of AMP-activated protein kinase

It has been brought to our attention following an investigation into the work of Bente Klarlund Pedersen by the Danish Committees for Scientific Dishonesty, that the erratum published in 2012 was insufficient to correct this article. Although the data published in the Diabetologia paper were previously unpublished, the data from the biological material collected from the additional eight healthy men presented in Fig. 1b and c originated from a previous study that was not referenced [1]. In addition, while these eight healthy subjects performed the same type of exercise at the same intensity, the duration was different. The following description of the methodology and Fig. 1 legend correct these oversights. The authors would like to reiterate that these methodological oversights in no way affect either the data presented in the paper or the conclusions reached. The authors also apologise to both the journal and its readership for these oversights.

Critical role of the disintegrin metalloprotease ADAM17 for intestinal inflammation and regeneration in mice.

The protease a disintegrin and metalloprotease (ADAM) 17 cleaves tumor necrosis factor (TNF), L-selectin, and epidermal growth factor receptor (EGF-R) ligands from the plasma membrane. ADAM17 is expressed in most tissues and is up-regulated during inflammation and cancer. ADAM17-deficient mice are not viable. Conditional ADAM17 knockout models demonstrated proinflammatory activities of ADAM17 in septic shock via shedding of TNF. We used a novel gene targeting strategy to generate mice with dramatically reduced ADAM17 levels in all tissues. The resulting mice called ADAM17ex/ex were viable, showed compromised shedding of ADAM17 substrates from the cell surface, and developed eye, heart, and skin defects as a consequence of impaired EGF-R signaling caused by failure of shedding of EGF-R ligands. Unexpectedly, although the intestine of unchallenged homozygous ADAM17ex/ex mice was normal, ADAM17ex/ex mice showed substantially increased susceptibility to inflammation in dextran sulfate sodium colitis. This was a result of impaired shedding of EGF-R ligands resulting in failure to phosphorylate STAT3 via the EGF-R and, consequently, in defective regeneration of epithelial cells and breakdown of the intestinal barrier. Besides regulating the systemic availability of the proinflammatory cytokine TNF, our results demonstrate that ADAM17 is needed for vital regenerative activities during the immune response. Thus, our mouse model will help investigate ADAM17 as a potential drug target.

Role of exercise-induced brain-derived neurotrophic factor production in the regulation of energy homeostasis in mammals

Brain‐derived neurotrophic factor (BDNF) has been shown to regulate neuronal development and plasticity and plays a role in learning and memory. Moreover, it is well established that BDNF plays a role in the hypothalamic pathway that controls body weight and energy homeostasis. Recent evidence identifies BDNF as a player not only in central metabolism, but also in regulating energy metabolism in peripheral organs. Low levels of BDNF are found in patients with neurodegenerative diseases, including Alzheimers disease and major depression. In addition, BDNF levels are low in obesity and independently so in patients with type 2 diabetes. Brain‐derived neurotrophic factor is expressed in non‐neurogenic tissues, including skeletal muscle, and exercise increases BDNF levels not only in the brain and in plasma, but in skeletal muscle as well. Brain‐derived neurotrophic factor mRNA and protein expression was increased in muscle cells that were electrically stimulated, and BDNF increased phosphorylation of AMP‐activated protein kinase (AMPK) and acetyl coenzyme A carboxylase‐beta (ACCβ) and enhanced fatty oxidation both in vitro and ex vivo. These data identify BDNF as a contraction‐inducible protein in skeletal muscle that is capable of enhancing lipid oxidation in skeletal muscle via activation of AMPK. Thus, BDNF appears to play a role both in neurobiology and in central as well as peripheral metabolism. The finding of low BDNF levels both in neurodegenerative diseases and in type 2 diabetes may explain the clustering of these diseases. Brain‐derived neurotrophic factor is likely to mediate some of the beneficial effects of exercise with regard to protection against dementia and type 2 diabetes.

PI3K(p110 alpha) Protects Against Myocardial Infarction-Induced Heart Failure Identification of PI3K-Regulated miRNA and mRNA

