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J. Biol. Chem., Vol. 283, Issue 39, 26705-26713, September 26, 2008

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

Koichiro Harada¹, Akiko Ogai^¶, Tomosaburo Takahashi^||, Masafumi Kitakaze, Hiroaki Matsubara^||, and Hidemasa Oh

From the Department of Experimental Therapeutics, Translational Research Center, Kyoto University Hospital, Kyoto 606-8507, ^||Department of Cardiovascular Medicine, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Cardiovascular Medicine, National Cardiovascular Center, Osaka 565-8565, and ^¶Department of Epidemiology, National Cardiovascular Center Research Institute, Osaka 565-8565, Japan

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 BMP-binding 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 down-stream 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.

Received for publication, February 25, 2008 , and in revised form, July 25, 2008.

^* This work was supported by grants from Japan Cardiovascular Research Foundation, grants-in-aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and grants-in-aid from the Ministry of Health, Labor, and Welfare of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: 5-7-1 Fujishiro-dai, Suita, Osaka 565-8565, Japan. Fax: 81-6-6833-9865; E-mail: kharada_kuhp{at}yahoo.co.jp.

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