Knockdown of CXCL1 improves ACLF by reducing neutrophil recruitment to attenuate ROS production and hepatocyte apoptosis.

BACKGROUND: Acute-on-chronic liver failure (ACLF) is an acute decompensated syndrome based on chronic liver disease, while neutrophil recruitment is the most critical early step. C-X-C motif chemokine ligand 1 (CXCL1), a cytokine that recruits neutrophils, was significantly upregulated in both ACLF mice and patients with ACLF. This present study aims to explore the role of CXCL1 in the pathogenesis of ACLF. METHODS: We established an ACLF mouse model induced by carbon tetrachloride, lipopolysaccharide, and D-galactosamine, and used adeno-associated virus to achieve overexpression and knockdown of Cxcl1. We employed mass cytometry, flow cytometry, multiplex cytokine and chemokine analysis, Western blot, and reactive oxygen species (ROS) detection in mice blood and liver. ACLF patients (n = 10) and healthy controls (n = 5) were included, and their liver samples were stained using multiplex immunohistochemistry techniques. RESULTS: CXCL1 was significantly elevated in both ACLF mice and patients. CXCL1 recruits neutrophils by binding to the C-X-C motif chemokine receptor 2 on the surface of neutrophils, affects ACLF prognosis by generating ROS and mitochondrial depolarization and modulating caspase3-related apoptotic pathways. We found that the knockdown of CXCL1 attenuated the infiltration of neutrophils in the mouse liver, reduced the expression of inflammatory cytokines, and also significantly downregulated ROS production and caspase3-related hepatocyte apoptosis, thereby ameliorating the liver injury of ACLF. CONCLUSIONS: CXCL1 is a core player in the mobilization of neutrophils in ACLF, and the knockdown of Cxcl1 improves neutrophil infiltration, reduces ROS levels, and reduces hepatocyte apoptosis, thereby attenuating inflammation and liver injury in ACLF. Our results revealed a previously unknown link between CXCL1-induced neutrophil recruitment and ACLF, providing evidencing for potential therapies targeting ACLF.

Potential mitigation of environmental impacts of intensive plum production in southeast China with maintenance of high yields: Evaluation using life cycle assessment.

Introduction: Intensive plum production usually involves high yields but also high environmental costs due to excessive fertilizer inputs. Quantitative analysis of the environmental effects of plum production is thereby required in the development of optimum strategies to promote sustainable fruit production. Methods: We collected survey questionnaires from 254 plum production farms in Zhao'an county, Fujian province, southeast China to assess the environmental impacts by life cycle assessment (LCA) methodology. The farms were categorized into four groups based on yield and environmental impacts, i.e., LL (low yield and low environmental impact), LH (low yield but high environmental impact), HL (high yield but low environmental impact), and HH (high yield and high environmental impact). Results: The environmental impacts, i.e., average energy depletion, global warming, acidification, and eutrophication potential in plum production were 18.17 GJ ha-1, 3.63 t CO2 eq ha-1, 42.18 kg SO2 eq ha-1, and 25.06 kg PO4 eq ha-1, respectively. Only 19.7% of farmers were in the HL group, with 13.3% in the HH group, 39.0% in LL, and 28.0% LH. Plum yields of the HL group were 109-114% higher than the mean value of all 254 farms. Additionally, the HL group had a lower environmental impact per unit area compared to the overall mean value, with a reduction ranging from 31.9% to 36.7%. Furthermore, on a per tonne of plum production basis, the energy depletion, global warming potential, acidification potential, and eutrophication potential of HL farms were lower by 75.4%, 75.0%, 75.6%, and 75.8%, respectively. Overall, the total environmental impact index of LL, LH, HL, and HH groups were 0.26, 0.42, 0.06, and 0.21, respectively. Discussion: Excessive fertilizer N application was the main source of the environmental impacts, the potential to reduce fertilizer N rate can be achieved without compromising plum yield by studying the HH group. The results provide an important foundation for enhancing the management of plum production, in order to promote 'green' agricultural development by reducing environmental impacts.

Transcriptome Analysis of Retinal and Choroidal Pathologies in Aged BALB/c Mice Following Systemic Neonatal Murine Cytomegalovirus Infection.

