ABRO1 arrests cardiomyocyte proliferation and myocardial repair by suppressing PSPH.

The role of Abraxas 2 (ABRO1 or KIAA0157), a component of the lysine63-linked deubiquitinating system, in the cardiomyocyte proliferation and myocardial regeneration is unknown. Here, we found that ABRO1 regulates cardiomyocyte proliferation and cardiac regeneration in the postnatal heart by targeting METTL3-mediated m6A methylation of Psph mRNA. The deletion of ABRO1 increased cardiomyocyte proliferation in hearts and restored the heart function after myocardial injury. On the contrary, ABRO1 overexpression significantly inhibited the neonatal cardiomyocyte proliferation and cardiac regeneration in mouse hearts. The mechanism by which ABRO1 regulates cardiomyocyte proliferation mainly involved METTL3-mediated Psph mRNA methylation and CDK2 phosphorylation. In the early postnatal period, METTL3-dependent m6A methylation promotes cardiomyocyte proliferation by hypermethylation of Psph mRNA and upregulating PSPH expression. PSPH dephosphorylates cyclin-dependent kinase 2 (CDK2), a positive regulator of cell cycle, at Thr14/Tyr15 and increases its activity. Upregulation of ABRO1 restricts METTL3 activity and halts the cardiomyocyte proliferation in the postnatal hearts. Thus, our study reveals that ABRO1 is an essential contributor in the cell cycle withdrawal and attenuation of proliferative response in the postnatal cardiomyocytes and could act as a potential target to accelerate cardiomyocyte proliferation and cardiac repair in the adult heart.

Long Noncoding RNA CPR (Cardiomyocyte Proliferation Regulator) Regulates Cardiomyocyte Proliferation and Cardiac Repair.

BACKGROUND: The adult mammalian cardiomyocytes lose their proliferative capacity, which is responsible for cardiac dysfunction and heart failure following injury. The molecular mechanisms underlying the attenuation of adult cardiomyocyte proliferation remain largely unknown. Because long noncoding RNAs (lncRNAs) have a critical role in the development of cardiovascular problems, we investigated whether lncRNAs have any role in the regulation of cardiomyocyte proliferation and cardiac repair. METHODS: Using bioinformatics and initial analysis, we identified an lncRNA, named CPR (cardiomyocyte proliferation regulator), that has a potential regulatory role in cardiomyocyte proliferation. For in vivo experiments, we generated CPR knockout and cardiac-specific CPR-overexpressing mice. In isolated cardiomyocytes, we used adenovirus for silencing (CPR-small interfering RNA) or overexpressing CPR. To investigate the mechanisms of CPR function in cardiomyocyte proliferation, we performed various analyses including quantitative reverse transcription-polymerase chain reaction, Western blot, histology, cardiac function (by echocardiography), transcriptome analyses (microarray assay), RNA pull-down assay, and chromatin immunoprecipitation assay. RESULTS: CPR level is comparatively higher in the adult heart than in the fetal stage. The silencing of CPR significantly increased cardiomyocyte proliferation in postnatal and adult hearts. Moreover, CPR deletion restored the heart function after myocardial injury, which was evident from increased cardiomyocyte proliferation, improvement of myocardial function, and reduced scar formation. In contrast, the neonatal cardiomyocyte proliferation and cardiac regeneration were remarkably suppressed in CPR-overexpressing mice or adeno-associated virus serotype 9-CPR-overexpressing heart. These results indicate that CPR acts as a negative regulator of cardiomyocyte proliferation and regeneration. Next, we found that CPR targets minichromosome maintenance 3, an initiator of DNA replication and cell cycle progression, to suppress cardiomyocyte proliferation. CPR silenced minichromosome maintenance 3 expression through directly interacting and recruiting DNMT3A to its promoter cysteine-phosphate-guanine sites, as evident from decreased minichromosome maintenance 3 promoter methylation and increased minichromosome maintenance 3 expression in CPR knocked-down cardiomyocytes and CPR knockout mouse heart. These results were confirmed in CPR-overexpressing cardiomyocytes and CPR-overexpressing mouse heart. CONCLUSIONS: Together, our findings identified that CPR is a suppressor of cardiomyocyte proliferation and indicated that lncRNAs take part in the regulation of cardiomyocyte proliferation and cardiac repair. Our study provides an lncRNA-based therapeutic strategy for effective cardiac repair and regeneration.

