The c-Abl-RACK1-FAK signaling axis promotes renal fibrosis in mice through regulating fibroblast-myofibroblast transition.

BACKGROUND: Renal fibrosis is a prevalent manifestation of chronic kidney disease (CKD), and effective treatments for this disease are currently lacking. Myofibroblasts, which originate from interstitial fibroblasts, aggregate in the renal interstitium, leading to significant accumulation of extracellular matrix and impairment of renal function. The nonreceptor tyrosine kinase c-Abl (encoded by the Abl1 gene) has been implicated in the development of renal fibrosis. However, the precise role of c-Abl in this process and its involvement in fibroblast-myofibroblast transition (FMT) remain poorly understood. METHODS: To investigate the effect of c-Abl in FMT during renal fibrosis, we investigated the expression of c-Abl in fibrotic renal tissues of patients with CKD and mouse models. We studied the phenotypic changes in fibroblast or myofibroblast-specific c-Abl conditional knockout mice. We explored the potential targets of c-Abl in NRK-49F fibroblasts. RESULTS: In this study, fibrotic mouse and cell models demonstrated that c-Abl deficiency in fibroblasts mitigated fibrosis by suppressing fibroblast activation, fibroblast-myofibroblast transition, and extracellular matrix deposition. Mechanistically, c-Abl maintains the stability of the RACK1 protein, which serves as a scaffold for proteins such as c-Abl and focal adhesion kinase at focal adhesions, driving fibroblast activation and differentiation during renal fibrosis. Moreover, specifically targeting c-Abl deletion in renal myofibroblasts could prove beneficial in established kidney fibrosis by reducing RACK1 expression and diminishing the extent of fibrosis. CONCLUSIONS: Our findings suggest that c-Abl plays a pathogenic role in interstitial fibrosis through the regulation of RACK1 protein stabilization and myofibroblast differentiation, suggesting a promising strategy for the treatment of CKD.

SMARCA4 (BRG1) activates ABCC3 transcription to promote hepatocellular carcinogenesis.

AIMS: Hepatocellular carcinoma (HCC) is a lead cause of cancer-related deaths. In the present study we investigated the role of Brahma-related gene 1 (BRG1), a chromatin remodeling protein, in HCC the pathogenesis focusing on identifying novel transcription targets. METHODS AND MATERIALS: Hepatocellular carcinogenesis was modeled in mice by diethylnitrosamine (DEN). Cellular transcriptome was evaluated by RNA-seq. RESULTS: Hepatocellular carcinoma was appreciably retarded in BRG1 knockout mice compared to wild type littermates. Transcriptomic analysis identified ATP Binding Cassette Subfamily C Member 3 (ABCC3) as a novel target of BRG1. BRG1 over-expression in BRG1low HCC cells (HEP1) up-regulated whereas BRG1 depletion in BRG1high HCC cells (SNU387) down-regulated ABCC3 expression. Importantly, BRG1 was detected to directly bind to the ABCC3 promoter to activate ABCC3 transcription. BRG1 over-expression in HEP1 cells promoted proliferation and migration, both of which were abrogated by ABCC3 silencing. On the contrary, BRG1 depletion in SNU387 cells decelerated proliferation and migration, both of which were rescued by ABCC3 over-expression. Importantly, high BRG1/ABCC3 expression predicted poor prognosis in HCC patients. Mechanistically, ABCC3 regulated hepatocellular carcinogenesis possibly by influencing lysosomal homeostasis. SIGNIFICANCE: In conclusion, our data suggest that targeting BRG1 and its downstream target ABCC3 can be considered as a reasonable approach for the intervention of hepatocellular carcinoma.

The insulin-like growth factor binding protein-microfibrillar associated protein-sterol regulatory element binding protein axis regulates fibroblast-myofibroblast transition and cardiac fibrosis.

