γ-Linolenic acid in maternal milk drives cardiac metabolic maturation.

Birth presents a metabolic challenge to cardiomyocytes as they reshape fuel preference from glucose to fatty acids for postnatal energy production1,2. This adaptation is triggered in part by post-partum environmental changes3, but the molecules orchestrating cardiomyocyte maturation remain unknown. Here we show that this transition is coordinated by maternally supplied γ-linolenic acid (GLA), an 18:3 omega-6 fatty acid enriched in the maternal milk. GLA binds and activates retinoid X receptors4 (RXRs), ligand-regulated transcription factors that are expressed in cardiomyocytes from embryonic stages. Multifaceted genome-wide analysis revealed that the lack of RXR in embryonic cardiomyocytes caused an aberrant chromatin landscape that prevented the induction of an RXR-dependent gene expression signature controlling mitochondrial fatty acid homeostasis. The ensuing defective metabolic transition featured blunted mitochondrial lipid-derived energy production and enhanced glucose consumption, leading to perinatal cardiac dysfunction and death. Finally, GLA supplementation induced RXR-dependent expression of the mitochondrial fatty acid homeostasis signature in cardiomyocytes, both in vitro and in vivo. Thus, our study identifies the GLA-RXR axis as a key transcriptional regulatory mechanism underlying the maternal control of perinatal cardiac metabolism.

DIDO is necessary for the adipogenesis that promotes diet-induced obesity.

The prevalence of overweight and obesity continues to rise in the population worldwide. Because it is an important predisposing factor for cancer, cardiovascular diseases, diabetes mellitus, and COVID-19, obesity reduces life expectancy. Adipose tissue (AT), the main fat storage organ with endocrine capacity, plays fundamental roles in systemic metabolism and obesity-related diseases. Dysfunctional AT can induce excess or reduced body fat (lipodystrophy). Dido1 is a marker gene for stemness; gene-targeting experiments compromised several functions ranging from cell division to embryonic stem cell differentiation, both in vivo and in vitro. We report that mutant mice lacking the DIDO N terminus show a lean phenotype. This consists of reduced AT and hypolipidemia, even when mice are fed a high-nutrient diet. DIDO mutation caused hypothermia due to lipoatrophy of white adipose tissue (WAT) and dermal fat thinning. Deep sequencing of the epididymal white fat (Epi WAT) transcriptome supported Dido1 control of the cellular lipid metabolic process. We found that, by controlling the expression of transcription factors such as C/EBPα or PPARγ, Dido1 is necessary for adipocyte differentiation, and that restoring their expression reestablished adipogenesis capacity in Dido1 mutants. Our model differs from other lipodystrophic mice and could constitute a new system for the development of therapeutic intervention in obesity.

Dido3-dependent SFPQ recruitment maintains efficiency in mammalian alternative splicing.

Alternative splicing is facilitated by accessory proteins that guide spliceosome subunits to the primary transcript. Many of these splicing factors recognize the RNA polymerase II tail, but SFPQ is a notable exception even though essential for mammalian RNA processing. This study reveals a novel role for Dido3, one of three Dido gene products, in alternative splicing. Binding of the Dido3 amino terminus to histones and to the polymerase jaw domain was previously reported, and here we show interaction between its carboxy terminus and SFPQ. We generated a mutant that eliminates Dido3 but preserves other Dido gene products, mimicking reduced Dido3 levels in myeloid neoplasms. Dido mutation suppressed SFPQ binding to RNA and increased skipping for a large group of exons. Exons bearing recognition sequences for alternative splicing factors were nonetheless included more efficiently. Reduced SFPQ recruitment may thus account for increased skipping of SFPQ-dependent exons, but could also generate a splicing factor surplus that becomes available to competing splice sites. Taken together, our data indicate that Dido3 is an adaptor that controls SFPQ utilization in RNA splicing. Distributing splicing factor recruitment over parallel pathways provides mammals with a simple mechanism to regulate exon usage while maintaining RNA splicing efficiency.

Filamin A interaction with the CXCR4 third intracellular loop regulates endocytosis and signaling of WT and WHIM-like receptors.

Warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome is a rare congenital immunodeficiency often caused by mutations in the last 10 to 19 C-terminal amino acids of CXCR4. These mutations impair CXCR4 internalization and increase responsiveness to CXCL12. The CXCR4 C-terminal domain (C-tail) also has a binding site for the actin-binding protein filamin A (FLNA); it is not known whether FLNA binds to WHIM CXCR4 mutants or whether this interaction is implicated in the hyperfunction of these receptors. Here we show that, in addition to interacting with the CXCR4 C-tail, FLNA interacted with a region in the receptor third intracellular loop (ICL3) spanning amino acids 238 to 246. This interaction involved specific FLNA repeats and was sensitive to Rho kinase inhibition. Deletion of the 238-246 motif accelerated CXCL12-induced wild-type (WT) receptor endocytosis but enabled CXCL12-mediated endocytosis and normalized signaling by the WHIM-associated receptor CXCR4(R334X). CXCL12 stimulation triggered CXCR4(R334X) internalization in FLNA-deficient M2 cells but not in the FLNA-expressing M2 subclone A7; this suggests a role for FLNA in stabilization of WHIM-like CXCR4 at the cell surface. FLNA increased ß-arrestin2 binding to CXCR4(R334X) in vivo, which provides a molecular basis for FLNA-mediated hyperactivation of WHIM receptor signaling. We propose that FLNA interaction with ICL3 is central for endocytosis and signaling of WT and WHIM-like CXCR4 receptors.

Autoimmune kidney disease and impaired engulfment of apoptotic cells in mice with macrophage peroxisome proliferator-activated receptor gamma or retinoid X receptor alpha deficiency.

Autoimmune glomerulonephritis is a common manifestation of systemic lupus erythematosus (SLE). In this study, we show that mice lacking macrophage expression of the heterodimeric nuclear receptors PPARγ or RXRα develop glomerulonephritis and autoantibodies to nuclear Ags, resembling the nephritis seen in SLE. These mice show deficiencies in phagocytosis and clearance of apoptotic cells, and they are unable to acquire an anti-inflammatory phenotype upon feeding of apoptotic cells, which is critical for the maintenance of self-tolerance. These results demonstrate that stimulation of PPARγ and RXRα in macrophages facilitates apoptotic cell engulfment, and they provide a potential strategy to avoid autoimmunity against dying cells and to attenuate SLE.

Retinoid X receptor alpha controls innate inflammatory responses through the up-regulation of chemokine expression.

The retinoid X receptor alpha (RXRalpha) plays a central role in the regulation of many intracellular receptor signaling pathways and can mediate ligand-dependent transcription by forming homodimers or heterodimers with other nuclear receptors. Although several members of the nuclear hormone receptor superfamily have emerged as important regulators of macrophage gene expression, the existence in vivo of an RXR signaling pathway in macrophages has not been established. Here, we provide evidence that RXRalpha regulates the transcription of the chemokines Ccl6 and Ccl9 in macrophages independently of heterodimeric partners. Mice lacking RXRalpha in myeloid cells exhibit reduced levels of CCL6 and CCL9, impaired recruitment of leukocytes to sites of inflammation, and lower susceptibility to sepsis. These studies demonstrate that macrophage RXRalpha plays key roles in the regulation of innate immunity and represents a potential target for immunotherapy of sepsis.

Discrimination between alternate membrane protein topologies in living cells using GFP/YFP tagging and pH exchange.

Membrane protein function is determined by the relative organization of the protein domains with respect to the membrane. We have experimentally verified the topology of a protein with diverse orientations arising from a single primary sequence (the cellular prion protein, PrP(C)), a novel somatostatin truncated receptor, and the Golgi-associated protein GPBP(91). Tagging with fluorescent proteins (FP) allows location of their expression at the plasma membrane or at endomembranes, but does not inform about their orientation. Exploiting the pH dependency of some FPs, we developed a pH exchange assay in which extracellularly exposed FPs are quenched by application of low pH buffer. We constructed standards to demonstrate and calibrate the assay, and the method was adapted for acidic organelle membrane proteins. This method can serve as a proof of concept, experimentally confirming and/or discriminating in living cells among theoretical topology predictions, providing the proportion of inside/outside orientation for proteins with multiple forms.

Hu antigen R is required for NOX-1 but not NOX-4 regulation by inflammatory stimuli in vascular smooth muscle cells.

OBJECTIVE: NOX-1 and NOX-4 are key enzymes responsible for reactive oxygen species (ROS) generation in vascular smooth muscle cells (VSMC). The RNA-binding protein Hu antigen R (HuR) is implicated in posttranscriptional regulation of gene expression; however, its role regulating NOX is unknown. We investigated transcriptional and posttranscriptional mechanisms underlying angiotensin II (AngII) and IL-1ß regulation of NOX-1 and NOX-4 in VSMC and their implications in cell migration. METHODS: Rat and human VSMC were stimulated with AngII (0.1 µmol/l) and/or IL-1ß (10 ng/ml). NOX-1 and NOX-4 mRNA and protein levels, NOX-1 and NOX-4 promoter and 3'UTR activities, NADPH oxidase activity, ROS production, and cell migration were studied. RESULTS: IL-1ß increased NOX-1 expression, NADPH oxidase activity and ROS production, and decreased NOX-4 expression and H2O2 production in VSMC. AngII potentiated the IL-1ß-mediated induction of NOX-1 expression, NADPH oxidase activity, ROS production, and cell migration. However, AngII did not influence IL-1ß-induced NOX-4 downregulation. AngII + IL-1ß interfered with the decay of NOX-1 mRNA and promoted HuR binding to NOX-1 mRNA. Moreover, HuR blockade reduced NOX-1 mRNA stability and AngII + IL-1ß-induced NOX-1 mRNA levels. IL-1ß decreased NOX-4 expression through a transcriptional mechanism that involved response elements situated in the proximal promoter. AngII and/or IL-1ß-induced cell migration were prevented by NOX-1 and HuR blockade and were augmented by NOX-4 overexpression. CONCLUSION: In VSMC HuR-mediated mRNA stabilization is partially responsible for AngII + IL-1ß-dependent NOX-1 expression, whereas transcriptional mechanisms are involved in decreased NOX-4 expression induced by IL-1ß. NOX4 and HuR regulation of NOX-1 contributes to VSMC migration, important in vascular inflammation and remodeling.

