Int J Biochem Cell Biol. 2016 Jul;76:51-63. doi: 10.1016/j.biocel.2016.04.010. Epub 2016 Apr 23.

Dynamin-dependent endocytosis of Bone Morphogenetic Protein 2 (BMP2) and its receptors is dispensable for the initiation of Smad signaling

Paarmann P1, Dörpholz G1, Fiebig J2, Amsalem AR3, Ehrlich M4, Henis YI3, Müller T2, Knaus P5.

  • 1Institute for Chemistry and Biochemistry, Freie Universität Berlin, Thielallee 63, 14195 Berlin, Germany.
  • 2Department for Molecular Plant Physiology and Biophysics, Biozentrum Universität Würzburg, Julius-von-Sachs Institute, Julius-von-Sachs Platz 2, 97082 Würzburg, Germany.
  • 3Department of Neurobiology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel.
  • 4Department of Cell Research and Immunology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel.
  • 5Institute for Chemistry and Biochemistry, Freie Universität Berlin, Thielallee 63, 14195 Berlin, Germany. Electronic address: knaus@chemie.fu-berlin.de.

 

Abstract:

Bone Morphogenetic Protein (BMP) signal transduction via the canonical Smad158 pathway has previously been linked to dynamin-dependent endocytosis, since the application of chemical inhibitors of clathrin or dynamin in functional cell culture based assays negatively affects initiation and propagation of the Smad response. More recent studies, however, demonstrated efficient Smad signaling by non-internalizable BMP2. The role of endocytosis in BMP signal transduction thus remained controversial. In our study we aimed to refine cell biological assays and to apply novel tools, including a new site-directed fluorescently labeled BMP2 ligand, to revisit key steps in BMP Smad signaling. We found that dynamin2 function was required for BMP2 uptake but was dispensable for C-terminal phosphorylation, nuclear translocation and transcriptional activity of BMP-dependent Smads. Furthermore, we demonstrated a role of dynamin2 in the regulation of steady-state and surface BMP receptor levels, as well as an impact on Smad1 protein level. Thus, dynamin2 allows for modulation of basal and ligand-dependent Smad signaling capacity. High levels of functional dynamin2 enhanced the myogenic differentiation of precursor cells. From our study we conclude that dynamin-dependent endocytosis serves as a regulatory mechanism to fine-tune Smad signaling, but it is not a prerequisite for signal initiation and propagation. Our findings contribute to the understanding of fundamental mechanisms of BMP signaling and thus provide important information for future consideration in the context of therapeutic applications of BMPs.

PMID: 27113717

 

Supplement:

Bone Morphogenetic Protein (BMP) signal transduction is involved in many cellular functions, such as proliferation, survival, migration and differentiation [1, 2]. Binding of BMP ligands to heteromeric complexes of transmembrane serine/threonine kinase receptors initiates intracellular signaling cascades by the activation of Smad proteins which form a transcriptional complex to regulate BMP target gene expression in the nucleus (Fig. 1). BMP receptors undergo internalization via clathrin-coated pits for canonical Smad signaling but also via caveolae to initiate non-Smad pathways [3, 4]. Both pathways require the action of the large GTPase dynamin for the detachment of intracellular vesicles.

There is controversial data on the role of dynamin-dependent endocytosis in BMP signaling. In our lab we work on the role of the BMP pathway in numerous different cellular contexts. We found that chemical inhibition of endocytosis reduced activation of Smad proteins as well as transcriptional response [3, 5], whereas surface-immobilized BMP ligand is incapable of undergoing internalization induced BMP response and BMP-dependent differentiation [6, 7]. We re-address the role of dynamin for BMP signal initiation and propagation by refined cell biological assays and application of novel tools.

To decipher if BMP ligand and receptor endocytosis is a pre-requisite for efficient initiation of BMP signaling, we made use of endocytosis-defective C2C12 cells overexpressing the GTPase domain dynamin2 mutant K44A.

First we analyzed Smad phosphorylation in C2C12 cells expressing wt and mutant dynamin as well as N-terminally YFP-fused Smad1, which can be distinguished from endogenous Smad1/5/8 by its different molecular weight. In the absence of BMP2, activation of Smad1 was detectable and was further enhanced after BMP2 addition for 15 or 60 min. Stronger Smad1 phosphorylation was observed in HA-dyn2 wt and in particular in HA-dyn2 K44A expressing cells compared to mock controls, indicating that dynamin-dependent endocytosis is dispensable for BMP-induced Smad phosphorylation.

