SARS-CoV-2 Spike Protein Stimulates Macropinocytosis in Murine and Human Macrophages via PKC-NADPH Oxidase Signaling.

Coronavirus disease 2019 (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). While recent studies have demonstrated that SARS-CoV-2 may enter kidney and colon epithelial cells by inducing receptor-independent macropinocytosis, it remains unknown whether this process also occurs in cell types directly relevant to SARS-CoV-2-associated lung pneumonia, such as alveolar epithelial cells and macrophages. The goal of our study was to investigate the ability of SARS-CoV-2 spike protein subunits to stimulate macropinocytosis in human alveolar epithelial cells and primary human and murine macrophages. Flow cytometry analysis of fluid-phase marker internalization demonstrated that SARS-CoV-2 spike protein subunits S1, the receptor-binding domain (RBD) of S1, and S2 stimulate macropinocytosis in both human and murine macrophages in an angiotensin-converting enzyme 2 (ACE2)-independent manner. Pharmacological and genetic inhibition of macropinocytosis substantially decreased spike-protein-induced fluid-phase marker internalization in macrophages both in vitro and in vivo. High-resolution scanning electron microscopy (SEM) imaging confirmed that spike protein subunits promote the formation of membrane ruffles on the dorsal surface of macrophages. Mechanistic studies demonstrated that SARS-CoV-2 spike protein stimulated macropinocytosis via NADPH oxidase 2 (Nox2)-derived reactive oxygen species (ROS) generation. In addition, inhibition of protein kinase C (PKC) and phosphoinositide 3-kinase (PI3K) in macrophages blocked SARS-CoV-2 spike-protein-induced macropinocytosis. To our knowledge, these results demonstrate for the first time that SARS-CoV-2 spike protein subunits stimulate macropinocytosis in macrophages. These results may contribute to a better understanding of SARS-CoV-2 infection and COVID-19 pathogenesis.

Receptor-independent fluid-phase macropinocytosis promotes arterial foam cell formation and atherosclerosis.

Accumulation of lipid-laden foam cells in the arterial wall plays a central role in atherosclerotic lesion development, plaque progression, and late-stage complications of atherosclerosis. However, there are still fundamental gaps in our knowledge of the underlying mechanisms leading to foam cell formation in atherosclerotic arteries. Here, we investigated the role of receptor-independent macropinocytosis in arterial lipid accumulation and pathogenesis of atherosclerosis. Genetic inhibition of fluid-phase macropinocytosis in myeloid cells (LysMCre+ Nhe1fl/fl) and repurposing of a Food and Drug Administration (FDA)-approved drug that inhibits macrophage macropinocytosis substantially decreased atherosclerotic lesion development in low-density lipoprotein (LDL) receptor-deficient and Apoe-/- mice. Stimulation of macropinocytosis using genetic (H-RASG12V) and physiologically relevant approaches promoted internalization of unmodified native (nLDL) and modified [e.g., acetylated (ac) and oxidized (ox) LDL] lipoproteins in both wild-type and scavenger receptor (SR) knockout (Cd36-/-/Sra-/-) macrophages. Pharmacological inhibition of macropinocytosis in hypercholesterolemic wild-type and Cd36-/-/Sra-/- mice identified an important role of macropinocytosis in LDL uptake by lesional macrophages and development of atherosclerosis. Furthermore, serial section high-resolution imaging, LDL immunolabeling, and three-dimensional (3D) reconstruction of subendothelial foam cells provide visual evidence of lipid macropinocytosis in both human and murine atherosclerotic arteries. Our findings complement the SR paradigm of atherosclerosis and identify a therapeutic strategy to counter the development of atherosclerosis and cardiovascular disease.

MEK inhibition exerts temporal and myeloid cell-specific effects in the pathogenesis of neurofibromatosis type 1 arteriopathy.

