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1.
Am J Pathol ; 193(10): 1501-1516, 2023 10.
Article in English | MEDLINE | ID: mdl-37676196

ABSTRACT

Chronic kidney disease (CKD) is characterized by the accumulation of uremic toxins and renal tubular damage. Tryptophan-derived uremic toxins [indoxyl sulfate (IS) and kynurenine (Kyn)] are well-characterized tubulotoxins. Emerging evidence suggests that transmembrane and immunoglobulin domain-containing 1 (TMIGD1) protects tubular cells and promotes survival. However, the direct molecular mechanism(s) underlying how these two opposing pathways crosstalk remains unknown. We posited that IS and Kyn mediate tubular toxicity through TMIGD1 and the loss of TMIGD1 augments tubular injury. Results from the current study showed that IS and Kyn suppressed TMIGD1 transcription in tubular cells in a dose-dependent manner. The wild-type CCAAT enhancer-binding protein ß (C/EBPß) enhanced, whereas a dominant-negative C/EBPß suppressed, TMIGD1 promoter activity. IS down-regulated C/EBPß in primary human renal tubular cells. The adenine-induced CKD, unilateral ureteric obstruction, and deoxycorticosterone acetate salt unilateral nephrectomy models showed reduced TMIGD1 expression in the renal tubules, which correlated with C/EBPß expression. C/EBPß levels negatively correlated with the IS and Kyn levels. Inactivation of TMIGD1 in mice significantly lowered acetylated tubulin, decreased tubular cell proliferation, caused severe tubular damage, and worsened renal function. Thus, the current results demonstrate that TMIGD1 protects renal tubular cells from renal injury in different models of CKD and uncovers a novel mechanism of tubulotoxicity of tryptophan-based uremic toxins.


Subject(s)
Renal Insufficiency, Chronic , Tryptophan , Humans , Animals , Mice , Uremic Toxins , Kidney/physiology , Immunoglobulin Domains , Membrane Glycoproteins
2.
Am J Pathol ; 192(8): 1167-1185, 2022 08.
Article in English | MEDLINE | ID: mdl-35710032

ABSTRACT

Aberrant hyperactivation of Wnt signaling, driven by nuclear ß-catenin in the colonic epithelium, represents the seminal event in the initiation and progression of colorectal cancer (CRC). Despite its established role in CRC tumorigenesis, clinical translation of Wnt inhibitors remains unsuccessful. Late SV40 factor (LSF; encoded by TFCP2) is a transcription factor and a potent oncogene. The current study identified a chemotype, named factor quinolinone inhibitors (FQIs), that specifically inhibits LSF DNA-binding, partner protein-binding, and transactivation activities. The role of LSF and FQIs in CRC tumor growth was examined. Herein, the study showed that LSF and ß-catenin interacted in several CRC cell lines irrespective of their mutational profile, which was disrupted by FQI2-34. FQI2-34 suppressed Wnt activity in CRC cells in a dose-dependent manner. Leveraging both allogeneic and syngeneic xenograft models showed that FQI2-34 suppressed CRC tumor growth, significantly reduced nuclear ß-catenin, and down-regulated Wnt targets such as axis inhibition protein 2 (AXIN-2) and SRY-box transcription factor 9, in the xenograft cells. FQI2-34 suppressed the proliferation of xenograft cells. Adenocarcinomas from a series of stage IV CRC patients revealed a positive correlation between LSF expression and Wnt targets (AXIN-2 and SRY-box transcription factor 9) within the CRC cells. Collectively, this study uncovers the Wnt inhibitory and CRC growth-suppressive effects of these LSF inhibitors in CRC cells, revealing a novel target in CRC therapeutics.


