Fabiana Passaro

Klf5 is involved in self-renewal of mouse embryonic stem cells.

Self-renewal of embryonic stem cells (ESCs) is maintained by a complex regulatory mechanism involving transcription factors Oct3/4 (Pou5f1), Nanog and Sox2. Here, we report that Klf5, a Zn-finger transcription factor of the Kruppel-like family, is involved in ESC self-renewal. Klf5 is expressed in mouse ESCs, blastocysts and primordial germ cells, and its knockdown by RNA interference alters the molecular phenotype of ESCs, thereby preventing their correct differentiation. The ability of Klf5 to maintain ESCs in the undifferentiated state is supported by the finding that differentiation of ESCs is prevented when Klf5 is constitutively expressed. Maintenance of the undifferentiated state by Klf5 is, at least in part, due to the control of Nanog and Oct3/4 transcription, because Klf5 directly binds to the promoters of these genes and regulates their transcription.

A set of miRNAs participates in the cellular senescence program in human diploid fibroblasts

Here we show that replicative senescence in normal human diploid IMR90 fibroblasts is accompanied by altered expression of a set of microRNAs (miRNAs) (senescence-associated miRNAs), with 14 and 10 miRNAs being either up or downregulated (>2-fold), respectively, in senescent with respect to young cells. The expression of most of these miRNAs was also deregulated upon senescence induced by DNA damage (etoposide) or mild oxidative stress (diethylmaleate). Four downregulated miRNAs were part of miRNA family-17, recently associated to human cell and tissue aging. Moreover, eight upregulated and six downregulated miRNAs mapped in specific chromosomal clusters, suggesting common transcriptional regulation. Upon adoptive overexpression, seven upregulated miRNAs induced the formation of senescence-associated heterochromatin foci and senescence-associated β-galactosidase staining (P<0.05), which was accompanied, in the case of five of them, by reduced cell proliferation. Finally, miR-210, miR-376a*, miR-486-5p, miR-494, and miR-542-5p induced double-strand DNA breaks and reactive oxygen species accumulation in transfected cells. In conclusion, we have identified a set of human miRNAs induced during replicative and chemically induced senescence that are able to foster the senescent phenotype by prompting DNA damage.

Fe65 is required for Tip60-directed histone H4 acetylation at DNA strand breaks.

Fe65 is a binding partner of the Alzheimers β-amyloid precursor protein APP. The possible involvement of this protein in the cellular response to DNA damage was suggested by the observation that Fe65 null mice are more sensitive to genotoxic stress than WT counterpart. Fe65 associated with chromatin under basal conditions and its involvement in DNA damage repair requires this association. A known partner of Fe65 is the histone acetyltransferase Tip60. Considering the crucial role of Tip60 in DNA repair, we explored the hypothesis that the phenotype of Fe65 null cells depended on its interaction with Tip60. We demonstrated that Fe65 knockdown impaired recruitment of Tip60-TRRAP complex to DNA double strand breaks and decreased histone H4 acetylation. Accordingly, the efficiency of DNA repair was decreased upon Fe65 suppression. To explore whether APP has a role in this mechanism, we analyzed a Fe65 mutant unable to bind to APP. This mutant failed to rescue the phenotypes of Fe65 null cells; furthermore, APP/APLP2 suppression results in the impairment of recruitment of Tip60-TRRAP complex to DNA double strand breaks, decreased histone H4 acetylation and repair efficiency. On these bases, we propose that Fe65 and its interaction with APP play an important role in the response to DNA damage by assisting the recruitment of Tip60-TRRAP to DNA damage sites.

Direct targets of Klf5 transcription factor contribute to the maintenance of mouse embryonic stem cell undifferentiated state.

BackgroundA growing body of evidence has shown that Krüppel-like transcription factors play a crucial role in maintaining embryonic stem cell (ESC) pluripotency and in governing ESC fate decisions. Krüppel-like factor 5 (Klf5) appears to play a critical role in these processes, but detailed knowledge of the molecular mechanisms of this function is still not completely addressed.ResultsBy combining genome-wide chromatin immunoprecipitation and microarray analysis, we have identified 161 putative primary targets of Klf5 in ESCs. We address three main points: (1) the relevance of the pathways governed by Klf5, demonstrating that suppression or constitutive expression of single Klf5 targets robustly affect the ESC undifferentiated phenotype; (2) the specificity of Klf5 compared to factors belonging to the same family, demonstrating that many Klf5 targets are not regulated by Klf2 and Klf4; and (3) the specificity of Klf5 function in ESCs, demonstrated by the significant differences between Klf5 targets in ESCs compared to adult cells, such as keratinocytes.ConclusionsTaken together, these results, through the definition of a detailed list of Klf5 transcriptional targets in mouse ESCs, support the important and specific functional role of Klf5 in the maintenance of the undifferentiated ESC phenotype.See: http://www.biomedcental.com/1741-7007/8/125

