The associations of IGF2, IGF2R and IGF2BP2 gene polymorphisms with gestational diabetes mellitus: A case-control study.

OBJECTIVE: To investigate the associations of Insulin-like growth factor-II (IGF2) gene, Insulin-like growth factor-II receptor (IGF2R) gene and Insulin-like growth factor-II binding protein 2 (IGF2BP2) gene polymorphisms with the susceptibility to gestational diabetes mellitus (GDM) in Chinese population. METHODS: A total of 1703 pregnant women (835 GDM and 868 Non-GDM) were recruited in this case-control study. All participants underwent prenatal 75 g oral glucose tolerance test (OGTT) examinations during 24-28 gestational weeks at the Maternal and Child Health Hospital of Hubei Province from January 15, 2018 to March 31, 2019. Genotyping of candidate SNPs (IGF2 rs680, IGF2R rs416572, IGF2BP2 rs4402960, rs1470579, rs1374910, rs11705701, rs6777038, rs16860234, rs7651090) was performed on Sequenom MassARRAY platform. Logistic regression analysis was conducted to investigate the associations between candidate SNPs and risk of GDM. In addition, multifactor dimensionality reduction (MDR) method was applied to explore the effects of gene-gene interactions on GDM risk. RESULTS: There were significant distribution differences between GDM group and non-GDM group in age, pre-pregnancy BMI, education level and family history of diabetes (P < 0.05). After adjusted for age, pre-pregnancy BMI, education level and family history of diabetes, there were no significant associations of the candidate SNPs polymorphisms and GDM risk (P > 0.05). Furthermore, there were no gene-gene interactions on the GDM risk among the candidate SNPs (P > 0.05). However, the fasting blood glucose (FBG) levels of rs6777038 CT carriers were significantly lower than TT carriers (4.69±0.69 vs. 5.03±1.57 mmol/L, P < 0.01), and the OGTT-2h levels of rs6777038 CC and CT genotype carriers were significantly lower than TT genotype carriers (8.10±1.91 and 8.08±1.87 vs. 8.99±2.90 mmol/L, P < 0.01). CONCLUSIONS: IGF2 rs680, IGF2R rs416572, IGF2BP2 rs4402960, rs1470579, rs11705701, rs6777038, rs16860234, rs7651090 polymorphisms were not significantly associated with GDM risk in Wuhan, China. Further lager multicenter researches are needed to confirm these results.

Collapse of late endosomal pH elicits a rapid Rab7 response via the V-ATPase and RILP.

Endosomal-lysosomal trafficking is accompanied by the acidification of endosomal compartments by the H+-V-ATPase to reach low lysosomal pH. Disruption of the correct pH impairs lysosomal function and the balance of protein synthesis and degradation (proteostasis). Here, we treated mammalian cells with the small dipeptide LLOMe, which is known to permeabilize lysosomal membranes, and find that LLOMe also impacts late endosomes (LEs) by neutralizing their pH without causing membrane permeabilization. We show that LLOMe leads to hyperactivation of Rab7 (herein referring to Rab7a), and disruption of tubulation and mannose-6-phosphate receptor (CI-M6PR; also known as IGF2R) recycling on pH-neutralized LEs. pH neutralization (NH4Cl) and expression of Rab7 hyperactive mutants alone can both phenocopy the alterations in tubulation and CI-M6PR trafficking. Mechanistically, pH neutralization increases the assembly of the V1G1 subunit (encoded by ATP6V1G1) of the V-ATPase on endosomal membranes, which stabilizes GTP-bound Rab7 via RILP, a known interactor of Rab7 and V1G1. We propose a novel pathway by which V-ATPase and RILP modulate LE pH and Rab7 activation in concert. This pathway might broadly contribute to pH control during physiologic endosomal maturation or starvation and during pathologic pH neutralization, which occurs via lysosomotropic compounds and in disease states.

Golgi-associated retrograde protein (GARP) complex-dependent endosomes to trans Golgi network retrograde trafficking is controlled by Rab4b.

