Teratogen-induced alterations in microRNA-34, microRNA-125b and microRNA-155 expression: correlation with embryonic p53 genotype and limb phenotype.

BACKGROUND: In a large number of studies, members of the microRNA (miRNA)-34 family such as miRNA-34a, miRNA-34b, miRNA-34c, as well as miRNA-125b and miRNA-155, have been shown to be regulators of apoptosis. The ability of these miRNAs to perform this function is mainly attributed to their ability to interact with the p53 tumor suppressor, which is a powerful regulator of the teratologic susceptibility of embryos. We chose to explore whether miRNA-34a/b/c, miRNA-125b and miRNA-155 may play a role in teratogenesis by using p53+/- pregnant mice treated with cyclophosphamide (CP) as a model. We evaluated how CP-induced alterations in the expression of these miRNAs in the embryonic limbs correlate with embryonic p53 genotype and CP-induced limb phenotypes. RESULTS: The limbs of p53 positive embryos were more sensitive to CP-induced teratogenic insult than the limbs of p53 negative embryos. The hindlimbs were more severely affected than the forelimbs. Robust miRNA-34a expression was observed in the fore- and hindlimbs of p53+/+ embryos exposed to 12.5 mg/kg CP. The dose of 20 mg/kg CP induced almost a two-fold increase in the level of miRNA-34a expression as compared to that exhibited by p53+/+ embryos exposed to a lower dose. Increased miRNA-34b and miRNA-34c expression was also observed. Of note, this dose activated miRNA-34a and miRNA-34c in the forelimbs of p53-/- embryos. When embryos were exposed to 40 mg/kg CP, the expression pattern of the miRNA-34a/b/c was identical to that registered in the limbs of embryos exposed to 20 mg/kg CP. However, this dose suppressed miRNA-125b and miRNA-155 expression in the fore- and hindlimbs of p53+/+ embryos. CONCLUSION: This study demonstrates that teratogen-induced limb dysmorphogenesis may be associated with alterations in miRNA-34, miRNA-125b and miRNA-155 expression. It also suggests for the first time that p53-independent mechanisms exist contributing to teratogen-induced activation of miRNA-34a and miRNA-34c. At the same time, teratogen-induced suppression of miRNA-125b and miRNA-155 expression may be p53 dependent. The analysis of correlations between the expression pattern of the tested miRNAs and CP induced limb phenotypes implies that miRNAs regulating apoptosis may differ from each other with respect to their functional role in teratogenesis: some miRNAs act to protect embryos, whereas other miRNAs boost a teratogen-induced process of maldevelopment to induce embryonic death.

Teratogen-induced distortions in the classical NF-kappaB activation pathway: correlation with the ability of embryos to survive teratogenic stress.

Studies with diverse teratogens implicated the transcription factor NF-kappaB in mechanisms determining teratological susceptibility of embryos. Here, a teratogen such as cyclophosphamide (CP) was used to test whether teratogenic insult alters the classical NF-kappaB activation pathway, and how these alterations correlate with the ability of mouse embryos to resist the teratogen-induced process of maldevelopment. We observed that embryos tested 24 h after the exposure of females to 40 mg/kg CP exhibited a dramatic decrease in the level of NF-kappaB (p65 subunit)-DNA binding, IkappaB kinase beta (IKKbeta) activity, expression of p65 and IKKbeta proteins, as well as NF-kappaB inhibitory proteins (IkappaBs) such as IkappaBalpha, IkappaBbeta, and IkappaBepsilon, and died within the next 24 h. Embryos of females exposed to 15 mg/kg CP exhibited only a decrease in NF-kappaB-DNA binding and IKKbeta activity at 24 h. However, at 48 h, a more prominent decrease in NF-kappaB activity was observed, accompanied by a decreased expression of p65 and IKKbeta proteins. These embryos died within the next 24 h. After treatment with 10 mg/kg CP, embryos survived until the end of the antenatal period of development, demonstrating a transient decrease in NF-kappaB-DNA binding activity and no alterations in NF-kappaB signaling. These results suggest that the classical NF-kappaB activation pathway may be among targets that teratogens engage to initiate abnormal development. Besides, the observation that embryos destined to be dead exhibited a dramatically decreased rate of cell proliferation suggests a pathway, whereby teratogen-induced alterations in NF-kappaB signaling may culminate in such a final effect as embryonic death.