A non-canonical role for a small nucleolar RNA in ribosome biogenesis and senescence.

Cellular senescence is an irreversible state of cell-cycle arrest induced by various stresses, including aberrant oncogene activation, telomere shortening, and DNA damage. Through a genome-wide screen, we discovered a conserved small nucleolar RNA (snoRNA), SNORA13, that is required for multiple forms of senescence in human cells and mice. Although SNORA13 guides the pseudouridylation of a conserved nucleotide in the ribosomal decoding center, loss of this snoRNA minimally impacts translation. Instead, we found that SNORA13 negatively regulates ribosome biogenesis. Senescence-inducing stress perturbs ribosome biogenesis, resulting in the accumulation of free ribosomal proteins (RPs) that trigger p53 activation. SNORA13 interacts directly with RPL23, decreasing its incorporation into maturing 60S subunits and, consequently, increasing the pool of free RPs, thereby promoting p53-mediated senescence. Thus, SNORA13 regulates ribosome biogenesis and the p53 pathway through a non-canonical mechanism distinct from its role in guiding RNA modification. These findings expand our understanding of snoRNA functions and their roles in cellular signaling.

Target-directed microRNA degradation regulates developmental microRNA expression and embryonic growth in mammals.

MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression that play critical roles in development and disease. Target-directed miRNA degradation (TDMD), a pathway in which miRNAs that bind to specialized targets with extensive complementarity are rapidly decayed, has emerged as a potent mechanism of controlling miRNA levels. Nevertheless, the biological role and scope of miRNA regulation by TDMD in mammals remains poorly understood. To address these questions, we generated mice with constitutive or conditional deletion of Zswim8, which encodes an essential TDMD factor. Loss of Zswim8 resulted in developmental defects in the heart and lungs, growth restriction, and perinatal lethality. Small RNA sequencing of embryonic tissues revealed widespread miRNA regulation by TDMD and greatly expanded the known catalog of miRNAs regulated by this pathway. These experiments also uncovered novel features of TDMD-regulated miRNAs, including their enrichment in cotranscribed clusters and examples in which TDMD underlies "arm switching," a phenomenon wherein the dominant strand of a miRNA precursor changes in different tissues or conditions. Importantly, deletion of two miRNAs, miR-322 and miR-503, rescued growth of Zswim8-null embryos, directly implicating the TDMD pathway as a regulator of mammalian body size. These data illuminate the broad landscape and developmental role of TDMD in mammals.

Target-directed microRNA degradation regulates developmental microRNA expression and embryonic growth in mammals.

MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression that play critical roles in development and disease. Target-directed miRNA degradation (TDMD), a pathway in which miRNAs that bind to specialized targets with extensive complementarity are rapidly decayed, has emerged as a potent mechanism of controlling miRNA levels. Nevertheless, the biological role and scope of miRNA regulation by TDMD in mammals remains poorly understood. To address these questions, we generated mice with constitutive or conditional deletion of Zswim8 , which encodes an essential TDMD factor. Loss of Zswim8 resulted in developmental defects in heart and lung, growth restriction, and perinatal lethality. Small RNA sequencing of embryonic tissues revealed widespread miRNA regulation by TDMD and greatly expanded the known catalog of miRNAs regulated by this pathway. These experiments also uncovered novel features of TDMD-regulated miRNAs, including their enrichment in co-transcribed clusters and examples in which TDMD underlies 'arm switching', a phenomenon wherein the dominant strand of a miRNA precursor changes in different tissues or conditions. Importantly, deletion of two miRNAs, miR-322 and miR-503, rescued growth of Zswim8 null embryos, directly implicating the TDMD pathway as a regulator of mammalian body size. These data illuminate the broad landscape and developmental role of TDMD in mammals.

miR-26 suppresses adipocyte progenitor differentiation and fat production by targeting Fbxl19.

