KLF2 in Myeloid Lineage Cells Regulates the Innate Immune Response during Skeletal Muscle Injury and Regeneration.

Skeletal muscle repair and regeneration after injury requires coordinated interactions between the innate immune system and the injured muscle. Myeloid cells predominate in these interactions. This study examined the role of KLF2, a zinc-finger transcription factor that regulates immune cell activation, in specifying myeloid cell functions during muscle regeneration. Loss of KLF2 in myeloid lineage cells (myeKlf2-/- mice) dramatically enhanced the initial inflammatory response to acute muscle injury (cardiotoxin). Injured muscles showed dramatically elevated expression of inflammatory mediators and greater numbers of infiltrating, pro-inflammatory monocytes that matured earlier into activated macrophages. Notably, the inflammatory phase resolved earlier and regeneration progressed to myogenesis, marked by elevated expression of factors that promote the formation of new fibers from satellite cells. Regeneration was completed earlier, with phenotypically normal adult fibers integrated into the muscle syncytium. These findings identify myeloid KLF2 as a key regulator of myeloid cell functions in adult skeletal muscle regeneration.

Stimulation of constitutive nitric oxide uniquely and compensatorily regulates intestinal epithelial cell brush border membrane Na absorption.

In the mammalian small intestine, sodium is primarily absorbed by Na+ /H+ exchange (NHE3) and Na-glucose cotransport (SGLT1) in the brush border membrane (BBM) of villus cells. However, how enhanced cellular constitutive nitric oxide (cNO) may affect NHE3 and SGLT1 remains unclear. Both in vivo in rabbit intestinal villus cells and in vitro IEC-18 cells, administration of NO donor, GSNAP, modestly increased cNO. GSNAP stimulated SGLT1 in villus and IEC-18 cells. The mechanism of stimulation was secondary to an increase in the affinity of SGLT1 for glucose. The change in SGLT1 was not secondary to altered Na-extruding capacity of the cell since Na+ /K+ -ATPase was decreased by GSNAP treatment. In contrast, GSNAP inhibited NHE3 activity in villus cell BBM. The mechanism of NHE3 inhibition was secondary to reduced BBM transporter numbers. These studies demonstrated that the physiological increase in cNO uniquely regulates mammalian small intestinal NHE3 and SGLT1 to maintain Na homeostasis.

Unique Regulation of Enterocyte Brush Border Membrane Na-Glutamine and Na-Alanine Co-Transport by Peroxynitrite during Chronic Intestinal Inflammation.

Na-amino acid co-transporters (NaAAcT) are uniquely affected in rabbit intestinal villus cell brush border membrane (BBM) during chronic intestinal inflammation. Specifically, Na-alanine co-transport (ASCT1) is inhibited secondary to a reduction in the affinity of the co-transporter for alanine, whereas Na-glutamine co-transport (B0AT1) is inhibited secondary to a reduction in BBM co-transporter numbers. During chronic intestinal inflammation, there is abundant production of the potent oxidant peroxynitrite (OONO). However, whether OONO mediates the unique alteration in NaAAcT in intestinal epithelial cells during chronic intestinal inflammation is unknown. In this study, ASCT1 and B0AT1 were inhibited by OONO in vitro. The mechanism of inhibition of ASCT1 by OONO was secondary to a reduction in the affinity of the co-transporter for alanine, and secondary to a reduction in the number of co-transporters for B0AT1, which were further confirmed by Western blot analyses. In conclusion, peroxynitrite inhibited both BBM ASCT1 and B0AT1 in intestinal epithelial cells but by different mechanisms. These alterations in the villus cells are similar to those seen in the rabbit model of chronic enteritis. Therefore, this study indicates that peroxynitrite may mediate the inhibition of ASCT1 and B0AT1 during inflammation, when OONO levels are known to be elevated in the mucosa.

Dilated cardiomyopathy-mediated heart failure induces a unique skeletal muscle myopathy with inflammation.

