The transforming-growth-factor-beta-activated kinase TAK1 is a member of the mitogen-activated protein kinase kinase kinase family, which couples extracellular stimuli to gene transcription. The in vivo function of TAK1 is not understood. Here, we investigated the potential involvement of TAK1 in cardiac hypertrophy. In adult mouse myocardium, TAK1 kinase activity was upregulated 7 days after aortic banding, a mechanical load that induces hypertrophy and expression of transforming growth factor beta. An activating mutation of TAK1 expressed in myocardium of transgenic mice was sufficient to produce p38 mitogen-activated protein kinase phosphorylation in vivo, cardiac hypertrophy, interstitial fibrosis, severe myocardial dysfunction, 'fetal' gene induction, apoptosis and early lethality. Thus, TAK1 activity is induced as a delayed response to mechanical stress, and can suffice to elicit myocardial hypertrophy and fulminant heart failure.

Cysteine-rich LIM-only proteins, CRP1 and CRP2, expressed during cardiovascular development act as bridging molecules that associate with serum response factor and GATA proteins. SRF-CRP-GATA complexes strongly activated smooth muscle gene targets. CRP2 was found in the nucleus during early stages of coronary smooth muscle differentiation from proepicardial cells. A dominant-negative CRP2 mutant blocked proepicardial cells from differentiating into smooth muscle cells. Together with SRF and GATA proteins, CRP1 and CRP2 converted pluripotent 10T1/2 fibroblasts into smooth muscle cells, while muscle LIM protein CRP3 inhibited the conversion. Thus, LIM-only proteins of the CRP family play important roles in organizing multiprotein complexes, both in the cytoplasm, where they participate in cytoskeletal remodeling, and in the nucleus, where they strongly facilitate smooth muscle differentiation.

Serum response factor (SRF), a member of an ancient family of DNA-binding proteins, is generally assumed to be a ubiquitous transcription factor involved in regulating growth factor-responsive genes. However, avian SRF was recently shown (Croissant, J. D., Kim, J.-H., Eichele, G., Goering, L., Lough, J., Prywes, R., and Schwartz, R. J. (1996) Dev. Biol. 177, 250 -264) to be preferentially expressed in myogenic lineages and is required for regulating post-replicative muscle gene expression. Given the central importance of SRF for the muscle tissue-restricted expression of the striated ␣-actin gene family, we wanted to determine how SRF might contribute to this muscle-restricted expression. Here we have characterized the murine SRF genomic locus, which has seven exons interrupted by six introns, with the entire locus spanning 11 kilobases. Murine SRF transcripts were processed to two 3 -untranslated region polyadenylation signals, yielding 4.5-and 2.5-kilobase mRNA species. Murine SRF mRNA levels were the highest in adult skeletal and cardiac muscle, but barely detected in liver, lung, and spleen tissues. During early mouse development, in situ hybridization analysis revealed enrichment of SRF transcripts in the myotomal portion of somites, the myocardium of the heart, and the smooth muscle media of vessels of mouse embryos. Likewise, murine SRF promoter activity was tissue-restricted, being 80-fold greater in primary skeletal myoblasts than in liver-derived HepG2 cells. In addition, SRF promoter activity increased 6-fold when myoblasts withdrew from the cell cycle and fused into differentiated myotubes. A 310-base pair promoter fragment depended upon multiple intact serum response elements in combination with Sp1 sites for maximal myogenic restricted activity. Furthermore, cotransfected SRF expression vector stimulated SRF promoter transcription, whereas dominant-negative SRF mutants blocked SRF promoter activity, demonstrating a positive role for an SRF-dependent autoregulatory loop. Earlier studies (8 -10) have demonstrated that SRF-binding sites, termed serum response elements (SRE; GG(A/T) 6 CC), play a primary role in regulating early response genes such as c-fos and egr-1 (reviewed in Ref. 11). Growth factor signaling through the c-fos SRE appeared to be mediated through the formation of ternary complexes with an accessory factor, p62 TCF , and through protein-DNA interactions with a purinerich sequence at the 5Ј-end of the c-fos SRE (12, 13). The ETS domain proteins Elk-1 (14) and SAP-1 (15) possess biochemical activities that are characteristic of p62 TCF (16) and interact with the C-terminal portion of the MADS box (17). Furthermore, another MADS box accessory factor, a paired-like homeodomain protein, Phox1 (18), has been shown to facilitate the DNA binding activity of SRF on the c-fos SRE. In addition, Phox1 and Elk-1 potentiate the ability of SRF to transcriptionally activate the c-fos promoter in response to growth-mediated events (18).SRF plays an obligatory role in regulating post-replicative muscl...

