Guoping Ai

ER stress induced by ionising radiation in IEC-6 cells

Purpose: Ionising radiation (IR) can evoke a series of biochemical events inside the cell. However, whether IR can directly induce endoplasmic reticulum (ER) stress is not clear. In our previous study, we found that there might be a causative link between IR and ER stress. In this study, we further characterised the type of ER stress induced by IR. Materials and methods: Rat intestinal epithelial cells IEC-6 were irradiated at a dose of 10 Gy, and total RNA and proteins were harvested at indicated time points. The mRNA and protein expression of immunoglobulin heavy chain binding protein (BiP) and glucose regulated protein 94 (GRP94) was detected along with proteins associated with ER stress signal pathways. Results: Our results indicated that IR induced up-regulation of ER stress marker including BiP and GRP94 at protein and mRNA levels in IEC-6 cells. Increased phosphorylation of eukaryotic translation initiation factor 2 (eIF2α) and induced mRNA splicing of X-box binding protein 1 (XBP1) suggested that PERK (interferon-induced double-stranded RNA-activated protein kinase (PRKR) -like endoplasmic reticulum kinase) and IRE1 (inositol requirement 1) signal transduction pathways were involved in this kind of ER stress. However, the active form of activating transcription factor 6 (ATF6) did not change significantly in irradiated cells, which suggested that the ATF6 pathway was not involved. Conclusions: Thus, we concluded that IR could induce moderate ER stress directly in IEC-6 cells.

Recruitment of Transplanted Dermal Multipotent Stem Cells to Sites of Injury in Rats with Combined Radiation and Wound Injury by Interaction of SDF-1 and CXCR4

Abstract Zong, Z-W., Cheng, T-M., Su, Y-P., Ran, X-Z., Shen, Y., Li, N., Ai, G-P., Dong, S-W. and Xu, H. Recruitment of Transplanted Dermal Multipotent stem Cells to the Sites of Injury in Rats with Combined Radiation and Wound Injury by Interaction of SDF-1 and CXCR4. Radiat. Res. 170, 444–450 (2008). Systemic transplantation of dermal multipotent stem cells has been shown to accelerate both hematopoietic recovery and wound healing in rats with combined radiation and wound injury. In the present study, we explored the mechanisms governing the recruitment of dermal multipotent stem cells to the sites of injury in rats with combined injury. Male dermal multipotent stem cells were transplanted into female rats, and using quantitative real-time PCR for the sex-determining region of Y chromosome, it was found that the amounts of dermal multipotent stem cells in irradiated bone marrow and wounded skin were far greater than those in normal bone marrow and skin (P < 0.01). However, incubation of dermal multipotent stem cells with AMD3100 before transplantation, which specifically blocks binding of stromal cell-derived factor 1 (SDF-1) to its receptor CXCR4, diminished the recruitment of dermal multipotent stem cells to the irradiated bone marrow and wounded skin by 58 ± 4% and 60 ± 4%, respectively (P < 0.05). In addition, it was confirmed that the expression of SDF-1 in irradiated bone marrow and wounded skin was up-regulated compared to that in their normal counterparts, and in vitro analysis revealed that irradiated bone marrow and wounded skin extracts had a strong chemotactic effect on dermal multipotent stem cells but that the effect decreased significantly when dermal multipotent stem cells were preincubated with AMD3100 (P < 0.05). These data suggest that transplanted dermal multipotent stem cells were recruited more frequently to the irradiated bone marrow and wounded skin than normal bone marrow and skin and that the interactions of SDF-1 and CXCR4 played a crucial role in this process.

Topical application of hPDGF-A-modified porcine BMSC and keratinocytes loaded on acellular HAM promotes the healing of combined radiation-wound skin injury in minipigs

Purpose: To evaluate the efficacy of cultured cutaneous substitute (CCS) in accelerating the healing of combined radiation-skin wound injury (CRWI) in minipigs. Material and methods: Autologous porcine bone marrow-derived mesenchymal stem cells (BMSC) and skin-derived keratinocytes (SK) were infected by recombinant retrovirus expressing human (h) platelet-derived growth factor-A (hPDGF-A). CCS was constructed by loading acellular human amniotic membrane (HAM) with normal porcine BMSC and SK (BMSC−/SK−CCS) or with hPDGF-A modified counterparts (BMSC+/SK+CCS). The expression of exogenous hPDGF-A in cells and CCS was assessed by reverse transcription polymerase chain reaction (RT-PCR) and enzyme linked immunosorbent assay (ELISA). The CCS or HAM were grafted to the dorsal CRWI sites (20 Gy local irradiation plus full-thickness skin removal, diameter = 40 mm) of minipigs. Wound healing rate and pathological changes were observed. Results: High levels of hPDGF-A expression were confirmed in gene-modified cells (3780 pg/ml), cultured CCS (506 pg/ml) and transplanted CCS (250 pg/ml). The transplantation of the BMSC+/SK+CCS resulted in a shorter healing time (16–18, days) (P < 0.05 vs. other groups). The healing rates ranked as BMSC+/SK+CCS > BMSC−/SK−CCS > HAM > wound control. Pathologically, there were better granulation formation and re-epithelialisation, and collagen deposition in BMSC+/SK+CCS-treated wound than those in other groups. The angiogenesis ability followed the same order as healing rate of different groups. At day 7, the area densities of vasculature in granulation tissue of group BMSC+/SK+CCS, BMSC−/SK−CCS, HAM, wound only were 15.4, 10.3, 6.0 and 5.7%, respectively, while the number densities of vasculature was 767, 691, 126 and 109 (number/mm2), respectively. Conclusions: Topical transplantation of hPDGF-A modified CCS may be applicable to the management of refractory wounds.

