miR‐9‐5p suppresses pro‐fibrogenic transformation of fibroblasts and prevents organ fibrosis by targeting NOX4 and TGFBR2

Uncontrolled extracellular matrix (ECM) production by fibroblasts in response to injury contributes to fibrotic diseases, including idiopathic pulmonary fibrosis (IPF). Reactive oxygen species (ROS) generation is involved in the pathogenesis of IPF. Transforming growth factor‐β1 (TGF‐β1) stimulates the production of NADPH oxidase 4 (NOX4)‐dependent ROS, promoting lung fibrosis (LF). Dysregulation of microRNAs (miRNAs) has been shown to contribute to LF. To identify miRNAs involved in redox regulation relevant for IPF, we performed arrays in human lung fibroblasts exposed to ROS. miR‐9‐5p was selected as the best candidate and we demonstrate its inhibitory effect on TGF‐β receptor type II (TGFBR2) and NOX4 expression. Increased expression of miR‐9‐5p abrogates TGF‐β1‐dependent myofibroblast phenotypic transformation. In the mouse model of bleomycin‐induced LF, miR‐9‐5p dramatically reduces fibrogenesis and inhibition of miR‐9‐5p and prevents its anti‐fibrotic effect both in vitro and in vivo. In lung specimens from patients with IPF, high levels of miR‐9‐5p are found. In omentum‐derived mesothelial cells (MCs) from patients subjected to peritoneal dialysis (PD), miR‐9‐5p also inhibits mesothelial to myofibroblast transformation. We propose that TGF‐β1 induces miR‐9‐5p expression as a self‐limiting homeostatic response.     

Hypoxia and loss of PHD2 inactivate stromal fibroblasts to decrease tumour stiffness and metastasis

Cancer‐associated fibroblasts (CAFs) interact with tumour cells and promote growth and metastasis. Here, we show that CAF activation is reversible: chronic hypoxia deactivates CAFs, resulting in the loss of contractile force, reduced remodelling of the surrounding extracellular matrix and, ultimately, impaired CAF‐mediated cancer cell invasion. Hypoxia inhibits prolyl hydroxylase domain protein 2 (PHD2), leading to hypoxia‐inducible factor (HIF)‐1α stabilisation, reduced expression of αSMA and periostin, and reduced myosin II activity. Loss of PHD2 in CAFs phenocopies the effects of hypoxia, which can be prevented by simultaneous depletion of HIF‐1α. Treatment with the PHD inhibitor DMOG in an orthotopic breast cancer model significantly decreases spontaneous metastases to the lungs and liver, associated with decreased tumour stiffness and fibroblast activation. PHD2 depletion in CAFs co‐injected with tumour cells similarly prevents CAF‐induced metastasis to lungs and liver. Our data argue that reversion of CAFs towards a less active state is possible and could have important clinical implications.     

MicroRNA‐455 regulates brown adipogenesis via a novel HIF1an‐AMPK‐PGC1α signaling network

Brown adipose tissue (BAT) dissipates chemical energy as heat and can counteract obesity. MicroRNAs are emerging as key regulators in development and disease. Combining microRNA and mRNA microarray profiling followed by bioinformatic analyses, we identified miR‐455 as a new regulator of brown adipogenesis. miR‐455 exhibits a BAT‐specific expression pattern and is induced by cold and the browning inducer BMP7. In vitro gain‐ and loss‐of‐function studies show that miR‐455 regulates brown adipocyte differentiation and thermogenesis. Adipose‐specific miR‐455 transgenic mice display marked browning of subcutaneous white fat upon cold exposure. miR‐455 activates AMPKα1 by targeting HIF1an, and AMPK promotes the brown adipogenic program and mitochondrial biogenesis. Concomitantly, miR‐455 also targets the adipogenic suppressors Runx1t1 and Necdin, initiating adipogenic differentiation. Taken together, the data reveal a novel microRNA‐regulated signaling network that controls brown adipogenesis and may be a potential therapeutic target for human metabolic disorders.     