BACKGROUND: The trafficking of cargoes from endosomes to the trans-Golgi network requires numerous sequential and coordinated steps. Cargoes are sorted into endosomal-derived carriers that are transported, tethered, and fused to the trans-Golgi network. The tethering step requires several complexes, including the Golgi-associated retrograde protein complex, whose localization at the trans-Golgi network is determined by the activity of small GTPases of the Arl and Rab family. However, how the Golgi-associated retrograde protein complex recognizes the endosome-derived carriers that will fuse with the trans-Golgi network is still unknown. METHODS: We studied the retrograde trafficking to the trans-Golgi network by using fluorescent cargoes in cells overexpressing Rab4b or after Rab4b knocked-down by small interfering RNA in combination with the downregulation of subunits of the Golgi-associated retrograde protein complex. We used immunofluorescence and image processing (Super Resolution Radial Fluctuation and 3D reconstruction) as well as biochemical approaches to characterize the consequences of these interventions on cargo carriers trafficking. RESULTS: We reported that the VPS52 subunit of the Golgi-associated retrograde protein complex is an effector of Rab4b. We found that overexpression of wild type or active Rab4b increased early endosomal to trans-Golgi network retrograde trafficking of the cation-independent mannose-6-phosphate receptor in a Golgi-associated retrograde protein complex-dependent manner. Conversely, overexpression of an inactive Rab4b or Rab4b knockdown attenuated this trafficking. In the absence of Rab4b, the internalized cation-independent mannose 6 phosphate receptor did not have access to VPS52-labeled structures that look like endosomal subdomains and/or endosome-derived carriers, and whose subcellular distribution is Rab4b-independent. Consequently, the cation-independent mannose-6-phosphate receptor was blocked in early endosomes and no longer had access to the trans-Golgi network. CONCLUSION: Our results support that Rab4b, by controlling the sorting of the cation-independent mannose-6-phosphate receptor towards VPS52 microdomains, confers a directional specificity for cargo carriers en route to the trans-Golgi network. Given the importance of the endocytic recycling in cell homeostasis, disruption of the Rab4b/Golgi-associated retrograde protein complex-dependent step could have serious consequences in pathologies.

Prenatal arsenic exposure alters keratinocyte stem cell fate through persistent activation of IGF2R-MAPK cascade leading to aggravated skin carcinogenesis in mice offspring.

Chronic exposure to arsenic (As) promotes skin carcinogenesis in humans and potentially disturbs resident stem cell dynamics, particularly during maternal and early life exposure. In the present study, we demonstrate how only prenatal arsenic exposure disturbs keratinocyte stem cell (KSC) conditioning using a BALB/c mice model. Prenatal As exposure alters the normal stemness (CD34, KRT5), differentiation (Involucrin), and proliferation (PCNA) program in skin of offspring with progression of age as observed at 2, 10, and 18 weeks. Primary KSCs isolated from exposed animal at Day-2 showed increased survival (Bax:Bcl-xL, TUNEL assay), proliferation (BrdU), and differentiation (KRT5, Involucrin) potential through the activation of pro-carcinogenic IGF2R-MAPK cascade (IGF2R-G(α)q-MEK1-ERK1/2). This was associated with reduced enrichment of histone H3K27me3 and its methylase, EZH2 along with increased binding of demethylase, KDM6A at Igf2r promoter. Altered KSCs conditioning through disturbed Igf2r imprint contributed to impaired proliferation and differentiation and an aggravated tumor response in offspring.

AP-1γ2 is an adaptor protein 1 variant required for endosome-to-Golgi trafficking of the mannose-6-P receptor (CI-MPR) and ATP7B copper transporter.

Selective retrograde transport from endosomes back to the trans-Golgi network (TGN) is important for maintaining protein homeostasis, recycling receptors, and returning molecules that were transported to the wrong compartments. Two important transmembrane proteins directed to this pathway are the Cation-Independent Mannose-6-phosphate receptor (CI-MPR) and the ATP7B copper transporter. Among CI-MPR functions is the delivery of acid hydrolases to lysosomes, while ATP7B facilitates the transport of cytosolic copper ions into organelles or the extracellular space. Precise subcellular localization of CI-MPR and ATP7B is essential for the proper functioning of these proteins. This study shows that both CI-MPR and ATP7B interact with a variant of the clathrin adaptor 1 (AP-1) complex that contains a specific isoform of the γ-adaptin subunit called γ2. Through synchronized anterograde trafficking and cell-surface uptake assays, we demonstrated that AP-1γ2 is dispensable for ATP7B and CI-MPR exit from the TGN while being critically required for ATP7B and CI-MPR retrieval from endosomes to the TGN. Moreover, AP-1γ2 depletion leads to the retention of endocytosed CI-MPR in endosomes enriched in retromer complex subunits. These data underscore the importance of AP-1γ2 as a key component in the sorting and trafficking machinery of CI-MPR and ATP7B, highlighting its essential role in the transport of proteins from endosomes.