BACKGROUND: Skeletal muscle myopathy and exercise intolerance are diagnostic hallmarks of heart failure (HF). However, the molecular adaptations of skeletal muscles during dilated cardiomyopathy (DCM)-mediated HF are not completely understood. METHODS: Skeletal muscle structure and function were compared in wild-type (WT) and cardiac myosin binding protein-C null mice (t/t), which develop DCM-induced HF. Cardiac function was examined by echocardiography. Exercise tolerance was measured using a graded maximum treadmill running test. Hindlimb muscle function was assessed in vivo from measurements of plantar flexor strength. Inflammatory status was evaluated from the expression of inflammatory markers and the presence of specific immune cell types in gastrocnemius muscles. Muscle regenerative capacityat days 3, 7, and 14 after eccentric contraction-induced injury was determined from the number of phenotypically new and adult fibers in the gastrocnemius, and functional recovery of plantar flexion torque. RESULTS: t/t mice developed DCM-induced HF in association with profound exercise intolerance, consistent with previous reports. Compared to WT, t/t mouse hearts show significant hypertrophy of the atria and ventricles and reduced fractional shortening, both systolic and diastolic. In parallel, the skeletal muscles of t/t mice exhibit weakness and myopathy. Compared to WT, plantar flexor muscles of t/t null mice produce less peak isometric plantar torque (Po), develop torque more slowly (+ dF/dt), and relax more slowly (- dF/dt, longer half-relaxation times,1/2RT). Gastrocnemius muscles of t/t mice have a greater number of fibers with smaller diameters and central nuclei. Oxidative fibers, both type I and type IIa, show significantly smaller cross-sectional areas and more central nuclei. These fiber phenotypes suggest ongoing repair and regeneration under homeostatic conditions. In addition, the ability of muscles to recover and regenerate after acute injury is impaired in t/t mice. CONCLUSIONS: Our studies concluded that DCM-induced HF induces a unique skeletal myopathy characterized by decreased muscle strength, atrophy of oxidative fiber types, ongoing inflammation and damage under homeostasis, and impaired regeneration after acute muscle injury. Furthermore, this unique myopathy in DCM-induced HF likely contributes to and exacerbates exercise intolerance. Therefore, efforts to develop therapeutic interventions to treat skeletal myopathy during DCM-induced HF should be considered.

Inducible Nitric Oxide Regulates Brush Border Membrane Na-Glucose Co-transport, but Not Na:H Exchange via p38 MAP Kinase in Intestinal Epithelial Cells.

During chronic intestinal inflammation in rabbit intestinal villus cells brush border membrane (BBM) Na-glucose co-transport (SGLT1), but not Na/H exchange (NHE3) is inhibited. The mechanism of inhibition is secondary to a decrease in the number of BBM co-transporters. In the chronic enteritis mucosa, inducible nitric oxide (iNO) and superoxide production are known to be increased and together they produce abundant peroxynitrite (OONO), a potent oxidant. However, whether OONO mediates the SGLT1 and NHE3 changes in intestinal epithelial cells during chronic intestinal inflammation is unknown. Thus, we determined the effect of OONO on SGLT1 and NHE3 in small intestinal epithelial cell (IEC-18) monolayers grown on trans well plates. In cells treated with 100 µM SIN-1 (OONO donor) for 24 h, SGLT1 was inhibited while NHE3 activity was unaltered. SIN-1 treated cells produced 40 times more OONO fluorescence compared to control cells. Uric acid (1mM) a natural scavenger of OONO prevented the OONO mediated SGLT1 inhibition. Na⁺/K⁺-ATPase which maintains the favorable trans-cellular Na gradient for Na-dependent absorptive processes was decreased by OONO. Kinetics studies demonstrated that the mechanism of inhibition of SGLT1 by OONO was secondary to reduction in the number of co-transporters (Vmax) without an alteration in the affinity. Western blot analysis showed a significant decrease in SGLT1 protein expression. Further, p38 mitogen-activated protein (MAP) kinase pathway appeared to mediate the OONO inhibition of SGLT1. Finally, at the level of the co-transporter, 3-Nitrotyrosine formation appears to be the mechanism of inhibition of SGLT1. In conclusion, peroxynitrite inhibited BBM SGLT1, but not NHE3 in intestinal epithelial cells. These changes and the mechanism of SGLT1 inhibition by OONO in IEC-18 cells is identical to that seen in villus cells during chronic enteritis. Thus, these data indicate that peroxynitrite, known to be elevated in the mucosa, may mediate the inhibition of villus cell BBM SGLT1 in vivo in the chronically inflamed intestine.

