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Breast cancer is one of the most lethal malignancies in women worldwide and is characterized by rapid growth and low survival rates, despite advances in tumor biology and therapies. Novel therapeutic approaches require new insights into the molecular mechanisms of malignant transformation and progression. To this end, here, we identified Prox1 as a negative regulator of proliferation and tumor-related metabolism in breast cancer. In particular, we showed that breast tumors from human patients exhibited reduced levels of Prox1 expression, while high expression levels of Prox1 were associated with a favorable prognosis in breast cancer patients. Moreover, we experimentally demonstrated that Prox1 was sufficient to strongly suppress proliferation, migration, and the Warburg effect in human breast cancer cells without inducing apoptosis. Most importantly, over-expression of Prox1 inhibited breast tumor growth in vivo in both heterotopic and orthotopic xenograft mouse models. The anti-tumorigenic effect of Prox1 was mediated by the direct repression of c-Myc transcription and its downstream target genes. Consistently, c-Myc over-expression from an artificial promoter that was not targeted by Prox1 reversed Prox1's anti-tumor effects. These findings suggest that Prox1 has a tumor suppressive role via direct transcriptional regulation of c-Myc, making it a promising therapeutic gene for breast cancer.

Chronic kidney diseases affect a substantial percentage of the adult population worldwide. This observation emphasizes the need for novel insights into the molecular mechanisms that control the onset and progression of renal diseases. Recent advances in genomics have uncovered a previously unanticipated link between the non-coding genome and human kidney diseases. Here we screened and analysed long non-coding RNAs (lncRNAs) previously identified in mouse kidneys by genome-wide transcriptomic analysis, for conservation in humans and differential expression in renal tissue from healthy and diseased individuals. Our data suggest that LINC01187 is strongly down-regulated in human kidney tissues of patients with diabetic nephropathy and rapidly progressive glomerulonephritis, as well as in murine models of kidney diseases, including unilateral ureteral obstruction, nephrotoxic serum-induced glomerulonephritis and ischemia/reperfusion. Interestingly, LINC01187 overexpression in human kidney cells in vitro inhibits cell death indicating an anti-apoptotic function. Collectively, these data suggest a negative association of LINC01187 expression with renal diseases implying a potential protective role., (© 2022 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Secreted wingless-interacting protein (Swim) is the Drosophila ortholog gene of the mammalian Tubulointerstitial Nephritis Antigen like 1 (TINAGL1), also known as lipocalin-7 (LCN7), or adrenocortical zonation factor 1 (AZ-1). Swim and TINAGL1 proteins share a significant homology, including the somatomedin B and the predictive inactive C1 cysteine peptidase domains. In mammals, both TINAGL1 and its closely related homolog TINAG have been identified in basement membranes, where they may function as modulators of integrin-mediated adhesion. In Drosophila , Swim was initially identified in the eggshell matrix and was subsequently detected in the culture medium of S2 cells. Further biochemical analysis indicated that Swim binds to wingless (wg) in a lipid-dependent manner. This observation, together with RNAi-knockdown studies, suggested that Swim is an essential cofactor of wg-signalling. However, recent elegant genetic studies ruled out the possibility that Swim is required alone to facilitate wg-signalling in Drosophila , because flies without Swim are viable and fertile. Here, we use the UAS/Gal4 expression system together with confocal imaging to analyze the in vivo localization of a chimeric Swim-GFP in the developing Drosophila embryo. Our data fully support the notion that Swim is an extracellular matrix component that is secreted upon ectopic expression and preferentially associates with the basement membranes of various organs and with the specialized tendon matrix at the muscle attachment sites (MAS). Interestingly, the accumulation of Swim at the MAS does not require integrins. In conclusion, Swim is an extracellular matrix component, and Swim may exhibit overlapping functions in concert with other undefined components.

