Cloning and characterization of the human USP22 gene promoter.

Ubiquitin-specific processing enzyme 22 (USP22) plays a direct role in regulating cell cycle, and its overexpression has been reported to be involved in tumor progression. However, little is known about the regulation of USP22 transcription. In this study, we cloned and characterized the human USP22 promoter. Using 5' RACE (rapid amplification of cDNA ends) analysis, the transcriptional initiation site was identified. Promoter deletion analysis showed that the sequence between -210 and -7 contains the basal promoter for USP22 in human fibroblast and tumor cells. Surprisingly, mutations in a putative Sp1 binding site immediately upstream of the USP22 transcriptional start site (-13 to -7) resulted in a significant induction of promoter activity. Further study revealed that Sp1 binds to this site in human normal fibroblast cells, and treatment with the Sp1 inhibitor mithramycin A led to a marked increase in USP22 transcript levels. Forced expression of exogenous Sp1 repressed the USP22 promoter activity in HeLa cells. In contrast, knockdown of Sp1 enhanced USP22 promoter activity and mRNA levels. These data suggest that Sp1 is a crucial regulator of USP22 transcription

Candidate genes responsible for lipid droplets formation during adipogenesis simultaneously affect osteoblastogenesis

Introduction. With cellular lipid storage varying, the balance between lipid intake and lipid degradation was a must to keep healthy and determined the level of lipid droplets. Although lipid droplets accumulation had been well demonstrated in adipocytes, gene expression profiling and gene function during adipogenesis and osteoblastogenesis remain unknown. Material and methods. Here, this work profiled gene transcriptional landscapes of lipid droplets formation during adipogenesis from human mesenchymal stem cells (hMSCs) using RNA-Seq technique. By using RNA interference (RNAi) we investigated the function of candidate genes during adipogenesis and osteoblastogenesis using Oil Red/Alizarin Red/alkaline phosphatase (ALPL) staining and qRT-PCR (quantitative real-time PCR). Results. Eleven differentially up-regulated genes associated with lipid droplets formation were identified at 3, 5, 7, 14, 21, and 28 days during adipogenesis. Unexpectedly, APOB per se inhibiting adipogenesis weakened osteoblastogenesis and METTL7A facilitating adipogenesis negligibly inhibited osteoblastogenesis according to the phenotypic characterization of adipocytes and osteoblasts and transcriptional condition of biomarkers through lentivirus transfection assays. Conclusions. The establishment of the gene transcriptional profiling of lipid droplets formation would providethe molecular switches of hMSCs cell fate determination and the study targets for fat metabolic diseases

The synergistic effect of dietary cholesterol with fruit tannins in forming kidney stones

Prevalence of kidney stones has increased continously over several decades worldwide, the major causes of which are largely unknown. To explore the dietary causes of kidney stones, and reveal mechanisms underlying dietary risk factors inducing kidney stones, animal experiments using mice as the disease model were performed. Eight-week old male CD-1 mice were treated by ethylene glycol, cholesterol or/and apple tannins for 3 d, respectively. In the present study, the crystalline analysis in urine and kidney tissues, HE staining kidney sections as well as observation of micro-stones, tannins and cholesterol deposition in kidneys of mice in different groups were conducted. We found that gavage with ethylene glycol, cholesterol and tannins resulted in mice urine solute supersaturation in renal tubules and forming kidney stones. Significant cholesterol and tannin deposits in mouse kidney were observed by laser confocal microscopy and crystals were shown either adhered with or co-deposited with cholesterol and tannin deposits. The primary crystals were found in renal cortex, medullar, especially papilla in the kidney sections under polarized microscope. These findings demonstrate that interaction of cholesterol and tannins in kidney plays a critical role in the formation of kidney stones

SIKVAV-Modified Chitosan Hydrogel as a Skin Substitutes for Wound Closure in Mice

Skin wound healing is a complex and dynamic process that involves angiogenesis and growth factor secretion. Newly formed vessels can provide nutrition and oxygen for skin wound healing. Growth factors in skin wounds are important for keratinocytes and fibroblasts proliferation, epithelialization, extracellular matrix remodeling, and angiogenesis, which accelerate skin wound healing. Therefore, treatment strategies that enhance angiogenesis and growth factors secretion in skin wounds can accelerate skin wound healing. This study investigated the effects of a SIKVAV (Ser-Ile-Lys-Val-Ala-Val) peptide-modified chitosan hydrogel on skin wound healing. Hematoxylin and eosin (H&E) staining demonstrated that the SIKVAV-modified chitosan hydrogel accelerated the re-epithelialization of wounds compared with that seen in the negative and positive controls. Masson’s trichrome staining showed that more collagen fibers were deposited in the skin wounds treated with the SIKVAV-modified chitosan hydrogel than in the negative and positive controls. Immunohistochemistry assays demonstrated that more myofibroblasts were deposited and more angiogenesis occurred in skin wounds treated with the SIKVAV-modified chitosan hydrogel than in the negative and positive controls. In addition, ELISA assays showed that the SIKVAV-modified chitosan hydrogels promoted the secretion of growth factors in skin wounds. Taken together, these results suggest that the SIKVAV-modified chitosan hydrogel has the potential to be developed as synthesized biomaterials for the treatment of skin wounds

