Dataset Information

AMOT130 drives BMP-SMAD signaling at the apical membrane in polarized cells.

ABSTRACT: The large isoform of the transmembrane protein angiomotin (AMOT130) controls cell proliferation and migration of many cell types. AMOT130 associates to the actin cytoskeleton and regulates tight-junction maintenance and signaling often via endosomal uptake of polarity proteins at tight junctions. AMOT130 is highly polarized and present only at the apical side of polarized cells. Here we show that bone morphogenetic protein (BMP) growth factor signaling and AMOT function are interlinked in apical-basal polarized cells. BMP6 controls AMOT internalization and endosomal trafficking in epithelial cells. AMOT130 interacts with the BMP receptor BMPR2 and facilitates SMAD activation and target gene expression. We further demonstrate that this effect of AMOT on BMP-SMAD signaling is dependent on endocytosis and specific to the apical side of polarized epithelial and endothelial cells. Knockdown of AMOT reduces SMAD signaling only from the apical side of polarized cells, while basolateral BMP-SMAD signaling is unaffected. This allows for the first time interference with BMP signaling in a polarized manner and identifies AMOT130 as a novel BMP signaling regulator.

SUBMITTER: Brunner P

PROVIDER: S-EPMC6960409 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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AMOT130 drives BMP-SMAD signaling at the apical membrane in polarized cells.

Brunner Patrizia P Hastar Nurcan N Kaehler Christian C Burdzinski Wiktor W Jatzlau Jerome J Knaus Petra P

Molecular biology of the cell 20191204 2

The large isoform of the transmembrane protein angiomotin (AMOT130) controls cell proliferation and migration of many cell types. AMOT130 associates to the actin cytoskeleton and regulates tight-junction maintenance and signaling often via endosomal uptake of polarity proteins at tight junctions. AMOT130 is highly polarized and present only at the apical side of polarized cells. Here we show that bone morphogenetic protein (BMP) growth factor signaling and AMOT function are interlinked in apical ...[more]

PMID: 31800378

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Project description:BackgroundBone morphogenetic proteins (BMPs) are involved in a plethora of cellular processes in embryonic development and adult tissue homeostasis. Signaling specificity is achieved by dynamic processes involving BMP receptor oligomerization and endocytosis. This allows for spatiotemporal control of Smad dependent and non-Smad pathways. In this study, we investigate the spatiotemporal regulation within the BMP-induced Smad transcriptional pathway.Methodology/principal findingsHere we discriminate between Smad signaling events that are dynamin-dependent (i.e., require an intact endocytic pathway) and dynamin-independent. Inhibition of dynamin-dependent endocytosis in fluorescence microscopy and fractionation studies revealed a delay in Smad1/5/8 phosphorylation and nuclear translocation after BMP-2 stimulation of C2C12 cells. Using whole genome microarray and qPCR analysis, we identified two classes of BMP-2 induced genes that are differentially affected by inhibition of endocytosis. Thus, BMP-2 induced gene expression of Id1, Id3, Dlx2 and Hey1 is endocytosis-dependent, whereas BMP-2 induced expression of Id2, Dlx3, Zbtb2 and Krt16 is endocytosis-independent. Furthermore, we demonstrate that short term inhibition of endocytosis interferes with osteoblast differentiation as measured by alkaline phosphatase (ALP) production and qPCR analysis of osteoblast marker gene expression.Conclusions/significanceOur study demonstrates that dynamin-dependent endocytosis is crucial for the concise spatial activation of the BMP-2 induced signaling cascade. Inhibition of endocytic processes during BMP-2 stimulation leads to altered Smad1/5/8 signaling kinetics and results in differential target gene expression. We show that interfering with the BMP-2 induced transcriptional network by endocytosis inhibition results in an attenuation of osteoblast differentiation. This implies that selective sensitivity of gene expression to endocytosis provides an additional mechanism for the cell to respond to BMP in a context specific manner. Moreover, we suggest a novel Smad dependent signal cascade induced by BMP-2, which does not require endocytosis.

Project description:Dentinogenesis, a formation of dentin by odontoblasts, is an essential process during tooth development. Bone morphogenetic proteins (BMPs) are one of the most crucial growth factors that contribute to dentin formation. However, it is still unclear how BMP signaling pathways regulate postnatal crown and root dentinogenesis. BMPs transduce signals through canonical Smad and non-Smad signaling pathways including p38 and ERK signaling pathways. To investigate the roles of Smad and non-Smad signaling pathways in dentinogenesis, we conditionally deleted Bmpr1a, which encodes the type 1A receptor for BMPs, to remove both Smad and non-Smad pathways in Osterix-expressing cells. We also expressed a constitutively activated form of Bmpr1a (caBmpr1a) to increase Smad1/5/9 signaling activity without altered non-Smad activity in odontoblasts. To understand the function of BMP signaling during postnatal dentin formation, Cre activity was induced at the day of birth. Our results showed that loss of BmpR1A in odontoblasts resulted in impaired dentin formation and short molar roots at postnatal day 21. Bmpr1a cKO mice displayed a reduction of dentin matrix production compared to controls associated with increased cell proliferation and reduced Osx and Dspp expression. In contrast, caBmpr1a mutant mice that show increased Smad1/5/9 signaling activity resulted in no overt tooth phenotype. To further dissect the functions of each signaling activity, we generated Bmpr1a cKO mice also expressing caBmpr1a to restore only Smad1/5/9 signaling activity. Restoring Smad activity in the compound mutant mice rescued impaired crown dentin formation in the Bmpr1a cKO mice; however, impaired root dentin formation and short roots were not changed. These results suggest that BMP-Smad signaling in odontoblasts is responsible for crown dentin formation, while non-Smad signaling may play a major role in root dentin formation and elongation. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Dataset Information

AMOT130 drives BMP-SMAD signaling at the apical membrane in polarized cells.

Publications

AMOT130 drives BMP-SMAD signaling at the apical membrane in polarized cells.

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