Overactivation of the mitogen-activated protein kinase (MAPK) pathway is a critical driver of many human cancers. However, therapies directly targeting this pathway lead to cancer drug resistance. ...Resistance has been linked to compensatory RAS overexpression, but the mechanisms underlying this response remain unclear. Here, we find that MEK inhibitors (MEKi) are associated with an increased translation of the KRAS and NRAS oncogenes through a mechanism involving dissolution of processing body (P-body) biocondensates. This effect is seen across different cell types and is extremely dynamic since removal of MEKi and ERK reactivation result in reappearance of P-bodies and reduced RAS-dependent signaling. Moreover, we find that P-body scaffold protein levels negatively impact RAS expression. Overall, we describe a new feedback loop mechanism involving biocondensates such as P-bodies in the translational regulation of RAS proteins and MAPK signaling.
Matrix remodeling is a salient feature of idiopathic pulmonary fibrosis (IPF). Targeting cells driving matrix remodeling could be a promising avenue for IPF treatment. Analysis of transcriptomic ...database identified the mesenchymal transcription factor PRRX1 as upregulated in IPF. PRRX1, strongly expressed by lung fibroblasts, was regulated by a TGF-β/PGE2 balance in vitro in control and IPF human lung fibroblasts, while IPF fibroblast-derived matrix increased
expression in a PDGFR-dependent manner in control ones. PRRX1 inhibition decreased human lung fibroblast proliferation by downregulating the expression of S phase cyclins. PRRX1 inhibition also impacted TGF-β driven myofibroblastic differentiation by inhibiting SMAD2/3 phosphorylation through phosphatase PPM1A upregulation and TGFBR2 downregulation, leading to TGF-β response global decrease. Finally, targeted inhibition of
attenuated fibrotic remodeling in vivo with intra-tracheal antisense oligonucleotides in bleomycin mouse model of lung fibrosis and ex vivo using human and mouse precision-cut lung slices. Our results identified PRRX1 as a key mesenchymal transcription factor during lung fibrogenesis.
The Fragile X-Related 1 gene (FXR1) is a paralog of the Fragile X Mental Retardation 1 gene (FMR1), whose absence causes the Fragile X syndrome, the most common form of inherited intellectual ...disability. FXR1P plays an important role in normal muscle development, and its absence causes muscular abnormalities in mice, frog, and zebrafish. Seven alternatively spliced FXR1 transcripts have been identified and two of them are skeletal muscle-specific. A reduction of these isoforms is found in myoblasts from Facio-Scapulo Humeral Dystrophy (FSHD) patients. FXR1P is an RNA-binding protein involved in translational control; however, so far, no mRNA target of FXR1P has been linked to the drastic muscular phenotypes caused by its absence. In this study, gene expression profiling of C2C12 myoblasts reveals that transcripts involved in cell cycle and muscular development pathways are modulated by Fxr1-depletion. We observed an increase of p21--a regulator of cell-cycle progression--in Fxr1-knocked-down mouse C2C12 and FSHD human myoblasts. Rescue of this molecular phenotype is possible by re-expressing human FXR1P in Fxr1-depleted C2C12 cells. FXR1P muscle-specific isoforms bind p21 mRNA via direct interaction with a conserved G-quadruplex located in its 3' untranslated region. The FXR1P/G-quadruplex complex reduces the half-life of p21 mRNA. In the absence of FXR1P, the upregulation of p21 mRNA determines the elevated level of its protein product that affects cell-cycle progression inducing a premature cell-cycle exit and generating a pool of cells blocked at G0. Our study describes a novel role of FXR1P that has crucial implications for the understanding of its role during myogenesis and muscle development, since we show here that in its absence a reduced number of myoblasts will be available for muscle formation/regeneration, shedding new light into the pathophysiology of FSHD.