Foxo3a-dependent miR-633 regulates chemotherapeutic sensitivity in gastric cancer by targeting Fas-associated death domain.

The development of chemotherapeutic drugs resistance such as doxorubicin (DOX) and cisplatin (DDP) is the major barrier in gastric cancer therapy. Emerging evidences reveal that microRNAs (miRNAs) contribute to chemosensitivity. In this study, we investigated the role of miR-633, an oncogenic miRNA, in gastric cancer chemoresistance. In gastric cancer tissue and cell lines, miR-633 expression was highly increased and correlated with down regulation of Fas-associated protein with death domain (FADD). Inhibition of miR-633 significantly increased FADD protein level and enhanced DOX/DDP induced apoptosis in vitro. MiR-633 antagomir administration remarkably decreased tumor growth in combination with DOX in vivo, suggesting that miR-633 targets FADD to block gastric cancer cell death. We found that the promoter region of miR-633 contained putative binding sites for forkhead box O 3 (Foxo3a), which can directly repress miR-633 transcription. In addition, we observed that DOX-induced nuclear accumulation of Foxo3a leaded to the suppression of miR-633 transcription. Together, our study revealed that miR-633/FADD axis played a significant role in the chemoresistance and Foxo3a regulated this pathway in gastric cancer. Thus, miR-633 antagomir resensitized gastric cancer cells to chemotherapy drug and had potentially therapeutic implication.

Role of noncoding RNAs in regulation of cardiac cell death and cardiovascular diseases.

Loss of functional cardiomyocytes is a major underlying mechanism for myocardial remodeling and heart diseases, due to the limited regenerative capacity of adult myocardium. Apoptosis, programmed necrosis, and autophagy contribute to loss of cardiac myocytes that control the balance of cardiac cell death and cell survival through multiple intricate signaling pathways. In recent years, non-coding RNAs (ncRNAs) have received much attention to uncover their roles in cell death of cardiovascular diseases, such as myocardial infarction, cardiac hypertrophy, and heart failure. In addition, based on the view that mitochondrial morphology is linked to three types of cell death, ncRNAs are able to regulate mitochondrial fission/fusion of cardiomyocytes by targeting genes involved in cell death pathways. This review focuses on recent progress regarding the complex relationship between apoptosis/necrosis/autophagy and ncRNAs in the context of myocardial cell death in response to stress. This review also provides insight into the treatment for heart diseases that will guide novel therapies in the future.

The role of K63-linked polyubiquitination in cardiac hypertrophy.

Ubiquitination, also known as ubiquitylation, is a vital post-translational modification of proteins that play a crucial role in the multiple biological processes including cell growth, proliferation and apoptosis. K63-linked ubiquitination is one of the vital post-translational modifications of proteins that are involved in the activation of protein kinases and protein trafficking during cell survival and proliferation. It also contributes to the development of various disorders including cancer, neurodegeneration and cardiac hypertrophy. In this review, we summarize the role of K63-linked ubiquitination signalling in protein kinase activation and its implications in cardiac hypertrophy. We have also provided our perspectives on therapeutically targeting K63-linked ubiquitination in downstream effector molecules of growth factor receptors for the treatment of cardiac hypertrophy.

Critical role of FOXO3a in carcinogenesis.

FOXO3a is a member of the FOXO subfamily of forkhead transcription factors that mediate a variety of cellular processes including apoptosis, proliferation, cell cycle progression, DNA damage and tumorigenesis. It also responds to several cellular stresses such as UV irradiation and oxidative stress. The function of FOXO3a is regulated by a complex network of processes, including post-transcriptional suppression by microRNAs (miRNAs), post-translational modifications (PTMs) and protein-protein interactions. FOXO3a is widely implicated in a variety of diseases, particularly in malignancy of breast, liver, colon, prostate, bladder, and nasopharyngeal cancers. Emerging evidences indicate that FOXO3a acts as a tumor suppressor in cancer. FOXO3a is frequently inactivated in cancer cell lines by mutation of the FOXO3a gene or cytoplasmic sequestration of FOXO3a protein. And its inactivation is associated with the initiation and progression of cancer. In experimental studies, overexpression of FOXO3a inhibits the proliferation, tumorigenic potential, and invasiveness of cancer cells, while silencing of FOXO3a results in marked attenuation in protection against tumorigenesis. The role of FOXO3a in both normal physiology as well as in cancer development have presented a great challenge to formulating an effective therapeutic strategy for cancer. In this review, we summarize the recent findings and overview of the current understanding of the influence of FOXO3a in cancer development and progression.