Fat storage in adult mammals is a highly regulated process that involves the mobilization of adipocyte progenitor cells (APCs) that differentiate to produce new adipocytes. Here we report a role for the broadly conserved miR-26 family of microRNAs (miR-26a-1, miR-26a-2, and miR-26b) as major regulators of APC differentiation and adipose tissue mass. Deletion of all miR-26-encoding loci in mice resulted in a dramatic expansion of adipose tissue in adult animals fed normal chow. Conversely, transgenic overexpression of miR-26a protected mice from high-fat diet-induced obesity. These effects were attributable to a cell-autonomous function of miR-26 as a potent inhibitor of APC differentiation. miR-26 blocks adipogenesis, at least in part, by repressing expression of Fbxl19, a conserved miR-26 target without a previously known role in adipocyte biology that encodes a component of SCF-type E3 ubiquitin ligase complexes. These findings have therefore revealed a novel pathway that plays a critical role in regulating adipose tissue formation in vivo and suggest new potential therapeutic targets for obesity and related disorders.

Loss of Dis3l2 partially phenocopies Perlman syndrome in mice and results in up-regulation of Igf2 in nephron progenitor cells.

Loss of function of the DIS3L2 exoribonuclease is associated with Wilms tumor and the Perlman congenital overgrowth syndrome. LIN28, a Wilms tumor oncoprotein, triggers the DIS3L2-mediated degradation of the precursor of let-7, a microRNA that inhibits Wilms tumor development. These observations have led to speculation that DIS3L2-mediated tumor suppression is attributable to let-7 regulation. Here we examine new DIS3L2-deficient cell lines and mouse models, demonstrating that DIS3L2 loss has no effect on mature let-7 levels. Rather, analysis of Dis3l2-null nephron progenitor cells, a potential cell of origin of Wilms tumors, reveals up-regulation of Igf2, a growth-promoting gene strongly associated with Wilms tumorigenesis. These findings nominate a new potential mechanism underlying the pathology associated with DIS3L2 deficiency.

Precise let-7 expression levels balance organ regeneration against tumor suppression.

The in vivo roles for even the most intensely studied microRNAs remain poorly defined. Here, analysis of mouse models revealed that let-7, a large and ancient microRNA family, performs tumor suppressive roles at the expense of regeneration. Too little or too much let-7 resulted in compromised protection against cancer or tissue damage, respectively. Modest let-7 overexpression abrogated MYC-driven liver cancer by antagonizing multiple let-7 sensitive oncogenes. However, the same level of overexpression blocked liver regeneration, while let-7 deletion enhanced it, demonstrating that distinct let-7 levels can mediate desirable phenotypes. let-7 dependent regeneration phenotypes resulted from influences on the insulin-PI3K-mTOR pathway. We found that chronic high-dose let-7 overexpression caused liver damage and degeneration, paradoxically leading to tumorigenesis. These dose-dependent roles for let-7 in tissue repair and tumorigenesis rationalize the tight regulation of this microRNA in development, and have important implications for let-7 based therapeutics.

Tumor suppression by miR-26 overrides potential oncogenic activity in intestinal tumorigenesis.

Down-regulation of miR-26 family members has been implicated in the pathogenesis of multiple malignancies. In some settings, including glioma, however, miR-26-mediated repression of PTEN promotes tumorigenesis. To investigate the contexts in which the tumor suppressor versus oncogenic activity of miR-26 predominates in vivo, we generated miR-26a transgenic mice. Despite measureable repression of Pten, elevated miR-26a levels were not associated with malignancy in transgenic animals. We documented reduced miR-26 expression in human colorectal cancer and, accordingly, showed that miR-26a expression potently suppressed intestinal adenoma formation in Apc(min/+) mice, a model known to be sensitive to Pten dosage. These studies reveal a tumor suppressor role for miR-26 in intestinal cancer that overrides putative oncogenic activity, highlighting the therapeutic potential of miR-26 delivery to this tumor type.

An essential mesenchymal function for miR-143/145 in intestinal epithelial regeneration.

Downregulation of the miR-143/145 microRNA (miRNA) cluster has been repeatedly reported in colon cancer and other epithelial tumors. In addition, overexpression of these miRNAs inhibits tumorigenesis, leading to broad consensus that they function as cell-autonomous epithelial tumor suppressors. We generated mice with deletion of miR-143/145 to investigate the functions of these miRNAs in intestinal physiology and disease in vivo. Although intestinal development proceeded normally in the absence of these miRNAs, epithelial regeneration after injury was dramatically impaired. Surprisingly, we found that miR-143/145 are expressed and function exclusively within the mesenchymal compartment of intestine. Defective epithelial regeneration in miR-143/145-deficient mice resulted from the dysfunction of smooth muscle and myofibroblasts and was associated with derepression of the miR-143 target Igfbp5, which impaired IGF signaling after epithelial injury. These results provide important insights into the regulation of epithelial wound healing and argue against a cell-autonomous tumor suppressor role for miR-143/145 in colon cancer.