ApoE4 upregulates the activity of mitochondria‐associated ER membranes

In addition to the appearance of senile plaques and neurofibrillary tangles, Alzheimer''s disease (AD) is characterized by aberrant lipid metabolism and early mitochondrial dysfunction. We recently showed that there was increased functionality of mitochondria‐associated endoplasmic reticulum (ER) membranes (MAM), a subdomain of the ER involved in lipid and cholesterol homeostasis, in presenilin‐deficient cells and in fibroblasts from familial and sporadic AD patients. Individuals carrying the ε4 allele of apolipoprotein E (ApoE4) are at increased risk for developing AD compared to those carrying ApoE3. While the reason for this increased risk is unknown, we hypothesized that it might be associated with elevated MAM function. Using an astrocyte‐conditioned media (ACM) model, we now show that ER–mitochondrial communication and MAM function—as measured by the synthesis of phospholipids and of cholesteryl esters, respectively—are increased significantly in cells treated with ApoE4‐containing ACM as compared to those treated with ApoE3‐containing ACM. Notably, this effect was seen with lipoprotein‐enriched preparations, but not with lipid‐free ApoE protein. These data are consistent with a role of upregulated MAM function in the pathogenesis of AD and may help explain, in part, the contribution of ApoE4 as a risk factor in the disease.     

Integrative genomics positions MKRN1 as a novel ribonucleoprotein within the embryonic stem cell gene regulatory network

In embryonic stem cells (ESCs), gene regulatory networks (GRNs) coordinate gene expression to maintain ESC identity; however, the complete repertoire of factors regulating the ESC state is not fully understood. Our previous temporal microarray analysis of ESC commitment identified the E3 ubiquitin ligase protein Makorin‐1 (MKRN1) as a potential novel component of the ESC GRN. Here, using multilayered systems‐level analyses, we compiled a MKRN1‐centered interactome in undifferentiated ESCs at the proteomic and ribonomic level. Proteomic analyses in undifferentiated ESCs revealed that MKRN1 associates with RNA‐binding proteins, and ensuing RIP‐chip analysis determined that MKRN1 associates with mRNAs encoding functionally related proteins including proteins that function during cellular stress. Subsequent biological validation identified MKRN1 as a novel stress granule‐resident protein, although MKRN1 is not required for stress granule formation, or survival of unstressed ESCs. Thus, our unbiased systems‐level analyses support a role for the E3 ligase MKRN1 as a ribonucleoprotein within the ESC GRN.     

CPAP promotes timely cilium disassembly to maintain neural progenitor pool

A mutation in the centrosomal‐P4.1‐associated protein (CPAP) causes Seckel syndrome with microcephaly, which is suggested to arise from a decline in neural progenitor cells (NPCs) during development. However, mechanisms of NPCs maintenance remain unclear. Here, we report an unexpected role for the cilium in NPCs maintenance and identify CPAP as a negative regulator of ciliary length independent of its role in centrosome biogenesis. At the onset of cilium disassembly, CPAP provides a scaffold for the cilium disassembly complex (CDC), which includes Nde1, Aurora A, and OFD1, recruited to the ciliary base for timely cilium disassembly. In contrast, mutated CPAP fails to localize at the ciliary base associated with inefficient CDC recruitment, long cilia, retarded cilium disassembly, and delayed cell cycle re‐entry leading to premature differentiation of patient iPS‐derived NPCs. Aberrant CDC function also promotes premature differentiation of NPCs in Seckel iPS‐derived organoids. Thus, our results suggest a role for cilia in microcephaly and its involvement during neurogenesis and brain size control.     