Understanding cardiomyocyte proliferation: an insight into cell cycle activity.

Cardiomyocyte proliferation and regeneration are key to the functional recovery of myocardial tissue from injury. In the recent years, studies on cardiomyocyte proliferation overturned the traditional belief that adult cardiomyocytes permanently withdraw from the cell cycle activity. Hence, targeting cardiomyocyte proliferation is one of the potential therapeutic strategies for myocardial regeneration and repair. To achieve this, a deep understanding of the fundamental mechanisms involved in cardiomyocyte cell cycle as well as differences between neonatal and adult cardiomyocytes' cell cycle activity is required. This review focuses on the recent progress in understanding of cardiomyocyte cell cycle activity at different life stages viz., gestation, birth, and adulthood. The temporal expression/activities of major cell cycle activators (cyclins and CDKs), inhibitors (p21, p27, p57, p16, and p18), and cell-cycle-associated proteins (Rb, p107, and p130) in cardiomyocytes during gestation and postnatal life are described in this review. The influence of different transcription factors and microRNAs on the expression of cell cycle proteins is demonstrated. This review also deals major pathways (PI3K/AKT, Wnt/ß-catenin, and Hippo-YAP) associated with cardiomyocyte cell cycle progression. Furthermore, the postnatal alterations in structure and cellular events responsible for the loss of cell cycle activity are also illustrated.

MiR-485-5p modulates mitochondrial fission through targeting mitochondrial anchored protein ligase in cardiac hypertrophy.

The pathogenesis of cardiac hypertrophy is tightly associated with mitochondrial dysfunction. Disequilibrium of mitochondrial dynamic is one of the main drivers in the pathological processes during development of various cardiac diseases. However, the effect of mitochondrial dynamics on cardiac hypertrophy remains largely unclear. MicroRNAs (miRNAs) are small noncoding RNAs that can switch off expression of many genes. Mitochondrial anchored protein ligase (MAPL) is a small ubiquitin-like modifier (SUMO) E3 ligase, which is an important contributor in mitochondrial fission process. In this study, we found that hypertrophic agonist phenylephrine (PE) enhanced the expression of MAPL and promoted mitochondrial fission, while it decreased the expression of mitochondrial fusion protein2 (Mfn2) in hypertrophic cardiomyocytes. Silencing expression of MAPL by siRNA attenuated PE-induced depletion of Mfn2 and increase of mitochondrial fission as well as hypertrophic response in cultured primary cardiomyocytes. MiR-485-5p is screened as a candidate inhibitor of MAPL. Overexpression of miR-485-5p blocked mitochondrial fission and hypertrophy induced by PE through inhibiting MAPL expression and increasing the level of Mfn2 in cultured primary cardiomyocytes. In mice model of cardiac hypertrophy induced by PE, the administration of miR-485-5p agomir significantly decreased the PE induced increase in the expression of MAPL and hypertrophic markers (ANP and ß-MHC) along with protection of cardiac structure and function. Together, this study exhibits a novel signaling axis composed of miR-485-5p/MAPL/Mfn2, which regulates mitochondrial machinery and cardiac hypertrophy.

Efficient production of recombinant glycoprotein D of herpes simplex virus type 2 in Pichia pastoris and its protective efficacy against viral challenge in mice.

Herpes simplex virus type 2 (HSV-2) infection is the leading cause of genital ulcer disease and a significant public health concern. However, there are no approved vaccines available to prevent HSV-2 infection. The glycoprotein D (gD) of HSV-2 is the most important candidate antigen for vaccine development. In this study, a truncated form of gD (codons 1-340, gD1-340) was produced as a secretory protein in the methylotrophic yeast Pichia pastoris. The recombinant gD1-340 with a His6 tag was purified to homogeneity by one-step affinity chromatography. Mice immunized with the recombinant gD1-340 developed high levels of antigen-specific antibody responses with HSV-2 neutralizing activity. Immunization with the recombinant gD1-340 conferred significant protection against lethal HSV-2 infection in mice. Moreover, measurement of the secretion of gD1-340-specific cytokines demonstrated that the recombinant gD1-340 induced mixed Th1/Th2 cellular immune responses. These findings indicated that P. pastoris-derived gD1-340 represents a promising HSV-2 vaccine candidate with strong immunogenicity and prophylactic efficacy.