Insulin protects apoptotic cardiomyocytes from hypoxia/reoxygenation injury through the sphingosine kinase/sphingosine 1-phosphate axis.

OBJECTIVE: Experimental and clinical studies have shown that administration of insulin during reperfusion is cardioprotective, but the mechanisms underlying this effect are still unknown. In this study, the ability of insulin to protect apoptotic cardiomyocytes from hypoxia/reoxygenation injury using the sphingosine kinase/sphingosine 1-phosphate axis was investigated. METHODS AND RESULTS: Rat cardiomyocytes were isolated and subjected to hypoxia and reoxygenation. [γ-32P] ATP was used to assess sphingosine kinase activity. Insulin was found to increase sphingosine kinase activity. Immunocytochemistry and Western blot analysis showed changes in the subcellular location of sphingosine kinase 1 from cytosol to the membrane in cardiomyocytes. Insulin caused cardiomyocytes to accumulate of S1P in a dose-dependent manner. FRET efficiency showed that insulin also transactivates the S1P1 receptor. TUNEL staining showed that administration of insulin during reoxygenation could to reduce the rate of reoxygenation-induced apoptosis, which is a requirement for SphK 1 activity. It also reduced the rate of activation of the S1P receptor and inhibited hypoxia/reoxygenation-induced cell death in cardiomyocytes. CONCLUSION: The sphingosine kinase 1/sphingosine 1-phosphate/S1P receptor axis is one pathway through which insulin protects rat cardiomyocytes from apoptosis induced by hypoxia/reoxygenation injury

Mutation analyses of the Sp1-binding site.

<p><b>A.</b> Nucleotide sequence and structural organization of the <i>USP22</i> gene core promoter region. Putative binding sites for the transcriptional factors are underlined. <b>B.</b> Luciferase activity expressed by the Sp1 site-directed mutant and deletion mutants relative to pGL3-basic activity.</p

Identification of the transcriptional start site of the <i>USP22</i> gene.

<p><b>A.</b> Results of 5′ RACE experiments on total RNA from HeLa cells; DNA sequences were detected by gel electrophoresis using 2% agarose. Lane 1. A single DNA band of 260 bp was detected; Lane 2. DL2000 marker. <b>B.</b> Sequence of <i>USP22</i> gene depicting the positions of the transcriptional start sites. <b>C.</b> Compared with the existing information in the human genome database, the DNA sequence of the HeLa clone contains two mutations, A–C and A–C, at positions 280 and 283 upstream from the transcriptional start site.</p

Primers used in the generation of promoter luciferase constructs.

<p>Primers used in the generation of promoter luciferase constructs.</p

Determination of Sp1 binding to the <i>USP22</i> gene promoter.

<p><b>A.</b> DNA was isolated from HFL1 cells in each group and immunoprecipitated with antibodies against Sp1, RNA polymerase II or nonspecific rat IgG. Input and immunoprecipitated DNAs were then PCR amplified using primer pairs covering the Sp1-binding site. <b>B.</b> HFL1 cells expressing p-210 or p-210/Sp1mut constructs were treated with 10 um of mithramycin A or vehicle. Luciferase activity was determined 24 h later (*, <i>p</i><0.05 <i>vs.</i> vehicle treatment).</p

SphK activity and insulin-induced inhibition of hypoxia-reoxygenation-induced cell death in cardiomyocytes.

<p>A: Effects of HACPT on insulin in cardiomyocytes, cardiomyocytes subjected to hypoxia-reoxygenation, and cardiomyocytes during reoxygenation, all cultured with empty vehicle (vehicle, 0.05% dimethylsulfoxide and 0.05% methanol) with 50 µM HACPT, as indicated by white and gray bars, respectively. Cells were treated with control or SphK1-siRNA and cultured for 72 h. Then cells were subjected to hypoxia and treated either with nothing, S1P, or insulin. B: Cardiomyocytes were transiently transfected and reoxygenated. Cells were stimulated with nothing, S1P, or insulin during reoxygenation. TUNEL-positive cardiomyocytes/total cardiomyocytes×100% (AI). Data are means ± S.E. of five independent experiments carried out in triplicate.</p