Mutations in the NF1 tumor suppressor gene are linked to arteriopathy. Nf1 heterozygosity (Nf1+/-) results in robust neointima formation, similar to humans, and myeloid-restricted Nf1+/- recapitulates this phenotype via MEK-ERK activation. Here we define the contribution of myeloid subpopulations to NF1 arteriopathy. Neutrophils from WT and Nf1+/- mice were functionally assessed in the presence of MEK and farnesylation inhibitors in vitro and neutrophil recruitment to lipopolysaccharide was assessed in WT and Nf1+/- mice. Littermate 12-15 week-old male wildtype and Nf1+/- mice were subjected to carotid artery ligation and provided either a neutrophil depleting antibody (1A8), liposomal clodronate to deplete monocytes/macrophages, or PD0325901 and neointima size was assessed 28 days after injury. Bone marrow transplant experiments assessed monocyte/macrophage mobilization during neointima formation. Nf1+/- neutrophils exhibit enhanced proliferation, migration, and adhesion via p21Ras activation of MEK in vitro and in vivo. Neutrophil depletion suppresses circulating Ly6Clow monocytes and enhances neointima size, while monocyte/macrophage depletion and deletion of CCR2 in bone marrow cells abolish neointima formation in Nf1+/- mice. Taken together, these findings suggest that neurofibromin-MEK-ERK activation in circulating neutrophils and monocytes during arterial remodeling is nuanced and points to important cross-talk between these populations in the pathogenesis of NF1 arteriopathy.

Role of R-spondin 2 in arterial lymphangiogenesis and atherosclerosis.

AIMS: Impaired lymphatic drainage of the arterial wall results in intimal lipid accumulation and atherosclerosis. However, the mechanisms regulating lymphangiogenesis in atherosclerotic arteries are not well understood. Our studies identified elevated levels of matrix protein R-spondin 2 (RSPO2) in atherosclerotic arteries. In this study, we investigated the role of RSPO2 in lymphangiogenesis, arterial cholesterol efflux into lesion-draining lymph nodes (LNs) and development of atherosclerosis. METHODS AND RESULTS: The effect of RSPO2 on lymphangiogenesis was investigated using human lymphatic endothelial cells (LEC) in vitro and implanted Matrigel plugs in vivo. Cellular and molecular approaches, pharmacological agents, and siRNA silencing of RSPO2 receptor LGR4 were used to investigate RSPO2-mediated signalling in LEC. In vivo low-density lipoprotein (LDL) tracking and perivascular blockade of RSPO2-LGR4 signalling using LGR4-extracellular domain (ECD) pluronic gel in hypercholesterolemic mice were utilized to investigate the role of RSPO2 in arterial reverse cholesterol transport and atherosclerosis. Immunoblotting and imaging experiments demonstrated increased RSPO2 expression in human and mouse atherosclerotic arteries compared to non-atherosclerotic controls. RSPO2 treatment inhibited lymphangiogenesis both in vitro and in vivo. LGR4 silencing and inhibition of RSPO2-LGR4 signalling abrogated RSPO2-induced inhibition of lymphangiogenesis. Mechanistically, we found that RSPO2 suppresses PI3K-AKT-endothelial nitric oxide synthase (eNOS) signalling via LGR4 and inhibits activation of the canonical Wnt-ß-catenin pathway. ApoE-/- mice treated with LGR4-ECD developed significantly less atherosclerosis compared with control treatment. Finally, increased arterial lymphatic vessel density and improved lymphatic drainage of fluorescently labelled LDL to deep cervical LNs were observed in LGR4-ECD-treated mice. CONCLUSION: These findings demonstrate that RSPO2 inhibits lymphangiogenesis via LGR4 and downstream impairment of AKT-eNOS-nitric oxide signalling. These results may also inform new therapeutic strategies to promote lymphangiogenesis and improve cholesterol efflux from atherosclerotic arteries.

Loss of GTPase activating protein neurofibromin stimulates paracrine cell communication via macropinocytosis.

