Margit Mahlapuu

Forkhead Transcription Factor FoxF2 Is Expressed in Mesodermal Tissues Involved in Epithelio-Mesenchymal Interactions

The growing family of forkhead transcription factors plays many important roles during embryonic development. In this study we have used in situ hybridization to explore the expression pattern of the forkhead transcription factor gene FoxF2 (FREAC‐2, LUN) during mouse and rat embryogenesis, postnatal development, and in adult tissues. We demonstrate that FoxF2 is expressed in the mesenchyme adjacent to the epithelium in alimentary, respiratory, and urinary tracts, similar to FoxF1 (FREAC‐1, HFH‐8). FoxF2 mRNA was also observed in organs that do not express FoxF1 during embryogenesis, e.g., in the central nervous system, eye, ear, and limb buds. In organs that express both FoxF2 and FoxF1, these transcription factors may have similar functions in epithelio‐mesenchymal crosstalk, but the fact that FoxF2 is more widely expressed than FoxF1 suggests that FoxF2 also has an independent role as a developmental regulator. Dev Dyn;218:136–149.

Antimicrobial Peptides: An Emerging Category of Therapeutic Agents

Antimicrobial peptides (AMPs), also known as host defense peptides, are short and generally positively charged peptides found in a wide variety of life forms from microorganisms to humans. Most AMPs have the ability to kill microbial pathogens directly, whereas others act indirectly by modulating the host defense systems. Against a background of rapidly increasing resistance development to conventional antibiotics all over the world, efforts to bring AMPs into clinical use are accelerating. Several AMPs are currently being evaluated in clinical trials as novel anti-infectives, but also as new pharmacological agents to modulate the immune response, promote wound healing, and prevent post-surgical adhesions. In this review, we provide an overview of the biological role, classification, and mode of action of AMPs, discuss the opportunities and challenges to develop these peptides for clinical applications, and review the innovative formulation strategies for application of AMPs.

Role of AMP-activated protein kinase in the coordinated expression of genes controlling glucose and lipid metabolism in mouse white skeletal muscle

Aims/hypothesisAMP-activated protein kinase (AMPK) regulates metabolic adaptations in skeletal muscle. The aim of this study was to investigate whether AMPK modulates the expression of skeletal muscle genes that have been implicated in lipid and glucose metabolism under fed or fasting conditions.MethodsTwo genetically modified animal models were used: AMPK γ3 subunit knockout mice (Prkag3−/−) and skeletal muscle-specific transgenic mice (Tg-Prkag3225Q) that express a mutant (R225Q) γ3 subunit. Levels of mRNA transcripts of genes involved in lipid and glucose metabolism in white gastrocnemius muscles of these mice (under fed or 16-h fasting conditions) were assessed by quantitative real-time PCR.ResultsWild-type mice displayed a coordinated increase in the transcription of skeletal muscle genes encoding proteins involved in lipid/oxidative metabolism (lipoprotein lipase, fatty acid transporter, carnitine palmitoyl transferase-1 and citrate synthase) and glucose metabolism (glycogen synthase and lactate dehydrogenase) in response to fasting. In contrast, these fasting-induced responses were impaired in Prkag3−/− mice. The transcription of genes involved in lipid and oxidative metabolism was increased in the skeletal muscle of Tg-Prkag3225Q mice compared with that in wild-type mice. Moreover, the expression of the genes encoding hexokinase II and 6-phosphofrucktokinase was decreased in Tg-Prkag3225Q mice after fasting.Conclusions/interpretationAMPK is involved in the coordinated transcription of genes critical for lipid and glucose metabolism in white glycolytic skeletal muscle.

AMP-activated protein kinase inhibits IL-6-stimulated inflammatory response in human liver cells by suppressing phosphorylation of signal transducer and activator of transcription 3 (STAT3)

Aim/hypothesisThe aim of the study was to examine the possible role of AMP-activated protein kinase (AMPK) in the regulation of the inflammatory response induced by cytokine action in human liver cells.MethodsIL-6-stimulated expression of the genes for acute-phase response markers serum amyloid A (SAA1, SAA2) and haptoglobin (HP) in the human hepatocarcinoma cell line HepG2 were quantified after modulation of AMPK activity by pharmacological agonists (5-amino-4-imidazole-carboxamideriboside [AICAR], metformin) or by using small interfering (si) RNA transfection. The intracellular signalling pathway mediating the effect of AMPK on IL-6-stimulated acute-phase marker expression was characterised by assessing the phosphorylation levels of the candidate protein signal transducer and activator of transcription 3 (STAT3) in response to AMPK agonists.ResultsAICAR and metformin markedly blunt the IL-6-stimulated expression of SAA cluster genes as well as of haptoglobin in a dose-dependent manner. Moreover, the repression of AMPK activity by siRNA significantly reversed the inhibition of SAA expression by both AICAR and metformin, indicating that the effect of the agonists is dependent on AMPK. For the first time we show that AMPK appears to regulate IL-6 signalling by directly inhibiting the activation of the main downstream target of IL-6, STAT3.Conclusions/interpretationWe provide evidence for a key function of AMPK in suppression of the acute-phase response caused by the action of IL-6 in liver, suggesting that AMPK may act as an intracellular link between chronic low-grade inflammation and metabolic regulation in peripheral metabolic tissues.