Objective—Myocardial infarction (MI) is a serious complication of atherosclerosis associated with increasing mortality attributable to heart failure. Activation of phosphoinositide 3-kinase [PI3K(p110&agr;)] is considered a new strategy for the treatment of heart failure. However, whether PI3K(p110&agr;) provides protection in a setting of MI is unknown, and PI3K(p110&agr;) is difficult to target because it has multiple actions in numerous cell types. The goal of this study was to assess whether PI3K(p110&agr;) is beneficial in a setting of MI and, if so, to identify cardiac-selective microRNA and mRNA that mediate the protective properties of PI3K(p110&agr;). Methods and Results—Cardiomyocyte-specific transgenic mice with increased or decreased PI3K(p110&agr;) activity (caPI3K-Tg and dnPI3K-Tg, respectively) were subjected to MI for 8 weeks. The caPI3K-Tg subjected to MI had better cardiac function than nontransgenic mice, whereas dnPI3K-Tg had worse function. Using microarray analysis, we identified PI3K-regulated miRNA and mRNA that were correlated with cardiac function, including growth factor receptor-bound 14. Growth factor receptor-bound 14 is highly expressed in the heart and positively correlated with PI3K(p110&agr;) activity and cardiac function. Mice deficient in growth factor receptor-bound 14 have cardiac dysfunction. Conclusion—Activation of PI3K(p110&agr;) protects the heart against MI-induced heart failure. Cardiac-selective targets that mediate the protective effects of PI3K(p110&agr;) represent new drug targets for heart failure.

Differential lymphotoxin-β and interferon gamma signaling during mouse liver regeneration induced by chronic and acute injury†

The liver regenerates after acute injury via hepatocyte cell division; during chronic injury, when hepatocyte replication is impaired or blocked, liver progenitor oval cells mediate liver regeneration. If both regeneration options are blocked in animal models, then liver failure and death ensues. The mechanisms underlying oval cell induction, proliferation, and subsequent liver regeneration remain poorly characterized. In particular, cell‐signaling pathways that distinguish the alternative pathways are unknown. This study shows that in a mouse model, hepatic expression of lymphotoxin‐β (LTβ) and interferon gamma (IFNγ) transcripts is increased in response to the choline‐deficient, ethionine‐supplemented (CDE) diet, which induces oval cell–mediated liver regeneration. Oval cells express LTβ and IFNγ transcripts, contributing to the increased expression in the liver of mice fed the CDE diet. An attenuated oval cell response to such a diet was observed in LTβ receptor‐, LTβ‐, and IFNγ‐gene targeted mice. Loss of LTβ and LTβ receptor signaling reduced the number of oval cells expressing A6 and muscle pyruvate kinase. The lack of IFNγ signaling reduced muscle pyruvate kinase+, but not A6+, oval cells. In contrast, partial hepatectomy suppressed LTβ and IFNγ transcripts. We also show that IFNγ induces STAT‐3 phosphorylation in an oval cell line. In conclusion, LTβ, LTβ receptor, and IFNγ are involved in oval cell–mediated, but not hepatocyte‐mediated, liver regeneration, and the absence of these pathways impairs the oval cell–dependent regenerative response. (HEPATOLOGY 2005;41:327–335.)

Liver inflammation and cytokine production, but not acute phase protein synthesis, accompany the adult liver progenitor (oval) cell response to chronic liver injury

Oval cells are facultative liver progenitor cells, which are invoked during chronic liver injury in order to replenish damaged hepatocytes and bile duct cells. Previous studies have observed inflammation and cytokine production in the liver during chronic injury. Further, it has been proposed that inflammatory growth factors may mediate the proliferation of oval cells during disease progression. We have undertaken a detailed examination of inflammation and cytokine production during a time course of liver injury and repair, invoked by feeding mice a choline‐deficient, ethionine‐supplemented (CDE) diet. We show that immediately following initial liver injury, B220‐expressing leucocytes transiently infiltrate the liver. This inflammatory response occurred immediately before oval cell numbers began to expand in the liver, suggesting that the two events may be linked. Two waves of liver cytokine production were observed during the CDE time course. The first occurred shortly following commencement of the diet, suggesting that it may represent a hepatic acute phase response. However, examination of acute phase marker expression in CDE‐fed mice did not support this hypothesis. The second wave of cytokine expression correlated with the expansion of oval cell numbers in the liver, suggesting that these factors may mediate oval cell proliferation. No inflammatory signalling was detected following withdrawal of the injury stimulus. In summary, our results document a close correlation between inflammation, cytokine production and the expansion of oval cells in the liver during experimental chronic injury.

