All-trans retinoic acid ameliorates glycemic control in diabetic mice via modulating pancreatic islet production of vascular endothelial growth factor-A.

Patients with type 1 diabetes mellitus are associated with impairment in vitamin A metabolism. This study evaluated whether treatment with retinoic acid, the biologically active metabolite of vitamin A, can ameliorate diabetes. All-trans retinoic acid (atRA) was used to treat streptozotocin (STZ)-induced diabetic mice which revealed atRA administration ameliorated blood glucose levels of diabetic mice. This hyperglycemic amelioration was accompanied by an increase in the amount of ß cells co-expressed Pdx1 and insulin and by restoration of the vascular laminin expression. The atRA-induced production of vascular endothelial growth factor-A from the pancreatic islets was possibly the key factor that mediated the restoration of islet vascularity and recovery of ß-cell mass. Furthermore, the combination of islet transplantation and atRA administration significantly rescued hyperglycemia in diabetic mice. These findings suggest that vitamin A derivatives can potentially be used as a supplementary treatment to improve diabetes management and glycemic control.

ABCG2 deficiency in skin impairs re-epithelialization in cutaneous wound healing.

The ATP-binding cassette transporter ABCG2 is expressed in the interfollicular epidermis and mediates the side-population phenotype in skin cells. However, the role of ABCG2 in skin is unclear. Increased expression levels of ABCG2 were found at the basal layer of transitional epidermis adjacent to cutaneous wounds in human patients, indicating that ABCG2 may be involved in regulating the wound healing process. To investigate the role of ABCG2 in cutaneous wound healing, full-thickness skin wounds were created in ABCG2 knockout (ABCG2-KO) and wild-type mice. The healing process was analysed and revealed that ABCG2 deficiency in skin results in delays in wound closure and impairments in re-epithelialization, as evidenced by reductions in both suprabasal differentiation and in p63-expressing keratinocytes migrating from transitional epidermis to epithelial tongues. The reduction in p63-expressing cells may be due to elevated levels of reactive oxygen species in ABCG2-KO epidermis, which can cause DNA damage and lead to proliferation arrest. To determine whether ABCG2 deficiency affects the potency of epidermal stem/progenitor cells (EPCs), transplantation studies were carried out, which demonstrated that ABCG2-KO EPCs display higher levels of γH2AX and lose the capacity to differentiate into suprabasal keratinocytes. A competitive repopulation assay confirmed that ABCG2 expression is critical for the proper expansion and differentiation of EPCs in cutaneous wounds. As EPCs are known to contribute to the healing of larger wounds, the current findings imply a functional role for ABCG2 in the expansion and differentiation of p63-expressing EPCs. Thus, ABCG2 deficiency in skin impairs re-epithelialization in cutaneous wound healing.

Label-free quantitative proteomics of CD133-positive liver cancer stem cells.

BACKGROUND: CD133-positive liver cancer stem cells, which are characterized by their resistance to conventional chemotherapy and their tumor initiation ability at limited dilutions, have been recognized as a critical target in liver cancer therapeutics. In the current work, we developed a label-free quantitative method to investigate the proteome of CD133-positive liver cancer stem cells for the purpose of identifying unique biomarkers that can be utilized for targeting liver cancer stem cells. Label-free quantitation was performed in combination with ID-based Elution time Alignment by Linear regression Quantitation (IDEAL-Q) and MaxQuant. RESULTS: Initially, IDEAL-Q analysis revealed that 151 proteins were differentially expressed in the CD133-positive hepatoma cells when compared with CD133-negative cells. We then analyzed these 151 differentially expressed proteins by MaxQuant software and identified 10 significantly up-regulated proteins. The results were further validated by RT-PCR, western blot, flow cytometry or immunofluorescent staining which revealed that prominin-1, annexin A1, annexin A3, transgelin, creatine kinase B, vimentin, and EpCAM were indeed highly expressed in the CD133-positive hepatoma cells. CONCLUSIONS: These findings confirmed that mass spectrometry-based label-free quantitative proteomics can be used to gain insights into liver cancer stem cells.

Elevation of ß-galactoside α2,6-sialyltransferase 1 in a fructoseresponsive manner promotes pancreatic cancer metastasis.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive type of pancreatic cancer with clinical characteristics of local invasion and early metastasis. Recent cohort studies indicate high fructose intake is associated with an increase in pancreatic cancer risk. However, the mechanisms by which fructose promotes pancreatic tumorigenesis remain unclear. Herein, Kras+/LSLG12D mice were crossed with Elas-CreER transgenic mice to determine whether fructose intake directly contributes to tumor formation. Orthotopic tumor-xenograft experiments were performed to determine whether fructose substitution enhances the metastatic potential of PDAC cells. The mechanisms underlying the effects of fructose were explored by RNAseq analysis in combination with high-performance anion exchange chromatography. Dietary fructose was initially found to promote the development of aggressive pancreatic cancer in mice conditionally expressing KrasG12D in the adult pancreas. We further revealed that fructose substitution enhanced the metastatic potential of human PDAC cell via selective outgrowth of aggressive ABCG2-positive subpopulations and elevating N-acetylmannosamine levels that upregulated ß-galactoside α2,6-sialyltransferase 1 (ST6Gal1), thereby promoting distant metastasis. Finally, we observed that PDAC patients expressing higher levels of ST6Gal1 and GLUT5 presented poorer prognosis compared to other groups. In conclusion, our findings have elucidated a crucial role of ST6Gal1 in regulating the invasiveness of PDACs in a fructose-responsive manner.