Receptor- and Non-Receptor Tyrosine Kinases Induce Processing of the Amyloid Precursor Protein: Role of the Low-Density Lipoprotein Receptor-Related Protein

The Alzheimer’s β-amyloid peptides derive from the proteolytic processing of the β-amyloid precursor protein, APP, by β- and γ-secretases. The regulation of this processing is not fully understood. Experimental evidence suggests that the activation of pathways involving protein tyrosine kinases, such as PDGFR and Src, could induce the cleavage of APP and in turn the generation of amyloid peptides. In this paper we addressed the effect of receptor and nonreceptor protein tyrosine kinases on the cleavage of APP and the mechanisms of their action. To this aim, we developed an in vitro system based on the APP-Gal4 fusion protein stably transfected in SHSY5Y neuroblastoma cell line. The cleavage of this molecule, induced by various stimuli, results in the activation of the transcription of the luciferase gene under the control of Gal4 cis-elements. By using this experimental system we demonstrated that, similarly to Src, three tyrosine kinases, TrkA, Ret and EGFR, induced the cleavage of APP-Gal4. We excluded that this effect was mediated by the activation of Ras-MAPK, PI3K-Akt and PLC-γ pathways. Furthermore, the direct phosphorylation of the APP cytosolic domain does not affect Aβ peptide generation. On the contrary, experiments in cells lacking the LDL-receptor related protein LRP support the hypothesis that the interaction of APP with LRP is required for the induction of APP cleavage by tyrosine kinases.

Comparative analysis of gene expression data reveals novel targets of senescence-associated microRNAs.

In the last decades, cellular senescence is viewed as a complex mechanism involved in different processes, ranging from tumor suppression to induction of age-related degenerative alterations. Senescence-inducing stimuli are myriad and, recently, we and others have demonstrated the role exerted by microRNAs in the induction and maintenance of senescence, by the identification of a subset of Senescence-Associated microRNAs (SAmiRs) up-regulated during replicative or stress-induced senescence and able to induce a premature senescent phenotype when over-expressed in human primary cells. With the intent to find novel direct targets of two specific SAmiRs, SAmiR-494 and -486-5p, and cellular pathways which they are involved in, we performed a comparative analysis of gene expression profiles available in literature to select genes down-regulated upon replicative senescence of human primary fibroblasts. Among them, we searched for SAmiR’s candidate targets by analyzing with different target prediction algorithms their 3’UTR for the presence of SAmiR-binding sites. The expression profiles of selected candidates have been validated on replicative and stress-induced senescence and the targeting of the 3’UTRs was assessed by luciferase assay. Results allowed us to identify Cell Division Cycle Associated 2 (CDCA2) and Inhibitor of DNA binding/differentiation type 4 (ID4) as novel targets of SAmiR-494 and SAmiR-486-5p, respectively. Furthermore, we demonstrated that the over-expression of CDCA2 in human primary fibroblasts was able to partially counteract etoposide-induced senescence by mitigating the activation of DNA Damage Response.

Angiotensin receptor I stimulates osteoprogenitor proliferation through TGFβ-mediated signaling.

Clinical studies of large human populations and pharmacological interventions in rodent models have recently suggested that anti‐hypertensive drugs that target angiotensin II (Ang II) activity may also reduce loss of bone mineral density. Here, we identified in a genetic screening the Ang II type I receptor (AT1R) as a potential determinant of osteogenic differentiation and, implicitly, bone formation. Silencing of AT1R expression by RNA interference severely impaired the maturation of a multipotent mesenchymal cell line (W20–17) along the osteoblastic lineage. The same effect was also observed after the addition of the AT1R antagonist losartan but not the AT2R inhibitor PD123,319. Additional cell culture assays traced the time of greatest losartan action to the early stages of W20–17 differentiation, namely during cell proliferation. Indeed, addition of Ang II increased proliferation of differentiating W20–17 and primary mesenchymal stem cells and this stimulation was reversed by losartan treatment. Cells treated with losartan also displayed an appreciable decrease of activated (phosphorylated)‐Smad2/3 proteins. Moreover, Ang II treatment elevated endogenous transforming growth factor β (TGFβ) expression considerably and in an AT1R‐dependent manner. Finally, exogenous TGFβ was able to restore high proliferative activity to W20–17 cells that were treated with both Ang II and losartan. Collectively, these results suggest a novel mechanism of Ang II action in bone metabolism that is mediated by TGFβ and targets proliferation of osteoblast progenitors. J. Cell. Physiol. 230: 1466–1474, 2015.

Lin28 is induced in primed embryonic stem cells and regulates let-7-independent events.