Elucidating the cellular determinants of targeted membrane protein degradation by lysosome-targeting chimeras.

Targeted protein degradation can provide advantages over inhibition approaches in the development of therapeutic strategies. Lysosome-targeting chimeras (LYTACs) harness receptors, such as the cation-independent mannose 6-phosphate receptor (CI-M6PR), to direct extracellular proteins to lysosomes. In this work, we used a genome-wide CRISPR knockout approach to identify modulators of LYTAC-mediated membrane protein degradation in human cells. We found that disrupting retromer genes improved target degradation by reducing LYTAC recycling to the plasma membrane. Neddylated cullin-3 facilitated LYTAC-complex lysosomal maturation and was a predictive marker for LYTAC efficacy. A substantial fraction of cell surface CI-M6PR remains occupied by endogenous M6P-modified glycoproteins. Thus, inhibition of M6P biosynthesis increased the internalization of LYTAC-target complexes. Our findings inform design strategies for next-generation LYTACs and elucidate aspects of cell surface receptor occupancy and trafficking.

Harnessing the Lysosomal Sorting Signals of the Cation-Independent Mannose-6-Phosphate Receptor for Targeted Degradation of Membrane Proteins.

Membrane proteins are a crucial class of therapeutic targets that remain challenging to modulate using traditional occupancy-driven inhibition strategies or current proteolysis-targeting degradation approaches. Here, we report that the inherent endolysosomal sorting machinery can be harnessed for the targeted degradation of membrane proteins. A new degradation technique, termed signal-mediated lysosome-targeting chimeras (SignalTACs), was developed by genetically fusing the signaling motif from the cation-independent mannose-6-phosphate receptor (CI-M6PR) to a membrane protein binder. Antibody-based SignalTACs were constructed with the CI-M6PR signal peptides fused to the C-terminus of both heavy and light chains of IgG. We demonstrated the scope of this platform technology by degrading five pathogenesis-related membrane proteins, including HER2, EGFR, PD-L1, CD20, and CD71. Furthermore, two simplified constructs of SignalTACs, nanobody-based and peptide-based SignalTACs, were created and shown to promote the lysosomal degradation of target membrane proteins. Compared to the parent antibodies, SignalTACs exhibited significantly higher efficiency in inhibiting tumor cell growth both in vitro and in vivo. This work provides a simple, general, and robust strategy for degrading membrane proteins with molecular precision and may represent a powerful platform with broad research and therapeutic applications.

Loss of the batten disease protein CLN3 leads to mis-trafficking of M6PR and defective autophagic-lysosomal reformation.

Batten disease, one of the most devastating types of neurodegenerative lysosomal storage disorders, is caused by mutations in CLN3. Here, we show that CLN3 is a vesicular trafficking hub connecting the Golgi and lysosome compartments. Proteomic analysis reveals that CLN3 interacts with several endo-lysosomal trafficking proteins, including the cation-independent mannose 6 phosphate receptor (CI-M6PR), which coordinates the targeting of lysosomal enzymes to lysosomes. CLN3 depletion results in mis-trafficking of CI-M6PR, mis-sorting of lysosomal enzymes, and defective autophagic lysosomal reformation. Conversely, CLN3 overexpression promotes the formation of multiple lysosomal tubules, which are autophagy and CI-M6PR-dependent, generating newly formed proto-lysosomes. Together, our findings reveal that CLN3 functions as a link between the M6P-dependent trafficking of lysosomal enzymes and lysosomal reformation pathway, explaining the global impairment of lysosomal function in Batten disease.

Sortilin knock-down alters the expression and distribution of cathepsin D and prosaposin and up-regulates the cation-dependent mannose-6-phosphate receptor in rat epididymal cells.