IGFBP1 increases β‐cell regeneration by promoting α‐ to β‐cell transdifferentiation

There is great interest in therapeutically harnessing endogenous regenerative mechanisms to increase the number of β cells in people with diabetes. By performing whole‐genome expression profiling of zebrafish islets, we identified 11 secreted proteins that are upregulated during β‐cell regeneration. We then tested the proteins'' ability to potentiate β‐cell regeneration in zebrafish at supraphysiological levels. One protein, insulin‐like growth factor (Igf) binding‐protein 1 (Igfbp1), potently promoted β‐cell regeneration by potentiating α‐ to β‐cell transdifferentiation. Using various inhibitors and activators of the Igf pathway, we show that Igfbp1 exerts its regenerative effect, at least partly, by inhibiting Igf signaling. Igfbp1''s effect on transdifferentiation appears conserved across species: Treating mouse and human islets with recombinant IGFBP1 in vitro increased the number of cells co‐expressing insulin and glucagon threefold. Moreover, a prospective human study showed that having high IGFBP1 levels reduces the risk of developing type‐2 diabetes by more than 85%. Thus, we identify IGFBP1 as an endogenous promoter of β‐cell regeneration and highlight its clinical importance in diabetes.     

Structure and function of the N‐terminal domain of the human mitochondrial calcium uniporter

The mitochondrial calcium uniporter (MCU) is responsible for mitochondrial calcium uptake and homeostasis. It is also a target for the regulation of cellular anti‐/pro‐apoptosis and necrosis by several oncogenes and tumour suppressors. Herein, we report the crystal structure of the MCU N‐terminal domain (NTD) at a resolution of 1.50 Å in a novel fold and the S92A MCU mutant at 2.75 Å resolution; the residue S92 is a predicted CaMKII phosphorylation site. The assembly of the mitochondrial calcium uniporter complex (uniplex) and the interaction with the MCU regulators such as the mitochondrial calcium uptake‐1 and mitochondrial calcium uptake‐2 proteins (MICU1 and MICU2) are not affected by the deletion of MCU NTD. However, the expression of the S92A mutant or a NTD deletion mutant failed to restore mitochondrial Ca²⁺ uptake in a stable MCU knockdown HeLa cell line and exerted dominant‐negative effects in the wild‐type MCU‐expressing cell line. These results suggest that the NTD of MCU is essential for the modulation of MCU function, although it does not affect the uniplex formation.     

The C9orf72 protein interacts with Rab1a and the ULK1 complex to regulate initiation of autophagy

A GGGGCC hexanucleotide repeat expansion in the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD). C9orf72 encodes two C9orf72 protein isoforms of unclear function. Reduced levels of C9orf72 expression have been reported in C9ALS/FTD patients, and although C9orf72 haploinsufficiency has been proposed to contribute to C9ALS/FTD, its significance is not yet clear. Here, we report that C9orf72 interacts with Rab1a and the Unc‐51‐like kinase 1 (ULK1) autophagy initiation complex. As a Rab1a effector, C9orf72 controls initiation of autophagy by regulating the Rab1a‐dependent trafficking of the ULK1 autophagy initiation complex to the phagophore. Accordingly, reduction of C9orf72 expression in cell lines and primary neurons attenuated autophagy and caused accumulation of p62‐positive puncta reminiscent of the p62 pathology observed in C9ALS/FTD patients. Finally, basal levels of autophagy were markedly reduced in C9ALS/FTD patient‐derived iNeurons. Thus, our data identify C9orf72 as a novel Rab1a effector in the regulation of autophagy and indicate that C9orf72 haploinsufficiency and associated reductions in autophagy might be the underlying cause of C9ALS/FTD‐associated p62 pathology.     

Reversible HuR‐microRNA binding controls extracellular export of miR‐122 and augments stress response

microRNAs (miRNAs), the tiny but stable regulatory RNAs in metazoan cells, can undergo selective turnover in presence of specific internal and external cues to control cellular response against the changing environment. We have observed reduction in cellular miR‐122 content, due to their accelerated extracellular export in human hepatic cells starved for small metabolites including amino acids. In this context, a new role of human ELAV protein HuR has been identified. HuR, a negative regulator of miRNA function, accelerates extracellular vesicle (EV)‐mediated export of miRNAs in human cells. In stressed cells, HuR replaces miRNPs from target messages and is both necessary and sufficient for the extracellular export of corresponding miRNAs. HuR could reversibly bind miRNAs to replace them from Ago2 and subsequently itself gets freed from bound miRNAs upon ubiquitination. The ubiquitinated form of HuR is predominantly associated with multivesicular bodies (MVB) where HuR‐unbound miRNAs also reside. These MVB‐associated pool of miRNAs get exported out via EVs thereby delimiting cellular miR‐122 level during starvation. Therefore, by modulating extracellular export of miR‐122, HuR could control stress response in starved human hepatic cells.     

