Quantitative kinetics analysis of BMP2 uptake into cells and its modulation by BMP antagonists

Abstract

SummaryBone morphogenetic proteins (BMPs) are members of the TGFb family of signaling proteins and play an important role during development and in tissue formation. BMP signaling is a well-studied process, which is initiated through binding of cognate receptors and processed through activation of Smad downstream mediators. A hallmark of BMP signaling is its modulation at the extracellular level through specific antagonists. Although it had been shown that BMP and TGFb receptors are internalized following activation, little is known about the fate of BMP ligands. We prepared biologically active fluorescently labeled BMP2 and quantitatively analyzed its binding and uptake in cells using flow cytometry and confocal microscopy. Exogenous BMP2 was rapidly bound to the cell surface and subsequently internalized in a time-dependent manner and accumulated in the cell center. Although binding to the cell surface was limited by binding sites at the beginning, internalization continously increased with time, after a short delay. Using different inhibitors we found that internalization of BMP2 through endosomal particles occurred in a clathrin-dependent pathway. Furthermore, uptake of BMP2 was modulated in strikingly different ways by BMP2 antagonists. Although Noggin and Gremlin increased BMP2 uptake, Chordin blocked BMP2 uptake, which was concentration dependent in both cases. In conclusion, our findings present interesting mechanisms for the modulation of BMP signaling by concentration gradients of BMP ligands and antagonists in a dose-and timedependent manner, which can provide an explanation of some properties of the BMP regulatory network.

Improving the production of recombinant human bone morphogenetic protein‐4 in Chinese hamster ovary cell cultures by inhibition of undesirable endocytosis

“…The association of dynamin with the clathrin‐dependent pathway is well known, though some evidence also implicates its association with the caveolin‐dependent pathway (Doherty & McMahon, ). These results are consistent with the previous studies showing that chlorpromazine and dynasore reduced rhBMP‐2 internalization (Alborzinia et al, ; Jortikka, Laitinen, Lindholm, & Marttinen, ). However, these endocytosis inhibitors could not be used for improving rhBMP‐4 production because they impaired cell growth and viability (data not shown).…”

Crimpy Enables Discrimination of Presynaptic and Postsynaptic Pools of a BMP at the Drosophila Neuromuscular Junction

“…4 H-L). We propose that total Gbb levels are not reduced as dramatically as neuronal Gbb-HA because the presynaptic terminal also likely contains internalized muscle-derived Gbb by virtue of trans-synaptic pro-growth signaling (Alborzinia et al, 2013; Kelley et al, 2009; von Einem et al, 2011). We next asked whether Gbb levels in presynaptic terminals decrease following stimulation in the absence of Cmpy.…”

“…S1). While Gbb in the presynaptic compartment may be neuron-derived, it may also represent muscle-derived ligand, as Gbb is present in internalized endosomes (Alborzinia et al, 2013; Kelley et al, 2009; von Einem et al, 2011). To test if at least some presynaptic Gbb arises within the presynaptic neuron, we asked whether presynaptic Gbb levels are increased following neuronal Gbb overexpression.…”

Abrus agglutinin stimulates BMP‐2‐dependent differentiation through autophagic degradation of β‐catenin in colon cancer stem cells

“…A previous study had demonstrated that BMP‐2 is localized to the late endosomal vesicles through a specific receptor that might be involved in autophagosome biogenesis. Besides, it is documented that hVps34 is essential for the formation of internal vesicles within multivesicular bodies .…”