During central nervous system (CNS) development, proper and timely induction of neurite elongation is critical for generating functional, mature neurons, and neuronal networks. Despite the wealth of information on the action of extracellular cues, little is known about the intrinsic gene regulatory factors that control this developmental decision. Here, we report the identification of Prox1, a homeobox transcription factor, as a key player in inhibiting neurite elongation. Although Prox1 promotes acquisition of early neuronal identity and is expressed in nascent post-mitotic neurons, it is heavily down-regulated in the majority of terminally differentiated neurons, indicating a regulatory role in delaying neurite outgrowth in newly formed neurons. Consistently, we show that Prox1 is sufficient to inhibit neurite extension in mouse and human neuroblastoma cell lines. More importantly, Prox1 overexpression suppresses neurite elongation in primary neuronal cultures as well as in the developing mouse brain, while Prox1 knock-down promotes neurite outgrowth. Mechanistically, RNA-Seq analysis reveals that Prox1 affects critical pathways for neuronal maturation and neurite extension. Interestingly, Prox1 strongly inhibits many components of Ca 2+ signaling pathway, an important mediator of neurite extension and neuronal maturation. In accordance, Prox1 represses Ca 2+ entry upon KCl-mediated depolarization and reduces CREB phosphorylation. These observations suggest that Prox1 acts as a potent suppressor of neurite outgrowth by inhibiting Ca 2+ signaling pathway. This action may provide the appropriate time window for nascent neurons to find the correct position in the CNS prior to initiation of neurites and axon elongation.

Transcriptome analysis has identified a plethora of long non-coding RNAs (lncRNAs) expressed in the human brain and associated with neurological diseases. However, whether lncRNAs expression levels correlate with Parkinson's disease (PD) pathogenesis remains unknown. Herein, we show that a number of lncRNA genes encompassing transcriptional units in close proximity to PD-linked protein-coding genes, including SNCA , LRRK2 , PINK1 , DJ-1 , UCH-L1 , MAPT and GBA1 , are expressed in human dopaminergic cells and post-mortem material, such as cortex, Substantia Nigra and cerebellum. Interestingly, these lncRNAs are upregulated during neuronal differentiation of SH-SY5Y cells and of dopaminergic neurons generated from human fibroblast-derived induced pluripotent stem cells. Importantly, six lncRNAs are found under-expressed in the nigra and three in the cerebellum of PD patients compared to controls. Simultaneously, SNCA mRNA levels are increased in the nigra, while LRRK2 and PINK1 mRNA levels are decreased both in the nigra and the cerebellum of PD subjects compared to controls, indicating a possible correlation between the expression profile of the respective lncRNAs with their adjacent coding genes. Interestingly, all dysregulated lncRNAs are also detected in human peripheral blood mononuclear cells and four of them in exosomes derived from human cerebrospinal fluid, providing initial evidence for their potential use as diagnostic tools for PD. Our data raise the intriguing possibility that these lncRNAs may be involved in disease pathogenesis by regulating their neighboring PD-associated genes and may thus represent novel targets for the diagnosis and/or treatment of PD or related diseases.

Background: Renal fibrosis is a common histological finding present in many pathologies; however, key signaling pathways and molecular determinants involved in the development of fibrosis are not fully known yet. Previous findings have established a causative role of calreticulin's up-regulation during the development of renal fibrosis while its down-regulation exhibited a protective effect against fibrosis. Therefore, the mechanism of its up-regulation needs to be explored., Methods: Bioinformatics analyses of the calreticulin gene promoter combined with transcriptional assays and in vivo chromatin immunoprecipitation experiments in the Unilateral Ureteric Obstruction (UUO) model of renal fibrosis, indicated that NR5A2 is a critical regulator of calreticulin expression. To confirm this finding, and further study post-translational modifications of NR5A2, real time RT-qPCR, immunohistochemistry and Western blotting experiments were performed., Results: NR5A2 is up-regulated at both mRNA and protein level during kidney fibrosis in the UUO model. The post-translational modification of SUMOylation was identified as a critical parameter in this phenomenon and SUMOylation was observed to be up-regulated during the development of renal fibrosis. The enzyme Ubc9, critical for the process of SUMOylation was also upregulated at mRNA and protein level during the process., Conclusion: These data establish for the first time a role for NR5A2 and its SUMOylation on the transcriptional regulation of the calreticulin gene in a rodent model of renal fibrosis and raise the possibility that NR5A2 might be a novel target for future anti-fibrotic interventions., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Fibrosis is a complex and multifactorial process, affecting the structure and compromising the function of several organs. Among those, renal fibrosis is an important pathological change, eventually leading to renal failure. Proteomic analysis of the renal parenchyma in the well-established rat model of unilateral ureteral obstruction (UUO model) suggested that transgelin was up-regulated during the development of fibrosis. Transgelin up-regulation was confirmed both at the protein and at the mRNA level. It was observed that at early stages of fibrosis transgelin was mainly expressed in the interstitial compartment and, more specifically, in cells surrounding the glomeruli. Subsequently, it was confirmed that transgelin expressing cells were activated fibroblasts, based on their extensive co-expression of α-SMA and their complete lack of co-distribution with markers of other cell types (endothelial, epithelial and cells of the immune system). These periglomerular fibroblasts exhibited staining for transgelin mainly cytoplasmic but occasionally nuclear as well. In addition, transgelin expression in periglomerular fibroblasts was absent in renal fibrosis developed in a hypertensive model, compared to the UUO model. Promoter analysis indicated that there are several conserved motifs for transcription factor binding. Among those, Kruppel-like factor 6 was found to be up-regulated in transgelin positive periglomerular activated fibroblasts, suggesting a possible involvement in the mechanism of transgelin up-regulation. These data strongly suggest that transgelin is up-regulated in the obstructive nephropathy and could be used as a novel marker for renal fibrosis in the future.