Neurofibromin, the protein product of the neurofibromatosis type 1 (NF1) tumor suppressor gene, is a negative regulator of Ras signaling. Patients with mutations in NF1 have a strong predisposition for cardiovascular disease, which contributes to their early mortality. Nf1 heterozygous (Nf1+/-) bone marrow to wild type chimeras and mice with heterozygous recombination of Nf1 in myeloid cells recapitulate many of the vascular phenotypes observed in Nf1+/- mutants. Although these results suggest that macrophages play a central role in NF1 vasculopathy, the underlying mechanisms are currently unknown. In the present study, we employed macrophages isolated from either Nf1+/- or Lysm Cre+/Nf1f/f mice to test the hypothesis that loss of Nf1 stimulates macropinocytosis in macrophages. Scanning electron microscopy and flow cytometry analysis of FITC-dextran internalization demonstrated that loss of Nf1 in macrophages stimulates macropinocytosis. We next utilized various cellular and molecular approaches, pharmacological inhibitors and genetically modified mice to identify the signaling mechanisms mediating macropinocytosis in Nf1-deficient macrophages. Our results indicate that loss of Nf1 stimulates PKCδ-mediated p47phox phosphorylation via RAS activation, leading to increased NADPH oxidase 2 activity, reactive oxygen species generation, membrane ruffling and macropinocytosis. Interestingly, we also found that Nf1-deficient macrophages internalize exosomes derived from angiotensin II-treated endothelial cells via macropinocytosis in vitro and in the peritoneal cavity in vivo. As a result of exosome internalization, Nf1-deficient macrophages polarized toward an inflammatory M1 phenotype and secreted increased levels of proinflammatory cytokines compared to controls. In conclusion, the findings of the present study demonstrate that loss of Nf1 stimulates paracrine endothelial to myeloid cell communication via macropinocytosis, leading to proinflammatory changes in recipient macrophages.

PKCδ stimulates macropinocytosis via activation of SSH1-cofilin pathway.

Macropinocytosis is an actin-dependent endocytic mechanism mediating internalization of extracellular fluid and associated solutes into cells. The present study was designed to identify the specific protein kinase C (PKC) isoform(s) and downstream effectors regulating actin dynamics during macropinocytosis. We utilized various cellular and molecular biology techniques, pharmacological inhibitors and genetically modified mice to study the signaling mechanisms mediating macropinocytosis in macrophages. The qRT-PCR experiments identified PKCδ as the predominant PKC isoform in macrophages. Scanning electron microscopy and flow cytometry analysis of FITC-dextran internalization demonstrated the functional role of PKCδ in phorbol ester- and hepatocyte growth factor (HGF)-induced macropinocytosis. Western blot analysis demonstrated that phorbol ester and HGF stimulate activation of slingshot phosphatase homolog 1 (SSH1) and induce cofilin Ser-3 dephosphorylation via PKCδ in macrophages. Silencing of SSH1 inhibited cofilin dephosphorylation and macropinocytosis stimulation. Interestingly, we also found that incubation of macrophages with BMS-5, a potent inhibitor of LIM kinase, does not stimulate macropinocytosis. In conclusion, the findings of the present study demonstrate a previously unidentified mechanism by which PKCδ via activation of SSH1 and cofilin dephosphorylation stimulates membrane ruffle formation and macropinocytosis. The results of the present study may contribute to a better understanding of the regulatory mechanisms during macrophage macropinocytosis.

Identification of novel macropinocytosis inhibitors using a rational screen of Food and Drug Administration-approved drugs.

BACKGROUND AND PURPOSE: Macropinocytosis is involved in many pathologies, including cardiovascular disorders, cancer, allergic diseases, viral and bacterial infections. Unfortunately, the currently available pharmacological inhibitors of macropinocytosis interrupt other endocytic processes and have non-specific endocytosis-independent effects. Here we have sought to identify new, clinically relevant inhibitors of macropinocytosis, using an FDA-approved drug library. EXPERIMENTAL APPROACH: In the present study, 640 FDA-approved compounds were tested for their ability to inhibit macropinocytosis. A series of secondary assays were performed to confirm inhibitory activity, determine IC50 values and investigate cell toxicity. The ability of identified hits to inhibit phagocytosis and clathrin-mediated and caveolin-mediated endocytosis was also investigated. Scanning electron microscopy and molecular biology techniques were utilized to examine the mechanisms by which selected compounds inhibit macropinocytosis. KEY RESULTS: The primary screen identified 14 compounds that at ~10 µM concentration inhibit >95% of macropinocytotic solute internalization. Three compounds - imipramine, phenoxybenzamine and vinblastine - potently inhibited (IC50  ≤ 131 nM) macropinocytosis without exerting cytotoxic effects or inhibiting other endocytic pathways. Scanning electron microscopy imaging indicated that imipramine inhibits membrane ruffle formation, a critical early step leading to initiation of macropinocytosis. Finally, imipramine has been shown to inhibit macropinocytosis in several cell types, including cancer cells, dendritic cells and macrophages. CONCLUSIONS AND IMPLICATIONS: Our results identify imipramine as a new pharmacological tool to study macropinocytosis in cellular and biological systems. This study also suggests that imipramine could be a good candidate for repurposing as a therapeutic agent in pathological processes involving macropinocytosis.

