Abstract:Keloids represent one extreme of aberrant dermal wound healing. One of the important characteristics of keloids is uncontrolled fibroblasts proliferation. However, the mechanism of excessive proliferation of fibroblasts in keloids remains elusive. In this study, we demonstrated that TRAF4 was highly expressed in keloid fibroblasts and promoted fibroproliferation. We investigated the underlying molecular mechanism and found that TRAF4 suppressed the p53 pathway independent of its E3 ubiquitin ligase activity. S… Show more
“… 65 Germane to this model is a recent study in which USP10/TRAF4 binding induced p53 ubiquitination and cytosolic degradation (similar to the USP10/G3BP interaction), leading to a fibroproliferative response and keloid formation. 66 Thus, we suggest that the switching of USP10 functions from pro-apoptotic to anti-apoptotic is context-dependent and depends on 3D environmental cues in the wound bed. Figure 8 Working Model for Divergent Roles of USP10 as Wound Healing/Scarring Progresses Left panel: immediately following a corneal stromal injury, resident keratocytes adjacent to the wound undergo apoptosis.…”
Section: Discussionmentioning
confidence: 82%
“…65 Germane to this model is a recent study in which USP10/TRAF4 binding induced p53 ubiquitination and cytosolic degradation (similar to the USP10/G3BP interaction), leading to a fibroproliferative response and keloid formation. 66 Thus, we suggest that the switching of USP10 functions from pro-apoptotic to anti-apoptotic is contextdependent and depends on 3D environmental cues in the wound bed.…”
Section: Discussionmentioning
confidence: 83%
“…This model is supported by data in prostate cancer cells and in keloid scars, in which USP10 switches from a pro-apoptotic role in the nucleus to binding to stress-related proteins in the cytoplasm. 64 , 66 Other studies have demonstrated a role for G3BP1/2 in regulating integrin signaling molecules (Src, FAK, and ERK). 65 Taken together, we predict that in the early stages of wounding, USP10 promotes apoptosis and subsequent immune cell infiltration, while in the later stages of wound healing (scar formation), USP10 is directed by its binding partners to promote myofibroblast survival (inhibition of apoptosis) and differentiation (α v integrin upregulation, enhanced cellular adhesion/contractility).…”
Ocular scarring after surgery, trauma, or infection leads to vision loss. The transparent cornea is an excellent model system to test anti-scarring therapies. Cholesterol-conjugated fully modified asymmetric small interfering RNAs (siRNAs) (selfdeliverable siRNAs [sdRNAs]) are a novel modality for in vivo gene knockdown, transfecting cells and tissues without any additional formulations. Myofibroblasts are a main contributor to scarring and fibrosis. a v integrins play a central role in myofibroblast pathological adhesion, overcontraction, and transforming growth factor b (TGF-b) activation. Previously, we demonstrated that a v integrins are protected from intracellular degradation after wounding by upregulation of the deubiquitinase (DUB) ubiquitin-specific protease 10 (USP10), leading to integrin cell surface accumulation. In this study, we tested whether knockdown of USP10 with a USP10-targeting sdRNA (termed US09) will reduce scarring after wounding a rabbit cornea in vivo. The wounded corneal stroma was treated once with US09 or non-targeting control (NTC) sdRNA. At 6 weeks US09 treatment resulted in faster wound closure, limited scarring, and suppression of fibrotic markers and immune response. Specifically, fibronectin-extra domain A (EDA), collagen III, and a-smooth muscle actin (p < 0.05), CD45 + cell infiltration (p < 0.01), and apoptosis at 24 (p < 0.01) and 48 h (p < 0.05) were reduced post-wounding. Corneal thickness and cell proliferation were restored to unwounded parameters. Targeting the DUB, USP10 is a novel strategy to reduce scarring. This study indicates that ubiquitin-mediated pathways should be considered in the pathogenesis of fibrotic healing.