In parallel, we also compared Smad1 nuclear translocation in these cells. Uptake of transferrin was abrogated with mutant dynamin as expected but nuclear Smad1 levels were even increased after BMP stimulation in mutant compared to wt dynamin expressing cells. BRE-luciferase assays showed comparable induction of reporter gene expression. Thus, dynamin-dependent endocytosis is neither required for Smad translocation nor for transcriptional activity.

Since activation of Smad1/5/8 is known to counteract myogenic differentiation in C2C12 myoblasts [8, 9], we analyzed in vitro myogenesis using a myogenin-luciferase reporter gene assay after three days of differentiation. We observed an expected decrease in reporter gene expression after stimulation with BMP2, which was most prominent in endocytosis-defective cells expressing HA-dyn2 K44A compared to mock controls. In contrast, reporter gene expression was rather increased by functional dynamin.

In addition, we analyzed myotube formation via immunofluorescence staining using myosin heavy chain (MHC) as a marker. Consistently, expression of HA-dyn2 K44A rather decreased myotube formation and further suppressed myogenesis upon BMP2 stimulation. These data indicate that dynamin is also dispensable for BMP effects on myogenic differentiation. In contrast, functional dyn2 promoted myogenic differentiation.   

To visualize BMP2 ligand internalization, we developed a functional labeled BMP2 (DL488-BMP2). We evaluated its biological activity on Smad1/5/8 phosphorylation and transcriptional activity. We demonstrate that DL488-BMP2 co-localized with internalized HA-BRIb or HA-BRII receptors indicating that also the ligand was fully internalized.

We applied DL488-BMP2 to study the role of dynamin in BMP2 ligand endocytosis. As expected, we observed a reduced ligand uptake in cells expressing HA-dyn2 K44A compared to wt. However, loss of ligand internalization was not accompanied with impaired nuclear translocation of Smad1, confirming that dynamin-dependent endocytosis is dispensable for the initiation and propagation of Smad signaling. We speculate that dynamin rather enables cellular adaptation of receptor and Smad protein levels by clearing the BMP ligand and part of the receptors to fine-tune BMP signal transduction.

Importance of the study:

This study provides new tools for the analysis of BMP ligand internalization that may facilitate further detailed mechanistic studies. Our findings thus contribute to the understanding of fundamental mechanisms of BMP signaling. Since BMPs are promising for therapeutic application in the context of bone regeneration and repair, this study also provides important information for future clinical consideration.

 

 

Figure 1: Dynamin2 mutant abolishes receptor internalization but does not compromise BMP downstream signaling.

 

References:

  1. Sieber, C., et al., Recent advances in BMP receptor signaling. Cytokine Growth Factor Rev, 2009. 20(5-6): p. 343-55.
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  5. Heining, E., et al., Spatial segregation of BMP/Smad signaling affects osteoblast differentiation in C2C12 cells. PLoS One, 2011. 6(10): p. e25163.
  6. Pohl, T.L., et al., Surface immobilization of bone morphogenetic protein 2 via a self-assembled monolayer formation induces cell differentiation. Acta Biomater, 2012. 8(2): p. 772-80.
  7. Schwab, E.H., et al., Nanoscale control of surface immobilized BMP-2: toward a quantitative assessment of BMP-mediated signaling events. Nano Lett, 2015. 15(3): p. 1526-34.
  8. Katagiri, T., et al., Bone morphogenetic protein-2 inhibits terminal differentiation of myogenic cells by suppressing the transcriptional activity of MyoD and myogenin. Exp Cell Res, 1997. 230(2): p. 342-51.
  9. Katagiri, T., et al., Bone morphogenetic protein-2 converts the differentiation pathway of C2C12 myoblasts into the osteoblast lineage. J Cell Biol, 1994. 127(6 Pt 1): p. 1755-66.

 

Acknowledgements:

This work was supported by the German Research Foundation (DFG)/ SFB958 and the Sonnenfeld Stiftung.

 

Contact: 

Dr. Christian Kähler,

Dr. Gina Dörpholz,

Prof. Dr. Petra Knaus 

Institute for Chemistry and Biochemistry

Freie Universität Berlin

Thielallee 63

14195 Berlin

Corresponding author: knaus@chemie.fu-berlin.de (P. Knaus)

 

 

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