Our previous studies have shown that systemic neonatal murine cytomegalovirus (MCMV) infection of BALB/c mice spread to the eye with subsequent establishment of latency in choroid/RPE. In this study, RNA sequencing (RNA-Seq) analysis was used to determine the molecular genetic changes and pathways affected by ocular MCMV latency. MCMV (50 pfu per mouse) or medium as control were injected intra-peritoneally (i.p.) into BALB/c mice at <3 days after birth. At 18 months post injection, the mice were euthanized, and the eyes were collected and prepared for RNA-Seq. Compared to three uninfected control eyes, we identified 321 differentially expressed genes (DEGs) in six infected eyes. Using the QIAGEN Ingenuity Pathway Analysis (QIAGEN IPA), we identified 17 affected canonical pathways, 10 of which function in neuroretinal signaling, with the majority of DEGs being downregulated, while 7 pathways function in upregulated immune/inflammatory responses. Retinal and epithelial cell death pathways involving both apoptosis and necroptosis were also activated. MCMV ocular latency is associated with upregulation of immune and inflammatory responses and downregulation of multiple neuroretinal signaling pathways. Cell death signaling pathways are also activated and contribute to the degeneration of photoreceptors, RPE, and choroidal capillaries.

MiR-623 links lncRNA RP11-89 and cyclin D1 to regulate the proliferation of glioblastoma cells.

PURPOSE: The tumorigenesis of bladder cancer has been proven to be related to the increased expression of lncRNA RP11-89, the participation of which in glioblastoma (GBM) is unknown. We predicted that RP11-89 could be targeted by miR-623, which targets cyclin D1. We then analyzed the role of RP11-89 in GBM. MATERIALS AND METHODS: Samples of both GBM and paired non-tumor tissue were obtained from 58 GBM patients to analyze the expression of RP11-89 and miR-623 through RT-qPCR. The direct binding of miR-623 to RP11-89 was analyzed with RNA-RNA pull down. The role of RP11-89 and miR-623 in regulating each other's expression was analyzed with overexpression assay. The role of RP11-89 and miR-623 in regulating the expression of cyclin D1 and GBM cell proliferation was analyzed by Western blot and BrdU assay, respectively. RESULTS: RP11-89 was expressed in high amounts in GBM, while miR-623 was expressed in low amounts in GBM. RP11-89 and miR-623 were not closely correlated, while miR-623 directly bound to RP11-89. RP11-89 and miR-623 showed no direct role in each other's expression. RP11-89 suppressed the role of miR-623 in downregulating cyclin D1 and GBM cell proliferation. CONCLUSIONS: Therefore, miR-623 may link lncRNA RP11-89 and cyclin D1 to regulate the proliferation of GBM cells.

The Role of Caspase-12 in Retinal Bystander Cell Death and Innate Immune Responses against MCMV Retinitis.

(1) Background: caspase-12 is activated during cytomegalovirus retinitis, although its role is presently unclear. (2) Methods: caspase-12-/- (KO) or caspase-12+/+ (WT) mice were immunosup eyes were analyzed by plaque assay, TUNEL assay, immunohistochemical staining, western blotting, and real-time PCR. (3) Results: increased retinitis and a more extensive virus spread were detected in the retina of infected eyes of KO mice compared to WT mice at day 14 p.i. Compared to MCMV injected WT eyes, mRNA levels of interferons α, ß and γ were significantly reduced in the neural retina of MCMV-infected KO eyes at day 14 p.i. Although similar numbers of MCMV infected cells, similar virus titers and similar numbers of TUNEL-staining cells were detected in injected eyes of both KO and WT mice at days 7 and 10 p.i., significantly lower amounts of cleaved caspase-3 and p53 protein were detected in infected eyes of KO mice at both time points. (4) Conclusions: caspase-12 contributes to caspase-3-dependent and independent retinal bystander cell death during MCMV retinitis and may also play an important role in innate immunity against virus infection of the retina.

Retinal and Choroidal Pathologies in Aged BALB/c Mice Following Systemic Neonatal Murine Cytomegalovirus Infection.

