[Aucubin combined with ADSCs-exos protects TBHP-induced nucleus pulposus cells via TLR4/NF-κB pathway].

This paper aims to investigate the effects and mechanisms of adipose-derived stem cells-exosomes(ADSCs-exos) toge-ther with aucubin in protecting human-derived nucleus pulposus cells(NPCs) from inflammatory injury, senescence, and apoptosis. The tert-butyl hydroperoxide(TBHP)-induced NPCs were assigned into normal, model, aucubin, ADSCs-exos, and aucubin+ADSCs-exos groups. The cell viability was examined by cell counting kit-8(CCK-8), cell proliferation by EdU staining, cell senescence by senescence-associated-ß-galactosidase(SA-ß-Gal), and cell cycle and apoptosis by flow cytometry. Enzyme-linked immunosorbent assay was employed to examine the expression of interleukin-1ß(IL-1ß), IL-10, and tumor necrosis factor-α(TNF-α). Real-time fluorescence quantitative PCR and Western blot were employed to determine the mRNA and protein levels of aggregated proteoglycan(aggrecan), type Ⅱ collagen alpha 1(COL2A1), Toll-like receptor 4(TLR4), and nuclear factor-kappa B(NF-κB). The results showed that compared with the model group, the aucubin or ADSCs-exos group showed enhanced viability and proliferation of NPCs, decreased proportion of G_0/G_1 phase cells, increased proportion of S phase cells, reduced apoptosis and proportion of cells in senescence, lowered IL-1ß and TNF-α levels, elevated IL-10 level, down-regulated mRNA and protein levels of TLR4 and NF-κB, and up-regulated mRNA and protein levels of aggrecan and COL2A1. Compared with the aucubin or ADSCs-exos group, the aucubin+ADSCs-exos combination further increased the viability and proliferation of NPCs, decreased the proportion of G_0/G_1 phase cells, increased the proportion of S phase cells, reduced the apoptosis and proportion of cells in senescence, lowered the IL-1ß and TNF-α levels, elevated the IL-10 level, down-regulated the mRNA and protein levels of TLR4 and NF-κB, and up-regulated the mRNA and protein levels of aggrecan and COL2A1. In summary, both aucubin and ADSCs-exos could exert protective effects by inhibiting inflammatory responses, reducing apoptosis and senescence of NPCs, improving cell viability and proliferation as well as extracellular matrix synthesis, which may be associated with the inhibition of TLR4/NF-κB signaling pathway activation. The combination of both plays a synergistic role in the protective effects.

Intricate gene regulatory networks of helix-loop-helix (HLH) proteins support regulation of bone-tissue related genes during osteoblast differentiation.

Helix-loop-helix (HLH) transcription factors are key regulators of neurogenesis, myogenesis and osteogenesis. Here the relative contributions of multiple classes of HLH factors to the expression of bone related genes during osteoblast maturation were compared. We examined the expression of a panel of HLH proteins (e.g., Twist1/2, USF1/2, c-Myc, Id1 approximately 4, E12/47, Stra13) and one Zn finger protein (Snail which recognizes a subset of E-boxes), during osteoblast differentiation and their functional contributions to bone phenotypic gene regulation. While expression of Twist1, Stra13, E12/47 and Snail transcripts remains relatively constant, expression of Twist2 as well as the inhibitory factors Id1, Id2, Id3, and Id4 decreases and USF1 is up-regulated during osteoblastic differentiation of MC3T3 cells. Forced expression of selected HLH transcription factors shows that Myc, Snail and USF factors increase expression of the bone markers osteocalcin (OC) and/or alkaline phosphatase (AP), while E12/47, Twist and Id factors decrease their expression. None of these factors affect Runx2 gene expression. Interestingly, Snail enhances expression of osteoblast markers, while Twist1 and Twist2 factors are cross-regulated and inhibit bone specific gene expression and other HLH proteins (e.g., Id) indirectly. Thus, our data suggest that the integrated activities of negative and positive E-box related regulatory factors control osteoblast differentiation.

Sequential recruitment of PCAF and BRG1 contributes to myogenin activation in 12-O-tetradecanoylphorbol-13-acetate-induced early differentiation of rhabdomyosarcoma-derived cells.

Myogenin and its upstream regulator MyoD are known to be required for myogenic cell differentiation. Although both of them can be expressed in rhabdomyosarcoma-derived RD cells, the cells are unable to undergo full-scale terminal myogenic differentiation. 12-O-Tetradecanoylphorbol-13-acetate (TPA) has been found to be functional in the induction of RD cell differentiation, whereas its mechanism is not fully understood. By using quantitative real-time-based chromatin immunoprecipitation and real-time reverse transcription-PCR-based promoter activity assays, we examined the activation mechanism of the myogenin gene during TPA-induced differentiation of the RD cells. We have shown that a histone acetyltransferase PCAF and ATPase subunit BRG1 of the SWI/SNF chromatin remodeling complex are sequentially recruited to the promoter of the myogenin gene. Both PCAF and BRG1 are also involved in the activation of the myogenin gene. In addition, we have found that the p38 mitogen-activated protein kinase is required for BRG1 recruitment in TPA-mediated myogenin induction. We propose that there are two distinct activation steps for the induction of myogenin in TPA-induced early differentiation of RD cells: 1) an early step that requires PCAF activity to acetylate core histones and MyoD to initiate myogenin gene expression, and 2) a later step that requires p38-dependent activity of the SWI/SNF remodeling complex to provide an open conformation for the induction of myogenin. Our studies reveal an essential role for epigenetic regulation in TPA-induced differentiation of RD cells and provide potential drug targets for future treatment of the rhabdomyosarcoma.

[Heat shock induced transcription of hsp90alpha gene on chromatin template].

OBJECTIVE: A CAT reporter plasmid (pBLCAT3alpha1) driven by the promoter of hsp90alpha was in vitro assembled into chromatin to investigate the transcription activity of the reporter gene upon heat shock. METHODS: A competitive RT-PCR-based technique was used to quantify the promoter activity of hsp90alpha gene on chromatin or naked DNA templates in vitro. RESULTS: The in vitro transcription efficiency was first optimized by using different amounts of whole cell extracts from heat shock-treated HeLa cells. In vitro chromatin assembly was carried out with purified components of chromatin assembly associated factors, core histones and CAT reporter gene driven by the promoter of hsp90alpha gene. Results showed that chromatin formation repressed the in vitro transcription of the gene. CONCLUSION: The heat shock induced transcription of hsp90alpha gene on chromatin template is more efficient than that on naked DNA in vitro.