The selective transport to lysosomes can be mediated by either mannose-6-phosphate receptors (CD-MPR and CI-MPR) or sortilin. In mammalian epididymis, some lysosomal proteins are secreted into the lumen through unknown mechanisms. To investigate the underlying mechanisms of lysosomal protein transport in epididymal cells we studied the expression and distribution of cathepsin D (CatD) and prosaposin (PSAP) in a sortilin knocked down RCE-1 epididymal cell line (RCE-1 KD) in comparison with non-transfected RCE-1 cells. In RCE-1 cells, CatD was found in the perinuclear zone and co-localize with sortilin, whereas in RCE-1 KD cells, the expression, distribution and processing of the enzyme were altered. In turn, PSAP accumulated intracellularly upon sortilin knock-down and redistributed from LAMP-1-positive compartment to a perinuclear location, remaining co-localized with CatD. Interestingly, the sortilin knock-down induced CD-MPR overexpression and a redistribution of the receptor from the perinuclear zone to a dispersed cytoplasmic location, accompanied by an increased co-localization with CatD. The increase in CD-MPR could result from a compensatory response for the proper delivery of CatD to lysosomes in epididymal cells. The intracellular pathway taken by lysosomal proteins could be an approach for addressing further studies to understand the mechanism of exocytosis and therefore the role of these proteins in the epididymis.

Characterization of IGF2R Molecular Expression in Canine Osteosarcoma as Part of a Novel Comparative Oncology Approach.

Progress in prognostic factors, treatments, and outcome for both canine and human osteosarcoma (OS) has been minimal over the last three decades. Surface overexpression of the cation independent mannose-6-phosphate/insulin-like growth factor receptor type 2 (IGF2R) has been proven to occur in human OS cells. Subsequently, radioimmunotherapy (RIT) targeting IGF2R has demonstrated promising preliminary results. The main aims of this study were to investigate the expression of IGF2R in spontaneously occurring canine OS cells using immunohistochemistry (IHC) on archived biopsy samples and to assess its prognostic significance. Thirty-four dogs were included in the study. All cases showed that 80-100% of OS cells stained positive for IGF2R. IGF2R overexpression alone was not shown to have prognostic significance using both visual and quantitative methods of IHC staining intensity. This study has established for the first time the consistent expression of IGF2R in spontaneously occurring canine OS. This comparative oncology approach will allow further investigation into RIT as a novel treatment modality; first in canines and then in humans with OS. In addition, further studies should be performed to assess the true prognostic significance of IGF2R overexpression.

LYSET/TMEM251- a novel key component of the mannose 6-phosphate pathway.

Degradation of macromolecules delivered to lysosomes by processes such as autophagy or endocytosis is crucial for cellular function. Lysosomes require more than 60 soluble hydrolases in order to catabolize such macromolecules. These soluble hydrolases are tagged with mannose6-phosphate (M6P) moieties in sequential reactions by the Golgi-resident GlcNAc-1-phosphotransferase complex and NAGPA/UCE/uncovering enzyme (N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase), which allows their delivery to endosomal/lysosomal compartments through trafficking mediated by cation-dependent and -independent mannose 6-phosphate receptors (MPRs). We and others recently identified TMEM251 as a novel regulator of the M6P pathway via independent genome-wide genetic screening strategies. We renamed TMEM251 to LYSET (lysosomal enzyme trafficking factor) to establish nomenclature reflective to this gene's function. LYSET is a Golgi-localized transmembrane protein important for the retention of the GlcNAc-1-phosphotransferase complex in the Golgi-apparatus. The current understanding of LYSET's importance regarding human biology is 3-fold: 1) highly pathogenic viruses that depend on lysosomal hydrolase activity require LYSET for infection. 2) The presence of LYSET is critical for cancer cell proliferation in nutrient-deprived environments in which extracellular proteins must be catabolized. 3) Inherited pathogenic alleles of LYSET can cause a severe inherited disease which resembles GlcNAc-1-phosphotransferase deficiency (i.e., mucolipidosis type II).Abbreviations: GlcNAc-1-PT: GlcNAc-1-phosphotransferase; KO: knockout; LSD: lysosomal storage disorder; LYSET: lysosomal enzyme trafficking factor; M6P: mannose 6-phosphate; MPRs: mannose-6-phosphate receptors, cation-dependent or -independent; MBTPS1/site-1 protease: membrane bound transcription factor peptidase, site 1; MLII: mucolipidosis type II; WT: wild-type.

Lower expression of the equine maternally imprinted gene IGF2R is related to the slow proliferation of hinny embryonic fibroblast in vitro.