Combinatorial interaction among cardiac tissue-restricted enriched transcription factors may facilitate the expression of cardiac tissue-restricted genes. Here we show that the MADS box factor serum response factor (SRF) cooperates with the zinc finger protein GATA-4 to synergistically activate numerous myogenic and nonmyogenic serum response element (SRE)-dependent promoters in CV1 fibroblasts. In the absence of GATA binding sites, synergistic activation depends on binding of SRF to the proximal CArG box sequence in the cardiac and skeletal ␣-actin promoter. GATA-4's C-terminal activation domain is obligatory for synergistic coactivation with SRF, and its N-terminal domain and first zinc finger are inhibitory. SRF and GATA-4 physically associate both in vivo and in vitro through their MADS box and the second zinc finger domains as determined by protein A pullout assays and by in vivo one-hybrid transfection assays using Gal4 fusion proteins. Other cardiovascular tissue-restricted GATA factors, such as GATA-5 and GATA-6, were equivalent to GATA-4 in coactivating SRE-dependent targets. Thus, interaction between the MADS box and C4 zinc finger proteins, a novel regulatory paradigm, mediates activation of SRF-dependent gene expression.Serum response factor (SRF) may play a leading role in the commitment of cardiac progenitors by virtue of its requirement for mesoderm formation and by its ability to activate target genes via specific protein-protein associations with other early cardiac enriched transcription factors. SRF, a member of an ancient DNA-binding protein family, shares a highly conserved DNA-binding and dimerization domain of 90 amino acids, termed the MADS box (reviewed in references 53 and 56). SRF, yeast transcription factors MCM1 and ARG80, and several plant proteins, such as Deficiens, all have a related MADS box and similar DNA sequence binding specificity. In addition, SRF-related proteins (RSRF and MEF-2) constitute a subfamily of the MADS box family of transcription factors (49, 59).

Herein, the restricted expression of serum response factors (SRF) closely overlapped with Nkx2-5 and GATA4 transcripts in early chick embryos coinciding with the earliest appearance of cardiac ␣-actin (␣CA) transcripts and nascent myocardial cells. The combinatorial expression of SRF, a MADS box factor Nkx2-5 (a NK4 homeodomain), and/or GATA4, a dual C4 zinc finger protein, in heterologous CV1 fibroblasts and Schneider 2 insect cells demonstrated synergistic induction of ␣CA promoter activity. These three factors induced endogenous ␣CA mRNA over a 100-fold in murine embryonic stem cells. In addition, the DNA-binding defective mutant Nkx2-5pm efficiently coactivated the ␣CA promoter in the presence of SRF and GATA4 in the presence of all four SREs and was substantially weakened when individual SREs were mutated and or serially deleted. In contrast, the introduction of SRFpm, a SRF DNA-binding mutant, blocked the activation with all of the ␣CA promoter constructions. These assays indicated a dependence upon cooperative SRF binding for facilitating the recruitment of Nkx2-5 and GATA4 to the ␣CA promoter. Furthermore, the recruitment of Nkx2-5 and GATA4 by SRF was observed to strongly enhance SRF DNA binding affinity. This mechanism allowed for the formation of higher ordered ␣CA promoter DNA binding complexes, led to a model of SRF physical association with Nkx2-5 and GATA4.