Fetal adrenal capsular cells serve as progenitor cells for steroidogenic and stromal adrenocortical cell lineages in M. musculus.

The lineage relationships of fetal adrenal cells and adrenal capsular cells to the differentiated adrenal cortex are not fully understood. Existing data support a role for each cell type as a progenitor for cells of the adult cortex. This report reveals that subsets of capsular cells are descendants of fetal adrenocortical cells that once expressed Nr5a1. These fetal adrenocortical cell descendants within the adrenal capsule express Gli1, a known marker of progenitors of steroidogenic adrenal cells. The capsule is also populated by cells that express Tcf21, a known inhibitor of Nr5a1 gene expression. We demonstrate that Tcf21-expressing cells give rise to Nr5a1-expressing cells but only before capsular formation. After the capsule has formed, capsular Tcf21-expressing cells give rise only to non-steroidogenic stromal adrenocortical cells, which also express collagen 1a1, desmin and platelet-derived growth factor (alpha polypeptide) but not Nr5a1. These observations integrate prior observations that define two separate origins of adult adrenocortical steroidogenic cells (fetal adrenal cortex and/or the adrenal capsule). Thus, these observations predict a unique temporal and/or spatial role of adult cortical cells that arise directly from either fetal cortical cells or from fetal cortex-derived capsular cells. Last, the data uncover the mechanism by which two populations of fetal cells (fetal cortex derived Gli1-expressing cells and mesenchymal Tcf21-expressing mesenchymal cells) participate in the establishment of the homeostatic capsular progenitor cell niche of the adult cortex.

Heart repair by reprogramming non-myocytes with cardiac transcription factors.

The adult mammalian heart possesses little regenerative potential following injury. Fibrosis due to activation of cardiac fibroblasts impedes cardiac regeneration and contributes to loss of contractile function, pathological remodelling and susceptibility to arrhythmias. Cardiac fibroblasts account for a majority of cells in the heart and represent a potential cellular source for restoration of cardiac function following injury through phenotypic reprogramming to a myocardial cell fate. Here we show that four transcription factors, GATA4, HAND2, MEF2C and TBX5, can cooperatively reprogram adult mouse tail-tip and cardiac fibroblasts into beating cardiac-like myocytes in vitro. Forced expression of these factors in dividing non-cardiomyocytes in mice reprograms these cells into functional cardiac-like myocytes, improves cardiac function and reduces adverse ventricular remodelling following myocardial infarction. Our results suggest a strategy for cardiac repair through reprogramming fibroblasts resident in the heart with cardiogenic transcription factors or other molecules.

The bHLH transcription factor Tcf21 is required for lineage-specific EMT of cardiac fibroblast progenitors.

The basic helix-loop-helix (bHLH) family of transcription factors orchestrates cell-fate specification, commitment and differentiation in multiple cell lineages during development. Here, we describe the role of a bHLH transcription factor, Tcf21 (epicardin/Pod1/capsulin), in specification of the cardiac fibroblast lineage. In the developing heart, the epicardium constitutes the primary source of progenitor cells that form two cell lineages: coronary vascular smooth muscle cells (cVSMCs) and cardiac fibroblasts. Currently, there is a debate regarding whether the specification of these lineages occurs early in the formation of the epicardium or later after the cells have entered the myocardium. Lineage tracing using a tamoxifen-inducible Cre expressed from the Tcf21 locus demonstrated that the majority of Tcf21-expressing epicardial cells are committed to the cardiac fibroblast lineage prior to initiation of epicardial epithelial-to-mesenchymal transition (EMT). Furthermore, Tcf21 null hearts fail to form cardiac fibroblasts, and lineage tracing of the null cells showed their inability to undergo EMT. This is the first report of a transcription factor essential for the development of cardiac fibroblasts. We demonstrate a unique role for Tcf21 in multipotent epicardial progenitors, prior to the process of EMT that is essential for cardiac fibroblast development.