Phospholemman is not required for the acute stimulation of Na⁺-K⁺-ATPase α2-activity during skeletal muscle fatigue.

The Na(+)-K(+)-ATPase α2-isoform in skeletal muscle is rapidly stimulated during muscle use and plays a critical role in fatigue resistance. The acute mechanisms that stimulate α2-activity are not completely known. This study examines whether phosphorylation of phospholemman (PLM/FXYD1), a regulatory subunit of Na(+)-K(+)-ATPase, plays a role in the acute stimulation of α2 in working muscles. Mice lacking PLM (PLM KO) have a normal content of the α2-subunit and show normal exercise capacity, in contrast to the greatly reduced exercise capacity of mice that lack α2 in the skeletal muscles. Nerve-evoked contractions in vivo did not induce a change in total PLM or PLM phosphorylated at Ser63 or Ser68, in either WT or PLM KO. Isolated muscles of PLM KO mice maintain contraction and resist fatigue as well as wild type (WT). Rb(+) transport by the α2-Na(+)-K(+)-ATPase is stimulated to the same extent in contracting WT and contracting PLM KO muscles. Phosphorylation of sarcolemmal membranes prepared from WT but not PLM KO skeletal muscles stimulates the activity of both α1 and α2 in a PLM-dependent manner. The stimulation occurs by an increase in Na(+) affinity without significant change in Vmax and is more effective for α1 than α2. These results demonstrate that phosphorylation of PLM is capable of stimulating the activity of both isozymes in skeletal muscle; however, contractile activity alone is not sufficient to induce PLM phosphorylation. Importantly, acute stimulation of α2, sufficient to support exercise and oppose fatigue, does not require PLM or its phosphorylation.

Chronic and selective inhibition of basolateral membrane Na-K-ATPase uniquely regulates brush border membrane Na absorption in intestinal epithelial cells.

Na-K-ATPase, an integral membrane protein in mammalian cells, is responsible for maintaining the favorable intracellular Na gradient necessary to promote Na-coupled solute cotransport processes [e.g., Na-glucose cotransport (SGLT1)]. Inhibition of brush border membrane (BBM) SGLT1 is, at least in part, due to the diminished Na-K-ATPase in villus cells from chronically inflamed rabbit intestine. The aim of the present study was to determine the effect of Na-K-ATPase inhibition on the two major BBM Na absorptive pathways, specifically Na-glucose cotransport and Na/H exchange (NHE), in intestinal epithelial (IEC-18) cells. Na-K-ATPase was inhibited using 1 mM ouabain or siRNA for Na-K-ATPase-α1 in IEC-18 cells. SGLT1 activity was determined as 3-O-methyl-D-[(3)H]glucose uptake. Na-K-ATPase activity was measured as the amount of inorganic phosphate released. Treatment with ouabain resulted in SGLT1 inhibition at 1 h but stimulation at 24 h. To further characterize this unexpected stimulation of SGLT1, siRNA silencing was utilized to inhibit Na-K-ATPase-α1. SGLT1 activity was significantly upregulated by Na-K-ATPase silencing, while NHE3 activity remained unaltered. Kinetics showed that the mechanism of stimulation of SGLT1 activity was secondary to an increase in affinity of the cotransporter for glucose without a change in the number of cotransporters. Molecular studies demonstrated that the mechanism of stimulation was not secondary to altered BBM SGLT1 protein levels. Chronic and direct silencing of basolateral Na-K-ATPase uniquely regulates BBM Na absorptive pathways in intestinal epithelial cells. Specifically, while BBM NHE3 is unaffected, SGLT1 is stimulated secondary to enhanced affinity of the cotransporter.

Molecular mechanism of regulation of villus cell Na-K-ATPase in the chronically inflamed mammalian small intestine.