Although pathologies associated with acute virus infections have been extensively studied, the effects of long-term latent virus infections are less well understood. Human cytomegalovirus, which infects 50% to 80% of humans, is usually acquired during early life and persists in a latent state for the lifetime. The purpose of this study was to determine whether systemic murine cytomegalovirus (MCMV) infection acquired early in life disseminates to and becomes latent in the eye and if ocular MCMV can trigger in situ inflammation and occurrence of ocular pathology. This study found that neonatal infection of BALB/c mice with MCMV resulted in dissemination of virus to the eye, where it localized principally to choroidal endothelia and pericytes and less frequently to the retinal pigment epithelium (RPE) cells. MCMV underwent ocular latency, which was associated with expression of multiple virus genes and from which MCMV could be reactivated by immunosuppression. Latent ocular infection was associated with significant up-regulation of several inflammatory/angiogenic factors. Retinal and choroidal pathologies developed in a progressive manner, with deposits appearing at both basal and apical aspects of the RPE, RPE/choroidal atrophy, photoreceptor degeneration, and neovascularization. The pathologies induced by long-term ocular MCMV latency share features of previously described human ocular diseases, such as age-related macular degeneration.

Cre/loxP approach-mediated downregulation of Pik3c3 inhibits the hypertrophic growth of renal proximal tubule cells.

Nephron loss stimulates residual functioning nephrons to undergo compensatory growth. Excessive nephron growth may be a maladaptive response that sets the stage for progressive nephron damage, leading to kidney failure. To date, however, the mechanism of nephron growth remains incompletely understood. Our previous study revealed that class III phosphatidylinositol-3-kinase (Pik3c3) is activated in the remaining kidney after unilateral nephrectomy (UNX)-induced nephron loss, but previous studies failed to generate a Pik3c3 gene knockout animal model. Global Pik3c3 deletion results in embryonic lethality. Given that renal proximal tubule cells make up the bulk of the kidney and undergo the most prominent hypertrophic growth after UNX, in this study we used Cre-loxP-based approaches to demonstrate for the first time that tamoxifen-inducible SLC34a1 promoter-driven CreERT2 recombinase-mediated downregulation of Pik3c3 expression in renal proximal tubule cells alone is sufficient to inhibit UNX- or amino acid-induced hypertrophic nephron growth. Furthermore, our mechanistic studies unveiled that the SLC34a1-CreERT2 recombinase-mediated Pik3c3 downregulation inhibited UNX- or amino acid-stimulated lysosomal localization and signaling activation of mechanistic target of rapamycin complex 1 (mTORC1) in the renal proximal tubules. Moreover, our additional cell culture experiments using RNAi confirmed that knocking down Pik3c3 expression inhibited amino acid-stimulated mTORC1 signaling and blunted cellular growth in primary cultures of renal proximal tubule cells. Together, both our in vivo and in vitro experimental results indicate that Pik3c3 is a major mechanistic mediator responsible for sensing amino acid availability and initiating hypertrophic growth of renal proximal tubule cells by activation of the mTORC1-S6K1-rpS6 signaling pathway.

Ocular cytomegalovirus latency exacerbates the development of choroidal neovascularization.

Age-related macular degeneration (AMD) is a complex, multifactorial, progressive disease which represents a leading cause of irreversible visual impairment and blindness in older individuals. Human cytomegalovirus (HCMV), which infects 50-80% of humans, is usually acquired during early life and persists in a latent state for the life of the individual. In view of its previously described pro-angiogenic properties, we hypothesized that cytomegalovirus might be a novel risk factor for progression to an advanced form, neovascular AMD, which is characterized by choroidal neovascularization (CNV). The purpose of this study was to investigate if latent ocular murine cytomegalovirus (MCMV) infection exacerbated the development of CNV in vascular endothelial growth factor (VEGF)-overexpressing VEGF-Ahyper mice. Here we show that neonatal infection with MCMV resulted in dissemination of virus to various organs throughout the body including the eye, where it localized principally to the choroid in both VEGF-overexpressingVEGF-Ahyper and wild-type(WT) 129 mice. By 6 months post-infection, no replicating virus was detected in eyes and extraocular tissues, although virus DNA was still present in all eyes and extraocular tissues of both VEGF-Ahyper and WT mice. Expression of MCMV immediate early (IE) 1 mRNA was detected only in latently infected eyes of VEGF-Ahyper mice, but not in eyes of WT mice. Significantly increased CNV was observed in eyes of MCMV-infected VEGF-Ahyper mice compared to eyes of uninfected VEGF-Ahyper mice, while no CNV lesions were observed in eyes of either infected or uninfected WT mice. Protein levels of several inflammatory/angiogenic factors, particularly VEGF and IL-6, were significantly higher in eyes of MCMV-infected VEGF-Ahyper mice, compared to uninfected controls. Initial studies of ocular tissue from human cadavers revealed that HCMV DNA was present in four choroid/retinal pigment epithelium samples from 24 cadavers. Taken together, our data suggest that ocular HCMV latency could be a significant risk factor for the development of AMD. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The expression level of class III phosphatidylinositol-3 kinase controls the degree of compensatory nephron hypertrophy.