DNA synthesis of rat bone marrow mesenchymal stem cells through α1-adrenergic receptors

Multipotential bone marrow mesenchymal stem cells (BMSCs) are important in maintaining the microenvironment of the bone marrow (BM). Sympathetic nerves histologically innervate the BM; however, their role remains unclear. In this study, the effects of norepinephrine on DNA synthesis and the related signaling molecules involved in rBMSCs were examined. mRNA levels of the alpha1-adrenergic receptor subtypes increased following norepinephrine stimulation (10(-5) M for 30 min). DNA synthesis increased in dose- and time-dependent manners as determined by [(3)H]thymidine incorporation. Intracellular Ca(2+) concentration and translocation of protein kinase C from the cytosol to the membrane were also found to be elevated in rBMSCs. Phentolamine was able to suppress translocation of PKC. Norepinephrine also induced phosphorylation of ERK1/2, which was prevented by staurosporine treatment. Pretreatment with PD98059 inhibited ERK1/2 phosphorylation and DNA synthesis in rBMSCs. These findings indicate that norepinephrine stimulates DNA synthesis via alpha1-adrenergic receptors and downstream Ca(2+)/PKC and ERK1/2 activation in rBMSCs.

The interaction between interferon-induced protein with tetratricopeptide repeats-1 and eukaryotic elongation factor-1A

It has been shown previously that in mammalian cells, interferon-induced protein with tetratricopeptide repeats-1(IFIT1) is rapidly synthesized in response to viral infection, functions as an inhibitor of translation by binding to the eukaryotic initiation factor-3, and consequently assigns resistive activity against viral invasion to cells. It has also been reported that IFIT1 is rapidly produced in response to other cell stress agents with no direct relation to virus such as bacterial lipopolysaccharide and interleukin-1, but its function under these non-viral infection cell stress conditions has yet to be elucidated. Here, we demonstrate an interaction between IFIT1 and eukaryotic elongation factor-1A (eEF1A) both in vitro, using recombinant proteins as bait in pull-down assays, and in vivo, using laser confocal microscopy and immunoprecipitation. In addition, we report the initial determination of the domain of IFIT1 that mediates this interaction. We also display that both IFIT1 and eEF1A protein levels are rapidly elevated, prolonged in tumor necrosis factor alpha pre-treated Raw264.7 cells, and most of those cells are induced to death by the end of investigations. Our results imply that under some stressful stimulations IFIT1 may participate in cell death pathways by interaction with eEF1A.

Recombinant adenoviral microRNA-206 induces myogenesis in C2C12 cells

Summary Background The expression of microRNA-206 (miR-206) is high in skeletal muscle but low in most other tissues. The expression of miR-206 is increased in muscular dystrophy, suggesting its involvement in the pathogenesis of muscle diseases. To determine the role of miR-206 in muscle cell differentiation and explore a possible gene therapy vector, we constructed a miR-206 adenoviral expression vector (AdvmiR-206) and tested for transfection into C2C12 stem cells. Material/Methods A 355-bp PCR amplicon from C57B6 mouse skeletal muscle genomic DNA was inserted into the adenoviral shuttle vector pAdTrack-CMV, which was then co-transformed with the adenoviral backbone plasmid pAdEasy-1 into competent E. coli BJ5183 bacteria. The specificity and function of this recombinant adenoviral MiR-206 were studied in C2C12 cells by Northern blot, immunofluorescence, Western blot, and flow cytometry. Results Increased expression of miR-206 in AdvmiR-206 transfected C2C12 cells (P<0.001) and resulted in morphological and biochemical changes over time that were similar to serum deprivation, including elongated cells and increased myosin heavy chain proteins. Even in the absence of serum deprivation, miR-206 overexpression accounted for a 50% reduction of S-phase cells (P<0.01). Moreover, in untransfected C2C12 cells, the introduction of miR-206-specific antisense oligoribonucleotides inhibited the normal response to serum deprivation. Twenty-four hours after lipofection of antisense oligoribonucleotides, the number of elongated cells was reduced by half (P<0.01). Conclusions Collectively, these data support a role for miR-206 in myoblast differentiation. We foresee potential applications for the AdvmiR-206 vector in research and therapy.