Melissa B. Rogers

Calcific Aortic Valve Disease A Consensus Summary From the Alliance of Investigators on Calcific Aortic Valve Disease

Calcific aortic valve disease (CAVD) is increasingly prevalent worldwide with significant morbidity and mortality. Therapeutic options beyond surgical valve replacement are currently limited. In 2011, the National Heart Lung and Blood Institute assembled a working group on aortic stenosis. This group identified CAVD as an actively regulated disease process in need of further study. As a result, the Alliance of Investigators on CAVD was formed to coordinate and promote CAVD research, with the goals of identifying individuals at risk, developing new therapeutic approaches, and improving diagnostic methods. The group is composed of cardiologists, geneticists, imaging specialists, and basic science researchers. This report reviews the current status of CAVD research and treatment strategies with identification of areas in need of additional investigation for optimal management of this patient population. # Significance {#article-title-72}Calcific aortic valve disease (CAVD) is increasingly prevalent worldwide with significant morbidity and mortality. Therapeutic options beyond surgical valve replacement are currently limited. In 2011, the National Heart Lung and Blood Institute assembled a working group on aortic stenosis. This group identified CAVD as an actively regulated disease process in need of further study. As a result, the Alliance of Investigators on CAVD was formed to coordinate and promote CAVD research, with the goals of identifying individuals at risk, developing new therapeutic approaches, and improving diagnostic methods. The group is composed of cardiologists, geneticists, imaging specialists, and basic science researchers. This report reviews the current status of CAVD research and treatment strategies with identification of areas in need of additional investigation for optimal management of this patient population.

Transcriptional Regulation of the Bmp2 Gene: Retinoic Acid Induction in F9 Embryonal Carcinoma Cells and Saccharomyces Cerevisiae

Bmp2, a highly conserved member of the transforming growth factor-β gene family, is crucial for normal development. Retinoic acid, combined with cAMP analogs, sharply induces the Bmp2 mRNA during the differentiation of F9 embryonal carcinoma cells into parietal endoderm. Retinoic acid (RA) also induces the Bmp2 gene in chick limb buds. Since normalBmp2 expression may require an endogenous retinoid signal and aberrant Bmp2 expression may cause some aspects of RA-induced teratogenesis, we studied the mechanism underlying the induction of Bmp2. Measurements of the Bmp2mRNA half-life and nuclear run-on assays indicated that RA stimulated the transcription rate of the Bmp2 gene. The results of ribonuclease protection and primer extension assays indicated that Bmp2 transcription started 2,127 nucleotides upstream of the translation start site in F9 cells. To identify genetic elements controlling this transcription rate increase, upstream and downstream genomic sequences flanking the Bmp2 gene were screened using chloramphenicol acetyltransferase reporter genes in F9 cells and β-galactosidase reporter genes in Saccharomyces cerevisiae that were cotransformed with retinoic acid receptor and retinoid X receptor expression plasmids. RA-dependent transcriptional activation was detected between base pairs −2,373 and −2,316 relative to the translation start site. We also identified a required Sp1 binding site between −2,308 and −2,298. The data indicate that Bmp2 is directly regulated by retinoic acid-bound receptors and Sp1.

In Situ Hybridization to Cellular RNA

In situ hybridization to cellular RNA is used to determine the cellular localization of specific messages within complex cell populations and tissues. In this unit, protocols are described for hybridizing slide‐mounted paraffin sections or cryosections with labeled probes. Support protocols describe synthesis of 35S‐labeled riboprobes and dsDNA probes, which are then detected using film autoradiography or emulsion autoradiography. Another support protocol describes synthesis of digoxigenin‐labeled RNA probes, which are non‐radioactive and thus have several advantages. They are easily synthesized in large quantities, they are stable for several months, and they can be reused up to three times. An additional advantage of RNA versus DNA probes is that they result in cleaner signals because nonspecifically bound probe is removed during ribonuclease treatment.

The oncofetal gene Pem encodes a homeodomain and is regulated in primordial and pre-muscle stem cells.

The oncofetal gene, Pem, is expressed in a stage specific manner during murine ontogeny. The carboxy terminal portion of the predicted Pem protein has significant similarity to homeodomains of the Drosophila prd family. The Pem gene is expressed in undifferentiated embryonal stem (ES) and embryonal carcinoma (EC) cell lines. Pem mRNA is induced 35-fold in ES cells differentiated in the absence of retinoic acid. Pem mRNA is increased in EC cells differentiated towards parietal or visceral endoderm, consistent with the abundant Pem expression in embryonic yolk sac. In 10T mesenchymal stem cells committed to muscle cell differentiation, Pem mRNA expression is dramatically increased. The elevation in Pem expression preceded the induction of the muscle master regulatory gene, myoD. We conclude that the Pem gene encodes a candidate transcription factor which is developmentally regulated.