Maternal vitamin A deficiency during pregnancy affects vascularized islet development.

Vitamin A deficiency is known to affect 20 million pregnant women worldwide. However, the prenatal effects of maternal vitamin A deficiency on pancreas development have not been clearly determined. The present study examined how maternal vitamin A deficiency affects fetal islet development. Vitamin A-deficient mice were generated by feeding female mice with a chemically defined diet lacking vitamin A prior to mating as well as during pregnancy. We found that maternal vitamin A deficiency during pregnancy affected fetal pancreas development. Although the exocrine differentiation appeared normal, development of islet tissue was impaired. In the pancreas of neonatal mice, only a few endocrine cell clusters were formed, and these cell clusters lacked capillary endothelial cells. To further determine how vitamin A metabolites, such as retinoic acid, regulate vascularized islet development, ex vivo culture of embryonic pancreas either in the presence of 4-diethylaminobenzaldehyde (DEAB; an inhibitor of retinaldehyde dehydrogenase), all-trans retinoic acid (atRA) or retinoic acid receptor agonist (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthylenyl)-1-propenyl] benzoic acid (TTNPB) was carried out. We found that the addition of DEAB blocked vascularization and suppressed ß-cell differentiation. Conversely, atRA or TTNPB promoted ß-cell differentiation accompanied by enhanced expression of vascular basement component, laminin. We further demonstrated that atRA regulated vascularization via upregulating vascular endothelial growth factor-A (VEGF-A) secretion in embryonic pancreas and treatment with VEGF-A was able to partially rescue vascularization and ß-cell differentiation in DEAB-treated embryonic pancreas cultures. The findings explain why maternal vitamin A deficiency affects fetal islet development and support an essential role of retinoid signaling in regulating vascularized islet development.

Protoporphyrin IX accumulation disrupts mitochondrial dynamics and function in ABCG2-deficient hepatocytes.

Targeted inhibition of multidrug ABCG2 transporter is believed to improve cancer therapeutics. However, the consequences of ABCG2 inhibition have not been systematically evaluated since ABCG2 is expressed in several organs including the liver. Here, we demonstrate that ABCG2-deficient hepatocytes have increased amounts of fragmental mitochondria accompanied by disruption of mitochondrial dynamics and functions. This disruption was due to ABCG2 knockout elevating intracellular protoporphyrin IX, which led to upregulation of DRP-1-mediated mitochondrial fission. The finding that ABCG2 deficiency can generate dysfunctional mitochondria in hepatocytes raises concerns regarding the systematic use of ABCG2 inhibitor in cancer patients.

Reduced 2,4-dinitro-1-fluorobenzene-induced contact hypersensitivity response in IL-15 receptor alpha-deficient mice correlates with diminished CCL5/RANTES and CXCL10/IP-10 expression.

Using a model of 2,4-dinitro-1-fluorobenzene-induced contact hypersensitivity (CHS) we found that, as compared with wild-type mice, IL-15 receptor alpha chain (IL-15Ralpha)-deficient mice showed significantly less ear swelling. This decreased response was associated with diminished expression of CCL5/RANTES and CXCL10/IP-10, chemokines critical for effector cell recruitment, in the inflamed tissue. We determined that both the number of CD8(+) T cells infiltrating the affected skin and the production of CCL5/RANTES by antigen-stimulated CD8(+) T cells were decreased in IL-15Ralpha(-/-) mice. The lower levels of CXCL10/IP-10 suggested that the IL-15Ralpha(-/-) mice had reduced production of IFN-gamma, the primary inducer of CXCL10/IP-10, which was in fact the case. However, by contrast with CCL5/RANTES, the diminished levels of IFN-gamma were likely due to the decreased number of skin-infiltrating CD8(+) T cells, since IFN-gamma production by antigen-stimulated CD8(+) T cells was comparable between wild-type and IL-15Ralpha(-/-) mice. Our data suggest a positive, pro-inflammatory feedback loop involving CCL5/RANTES, IFN-gamma and CXCL10/IP-10 that underlies the CHS reaction and that is disrupted, likely primarily by a defect in CCL5/RANTES production, in mice lacking IL-15Ralpha, resulting in impaired leukocyte recruitment and inflammation. Moreover, it is particularly noteworthy that the defect in CCL5/RANTES expression in CD8(+) T cells is intrinsic to the absence of IL-15Ralpha, indicating that IL-15Ralpha is critical for CCL5/RANTES expression in CD8(+) T cells.