Elamipretide Promotes Mitophagosome Formation and Prevents Its Reduction Induced by Nutrient Excess in INS1 β-cells

Elamipretide is a tetrapeptide that restores defects in mitochondrial function, binds to cardiolipin, and is being tested in clinical trials for mitochondria-related diseases. However, whether elamipretide modulates mitochondrial quality control and dynamics, processes essential to preserve mitochondrial function, is unclear. Thus, we tested the effects of elamipretide on mitochondrial morphology, mitophagosome formation, and their early disruption induced by excess nutrients in INS1 β-cells. Elamipretide treatment was sufficient to increase engulfment of mitochondria into autophagosomes in control INS1 β-cells, without inducing widespread changes in mitochondrial morphology or membrane potential. In an early pathogenic context mimicked by short-term exposure to nutrient excess, elamipretide treatment prevented both mitochondrial fragmentation and defects in the engulfment of mitochondria into autophagosomes. On the other hand, elamipretide did not prevent lysosomal defects induced by nutrient excess. Accordingly, elamipretide treatment did not entail benefits on pathogenic p62 and LC3II accumulation or on insulin secretory function. In conclusion, our data show that elamipretide selectively stimulates the engulfment of mitochondria into autophagosomes and prevents its defects induced by nutrient excess. Thus, we propose that improved selectivity of mitochondrial quality control processes might contribute to the benefits stemming from elamipretide treatments in other disease models.     

Fas-associated Factor 1 Is a Scaffold Protein That Promotes β-Transducin Repeat-containing Protein (β-TrCP)-mediated β-Catenin Ubiquitination and Degradation

FAS-associated factor 1 (FAF1) antagonizes Wnt signaling by stimulating β-catenin degradation. However, the molecular mechanism underlying this effect is unknown. Here, we demonstrate that the E3 ubiquitin ligase β-transducin repeat-containing protein (β-TrCP) is required for FAF1 to suppress Wnt signaling and that FAF1 specifically associates with the SCF (Skp1-Cul1-F-box protein)-β-TrCP complex. Depletion of β-TrCP reduced FAF1-mediated β-catenin polyubiquitination and impaired FAF1 in antagonizing Wnt/β-catenin signaling. FAF1 was shown to act as a scaffold for β-catenin and β-TrCP and thereby to potentiate β-TrCP-mediated β-catenin ubiquitination and degradation. Data mining revealed that FAF1 expression is statistically down-regulated in human breast carcinoma compared with normal breast tissue. Consistent with this, FAF1 expression is higher in epithelial-like MCF7 than mesenchymal-like MDA-MB-231 human breast cancer cells. Depletion of FAF1 in MCF7 cells resulted in increased β-catenin accumulation and signaling. Importantly, FAF1 knockdown promoted a decrease in epithelial E-cadherin and an increase in mesenchymal vimentin expression, indicative for an epithelial to mesenchymal transition. Moreover, ectopic FAF1 expression reduces breast cancer cell migration in vitro and invasion/metastasis in vivo. Thus, our studies strengthen a tumor-suppressive function for FAF1.     

The Nesprin Family Member ANC-1 Regulates Synapse Formation and Axon Termination by Functioning in a Pathway with RPM-1 and β-Catenin

Mutations in Nesprin-1 and 2 (also called Syne-1 and 2) are associated with numerous diseases including autism, cerebellar ataxia, cancer, and Emery-Dreifuss muscular dystrophy. Nesprin-1 and 2 have conserved orthologs in flies and worms called MSP-300 and abnormal nuclear Anchorage 1 (ANC-1), respectively. The Nesprin protein family mediates nuclear and organelle anchorage and positioning. In the nervous system, the only known function of Nesprin-1 and 2 is in regulation of neurogenesis and neural migration. It remains unclear if Nesprin-1 and 2 regulate other functions in neurons. Using a proteomic approach in C. elegans, we have found that ANC-1 binds to the Regulator of Presynaptic Morphology 1 (RPM-1). RPM-1 is part of a conserved family of signaling molecules called Pam/Highwire/RPM-1 (PHR) proteins that are important regulators of neuronal development. We have found that ANC-1, like RPM-1, regulates axon termination and synapse formation. Our genetic analysis indicates that ANC-1 functions via the β-catenin BAR-1, and the ANC-1/BAR-1 pathway functions cell autonomously, downstream of RPM-1 to regulate neuronal development. Further, ANC-1 binding to the nucleus is required for its function in axon termination and synapse formation. We identify variable roles for four different Wnts (LIN-44, EGL-20, CWN-1 and CWN-2) that function through BAR-1 to regulate axon termination. Our study highlights an emerging, broad role for ANC-1 in neuronal development, and unveils a new and unexpected mechanism by which RPM-1 functions.     

