Endothelial PERK-ATF4-JAG1 axis activated by T-ALL remodels bone marrow vascular niche.

The endoplasmic reticulum unfolded protein response (UPR) is a conserved adaptive signaling in ER homeostasis and has emerged as critical in highly proliferating cells and potential treatment target for acute T-cell lymphoblastic leukemia (T-ALL). Methods: in this study, we assessed the transcriptomic and phenotypic alterations in UPR response of the bone marrow endothelial cells (ECs) in mice engrafted with T-ALL and in bone marrow specimens from patients who have T-ALL. We used PERK inhibitor and generated endothelial specific PERK knockout mice to study the impact of PERK on leukemia progression and hematopoiesis. We performed chromatin immunoprecipitation (ChIP) to study the mechanistic regulation of JAG1 by ATF4. We characterized small extracellular vesicles (SEV) from leukemia-developing mice and studied the effect of SEVs on EC function. Results: we found that T-ALL development induced a robust activation of protein kinase RNA-like endoplasmic reticulum kinase (PERK)-dominant UPR in the bone marrow endothelial vascular niche. The activation of PERK-eIF2a-ATF4 axis remodels the vascular niche, upregulates angiogenic factors including VEGFα and ATF4-regulated JAG1, and suppresses the expression of SCF and CXCL12, which are important to HSC maintenance and regeneration. Further, targeting endothelial PERK significantly improved T-ALL outcome. EC-specific deletion of PERK abolished the aberrant JAG1 up-regulation, improved HSC maintenance, promoted leukemia apoptosis, and improved overall survival. Finally, we showed that small extracellular vesicles are critical mediators of endothelial PERK-eIF2a-ATF4 activation and JAG1 up-regulation in leukemia. Corroborating animal model studies, activation of PERK-ATF4-JAG1 is prominent in human T-ALL bone marrow and T-ALL xenografts. Conclusion: our studies thus revealed for the first time that the leukemia-initiated PERK-ATF4-JAG1 axis plays a critical role in the remodeling of the bone marrow vascular niche and that targeting vascular niche UPR is a potential therapeutic opportunity in T-ALL.

Targeting CRABP-II overcomes pancreatic cancer drug resistance by reversing lipid raft cholesterol accumulation and AKT survival signaling.

BACKGROUND: Resistance to standard therapy is a major reason for the poor prognosis of pancreatic ductal adenocarcinoma (PDAC). Developing novel therapy to overcome PDAC drug-resistance is urgently needed. CRABP-II was highly expressed in all PDAC but not expressed in normal pancreatic tissues and chronic pancreatitis. CRABP-II was shown to promote PDAC migration and metastasis while its potential role in promoting PDAC drug-resistance was not known. METHODS: A paired cohort of human primary and relapsing PDAC tissues was assessed for CRABP-II expression by immunohistochemistry. CRISPR/cas9 gene editing was used to establish CRABP-II knockout cell lines and MTT assays were performed to assess gemcitabine sensitivity in vitro. Cleaved caspase-3/PARP blots and Annexin V staining were conducted to detect cell apoptosis. Gene expression microarray, Q-PCR, western blots, Co-IP and RNA-IP were used to study the molecular function of CRABP-II. Sucrose gradient ultracentrifugation was applied to isolate lipid rafts and LC-MS-MS was used to assess cholesterol content. Both subcutaneous CDX models and orthotopic PDX models were established to examine the efficacy of SNIPER-11 and the synergistic effect between SNIPER-11 and gemcitabine in vivo. RESULTS: A higher expression of CRABP-II was found in relapsing PDAC tissue and was associated with poor prognosis. Gemcitabine-resistant cell lines exhibited increased level of CRABP-II, while CRABP-II knockout resensitized PDAC cells to gemcitabine. Mechanistically, aberrant expression of CRABP-II increased the stability of SREBP-1c mRNA through cooperation with HuR and upregulated the downstream genes of SREBP-1c to favor cholesterol uptake and accumulation in lipid rafts. Increased lipid raft cholesterol accumulation facilitated ATK survival signaling and PDAC drug resistance. The small compound SNIPER-11 treatment effectively induced CRABP-II protein degradation, induced apoptosis, and suppressed tumor growth. Combination of SNIPER-11 and gemcitabine significantly reduced the lipid raft cholesterol content in CDX/PDX and profoundly inhibited tumor progression. CONCLUSIONS: These findings identified CRABP-II as a novel regulator of cholesterol metabolism and suggested that CRABP-II is a selective target for overcoming PDAC drug resistance.

Vascular thrombosis and vasculitis in the gastrointestinal tract are associated with poor prognosis in patients with COVID-19.

