Koshiro Monzen

Bone Morphogenetic Proteins Induce Cardiomyocyte Differentiation through the Mitogen-Activated Protein Kinase Kinase Kinase TAK1 and Cardiac Transcription Factors Csx/Nkx-2.5 and GATA-4

ABSTRACT Bone morphogenetic proteins (BMPs) have been shown to induce ectopic expression of cardiac transcription factors and beating cardiomyocytes in nonprecardiac mesodermal cells in chicks, suggesting that BMPs are inductive signaling molecules that participate in the development of the heart. However, the precise molecular mechanisms by which BMPs regulate cardiac development are largely unknown. In the present study, we examined the molecular mechanisms by which BMPs induce cardiac differentiation by using the P19CL6 in vitro cardiomyocyte differentiation system, a clonal derivative of P19 embryonic teratocarcinoma cells. We established a permanent P19CL6 cell line, P19CL6noggin, which constitutively overexpresses the BMP antagonist noggin. Although almost all parental P19CL6 cells differentiate into beating cardiomyocytes when treated with 1% dimethyl sulfoxide, P19CL6noggin cells did not differentiate into beating cardiomyocytes nor did they express cardiac transcription factors or contractile protein genes. The failure of differentiation was rescued by overexpression of BMP-2 or addition of BMP protein to the culture media, indicating that BMPs were indispensable for cardiomyocyte differentiation in this system. Overexpression of TAK1, a member of the mitogen-activated protein kinase kinase kinase superfamily which transduces BMP signaling, restored the ability of P19CL6noggin cells to differentiate into cardiomyocytes and concomitantly express cardiac genes, whereas overexpression of the dominant negative form of TAK1 in parental P19CL6 cells inhibited cardiomyocyte differentiation. Overexpression of both cardiac transcription factors Csx/Nkx-2.5 and GATA-4 but not of Csx/Nkx-2.5 or GATA-4 alone also induced differentiation of P19CL6noggin cells into cardiomyocytes. These results suggest that TAK1, Csx/Nkx-2.5, and GATA-4 play a pivotal role in the cardiogenic BMP signaling pathway.

Context-dependent Transcriptional Cooperation Mediated by Cardiac Transcription Factors Csx/Nkx-2.5 and GATA-4

Although the cardiac homeobox geneCsx/Nkx-2.5 is essential for normal heart development, little is known about its regulatory mechanisms. In a search for the downstream target genes of Csx/Nkx-2.5, we found that the atrial natriuretic peptide (ANP) gene promoter was strongly transactivated by Csx/Nkx-2.5. Deletion and mutational analyses of the ANP promoter revealed that the Csx/Nkx-2.5-binding element (NKE2) located at −240 was required for high level transactivation by Csx/Nkx-2.5. We also found that Csx/Nkx-2.5 and GATA-4 displayed synergistic transcriptional activation of the ANP promoter, and in contrast to previous reports (Durocher, D., Charron, F., Warren, R., Schwartz, R. J., and Nemer, M. (1997) EMBO J. 16, 5687–5696; Lee, Y., Shioi, T., Kasahara, H., Jobe, S. M., Wiese, R. J., Markham, B., and Izumo, S (1998) Mol. Cell. Biol. 18, 3120–3129), this synergism was dependent on binding of Csx/Nkx-2.5 to NKE2, but not on GATA-4-DNA interactions. Although GATA-4 also potentiated the Csx/Nkx-2.5-induced transactivation of the artificial promoter that contains multimerized Csx/Nkx-2.5-binding sites, Csx/Nkx-2.5 reduced the GATA-4-induced transactivation of the GATA-4-dependent promoters. These findings indicate that the cooperative transcriptional regulation mediated by Csx/Nkx-2.5 and GATA-4 is promoter context-dependent and suggest that the complexcis-trans interactions may fine-tune gene expression in cardiac myocytes.

UTF1 is a chromatin-associated protein involved in ES cell differentiation

Embryonic stem (ES) cells are able to grow indefinitely (self-renewal) and have the potential to differentiate into all adult cell types (pluripotency). The regulatory network that controls pluripotency is well characterized, whereas the molecular basis for the transition from self-renewal to the differentiation of ES cells is much less understood, although dynamic epigenetic gene silencing and chromatin compaction are clearly implicated. In this study, we report that UTF1 (undifferentiated embryonic cell transcription factor 1) is involved in ES cell differentiation. Knockdown of UTF1 in ES and carcinoma cells resulted in a substantial delay or block in differentiation. Further analysis using fluorescence recovery after photobleaching assays, subnuclear fractionations, and reporter assays revealed that UTF1 is a stably chromatin-associated transcriptional repressor protein with a dynamic behavior similar to core histones. An N-terminal Myb/SANT domain and a C-terminal domain containing a putative leucine zipper are required for these properties of UTF1. These data demonstrate that UTF1 is a strongly chromatin-associated protein involved in the initiation of ES cell differentiation.