BACKGROUND: Proliferation of embryonic fibroblasts under the same cell culture conditions, hinny embryonic fibroblasts (HiEFs) was slower than horse embryonic fibroblast (HEFs), donkey embryonic fibroblasts (DEFs) and mule embryonic fibroblasts (MuEFs). The imprinted genes IGF2 and IGF2R are important for cell proliferation. Therefore, we investigated whether the slower proliferation of HiEFs is related to an aberrant gene expression of IGF2 or its receptors or genes influencing the expression of the IGF2 system. METHODS AND RESULTS: Real-time polymerase chain reaction, immunofluorescence and cell starving experiment in HEFs, DEFs, MuEFs and HiEFs revealed that the slower proliferation of HiEF in vitro was related to its lower expression of IGF2R (P < 0.001). Moreover, quantification of allele-specific expression and bisulfate assay confirmed that in both MuEFs and HiEFs, IGF2R had normal maternal imprinting, implying that the imprint aberrant was not involved in the lower IGF2R expression in HiEFs. CONCLUSIONS: The reduction of IGF2R expression in HiEFs is associated with its slower proliferation in vitro.

Gimap5 promoted RSV degradation through interaction with M6PR.

Respiratory syncytial virus (RSV) is one of the main pathogens of viral pneumonia and bronchiolitis in infants and young children and life-threatening diseases among infants and young children. GTPases of the immune-associated protein family (GIMAP) are new family members of immune-associated GTPases. In recent years, much attention has been paid to the function of the GIMAP family in coping with infection and stress. Gimap5 is a member of the GIMAP family, which may be correlated with anti-infectious immunity. RT-qPCR, Western blot, and indirect immunofluorescence (IFA) were used to detect the expression of Gimap5, M6PR and IGF1R(the major RSV receptor). Transmission electron microscopy (TEM) was used to detect the degradation of RSV in Gimap5-overexpressed or -silent cell lines. Computer virtual screening was used to screen small molecule compounds targeting Gimap5 and the anti-RSV effects were explored through in vivo and in vitro experiments. GIMAP5 and M6PR were significantly downregulated after RSV infection. Gimap5 accelerated RSV degradation in lysosomes by interacting with M6PR, and further prevented RSV invasion by downregulating the expression of RSV surface receptor IGF1R. Three small molecule compounds targeting Gimap5 were confirmed to be the agonists of Gimap5. The three compounds effectively inhibited RSV infection and RSV-induced complications. Gimap5 promotes the degradation of RSV and its receptor through interacting with M6PR. Gimap5 agonists can effectively reduce RSV infection and RSV-induced complication in vivo and in vitro, which provides a new choice for the treatment of RSV.

The uncoating of EV71 in mature late endosomes requires CD-M6PR.

Enterovirus 71 (EV71) is one of the causative agents of hand-foot-and-mouth disease, which in some circumstances could lead to severe neurological diseases. Despite of its importance for human health, little is known about the early stages of EV71 infection. EV71 starts uncoating with its receptor, human scavenger receptor B2 (hSCARB2), at low pH. We show that EV71 was not targeted to lysosomes in human rhabdomyosarcoma cells overexpressing hSCARB2 and that the autophagic pathway is not essential for EV71 productive uncoating. Instead, EV71 was efficiently uncoated 30 min after infection in late endosomes (LEs) containing hSCARB2, mannose-6-phosphate receptor (M6PR), RAB9, bis(monoacylglycero)phosphate and lysosomal associated membrane protein 2 (LAMP2). Furthering the notion that mature LEs are crucial for EV71 uncoating, cation-dependent (CD)-M6PR knockdown impairs EV71 infection. Since hSCARB2 interacts with cation-independent (CI)-M6PR through M6P-binding sites and CD-M6PR also harbor a M6P-binding site, CD-M6PR is likely to play important roles in EV71 uncoating in LEs.

Aqueous extract of Solanum nigrum attenuates Angiotensin-II induced cardiac hypertrophy and improves cardiac function by repressing protein kinase C-ζ to restore HSF2 deSUMOlyation and Mel-18-IGF-IIR signaling suppression.