Excessive compensatory nephron hypertrophy (CNH) has been implicated in setting the stage for progressive nephron damage. Lack of a class III phosphatidylinositol 3-kinase (Pik3c3) inhibitor suitable for using in animals and lack of a Pik3c3-deficient animal model preclude the possibility of conclusively defining a role for Pik3c3 in CNH in previous studies. Here, we report that insertion of an Frt-flanked PGK-Neo cassette into intron 19 of the mouse Pik3c3 gene resulted in a hypomorphic allele. This allowed us to create a unique mouse model and provide the first definitive genetic evidence demonstrating whether Pik3c3 is essential for the regulation of CNH. Our results indicate that homozygous Pik3c3 hypomorphic (Pik3c3Hypo/Hypo) mice express significantly low levels of Pik3c3 than heterozygous Pik3c3 hypomorphic (Pik3c3Hypo/WT) littermates, which already express a lower level of Pik3c3 than wild-type (Pik3c3WT/WT) littermates. Interestingly, after unilateral nephrectomy (UNX), Pik3c3Hypo/Hypo mice develop a significantly lower degree of CNH than Pik3c3WT/WT mice and Pik3c3Hypo/WT mice, as revealed by measurement of kidney weight, kidney-to-body weight ratio, renal protein-to-DNA ratio, and morphometric analysis of proximal tubular and glomerular size. Mechanistically, UNX-induced mammalian target of rapamycin complex 1 (mTORC1) signaling to phosphorylation of ribosomal protein S6 (rpS6) in the remaining kidney was markedly inhibited in Pik3c3 hypomorphic mice. In conclusion, the present study reports a Pik3c3 hypomorphic mouse model and provides the first definitive evidence that Pik3c3 controls the degree of compensatory nephron hypertrophy. In addition, our signaling data provide the first definitive in vivo proof that Pik3c3 functions upstream of the mTORC1-S6 kinase 1-rpS6 pathway in the regulation of compensatory nephron hypertrophy.

Oral Pathobiont Activates Anti-Apoptotic Pathway, Promoting both Immune Suppression and Oncogenic Cell Proliferation.

Chronic periodontitis (CP) is a microbial dysbiotic disease linked to increased risk of oral squamous cell carcinomas (OSCCs). To address the underlying mechanisms, mouse and human cell infection models and human biopsy samples were employed. We show that the 'keystone' pathogen Porphyromonas gingivalis, disrupts immune surveillance by generating myeloid-derived dendritic suppressor cells (MDDSCs) from monocytes. MDDSCs inhibit CTLs and induce FOXP3 + Tregs through an anti-apoptotic pathway. This pathway, involving pAKT1, pFOXO1, FOXP3, IDO1 and BIM, is activated in humans with CP and in mice orally infected with Mfa1 expressing P. gingivalis strains. Mechanistically, activation of this pathway, demonstrating FOXP3 as a direct FOXO1-target gene, was demonstrated by ChIP-assay in human CP gingiva. Expression of oncogenic but not tumor suppressor markers is consistent with tumor cell proliferation demonstrated in OSCC-P. gingivalis cocultures. Importantly, FimA + P. gingivalis strain MFI invades OSCCs, inducing inflammatory/angiogenic/oncogenic proteins stimulating OSCCs proliferation through CXCR4. Inhibition of CXCR4 abolished Pg-MFI-induced OSCCs proliferation and reduced expression of oncogenic proteins SDF-1/CXCR4, plus pAKT1-pFOXO1. Conclusively, P. gingivalis, through Mfa1 and FimA fimbriae, promotes immunosuppression and oncogenic cell proliferation, respectively, through a two-hit receptor-ligand process involving DC-SIGN+hi/CXCR4+hi, activating a pAKT+hipFOXO1+hiBIM-lowFOXP3+hi and IDO+hi- driven pathway, likely to impact the prognosis of oral cancers in patients with periodontitis.