PKCδ-Mediated Nox2 Activation Promotes Fluid-Phase Pinocytosis of Antigens by Immature Dendritic Cells.

Aims: Macropinocytosis is a major endocytic pathway by which dendritic cells (DCs) internalize antigens in the periphery. Despite the importance of DCs in the initiation and control of adaptive immune responses, the signaling mechanisms mediating DC macropinocytosis of antigens remain largely unknown. The goal of the present study was to investigate whether protein kinase C (PKC) is involved in stimulation of DC macropinocytosis and, if so, to identify the specific PKC isoform(s) and downstream signaling mechanisms involved. Methods: Various cellular, molecular and immunological techniques, pharmacological approaches and genetic knockout mice were utilized to investigate the signaling mechanisms mediating DC macropinocytosis. Results: Confocal laser scanning microscopy confirmed that DCs internalize fluorescent antigens (ovalbumin) using macropinocytosis. Pharmacological blockade of classical and novel PKC isoforms using calphostin C abolished both phorbol ester- and hepatocyte growth factor-induced antigen macropinocytosis in DCs. The qRT-PCR experiments identified PKCδ as the dominant PKC isoform in DCs. Genetic studies demonstrated the functional role of PKCδ in DC macropinocytosis of antigens, their subsequent maturation, and secretion of various T-cell stimulatory cytokines, including IL-1α, TNF-α and IFN-ß. Additional mechanistic studies identified NADPH oxidase 2 (Nox2) and intracellular superoxide anion as important players in DC macropinocytosis of antigens downstream of PKCδ activation. Conclusion: The findings of the present study demonstrate a novel mechanism by which PKCδ activation via stimulation of Nox2 activity and downstream redox signaling promotes DC macropinocytosis of antigens. PKCδ/Nox2-mediated antigen macropinocytosis stimulates maturation of DCs and secretion of T-cell stimulatory cytokines. These findings may contribute to a better understanding of the regulatory mechanisms in DC macropinocytosis and downstream regulation of T-cell-mediated responses.

CD47 and Nox1 Mediate Dynamic Fluid-Phase Macropinocytosis of Native LDL.

AIMS: Macropinocytosis has been implicated in cardiovascular and other disorders, yet physiological factors that initiate fluid-phase internalization and the signaling mechanisms involved remain poorly identified. The present study was designed to examine whether matrix protein thrombospondin-1 (TSP1) stimulates macrophage macropinocytosis and, if so, to investigate the potential signaling mechanism involved. RESULTS: TSP1 treatment of human and murine macrophages stimulated membrane ruffle formation and pericellular solute internalization by macropinocytosis. Blockade of TSP1 cognate receptor CD47 and NADPH oxidase 1 (Nox1) signaling, inhibition of phosphoinositide 3-kinase, and transcriptional knockdown of myotubularin-related protein 6 abolished TSP1-induced macropinocytosis. Our results demonstrate that Nox1 signaling leads to dephosphorylation of actin-binding protein cofilin at Ser-3, actin remodeling, and macropinocytotic uptake of unmodified native low-density lipoprotein (nLDL), leading to foam cell formation. Finally, peritoneal chimera studies suggest the role of CD47 in macrophage lipid macropinocytosis in hypercholesterolemic ApoE-/- mice in vivo. INNOVATION: Activation of a previously unidentified TSP1-CD47 signaling pathway in macrophages stimulates direct receptor-independent internalization of nLDL, leading to significant lipid accumulation and foam cell formation. These findings reveal a new paradigm in which delimited Nox1-mediated redox signaling, independent of classical lipid oxidation, contributes to early propagation of vascular inflammatory disease. CONCLUSIONS: The findings of the present study demonstrate a new mechanism of solute uptake with implications for a wide array of cell types, including macrophages, dendritic cells, and cancer cells, and multiple pathological conditions in which matrix proteins are upregulated. Antioxid. Redox Signal. 26, 886-901.