Effects of miRNAs on myocardial apoptosis by modulating mitochondria related proteins.

Myocardial apoptosis play a vital role in pathogenesis of cardiovascular diseases. The intrinsic pathway of apoptosis (mitochondrial apoptosis pathway) and abnormal mitochondrial fission and fusion have a detrimental effect on cells under a variety of intracellular stresses including hypoxia, oxidative stress, drug toxicity or DNA damage and contributes to the development of heart failure (HF), myocardial infarction (MI), diabetic cardiomyopathy and ischaemia/reperfusion injury (I/R). MicroRNAs (miRNAs) are endogenous short non-coding RNAs, which target 3'-untranslated region of mRNA to switch off gene expression. They play crucial roles in regulating complicated cardiac signalling and transcriptional events during cardiac development as well as in diseased condition. In this review, we summarize the molecular mechanism of mitochondrial apoptosis in cardiac cells and influence of miRNAs on them. MiRNAs regulate cardiac mitochondrial apoptosis by exert their effects on mitochondrial fission and fusion, reactive oxygen species (ROS) generation and Ca2+ homeostasis, Bcl-2 family members, and other mitochondrial function proteins. This advancement in understanding mechanism of cardiac cells death provides us new therapy targets for cardiovascular diseases associated with mitochondrial dysfunctions.

Enhancer of Zeste Homolog 2 Inhibition Attenuates Renal Fibrosis by Maintaining Smad7 and Phosphatase and Tensin Homolog Expression.

Enhancer of zeste homolog 2 (EZH2) is a methyltransferase that induces histone H3 lysine 27 trimethylation (H3K27me3) and functions as an oncogenic factor in many cancer types. However, the role of EZH2 in renal fibrogenesis remains unexplored. In this study, we found high expression of EZH2 and H3K27me3 in cultured renal fibroblasts and fibrotic kidneys from mice with unilateral ureteral obstruction and humans with CKD. Pharmacologic inhibition of EZH2 with 3-deazaneplanocin A (3-DZNeP) or GSK126 or siRNA-mediated silencing of EZH2 inhibited serum- and TGFß1-induced activation of renal interstitial fibroblasts in vitro, and 3-DZNeP administration abrogated deposition of extracellular matrix proteins and expression of α-smooth muscle actin in the obstructed kidney. Injury to the kidney enhanced Smad7 degradation, Smad3 phosphorylation, and TGFß receptor 1 expression, and 3-DZNeP administration prevented these effects. 3-DZNeP also suppressed phosphorylation of the renal EGF and PDGFß receptors and downstream signaling molecules signal transducer and activator of transcription 3 and extracellular signal-regulated kinase 1/2 after injury. Moreover, EZH2 inhibition increased the expression of phosphatase and tensin homolog (PTEN), a protein previously associated with dephosphorylation of tyrosine kinase receptors in the injured kidney and serum-stimulated renal interstitial fibroblasts. Finally, blocking PTEN with SF1670 largely diminished the inhibitory effect of 3-DZNeP on renal myofibroblast activation. These results uncovered the important role of EZH2 in mediating the development of renal fibrosis by downregulating expression of Smad7 and PTEN, thus activating profibrotic signaling pathways. Targeted inhibition of EZH2, therefore, could be a novel therapy for treating CKD.

Src inhibition blocks renal interstitial fibroblast activation and ameliorates renal fibrosis.

Increased Src activity has been associated with the pathogenesis of renal tumors and some glomerular diseases, but its role in renal interstitial fibrosis remains elusive. To evaluate this, cultured renal interstitial fibroblasts (NRK-49F) were treated with PP1, a selective inhibitor of Src. This resulted in decreased expression of α-smooth muscle actin, fibronectin, and collagen I in response to serum, angiotension II, or transforming growth factor-ß1 (TGF-ß1). Silencing Src with siRNA also inhibited expression of those proteins. Furthermore, inhibition of Src activity blocked renal fibroblast proliferation. In a murine model of renal interstitial fibrosis induced by unilateral ureteral obstruction, the active form of Src (phopsho-Src Tyr416) was upregulated in both renal interstitial fibroblasts and renal tubular cells of the fibrotic kidney. Its inactivation reduced renal fibroblast activation and attenuated extracellular matrix protein deposition. Src inhibition also suppressed activation of TGF-ß1 signaling, activation of the epidermal growth factor receptor and STAT3, and reduced the number of renal epithelial cells arrested at the G2/M phase of the cell cycle after ureteral obstruction. Thus, Src is an important mediator of renal interstitial fibroblast activation and renal fibrosis, and we suggest that Src is a potential therapeutic target for treatment of chronic renal fibrosis.

