Molecular profiles of mitogen activated protein kinase signaling pathways in orofacial development

Abstract

Background: Formation of the mammalian orofacial region involves multiple signaling pathways regulating sequential expression of and interaction between molecular signals during embryogenesis. The present study examined the expression patterns of members of the MAPK family in developing murine orofacial tissue.

Methods: Total RNA was extracted from developing embryonic orofacial tissue during gestational days (GDs) 12-14 and used to prepare biotinylated cDNA probes, which were then denatured and hybridized to murine MAPK signaling pathways gene arrays.

Results: Expression of a number of genes involved in the (ERK1/2) cascade transiently increased in the embryonic orofacial tissue over the developmental period examined. Numerous members of the SAPK/JNK cascade were constitutively expressed in the tissue. Genes known to play a role in p38 MAPK signaling exhibited constitutive expression during orofacial development. Western blot analysis demonstrated that ERK2/1, p38, and SAPK/JNK kinases are present in embryonic orofacial tissue on each of GD 12, 13, and 14. By using phospho-specific antibodies, active ERK was shown to be temporally regulated during orofacial development. Minimal amounts of active p38 and active SAPK/JNK were detected in orofacial tissue during GDs 12-14.

Conclusions: Our study documents specific expression patterns of genes coding for proteins belonging to the ERK1/2, p38, and SAPK/JNK MAPK families in embryonic orofacial tissue. We also demonstrate that active, phosphorylated forms of ERK1/2 only were detected in the embryonic tissue investigated, suggesting a more central role for members of this family in embryonic orofacial development.

Figure 1

Immunoblot analysis of p42/44 (ERK1/2) in embryonic orofacial tissue on GDs 12, 13 and 14. A total of 60 µg of total protein lysate per lane was separated by SDS-PAGE electrophoresis on 8–16% polyacrylamide Tris-Glycine gels. Western blot analysis was carried out using antibodies specific for phospho-p42/44 (upper panel) and total p42/44 (middle panel). Note that ERKs are temporally regulated with higher activity on GD 13 and 14 as compared to GD 12 (upper panel). Lower panel shows the western blot of the loading control β-actin.

Figure 2

Immunoblot analysis of total and active p38 MAPK and SAPK/JNK in embryonic orofacial tissue on GDs 12, 13 and 14. A total of 60 µg of total protein lysate was separated by SDS-PAGE electrophoresis on 8–16% Tris-Glycine gels. A) Active, phosphorylated (upper panel) and total (lower panel) SAPK/JNK were analyzed by immunoblotting with specific antibodies. B) Active, phosphorylated (upper panel) and total (lower panel) p38 MAPK was analyzed by immunoblotting with specific antibodies. Minimal amounts of active, phosphorylated p38 were detected in embryonic orofacial tissue during GDs 12–14.