ETHNOPHARMACOLOGICAL RELEVANCE: Solanum nigrum, commonly known as Makoi or black shade has been traditionally used in Asian countries and other regions of world to treat liver disorders, diarrhoea, inflammatory conditions, chronic skin ailments (psoriasis and ringworm), fever, hydrophobia, painful periods, eye diseases, etc. It has been observed that S. nigrum contains substances, like steroidal saponins, total alkaloid, steroid alkaloid, and glycoprotein, which show anti-tumor activity. However; there is no scientific evidence of the efficacy of S. nigrum in the treatment of cardiac hypertrophy. AIM: To investigate the ability of S. nigrum to attenuate Angiotensin II - induced cardiac hypertrophy and improve cardiac function through the suppression of protein kinase PKC-ζ and Mel-18-IGF-IIR signaling leading to the restoration of HSF2 desumolyation. MATERIALS AND METHODS: Cardiomyoblast cells (H9c2) were challenged with 100 nM Angiotensin-II (AngII) for 24 h and were then treated with different concentration of S.nigrum or Calphostin C for 24 h. The hypertrophic effect in cardiomyoblast cells were determined by immunofluorescence staining and the modulations in hypertrophic protein marker along with Protein Kinase C-ζ, MEL18, HSF2, and Insulin like growth factor II (IGFIIR), markers were analyzed by western blotting. In vivo experiments were performed using 12 week old male Wistar Kyoto rats (WKY) and Spontaneously hypertensive rats (SHR) separated into five groups. [1]Control WKY, [2] WKY -100 mg/kg of S.nigrum treatment, [3] SHR, [4] SHR-100 mg/kg of S.nigrum treatment, [5] SHR-300 mg/kg of S.nigrum treatment. S. nigrum was administered intraperitoneally for 8 week time interval. RESULTS: Western blotting results indicate that S. nigrum significantly attenuates AngII induced cardiac hypertrophy. Furthermore, actin staining confirmed the ability of S. nigrum to ameliorate AngII induced cardiac hypertrophy. Moreover, S. nigrum administration suppressed the hypertrophic signaling mediators like Protein Kinase C-ζ, Mel-18, and IGFIIR in a dose-dependent manner and HSF2 activation (restore deSUMOlyation) that leads to downregulation of IGF-IIR expression. Additionally in vivo experiments demonstrate the reduced heart sizes of S. nigrum treated SHRs rats when compared to control WKY rats. CONCLUSION: Collectively, the data reveals the cardioprotective effect of S. nigrum inhibiting PKC-ζ with alleviated IGF IIR level in the heart that profoundly remits cardiac hypertrophy for hypertension-induced heart failure.

Association between genetic variants in oxidative stress-related genes and osteoporotic bone fracture. The Hortega follow-up study.

The most widely accepted etiopathogenesis hypothesis of the origin of osteoporosis and its complications is that they are a consequence of bone aging and other environmental factors, together with a genetic predisposition. Evidence suggests that oxidative stress is crucial in bone pathologies associated with aging. The aim of this study was to determine whether genetic variants in oxidative stress-related genes modified the risk of osteoporotic fracture. We analysed 221 patients and 354 controls from the HORTEGA sample after 12-14 years of follow up. We studied the genotypic and allelic distribution of 53 SNPs in 24 genes involved in oxidative stress. The results showed that being a carrier of the variant allele of the SNP rs4077561 within TXNRD1 was the principal genetic risk factor associated with osteoporotic fracture and that variant allele of the rs1805754 M6PR, rs4964779 TXNRD1, rs406113 GPX6, rs2281082 TXN2 and rs974334 GPX6 polymorphisms are important genetic risk factors for fracture. This study provides information on the genetic factors associated with oxidative stress which are involved in the risk of osteoporotic fracture and reinforces the hypothesis that genetic factors are crucial in the etiopathogenesis of osteoporosis and its complications.

Perturbed ER homeostasis by IGF-IIRα promotes cardiac damage under stresses.