A crucial role of a high mobility group protein HMGA2 in cardiogenesis

The high mobility group (HMG) of nuclear proteins regulates expression of many genes through architectural remodelling of the chromatin structure, and formation of multiprotein complexes on promoter/enhancer regions. This leads to the active transcription of their target genes. Here we show that HMGA2, a member of the HMGA sub-family of HMG proteins, has a critical function in cardiogenesis. Overexpression of HMGA2 enhanced, whereas siRNA-mediated knockdown of HMGA2 blocked, cardiomyocyte differentiation of the embryonal carcinoma cell line P19CL6. Moreover, overexpression of a dominant-negative HMGA2 or morpholino-mediated knockdown of HMGA2 expression blocked normal heart formation in Xenopus laevis embryos, suggesting that HMGA2 has an important role in cardiogenesis both in vitro and in vivo. Mechanistically, HMGA2 associated with Smad1/4 and showed synergistic trans-activation of the gene for a cardiac transcription factor Nkx2.5; a conserved HMGA2 binding site was required for the promoter activity of Nkx2.5 gene, both in P19CL6 cells and in transgenic Xenopus embryos. Thus, HMGA2 is a positive regulator of Nkx2.5 gene expression and is essential for normal cardiac development.

A common Ile 823 Met variant of ATP-binding cassette transporter A1 gene (ABCA1) alters high density lipoprotein cholesterol level in Japanese population

Recently, variants in ATP-binding cassette transporter A1 (ABCA1) were demonstrated to be associated with increased level of high density lipoprotein cholesterol (HDL-C) and decreased risk of coronary artery disease (CAD) in Caucasians. However, this is not universally applicable due to the ethnic or environmental differences. In this context, to clarify the effect of ABCA1 in Japanese, we evaluated the phenotypic effects of I/M 823 and R/K 219 variants on the plasma level of HDL-C in 410 patients recruited in our hospital. Subjects with M 823 allele had significantly higher level of HDL-C than those without M823 allele (49.0+/-15.1 vs. 44.9+/-11.5 mg/dl, respectively, P<0.05). This statistical significance did not change even after multiple regression analysis. In contrast, there was no difference in HDL-C level among the genotypes in R/K 219 polymorphism. Further, in our study population an inverse relationship was shown to exist between HDL-C level and incidence of CAD. However, no positive association was observed between those variants and susceptibility to CAD. In this study, we provide evidence that I/M 823 variant, not R/K 219 variant, in ABCA1 is one of the determinants of HDL-C level, suggesting the importance of this gene on lipid metabolism in Japanese.

Calcineurin Inhibitor Attenuates the Development and Induces the Regression of Cardiac Hypertrophy in Rats With Salt-Sensitive Hypertension

BackgroundIt remains unclear how hemodynamic overload induces cardiac hypertrophy. Recently, activation of calcium-dependent phosphatase, calcineurin, has been elucidated to induce cardiac hypertrophy. In the present study, we examined the role of calcineurin in load-induced cardiac hypertrophy by using Dahl salt-sensitive (DS) rats, which develop both pressure and volume overload when fed a high salt diet. Methods and ResultsIn the DS rat heart, the activity of calcineurin was increased and cardiac hypertrophy was induced by high salt diet. Treatment of DS rats with the calcineurin inhibitor FK506 (0.1 or 0.01 mg/kg twice daily) from the age of 6 weeks to 12 weeks inhibited the activation of calcineurin in the heart in a dose-dependent manner and attenuated the development of load-induced cardiac hypertrophy and fibrosis without change of hemodynamic parameters. Additionally, treatment with 0.1 mg/kg twice daily but not with 0.01 mg/kg twice daily of FK506 from the age of 12 weeks to 16 weeks induced regression of cardiac hypertrophy in DS rats. Load-induced reprogramming of gene expression was also suppressed by the FK506 treatment. ConclusionsThese results suggest that calcineurin is involved in the development of cardiac hypertrophy in rats with salt-sensitive hypertension and that inhibition of calcineurin could induce regression of cardiac hypertrophy.

Early stage-specific inhibitions of cardiomyocyte differentiation and expression of Csx/Nkx-2.5 and GATA-4 by phosphatidylinositol 3-kinase inhibitor LY294002

Inhibition of phosphatidylinositol 3-kinase (PI3-kinase) has been reported to block cardiomyocyte differentiation. However, at which stage PI3-kinase plays this important role and what its molecular targets are remain unknown. To answer these questions, we induced cardiomyocyte differentiation of P19CL6 mouse embryonal carcinoma cells and investigated the activation of PI3-kinase by analyzing phospho-Akt. We also treated P19CL6 cells with the PI3-kinase-specific inhibitor LY294002 either continuously or at various time points and monitored the expression of cardiac contractile proteins and transcription factors. Most cells differentiated into sarcomeric myosin heavy chain (MHC)-positive cardiomyocytes on day 16 after induction. An increase in phospho-Akt was observed after induction and was maintained throughout the differentiation. LY294002 treatment restricted to the phase from days 0 to 4 was sufficient to inhibit cardiomyocyte differentiation in a dose-dependent manner. In contrast, LY294002 treatment either from days 4 to 8 or from days 8 to 12 did not cause significant changes in sarcomeric MHC expression. LY294002 treatment from days 0 to 4 also suppressed Csx/Nkx-2.5 and GATA-4 expression. These results demonstrate that PI3-kinase becomes activated and plays a pivotal role at a very early stage of cardiomyocyte differentiation, possibly by modulating the expression of the cardiac transcription factors.

