Crossveinless-2 Controls Bone Morphogenetic Protein Signaling during Early Cardiomyocyte Differentiation in P19 Cells

Abstract

Increasing evidence indicates that bone morphogenetic proteins (BMPs) are crucial for cardiac induction, specification, and development. Although signaling of BMPs is tightly regulated through soluble BMP-binding proteins, how they regulate BMP signaling during cardiac differentiation remains unknown. To identify molecules responsible for BMP signaling during early cardiomyocyte differentiation of P19 cells, cDNA subtraction was performed. We found a bimodal expression of the BMPbinding protein Crossveinless-2 (Cv2) during cardiomyocyte differentiation; Cv2 is temporally expressed earlier than cardiac transcription factors such as Nkx2.5 and Tbx5 and acts as a suppressor for BMP signaling in P19 cells. We established a P19 clonal cell line harboring a cardiac alpha-myosin heavy chain promoter-driven enhanced green fluorescent protein gene to monitor cardiac differentiation by flow cytometry. Treatment with BMP2 during the first 2 days of differentiation suppressed cardiomyocyte differentiation through activation of downstream targets Smad1/5/8 protein and Id1 gene, whereas treatment with Cv2 conversely inhibited Smad1/5/8 activation and Id1 expression, leading to increased generation of cardiac cells. RNA interference-mediated knockdown (KD) of endogenous Cv2 showed increased Smad1/5/8 activation and impaired cardiomyocyte differentiation. Expression of cardiac mesoderm markers was reduced, whereas expression of Id1 and endoderm markers such as Sox7, Hnf4, and E-cadherin was induced in Cv2-kinase dead cells. These phenotypes were rescued by the addition of Cv2 protein to the culture media during the first 2 days of differentiation or co-culture with parental cells. These data suggest that Cv2 may specify cardiac mesodermal lineage through inhibition of BMP signaling at early stage of cardiogenesis.Heart development during embryogenesis is a multistep process that involves cardiac induction of mesodermal progenitor cells into the cardiac lineage. Although the genetic blueprint for cardiac differentiation and development is rapidly being elucidated (1-3), there is still uncertainty about cardiac-inducing factors which might be involved in cardiogenic induction and specification.Increasing evidence indicates that BMPs 2 are crucial for cardiac induction, specification, and development (2-7). Conventional deletion of BMP receptor 1a in mice showed no formation of mesoderm and heart (8). Studies with an in vitro cardiac differentiation model demonstrated an essential role of BMP signals in cardiomyogenesis (9). In contrast, functional disruption of the different BMPs in mice displayed a role in late but not in early cardiogenesis (10). For example, although deletion of BMP2 in the heart resulted in abnormal heart development, specification of cardiac mesoderm occurred normally (11). Explant cultures in chicken revealed that activation of BMP signaling inhibits cardiogenesis at early developmental stages (7). Until now, whether activation or suppression of BMP signaling is required during early cardiogenesis has not b...

Tbx20 Transcription Factor Is a Downstream Mediator for Bone Morphogenetic Protein-10 in Regulating Cardiac Ventricular Wall Development and Function

“…4A). P19 is a well characterized mouse embryo-derived teratocarcinoma cell line that can be differentiated into cardiomyocytes and is widely used to study BMP-mediated signaling (27,28). We found that Smad1 activation reached a plateau level at 50 ng/ml concentration for both of rhBMP10 and rhBmP2 in culture (Fig.…”

BMP activity controlled by BMPER regulates the proinflammatory phenotype of endothelium

“…24,26 Based on this data, here we hypothesized that BMPER exerts its anti-inflammatory effects by inhibiting the binding of proinflammatory BMP2 to its receptor. Vice versa, loss of BMPER may cause inflammation by allowing enhanced BMP activity.…”

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The extracellular regulation of bone morphogenetic protein signaling

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