BACKGROUND AND PURPOSE: Excessive fibrogenesis is associated with adverse cardiac remodelling and heart failure. The myofibroblast, primarily derived from resident fibroblast, is the effector cell type in cardiac fibrosis. Megakaryocytic leukaemia 1 (MKL1) is considered the master regulator of fibroblast-myofibroblast transition (FMyT). The underlying transcriptional mechanism is not completely understood. Our goal was to identify novel transcriptional targets of MKL1 that might regulate FMyT and contribute to cardiac fibrosis. EXPERIMENTAL APPROACH: RNA sequencing (RNA-seq) performed in primary cardiac fibroblasts identified insulin-like growth factor binding protein 5 (IGFBP5) as one of the genes most significantly up-regulated by constitutively active (CA) MKL1 over-expression. IGFBP5 expression was detected in heart failure tissues using RT-qPCR and western blots. KEY RESULTS: Once activated, IGFBP5 translocated to the nucleus to elicit a pro-FMyT transcriptional programme. Consistently, IGFBP5 knockdown blocked FMyT in vitro and dampened cardiac fibrosis in mice. Of interest, IGFBP5 interacted with nuclear factor of activated T-cell 4 (NFAT4) to stimulate the transcription of microfibril-associated protein 5 (MFAP5). MFAP5 contributed to FMyT and cardiac fibrosis by enabling sterol response element binding protein 2 (SREBP2)-dependent cholesterol synthesis. CONCLUSIONS AND IMPLICATIONS: Our data unveil a previously unrecognized transcriptional cascade, initiated by IGFBP5, that promotes FMyT and cardiac fibrosis. Screening for small-molecule compounds that target this axis could yield potential therapeutics against adverse cardiac remodelling.

DNA from macrophages induces fibrosis and vasculopathy through POLR3A/STING/type I interferon axis in systemic sclerosis.

OBJECTIVE: To clarify the role of RNA polymerase III A (POLR3A)/type I IFN in the pathogenesis of SSc. METHODS: Cytosolic DNA and stimulator of IFN genes (STING) pathway in skin or serum of SSc patients were detected by immunofluorescence, immunohistochemistry and western blotting. DNA from human macrophages was transfected to SSc fibroblasts or human umbilical vein endothelial cells (HUVECs) and then markers of POLR3A/STING pathway were detected by real-time qPCR, western blotting and confocal microscopy. After H151 treatment or knocking down POLR3A/STING, type I IFN response, monocytes adhesion and activation of fibroblasts and HUVECs were evaluated. Regulation of IFN regulatory factor 3 (IRF3) on monocyte chemoattractant protein-1 (MCP-1) was determined by chromatin immunoprecipitation. In bleomycin (BLM)-induced SSc mice, the effect of STING knockout or H151 on vasculopathy and fibrosis was assessed. RESULTS: Cytosolic DNA, colocalization of STING with alpha-smooth muscle actin (α-SMA) or CD31 in the skin, and STING pathway in the serum of SSc patients were increased. Macrophage-derived DNA stimulated the translocation of POLR3A from nucleus to the perinuclear region near STING and activated POLR3A/STING/type I IFN response, monocytes adhesion and MCP-1 expression in fibroblasts/HUVECs and collagen overproduction of fibroblasts. The activated IRF3 bound to the promoter of MCP-1. STING deficiency or H151 administration ameliorated fibrosis and vasculopathy both in vitro and in BLM-induced SSc mice. CONCLUSIONS: SSc presented increased DNA leakage and STING pathway activation. DNA from macrophages induced type I IFN signature of fibroblasts and ECs through POLR3A/STING pathway. Blocking POLR3A/STING axis provides a new therapeutic target for SSc.

BRG1 Mediates Nephronectin Activation in Hepatocytes to Promote T Lymphocyte Infiltration in ConA-Induced Hepatitis.

