Differential regulation of Krüppel-like factor family transcription factor expression in neonatal rat cardiac myocytes: effects of endothelin-1, oxidative stress and cytokines

Krüppel-like transcription factors (Klfs) modulate fundamental cell processes. Cardiac myocytes are terminally-differentiated, but hypertrophy in response to stimuli such as endothelin-1. H2O2 or cytokines promote myocyte apoptosis. Microarray studies of neonatal rat myocytes identified several Klfs as endothelin-1-responsive genes. We used quantitative PCR for further analysis of Klf expression in neonatal rat myocytes. In response to endothelin-1, Klf2 mRNA expression was rapidly increased ( approximately 9-fold; 15-30 min) with later increases in expression of Klf4 and Klf6 ( approximately 5-fold; 30-60 min). All were regulated as immediate early genes (cycloheximide did not inhibit the increases in expression). Klf5 expression was increased at 1-2 h ( approximately 13-fold) as a second phase response (cycloheximide inhibited the increase). These increases were transient and attenuated by U0126. H2O2 increased expression of Klf2, Klf4 and Klf6, but interleukin-1beta or tumor necrosis factor alpha downregulated Klf2 expression with no effect on Klf4 or Klf6. Of the Klfs which repress transcription, endothelin-1 rapidly downregulated expression of Klf3, Klf11 and Klf15. The dynamic regulation of expression of multiple Klf family members in cardiac myocytes suggests that, as a family, they are actively involved in regulating phenotypic responses (hypertrophy and apoptosis) to extracellular stimuli

Tcf15 Primes Pluripotent Cells for Differentiation

SummaryThe events that prime pluripotent cells for differentiation are not well understood. Inhibitor of DNA binding/differentiation (Id) proteins, which are inhibitors of basic helix-loop-helix (bHLH) transcription factor activity, contribute to pluripotency by blocking sequential transitions toward differentiation. Using yeast-two-hybrid screens, we have identified Id-regulated transcription factors that are expressed in embryonic stem cells (ESCs). One of these, Tcf15, is also expressed in the embryonic day 4.5 embryo and is specifically associated with a novel subpopulation of primed ESCs. An Id-resistant form of Tcf15 rapidly downregulates Nanog and accelerates somatic lineage commitment. We propose that because Tcf15 can be held in an inactive state through Id activity, it may prime pluripotent cells for entry to somatic lineages upon downregulation of Id. We also find that Tcf15 expression is dependent on fibroblast growth factor (FGF) signaling, providing an explanation for how FGF can prime for differentiation without driving cells out of the pluripotent state

PIAS1 regulates CP2c localization and active promoter complex formation in erythroid cell-specific α-globin expression

Data presented here extends our previous observations on α-globin transcriptional regulation by the CP2 and PIAS1 proteins. Using RNAi knockdown, we have now shown that CP2b, CP2c and PIAS1 are each necessary for synergistic activation of endogenous α-globin gene expression in differentiating MEL cells. In this system, truncated PIAS1 mutants lacking the ring finger domain recruited CP2c to the nucleus, as did wild-type PIAS1, demonstrating that this is a sumoylation-independent process. In vitro, recombinant CP2c, CP2b and PIAS1 bound DNA as a stable CBP (CP2c/CP2b/PIAS1) complex. Following PIAS1 knockdown in MEL cells, however, the association of endogenous CP2c and CP2b with the α-globin promoter simultaneously decreased. By mapping the CP2b- and CP2c-binding domains on PIAS1, and the PIAS1-binding domains on CP2b and CP2c, we found that two regions of PIAS1 that interact with CP2c/CP2b are required for its co-activator function. We propose that CP2c, CP2b, and PIAS1 form a hexametric complex with two units each of CP2c, CP2b, and PIAS1, in which PIAS1 serves as a clamp between two CP2 proteins, while CP2c binds directly to the target DNA and CP2b mediates strong transactivation