Differences in Fat and Muscle Mass Associated With a Functional Human Polymorphism in a Post-Transcriptional BMP2 Gene Regulatory Element

A classic morphogen, bone morphogenetic protein 2 (BMP2) regulates the differentiation of pluripotent mesenchymal cells. High BMP2 levels promote osteogenesis or chondrogenesis and low levels promote adipogenesis. BMP2 inhibits myogenesis. Thus, BMP2 synthesis is tightly controlled. Several hundred nucleotides within the 3′ untranslated regions of BMP2 genes are conserved from mammals to fishes indicating that the region is under stringent selective pressure. Our analyses indicate that this region controls BMP2 synthesis by post‐transcriptional mechanisms. A common A to C single nucleotide polymorphism (SNP) in the BMP2 gene (rs15705, +A1123C) disrupts a putative post‐transcriptional regulatory motif within the human ultra‐conserved sequence. In vitro studies indicate that RNAs bearing the A or C alleles have different protein binding characteristics in extracts from mesenchymal cells. Reporter genes with the C allele of the ultra‐conserved sequence were differentially expressed in mesenchymal cells. Finally, we analyzed MRI data from the upper arm of 517 healthy individuals aged 18–41 years. Individuals with the C/C genotype were associated with lower baseline subcutaneous fat volumes (P = 0.0030) and an increased gain in skeletal muscle volume (P = 0.0060) following resistance training in a cohort of young males. The rs15705 SNP explained 2–4% of inter‐individual variability in the measured parameters. The rs15705 variant is one of the first genetic markers that may be exploited to facilitate early diagnosis, treatment, and/or prevention of diseases associated with poor fitness. Furthermore, understanding the mechanisms by which regulatory polymorphisms influence BMP2 synthesis will reveal novel pharmaceutical targets for these disabling conditions. J. Cell. Biochem. 107: 1073–1082, 2009.

Mycoplasma infection transforms normal lung cells and induces bone morphogenetic protein 2 expression by post-transcriptional mechanisms

Bone morphogenetic protein 2 (BMP2) is an essential growth factor and morphogen, whose pattern and level of expression profoundly influences development and physiology. We present the novel finding that mycoplasma infection induces BMP2 RNA production in six cell lines of diverse types (mesenchymal, epithelial, and myeloid). Mycoplasma infection triggered the expression of mature secreted BMP2 protein in BEAS‐2B cells (immortalized human bronchial epithelial cells), which normally do not express BMP2, and further increased BMP2 production in A549 cells (lung adenocarcinoma cells). Indeed, mycoplasma is as strong an experimental inducer as inflammatory cytokines and retinoic acid. Second, we showed that post‐transcriptional mechanisms including regulation of RNA stability, rather than transcriptional mechanisms, contributed to the increased BMP2 expression in mycoplasma‐infected cells. Furthermore, a novel G‐rich oligonucleotide, AS1411 that binds the post‐transcriptional regulator nucleolin induced BMP2 exclusively in infected cells. Finally, BMP2 stimulated proliferation in BEAS‐2B cells transformed by chronic mycoplasma infection, as demonstrated by treatment with Noggin, a BMP2 antagonist. These findings have important implications regarding the effects of mycoplasma on BMP2‐regulated processes, including cell proliferation, differentiation, and apoptosis. J. Cell. Biochem. 104: 580–594, 2008.

Life-and-Death Decisions Influenced by Retinoids

Publisher Summary This chapter discusses the interactions between retinoids and their receptors as well as the regulators of cell proliferation and death. Retinoids exert many effects via transcriptional regulation, and so far over 200 known genes have been found to be retinoidresponsive. Some retinoid-responsive genes may act directly in the paths, affecting cell proliferation and death. Retinoid-induced decreases in cell proliferation rate are tightly linked to the well-known ability of retinoids to induce differentiation. In some cells, apoptosis follows this differentiation. Paradoxically, retinoids promote proliferation and survival in other cells. This chapter discusses, primarily, the effects of naturally occurring retinoids, except where the synthetic retinoids provide specific information, regarding retinoid signaling mechanisms. Intertwined cellular signals confront cells, with three primary choices: stop growing (desirable in most adult cells), proliferate, or die, by apoptosis. Although the biochemical mechanisms underlying a cells ultimate choice are inextricably linked to differentiation and many details are unique to a cell type, certain basic principles exist. This chapter describes the cell cycle as understood from a few well-characterized cell types, for example, fibroblasts. It discusses the retinoid signaling molecules involved in transmitting a signal to stop growing or to undergo apoptosis. Apoptosis has two distinct phases. During the first phase, a cell becomes committed to undergoing apoptosis but does not exhibit any of the characteristic features of apoptosis.

Turning Bone Morphogenetic Protein 2 (BMP2) on and off in Mesenchymal Cells.