SUMO-SIM interactions regulate the activity of RGSZ2 proteins.

The RGSZ2 gene, a regulator of G protein signaling, has been implicated in cognition, Alzheimer's disease, panic disorder, schizophrenia and several human cancers. This 210 amino acid protein is a GTPase accelerating protein (GAP) on Gαi/o/z subunits, binds to the N terminal of neural nitric oxide synthase (nNOS) negatively regulating the production of nitric oxide, and binds to the histidine triad nucleotide-binding protein 1 at the C terminus of different G protein-coupled receptors (GPCRs). We now describe a novel regulatory mechanism of RGS GAP function through the covalent incorporation of Small Ubiquitin-like MOdifiers (SUMO) into RGSZ2 RGS box (RH) and the SUMO non covalent binding with SUMO-interacting motifs (SIM): one upstream of the RH and a second within this region. The covalent attachment of SUMO does not affect RGSZ2 binding to GPCR-activated GαGTP subunits but abolishes its GAP activity. By contrast, non-covalent binding of SUMO with RH SIM impedes RGSZ2 from interacting with GαGTP subunits. Binding of SUMO to the RGSZ2 SIM that lies outside the RH does not affect GαGTP binding or GAP activity, but it could lead to regulatory interactions with sumoylated proteins. Thus, sumoylation and SUMO-SIM interactions constitute a new regulatory mechanism of RGS GAP function and therefore of GPCR cell signaling as well.

Calnuc plays a role in dynamic distribution of Galphai but not Gbeta subunits and modulates ACTH secretion in AtT-20 neuroendocrine secretory cells.

In AtT-20 cells ACTH secretion is regulated by both Ca2+ and G proteins. We previously demonstrated that calnuc, an EF-hand Ca2+ binding protein which regulates Alzheimer's beta-amyloid precursor protein (APP) biogenesis, binds both Ca2+ as well as Galpha subunits. Here we investigate calnuc's role in G protein-mediated regulation of ACTH secretion in AtT-20 neuroendocrine secretory cells stably overexpressing calnuc-GFP. Similar to endogenous calnuc, calnuc-GFP is mainly found in the Golgi, on the plasma membrane (PM), and associated with regulated secretion granules (RSG). By deconvolution immunofluorescence, calnuc-GFP partially colocalizes with Galphai1/2 and Galphai3 at the PM and on RSG. Cytosolic calnuc(DeltaSS)-CFP with the signal sequence deleted also partially colocalizes with RSG and partially cosediments with Galphai1/2 in fractions enriched in RSG. Overexpression of calnuc-GFP specifically increases the distribution of Galphai1/2 on the PM whereas the distribution of Gbeta subunits and synaptobrevin 2 (Vamp 2) is unchanged. Overexpression of calnuc-GFP or cytosolic calnuc(DeltaSS)-CFP enhances ACTH secretion two-fold triggered by mastoparan or GTPgammaS but does not significantly affect glycosaminoglycan (GAG) chain secretion along the constitutive pathway or basal secretion of ACTH. Calnuc's facilitating effects on ACTH secretion are decreased after introducing anti-Galphai1/2, Galphai3, Gbeta or calnuc IgG into permeabilized cells but not when Galpha12 or preimmune IgG is introduced. The results suggest that calnuc binds to Galpha subunits on the Golgi and on RSG and that overexpression of calnuc causes redistribution of Galphai subunits to the PM and RSG, indicating that calnuc plays a role in dynamic distribution of only Galpha but not Gbeta subunits. Thus calnuc may connect G protein signaling and calcium signaling during regulated secretion.

Identification and characterization of GIV, a novel Galpha i/s-interacting protein found on COPI, endoplasmic reticulum-Golgi transport vesicles.