Na-K-ATPase located on the basolateral membrane (BLM) of intestinal epithelial cells provides a favorable intracellular Na+ gradient to promote all Na dependent co-transport processes across the brush border membrane (BBM). Down-regulation of Na-K-ATPase activity has been postulated to alter the absorption via Na-solute co-transporters in human inflammatory bowel disease (IBD). Further, the altered activity of a variety of Na-solute co-transporters in intact villus cells has been reported in animal models of chronic enteritis. But the molecular mechanism of down-regulation of Na-K-ATPase is not known. In the present study, using a rabbit model of chronic intestinal inflammation, which resembles human IBD, Na-K-ATPase in villus cells was shown to decrease. The relative mRNA abundance of α-1 and ß-1 subunits was not altered in villus cells during chronic intestinal inflammation. Similarly, the protein levels of these subunits were also not altered in villus cells during chronic enteritis. However, the BLM concentration of α-1 and ß-1 subunits was diminished in the chronically inflamed intestinal villus cells. An ankyrin-spectrin skeleton is necessary for the proper trafficking of Na-K-ATPase to the BLM of the cell. In the present study, ankyrin expression was markedly diminished in villus cells from the chronically inflamed intestine resulting in depolarization of ankyrin-G protein. The decrease of Na-K-ATPase activity was comparable to that seen in ankyrin knockdown IEC-18 cells. Therefore, altered localization of Na-K-ATPase as a result of transcriptional down-regulation of ankyrin-G mediates the down-regulation of Na-K-ATPase activity during chronic intestinal inflammation.

Reduced levels of microRNAs miR-124a and miR-150 are associated with increased proinflammatory mediator expression in Krüppel-like factor 2 (KLF2)-deficient macrophages.

Previous studies have shown that the myeloid-specific deficiency of the transcription factor Krüppel-like factor 2 (KLF2) accelerates atherosclerosis in hypercholesterolemic Ldlr(-/-) mice due to the enhanced adhesion of myeloid cells to activated endothelial cells in the vessel wall. This study revealed elevated basal inflammation with elevated plasma levels of Ccl2, Ccl4, Ccl5, and Ccl11 in the myeloid-specific KLF2 knock-out (myeKlf2(-/-)) mice. Peritoneal macrophages isolated from myeKlf2(-/-) mice showed increased mRNA levels of several inflammatory mediators, including Ccl2, Ccl5, Ccl7, Cox-2, Cxcl1, and IL-6. In contrast, the levels of two microRNAs, miR-124a and miR-150, were lower in Klf2(-/-) macrophages compared with Klf2(+/+) macrophages. Additional studies showed a direct inverse relationship between miR-124a levels with Ccl2 expression, with anti-miR-124a increasing Ccl2 mRNA levels in Klf2(+/+) macrophages, whereas the restoration of miR-124a levels in Klf2(-/-) macrophages significantly reduced Ccl2 mRNA expression. Likewise, the inverse relationship was observed between miR-150 levels and Cxcl1 expression in Klf2(+/+) and Klf2(-/-) mice. Moreover, miR150 likely regulates the miR124a expression and thus augments expression of inflammatory mediators in myeKlf2(-/-) macrophages. This study documented that the transcription factor KLF2 modulates inflammatory chemokine production via regulation of microRNA expression levels in immune cells.

Monocarboxylate 4 mediated butyrate transport in a rat intestinal epithelial cell line.

BACKGROUND: Short chain fatty acids (SCFA) are absorbed by carrier mediated uptake in the small intestine by pH-dependent SCFA/HCO3 (-) exchangers on the apical membrane of epithelial cells. Conventional assumption is that MCT1 mediates SCFA/HCO3 (-) exchange in the intestine. Further, due to the presence of multiple such anion exchangers, the identity of the intestinal SCFA/HCO3 (-) has been controversial. AIMS: The aim of this study was to determine the identities of the butyrate transporter in the intestinal epithelial cells (IEC-18). METHODS: IEC-18 cells were treated with specific siRNAs for MCT1 and MCT4, and butyrate and lactate uptake studies were performed. RESULTS: Alpha-cyano-4-hydroxycinnamic acid inhibited lactate uptake but not butyrate uptake in IEC-18 cells, indicating that these two substrates are transported via two different transporter systems. MCT1 siRNA treatment abolished both MCT1 mRNA by more than 95 % and protein expression by 83 % as evidenced by RTQ-PCR and western blotting experiments. However, MCT1 siRNA treatment inhibited butyrate uptake upto 24 %, whereas it inhibited lactate uptake significantly by 70 %. Treatment with MCT4 siRNA inhibited MCT4 mRNA expression by 75 % and protein expression by 85 % in these cells. MCT4 siRNA inhibited butyrate uptake by 40 %. Further, several non-steroidal anti-inflammatory drugs (NSAIDs) are transported by the butyrate transporter. Finally, MCT4 siRNA inhibited salicylate uptake by 27 % indicating direct evidence for the transport of salicylate by MCT4. CONCLUSIONS: These data indicate that MCT1 is the high affinity lactate transporter and MCT4 is the high affinity butyrate transporter in the intestinal epithelial cell line IEC-18.