AIM: To report pathologic findings in the gastrointestinal (GI) tract of coronavirus disease 2019 (COVID-19) patients. MATERIAL AND METHODS: we evaluated clinical and GI tract histologic findings in six COVID-19 patients that presented with GI symptoms like diarrhea, and abdominal pain. This study includes surgical resection specimens from five patients and two sets of biopsy specimens from one patient. RESULTS: Idiopathic inflammatory bowel disease was considered in three of six cases based on clinical, radiologic, and endoscopic presentation. Histologically, the enteric mucosa had a spectrum of histologic changes, including active enteritis, chronic active enteritis, and transmural necrosis. Extensive thrombi in vessels and/or vasculitis were identified in three out of the six cases. The presence of extensive vascular thrombi is associated with poor prognosis, and the three patients deceased in a short period of time (ranges from 7-67 days, median 14 days) after admission for GI symptoms. Severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) RNA was detected in bowel tissue of one case. The other three patients recovered and were discharged and free of GI symptoms (follow-up period ranges from 235 days to 270 days, median 237 days). CONCLUSION: COVID-19 associated enteritis may mimic Crohn's disease clinically, radiologically and endoscopically, and these two entities can be differentiated by pathologic findings. COVID-19 patients with GI symptoms may warrant a workup to evaluate for pathologic changes, as the presence of vasculitis and microthrombi may predict poor clinical outcome.

Notch-Regulated Dendritic Cells Restrain Inflammation-Associated Colorectal Carcinogenesis.

Conventional dendritic cells (cDC) play a central role in T-cell antitumor responses. We studied the significance of Notch-regulated DC immune responses in a mouse model of colitis-associated colorectal cancer in which there is epithelial downregulation of Notch/Hes1 signaling. This defect phenocopies that caused by GMDS (GDP-mannose 4,6-dehydratase) mutation in human colorectal cancers. We found that, although wild-type immune cells restrained dysplasia progression and decreased the incidence of adenocarcinoma in chimeric mice, the immune system with Notch2 deleted in all blood lineages or in only DCs promoted inflammation-associated transformation. Notch2 signaling deficiency not only impaired cDC terminal differentiation, but also downregulated CCR7 expression, reduced DC migration, and suppressed antigen cross-presentation to CD8+ T cells. Transfer of Notch-primed DCs restrained inflammation-associated dysplasia progression. Consistent with the mouse data, we observed a correlation between infiltrating cDC1 and Notch2 signaling in human colorectal cancers and found that GMDS-mutant colorectal cancers showed decreased CCR7 expression and suppressed cDC1 signature gene expression. Suppressed cDC1 gene signature expression in human colorectal cancer was associated with a poor prognosis. In summary, our study supports an important role for Notch2 signaling in cDC1-mediated antitumor immunity and indicates that Notch2-controlled DCs restrain inflammation-associated colon cancer development in mice.

MUPP1 complexes renal K+ channels to alter cell surface expression and whole cell currents.

We previously found that the Ca(2+)-sensing receptor (CaR) interacts with and inactivates the inwardly rectifying K(+) channel Kir4.2 that is expressed in the kidney cortex and that has a COOH-terminal PDZ domain. To identify potential scaffolding proteins that could organize a macromolecular signaling complex involving the CaR and Kir4.2, we used yeast two-hybrid cloning with the COOH-terminal 125 amino acids (AA) of Kir4.2 as bait to screen a human kidney cDNA library. We identified two independent partial cDNAs corresponding to the COOH-terminal 900 AA of MUPP1, a protein containing 13 PDZ binding domains that is expressed in the kidney in tight junctions and lateral borders of epithelial cells. When expressed in human embryonic kidney (HEK)-293 cells, Kir4.2 coimmunoprecipitates reciprocally with MUPP1 but not with a Kir4.2 construct lacking the four COOH-terminal amino acids, Kir5.1, or the CaR. MUPP1 and Kir4.2 coimmunoprecipitate reciprocally from rat kidney cortex extracts. Coexpression of MUPP1 with Kir4.2 in HEK-293 cells leads to reduced cell surface expression of Kir4.2 as assessed by cell surface biotinylation. Coexpression of MUPP1 and Kir4.2 in Xenopus oocytes results in reduced whole cell currents compared with expression of Kir4.2 alone, whereas expression of Kir4.2DeltaPDZ results in minimal currents and is not affected by coexpression with MUPP1. Immunofluorescence studies of oocytes demonstrate that MUPP1 reduces Kir4.2 membrane localization. These results indicate that Kir4.2 interacts selectively with MUPP1 to affect its cell surface expression. Thus MUPP1 and Kir4.2 may participate in a protein complex in the nephron that could regulate transport of K(+) as well as other ions.

Inhibition of Nox-4 activity by plumbagin, a plant-derived bioactive naphthoquinone.

NAD(P)H oxidase contributes to the pathogenesis of cancer and cardiovascular diseases such as hypertension, atherosclerosis, restenosis, cardiac hypertrophy and heart failure. Plumbagin, a plant-derived naphthoquinone, has been shown to exert anticarcinogenic and anti-atherosclerosis effects in animals. However, the molecular mechanisms underlying these effects remain unknown. It is possible that the beneficial effect of plumbagin is due to the inhibition of NAD(P)H oxidase. Human embryonic kidney 293 (HEK293) and brain tumour LN229 cells express mainly Nox-4, a renal NAD(P)H oxidase. We have examined the effect of plumbagin on Nox-4 activity in HEK293 and LN229 cells using lucigenin-dependent chemiluminescence assay. Plumbagin inhibited the activity of Nox-4 in a time- and dose-dependent manner in HEK293 and LN229 cells. Production of superoxide in HEK293 cells was inhibited by diphenyleneiodonium (DPI), a NAD(P)H oxidase inhibitor. The superoxide production in HEK293 cells was NADPH- and NADH-dependent indicating that the superoxide was generated by a NAD(P)H oxidase in HEK293 cells, but not by the redox-cycling of lucigenin. Furthermore, plumbagin inhibited the superoxide production in Nox-4 transfected COS-7 cells. These results indicated that plumbagin directly interacted with Nox-4 and inhibited its activity.

