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Background and Aims Obesity‐induced chronic inflammation is a key component in the pathogenesis of nonalcoholic fatty liver disease (NAFLD) and insulin resistance. Increased secretion of proinflammatory cytokines by macrophages in metabolic tissues promotes disease progression. In the diet‐induced obesity (DIO) mouse model, activation of liver resident macrophages, or Kupffer cells (KCs), drives inflammatory responses, which recruits circulating macrophages and promotes fatty liver development, and ultimately contributes to impaired hepatic insulin sensitivity. Hepatic macrophages express the highest level of vitamin D receptors (VDRs) among nonparenchymal cells, whereas VDR expression is very low in hepatocytes. VDR activation exerts anti‐inflammatory effects in immune cells. Approach and Results Here we found that VDR activation exhibits strong anti‐inflammatory effects in mouse hepatic macrophages, including those isolated from DIO livers, and mice with genetic loss of Vdr developed spontaneous hepatic inflammation at 6 months of age. Under the chronic inflammation conditions of the DIO model, VDR activation by the vitamin D analog calcipotriol reduced liver inflammation and hepatic steatosis, significantly improving insulin sensitivity. The hyperinsulinemic euglycemic clamp revealed that VDR activation greatly increased the glucose infusion rate, while hepatic glucose production was remarkably decreased. Glucose uptake in muscle and adipose did not show similar effects, suggesting that improved hepatic insulin sensitivity is the primary contributor to the beneficial effects of VDR activation. Finally, specifically ablating liver macrophages by treatment with clodronate liposomes largely abolished the beneficial metabolic effects of calcipotriol, confirming that VDR activation in liver macrophages is required for the antidiabetic effect. Conclusions Activation of liver macrophage VDRs by vitamin D ligands ameliorates liver inflammation, steatosis and insulin resistance. Our results suggest therapeutic paradigms for treatment of NAFLD and type 2 diabetes mellitus.

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Vasculogenic mimicry (VM) is an alternative type of blood supplement and is responsible for aggressive tumor biology and increased tumor-related mortality. Tumor cells obtain oxygen and nutriment through VM channels in the early, rapid-growth stage when blood vessels are insufficient. VM channels are characterized by tubular structures with tumor cells. Autophagy is a catabolic process, by which the cell digests damaged components or organelles of its own cytoplasm in response to nutrient deprivation, hypoxia, and the presence of non-functional protein aggregates. In fact, autophagy plays an important role in normal cell growth, development, and homeostasis. However, it is still controversial whether autophagy is also involved in cell death or cell survival in malignancy. In the present study, we therefore investigated the expression levels of two autophagy-related proteins, microtubule-associated protein-1 light chain 3 (LC3) and beclin-1, with respect to melanoma metastasis and vasculogenic mimicry. Melanoma is characterized by rapid growth, high-metastasis rate, and unpredictable behavior. A total of 70 human melanoma tissues were analyzed, showing that VM was present in 31 melanoma specimens (44.3%). Melanoma cells displayed high levels of autophagy when VM was present. Real-time quantitative PCR and immunohistochemical analyses showed that the expression levels of beclin-1 and LC3 mRNAs and proteins were both higher in the VM-positive melanoma than those in the VM-negative melanoma (p < 0.05). Moreover, the expression of LC3, rather than beclin-1, was strongly associated with metastasis and poor clinical prognosis of human melanoma. Therefore, the enhanced autophagic activity may be related to VM and metastasis of melanoma.Keywords: melanoma; vasculogenic mimicry; microtubule-associated protein 1 light chain 3; beclin-1; melanoma metastasis Tohoku J. Exp. Med., 2011, 223 (4), 243-251. © 2011 Tohoku University Medical Press Melanoma is characterized by poorly differentiated rapid growth, high-metastasis rate, and unpredictable behavior (McKinnon et al. 2003). It continues to be one of the most aggressive and fatal malignancies once metastasized. When distant metastasis occurs, the cancer is generally considered incurable. The five-year survival rate is less than 10%, whereas the median survival is 6-12 months (Balch et al. 2001). Accordingly, inhibiting metastasis can be an effective therapeutic strategy and benefit patients with melanoma. Despite many intensive laboratories and clinical researches, the mechanism involved in metastasis of melanoma still remains unclear.Some studies have confirmed that the presence of vasculogenic mimicry (VM) channels, a matrix-rich vascularlike network, and the development of fluid-conducting pathways by highly invasive and genetically dysregulated tumor cells are associated with more aggressive and metastatic tumor biology (Maniotis et al. 1999;Hess et al. 2001). VM has been described in highly metastatic malignant tumors, including melanoma (Folberg and Maniotis 2004), in...

Cellular uptake of organic solutes is mediated in large part by a gene family of membrane transporters called OATPs (SLC21A). To study the structural determinants and evolutionary development of the SLC21A family, we have cloned and functionally characterized a highly expressed evolutionarily primitive Oatp from the liver of the small skate, Raja erinacea. A full-length cDNA (2.3 kb) was obtained that encodes a protein of 689 amino acids. The characteristics of this novel skate Oatp, including tissue expression, subcellular localization, substrate selectivity, Na+ dependence, and inhibitor selectivity were generally similar to liver-specific human OATP-C and rat Oatp4. However, sequence comparisons with other OATPs indicate that this skate Oatp shares only ∼40–50% amino acid identity with the liver-specific OATPs/Oatps and with human OATP-F. Further computer analysis revealed that the highest amino acid identities reside in the first external (78%) and internal loops (75%) and transmembrane domains 2 (76%), 3 (62%), 4 (70%), and 11 (64%). We propose that the conserved regions of the SLC21A transporter family may be critical structural determinants of substrate specificity and function.

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BackgroundAmino acid sequence alignment of phage phiC31 integrase with the serine recombinases family revealed highly conserved regions outside the catalytic domain. Until now, no system mutational or biochemical studies have been carried out to assess the roles of these conserved residues in the recombinaton of phiC31 integrase.Methodology/Principal FindingsTo determine the functional roles of these conserved residues, a series of conserved residues were targeted by site-directed mutagenesis. Out of the 17 mutants, 11 mutants showed impaired or no recombination ability, as analyzed by recombination assay both in vivo and in vitro. Results of DNA binding activity assays showed that mutants (R18A, I141A, L143A,E153A, I432A and V571A) exhibited a great decrease in DNA binding affinity, and mutants (G182A/F183A, C374A, C376A/G377A, Y393A and V566A) had completely lost their ability to bind to the specific target DNA attB as compared with wild-type protein. Further analysis of mutants (R18A, I141A, L143A and E153A) synapse and cleavage showed that these mutants were blocked in recombination at the stage of strand cleavage.Conclusions/SignificanceThis data reveals that some of the highly conserved residues both in the N-terminus and C-terminus region of phiC31 integrase, play vital roles in the substrate binding and cleavage. The cysteine-rich motif and the C-tail val-rich region of phiC31 integrase may represent the major DNA binding domains of phiC31 integrase.

MAPK6-AKT signaling promotes tumor growth and resistance to mTOR kinase inhibitors.