Trophoblasts regulate the placental hematopoietic niche through PDGF-B signaling.

The placenta is a hematopoietic organ that supports hematopoietic stem/progenitor cell (HSPC) generation and expansion without promoting differentiation. We identified PDGF-B signaling in trophoblasts as a key component of the unique placental hematopoietic microenvironment that protects HSPCs from premature differentiation. Loss of PDGF-B or its receptor, PDGFRß, induced definitive erythropoiesis in placental labyrinth vasculature. This was evidenced by accumulation of CFU-Es and actively proliferating definitive erythroblasts that clustered around central macrophages, highly reminiscent of erythropoiesis in the fetal liver. Ectopic erythropoiesis was not due to a requirement of PDGF-B signaling in hematopoietic cells but rather in placental trophoblasts, which upregulated Epo in the absence of PDGF-B signaling. Furthermore, overexpression of hEPO specifically in the trophoblasts in vivo was sufficient to convert the placenta into an erythropoietic organ. These data provide genetic evidence of a signaling pathway that is required to restrict erythroid differentiation to specific anatomical niches during development.

Antioxidative effects of the spice cardamom against non-melanoma skin cancer by modulating nuclear factor erythroid-2-related factor 2 and NF-κB signalling pathways.

The role of dietary factors in inhibiting or delaying the development of non-melanoma skin cancer (NMSC) has been investigated for many years. Cardamom, which is a dietary phytoproduct, has been commonly used in cuisines for flavour and has numerous health benefits, such as improving digestion and stimulating metabolism and having antitumorigenic effects. We have investigated the efficacy of dietary cardamom against 7,12-dimethylbenz[a]anthracene (DMBA)-induced skin papillomatogenesis in Swiss albino mice that closely resembles human NMSC. Mice were grouped into normal wild type (untreated), vehicle-treated (acetone), carcinogen-treated (DMBA), and DMBA and cardamom-treated (DMBA+CARD) to delineate the role of cardamom against DMBA-induced papillomatogenesis. Oral administration of cardamom to DMBA-treated mice up-regulated the phase II detoxification enzymes, such as glutathione-S-transferase and glutathione peroxidase, probably via activation of nuclear factor erythroid-2-related factor 2 transcription factor in 'DMBA+CARD' mice. Furthermore, reduced glutathione, glutathione reductase, superoxide dismutase and catalase were also up-regulated by cardamom in the same 'DMBA+CARD' group of mice compared with DMBA-treated mice. Cardamom ingestion in DMBA-treated mice blocked NF-κB activation and down-regulated cyclo-oxygenase-2 expression. As a consequence, both the size and the number of skin papillomas generated on the skin due to the DMBA treatment were reduced in the 'DMBA+CARD' group. Thus, the results from the present study suggest that cardamom has a potential to become a pivotal chemopreventive agent to prevent papillomagenesis on the skin.

Inhibitory role of Notch1 in calcific aortic valve disease.

Aortic valve calcification is the most common form of valvular heart disease, but the mechanisms of calcific aortic valve disease (CAVD) are unknown. NOTCH1 mutations are associated with aortic valve malformations and adult-onset calcification in families with inherited disease. The Notch signaling pathway is critical for multiple cell differentiation processes, but its role in the development of CAVD is not well understood. The aim of this study was to investigate the molecular changes that occur with inhibition of Notch signaling in the aortic valve. Notch signaling pathway members are expressed in adult aortic valve cusps, and examination of diseased human aortic valves revealed decreased expression of NOTCH1 in areas of calcium deposition. To identify downstream mediators of Notch1, we examined gene expression changes that occur with chemical inhibition of Notch signaling in rat aortic valve interstitial cells (AVICs). We found significant downregulation of Sox9 along with several cartilage-specific genes that were direct targets of the transcription factor, Sox9. Loss of Sox9 expression has been published to be associated with aortic valve calcification. Utilizing an in vitro porcine aortic valve calcification model system, inhibition of Notch activity resulted in accelerated calcification while stimulation of Notch signaling attenuated the calcific process. Finally, the addition of Sox9 was able to prevent the calcification of porcine AVICs that occurs with Notch inhibition. In conclusion, loss of Notch signaling contributes to aortic valve calcification via a Sox9-dependent mechanism.

