Jay W. Schneider

Cell-free Formation of RNA Granules: Low Complexity Sequence Domains Form Dynamic Fibers within Hydrogels

Eukaryotic cells contain assemblies of RNAs and proteins termed RNA granules. Many proteins within these bodies contain KH or RRM RNA-binding domains as well as low complexity (LC) sequences of unknown function. We discovered that exposure of cell or tissue lysates to a biotinylated isoxazole (b-isox) chemical precipitated hundreds of RNA-binding proteins with significant overlap to the constituents of RNA granules. The LC sequences within these proteins are both necessary and sufficient for b-isox-mediated aggregation, and these domains can undergo a concentration-dependent phase transition to a hydrogel-like state in the absence of the chemical. X-ray diffraction and EM studies revealed the hydrogels to be composed of uniformly polymerized amyloid-like fibers. Unlike pathogenic fibers, the LC sequence-based polymers described here are dynamic and accommodate heterotypic polymerization. These observations offer a framework for understanding the function of LC sequences as well as an organizing principle for cellular structures that are not membrane bound.

Interaction of myogenic factors and the retinoblastoma protein mediates muscle cell commitment and differentiation

The experiments reported here document that the tumor suppressor retinoblastoma protein (pRB) plays an important role in the production and maintenance of the terminally differentiated phenotype of muscle cells. We show that pRB inactivation, through either phosphorylation, binding to T antigen, or genetic alteration, inhibits myogenesis. Moreover, inactivation of pRB in terminally differentiated cells allows them to reenter the cell cycle. In addition to its involvement in the myogenic activities of MyoD, pRB is also required for the cell growth-inhibitory activity of this myogenic factor. We also show that pRB and MyoD directly bind to each other, both in vivo and in vitro, through a region that involves the pocket and the basic-helix-loop-helix domains, respectively. All the results obtained are consistent with the proposal that the effects of MyoD on the cell cycle and of pRB on the myogenic pathway result from the direct binding of the two molecules.

The Distinct Metabolic Profile of Hematopoietic Stem Cells Reflects Their Location in a Hypoxic Niche

Bone marrow transplantation is the primary therapy for numerous hematopoietic disorders. The efficiency of bone marrow transplantation depends on the function of long-term hematopoietic stem cells (LT-HSCs), which is markedly influenced by their hypoxic niche. Survival in this low-oxygen microenvironment requires significant metabolic adaptation. Here, we show that LT-HSCs utilize glycolysis instead of mitochondrial oxidative phosphorylation to meet their energy demands. We used flow cytometry to identify a unique low mitochondrial activity/glycolysis-dependent subpopulation that houses the majority of hematopoietic progenitors and LT-HSCs. Finally, we demonstrate that Meis1 and Hif-1alpha are markedly enriched in LT-HSCs and that Meis1 regulates HSC metabolism through transcriptional activation of Hif-1alpha. These findings reveal an important transcriptional network that regulates HSC metabolism.

Cardiogenic small molecules that enhance myocardial repair by stem cells

The clinical success of stem cell therapy for myocardial repair hinges on a better understanding of cardiac fate mechanisms. We have identified small molecules involved in cardiac fate by screening a chemical library for activators of the signature gene Nkx2.5, using a luciferase knockin bacterial artificial chromosome (BAC) in mouse P19CL6 pluripotent stem cells. We describe a family of sulfonyl-hydrazone (Shz) small molecules that can trigger cardiac mRNA and protein expression in a variety of embryonic and adult stem/progenitor cells, including human mobilized peripheral blood mononuclear cells (M-PBMCs). Small-molecule-enhanced M-PBMCs engrafted into the rat heart in proximity to an experimental injury improved cardiac function better than control cells. Recovery of cardiac function correlated with persistence of viable human cells, expressing human-specific cardiac mRNAs and proteins. Shz small molecules are promising starting points for drugs to promote myocardial repair/regeneration by activating cardiac differentiation in M-PBMCs.