Subject(s)
Colonic Neoplasms , Colorectal Neoplasms , Hematopoietic Stem Cell Transplantation , Axin Protein/metabolism , Cell Line, Tumor , Cell Proliferation , Colonic Neoplasms/drug therapy , Colorectal Neoplasms/pathology , DNA-Binding Proteins/metabolism , Gene Expression Regulation, Neoplastic , Heterografts , Humans , Transcription Factors/metabolism , Wnt Signaling Pathway , beta Catenin/metabolism
3.
Proc Natl Acad Sci U S A ; 119(6)2022 02 08.
Article in English | MEDLINE | ID: mdl-35078919

ABSTRACT

SARS-CoV-2 entry into host cells is a crucial step for virus tropism, transmission, and pathogenesis. Angiotensin-converting enzyme 2 (ACE2) has been identified as the primary entry receptor for SARS-CoV-2; however, the possible involvement of other cellular components in the viral entry has not yet been fully elucidated. Here we describe the identification of vimentin (VIM), an intermediate filament protein widely expressed in cells of mesenchymal origin, as an important attachment factor for SARS-CoV-2 on human endothelial cells. Using liquid chromatography-tandem mass spectrometry, we identified VIM as a protein that binds to the SARS-CoV-2 spike (S) protein. We showed that the S-protein receptor binding domain (RBD) is sufficient for S-protein interaction with VIM. Further analysis revealed that extracellular VIM binds to SARS-CoV-2 S-protein and facilitates SARS-CoV-2 infection, as determined by entry assays performed with pseudotyped viruses expressing S and with infectious SARS-CoV-2. Coexpression of VIM with ACE2 increased SARS-CoV-2 entry in HEK-293 cells, and shRNA-mediated knockdown of VIM significantly reduced SARS-CoV-2 infection of human endothelial cells. Moreover, incubation of A549 cells expressing ACE2 with purified VIM increased pseudotyped SARS-CoV-2-S entry. CR3022 antibody, which recognizes a distinct epitope on SARS-CoV-2-S-RBD without interfering with the binding of the spike with ACE2, inhibited the binding of VIM with CoV-2 S-RBD, and neutralized viral entry in human endothelial cells, suggesting a key role for VIM in SARS-CoV-2 infection of endothelial cells. This work provides insight into the pathogenesis of COVID-19 linked to the vascular system, with implications for the development of therapeutics and vaccines.


Subject(s)
Endothelial Cells/virology , Extracellular Space/metabolism , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/metabolism , Vimentin/metabolism , Virus Internalization , A549 Cells , Angiotensin-Converting Enzyme 2/metabolism , Coculture Techniques , Endothelium, Vascular/cytology , Endothelium, Vascular/metabolism , Endothelium, Vascular/virology , HEK293 Cells , Humans , Protein Binding
4.
J Am Soc Nephrol ; 32(11): 2834-2850, 2021 11.
Article in English | MEDLINE | ID: mdl-34716244

ABSTRACT

BACKGROUND: CKD, characterized by retained uremic solutes, is a strong and independent risk factor for thrombosis after vascular procedures . Urem ic solutes such as indoxyl sulfate (IS) and kynurenine (Kyn) mediate prothrombotic effect through tissue factor (TF). IS and Kyn biogenesis depends on multiple enzymes, with therapeutic implications unexplored. We examined the role of indoleamine 2,3-dioxygenase-1 (IDO-1), a rate-limiting enzyme of kynurenine biogenesis, in CKD-associated thrombosis after vascular injury. METHODS: IDO-1 expression in mice and human vessels was examined. IDO-1-/- mice, IDO-1 inhibitors, an adenine-induced CKD, and carotid artery injury models were used. RESULTS: Both global IDO-1-/- CKD mice and IDO-1 inhibitor in wild-type CKD mice showed reduced blood Kyn levels, TF expression in their arteries, and thrombogenicity compared with respective controls. Several advanced IDO-1 inhibitors downregulated TF expression in primary human aortic vascular smooth muscle cells specifically in response to uremic serum. Further mechanistic probing of arteries from an IS-specific mouse model, and CKD mice, showed upregulation of IDO-1 protein, which was due to inhibition of its polyubiquitination and degradation by IS in vascular smooth muscle cells. In two cohorts of patients with advanced CKD, blood IDO-1 activity was significantly higher in sera of study participants who subsequently developed thrombosis after endovascular interventions or vascular surgery. CONCLUSION: Leveraging genetic and pharmacologic manipulation in experimental models and data from human studies implicate IS as an inducer of IDO-1 and a perpetuator of the thrombotic milieu and supports IDO-1 as an antithrombotic target in CKD.