Activation of Sirtuin-1 Promotes Renal Fibroblast Activation and Aggravates Renal Fibrogenesis.

Although activation of sirtuin-1 (SIRT1) has been shown to protect the kidney from acute injury, its role in renal fibrosis remains controversial since both inhibition and activation of SIRT1 have been reported to attenuate renal fibrosis. To resolve this conflict, we further examined the effect of SIRT1 activators on the activation of renal interstitial fibroblasts and development of renal fibrosis in vivo and in vitro. In a murine model of renal fibrosis induced by unilateral ureteral obstruction, administration of SRT1720 (N-[2-[3-(piperazin-1-ylmethyl)imidazo[2,1-b][1,3]thiazol-6-yl]phenyl]quinoxaline-2-carboxamide), a potent activator of SIRT1, accelerated deposition of collagen fibrils and increased expression of fibroblast activation markers (α-smooth muscle actin [α-SMA], collagen I, and fibronectin) in the obstructive kidney of mice. In cultured rat renal interstitial fibroblasts (NRK-49F), exposure of cells to SRT1720 or YK-3-237 (B-[2-methoxy-5-[(1E)-3-oxo-3-(3,4,5-trimethoxyphenyl)-1-propen-1-yl]phenyl]-boronic acid), another SIRT1 activator, also resulted in enhanced expression of α-SMA and fibronectin. Mechanistic studies showed that augmentation of renal fibrogenesis by SRT1720 is associated with elevated phosphorylation of epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor ß (PDGFRß). SRT1720 treatment also increased the phosphorylation of signal transducer and activator of transcription 3 and protein kinase B in the fibrotic kidney and NRK-49F cells. However, SRT1720 treatment did not affect expression of proliferating cell nuclear protein, a proliferation marker and activation of extracellular signal regulated kinase 1/2 in vitro and in vivo. These results indicate that SIRT1-activating compounds can provoke renal fibrogenesis through a mechanism involved in the activation of EGFR and PDGFR signaling pathways and suggest that long-term use of SIRT1 activators risks the development and progression of chronic kidney disease.

Blocking sirtuin 1 and 2 inhibits renal interstitial fibroblast activation and attenuates renal interstitial fibrosis in obstructive nephropathy.

Our recent studies revealed that blocking class I/II histone deacetylases (HDACs) inhibits renal interstitial fibroblast activation and proliferation and alleviates development of renal fibrosis. However, the effect of class III HDAC, particularly sirtuin 1 and 2 (SIRT1 and SIRT2), inhibition on renal fibrogenesis remains elusive. Here, we demonstrate that both SIRT1 and SIRT2 were expressed in cultured renal interstitial fibroblasts (NRK-49F). Exposure of NRK-49F to sirtinol, a selective inhibitor of SIRT1/2, or EX527 (6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxamide), an inhibitor for SIRT1, resulted in reduced expression of fibroblast activation markers (α-smooth muscle actin, fibronectin, and collagen I) as well as proliferation markers (proliferating cell nuclear antigen, cyclin D1, cyclin E) in dose- and time-dependent manners. Treatment with a SIRT2 inhibitor, AGK2 (2-cyano-3-[5-(2,5-dichlorophenyl)-2-furanyl]-N-5-quinolinyl-2-propenamide), also dose- and time-dependently inhibited renal fibroblast activation and, to a lesser extent, cell proliferation. Furthermore, silencing of either SIRT1 or SIRT2 by small interfering RNA exhibited similar inhibitory effects. In a mouse model of obstructive nephropathy, administration of sirtinol attenuated deposition of collagen fibrils as well as reduced expression of α-smooth muscle actin, collagen I, and fibronectin in the injured kidney. SIRT1/2 inhibition-mediated antifibrotic effects are associated with dephosphorylation of epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor-ß (PDGFRß), and signal transducer and activator of transcription 3. Thus, SIRT1/2 activity may contribute to renal fibroblast activation and proliferation as well as renal fibrogenesis through activation of at least EGFR and PDGFRß signaling. Blocking SIRT1/2 activation may have therapeutic potential for the treatment of chronic kidney disease.

