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. 2008 Sep;216(3):771-9.
doi: 10.1002/jcp.21455.

BMP signaling dynamics in embryonic orofacial tissue

Partha Mukhopadhyay  1 , Cynthia L Webb, Dennis R Warner, Robert M Greene, M Michele Pisano
Affiliations

BMP signaling dynamics in embryonic orofacial tissue

Partha Mukhopadhyay et al. J Cell Physiol. 2008 Sep.

Abstract

The bone morphogenetic protein (BMP) family represents a class of signaling molecules, that plays key roles in morphogenesis, cell proliferation, survival and differentiation during normal development. Members of this family are essential for the development of the mammalian orofacial region where they regulate cell proliferation, extracellular matrix synthesis, and cellular differentiation. Perturbation of any of these processes results in orofacial clefting. Embryonic orofacial tissue expresses BMP mRNAs, their cognate proteins, and BMP-specific receptors in unique temporo-spatial patterns, suggesting functional roles in orofacial development. However, specific genes that function as downstream mediators of BMP action during orofacial ontogenesis have not been well defined. In the current study, elements of the Smad component of the BMP intracellular signaling system were identified and characterized in embryonic orofacial tissue and functional activation of the Smad pathway by BMP2 and BMP4 was demonstrated. BMP2 and BMP4-initiated Smad signaling in cells derived from embryonic orofacial tissue was found to result in: (1) phosphorylation of Smads 1 and 5; (2) nuclear translocation of Smads 1, 4, and 5; (3) binding of Smads 1, 4, and 5 to a consensus Smad binding element (SBE)-containing oligonucleotide; (4) transactivation of transfected reporter constructs, containing BMP-inducible Smad response elements; and (5) increased expression at transcriptional as well as translational levels of Id3 (endogenous gene containing BMP receptor-specific Smad response elements). Collectively, these data document the existence of a functional Smad-mediated BMP signaling system in cells of the developing murine orofacial region.

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Figures

Fig. 1
Fig. 1
Immunoblot analysis of steady state levels of Smad 1 (a), Smad 5 (b), phospho-Smad 1 (c), and phospho-Smad 1/5 (d) proteins in cell extracts of murine embryonic orofacial tissue from gestational days (GD) 12, 13, and 14 of development. Equal amounts of protein (50 μg) were resolved by SDS–PAGE on 8% polyacrylamide Tris/glycine gels, transferred to PVDF membranes, probed with Smad-specific antibodies and immunoreactive species detected by chemiluminescence, as detailed in the Materials and Methods Section. Molecular weights of the marker proteins are indicated to the left of each part. Each immunoblot is representative of no less than three independent blots of unique tissue extracts from embryonic orofacial tissue from each of GD 12, 13, and 14. Lower part (e) shows one representative western blot of the loading control β-actin.
Fig. 2
Fig. 2
Immunoblot analysis of steady state levels of Smad 1 (a), Smad 5 (b), phospho-Smad 1 (c), phospho-Smad 1/5 (d), and Smad 4 (e) proteins in nuclear extracts of MEMM cells treated with 0.1–100 ng/ml BMP2. Proteins were separated and immunoreactive species detected as described in Figure 1. Each immunoblot is representative of no less than three independent blots of unique extracts from BMP2-treated MEMM cells. Lower part (f) shows one representative western blot of the loading control β-actin.
Fig. 3
Fig. 3
Immunoblot analysis of steady state levels of Smad 1 (a), Smad 5 (b), phospho-Smad 1 (c), phospho-Smad 1/5 (d), and Smad 4 (e) proteins in nuclear extracts of MEMM cells treated with 0.1–100 ng/ml BMP4. Proteins were separated and immunoreactive species detected as described in Figure 1. Each immunoblot is representative of no less than three independent blots of unique extracts from BMP4-treated MEMM cells. Lower part (f) shows one representative western blot of the loading control β-actin.
Fig. 4
Fig. 4
Combined DNA affinity purification and immunoblot analysis of phospho-Smad 1 (a and d), phospho-Smad 1/5 (b and e), and Smad 4 (c and f), binding to a BR-Smad binding element (BRE)-containing oligonucleotide in MEMM cells in response to various concentrations of BMP2 (a–c) and BMP4 (d–f). Cell extracts derived from BMP2-, BMP4-, or vehicle-treated MEMM cells were incubated with a biotinylated BR-SBE oligonucleotide. Protein–oligonucleotide complexes were retrieved on streptavidin–agarose beads, resolved by SDS–PAGE, and subjected to immunoblotting. Specificity of Smad binding to the BR-SBE is demonstrated by the absence of binding when a mutant oligonucleotide was utilized that differed at several nucleotide positions within the BR-Smad binding response element. BMP-treated (10 ng/ml) utilizing mutated BR-SBE (mO); Vehicle control utilizing wild-type BR-SBE (V); BMP2- or BMP4-treated (0.1–10 ng/ml) utilizing wild-type BR-SBE (0.1, 1, 10). Each immunoblot is representative of no less than three independent blots from three unique sets of DAPA-purified MEMM cell extracts.
Fig. 5
Fig. 5
Transcriptional activation of a BMP-inducible luciferase reporter plasmid in MEMM cells. Primary cultures of MEMM cells were co-transfected by the Effectene transfection method with 1.0 μg of the BMP-responsive plasmid BRE-luc and 0.1 μg pRL-CMV for 24 h followed by 24 h exposure to either vehicle, 2.0 ng/ml TGFβ1, or 25, 50, 100 ng/ml BMP2 or BMP4. Cells were extracted and luciferase activity was measured as described in Materials and Methods Section. Firefly luciferase activity was assayed in cell extracts and expressed relative to Renilla luciferase activity to control for variability in transfection efficiencies. Mean luciferase activities and standard errors were calculated from triplicate cell culture samples, and treatments compared by analysis of variance (ANOVA), followed by Student’s t-test. P values <0.05 were considered statistically significant. *Indicates luciferase activity significantly different from control.
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