Fulminant hepatitis (FH) is a major cause of acute liver failure. Concanavalin A (ConA) belongs to the lectin family and is frequently used as an inducer of FH in animal models. ConA induced FH is characterized by massive accumulation of T lymphocytes in the liver. A host of chemoattractive substances are known to promote T cell homing to the liver during acute hepatitis. Here we investigated the involvement of Brahma-related gene 1 (BRG1), a chromatin remodeling protein, in FH. BRG1-flox mice were crossed to Alb-Cre mice to generate hepatocyte conditional BRG1 knockout (LKO) mice. The mice were peritoneally injected with a single dose of ConA to induce FH. BRG1 deficiency mitigated ConA-induced FH in mice. Consistently, there were fewer T lymphocyte infiltrates in the LKO livers compared to the wild type (WT) livers paralleling downregulation of T cell specific cytokines. Further analysis revealed that BRG1 deficiency repressed the expression of several chemokines critical for T cell homing including nephronectin (Npnt). BRG1 knockdown blocked the induction of Npnt in hepatocytes and attenuated T lymphocyte migration in vitro, which was reversed by the addition of recombinant nephronectin. Mechanistically, BRG1 interacted with ß-catenin to directly bind to the Npnt promoter and activate Npnt transcription. Importantly, a positive correlation between infiltration of CD3+ T lymphocyes and nephronectin expression was detected in human acute hepatitis biopsy specimens. In conclusion, our data identify a novel role for BRG1 as a promoter of T lymphocyte trafficking by activating Npnt transcription in hepatocytes. Targeting the BRG1-Npnt axis may yield novel therapeutic solutions for FH.

The chromatin remodeling protein BRG1 regulates APAP-induced liver injury by modulating CYP3A11 transcription in hepatocyte.

Acetaminophen (APAP) overdose represents the most frequent cause of acute liver failure. The underlying epigenetic mechanism is not fully understood. In the present study we investigated the mechanism whereby the chromatin remodeling protein brahma related gene 1 (Brg1) regulates APAP induced liver injury in mice. We report that hepatocyte-specific deletion of Brg1 attenuated APAP induced liver injury in mice as evidenced by reduced plasma ALT and AST levels, decreased liver necrosis, amelioration of GSH depletion, and prolonged survival. Brg1 regulated APAP-induced liver injury likely by stimulating the transcription of Cyp3a11, a key cytochrome enzyme involved in APAP metabolism. Immunoprecipitation coupled with DNA affinity microarray identified hepatocyte nuclear factor 4 (HNF4) as a novel binding partner for Brg1. HNF4 recruited Brg1 to the Cyp3a11 promoter and formed a complex with Brg1 to trans-activate Cyp3a11. In contrast, BRG1 deficiency attenuated HNF4 binding to the Cyp3a11 promoter and dampened Cyp3a11 transcription. Therefore, our data suggest that Brg1 might play an essential role mediating APAP induced liver injury in vivo.

Hepatic stellate cell-specific deletion of SIRT1 exacerbates liver fibrosis in mice.

Liver fibrosis is widely perceived as a host defense mechanism that aids tissue repair following liver injury. Excessive fibrogenesis, however, serves to disrupt normal liver structure and precedes such irrevocable human pathologies as cirrhosis and hepatocellular carcinoma. Activation of hepatic stellate cells (HSCs) is a hallmark event during liver fibrosis. In the present study we investigated the mechanism by which the lysine deacetylase SIRT1 regulates HSC activation. We report here that SIRT1 levels were decreased in the liver in different mouse models and in cultured HSCs undergoing activation. SIRT1 down-regulation paralleled HDAC4 up-regulation. HDAC4 was recruited to the SIRT1 promoter during HSC activation and removed acetylated histones H3 and H4 from the SIRT1 promoter leading to SIRT1 trans-repression. HDAC4 silencing restored SIRT1 expression and attenuated HSC activation in SIRT1-dependent manner. More important, selective deletion of SIRT1 in HSCs exacerbated CCl4-induced liver fibrosis in mice. Mechanistically, SIRT1 deacetylated PPARγ to block HSC activation. Together, our data reveal an HDAC4-SIRT1-PPARγ axis that contributes to the regulation of HSC activation and liver fibrosis.