Sonic hedgehog supplementation of oocyte and embryo culture media enhances development of IVF porcine embryos.

We investigated the expression of sonic hedgehog (SHH) receptor PTCH1 and its co-receptor smoothened (SMO) in fertilized porcine embryos. Effects of exogenous SHH on embryonic development and expressions of survival- and pluripotency-related genes were also determined. We found that PTCH1 and SMO are expressed from two-cell to blastocyst embryos. When oocytes or fertilized embryos were respectively cultured in the maturation or embryo culture medium supplemented with SHH (0.5 µg/ml), their blastocyst rates and total cell numbers increased (P<0.05) compared with the untreated control. When cultured simultaneously in the in vitro maturation (IVM) and in vitro culture (IVC) media supplemented with SHH, the oocytes gained increased blastocyst rates and total cell numbers in an additive manner, with reduced apoptotic indices (P<0.05). Interestingly, SHH treatment did not affect the expression of the BCL2L1 (BCL-XL) gene, yet reduced BAX expression. Blastocysts cultured with various SHH regimes had similar pluripotency-related gene (POU5F1 (OCT-4) and CDX2) expression levels, but blastocysts derived from SHH treatment during IVM had higher ZPF42 (REX01) expression (P<0.05). The highest ZPF42 expression was observed in the blastocysts derived from SHH-supplemented IVC and from dual IVM and IVC treatments. The levels of acetylated histone 3 (AcH3K9/K14) increased in the two-cell and the four-cell embryos when IVM and/or IVC media were supplemented with SHH (P<0.05). Our findings indicate that SHH conferred a beneficial effect on preimplantation development of porcine embryos, particularly when both IVM and IVC media were supplemented with SHH, and the effects may be further carried over from IVM to the subsequent embryonic development.

Regulation of Toll-like receptor 4-induced proasthmatic changes in airway smooth muscle function by opposing actions of ERK1/2 and p38 MAPK signaling.

Activation of Toll-like receptors (TLRs) on immune surveillance cells in the lung has been implicated in the pathobiology of allergic asthma, a condition associated with altered airway smooth muscle (ASM) contractility. Because ASM is known to directly respond to various proasthmatic stimuli, the potential role of TLR signaling in ASM in regulating airway expression of the proasthmatic phenotype was investigated. Cultured human ASM cells were found to express TLR4 and TLR9 mRNA transcripts and, whereas TLR9 stimulation had little effect, TLR4 activation with LPS elicited significant increases in IL-6 release and evoked proasthmatic-like changes in the constrictor and relaxation responsiveness of isolated rabbit ASM tissues. Complementary studies further demonstrated that the ASM responses to LPS were associated with activation of the ERK1/2 and p38 MAPK signaling pathways, IKK-mediated activation of NF-kappaB, and coupling of phosphorylated ERK1/2 with the p65 subunit of NF-kappaB. Moreover, the induced NF-kappaB activity and changes in ASM responsiveness were prevented in LPS-exposed ASM that were pretreated with inhibitors of ERK1/2 signaling, whereas inhibition of p38 MAPK augmented the proasthmatic responses to LPS. Finally, activation of p38 MAPK with anisomycin prevented both the LPS-induced stimulation of ERK1/2-mediated NF-kappaB activity and associated changes in ASM responsiveness. Collectively, these data support the novel concept that TLR4 activation in ASM elicits changes in ASM function that are regulated by opposing effects of MAPK signaling, wherein LPS-induced ERK1/2 activation mediates NF-kappaB-dependent proasthmatic-like changes in ASM function, whereas coactivation of p38 MAPK serves to homeostatically downregulate the proasthmatic effects of ERK1/2 activation.

Proasthmatic effects and mechanisms of action of the dust mite allergen, Der p 1, in airway smooth muscle.

BACKGROUND: House dust mite allergen exposure is a key risk factor for the development of allergic asthma. Beyond provoking immune cell-mediated allergic responses, house dust mite allergens were recently shown to exert direct effects on airway structural cells secondary to their intrinsic protease activities. OBJECTIVE: This study tested the hypothesis that house dust mite allergen exposure can produce changes in airway responsiveness through a direct effect on airway smooth muscle (ASM). METHODS: Isolated rabbit ASM tissues were exposed to the house dust mite allergen, Der p 1, and induced changes in ASM responsiveness and activation of mitogen-activated protein kinase (MAPK) signaling pathways were examined under different experimental conditions. RESULTS: The observations demonstrated the following: (1) Der p 1 exposure elicited enhanced constrictor responses and impaired relaxation responses in the ASM tissues, (2) these proasthmatic-like effects of Der p 1 were attributed to its intrinsic cysteine protease activity, and (3) the induced changes in ASM responsiveness were associated with activation of both the extracellular signal-regulated kinase (ERK) 1/2 and the p38 MAPK signaling pathways. Additionally, specific blockade of ERK1/2 signaling was found to prevent the Der p 1-induced changes in ASM responsiveness, whereas inhibition of p38 MAPK signaling enhanced the proasthmatic-like action of Der p 1, with the latter effect a result of augmented activation of ERK1/2. CONCLUSION: These findings are the first to demonstrate that the dust mite allergen, Der p 1, can directly elicit changes in ASM responsiveness that are associated with activation of MAPK signaling, wherein proasthmatic effects induced by Der p 1 are attributed to activation of ERK1/2, whereas coactivation of p38 MAPK exerts a homeostatic action by negatively regulating ERK1/2 signaling.