Nox2-Mediated PI3K and Cofilin Activation Confers Alternate Redox Control of Macrophage Pinocytosis.

AIMS: Internalization of extracellular fluid and its solute by macropinocytosis requires dynamic reorganization of actin cytoskeleton, membrane ruffling, and formation of large endocytic vacuolar compartments, called macropinosomes, inside the cell. Although instigators of macropinocytosis, such as growth factors and phorbol esters, stimulate NADPH oxidase (Nox) activation and signal transduction mediators upstream of Nox assembly, including Rac1 and protein kinase C (PKC), are involved in macropinocytosis, the role of Nox enzymes in macropinocytosis has never been investigated. This study was designed to examine the role of Nox2 and the potential downstream redox signaling involved in macropinocytosis. RESULTS: Phorbol myristate acetate activation of human and murine macrophages stimulated membrane ruffling, macropinosome formation, and subsequent uptake of macromolecules by macropinocytosis. Mechanistically, we found that pharmacological blockade of PKC, transcriptional knockdown of Nox2, and scavenging of intracellular superoxide anion abolished phorbol ester-induced macropinocytosis. We observed that Nox2-derived reactive oxygen species via inhibition of phosphatase and tensin homolog and activation of the phosphoinositide-3-kinase (PI3K)/Akt pathway lead to activation of actin-binding protein cofilin, membrane ruffling, and macropinocytosis. Similarly, activation of macropinocytosis by macrophage colony-stimulating factor involves Nox2-mediated cofilin activation. Furthermore, peritoneal chimera experiments indicate that macropinocytotic uptake of lipids in hypercholesterolemic ApoE-/- mice was attenuated in Nox2y/- macrophages compared with wild-type controls. Innovation and Conclusion: In summary, these findings demonstrate a novel Nox2-mediated mechanism of solute uptake via macropinocytosis, with broad implications for both general cellular physiology and pathological processes. The redox mechanism described here may also identify new targets in atherosclerosis and other disease conditions involving macropinocytosis. Antioxid. Redox Signal. 26, 902-916.

Glycosylation inhibitors efficiently inhibit P-selectin-mediated cell adhesion to endothelial cells.

Adhesion molecules play a critical role in the adhesive interactions of multiple cell types in sickle cell disease (SCD). We previously showed that anti-P-selectin aptamer efficiently inhibits cell adhesion to endothelial cells (ECs) and permits SCD mice to survive hypoxic stress. In an effort to discover new mechanisms with which to inhibit P-selectin, we examined the role of glycosylation. P-selectin is a 90 kDa protein but was found to migrate as 90 and 140 kDa bands on gel electrophoresis. When P-selectin isolated from ECs was digested with peptide N-glycosidase F, but not O-glycosidase, the 140 kDa band was lost and the 90 kDa band was enhanced. Treatment of ECs with tunicamycin, an N-glycosylation inhibitor, suppressed CD62P (P-selectin) expression on the cell surface as well as the 140 kDa form in the cytoplasm. These results indicate that the 140 kDa band is N-glycosylated and glycosylation is critical for cell surface expression of P-selectin in ECs. Thrombin, which stimulates P-selectin expression on ECs, induced AKT phosphorylation, whereas tunicamycin inhibited AKT phosphorylation, suggesting that AKT signaling is involved in the tunicamycin-mediated inhibition of P-selectin expression. Importantly, the adhesion of sickle red blood cells (sRBCs) and leukocytes to ECs induced by thrombin or hypoxia was markedly inhibited by two structurally distinct glycosylation inhibitors; the levels of which were comparable to that of a P-selectin monoclonal antibody which most strongly inhibited cell adhesion in vivo. Knockdown studies of P-selectin using short-hairpin RNAs in ECs suppressed sRBC adhesion, indicating a legitimate role for P-selectin in sRBC adhesion. Together, these results demonstrate that P-selectin expression on ECs is regulated in part by glycosylation mechanisms and that glycosylation inhibitors efficiently reduce the adhesion of sRBCs and leukocytes to ECs. Glycosylation inhibitors may lead to a novel therapy which inhibits cell adhesion in SCD.