Opposing roles of gp130-mediated STAT-3 and ERK-1/2 signaling in liver progenitor cell migration and proliferation†

Gp130‐mediated IL‐6 signaling may play a role in oval cell proliferation in vivo. Levels of IL‐6 are elevated in livers of mice treated with a choline‐deficient ethionine‐supplemented (CDE) diet that induces oval cells, and there is a reduction of oval cells in IL‐6 knockout mice. The CDE diet recapitulates characteristics of chronic liver injury in humans. In this study, we determined the impact of IL‐6 signaling on oval cell‐mediated liver regeneration in vivo. Signaling pathways downstream of gp130 activation were also dissected. Numbers of A6+ve liver progenitor oval cells (LPCs) in CDE‐treated murine liver were detected by immunohistochemistry and quantified. Levels of oval cell migration and proliferation were compared in CDE‐treated mouse strains that depict models of gp130‐mediated hyperactive ERK‐1/2 signaling (gp130ΔSTAT), hyperactive STAT‐3 signaling (gp130Y757F and Socs‐3−/ΔAlb) or active ERK‐1/2 as well as active STAT‐3 signaling (wild‐type). The A6+ve LPC numbers were increased with IL‐6 treatment in vivo. The gp130Y757F mice displayed increased A6+ve LPCs numbers compared with wild‐type and gp130ΔSTAT mice. Numbers of A6+ve LPCs were also increased in the livers of CDE treated Socs‐3−/ΔAlbmice compared with their control counterparts. Lastly, inhibition of ERK‐1/2 activation in cultured oval cells increased hyper IL‐6‐induced cell growth. For the first time, we have dissected the gp130‐mediated signaling pathways, which influence liver progenitor oval cell proliferation. Conclusion: Hyperactive STAT‐3 signaling results in enhanced oval cell numbers, whereas ERK‐1/2 activation suppresses oval cell proliferation. (HEPATOLOGY 2007;45:486–494.)

Supplementation of a High-Fat Diet with Chlorogenic Acid Is Associated with Insulin Resistance and Hepatic Lipid Accumulation in Mice

The increasing prevalence of the metabolic syndrome requires a greater need for therapeutic and prevention strategies. Higher coffee consumption is consistently associated with a lower risk of type 2 diabetes in population studies. Dietary polyphenols have been linked to benefits on several features of the metabolic syndrome. Chlorogenic acid (CGA), a major component of coffee, is one of the most consumed polyphenols in the diet. In our study, we conducted a controlled dietary intervention over 12 weeks in male mice. There were three dietary groups: (i) normal diet, (ii) high-fat diet, and (iii) high-fat diet + CGA. We assessed the effect of CGA at a physiologically obtainable dose (1 g/kg of diet) on high-fat-diet-induced obesity, glucose intolerance, insulin resistance, and also fatty acid oxidation and insulin signaling in C57BL/6 male mice. Supplementation of CGA in the high-fat diet did not reduce body weight compared to mice fed the high-fat diet alone (p = 0.32). CGA resulted in increased insulin resistance compared to mice fed a high-fat diet only (p < 0.05). CGA resulted in decreased phosphorylation of AMP-activated protein kinase (AMPK) (p < 0.001) and acetyl carboxylase β (ACCβ), a downstream target of AMPK (p < 0.05), in liver. The liver of mice fed a high-fat diet supplemented with CGA had a higher lipid content (p < 0.05) and more steatosis relative to mice fed a high-fat diet only, indicating impaired fatty acid oxidation. This study suggests that CGA supplementation in a high-fat diet does not protect against features of the metabolic syndrome in diet-induced obese mice.

Contraction-induced interleukin-6 gene transcription in skeletal muscle is regulated by c-Jun terminal kinase/activator protein-1

Background: IL-6 is up-regulated by contraction in skeletal muscle. Results: Contraction-induced IL-6 expression is blunted by JNK inhibition. Conclusion: The JNK/AP-1 pathway regulates IL-6 expression in contracting muscle. Significance: This highlights a novel contraction-mediated transcriptional pathway for IL-6 in skeletal muscle. Exercise increases the expression of the prototypical myokine IL-6, but the precise mechanism by which this occurs has yet to be identified. To mimic exercise conditions, C2C12 myotubes were mechanically stimulated via electrical pulse stimulation (EPS). We compared the responses of EPS with the pharmacological Ca2+ carrier calcimycin (A23187) because contraction induces marked increases in cytosolic Ca2+ levels or the classical IκB kinase/NFκB inflammatory response elicited by H2O2. We demonstrate that, unlike H2O2-stimulated increases in IL-6 mRNA, neither calcimycin- nor EPS-induced IL-6 mRNA expression is under the transcriptional control of NFκB. Rather, we show that EPS increased the phosphorylation of JNK and the reporter activity of the downstream transcription factor AP-1. Furthermore, JNK inhibition abolished the EPS-induced increase in IL-6 mRNA and protein expression. Finally, we observed an exercise-induced increase in both JNK phosphorylation and IL-6 mRNA expression in the skeletal muscles of mice after 30 min of treadmill running. Importantly, exercise did not increase IL-6 mRNA expression in skeletal muscle-specific JNK-deficient mice. These data identify a novel contraction-mediated transcriptional regulatory pathway for IL-6 in skeletal muscle.