Current challenges in microRNA (miRNA) research are to improve the identification of in vivo mRNA targets and clarify the complex interplay existing between a specific miRNA and multiple biological ...networks. MiRonTop is an online java web tool that integrates DNA microarrays or high-throughput sequencing data to identify the potential implication of miRNAs on a specific biological system. It allows a rapid characterization of the most pertinent mRNA targets according to several existing miRNA target prediction approaches. It also provides useful representations of the enrichment scores according to the position of the target site along the 3′-UTR, where the contribution of the sites located in the vicinity of the stop codon and of the polyA tail can be clearly highlighted. It provides different graphs of miRNA enrichment associated with up- or down-regulated transcripts and different summary tables about selections of mRNA targets and their functional annotations by Gene Ontology. Availability: http://www.microarray.fr:8080/miRonTop/index Contact: barbry@ipmc.cnrs.fr
Abstract
Non-small cell lung cancer is characterized by a dismal prognosis largely owing to inefficient diagnosis and tenacious drug resistance. Therefore, the identification of new molecular ...determinants underlying sensitivity of cancer cells to existing therapy is of particular importance to develop new effective combinatorial treatment strategy. MicroRNAs (miRNAs), a class of small non-coding RNAs, have been established as master regulators of a variety of cellular processes that play a key role in tumor initiation, progression and metastasis. This, along with their widespread deregulation in many distinct cancers, has triggered enthusiasm for miRNAs as novel therapeutic targets for cancer management, in particular in patients with refractory cancers such as those harboring KRAS mutations. In this study, we performed a loss-of-function screening approach to identify miRNAs whose silencing promotes sensitivity of lung adenocarcinoma (LUAD) cells to cisplatin. Our results showed in particular that antisense oligonucleotides directed against miR-92a-3p, a member of the oncogenic miR-17 ~ 92 cluster, caused the greatest increase in the sensitivity of KRAS-mutated LUAD cells to cisplatin. In addition, we demonstrated that this miRNA finely regulates the apoptotic threshold and the proliferative capacity of various tumor cell lines with distinct genetic alterations. Collectively, these data suggest that targeting miR-92a-3p may serve as an effective strategy to overcome treatment resistance of solid tumors.
Tissue regeneration requires coordinated and dynamic remodeling of stem and progenitor cells and the surrounding niche. Although the plasticity of epithelial cells has been well explored in many ...tissues, the dynamic changes occurring in niche cells remain elusive. Here, we show that, during lung repair after naphthalene injury, a population of PDGFRα+ cells emerges in the non-cartilaginous conducting airway niche, which is normally populated by airway smooth muscle cells (ASMCs). This cell population, which we term “repair-supportive mesenchymal cells” (RSMCs), is distinct from conventional ASMCs, which have previously been shown to contribute to epithelial repair. Gene expression analysis on sorted lineage-labeled cells shows that RSMCs express low levels of ASMC markers, but high levels of the pro-regenerative marker Fgf10. Organoid co-cultures demonstrate an enhanced ability for RSMCs in supporting club-cell growth. Our study highlights the dynamics of mesenchymal cells in the airway niche and has implications for chronic airway-injury-associated diseases.
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•The mesenchymal niche is critical for airway epithelial regeneration•β-catenin signaling and FGF10 production are pivotal for proper repair after injury•RSMCs, which are distinct from ASMCs, emerge after club-cell depletion•RSMCs display high potential to support bronchiolosphere formation in vitro
Moiseenko et al. explore the dynamics of mesenchymal cells in the peribronchial niche in response to airway injury. They identify a population of mesenchymal cells located in close proximity to airway smooth muscle cells (ASMCs). This population, termed “repair-supportive mesenchymal cells” (RSMCs), is recruited to facilitate airway epithelial regeneration.
Dupuytren disease (DD) is a hand-localized fibrotic disorder characterized by a scar-like, collagen-rich cord. Treatment usually comprises surgical removal of the cord, but is associated with a high ...relapse rate, in some cases requiring finger amputation. There is currently no consensual medical approach for treating DD. Numerous preclinical studies have highlighted antifibrotic properties of metformin, and the aim of this study was to assess a potential antifibrotic role of metformin in DD. Fibroblasts from DD cords (DF) and phenotypically normal palmar fascia (PF) were extracted from surgical specimens and cultured. The fibrotic status of DF and PF was compared at baseline, and under profibrotic (TGF-β stimulation) and antifibrotic (metformin stimulation) conditions, using quantitative RT-PCR, western blot, immunocytochemistry, and a functional fibroblast contraction assay. At baseline, DF showed higher levels of fibrotic markers and contraction capacity compared with PF. Both types of fibroblasts responded to TGF-β stimulation. Treatment of DF and PF with metformin did not affect basal levels of fibrotic markers and contraction but largely prevented their induction by TGF-β. In conclusion, our data show that metformin inhibits TGF-β-induced expression of fibrotic markers and contraction in hand-derived fibroblasts. This supports the case for a clinical trial to assess the repurposing of metformin as an adjuvant to surgery, to prevent, reduce, or delay recurrence in at-risk DD patients.
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•Metformin cannot revert the fibrotic phenotype of myofibroblasts in DD patients.•Metformin can prevent TGF-β induced differentiation of hand fibroblast into myofibroblasts.•The role of metformin as an adjuvant treatment to surgery should be evaluated in clinical trials.