Relationship between AMPK and the transcriptional balance of clock-related genes in skeletal muscle

Circadian clocks coordinate physiological, behavioral, and biochemical events with predictable daily environmental changes by a self-sustained transcriptional feedback loop. CLOCK and ARNTL are transcriptional activators that regulate Per and Cry gene expression. PER and CRY inhibit their own transcription, and their turnover allows this cycle to restart. The transcription factors BHLHB2 and BHLHB3 repress Per activation, whereas orphan nuclear receptors of the NR1D and ROR families control Arntl expression. Here we show the AMP-activated protein kinase (AMPK)gamma(3) subunit is involved in the regulation of peripheral circadian clock function. AMPKgamma3 knockout (Prkag3(-/-)) mice or wild-type littermates were injected with saline or an AMPK activator, 5-amino-4-imidazole-carboxamide riboside (AICAR), and white glycolytic gastrocnemius muscle was removed for gene expression analysis. Genes involved in the regulation of circadian rhythms (Cry2, Nr1d1, and Bhlhb2) were differentially regulated in response to AICAR in wild-type mice but remained unaltered in Prkag3(-/-) mice. Basal expression of Per1 was higher in Prkag3(-/-) mice compared with wild-type mice. Distinct diurnal changes in the respiratory exchange ratio (RER) between the light and dark phase of the day were observed in wild-type mice but not Prkag3(-/-) mice. In summary, the expression profile of clock-related genes in skeletal muscle in response to AICAR, as well as the diurnal shift in energy utilization, is impaired in AMPKgamma(3) subunit knockout mice. Our results indicate AMPK heterotrimeric complexes containing the AMPKgamma(3) subunit may play a specific role in linking circadian oscillators and energy metabolism in skeletal muscle.

Opposite Transcriptional Regulation in Skeletal Muscle of AMP-activated Protein Kinase γ3 R225Q Transgenic Versus Knock-out Mice

AMP-activated protein kinase (AMPK) is an evolutionarily conserved heterotrimer important for metabolic sensing in all eukaryotes. The muscle-specific isoform of the regulatory γ-subunit of the kinase, AMPK γ3, has an important role in glucose uptake, glycogen synthesis, and fat oxidation in white skeletal muscle, as previously demonstrated by physiological characterization of AMPK γ3 mutant (R225Q) transgenic (TgPrkag3225Q) and γ3 knock-out (Prkag3-/-) mice. We determined AMPK γ3-dependent regulation of gene expression by analyzing global transcription profiles in glycolytic skeletal muscle from γ3 mutant transgenic and knock-out mice using oligonucleotide microarray technology. Evidence is provided for coordinated and reciprocal regulation of multiple key components in glucose and fat metabolism, as well as skeletal muscle ergogenics in TgPrkag3225Q and Prkag3-/- mice. The differential gene expression profile was consistent with the physiological differences between the models, providing a molecular mechanism for the observed phenotype. The striking pattern of opposing transcriptional changes between TgPrkag3225Q and Prkag3-/- mice identifies differentially expressed targets being truly regulated by AMPK and is consistent with the view that R225Q is an activating mutation, in terms of its downstream effects. Additionally, we identified a wide array of novel targets and regulatory pathways for AMPK in skeletal muscle.

The Human Forkhead Protein FREAC-2 Contains Two Functionally Redundant Activation Domains and Interacts with TBP and TFIIB

Forkhead-related activator 2 (FREAC-2) is a human transcription factor expressed in lung and placenta that binds tocis-elements in several lung-specific genes. We have identified the parts of FREAC-2 responsible fortrans-activation and found two functionally redundant activation domains on the C-terminal side of the DNA binding forkhead domain. Activation domain 1 consists of the most C-terminal 23 amino acids of FREAC-2 and contains a sequence motif conserved in an activation domain of another forkhead protein, FREAC-1. Activation domain 2 is built up by three synergistic subdomains in the central part of the FREAC-2 protein. FREAC-2 was shown to interact in vitro with TBP and TFIIB. The target site for FREAC-2 on TBP was localized to the N-terminal repeat in the core domain of TBP. TFIIB binds FREAC-2 close to the cleft between its two globular domains. The part of FREAC-2 that binds TBP was mapped to 21 amino acids in the C-terminal end of the forkhead domain. This sequence is well conserved among forkhead proteins, raising the possibility that interaction with TBP may be a general characteristic of this family of transcription factors. Overexpression of TFIIB potentiates activation by FREAC-2 in a manner dependent on the FREAC-2 activation domains. Nuclear localization of FREAC-2 was found to depend on sequences from both ends of the forkhead domain.

