Fbxo25 controls Tbx5 and Nkx2-5 transcriptional activity to regulate cardiomyocyte development.
The ubiquitin-proteasome system (UPS) plays an important role in protein quality control, cellular signalings, and cell differentiation through the regulated turnover of key transcription factors in cardiac tissue. However, the molecular mechanism underlying Fbxo25-mediated ubiquitination of cardiac transcription factors remains elusive. We report that an Fbxo25-mediated SCF ubiquitination pathway regulates the protein levels and activities of Tbx5 and Nkx2-5 based on our studies using MG132, proteasome inhibitor, and the temperature sensitive ubiquitin system in ts20 cells. Our data indicate that Fbxo25 directly interacts with Tbx5 and Nkx2-5 in vitro and in vivo. In support of our findings, a dominant-negative mutant of Fbxo25, Fbxo251-236, prevents Tbx5 degradation and increases Tbx5 transcriptional activity in a Tbx5 responsive luciferase assay. Therefore, Fbxo25 facilitates Tbx5 degradation in an SCF-dependent manner. In addition, the silencing of endogenous Fbxo25 increases Tbx5 and Nkx2-5 mRNA levels and suppresses mESC-derived cardiomyocyte differentiation. Likewise, the exogenous expression of FBXO25 downregulates NKX2-5 level in human ESC-derived cardiomyocytes. In myocardial infarction model, Fbxo25 mRNA decreases, whereas the mRNA and protein levels of Tbx5 and Nkx2-5 increase. The protein levels of Tbx5 and Nkx2-5 are regulated negatively by Fbxo25-mediated SCF ubiquitination pathway. Thus, our findings reveal a novel mechanism for regulation of SCFFbox25-dependent Nkx2-5 and Tbx5 ubiquitination in cardiac development and provide a new insight into the regulatory mechanism of Nkx2-5 and Tbx5 transcriptional activity.
Department of Biomedical Science, Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul 133-791, Republic of Korea.
Hoe-Su
HS
Jeong
Department of Biomedical Science, Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul 133-791, Republic of Korea.
Eun-Shil
ES
Jung
Department of Biomedical Science, Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul 133-791, Republic of Korea.
Ye-Ji
YJ
Sim
Department of Biomedical Science, Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul 133-791, Republic of Korea.
Su-Jin
SJ
Kim
Department of Developmental Biology, CHA University, Seoul 135-907, Republic of Korea.
Jae-Woo
JW
Jang
Department of Developmental Biology, CHA University, Seoul 135-907, Republic of Korea.
Ki-Sung
KS
Hong
Major of Animal Science, College of Natural Science, Konkuk University, Chungju 380-701, Republic of Korea.
Won-Young
WY
Lee
Department of Stem Cell Biology, School of Medicine, Konkuk University, Seoul 143-701, Republic of Korea.
Hyung-Min
HM
Chung
Center for Cancer Research, Department of Pathology and Laboratory Medicine, University of Tennessee Health Science Center, Memphis, TN, USA.
Kyung-Tae
KT
Park
College of Pharmacy, Ajou University, Suwon 443-749, Republic of Korea.
Yi-Sook
YS
Jung
Department of Biomedical Science, Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul 133-791, Republic of Korea. Electronic address: chakimster@gmail.com.
Chang-Hoon
CH
Kim
Department of Biomedical Science, Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul 133-791, Republic of Korea. Electronic address: ks66kim@hanyang.ac.kr.
Kye-Seong
KS
Kim
D000818:Animals; D002454:Cell Differentiation / Q000235:genetics*; D053595:Embryonic Stem Cells; D044783:F-Box Proteins / Q000235:genetics* / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D000072576:Homeobox Protein Nkx-2.5; D018398:Homeodomain Proteins / Q000096:biosynthesis / Q000235:genetics*; D006801:Humans; D007976:Leupeptins / Q000008:administration & dosage; D051379:Mice; D032383:Myocytes, Cardiac / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000187:drug effects / Q000235:genetics; D059748:Proteolysis; D044843:SKP Cullin F-Box Protein Ligases; D020825:T-Box Domain Proteins / Q000096:biosynthesis / Q000235:genetics*; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics*; D015533:Transcriptional Activation / Q000187:drug effects
D044783:F-Box Proteins; C512115:FBXO25 protein, mouse; D000072576:Homeobox Protein Nkx-2.5; D018398:Homeodomain Proteins; D007976:Leupeptins; C484469:Nkx2-5 protein, mouse; D020825:T-Box Domain Proteins; C103172:T-box transcription factor 5; D014157:Transcription Factors; D044843:SKP Cullin F-Box Protein Ligases; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde
Cardiomyocyte; E3 ubiquitin ligase; Fbxo25; Nkx2–5; Tbx5