Subject(s)
Indican/physiology , Indoleamine-Pyrrole 2,3,-Dioxygenase/antagonists & inhibitors , Indoleamine-Pyrrole 2,3,-Dioxygenase/blood , Kynurenine/physiology , Molecular Targeted Therapy , Postoperative Complications/enzymology , Renal Insufficiency, Chronic/enzymology , Thrombosis/enzymology , Vascular Surgical Procedures/adverse effects , Animals , Aorta , Carotid Artery Injuries/complications , Carotid Artery Thrombosis/etiology , Carotid Artery Thrombosis/prevention & control , Culture Media/pharmacology , Enzyme Induction/drug effects , Feedback, Physiological , Female , HEK293 Cells , Humans , Indoleamine-Pyrrole 2,3,-Dioxygenase/deficiency , Indoleamine-Pyrrole 2,3,-Dioxygenase/genetics , Kynurenine/blood , Mice , Mice, Inbred C57BL , Mice, Knockout , Myocytes, Smooth Muscle/drug effects , Postoperative Complications/blood , Postoperative Complications/etiology , Postoperative Complications/prevention & control , Renal Insufficiency, Chronic/drug therapy , Thromboplastin/metabolism , Thrombosis/blood , Thrombosis/etiology , Thrombosis/prevention & control , Tryptophan/metabolism , Uremia/blood
5.
ACS Cent Sci ; 7(7): 1156-1165, 2021 Jul 28.
Article in English | MEDLINE | ID: mdl-34341769

ABSTRACT

As the COVID-19 pandemic continues to spread, investigating the processes underlying the interactions between SARS-CoV-2 and its hosts is of high importance. Here, we report the identification of CD209L/L-SIGN and the related protein CD209/DC-SIGN as receptors capable of mediating SARS-CoV-2 entry into human cells. Immunofluorescence staining of human tissues revealed prominent expression of CD209L in the lung and kidney epithelia and endothelia. Multiple biochemical assays using a purified recombinant SARS-CoV-2 spike receptor-binding domain (S-RBD) or S1 encompassing both N termal domain and RBD and ectopically expressed CD209L and CD209 revealed that CD209L and CD209 interact with S-RBD. CD209L contains two N-glycosylation sequons, at sites N92 and N361, but we determined that only site N92 is occupied. Removal of the N-glycosylation at this site enhances the binding of S-RBD with CD209L. CD209L also interacts with ACE2, suggesting a role for heterodimerization of CD209L and ACE2 in SARS-CoV-2 entry and infection in cell types where both are present. Furthermore, we demonstrate that human endothelial cells are permissive to SARS-CoV-2 infection, and interference with CD209L activity by a knockdown strategy or with soluble CD209L inhibits virus entry. Our observations demonstrate that CD209L and CD209 serve as alternative receptors for SARS-CoV-2 in disease-relevant cell types, including the vascular system. This property is particularly important in tissues where ACE2 has low expression or is absent and may have implications for antiviral drug development.