A novel myocyte-specific gene Midori promotes the differentiation of P19CL6 cells into cardiomyocytes.

Although several cardiac-specific transcription factors have been shown to play vital roles in various steps during the heart formation, the precise mechanism of the early stage of cardiogenesis has yet to be elucidated. By differential display technique, we tried to identify molecules that are expressed earlier than cardiac transcription factors such as CSX/NKX2-5 and GATA-4 and are involved in cardiomyocyte differentiation using the P19CL6 cell line, which efficiently differentiates into cardiomyocytes when treated with dimethyl sulfoxide. We isolated a novel gene designatedMidori. Its deduced amino acid sequence contained an ATP/GTP-binding site, Ig-like domain, and Kringle-like domain. Northern blot analysis revealed that expression of Midori was restricted to the fetal and adult heart and adult skeletal muscle in mice. In whole mount in situ hybridization,Midori was expressed in cardiac crescent and developing heart but not in somites. The MIDORI protein was localized in the nucleus and overexpression of Midori induced expression of endogenous Midori itself, suggesting that MIDORI may act as a transcriptional regulator. Permanent P19CL6 cell lines overexpressing Midori more efficiently differentiated into cardiomyocytes than did parental cells, whereas those overexpressing the antisense Midori less efficiently differentiated. These results suggest that Midori may promote the differentiation of P19CL6 into cardiomyocytes.

Dual effects of the homeobox transcription factor Csx/Nkx2-5 on cardiomyocytes

A homeobox-containing transcription factor Csx/Nkx2-5 is an important regulator of cardiac development. Many different human CSX/NKX2-5 mutations have been reported to cause congenital heart disease. We here examined the effects of three representative CSX/NKX2-5 mutations on cardiomyocyte differentiation and death with the use of the P19CL6 cardiomyogenic cell lines. Stable overexpression of wild-type CSX/NKX2-5 enhanced expression of cardiac-specific genes such as MEF2C and MLC2v, the promoter activity of the atrial natriuretic peptide gene, and the terminal differentiation of P19CL6 into cardiomyocytes, while all CSX/NKX2-5 mutants attenuated them by different degrees. When exposed to H(2)O(2) or cultured without change of the medium, many differentiated P19CL6 cells overexpressing the mutants, especially the mutant which lacks the carboxyl terminal region just after the homeodomain, were dead, while most of the cells overexpressing wild-type CSX/NKX2-5 survived. Overexpression of the carboxyl terminus-deleted mutant down-regulated expression of an anti-apoptotic protein Bcl-x(L) and up-regulated that of a pro-apoptotic protein CAS, while in the cells overexpressing wild-type CSX/NKX2-5, expression of a pro-apoptotic protein RIP was reduced. Furthermore, overexpression of wild-type CSX/NKX2-5 decreased the number of H(2)O(2)-induced TUNEL-positive cultured cardiomyocytes of neonatal rats, whereas overexpression of the mutants enhanced it. These results suggest that Csx/Nkx2-5 not only regulates expression of cardiac-specific genes but protects cardiomyocytes from stresses and that cell death may be another cause for the cardiac defects induced by human CSX/NKX2-5 mutations.

Overexpression of TNNI3K, a cardiac-specific MAP kinase, promotes P19CL6-derived cardiac myogenesis and prevents myocardial infarction-induced injury

TNNI3K is a new cardiac-specific MAP kinase whose gene is localized to 1p31.1 and that belongs to a tyrosine kinase-like branch in the kinase tree of the human genome. In the present study we investigated the role of TNNI3K in the cardiac myogenesis process and in the repair of ischemic injury. Pluripotent P19CL6 cells with or without transfection by pcDNA6-TNNI3K plasmid were used to induce differentiation into beating cardiomyocytes. TNNI3K promoted the differentiation process, judging from the increasing beating mass and increased number of alpha-actinin-positive cells. TNNI3K improved cardiac function by enhancing beating frequency and increasing the contractile force and epinephrine response of spontaneous action potentials without an increase of the single-cell size. TNNI3K suppressed phosphorylation of cardiac troponin I, annexin-V(+) cells, Bax protein, and p38/JNK-mediated apoptosis. Intramyocardial administration of TNNI3K-overexpressing P19CL6 cells in mice with myocardial infarction improved cardiac performance and attenuated ventricular remodeling compared with injection of wild-type P19CL6 cells. In conclusion, our study clearly indicates that TNNI3K promotes cardiomyogenesis, enhances cardiac performance, and protects the myocardium from ischemic injury by suppressing p38/JNK-mediated apoptosis. Therefore, modulation of TNNI3K activity would be a useful therapeutic approach for ischemic cardiac disease.