The concentration, location, and timing of bone morphogenetic protein 2 (BMP2, HGNC:1069, GeneID: 650) gene expression must be precisely regulated. Abnormal BMP2 levels cause congenital anomalies and diseases involving the mesenchymal cells that differentiate into muscle, fat, cartilage, and bone. The molecules and conditions that influence BMP2 synthesis are diverse. Understandably, complex mechanisms control Bmp2 gene expression. This review includes a compilation of agents and conditions that can induce Bmp2. The currently known trans‐regulatory factors and cis‐regulatory elements that modulate Bmp2 expression are summarized and discussed. Bone morphogenetic protein 2 (BMP2, HGNC:1069, GeneID: 650) is a classical morphogen; a molecule that acts at a distance and whose concentration influences cell behavior. In mesenchymal cells, the concentration of BMP2 influences myogenesis, adipogenesis, chondrogenesis, and osteogenesis. Because the amount, timing, and location of BMP2 synthesis influence the allocation of cells to muscle, fat, cartilage, and bone, the mechanisms that regulate the Bmp2 gene are crucial. Key early mesodermal events that require precise Bmp2 regulation include heart specification and morphogenesis. Originally named for its osteoinductive properties, healing fractures requires BMP2. The human Bmp2 gene also has been linked to osteoporosis and osteoarthritis. In addition, all forms of pathological calcification in the vasculature and in cardiac valves involve the pro‐osteogenic BMP2. The diverse tissues, mechanisms, and diseases influenced by BMP2 are too numerous to list here (see OMIM: 112261). However, in all BMP2‐influenced pathologies, changes in the behavior and differentiation of pluripotent mesenchymal cells are a recurring theme. Consequently, much effort has been devoted to identifying the molecules and conditions that influence BMP2 synthesis and the complex mechanisms that control Bmp2 gene expression. This review begins with an overview of the Bmp2 genes chromosomal neighborhood and then summarizes and evaluates known regulatory mechanisms and inducers. J. Cell. Biochem. 116: 2127–2138, 2015.

Utilization of DR1 as true RARE in regulating the Ssm, a novel retinoic acid-target gene in the mouse testis

Various nuclear receptors form dimers to activate target genes via specific response elements located within promoters or enhancers. Retinoid X receptor (RXR) serves as a dimerization partner for many nuclear receptors including retinoic acid receptor (RAR) and peroxisome proliferator-activated receptor (PPAR). Dimers show differential preference towards directly repeated response elements with 1-5 nucleotide spacing, and direct repeat 1 (DR1) is a promiscuous element which recruits RAR/RXR, RXR/RXR, and PPAR/RXR in vitro. In the present investigation, we report identification of a novel RAR/RXR target gene which is regulated by DR1s in the promoter region. This gene, namely spermatocyte-specific marker (Ssm), recruits all the three combinations of nuclear receptors in vitro, but in vivo regulation is observed by trans-retinoic acid-activated RAR/RXR dimer. Indeed, chromatin immunoprecipitation experiment demonstrates binding of RARbeta and RXRalpha in the promoter region of the Ssm. Interestingly, expression of Ssm is almost exclusively observed in spermatocytes in the adult mouse testis, where RA signaling is known to regulate developmental program of male germ cells. The results show that Ssm is a RAR/RXR target gene uniquely using DR1 and exhibits stage-specific expression in the mouse testis with potential function in later stages of spermatogenesis. This finding exemplifies usage of DR1s as retinoic acid response element (RARE) under a specific in vivo context.

Species-specific cis-Regulatory Elements in the 3′-Untranslated Region Direct Alternative Polyadenylation of Bone Morphogenetic Protein 2 mRNA

BMP2 (bone morphogenetic protein 2) is a multifunctional member of the transforming growth factor-β family of growth factors. Disruption of BMP2 signaling results in developmental defects, cancers, and other diseases. BMP2 mRNAs are alternatively polyadenylated, resulting in mRNAs with distinct 3′-untranslated regions. The longer mRNA contains additional putative binding sites for post-transcriptional regulatory factors, including micro-RNAs. We combined functional assays with computational analyses of emerging genome data to define site- and species-specific polyadenylation determinants. In all mouse and human cell lines tested, shorter mRNAs resulting from using the first polyadenylation signal (PA1) were more abundant than mRNAs from the second signal (PA2). However, the PA1/PA2 usage ratios were 2–3-fold higher in human than in mouse cells. Expression of human BMP2 constructs in mouse cells and mouse constructs in human cells showed that cis-regulatory elements direct species-specific 3′ processing of BMP2 transcripts. A 72-nucleotide region downstream of PA2 in the mouse sequence contains two novel cis-acting elements previously hypothesized to regulate polyadenylation in a bioinformatics analysis. Mutations that humanized the mouse-specific elements lowered the affinity for cleavage stimulation factor CstF64 and significantly weakened the PA2 signal relative to the PA1 signal. Thus, we have experimentally defined for the first time cis-regulatory elements that control a species-specific difference in the 3′-end processing of BMP2 and potentially of other genes.