A Dynamic Notch Injury Response Activates Epicardium and Contributes to Fibrosis Repair

Rationale: Transgenic Notch reporter mice express enhanced green fluorescent protein in cells with C-promoter binding factor-1 response element transcriptional activity (CBF1-REx4-EGFP), providing a unique and powerful tool for identifying and isolating “Notch-activated” progenitors. Objective: We asked whether, as in other tissues of this mouse, EGFP localized and functionally tagged adult cardiac tissue progenitors, and, if so, whether this cell-based signal could serve as a quantitative and qualitative biosensor of the injury repair response of the heart. Methods and Results: In addition to scattered endothelial and interstitial cells, Notch-activated (EGFP+) cells unexpectedly richly populated the adult epicardium. We used fluorescence-activated cell sorting to isolate EGFP+ cells and excluded hematopoietic (CD45+) and endothelial (CD31+) subsets. We analyzed EGFP+/CD45−/CD31− cells, a small (<2%) but distinct subpopulation, by gene expression profiling and functional analyses. We called this mixed cell pool, which had dual multipotent stromal cell and epicardial lineage signatures, Notch-activated epicardial-derived cells (NECs). Myocardial infarction and thoracic aortic banding amplified the NEC pool, increasing fibroblast differentiation. Validating the functional vitality of clonal NEC lines, serum growth factors triggered epithelial–mesenchymal transition and the immobilized Notch ligand Delta-like 1–activated downstream target genes. Moreover, cardiomyocyte coculture and engraftment in NOD-SCID (nonobese diabetic–severe combined immunodeficiency) mouse myocardium increased cardiac gene expression in NECs. Conclusions: A dynamic Notch injury response activates adult epicardium, producing a multipotent cell population that contributes to fibrosis repair.

Small-molecule activation of neuronal cell fate

We probed an epigenetic regulatory path from small molecule to neuronal gene activation. Isoxazole small molecules triggered robust neuronal differentiation in adult neural stem cells, rapidly signaling to the neuronal genome via Ca(2+) influx. Ca(2+)-activated CaMK phosphorylated and mediated nuclear export of the MEF2 regulator HDAC5, thereby de-repressing neuronal genes. These results provide new tools to explore the epigenetic signaling circuitry specifying neuronal cell fate and new leads for neuro-regenerative drugs.

Rat-brain Na,K-ATPase beta-chain gene: primary structure, tissue-specific expression, and amplification in ouabain-resistant HeLa C+ cells.

We deduced the complete amino acid sequence of the rat brain Na,K-ATPase beta-subunit from cDNA. The rat brain beta-subunit exhibits a high degree of primary sequence and secondary structural homology with the human and Torpedo beta-subunit polypeptides. Analysis of rat tissue RNA reveals that the beta-subunit gene encodes four separate mRNA species which are expressed in a tissue-specific fashion. In ouabain-resistant HeLa C+ cells, beta-subunit DNA sequences are amplified (approximately 20-fold) and beta-subunit mRNAs are overproduced relative to levels in parental HeLa cells. These results suggest that the beta-subunit plays an important role in Na,K-ATPase structure-function and in the mechanism underlying cellular resistance to the cardiac glycosides.

Activation of MRTF-A–dependent gene expression with a small molecule promotes myofibroblast differentiation and wound healing