Inflammation and outer blood-retina barrier (BRB) compromise following choroidal murine cytomegalovirus (MCMV) infections.

Purpose: The purpose of this study was to determine whether the blood-retina barrier is compromised by choroidal murine cytomegalovirus (MCMV) infection, using electron microscopy. Methods: BALB/c mice were immunosuppressed with methylprednisolone and monoclonal antibodies to CD4 and CD8. At several time points post-MCMV intraperitoneal inoculation, the eyes were removed and analyzed with western blotting and immunoelectron microscopy for the presence of MCMV early antigen (EA) and the host protein RIP3. Posterior eyecups from RIP3-/- and RIP3+/+ mice were cultured and inoculated with MCMV. At days 4, 7, and 11 post-infection, cultures were collected and analyzed with plaque assay, immunohistochemical staining, and real-time PCR (RT-PCR). Results: MCMV EA was observed in the nuclei of vascular endothelial cells and pericytes in the choriocapillaris. Disruption of Bruch's membrane was observed, especially at sites adjacent to activated platelets, and a few RPE cells containing some enlarged vesicles were found directly beneath disrupted Bruch's membrane. Some virus particles were also observed in the enlarged vesicles of RPE cells. Levels of the RIP3 protein, which was observed mainly in the RPE cells and the basement membrane of the choriocapillaris, were greatly increased following MCMV infection, while depletion of RIP3 resulted in greatly decreased inflammasome formation, as well as expression of downstream inflammation factors. Conclusions: The results suggest that systemic MCMV spreads to the choroid and replicates in vascular endothelia and pericytes of the choriocapillaris during immunosuppression. Choroidal MCMV infection is associated with in situ inflammation and subsequent disruption of Bruch's membrane and the outer blood-retina barrier.

Depletion of the Receptor-Interacting Protein Kinase 3 (RIP3) Decreases Photoreceptor Cell Death During the Early Stages of Ocular Murine Cytomegalovirus Infection.

Purpose: The purpose of this study was to determine if the receptor-interacting protein kinase 3 (RIP3) plays a significant role in innate immune responses and death of bystander retinal neurons during murine cytomegalovirus (MCMV) retinal infection, by comparing the innate immune response and cell death in RIP3-depleted mice (Rip3-/-) and Rip3+/+ control mice. Methods: Rip3-/- and Rip3+/+ mice were immunosuppressed (IS) and inoculated with MCMV via the supraciliary route. Virus-injected and mock-injected control eyes were removed at days 4, 7, and 10 post infection (p.i.) and markers of innate immunity and cell death were analyzed. Results: Compared to Rip3+/+ mice, significantly more MCMV was recovered and more MCMV-infected RPE cells were observed in injected eyes of Rip3-/- mice at days 4 and 7 p.i. In contrast, fewer TUNEL-stained photoreceptors were observed in Rip3-/- eyes than in Rip3+/+ eyes at these times. Electron microscopy showed that significantly more apoptotic photoreceptor cells were present in Rip3+/+ mice than in Rip3-/- mice. Immunohistochemistry showed that the majority of TUNEL-stained photoreceptors died via mitochondrial flavoprotein apoptosis-inducing factor (AIF)-mediated, caspase 3-independent apoptosis. The majority of RIP3-expressing cells in infected eyes were RPE cells, microglia/macrophages, and glia, whereas retinal neurons contained much lower amounts of RIP3. Western blots showed significantly higher levels of activated nuclear factor-κB and caspase 1 were present in Rip3+/+ eyes compared to Rip3-/- eyes. Conclusions: Our results suggest that RIP3 enhances innate immune responses against ocular MCMV infection via activation of the inflammasome and nuclear factor-κB, which also leads to inflammation and death of bystander cells by multiple pathways including apoptosis and necroptosis.