Cyclophilin A functions as an endogenous inhibitor for membrane-bound guanylate cyclase-A.

Cyclophilin A (CypA), a receptor for the immunosuppressive agent cyclosporin A, is a cis-trans-peptidyl-prolyl isomerase (PPIase). It accelerates the cis-trans isomerization of prolyl-peptide bonds. CypA binds and regulates the activity of a variety of proteins. Atrial natriuretic factor (ANF) and its receptor membrane-bound guanylate cyclase-A (GC-A) are involved in the regulation of blood pressure. We examined whether CypA affects the activation of GC-A by ANF. The results showed that CypA associated with GC-A. Interestingly, binding of ANF to GC-A released CypA. Transfection of CypA inhibited ANF-stimulated GC-A activity, indicating that CypA functions as an endogenous inhibitor for GC-A activation. CypA also inhibits the activity of guanylate cyclase-C (GC-c), the catalytic domain of GC-A, indicating that CypA interacts with the catalytic domain of GC-A. In contrast, transfection of CypA R55A, a CypA mutant expressing low PPIase activity, did not significantly attenuate the activity of GC-c and the activation of GC-A. Inhibition of PPIase activity of CypA with cyclosporin A also blocks the inhibitory effect of CypA on GC-c activity. These results demonstrate that PPIase activity is required for CypA to inhibit GC-c activity and GC-A activation by ANF. Furthermore, mutation of Pro 822, 902, or 958 in GC-c abolished its activity. Therefore, it is likely that CypA binds to GC-A and catalyzes the cis-trans isomerization of Pro 822, 902, or 958, which keeps GC-A in the inactive state, and that binding of ANF to GC-A alters the conformation of the catalytic domain that releases CypA from GC-A leading to enzyme activation.

Cyclosporin A disrupts bradykinin signaling through superoxide.

Cyclosporin A (CsA) is used to reduce transplant rejection rates. Chronic use, however, has a destructive toxic effect on the kidney, resulting in hypertension. In this study, we investigated the effects of CsA treatment on the bradykinin/soluble guanylate cyclase signaling cascade and the involvement of superoxide in LLC-PK1 porcine kidney proximal tubule cells. Treatment with 1 micromol/L CsA for 24 hours increased basal cGMP levels by 41%, whereas CsA inhibited bradykinin-stimulated cGMP production by 26%. Western blotting showed increased expression of eNOS, but no other protein in the bradykinin/soluble guanylate cyclase (sGC) pathway was affected. Using lucigenin-dependent chemiluminescence, we found that CsA treatment significantly increased superoxide production. Production of O2- was not significantly reduced by 10 micromol/L oxypurinol or 30 micromol/L ketoconazole. However, it was inhibited by the NADPH oxidase inhibitor diphenyleneiodonium chloride (10 micromol/L) as well as the O2- scavenger superoxide dismutase (SOD) (100 U). On treatment with 50 micromol/L quercetin, 10 mmol/L N-acetyl-cysteine, both antioxidants, as well as the O2- scavenger Tiron (10 mmol/L), concomitant with 1 micromol/L CsA for 24 hours the activation of cGMP production, was restored in combination with a reduction in O2-. Incubation with 100 micromol/L menadione, a reactive oxygen generator, and 10 nmol/L bradykinin showed similar effects on the level of cGMP as with CsA. CsA treatment was found to increase nitrotyrosine levels. These findings suggest that CsA activates a NADPH oxidase that releases O2- and disrupts the bradykinin/soluble guanylate cyclase pathway, probably by binding with NO to form peroxynitrite (ONOO-).

The bradykinin/soluble guanylate cyclase signaling pathway is impaired in androgen-independent prostate cancer cells.

The activation of soluble guanylate cyclase by bradykinin and sodium nitroprusside (SNP), a direct activator of soluble guanylate cyclase, was evaluated in androgen-sensitive LNCaP and androgen-independent PC3 and DU145 prostate cancer cells. Bradykinin and SNP activated soluble guanylate cyclase in LNCaP cells, but not in PC3 and DU145 cells. Western blot analysis revealed that the bradykinin B2 receptor, Gqalpha, phospholipase Cgamma and endothelial nitric oxide synthase were expressed in LNCaP, PC3 and DU145 cells. However, both Western blotting and reverse transcriptase--polymerase chain reaction indicated that soluble guanylate cyclase was only expressed in LNCaP cells. These results demonstrate that the impaired bradykinin-soluble guanylate cyclase pathway in PC3 and DU145 cells is likely due to lack of expression of soluble guanylate cyclase.