Significance Myofibroblasts are contractile smooth muscle-like cells that control tissue repair and remodeling. Inappropriate myofibroblast differentiation can cause organ fibrosis or inefficient wound healing. In this article, we demonstrate that myocardin-related transcription factor (MRTF)-A is necessary for myofibroblast differentiation. We also identify an isoxazole ring-containing small molecule that stimulates MRTF-A–dependent gene expression, myofibroblast differentiation, and wound healing. These findings suggest that targeting MRTFs pharmacologically may prove useful in treating fibrotic diseases. Myocardin-related transcription factors (MRTFs) regulate cellular contractility and motility by associating with serum response factor (SRF) and activating genes involved in cytoskeletal dynamics. We reported previously that MRTF-A contributes to pathological cardiac remodeling by promoting differentiation of fibroblasts to myofibroblasts following myocardial infarction. Here, we show that forced expression of MRTF-A in dermal fibroblasts stimulates contraction of a collagen matrix, whereas contractility of MRTF-A null fibroblasts is impaired under basal conditions and in response to TGF–β1 stimulation. We also identify an isoxazole ring-containing small molecule, previously shown to induce smooth muscle α-actin gene expression in cardiac progenitor cells, as an agonist of myofibroblast differentiation. Isoxazole stimulates myofibroblast differentiation via induction of MRTF-A–dependent gene expression. The MRTF-SRF signaling axis is activated in response to skin injury, and treatment of dermal wounds with isoxazole accelerates wound closure and suppresses the inflammatory response. These results reveal an important role for MRTF-SRF signaling in dermal myofibroblast differentiation and wound healing and suggest that targeting MRTFs pharmacologically may prove useful in treating diseases associated with inappropriate myofibroblast activity.

Trastuzumab cardiotoxicity: Speculations regarding pathophysiology and targets for further study

Trastuzumab, a monoclonal antibody that is selective for cells that overexpress the erbB2 receptor protein tyrosine kinase, is a promising targeted therapy for the treatment of breast cancer. Surprisingly, toxic cardiovascular side effects were discovered in late-phase clinical trials, and these effects were most prominent when trastuzumab was combined with anthracycline chemotherapy. We review recent data focusing on how erbB2 monoclonal antibodies could exert a cardiotoxic effect through unique cardiomyocyte cell surface and intracellular structural features, and how an individuals cardiac susceptibility to erbB2 monoclonal antibodies may be dictated by the ability of erbB2 monoclonal antibodies to bind cardiomyocytes. In addition, we discuss ways that anthracyclines may also affect erbB2/erbB4/neuregulin receptor signaling, explaining the apparent synergistic effect. Further investigation of the role of normal and aberrant erbB2 signaling in the development of cardiac dysfunction could lead to an improved understanding of the pathophysiology of cardiac dysfunction and may lead to novel therapies for the treatment of heart failure, regardless of etiology. Understanding the nature and specificity of trastuzumabs cardiotoxic effects is important in better defining clinical criteria for inclusion and exclusion of patients who can safely receive trastuzumab for the treatment of breast cancer, or possibly other malignancies.

Doxorubicin Blocks Cardiomyocyte Autophagic Flux by Inhibiting Lysosome Acidification

Background— The clinical use of doxorubicin is limited by cardiotoxicity. Histopathological changes include interstitial myocardial fibrosis and the appearance of vacuolated cardiomyocytes. Whereas dysregulation of autophagy in the myocardium has been implicated in a variety of cardiovascular diseases, the role of autophagy in doxorubicin cardiomyopathy remains poorly defined. Methods and Results— Most models of doxorubicin cardiotoxicity involve intraperitoneal injection of high-dose drug, which elicits lethargy, anorexia, weight loss, and peritoneal fibrosis, all of which confound the interpretation of autophagy. Given this, we first established a model that provokes modest and progressive cardiotoxicity without constitutional symptoms, reminiscent of the effects seen in patients. We report that doxorubicin blocks cardiomyocyte autophagic flux in vivo and in cardiomyocytes in culture. This block was accompanied by robust accumulation of undegraded autolysosomes. We go on to localize the site of block as a defect in lysosome acidification. To test the functional relevance of doxorubicin-triggered autolysosome accumulation, we studied animals with diminished autophagic activity resulting from haploinsufficiency for Beclin 1. Beclin 1 +/− mice exposed to doxorubicin were protected in terms of structural and functional changes within the myocardium. Conversely, animals overexpressing Beclin 1 manifested an amplified cardiotoxic response. Conclusions— Doxorubicin blocks autophagic flux in cardiomyocytes by impairing lysosome acidification and lysosomal function. Reducing autophagy initiation protects against doxorubicin cardiotoxicity.