Expression of Connexin43 in Cerebral Arteries of Patients with Moyamoya Disease.

BACKGROUND AND PURPOSE: Moyamoya disease has a high incidence of cerebral vascular accident in children and adolescents, which can endanger the physical and mental health of children and adults seriously. However, the etiology and the pathogenesis of moyamoya disease remain unclear. Connexin43 (Cx43) is a predominant intercellular gap junction protein that plays an important role in the normal function of arteries and the development of several cardiovascular diseases. We aimed to preliminarily investigate pathological changes and the expression of Cx43 in cerebral arteries of patients with moyamoya disease. MATERIALS AND METHODS: This study collected 10 experimental cerebral artery specimens from patients with moyamoya disease and 10 control cerebral artery specimens from patients without moyamoya disease during surgery, then pathological changes and change in Cx43 expression of cerebral artery specimens were investigated in the 2 groups by hematoxylin and eosin staining and immunofluorescence. RESULTS: The intima of cerebral arteries was thin with monolayer endothelial cells in the control group but had asymmetrical thickening for the cerebral arteries in the experimental group. The mean ± standard deviation of the mean optical density of Cx43 in the experimental group was .065 ± .011 (range, .045-.081), whereas that in the control group was .035 ± .005 (range, .028-.042). The expression of Cx43 in the experimental group was statistically significantly higher in comparison with the control group (P < .01). CONCLUSION: The abnormal expression of Cx43 in the cerebral arteries may play an important role in the formation of vascular intima thickening in patients with moyamoya disease.

Blocking rpS6 Phosphorylation Exacerbates Tsc1 Deletion-Induced Kidney Growth.

The molecular mechanisms underlying renal growth and renal growth-induced nephron damage remain poorly understood. Here, we report that in murine models, deletion of the tuberous sclerosis complex protein 1 (Tsc1) in renal proximal tubules induced strikingly enlarged kidneys, with minimal cystogenesis and occasional microscopic tumorigenesis. Signaling studies revealed hyperphosphorylation of eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) and increased phosphorylation of ribosomal protein S6 (rpS6) in activated renal tubules. Notably, knockin of a nonphosphorylatable rpS6 in these Tsc1-mutant mice exacerbated cystogenesis and caused drastic nephron damage and renal fibrosis, leading to kidney failure and a premature death rate of 67% by 9 weeks of age. In contrast, Tsc1 single-mutant mice were all alive and had far fewer renal cysts at this age. Mechanistic studies revealed persistent activation of mammalian target of rapamycin complex 1 (mTORC1) signaling causing hyperphosphorylation and consequent accumulation of 4E-BP1, along with greater cell proliferation, in the renal tubules of Tsc1 and rpS6 double-mutant mice. Furthermore, pharmacologic treatment of Tsc1 single-mutant mice with rapamycin reduced hyperphosphorylation and accumulation of 4E-BP1 but also inhibited phosphorylation of rpS6. Rapamycin also exacerbated cystic and fibrotic lesions and impaired kidney function in these mice, consequently leading to a premature death rate of 40% within 2 weeks of treatment, despite destroying tumors and decreasing kidney size. These findings indicate that Tsc1 prevents aberrant renal growth and tumorigenesis by inhibiting mTORC1 signaling, whereas phosphorylated rpS6 suppresses cystogenesis and fibrosis in Tsc1-deleted kidneys.

Phosphatidylinositol 3-kinase signaling determines kidney size.

Kidney size adaptively increases as mammals grow and in response to the loss of 1 kidney. It is not clear how kidneys size themselves or if the processes that adapt kidney mass to lean body mass also mediate renal hypertrophy following unilateral nephrectomy (UNX). Here, we demonstrated that mice harboring a proximal tubule-specific deletion of Pten (Pten(ptKO)) have greatly enlarged kidneys as the result of persistent activation of the class I PI3K/mTORC2/AKT pathway and an increase of the antiproliferative signals p21(Cip1/WAF) and p27(Kip1). Administration of rapamycin to Pten(ptKO) mice diminished hypertrophy. Proximal tubule-specific deletion of Egfr in Pten(ptKO) mice also attenuated class I PI3K/mTORC2/AKT signaling and reduced the size of enlarged kidneys. In Pten(ptKO) mice, UNX further increased mTORC1 activation and hypertrophy in the remaining kidney; however, mTORC2-dependent AKT phosphorylation did not increase further in the remaining kidney of Pten(ptKO) mice, nor was it induced in the remaining kidney of WT mice. After UNX, renal blood flow and amino acid delivery to the remaining kidney rose abruptly, followed by increased amino acid content and activation of a class III PI3K/mTORC1/S6K1 pathway. Thus, our findings demonstrate context-dependent roles for EGFR-modulated class I PI3K/mTORC2/AKT signaling in the normal adaptation of kidney size and PTEN-independent, nutrient-dependent class III PI3K/mTORC1/S6K1 signaling in the compensatory enlargement of the remaining kidney following UNX.