Stimulation of Bone Formation in Cortical Bone of Mice Treated with a Receptor Activator of Nuclear Factor-κB Ligand (RANKL)-binding Peptide That Possesses Osteoclastogenesis Inhibitory Activity

To date, parathyroid hormone is the only clinically available bone anabolic drug. The major difficulty in the development of such drugs is the lack of clarification of the mechanisms regulating osteoblast differentiation and bone formation. Here, we report a peptide (W9) known to abrogate osteoclast differentiation in vivo via blocking receptor activator of nuclear factor-κB ligand (RANKL)-RANK signaling that we surprisingly found exhibits a bone anabolic effect in vivo. Subcutaneous administration of W9 three times/day for 5 days significantly augmented bone mineral density in mouse cortical bone. Histomorphometric analysis showed a decrease in osteoclastogenesis in the distal femoral metaphysis and a significant increase in bone formation in the femoral diaphysis. Our findings suggest that W9 exerts bone anabolic activity. To clarify the mechanisms involved in this activity, we investigated the effects of W9 on osteoblast differentiation/mineralization in MC3T3-E1 (E1) cells. W9 markedly increased alkaline phosphatase (a marker enzyme of osteoblasts) activity and mineralization as shown by alizarin red staining. Gene expression of several osteogenesis-related factors was increased in W9-treated E1 cells. Addition of W9 activated p38 MAPK and Smad1/5/8 in E1 cells, and W9 showed osteogenesis stimulatory activity synergistically with BMP-2 in vitro and ectopic bone formation. Knockdown of RANKL expression in E1 cells reduced the effect of W9. Furthermore, W9 showed a weak effect on RANKL-deficient osteoblasts in alkaline phosphatase assay. Taken together, our findings suggest that this peptide may be useful for the treatment of bone diseases, and W9 achieves its bone anabolic activity through RANKL on osteoblasts accompanied by production of several autocrine factors.     

Maternal levels of pregnancy-specific β1 (SP-1) are elevated in pregnancies affected by Down's syndrome

Summary Concentrations of pregnancy-specific ₁-glycoprotein (SP-1) were measured in maternal blood and amniotic fluid of patients with a trisomic fetus and compared with that of a cytogenetically normal fetus at weeks 16–19 of pregnancy. The SP-1 concentrations were significantly elevated in the sera of women with a Down's syndrome fetus, whereas amniotic fluid levels were only slightly increased. It is suggested that high levels of maternal SP-1 in the second trimester of pregnancy may be a valuable indicator in the prenatal detection of fetal trisomy 21.     

Commensal Bacteria-induced Interleukin 1β (IL-1β) Secreted by Macrophages Up-regulates Hepcidin Expression in Hepatocytes by Activating the Bone Morphogenetic Protein Signaling Pathway

The liver hormone hepcidin is the central regulator of systemic iron metabolism. Its increased expression in inflammatory states leads to hypoferremia and anemia. Elucidation of the mechanisms that up-regulate hepcidin during inflammation is essential for developing rational therapies for this anemia. Using mouse models of inflammatory bowel disease, we have shown previously that colitis-associated hepcidin induction is influenced by intestinal microbiota composition. Here we investigate how two commensal bacteria, Bifidobacterium longum and Bacteroides fragilis, representative members of the gut microbiota, affect hepcidin expression. We found that supernatants of a human macrophage cell line infected with either of the bacteria up-regulated hepcidin when added to a human hepatocyte cell line. This activity was abrogated by neutralization of IL-1β. Moreover, purified IL-1β increased hepcidin expression when added to the hepatocyte line or primary human hepatocytes and when injected into mice. IL-1β activated the bone morphogenetic protein (BMP) signaling pathway in hepatocytes and in mouse liver, as indicated by increased phosphorylation of small mothers against decapentaplegic proteins. Activation of BMP signaling correlated with IL-1β-induced expression of BMP2 in human hepatocytes and activin B in mouse liver. Treatment of hepatocytes with two different chemical inhibitors of BMP signaling or with a neutralizing antibody to BMP2 prevented IL-1β-induced up-regulation of hepcidin. Our results clarify how commensal bacteria affect hepcidin expression and reveal a novel connection between IL-1β and activation of BMP signaling. They also suggest that there may be differences between mice and humans with respect to the mechanism by which IL-1β up-regulates hepcidin.     

Loss of cleavage at β'-site contributes to apparent increase in β-amyloid peptide (Aβ) secretion by β-secretase (BACE1)-glycosylphosphatidylinositol (GPI) processing of amyloid precursor protein