“… 65 Germane to this model is a recent study in which USP10/TRAF4 binding induced p53 ubiquitination and cytosolic degradation (similar to the USP10/G3BP interaction), leading to a fibroproliferative response and keloid formation. 66 Thus, we suggest that the switching of USP10 functions from pro-apoptotic to anti-apoptotic is context-dependent and depends on 3D environmental cues in the wound bed. Figure 8 Working Model for Divergent Roles of USP10 as Wound Healing/Scarring Progresses Left panel: immediately following a corneal stromal injury, resident keratocytes adjacent to the wound undergo apoptosis.…”
Section: Discussionmentioning
confidence: 82%
“…65 Germane to this model is a recent study in which USP10/TRAF4 binding induced p53 ubiquitination and cytosolic degradation (similar to the USP10/G3BP interaction), leading to a fibroproliferative response and keloid formation. 66 Thus, we suggest that the switching of USP10 functions from pro-apoptotic to anti-apoptotic is contextdependent and depends on 3D environmental cues in the wound bed.…”
Section: Discussionmentioning
confidence: 83%
“…This model is supported by data in prostate cancer cells and in keloid scars, in which USP10 switches from a pro-apoptotic role in the nucleus to binding to stress-related proteins in the cytoplasm. 64 , 66 Other studies have demonstrated a role for G3BP1/2 in regulating integrin signaling molecules (Src, FAK, and ERK). 65 Taken together, we predict that in the early stages of wounding, USP10 promotes apoptosis and subsequent immune cell infiltration, while in the later stages of wound healing (scar formation), USP10 is directed by its binding partners to promote myofibroblast survival (inhibition of apoptosis) and differentiation (α v integrin upregulation, enhanced cellular adhesion/contractility).…”
Ocular scarring after surgery, trauma, or infection leads to vision loss. The transparent cornea is an excellent model system to test anti-scarring therapies. Cholesterol-conjugated fully modified asymmetric small interfering RNAs (siRNAs) (selfdeliverable siRNAs [sdRNAs]) are a novel modality for in vivo gene knockdown, transfecting cells and tissues without any additional formulations. Myofibroblasts are a main contributor to scarring and fibrosis. a v integrins play a central role in myofibroblast pathological adhesion, overcontraction, and transforming growth factor b (TGF-b) activation. Previously, we demonstrated that a v integrins are protected from intracellular degradation after wounding by upregulation of the deubiquitinase (DUB) ubiquitin-specific protease 10 (USP10), leading to integrin cell surface accumulation. In this study, we tested whether knockdown of USP10 with a USP10-targeting sdRNA (termed US09) will reduce scarring after wounding a rabbit cornea in vivo. The wounded corneal stroma was treated once with US09 or non-targeting control (NTC) sdRNA. At 6 weeks US09 treatment resulted in faster wound closure, limited scarring, and suppression of fibrotic markers and immune response. Specifically, fibronectin-extra domain A (EDA), collagen III, and a-smooth muscle actin (p < 0.05), CD45 + cell infiltration (p < 0.01), and apoptosis at 24 (p < 0.01) and 48 h (p < 0.05) were reduced post-wounding. Corneal thickness and cell proliferation were restored to unwounded parameters. Targeting the DUB, USP10 is a novel strategy to reduce scarring. This study indicates that ubiquitin-mediated pathways should be considered in the pathogenesis of fibrotic healing.
“…Keloid scarring is an aggressive skin disease with unclear pathogenesis. It is characterized by excessive fibroblast proliferation and collagen accumulation in the ECM that does not regress over time [ 31 ]. Keloid scars are an ongoing clinical challenge with no single effective treatment regimen.…”
As a part of an abnormal healing process of dermal injuries and irritation, keloid scars arise on the skin as benign fibroproliferative tumors. Although the etiology of keloid scarring remains unsettled, considerable recent evidence suggested that keloidogenesis may be driven by epigenetic changes, particularly, DNA methylation. Therefore, genome-wide scanning of methylated cytosine-phosphoguanine (CpG) sites in extracted DNA from 12 keloid scar fibroblasts (KF) and 12 control skin fibroblasts (CF) (six normal skin fibroblasts and six normotrophic fibroblasts) was conducted using the Illumina Human Methylation 450K BeadChip in two replicates for each sample. Comparing KF and CF used a Linear Models for Microarray Data (Limma) model revealed 100,000 differentially methylated (DM) CpG sites, 20,695 of which were found to be hypomethylated and 79,305 were hypermethylated. The top DM CpG sites were associated with TNKS2, FAM45B, LOC723972, GAS7, RHBDD2 and CAMKK1. Subsequently, the most functionally enriched genes with the top 100 DM CpG sites were significantly (p ≤ 0.05) associated with SH2 domain binding, regulation of transcription, DNA-templated, nucleus, positive regulation of protein targeting to mitochondrion, nucleoplasm, Swr1 complex, histone exchange, and cellular response to organic substance. In addition, NLK, CAMKK1, LPAR2, CASP1, and NHS showed to be the most common regulators in the signaling network analysis. Taken together, these findings shed light on the methylation status of keloids that could be implicated in the underlying mechanism of keloid scars formation and remission.
“…Western blotting was performed according to previously described standard protocols with minor alteration ( 36 ). Briefly, cells of iKA-CRISPR clone #17 and MCF cells transfected with Cas9, Cas9-SB, Cas9-N123 or Cas9-N57 were lysed, boiled for 10 min at 95°C, separated on a 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis gel, transferred to a nitrocellulose membrane and detected with sequential blotting with primary antibodies against GAPDH (6004–1-Ig, Proteintech), Cas9 (ab191468, Abcam), Flag tag (66008–3, Proteintech) and anti-mouse IgG HRP secondary antibody (7076S, Cell Signaling Technology).…”
The CRISPR/Cas system is widely used for genome editing. However, robust and targeted insertion of a DNA segment remains a challenge. Here, we present a fusion nuclease (Cas9-N57) to enhance site-specific DNA integration via a fused DNA binding domain of Sleeping Beauty transposase to tether the DNA segment to the Cas9/sgRNA complex. The insertion was unidirectional and specific, and DNA fragments up to 12 kb in length were successfully integrated. As a test of the system, Cas9-N57 mediated the insertion of a CD19-specific chimeric antigen receptor (CD19-CAR) cassette into the AAVS1 locus in human T cells, and induced intrahepatic cholangiocarcinoma in mice by simultaneously mediating the insertion of oncogenic KrasG12D into the Rosa26 locus and disrupting Trp53 and Pten. Moreover, the nuclease-N57 fusion proteins based on AsCpf1 (AsCas12a) and CjCas9 exhibited similar activity. These findings demonstrate that CRISPR-associated nuclease-N57 protein fusion is a powerful tool for targeted DNA insertion and holds great potential for gene therapy applications.
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