Phosphorylation of ribosomal protein S6 mediates compensatory renal hypertrophy.

The molecular mechanism underlying renal hypertrophy and progressive nephron damage remains poorly understood. Here we generated congenic ribosomal protein S6 (rpS6) knock-in mice expressing nonphosphorylatable rpS6 and found that uninephrectomy-induced renal hypertrophy was significantly blunted in these knock-in mice. Uninephrectomy-induced increases in cyclin D1 and decreases in cyclin E in the remaining kidney were attenuated in the knock-in mice compared with their wild-type littermates. Uninephrectomy induced rpS6 phosphorylation in the wild-type mice; however, no rpS6 phosphorylation was detected in uninephrectomized or sham-operated knock-in mice. Nonetheless, uninephrectomy stimulated comparable 4E-BP1 phosphorylation in both knock-in and wild-type mice, indicating that mTORC1 was still activated in the knock-in mice. Moreover, the mTORC1 inhibitor rapamycin prevented both rpS6 and 4E-BP1 phosphorylation, significantly blunted uninephrectomy-induced renal hypertrophy in wild-type mice, but did not prevent residual renal hypertrophy despite inhibiting 4E-BP1 phosphorylation in uninephrectomized knock-in mice. Thus, both genetic and pharmacological approaches unequivocally demonstrate that phosphorylated rpS6 is a downstream effector of the mTORC1-S6K1 signaling pathway mediating renal hypertrophy. Hence, rpS6 phosphorylation facilitates the increase in cyclin D1 and decrease in cyclin E1 that underlie the hypertrophic nature of uninephrectomy-induced kidney growth.

IKKß links vascular inflammation to obesity and atherosclerosis.

IκB kinase ß (IKKß), a central coordinator of inflammatory responses through activation of NF-κB, has been implicated in vascular pathologies, but its role in atherogenesis remains elusive. Here, we demonstrate that IKKß functions in smooth muscle cells (SMCs) to regulate vascular inflammatory responses and atherosclerosis development. IKKß deficiency in SMCs driven by a SM22Cre-IKKß-flox system rendered low density lipoprotein receptor-null mice resistant to vascular inflammation and atherosclerosis induced by high-fat feeding. Unexpectedly, IKKß-deficient mice were also resistant to diet-induced obesity and metabolic disorders. Cell lineage analysis revealed that SM22Cre is active in primary adipose stromal vascular cells and deficiency of IKKß diminished the ability of these cells to differentiate, leading to accumulation of adipocyte precursor cells in adipose tissue. Mechanistically, reduction of IKKß expression or pharmacological inhibition of IKKß inhibited proteasome-mediated ß-catenin ubiquitination and degradation in murine preadipocytes, resulting in elevated ß-catenin levels and impaired adipocyte differentiation. Further, chronic treatment of mice with a potent IKKß inhibitor decreased adipogenesis and ameliorated diet-induced obesity. Our findings demonstrate a pivotal role of IKKß in linking vascular inflammation to atherosclerosis and adipose tissue development, and provide evidence for using appropriate IKKß inhibitors in the treatment of obesity and metabolic disorders.

Dual role of C/EBPα as an activator and repressor of Gαi2 gene transcription.