Although Tacrolimus is an immunosuppressive drug widely used in renal transplantation, its chronic use paradoxically induces nephrotoxic effects, in particular renal fibrosis, which is responsible ...for chronic allograft dysfunction and represents a major prognostic factor of allograft survival. As molecular pathways and mechanisms involved in Tacrolimus-induced fibrogenic response are poorly elucidated, we assessed whether miRNAs are involved in the nephrotoxic effects mediated by Tacrolimus. Treatment of CD-1 mice with Tacrolimus (1 mg/kg/d for 28 days) resulted in kidney injury and was associated with alteration of a gene expression signature associated with cellular stress, fibrosis and inflammation. Tacrolimus also affected renal miRNA expression, including miRNAs previously involved in fibrotic and inflammatory processes as “fibromirs” such as miR-21-5p, miR-199a-5p and miR-214-3p. In agreement with in vivo data, Renal Proximal Tubular Epithelial cells exposed to Tacrolimus (25 and 50 µM) showed upregulation of miR-21-5p and the concomitant induction of epithelial phenotypic changes, inflammation and oxidative stress. In conclusion, this study suggests for the first time that miRNAs, especially fibromiRs, participate to Tacrolimus-induced nephrotoxic effects. Therefore, targeting miRNAs may be a new therapeutic option to counteract Tacrolimus deleterious effects on kidney.
ABSTRACT
The mechanisms that regulate keratinocyte migration and proliferation in wound healing remain largely unraveled, notably regarding possible involvements of microRNAs (miRNAs). Here we ...disclose up‐regulation of miR‐483‐3p in 2 distinct models of wound healing: scratch‐injured cultures of human keratinocytes and wounded skin in mice. miR‐483‐3p accumulation peaks at the final stage of the wound closure process, consistent with a role in the arrest of “healing” progression. Using an in vitro wound‐healing model, videomicroscopy, and 5‐bromo‐2'‐uridine incorporation, we observed that overexpression of miR‐483‐3p inhibits keratinocyte migration and proliferation, whereas delivery of anti‐miR‐483‐3p oligonucleotides sustains keratinocyte proliferation beyond the closure of the wound, compared with irrelevant anti‐miR treatment. Expression profiling of keratinocytes transfected with miR‐483‐3p identified 39 transcripts that were both predicted targets of miR‐483‐3p and down‐regulated after miR‐483‐3p overexpression. Luciferase reporter assays, Western blot analyses, and silencing by specific siRNAs finally established that kinase MK2, cell proliferation marker MKI67, and transcription factor YAP1 are direct targets of miR‐483‐3p that control keratinocyte proliferation. miR‐483‐3p‐mediated down‐regulation of MK2, MKI67, and YAP1 thus represents a novel mechanism controlling keratinocyte growth arrest at the final steps of reepithelialization.—Bertero, T., Gastaldi, C., Bourget‐Ponzio, I., Imbert, V., Loubat, A., Selva, E., Busca, R., Mari, B., Hofman, P., Barbry, P., Meneguzzi, G., Ponzio, G., Rezzonico, R. miR‐483‐3p controls proliferation in wounded epithelial cells. FASEB J. 25, 3092‐3105 (2011). www.fasebj.org
Matrix metalloproteinases (MMPs) are essential for proper extracellular matrix remodeling, a process that takes place during obesity-mediated adipose tissue formation. Here, we examine expression ...profiles and the potential role of MMPs and their tissue inhibitors (TIMPs) in adipose tissue remodeling during obesity. Expression patterns are studied by Northern blot and real-time PCR in two genetic models of obesity (ob/ob and db/db mice) and in a diet-induced model of obesity (AKR mice). Of the MMPs and TIMPs studied, mRNA levels for MMP-2, MMP-3, MMP-12, MMP-14, MMP-19, and TIMP-1 are strongly induced in obese adipose tissues compared with lean tissues. In contrast, MMP-7 and TIMP-3 mRNAs are markedly decreased in obesity. Interestingly, enzymatic activities of MMP-12 and of a new identified adipocyte-derived 30-kDa metalloproteinase are enhanced in obese adipose tissue fractions, demonstrating that MMP/TIMP balance is shifted toward increased matrix degradation in obesity. Finally, we analyze the modulation of MMP-2, MMP-19, and TIMP-1 during 3T3-L1 preadipocyte differentiation, and we explore the effect of inhibition of MMP activity on in vitro adipogenesis. We find that the synthetic MMP inhibitor BB-94 (Batimastat) decreases adipose conversion of 3T3-L1 and primary rat preadipocytes. BB-94 represses differentiation without affecting mitotic clonal expansion but prevents the early expression of CCAAT/enhancer-binding protein beta, a transcription factor that is thought to play a major role in the adipogenic program. Such findings support a role for the MMP/TIMP system in the control of proteolytic events and adipogenesis during obesity-mediated fat mass development.
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