Extraneous E-Cadherin Engages the Deterministic Process of Somatic Reprogramming through Modulating STAT3 and Erk1/2 Activity.

Although several modes of reprogramming have been reported in different cell types during iPSC induction, the molecular mechanism regarding the selection of different modes of action is still mostly unknown. The present study examined the molecular events that participate in the selection of such processes at the onset of somatic reprogramming. The activity of STAT3 versus that of Erk1/2 reversibly determines the reprogramming mode entered; a lower activity ratio favors the deterministic process and vice versa. Additionally, extraneous E-cadherin facilitates the early events of somatic reprogramming, potentially by stabilizing the LIF/gp130 and EGFR/ErbB2 complexes to promote entry into the deterministic process. Our current findings demonstrated that manipulating the pSTAT3/pErk1/2 activity ratio in the surrounding milieu can drive different modes of action toward either the deterministic or the stochastic process in the context of OSKM-mediated somatic reprogramming.

Optimization of reprogramming culture conditions for the generation of induced pluripotent stem cells from Col1a1 4F2A-Oct4-GFP mice with high efficiency.

A reprogrammable transgenic mouse strain, called Col1a1 4F2A-Oct4-GFP, was bred for the present study. Because the somatic cells of this mouse strain contain only two copies of each Yamanaka factor, these animals are inefficient at producing induced pluripotent stem cells (iPSCs; approx. 0.005%) under traditional culture conditions. With an optimized culture condition, the iPSC production rate of mouse embryonic fibroblasts (MEFs) of Col1a1 4F2A-Oct4-GFP mice (MEFCol1a14F2A-Oct4-GFP ) was increased to approximately 8%. Further, promotion of cell proliferation by serum supplementation did not enhance iPSC production. Inhibition of transforming growth factor ß (TGF-ß) in the serum by SB431542 neither affected the growth rate of MEFCol1a14F2A-Oct4-GFP nor promoted iPSC production. However, the use of the gamma-irradiated STO-NEO-LIF (γSNL) cells to serve as feeders for iPSC production resulted in a 5-fold higher rate of iPSC production than the use of γMEFICR feeders. Interestingly, the use of SB431542 with the γMEFICR -adopted system could eliminate this difference. RT-PCR-based comparative analysis further demonstrated that TGF-ß expression was 10-fold higher in γMEFICR than in γSNL cells. Consistent with previous reports, mesenchymal to epithelial transition was found to participate in the initial steps of reprogramming in the specific context of MEFCol1a14F2A-Oct4-GFP . Moreover, we found that the initial seeding density is one of the pivotal factors for determining a high efficiency of iPSC generation. The iPSCs efficiently generated from our MEFCol1a14F2A-Oct4-GFP resembled mouse embryonic stem cells (mESCs) in aspects of teratoma formation and germline transmission. Depending on the culture conditions, our Col1a1 4F2A-Oct4-GFP mouse system can generate bona fide iPSCs with variable efficiencies, which can serve as a tool for interrogating the route taken by cells during somatic reprogramming.

Casticin inhibits COX-2 and iNOS expression via suppression of NF-κB and MAPK signaling in lipopolysaccharide-stimulated mouse macrophages.

ETHNOPHARMACOLOGICAL RELEVANCE: The fruits of Vitex rotundifolia L. are widely used to treat inflammation of the airway in Traditional Chinese medicine. Previous studies found that casticin, isolated from Vitex rotundifolia, could induce apoptosis of tumor cells. In this study, we evaluated the anti-inflammatory effects of casticin and its underlying molecular mechanism in lipopolysaccharide (LPS)-stimulated macrophages. MATERIALS AND METHODS: RAW264.7 cells were pretreated with various concentrations of casticin (0.3-10µM), and then treated with LPS to induce inflammation. We assayed the levels of proinflammatory cytokines and prostaglandin E2 (PGE2) using ELISA, and examined the protein expression of inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, and heme oxygenase (HO)-1 by Western blot. We also investigated the anti-inflammatory molecular mechanism by analyzing inflammatory-associated signaling pathways, including the nuclear transcription factor kappa-B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways. RESULTS: We found casticin inhibited the levels of nitric oxide and PGE2, and decreased the production of proinflammatory cytokines such as interleukin (IL)-1ß, IL-6, and tumor necrosis factor α (TNF-α). In addition, iNOS and COX-2 expression levels were suppressed and casticin increased HO-1 and Nrf2 production in a concentration-dependent manner. Furthermore, casticin significantly inhibited NF-κB subunit p65 proteins in the nucleus and decreased Akt and MAPK activation. CONCLUSION: These results suggest that the anti-inflammatory effect of casticin is due to inhibition of proinflammatory cytokines and mediators by blocking the NF-κB, Akt, and MAPK signaling pathways.

Effect of dehydroepiandrosterone on atopic dermatitis-like skin lesions induced by 1-chloro-2,4-dinitrobenzene in mouse.

BACKGROUND: Th2 cells are overexpressed in the skin and serum of atopic dermatitis (AD) patients. Previously, we found that dehydroepiandrosterone (DHEA) decreased eosinophil infiltration in asthmatic mice through the suppression of Th2-associated cytokines. Therefore, we hypothesized that DHEA might improve the symptoms of AD syndrome. OBJECTIVE: In this study, we evaluated the symptom improvement and anti-inflammatory response that result from the modulation of immunity by DHEA modulated in AD-like mice. METHODS: Female BALB/c mice were sensitized and challenged with 1-chloro-2,4-dinitrobenzene. On days 14-29 after sensitization, mice were treated with cutaneous (skin smear) or oral administration of DHEA. In addition, human keratinocyte (HaCat) cells were used to evaluate the effect of DHEA on the in vitro production of proinflammatory cytokines and chemokines. RESULTS: Both cutaneous and oral DHEA were able to decrease ear swelling and skin inflammation in AD-like mice. DHEA also attenuated eosinophil and mast cell infiltration into ear and skin tissue. Additionally, Th2-associated cytokines were inhibited in splenocyte culture, and suppressed the levels of IgE and interleukin 4 in serum. Oral and cutaneous administration of DHEA reduced the inflammatory response, as evidenced by AD-like skin lesions, in a similar manner. DHEA significantly reduced inflammatory cytokines and chemokines through the nuclear factor-κB and mitogen-activated protein kinases pathways in tumor necrosis factor-α activated HaCat cells. CONCLUSION: DHEA ameliorates AD-like mouse skin inflammation and reduces eosinophil and mast cell infiltration by reducing the production of Th2-associated cytokines and chemokines.