Sustained activation of EGFR triggers renal fibrogenesis after acute kidney injury.

Severe acute kidney injury (AKI) is frequently accompanied by maladaptive repair and renal fibrogenesis; however, the molecular mechanisms that mediate these acute and chronic consequences of AKI remain poorly understood. In this study, we examined the role of epidermal growth factor receptor (EGFR) in these processes using waved-2 (Wa-2) mice, which have reduced EGFR activity, and their wild-type (WT) littermates after renal ischemia. Renal EGFR phosphorylation was induced within 2 days after ischemia, increased over time, and remained elevated at 28 days in WT mice, but this was diminished in Wa-2 mice. At the early stage of postischemia (2 days), Wa-2 mice developed more severe acute renal tubular damage with less reparative responses as indicated by enhanced tubular cell apoptosis, and reduced dedifferentiation and proliferation as compared to WT animals. At the late stage of postischemia (28 days), Wa-2 mice exhibited a less severe renal interstitial fibrosis as shown by reduced activation/proliferation of renal myofibroblasts and decreased deposition of extracellular matrix proteins. EGFR activation also contributed to cell cycle arrest at the G2/M phase, a cellular event associated with production of profibrogenetic factors, in the injured kidney. Collectively, these results indicate that severe AKI results in sustained activation of EGFR, which is required for reparative response of renal tubular cells initially, but eventually leads to fibrogenesis.

Necrotic renal epithelial cell inhibits renal interstitial fibroblast activation: role of protein tyrosine phosphatase 1B.

Our recent studies showed that contents of necrotic renal proximal tubular cells (RPTC) from 2 × 10(6) cells/ml directly induced death of cultured renal interstitial fibroblasts. However, it remains unknown whether nonlethal number of necrotic RPTC would also alter the fate of renal interstitial fibroblasts. To address this issue, renal interstitial fibroblasts (NRK-49F) were exposed to necrotic RPTC supernatant (RPTC-Sup) obtained from 2 × 10(4) to 5 × 10(5) cells/ml. These concentrations of RPTC did not induce cell death, but led to inactivation of renal fibroblasts as indicated by reduced expression of α-smooth muscle actin and fibronectin, two hallmarks of activated fibroblasts. Concurrently, the same doses of necrotic RPTC-Sup suppressed phosphorylation of epidermal growth factor receptor (EGFR) and signal transducers and activators of transcription-3 (STAT3) in a time- and dose-dependent manner, but did not affect phosphorylation of platelet-derived growth factor receptor-ß, AKT, and extracellular signal-regulated kinase 1/2. The presence of sodium orthovanadate, a general protein tyrosine phosphatase (PTP) inhibitor or TCS-401 (a selective PTP1B inhibitor), abrogated those effects of RPTC-Sup, whereas coincubation with the EGFR inhibitor (Gefitinib) or silencing of EGFR with siRNA preserved the ability of RPTC-Sup in suppressing renal fibroblast activation and STAT3 phosphorylation. Moreover, RPTC-Sup treatment induced PTP1B phosphorylation and its interaction with EGFR. Collectively, these results indicate that nonlethal necrotic RPTC-Sup can induce inactivation of renal interstitial fibroblasts, which occurs through a mechanism involved in PTP1B-mediated inhibition of EGFR signaling.

Genetic or pharmacologic blockade of EGFR inhibits renal fibrosis.

Although enhanced activation of the EGF receptor (EGFR) associates with the development and progression of renal fibrosis, the mechanisms linking these observations are not completely understood. Here, after unilateral ureteral obstruction (UUO), wild-type mice exhibited sustained EGFR phosphorylation in the kidney and developed renal fibrosis that was more severe than the renal fibrosis observed in waved-2 mice, which have reduced EGFR tyrosine kinase activity. Waved-2 mice also showed fewer renal tubular cells arrested at G2/M, reduced expression of α-smooth muscle actin (α-SMA), downregulation of multiple genes encoding profibrogenic cytokines, including TGF-ß1, and dephosphorylation of Smad3, STAT3, and ERK1/2. Administration of the specific EGFR inhibitor gefitinib recapitulated this phenotype in wild-type mice after UUO. Furthermore, inactivation of either EGFR or STAT3 reduced UUO-induced expression of lipocalin-2, a molecule associated with the pathogenesis of CKD. In cultured renal interstitial fibroblasts, inhibition of EGFR also abrogated TGF-ß1- or serum-induced phosphorylation of EGFR, STAT3, ERK1/2, and Smad3 as well as expression of α-SMA and extracelluar matrix proteins. Taken together, these data suggest that EGFR may mediate renal fibrogenesis by promoting transition of renal epithelial cells to a profibrotic phenotype, increased production of inflammatory factors, and activation of renal interstitial fibroblasts. Inhibition of EGFR may have therapeutic potential for fibrotic kidney disease.