The G-protein Gαi2 mediates signaling in a variety of processes. Induced expression of Gαi2 by butyrate and various transcription factors has been established, but transcriptional suppression has not previously been explored. Using HepG2 and K562 cells in culture, we show here that whereas both C/EBPα and C/EBPß induced transcription from the Gαi2 gene promoter, C/EBPα, but not C/EBPß, inhibited butyrate-induced Gαi2 expression. Because the transcriptional effect of butyrate on this gene promoter is largely mediated by the transcription factor Sp1, we investigated whether C/EBPα influenced Sp1-induced Gαi2 gene transcription. Binding of C/EBPα to a C/EBP response element in Gαi2 gene promoter inhibited Sp1-induced promoter activity. ChIP analysis showed decreased butyrate-induced recruitment of Sp1 to the Gαi2 gene promoter in response to C/EBPα treatment. Incubating cells with acetate or transfecting them with expression plasmid for either the acetyltransferase p300 or CREB-binding protein (CBP) reversed the antagonistic effect of C/EBPα on Sp1-dependent gene transcription, suggesting that the mechanistic basis for the antagonism is related to the squelching of co-activator acetyltransferase(s) by C/EBPα or the acetylation of Sp1 and/or C/EBPα. This work reveals that C/EBPα plays a dual role as an activator and as a repressor of Gαi2 gene transcription.

Myeloid-specific IκB kinase ß deficiency decreases atherosclerosis in low-density lipoprotein receptor-deficient mice.

OBJECTIVE: Inflammatory responses are the driving force of atherosclerosis development. IκB kinase ß (IKKß), a central coordinator in inflammation through regulation of nuclear factor-κB, has been implicated in the pathogenesis of atherosclerosis. Macrophages play an essential role in the initiation and progression of atherosclerosis, yet the role of macrophage IKKß in atherosclerosis remains elusive and controversial. This study aims to investigate the impact of IKKß expression on macrophage functions and to assess the effect of myeloid-specific IKKß deletion on atherosclerosis development. METHODS AND RESULTS: To explore the issue of macrophage IKKß involvement of atherogenesis, we generated myeloid-specific IKKß-deficient low-density lipoprotein receptor-deficient mice (IKKß(ΔMye)LDLR(-/-)). Deficiency of IKKß in myeloid cells did not affect plasma lipid levels but significantly decreased diet-induced atherosclerotic lesion areas in the aortic root, brachiocephalic artery, and aortic arch of low-density lipoprotein receptor-deficient mice. Ablation of myeloid IKKß attenuated macrophage inflammatory responses and decreased atherosclerotic lesional inflammation. Furthermore, deficiency of IKKß decreased adhesion, migration, and lipid uptake in macrophages. CONCLUSIONS: The present study demonstrates a pivotal role for myeloid IKKß expression in atherosclerosis by modulating macrophage functions involved in atherogenesis. These results suggest that inhibiting nuclear factor-κB activation in macrophages may represent a feasible approach to combat atherosclerosis.

Deficiency of PXR decreases atherosclerosis in apoE-deficient mice.

The pregnane X receptor (PXR, also known as SXR) is a nuclear hormone receptor activated by xenobiotics as well as diverse sterols and their metabolites. PXR functions as a xenobiotic sensor to coordinately regulate xenobiotic metabolism via transcriptional regulation of xenobiotic-detoxifying enzymes and transporters. Recent evidence indicates that PXR may also play an important role in lipid homeostasis and atherosclerosis. To define the role of PXR in atherosclerosis, we generated PXR and apoE double knockout (PXR(-/-)apoE(-/-)) mice. Here we show that deficiency of PXR did not alter plasma triglyceride and cholesterol levels in apoE(-/-) mice. However, PXR(-/-)apoE(-/-) mice had significantly decreased atherosclerotic cross-sectional lesion area in both the aortic root and brachiocephalic artery by 40% (P < 0.01) and 60% (P < 0.001), respectively. Interestingly, deficiency of PXR reduced the expression levels of CD36, lipid accumulation, and CD36-mediated oxidized LDL uptake in peritoneal macrophages of PXR(-/-)apoE(-/-) mice. Furthermore, immunofluorescence staining showed that PXR and CD36 were expressed in the atherosclerotic lesions of apoE(-/-) mice, and the expression levels of PXR and CD36 were diminished in the lesions of PXR(-/-)apoE(-/-) mice. Our findings indicate that deficiency of PXR attenuates atherosclerosis development, which may result from decreased CD36 expression and reduced lipid uptake in macrophages.