6.
bioRxiv ; 2021 Jun 14.
Article in English | MEDLINE | ID: mdl-32607506

ABSTRACT

As the COVID-19 pandemic continues to spread, investigating the processes underlying the interactions between SARS-CoV-2 and its hosts is of high importance. Here, we report the identification of CD209L/L-SIGN and the related protein CD209/DC-SIGN as receptors capable of mediating SARS-CoV-2 entry into human cells. Immunofluorescence staining of human tissues revealed prominent expression of CD209L in the lung and kidney epithelium and endothelium. Multiple biochemical assays using a purified recombinant SARS-CoV-2 spike receptor binding domain (S-RBD) or S1 encompassing both NTB and RBD and ectopically expressed CD209L and CD209 revealed that CD209L and CD209 interact with S-RBD. CD209L contains two N-glycosylation sequons, at sites N92 and N361, but we determined that only site N92 is occupied. Removal of the N-glycosylation at this site enhances the binding of S-RBD with CD209L. CD209L also interacts with ACE2, suggesting a role for heterodimerization of CD209L and ACE2 in SARS-CoV-2 entry and infection in cell types where both are present. Furthermore, we demonstrate that human endothelial cells are permissive to SARS-CoV-2 infection and interference with CD209L activity by knockdown strategy or with soluble CD209L inhibits virus entry. Our observations demonstrate that CD209L and CD209 serve as alternative receptors for SARS-CoV-2 in disease-relevant cell types, including the vascular system. This property is particularly important in tissues where ACE2 has low expression or is absent, and may have implications for antiviral drug development.

7.
Am J Pathol ; 190(3): 602-613, 2020 03.
Article in English | MEDLINE | ID: mdl-32113662

ABSTRACT

Casitas B-lineage lymphoma (c-Cbl) is a recently identified ubiquitin ligase of nuclear ß-catenin and a suppressor of colorectal cancer (CRC) growth in cell culture and mouse tumor xenografts. We hypothesized that reduction in c-Cbl in colonic epithelium is likely to increase the levels of nuclear ß-catenin in the intestinal crypt, augmenting CRC tumorigenesis in an adenomatous polyposis coli (APCΔ14/+) mouse model. Haploinsufficient c-Cbl mice (APCΔ14/+ c-Cbl+/-) displayed a significant (threefold) increase in atypical hyperplasia and adenocarcinomas in the small and large intestines; however, no differences were noted in the adenoma frequency. In contrast to the APCΔ14/+ c-Cbl+/+ mice, APCΔ14/+ c-Cbl+/- crypts showed nuclear ß-catenin throughout the length of the crypts and up-regulation of Axin2, a canonical Wnt target gene, and SRY-box transcription factor 9, a marker of intestinal stem cells. In contrast, haploinsufficiency of c-Cbl+/- alone was insufficient to induce tumorigenesis regardless of an increase in the number of intestinal epithelial cells with nuclear ß-catenin and SRY-box transcription factor 9 in APC+/+ c-Cbl+/- mice. This study demonstrates that haploinsufficiency of c-Cbl results in Wnt hyperactivation in intestinal crypts and accelerates CRC progression to adenocarcinoma in the milieu of APCΔ14/+, a phenomenon not found with wild-type APC. While emphasizing the role of APC as a gatekeeper in CRC, this study also demonstrates that combined partial loss of c-Cbl and inactivation of APC significantly contribute to CRC tumorigenesis.


Subject(s)
Adenocarcinoma/genetics , Adenomatous Polyposis Coli Protein/metabolism , Colonic Neoplasms/genetics , Colorectal Neoplasms/genetics , Haploinsufficiency , Lymphoma/genetics , Proto-Oncogene Proteins c-cbl/metabolism , Adenocarcinoma/pathology , Adenomatous Polyposis Coli Protein/genetics , Animals , Carcinogenesis , Colonic Neoplasms/pathology , Colorectal Neoplasms/pathology , Disease Progression , Female , Gene Expression Regulation, Neoplastic , Lymphoma/pathology , Male , Mice , Mice, Inbred C57BL , Proto-Oncogene Proteins c-cbl/genetics , Wnt Proteins/genetics , Wnt Proteins/metabolism , beta Catenin/genetics , beta Catenin/metabolism
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