Background: Increased intestinal permeability in cirrhosis exerts a pivotal role in the pathogenesis of spontaneous bacterial peritonitis and other complications of cirrhosis through promotion of systemic endotoxemia. This study was designed to investigate whether the expression of tight junction (TJ) proteins, which regulate gut paracellular permeability, is altered in the intestinal mucosa of patients with liver cirrhosis and study its potential association with the stage of liver disease and the development of systemic endotoxemia., Design: Twenty-four patients with cirrhosis at a decompensated (n = 12, group A) or compensated condition (n = 12, group B) and 12 healthy controls (group C) were subjected to duodenal biopsy. The expression of the TJ proteins occludin and claudin-1 in the intestinal epithelium was evaluated by immunohistochemistry. Plasma endotoxin concentrations were also determined., Results: Patients with cirrhosis presented significantly higher serum endotoxin concentrations as compared to healthy controls (P < 0·001), whilst endotoxemia was higher in decompensated disease (P < 0·05 vs. compensated cirrhosis). Patients with decompensated and compensated cirrhosis presented significantly reduced expression of occludin and claudin-1 as compared to controls (P < 0·01, respectively). These alterations were significantly more pronounced in decompensated patients as compared to compensated (P < 0·05). Regarding occludin, in patients with cirrhosis, a specific pattern of expression in the intestinal epithelium was observed, with a gradually increasing loss of expression from crypt to tip of the villi. Occludin and claudin-1 expression were inversely correlated with Child-Pugh score (P < 0·001), the grade of oesophageal varices (P < 0·01) and endotoxin concentrations (P < 0·001)., Conclusions: This study demonstrates for the first time that human liver cirrhosis induces significant alterations in enterocytes' TJs. These changes might represent an important cellular mechanism for intestinal barrier dysfunction and hyperpermeability in patients with liver cirrhosis., (© 2011 The Authors. European Journal of Clinical Investigation © 2011 Stichting European Society for Clinical Investigation Journal Foundation.)

Activation of Notch1 signaling in neural progenitor cells (NPCs) induces self-renewal and inhibits neurogenesis. Upon neuronal differentiation, NPCs overcome this inhibition, express proneural genes to induce Notch ligands, and activate Notch1 in neighboring NPCs. The molecular mechanism that coordinates Notch1 inactivation with initiation of neurogenesis remains elusive. Here, we provide evidence that Prox1, a transcription repressor and downstream target of proneural genes, counteracts Notch1 signaling via direct suppression of Notch1 gene expression. By expression studies in the developing spinal cord of chick and mouse embryo, we showed that Prox1 is limited to neuronal precursors residing between the Notch1+ NPCs and post-mitotic neurons. Physiological levels of Prox1 in this tissue are sufficient to allow binding at Notch1 promoter and they are critical for proper Notch1 transcriptional regulation in vivo. Gain-of-function studies in the chick neural tube and mouse NPCs suggest that Prox1-mediated suppression of Notch1 relieves its inhibition on neurogenesis and allows NPCs to exit the cell cycle and differentiate. Moreover, loss-of-function in the chick neural tube shows that Prox1 is necessary for suppression of Notch1 outside the ventricular zone, inhibition of active Notch signaling, down-regulation of NPC markers, and completion of neuronal differentiation program. Together these data suggest that Prox1 inhibits Notch1 gene expression to control the balance between NPC self-renewal and neuronal differentiation., Competing Interests: The authors have declared that no competing interests exist.