Efficient inducible Cre-mediated recombination in Tcf21 cell lineages in the heart and kidney.

Tcf21 is a Class II bHLH family member with essential roles in the formation of the lungs, kidneys, gonads, spleen, and heart. Here, we report the utility of a mouse line with targeted insertion of a tamoxifen-inducible Cre recombinase, MerCreMer at the Tcf21 locus. This mouse line will permit the inducible expression of Cre recombinase in Tcf21-expressing cells. Using ROSA26 reporter mice, we show that Cre recombinase is specifically and robustly activated in multiple Tcf21-expressing tissues during embryonic and postnatal development. The expression profile in the kidney is particularly dynamic with the ability to cause recombination in mesangial cells at one time of induction and podocytes at another time. These features make the Tcf21-driven inducible Cre line (Tcf21(iCre) ) a valuable genetic tool for spatiotemporal gene function analysis and lineage tracing of cells in the heart, kidney, cranial muscle, and gonads.

Redox regulation in cancer: a double-edged sword with therapeutic potential.

Oxidative stress, implicated in the etiology of cancer, results from an imbalance in the production of reactive oxygen species (ROS) and cell's own antioxidant defenses. ROS deregulate the redox homeostasis and promote tumor formation by initiating an aberrant induction of signaling networks that cause tumorigenesis. Ultraviolet (UV) exposures, gamma-radiation and other environmental carcinogens generate ROS in the cells, which can exert apoptosis in the tumors, thereby killing the malignant cells or induce the progression of the cancer growth by blocking cellular defense system. Cancer stem cells take the advantage of the aberrant redox system and spontaneously proliferate. Oxidative stress and gene-environment interactions play a significant role in the development of breast, prostate, pancreatic and colon cancer. Prolonged lifetime exposure to estrogen is associated with several kinds of DNA damage. Oxidative stress and estrogen receptor-associated proliferative changes are suggested to play important roles in estrogen-induced breast carcinogenesis. BRCA1, a tumor suppressor against hormone responsive cancers such as breast and prostate cancer, plays a significant role in inhibiting ROS and estrogen mediated DNA damage; thereby regulate the redox homeostasis of the cells. Several transcription factors and tumor suppressors are involved during stress response such as Nrf2, NF-kappaB and BRCA1. A promising strategy for targeting redox status of the cells is to use readily available natural substances from vegetables, fruits, herbs and spices. Many of the phytochemicals have already been identified to have chemopreventive potential, capable of intervening in carcinogenesis.

Chemopreventive properties of indole-3-carbinol, diindolylmethane and other constituents of cardamom against carcinogenesis.

Oxidative stress results from an imbalance in the production of reactive oxygen species (ROS) and cell's own antioxidant defenses that in part lead to numerous carcinogenesis. Several phytochemicals, derived from vegetables, fruits, herbs and spices, have demonstrated excellent chemopreventive properties against carcinogenesis by regulating the redox status of the cells during oxidative stress. I3C (indole-3-carbinol) and DIM (diindolylmethane) are the phytochemicals that are found in all types of cruciferous vegetables and demonstrated exceptional anti-cancer effects against hormone responsive cancers like breast, prostate and ovarian cancers. Novel analogs of I3C were designed to enhance the overall efficacy, particularly with respect to the therapeutic activity and oral bioavailability and that results in several patent applications on symptoms associated with endometriosis, vaginal neoplasia, cervical dysplasia and mastalgia. Likewise, DIM and its derivatives are patented for treatment and prevention of leiomyomas, HPV infection, respiratory syncytial virus, angiogenesis, atherosclerosis and anti-proliferative actions. On the other hand, phytochemicals in cardamom have not been explored in great details but limonene and cineole demonstrate promising effects against carcinogenesis. Thus studies with selected phytochemicals of cardamom and bioavailability research might lead to many patent applications. This review is focused on the patents generated on the effects of I3C, DIM and selected phytochemicals of cardamom on carcinogenesis.

Stability of 100 homo and heterotypic coiled-coil a-a' pairs for ten amino acids (A, L, I, V, N, K, S, T, E, and R).