Critical role of the WASF3 gene in JAK2/STAT3 regulation of cancer cell motility.

WASF3 has been shown to be required for invasion and metastasis in different cancer cell types and knockdown of WASF3 leads to suppression of invasion/metastasis. Aberrant signaling through the interleukin 6/Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) axis in cancer cells has emerged as a major mechanism for cancer progression. In this study, we demonstrate that interleukin 6 induces both WASF3 expression and phosphoactivation in breast and prostate cancer cell lines through the JAK2/STAT3 pathway in two different ways. First, we show that STAT3 binds directly to the WASF3 promoter and increases transcription levels, which correlates with increased migration potential. Inactivation of STAT3 with short hairpin RNA, dominant negative constructs or S3I-201 leads to reduced WASF3 levels and reduced migration. Second, we have shown that JAK2, while activating STAT3, also interacts with and activates WASF3. Inhibition of JAK2 with short hairpin RNA or AG490 leads to loss of migration due to reduced WASF3 activation levels and prevention of its membrane localization. Together, these results define a novel signaling network whereby JAK2/STAT3 signaling creates a feed-forward loop to raise activated WASF3 levels that promote cancer cell motility.

HIF1A induces expression of the WASF3 metastasis-associated gene under hypoxic conditions.

The WASF3 (WAVE3) gene is an important mediator of cell motility, invasion and metastasis and is expressed at high levels in some advanced stage tumors. In our survey of breast cancer cells, we now demonstrate that exposure to hypoxic conditions increases WASF3 expression levels in MDA231, SKBR3 and MCF7 cells. The WASF3 promoter region contains HIF1A response elements (HRE). ChIP assays demonstrate that HIF1A binds to these HRE elements in the promoter region, and luciferase reporter assays using the WASF3 gene minimal promoter shows that hypoxia results in its upregulation. Phosphorylation of WASF3 is required for its ability to affect invasion and increased phosphoactivation of WASF3 is also seen in cells challenged with hypoxia. These cells also show increased motility in the scratch wound assay. Cells in which WASF3 has been knocked down show no response to hypoxia as expected, implicating the specificity of the hypoxic response to WASF3. Overall, these experiments demonstrate WASF3 is a HIF1A-regulated gene and suggests a mechanism to explain the observation of elevated expression of WASF3 in advanced stage tumors.

ß-Cell-specific pyruvate dehydrogenase deficiency impairs glucose-stimulated insulin secretion.

Glucose-stimulated insulin secretion (GSIS) by ß-cells requires the generation of ATP from oxidation of pyruvate as well as generation of coupling factors involving three different pyruvate cycling shuttles. The roles of several key enzymes involved in pyruvate cycling in ß-cells have been documented using isolated islets and ß-cell clonal lines. To investigate the role of the pyruvate dehydrogenase (PDH) complex (PDC) in GSIS, a murine model of ß-cell-specific PDH deficiency (ß-PDHKO) was created. Pancreatic insulin content was decreased in 1-day-old ß-PDHKO male pups and adult male mice. The plasma insulin levels were decreased and blood glucose levels increased in ß-PDHKO male mice from neonatal life onward. GSIS was reduced in isolated islets from ß-PDHKO male mice with about 50% reduction in PDC activity. Impairment in a glucose tolerance test and in vivo insulin secretion during hyperglycemic clamp was evident in ß-PDHKO adults. No change in the number or size of islets was found in pancreata from 4-wk-old ß-PDHKO male mice. However, an increase in the mean size of individual ß-cells in islets of these mice was observed. These findings show a key role of PDC in GSIS by pyruvate oxidation. This ß-PDHKO mouse model represents the first mouse model in which a mitochondrial oxidative enzyme deletion by gene knockout has been employed to demonstrate an altered GSIS by ß-cells.