Autophagy protects against necrotic renal epithelial cell-induced death of renal interstitial fibroblasts.

We recently reported that necrotic renal proximal tubular cells (RPTC) can induce the death of renal interstitial fibroblasts. Since autophagy plays either cytoprotective or cytodestructive roles depending on the experimental condition, the present study was carried out to investigate whether necrotic RPTC would induce autophagy of renal interstitial fibroblasts and, if so, whether autophagy would contribute to cell death or exert a protective effect. Exposure of necrotic RPTC supernatant (RPTC-Sup) induced autophagy in renal interstitial fibroblast cells (NRK-49F) in a time- and dose-dependent manner, and its induction was earlier than caspase-3 activation. Inhibition of autophagy with 3-methyladenine (3-MA) or knockdown of Beclin-1, a molecule involved in the initiation of autophagosome formation, with small interference RNA (siRNA) significantly enhanced necrotic RPTC-Sup-induced cell death. Necrotic RPTC-Sup induced phosphorylation of extracellular signal-regulated kinases (ERK1/2), p38, c-Jun NH(2)-terminal kinases (JNKs), and AKT. Treatment with an ERK1/2 pathway inhibitor, but not with specific inhibitors for p38, JNKs, or AKT pathways, blocked NRK-49F autophagy and cell death upon exposure to necrotic RPTC-Sup. Furthermore, knockdown of MEK1 with siRNA also reduced autophagy along with cell death in NRK-49F exposed to necrotic RPTC-Sup. In contrast, overexpression of MEK1/2 increased RPTC-Sup-induced fibroblast cell death without enhancing autophagy. Collectively, this study demonstrates that necrotic RPTC induce both autophagy and cell death and that autophagy plays a cytoprotective or prosurvival role in renal fibroblasts. Furthermore, necrotic RPTC-induced autophagy and cell death in renal fibroblasts is mediated by the activation of the MEK1-ERK1/2 signaling pathway.

Suramin inhibits renal fibrosis in chronic kidney disease.

The activation of cytokine and growth factor receptors associates with the development and progression of renal fibrosis. Suramin is a compound that inhibits the interaction of several cytokines and growth factors with their receptors, but whether suramin inhibits the progression of renal fibrosis is unknown. Here, treatment of cultured renal interstitial fibroblasts with suramin inhibited their activation induced by TGF-ß1 and serum. In a mouse model of obstructive nephropathy, administration of a single dose of suramin immediately after ureteral obstruction abolished the expression of fibronectin, largely suppressed expression of α-SMA and type I collagen, and reduced the deposition of extracellular matrix proteins. Suramin also decreased the expression of multiple cytokines including TGF-ß1 and reduced the interstitial infiltration of leukocytes. Moreover, suramin decreased expression of the type II TGF-ß receptor, blocked phosphorylation of the EGF and PDGF receptors, and inactivated several signaling pathways associated with the progression of renal fibrosis. In a rat model of CKD, suramin abrogated proteinuria, limited the decline of renal function, and prevented glomerular and tubulointerstitial damage. Collectively, these findings indicate that suramin is a potent antifibrotic agent that may have therapeutic potential for patients with fibrotic kidney diseases.

Crosstalk among long non-coding RNA, tumor-associated macrophages and small extracellular vesicles in tumorigenesis and dissemination.

Long noncoding RNAs (lncRNAs), which lack protein-coding ability, can regulate cancer cell growth, proliferation, invasion, and metastasis. Tumor-associated macrophages (TAMs) are key components of the tumor microenvironment that have a significant impact on cancer progression. Small extracellular vesicles (sEV) are crucial mediators of intercellular communications. Cancer cell and macrophage-derived sEV can carry lncRNAs that influence the onset and progression of cancer. Dysregulation of lncRNAs, TAMs, and sEV is widely observed in tumors which makes them valuable targets for cancer immunotherapy. In this review, we summarize current updates on the interactions among sEV, lncRNAs, and TAMs in tumors and provide new perspectives on cancer diagnosis and treatment.