Phosphoproteomic screening identifies Rab GTPases as novel downstream targets of PINK1

Mutations in the PTEN‐induced kinase 1 (PINK1) are causative of autosomal recessive Parkinson''s disease (PD). We have previously reported that PINK1 is activated by mitochondrial depolarisation and phosphorylates serine 65 (Ser⁶⁵) of the ubiquitin ligase Parkin and ubiquitin to stimulate Parkin E3 ligase activity. Here, we have employed quantitative phosphoproteomics to search for novel PINK1‐dependent phosphorylation targets in HEK (human embryonic kidney) 293 cells stimulated by mitochondrial depolarisation. This led to the identification of 14,213 phosphosites from 4,499 gene products. Whilst most phosphosites were unaffected, we strikingly observed three members of a sub‐family of Rab GTPases namely Rab8A, 8B and 13 that are all phosphorylated at the highly conserved residue of serine 111 (Ser¹¹¹) in response to PINK1 activation. Using phospho‐specific antibodies raised against Ser¹¹¹ of each of the Rabs, we demonstrate that Rab Ser¹¹¹ phosphorylation occurs specifically in response to PINK1 activation and is abolished in HeLa PINK1 knockout cells and mutant PINK1 PD patient‐derived fibroblasts stimulated by mitochondrial depolarisation. We provide evidence that Rab8A GTPase Ser¹¹¹ phosphorylation is not directly regulated by PINK1 in vitro and demonstrate in cells the time course of Ser¹¹¹ phosphorylation of Rab8A, 8B and 13 is markedly delayed compared to phosphorylation of Parkin at Ser⁶⁵. We further show mechanistically that phosphorylation at Ser¹¹¹ significantly impairs Rab8A activation by its cognate guanine nucleotide exchange factor (GEF), Rabin8 (by using the Ser111Glu phosphorylation mimic). These findings provide the first evidence that PINK1 is able to regulate the phosphorylation of Rab GTPases and indicate that monitoring phosphorylation of Rab8A/8B/13 at Ser¹¹¹ may represent novel biomarkers of PINK1 activity in vivo. Our findings also suggest that disruption of Rab GTPase‐mediated signalling may represent a major mechanism in the neurodegenerative cascade of Parkinson''s disease.     

Human TLR8 senses UR/URR motifs in bacterial and mitochondrial RNA

Toll‐like receptor (TLR) 13 and TLR2 are the major sensors of Gram‐positive bacteria in mice. TLR13 recognizes Sa19, a specific 23S ribosomal (r) RNA‐derived fragment and bacterial modification of Sa19 ablates binding to TLR13, and to antibiotics such as erythromycin. Similarly, RNase A‐treated Staphylococcus aureus activate human peripheral blood mononuclear cells (PBMCs) only via TLR2, implying single‐stranded (ss) RNA as major stimulant. Here, we identify human TLR8 as functional TLR13 equivalent that promiscuously senses ssRNA. Accordingly, Sa19 and mitochondrial (mt) 16S rRNA sequence‐derived oligoribonucleotides (ORNs) stimulate PBMCs in a MyD88‐dependent manner. These ORNs, as well as S. aureus‐, Escherichia coli‐, and mt‐RNA, also activate differentiated human monocytoid THP‐1 cells, provided they express TLR8. Moreover, Unc93b1 ^−/−‐ and Tlr8 ^−/−‐THP‐1 cells are refractory, while endogenous and ectopically expressed TLR8 confers responsiveness in a UR/URR RNA ligand consensus motif‐dependent manner. If TLR8 function is inhibited by suppression of lysosomal function, antibiotic treatment efficiently blocks bacteria‐driven inflammatory responses in infected human whole blood cultures. Sepsis therapy might thus benefit from interfering with TLR8 function.