Several lines of evidence implicate lipid raft microdomains in Alzheimer disease-associated β-amyloid peptide (Aβ) production. Notably, targeting β-secretase (β-site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1)) exclusively to lipid rafts by the addition of a glycosylphosphatidylinositol (GPI) anchor to its ectodomain has been reported to elevate Aβ secretion. Paradoxically, Aβ secretion is not reduced by the expression of non-raft resident S-palmitoylation-deficient BACE1 (BACE1-4C/A (C474A/C478A/C482A/C485A)). We addressed this apparent discrepancy in raft microdomain-associated BACE1 processing of APP in this study. As previously reported, we found that expression of BACE1-GPI elevated Aβ secretion as compared with wild-type BACE1 (WTBACE1) or BACE1-4C/A. However, this increase occurred without any difference in the levels of APP ectodomain released following BACE1 cleavage (soluble APPβ), arguing against an overall increase in BACE1 processing of APP per se. Further analysis revealed that WTBACE1 cleaves APP at β- and β'-sites, generating +1 and +11 β-C-terminal fragments and secreting intact as well as N-terminally truncated Aβ. In contrast, three different BACE1-GPI chimeras preferentially cleaved APP at the β-site, mainly generating +1 β-C-terminal fragment and secreting intact Aβ. As a consequence, cells expressing BACE1-GPI secreted relatively higher levels of intact Aβ without an increase in BACE1 processing of APP. Markedly reduced cleavage at β'-site exhibited by BACE1-GPI was cell type-independent and insensitive to subcellular localization of APP or the pathogenic KM/NL mutant. We conclude that the apparent elevation in Aβ secretion by BACE1-GPI is mainly attributed to preferential cleavage at the β-site and failure to detect +11 Aβ species secreted by cells expressing WTBACE1.     

Roles of N-acetylglucosaminyltransferase III in epithelial-to-mesenchymal transition induced by transforming growth factor β1 (TGF-β1) in epithelial cell lines

The epithelial-to-mesenchymal transition (EMT) plays crucial roles in embryonic development, wound healing, tissue repair, and cancer progression. Results of this study show how transforming growth factor β1 (TGF-β1) down-regulates expression of N-acetylglucosaminyltransferase III (GnT-III) during EMT-like changes. Treatment with TGF-β1 resulted in a decrease in E-cadherin expression and GnT-III expression, as well as its product, the bisected N-glycans, which was confirmed by erythro-agglutinating phytohemagglutinin lectin blot and HPLC analysis in human MCF-10A and mouse GE11 cells. In contrast with GnT-III, the expression of N-acetylglucosaminyltransferase V was slightly enhanced by TGF-β1 treatment. Changes in the N-glycan patterns on α3β1 integrin, one of the target proteins for GnT-III, were also confirmed by lectin blot analysis. To understand the roles of GnT-III expression in EMT-like changes, the MCF-10A cell was stably transfected with GnT-III. It is of particular interest that overexpression of GnT-III influenced EMT-like changes induced by TGF-β1, which was confirmed by cell morphological changes of phase contrast, immunochemical staining patterns of E-cadherin, and actin. In addition, GnT-III modified E-cadherin, which served to prolong E-cadherin turnover on the cell surface examined by biotinylation and pulse-chase experiments. GnT-III expression consistently inhibited β-catenin translocation from cell-cell contact into the cytoplasm and nucleus. Furthermore, the transwell assay showed that GnT-III expression suppressed TGF-β1-induced cell motility. Taken together, these observations are the first to clearly demonstrate that GnT-III affects cell properties, which in turn influence EMT-like changes, and to explain a molecular mechanism for the inhibitory effects of GnT-III on cancer metastasis.     

The smooth muscle-type β1 subunit potentiates activation by DiBAC4(3) in recombinant BK channels

Large-conductance Ca²⁺-activated (BK) channels, expressed in a variety of tissues, play a fundamental role in regulating and maintaining arterial tone. We recently demonstrated that the slow voltage indicator DiBAC₄(3) does not depend, as initially proposed, on the β₁ or β₄ subunits to activate native arterial smooth muscle BK channels. Using recombinant mslo BK channels, we now show that the β₁ subunit is not essential to this activation but exerts a large potentiating effect. DiBAC₄(3) promotes concentration-dependent activation of BK channels and slows deactivation kinetics, changes that are independent of Ca²⁺. K_d values for BK channel activation by DiBAC₄(3) in 0 mM Ca²⁺ are approximately 20 μM (α) and 5 μM (α+β₁), and G-V curves shift up to −40mV and −110 mV, respectively. β₁ to β₂ mutations R11A and C18E do not interfere with the potentiating effect of the subunit. Our findings should help refine the role of the β₁ subunit in cardiovascular pharmacology.     

The smooth muscle-type β1 subunit potentiates activation by DiBAC4(3) in recombinant BK channels

Large-conductance Ca²⁺-activated (BK) channels, expressed in a variety of tissues, play a fundamental role in regulating and maintaining arterial tone. We recently demonstrated that the slow voltage indicator DiBAC₄(3) does not depend, as initially proposed, on the β₁ or β₄ subunits to activate native arterial smooth muscle BK channels. Using recombinant mslo BK channels, we now show that the β₁ subunit is not essential to this activation but exerts a large potentiating effect. DiBAC₄(3) promotes concentration-dependent activation of BK channels and slows deactivation kinetics, changes that are independent of Ca²⁺. K_d values for BK channel activation by DiBAC₄(3) in 0 mM Ca²⁺ are approximately 20 μM (α) and 5 μM (α+β₁), and G-V curves shift up to ?40mV and ?110 mV, respectively. β₁ to β₂ mutations R11A and C18E do not interfere with the potentiating effect of the subunit. Our findings should help refine the role of the β₁ subunit in cardiovascular pharmacology.