Lin28 RNA‐binding proteins play important roles in pluripotent stem cells, but the regulation of their expression and the mechanisms underlying their functions are still not definitively understood. Here we address the above‐mentioned issues in the first steps of mouse embryonic stem cell (ESC) differentiation. We observed that the expression of Lin28 genes is transiently induced soon after the exit of ESCs from the naive ground state and that this induction is due to the Hmga2‐dependent engagement of Otx2 with enhancers present at both Lin28 gene loci. These mechanisms are crucial for Lin28 regulation, as demonstrated by the abolishment of the Lin28 accumulation in Otx2‐or Hmga2‐knockout cells compared to the control cells. We have also found that Lin28 controls Hmga2 expression levels during ESC differentiation through a let‐7‐independent mechanism. Indeed, we found that Lin28 proteins bind a highly conserved element in the 3′ UTR of Hmga2 mRNA, and this provokes a down‐regulation of its translation. This mechanism prevents the inappropriate accumulation of Hmga2 that would modify the proliferation and physiological apoptosis of differentiating ESCs. In summary, we demonstrated that during ESC differentiation, Lin28 transient induction is dependent on Otx2 and Hmga2 and prevents an inappropriate excessive rise of Hmga2 levels.—Parisi, S., Passaro, F., Russo, L., Musto, A., Navarra, A., Romano, S., Petrosino, G., Russo, T. Lin28 is induced in primed embryonic stem cells and regulates let‐7‐independent events. FASEB J. 31, 1046–1058 (2017). www.fasebj.org

Serum withdrawal after embryoid body formation does not impair cardiomyocyte development from mouse embryonic stem cells

BACKGROUND AIMS Procedures for cardiomyocyte differentiation of mouse embryonic stem cells (mESCs) utilize different amounts of serum. Because the serum composition is unknown, unambiguous characterization of the differentiation process is biased. All reported serum-free protocols used for compound testing provide serum throughout the differentiation process. We report on an embryoid body (EB)-based procedure for cardiomyocyte differentiation of mESCs in which serum is provided only in the earliest step (hanging drop, 0-2 days). METHODS To assess cardiomyocyte differentiation, we generated an mESCs clone that expressed green fluorescence protein (GFP) under the control of the myosin light chain 2v (MLC2v) promoter. To define the lowest serum concentration required for efficient induction of cardiomyocyte differentiation, EBs were formed in presence of 5% (S5), 10% (S10) and 15% (S15) serum until day 2, then switched to a serum-free medium. RESULTS Analysis of cardiac-specific transcripts on day 6 of differentiation showed that 10% (S10) was the minimum amount of serum for efficient continuation of cultures under serum-free conditions. Spontaneously beating foci were detected in 90.0 ± 5.5% of S10 EBs on day 7 of differentiation, and cardiomyocyte markers were expressed from day 8 of differentiation (MLC2v-driven GFP; α-myosin heavy chain). Dose-response curves to isoproterenol showed that the beating rate increased by 113.0 ± 39.4%, with a concentration for half-maximal effect (EC(50)) of 25.7 nm. CONCLUSIONS The development of functional cardiomyocytes from mESCs is not affected by serum withdrawal after EBs formation. This culture system represents a new model for cardiomyocyte differentiation of mESCs to assess the effects of compounds on the process of cardiomyogenesis.

Implications of Cellular Aging in Cardiac Reprogramming

Aging is characterized by a chronic functional decline of organ systems which leads to tissue dysfunction over time, representing a risk factor for diseases development, including cardiovascular. The aging process occurring in the cardiovascular system involves heart and vessels at molecular and cellular level, with subsequent structural modifications and functional impairment. Several modifications involved in the aging process can be ascribed to cellular senescence, a biological response that limits the proliferation of damaged cells. In physiological conditions, the mechanism of cellular senescence is involved in regulation of tissue homeostasis, remodeling, and repair. However, in some conditions senescence-driven tissue repair may fail, leading to the tissue accumulation of senescent cells which in turn may contribute to tumor promotion, aging, and age-related diseases. Cellular reprogramming processes can reverse several age-associated cell features, such as telomere length, DNA methylation, histone modifications and cell-cycle arrest. As such, induced Pluripotent Stem Cells (iPSCs) can provide models of progeroid and physiologically aged cells to gain insight into the pathogenesis of such conditions, to drive the development of new therapies for premature aging and to further explore the possibility of rejuvenating aged cells. An emerging picture is that the tissue remodeling role of cellular senescence could also be crucial for the outcomes of in vivo reprogramming processes. Experimental evidence has demonstrated that, on one hand, senescence represents a cell-autonomous barrier for a cell candidate to reprogramming, but, on the other hand, it may positively sustain the reprogramming capability of surrounding cells to generate fully proficient tissues. This review fits into this conceptual framework by highlighting the most prominent concepts that characterize aging and reprogramming and discusses how the aging tissue might provide a favorable microenvironment for in vivo cardiac reprogramming.