Liver-specific pyruvate dehydrogenase complex deficiency upregulates lipogenesis in adipose tissue and improves peripheral insulin sensitivity.

The pyruvate dehydrogenase complex (PDC) plays a critical role in lipid synthesis and glucose homeostasis in the fed and fasting states. The central role of the liver in the maintenance of glucose homeostasis has been established by studying changes in key enzymes (including PDC) and the carbon-flux via several pathways under different metabolic states. In the present study we have developed a murine model of liver-specific PDC deficiency using Cre-loxP technology to investigate its consequences on lipid and carbohydrate metabolism. There was no incorporation of glucose-carbon into fatty acids by liver in vitro from liver-specific Pdha1 knockout (L-PDHKO) male mice due to absence of hepatic PDC activity. Interestingly, there was a compensatory increase in lipogenic capacity in epididymal adipose tissue from L-PDHKO mice. Both fat and lean body mass were significantly reduced in L-PDHKO mice, which might be explained by an increase in total energy expenditure compared with wild-type littermate mice. Furthermore, both liver and peripheral insulin sensitivities measured during a hyperinsulinemic-euglycemic clamp were improved in L-PDHKO mice. The findings presented here demonstrate (i) the indispensable role of PDC for lipogenesis from glucose in liver and (ii) specific adaptations in lipid and glucose metabolism in the liver and adipose tissue to compensate for loss of PDC activity in liver only.

Loss of the SMRT/NCoR2 corepressor correlates with JAG2 overexpression in multiple myeloma.

Multiple myeloma (MM) is a clonal B-cell neoplasm that accounts for 10% of all malignant hematologic neoplasms and that affects terminally differentiated B cells (i.e., plasma cells). It is now well recognized that the cytokine interleukin-6 (IL-6) is a major cytokine that promotes the proliferation of malignant plasma cells in MM. The IL-6 gene can be regulated by the NOTCH genes products. We have previously shown that the NOTCH ligand, JAG2, is overexpressed in MM. To investigate the mechanism(s) leading to JAG2 overexpression in MM, we assessed potential epigenetic modifications of the JAG2 promoter. We showed that the JAG2 promoter region is aberrantly acetylated in MM cell lines and patient samples. The acetylation state of histones is regulated by the recruitment of histone deacetylases (HDAC). HDACs are typically recruited to promoter regions through interaction with nuclear corepressors such as SMRT. SMRT levels were therefore investigated. Interestingly, MM cell lines and patient samples presented significantly reduced SMRT levels. The experiments suggest a correlation between constitutive acetylation of the JAG2 core promoter in the MM cell lines and reduced levels of the SMRT corepressor that recruits HDAC to promoter regions. Finally, SMRT function restoration induced JAG2 down-regulation as well as MM cell apoptosis.

Alteration of SMRT tumor suppressor function in transformed non-Hodgkin lymphomas.

Indolent non-Hodgkin lymphomas are characterized by a prolonged phase that is typically followed by a clinical progression associated with an accelerated clinical course and short survival time. Previous studies have not identified a consistent cytogenetic or molecular abnormality associated with transformation. The development of a transformed phenotype, evolving from the original low-grade component, most likely depends on multiple genetic events, including the activation of synergistic dominant oncogenes and a loss of tumor suppressor gene functions. Complex karyotypes and relatively bad chromosome morphology are typical of transformed non-Hodgkin lymphomas, rendering complete cytogenetic analysis difficult. Here, we report the use of transformed non-Hodgkin lymphoma cell lines and primary samples to identify the involvement of the silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) gene that maps at chromosome 12q24 in transformed non-Hodgkin lymphomas. We also show that down-regulation of SMRT in the immortalized "Weinberg's model" cell lines induces transformation of the cells. Assessment of cDNA array profiles should further help us to design a working model for SMRT involvement in non-Hodgkin lymphoma transformation as a novel, nonclassical tumor suppressor.