Administration of Lactobacillus evokes coordinated changes in the intestinal expression profile of genes regulating energy homeostasis and immune phenotype in mice

Lactic acid bacteria are probiotics widely used in functional food products, with a variety of beneficial effects reported. Recently, intense research has been carried out to provide insight into the mechanism of the action of probiotic bacteria. We have used gene array technology to map the pattern of changes in the global gene expression profile of the host caused by Lactobacillus administration. Affymetrix microarrays were applied to comparatively characterize differences in gene transcription in the distal ileum of normal microflora (NMF) and germ-free (GF) mice evoked by oral administration of two Lactobacillus strains used in fermented dairy products today - Lactobacillus paracasei ssp. paracasei F19 (L. F19) or Lactobacillus acidophilus NCFB 1748. We show that feeding either of the two strains caused very similar effects on the transcriptional profile of the host. Both L. F19 and L. acidophilus NCFB 1748 evoked a complex response in the gut, reflected by differential regulation of a number of genes involved in essential physiological functions such as immune response, regulation of energy homeostasis and host defence. Notably, the changes in intestinal gene expression caused by Lactobacillus were different in the mice raised under GF v. NMF conditions, underlying the complex and dynamic nature of the host-commensal relationship. Differential expression of an array of genes described in this report evokes novel hypothesis of possible interactions between the probiotic bacteria and the host organism and warrants further studies to evaluate the functional significance of these transcriptional changes on the metabolic profile of the host.

Treatment with LL-37 is safe and effective in enhancing healing of hard-to-heal venous leg ulcers : a randomized, placebo-controlled clinical trial

Venous leg ulcers (VLUs) are one of the most prevalent types of chronic wounds. The aim of this study was to determine the safety and dose–response efficacy of the human synthetic peptide LL‐37 in the treatment of hard‐to‐heal VLUs. This first‐in‐man trial included 34 participants with VLUs and comprised a 3‐week, open‐label, run‐in period on placebo, followed by a 4‐week randomized double‐blind treatment phase with twice weekly applications of LL‐37 (0.5, 1.6, or 3.2 mg/mL) or placebo, and a 4‐week follow‐up. The healing rate constants for 0.5 and 1.6 mg/mL of LL‐37 were approximately six‐ and threefold higher than for placebo (p = 0.003 for 0.5 mg/mL and p = 0.088 for 1.6 mg/mL). Square‐root transformed wound area data showed improved healing for the 0.5 and 1.6 mg/mL dose groups compared with pretreatment values (p < 0.001 and p = 0.011, respectively). Consistently, treatment with the two lower doses markedly decreased the mean ulcer area (68% for 0.5 mg/mL and 50% for 1.6 mg/mL groups). No difference in healing was observed between the groups receiving 3.2 mg/mL of LL‐37 and placebo. There were no safety concerns regarding local or systemic adverse events. In conclusion, topical treatment with LL‐37 for chronic leg ulcers was safe and well tolerated with the marked effect on healing predictors at the two lower doses warranting further investigations.

Protein kinase STK25 regulates hepatic lipid partitioning and progression of liver steatosis and NASH

Nonalcoholic fatty liver disease (NAFLD) is the most common form of liver disease, and 10% to 20% of NAFLD patients progress to nonalcoholic steatohepatitis (NASH). The molecular pathways controlling progression to NAFLD/NASH remain poorly understood. We recently identified serine/threonine protein kinase 25 (STK25) as a regulator of whole‐body insulin and glucose homeostasis. This study investigates the role of STK25 in liver lipid accumulation and NASH. Stk25 transgenic mice challenged with a high‐fat diet displayed a dramatic increase in liver steatosis and hepatic insulin resistance compared to wild‐type siblings. Focal fibrosis, hepatocellular damage, and inflammation were readily seen in transgenic but not wild‐type livers. Transgenic livers displayed reduced β‐oxidation and triacylglycerol secretion, while lipid uptake and synthesis remained unchanged. STK25 was associated with lipid droplets, colocalizing with the main hepatic lipid droplet‐coating protein adipose differentiation‐related protein, the level of which was increased 3.8 ± 0.7‐fold in transgenic livers (P < 0.01), while a key hepatic lipase, adipose triacylglycerol lipase, was translocated from the lipid droplets surface to the cytoplasm, providing the likely mechanism underlying the effect of STK25. In summary, STK25 is a lipid droplet‐associated protein that promotes NAFLD through control of lipid release from the droplets for β‐oxidation and triacylglycerol secretion. STK25 also drives pathogenesis of NASH.—Amrutkar, M., Cansby, E., Nuñez‐Durán, E., Pirazzi, C., Ståhlman, M., Stenfeldt, E., Smith, U., Borén, J., Mahlapuu, M. Protein kinase STK25 regulates hepatic lipid partitioning and progression of liver steatosis and NASH. FASEB J. 29, 1564‐1576 (2015). www.fasebj.org