In this report, we characterize GIV (Galpha-interacting vesicle-associated protein), a novel protein that binds members of the Galpha(i) and Galpha subfamilies of heterotrimeric G proteins. The Galpha(s) interaction site was mapped to an 83-amino acid region of GIV that is enriched in highly charged amino acids. BLAST searches revealed two additional mammalian family members, Daple and an uncharacterized protein, FLJ00354. These family members share the highest homology at the Galpha binding domain, are homologous at the N terminus and central coiled coil domain but diverge at the C terminus. Using affinity-purified IgG made against two different regions of the protein, we localized GIV to COPI, endoplasmic reticulum (ER)-Golgi transport vesicles concentrated in the Golgi region in GH3 pituitary cells and COS7 cells. Identification as COPI vesicles was based on colocalization with beta-COP, a marker for these vesicles. GIV also codistributes in the Golgi region with endogenous calnuc and the KDEL receptor, which are cis Golgi markers and with Galpha(i3)-yellow fluorescent protein expressed in COS7 cells. By immunoelectron microscopy, GIV colocalizes with beta-COP and Galpha(i3) on vesicles found in close proximity to ER exit sites and to cis Golgi cisternae. In cell fractions prepared from rat liver, GIV is concentrated in a carrier vesicle fraction (CV2) enriched in ER-Golgi transport vesicles. beta-COP and several Galpha subunits (Galpha(i1-3), Galpha(s)) are also most enriched in CV2. Our results demonstrate the existence of a novel Galpha-interacting protein associated with COPI transport vesicles that may play a role in Galpha-mediated effects on vesicle trafficking within the Golgi and/or between the ER and the Golgi.

Promotion of G alpha i3 subunit down-regulation by GIPN, a putative E3 ubiquitin ligase that interacts with RGS-GAIP.

We have isolated an RGS-GAIP interacting protein that links RGS proteins to protein degradation. GIPN (GAIP interacting protein N terminus) is a 38-kDa protein with an N-terminal leucine-rich region, a central RING finger-like domain, and a putative C-terminal transmembrane domain. GIPN binds exclusively to RGS proteins of subfamily A, RGS-GAIP, RGSZ1, and RGSZ2. The N-terminal leucine-rich region of GIPN interacts with the cysteine-rich motif of RGS-GAIP. GIPN mRNA is ubiquitously expressed, and GIPN is found on the plasma membrane of transfected HEK293 cells. Endogenous GIPN is concentrated along the basolateral plasma membrane of proximal and distal tubules in rat kidney, where many G protein-coupled receptors and some G proteins are also located. Two immunoreactive species are found in rat kidney, a 38-kDa cytosolic form and an approximately 94-kDa membrane form. GIPN shows Zn2+- and E1/E2-dependent autoubiquitination in vitro, suggesting that it has E3 ubiquitin ligase activity. Overexpression of GIPN stimulates proteasome-dependent reduction of endogenous G alpha i3 in HEK293 cells and reduces the half-life of overexpressed G alpha i3-YFP. Thus, our findings suggest that GIPN is involved in the degradation of G alpha i3 subunits via the proteasome pathway. RGS-GAIP functions as a bifunctional adaptor that binds to G alpha subunits through its RGS domain and to GIPN through its cysteine string motif.

Spatial regulation of Galphai protein signaling in clathrin-coated membrane microdomains containing GAIP.

Regulators of G-protein signaling (RGS) proteins are GTPase-activating proteins (GAPs) that bind to Galpha subunits and attenuate G protein signaling, but where these events occur in the cell is not yet established. Here we investigated, by immunofluorescence labeling and deconvolution analysis, the site at which endogenous Galpha-interacting protein (GAIP) (RGS19) binds to Galphai3-YFP and its fate after activation of delta-opioid receptor (DOR). In the absence of agonist, GAIP is spatially segregated from Galphai3 and DOR in clathrin-coated domains (CCPs) of the cell membrane (PM), whereas Galphai3-YPF and DOR are located in non-clathrin-coated microdomains of the PM. Upon addition of agonist, Galphai3 partially colocalizes with GAIP in CCPs at the PM. When endocytosis is blocked by expression of a dynamin mutant [dyn(K44A)], there is a striking overlap in the distribution of DOR and Galphai3-YFP with GAIP in CCPs. Moreover, Galphai3-YFP and GAIP form a coprecipitable complex. Our results support a model whereby, after agonist addition, DOR and Galphai3 move together into CCPs where Galphai3 and GAIP meet and turn off G protein signaling. Subsequently, Galphai3 returns to non-clathrin-coated microdomains of the PM, GAIP remains stably associated with CCPs, and DOR is internalized via clathrin-coated vesicles. This constitutes a novel mechanism for regulation of Galpha signaling through spatial segregation of a GAP in clathrin-coated pits.