Prostaglandins, not the leukotrienes, regulate Cl(-)/HCO(3)(-) exchange (DRA, SLC26A3) in villus cells in the chronically inflamed rabbit ileum.

Previously studies have demonstrated that Cl(-)/HCO(3)(-) exchange was inhibited during chronic intestinal inflammation secondary to decrease in the affinity of the exchanger for Cl(-) rather than the number of transporters. Arachidonic acid metabolites (AAM) are elevated in the mucosa of the chronically inflamed small intestine. However, their role in the alteration of Cl(-)/HCO(3)(-) during chronic enteritis was unknown. Inhibition of AAM formation with arachidonyl trifluoro methylketone (ATMK) in chronically inflamed rabbit intestine reversed the diminished Cl(-)/HCO(3)(-) exchange activity. Kinetics studies showed that the reversal was secondary to restoration of the altered affinity of transporter. Downstream regulation of Cl(-)/HCO(3)(-) inhibition by AAM was determined to be by the cyclooxygenase pathway since only inhibition of cyclooxygenase with piroxicam treatment reversed the inhibited Cl(-)/HCO(3)(-) exchange. Further, DRA was shown to be the primary Cl(-)/HCO(3)(-) exchanger in villus cells. Kinetics and molecular studies indicated that the mechanism of inhibition of Cl(-)/HCO(3)(-) exchange by cyclooxygenase pathway metabolites was secondary to diminished affinity of the transporter for Cl(-) without a change in DRA BBM expression. Thus our data indicated that cyclooxygenase pathway metabolites mediate the inhibition of DRA during chronic intestinal inflammation.

Myeloid-specific Krüppel-like factor 2 inactivation increases macrophage and neutrophil adhesion and promotes atherosclerosis.

RATIONALE: Hemizygous deficiency of the transcription factor Krüppel-like factor 2 (KLF2) has been shown previously to augment atherosclerosis in hypercholesterolemic mice. However, the cell type responsible for the increased atherosclerosis due to KLF2 deficiency has not been identified. This study examined the consequence of myeloid cell-specific KLF2 inactivation in atherosclerosis. METHODS AND RESULTS: Cell-specific knockout mice were generated by Cre/loxP recombination. Macrophages isolated from myeloid-specific Klf2 knockout (myeKlf2(-/-)) mice were similar to myeKlf2(+/+) macrophages in response to activation, polarization, and lipid accumulation. However, in comparison to myeKlf2(+/+) macrophages, myeKlf2(-/-) macrophages adhered more robustly to endothelial cells. Neutrophils from myeKlf2(-/-) mice also adhered more robustly to endothelial cells, and fewer myeKlf2(-/-) neutrophils survived in culture over a 24-hour period in comparison with myeKlf2(+/+) neutrophils. When myeKlf2(-/-) mice were mated to Ldlr(-/-) mice and then fed a high fat and high cholesterol diet, significant increase in atherosclerosis was observed in the myeKlf2(-/-)Ldlr(-/-) mice compared with myeKlf2(+/+)Ldlr(-/-) littermates. The increased atherosclerosis in myeKlf2(-/-)Ldlr(-/-) mice was associated with elevated presence of neutrophils and macrophages, with corresponding increase of myeloperoxidase as well as chlorinated and nitrosylated tyrosine epitopes in their lesion areas compared with myeKlf2(+/+)Ldlr(-/-) mice. CONCLUSIONS: This study documents a role for myeloid KLF2 expression in modulating atherosclerosis. The increased neutrophil accumulation and atherosclerosis progression with myeloid-specific KLF2 deficiency also underscores the importance of neutrophils in promoting vascular oxidative stress and atherosclerosis. Collectively, these results suggest that elevating KLF2 expression may be a novel strategy for prevention and treatment of atherosclerosis.