Thyroid Hormone Promotes Remodeling of Coronary Resistance Vessels

Low thyroid hormone (TH) function has been linked to impaired coronary blood flow, reduced density of small arterioles, and heart failure. Nonetheless, little is known about the mechanisms by which THs regulate coronary microvascular remodeling. The current study examined the initial cellular events associated with coronary remodeling induced by triiodothyronine (T3) in hypothyroid rats. Rats with established hypothyroidism, eight weeks after surgical thyroidectomy (TX), were treated with T3 for 36 or 72 hours. The early effects of T3 treatment on coronary microvasculature were examined morphometrically. Gene expression changes in the heart were assessed by quantitative PCR Array. Hypothyroidism resulted in arteriolar atrophy in the left ventricle. T3 treatment rapidly induced small arteriolar muscularization and, within 72 hours, restored arteriolar density to control levels. Total length of the capillary network was not affected by TX or T3 treatment. T3 treatment resulted in the coordinate regulation of Angiopoietin 1 and 2 expression. The response of Angiopoietins was consistent with vessel enlargement. In addition to the well known effects of THs on vasoreactivity, these results suggest that THs may affect function of small resistance arteries by phenotypic remodeling of vascular smooth muscle cells (VSMC)

Smooth Muscle miRNAs Are Critical for Post-Natal Regulation of Blood Pressure and Vascular Function

Phenotypic modulation of smooth muscle cells (SMCs) plays a key role in vascular disease, including atherosclerosis. Several transcription factors have been suggested to regulate phenotypic modulation of SMCs but the decisive mechanisms remain unknown. Recent reports suggest that specific microRNAs (miRNAs) are involved in SMC differentiation and vascular disease but the global role of miRNAs in postnatal vascular SMC has not been elucidated. Thus, the objective of this study was to identify the role of Dicer-dependent miRNAs for blood pressure regulation and vascular SMC contractile function and differentiation in vivo. Tamoxifen-inducible and SMC specific deletion of Dicer was achieved by Cre-Lox recombination. Deletion of Dicer resulted in a global loss of miRNAs in aortic SMC. Furthermore, Dicer-deficient mice exhibited a dramatic reduction in blood pressure due to significant loss of vascular contractile function and SMC contractile differentiation as well as vascular remodeling. Several of these results are consistent with our previous observations in SM-Dicer deficient embryos. Therefore, miRNAs are essential for maintaining blood pressure and contractile function in resistance vessels. Although the phenotype of miR-143/145 deficient mice resembles the loss of Dicer, the phenotypes of SM-Dicer KO mice were far more severe suggesting that additional miRNAs are involved in maintaining postnatal SMC differentiation

PIAS1 Activates the Expression of Smooth Muscle Cell Differentiation Marker Genes by Interacting with Serum Response Factor and Class I Basic Helix-Loop-Helix Proteins

Although a critical component of vascular disease is modulation of the differentiated state of vascular smooth muscle cells (SMC), the mechanisms governing SMC differentiation are relatively poorly understood. We have previously shown that E-boxes and the ubiquitously expressed class I basic helix-loop-helix (bHLH) proteins, including E2-2 and E12, are important in regulation of the SMC differentiation marker gene, the SM α-actin gene. The aim of the present study was to identify proteins that bind to class I bHLH proteins in SMC and modulate transcriptional regulation of SMC differentiation marker genes. Herein we report that members of the protein inhibitor of activated STAT (PIAS) family interact with class I bHLH factors as well as serum response factor (SRF). PIAS1 interacted with E2-2 and E12 based on yeast two-hybrid screens, mammalian two-hybrid assays, and/or coimmunoprecipitation assays. Overexpression of PIAS1 significantly activated the SM α-actin promoter and mRNA expression, as well as SM myosin heavy chain and SM22α, whereas a small interfering RNA for PIAS1 decreased activity of these promoters, as well as endogenous mRNA expression, and SRF binding to SM α-actin promoter within intact chromatin in cultured SMC. Of significance, PIAS1 bound to SRF and activated SM α-actin promoter expression in wild-type but not SRF(−/−) embryonic stem cells. These results provide novel evidence that PIAS1 modulates transcriptional activation of SMC marker genes through cooperative interactions with both SRF and class I bHLH proteins