We present the thermal stability monitored by circular dichroism (CD) spectroscopy at 222 nm of 100 heterodimers that contain all possible coiled-coil a-a' pairs for 10 amino acids (I, V, L, N, A, K S, T, E, and R). This includes the stability of 36 heterodimers for 6 amino acids (I, V, L, N, A, and K) previously described and 64 new heterodimers including the 4 amino acids (S, T, E, and R). We have calculated a double mutant alanine thermodynamic cycle to determine a-a' pair coupling energies to evaluate which a-a' pairs encourage specific dimerization partners. The four new homotypic a-a' pairs (T-T, S-S, R-R, E-E) are repulsive relative to A-A and have destabilizing coupling energies. Among the 90 heterotypic a-a' pairs, the stabilizing coupling energies contain lysine or arginine paired with either an aliphatic or a polar amino acid. The range in coupling energies for each amino acid reveals its potential to regulate dimerization specificity. The a-a' pairs containing isoleucine and asparagine have the greatest range in coupling energies and thus contribute dramatically to dimerization specificity, which is to encourage homodimerization. In contrast, the a-a' pairs containing charged amino acids (K, R, and E) show the least range in coupling energies and promiscuously encourage heterodimerization.

Activator protein-1 activity regulates epithelial tumor cell identity.

To examine the consequences of inhibiting activator protein-1 (AP-1) transcription factors in skin, transgenic mice were generated, which use the tetracycline system to conditionally express A-FOS, a dominant negative that inhibits AP-1 DNA binding. Older mice develop mild alopecia and hyperplasia of sebaceous glands, particularly around the eyes. When A-FOS was expressed during chemical-induced skin carcinogenesis, mice do not develop characteristic benign and malignant squamous lesions but instead develop benign sebaceous adenomas containing a signature mutation in the H-ras proto-oncogene. Inhibiting AP-1 activity after tumor formation caused squamous tumors to transdifferentiate into sebaceous tumors. Furthermore, reactivating AP-1 in sebaceous tumors results in a reciprocal transdifferentiation into squamous tumors. In both cases of transdifferentiation, individual cells express molecular markers for both cell types, indicating individual tumor cells have the capacity to express multiple lineages. Molecular characterization of cultured keratinocytes and tumor material indicates that AP-1 regulates the balance between the wnt/beta-catenin and hedgehog signaling pathways that determine squamous and sebaceous lineages, respectively. Chromatin immunoprecipitation analysis indicates that c-Jun binds several wnt promoters, which are misregulated by A-FOS expression, suggesting that members of the wnt pathway can be a primary targets of AP-1 transcriptional regulation. Thus, AP-1 activity regulates tumor cell lineage and is essential to maintain the squamous tumor cell identity.

Experimental identification of homodimerizing B-ZIP families in Homo sapiens.

B-ZIP transcription factors dimerization is mediated by a parallel coiled-coil termed the leucine zipper. We have evaluated the dimerization specificity of the seven coiled-coil B-ZIP proteins (ATF6, XBP, LZIP, NFIL3, TEF, CREB, and C/EBPalpha) with themselves and each other. To do this, we designed dominant negative proteins, termed A-ZIPs, that contain the leucine zipper dimerization domain of a B-ZIP protein and an acidic amphipathic N-terminal extension. The A-ZIPs heterodimerize with B-ZIP proteins in a leucine zipper-dependent manner. The acidic N-terminal extension is hypothesized to form an heterodimeric coiled-coil structure with the basic region, essentially zippering the leucine zipper into the basic region. We now present a new acidic extension design that stabilizes heterodimerization with B-ZIP proteins up to 11 kcal mol(-1). We have used these A-ZIP proteins in a competition EMSA to evaluate which A-ZIP can prevent DNA binding of which B-ZIP domain. Inhibition of DNA binding is interpreted to indicate that the A-ZIP is forming a heterodimer with the B-ZIP domain and thus prevents the B-ZIP from binding to DNA. All leucine zippers examined can homodimerize and two pairs (CREB & NFIL3 and ATF6 & XBP) can heterodimerize. We discuss these results with reference to the amino acid sequence of the leucine zipper region. These A-ZIP reagents may be of value in biological systems to inhibit the DNA binding and transcriptional potential of specific B-ZIP families.