The heart is a very dynamic pumping organ working perpetually to maintain a constant blood supply to the whole body to transport oxygen and nutrients. Unfortunately, it is also subjected to various stresses based on physiological or pathological conditions, particularly more vulnerable to damages caused by oxidative stress. In this study, we investigate the molecular mechanism and contribution of IGF-IIRα in endoplasmic reticulum stress induction in the heart under doxorubicin-induced cardiotoxicity. Using in vitro H9c2 cells, in vivo transgenic rat cardiac tissues, siRNAs against CHOP, chemical ER chaperone PBA, and western blot experiments, we found that IGF-IIRα overexpression enhanced ER stress markers ATF4, ATF6, IRE1α, and PERK which were further aggravated by DOX treatment. This was accompanied by a significant perturbation in stress-associated MAPKs such as p38 and JNK. Interestingly, PARKIN, a stress responsive cellular protective mediator was significantly downregulated by IGF-IIRα concomitant with decreased expression of ER chaperone GRP78. Furthermore, ER stress-associated pro-apoptotic factor CHOP was increased considerably in a dose-dependent manner followed by elevated c-caspase-12 and c-caspase-3 activities. Conversely, treatment of H9c2 cells with chemical ER chaperone PBA or siRNA against CHOP abolished the IGF-IIRα-induced ER stress responses. Altogether, these findings suggested that IGF-IIRα contributes to ER stress induction and inhibits cellular stress coping proteins while increasing pro-apoptotic factors feeding into a cardio myocyte damage program that eventually paves the way to heart failure.

Tanshinone IIA inhibits Leu27IGF-II-induced insulin-like growth factor receptor II signaling and myocardial apoptosis via estrogen receptor-mediated Akt activation.

Different stress condition stimulates the expression level of insulin-like growth factor receptor II (IGF-IIR) in cardiomyoblasts that lead to apoptosis. Tanshinone IIA (TSN), a pharmacologically active component from Danshen, has been shown cardioprotective effects against cardiac apoptosis induced by several stress conditions. Therefore, this study was conducted to assess the cardioprotective effects of TSN IIA mediated through the estrogen receptor (ER) in order to inhibit the Leu27IGF-II-enhanced IGF-IIR-mediated cardiac apoptosis. The estrogenic activity of TSN IIA was examined after myocardial cells were pretreated with the ER antagonist, and inhibited the phospho-inositide-3 kinase (PI3K). Here, we found that TSN IIA significantly induced ER that phosphorylated Akt. Further, Akt activation considerably suppressed the Leu27IGF-II induced IGF-IIR expression level and the downstream effectors, including Gαq and calcineurin as well as mitochondrial dependent apoptosis proteins including Bad, cytochrome c, and active caspase-3 that result in cardiac apoptosis resistance. However, the western blot analysis, JC-1 staining, and terminal deoxynucleotide transferase-mediated dUTP nick end labeling assay revealed that TSN IIA attenuated Leu27IGF-II-induced IGF-IIR mediated cardiac apoptosis was reversed by an ER antagonist such as ICI 182780, and PI3K inhibition. All these findings demonstrate that TSN IIA exerts estrogenic activity, which can activate PI3K-Akt pathway, and thereby inhibits Leu27IGFII induced IGF-IIR mediated cardiac apoptosis. Thus, TSN IIA can be considered as an effective therapeutic strategy against IGF-IIR signaling cascade to suppress cardiac apoptosis.

PI4P/PS countertransport by ORP10 at ER-endosome membrane contact sites regulates endosome fission.

Membrane contact sites (MCSs) serve as a zone for nonvesicular lipid transport by oxysterol-binding protein (OSBP)-related proteins (ORPs). ORPs mediate lipid countertransport, in which two distinct lipids are transported counterdirectionally. How such lipid countertransport controls specific biological functions, however, remains elusive. We report that lipid countertransport by ORP10 at ER-endosome MCSs regulates retrograde membrane trafficking. ORP10, together with ORP9 and VAP, formed ER-endosome MCSs in a phosphatidylinositol 4-phosphate (PI4P)-dependent manner. ORP10 exhibited a lipid exchange activity toward its ligands, PI4P and phosphatidylserine (PS), between liposomes in vitro, and between the ER and endosomes in situ. Cell biological analysis demonstrated that ORP10 supplies a pool of PS from the ER, in exchange for PI4P, to endosomes where the PS-binding protein EHD1 is recruited to facilitate endosome fission. Our study highlights a novel lipid exchange at ER-endosome MCSs as a nonenzymatic PI4P-to-PS conversion mechanism that organizes membrane remodeling during retrograde membrane trafficking.

The imprinted Igf2-Igf2r axis is critical for matching placental microvasculature expansion to fetal growth.

In all eutherian mammals, growth of the fetus is dependent upon a functional placenta, but whether and how the latter adapts to putative fetal signals is currently unknown. Here, we demonstrate, through fetal, endothelial, hematopoietic, and trophoblast-specific genetic manipulations in the mouse, that endothelial and fetus-derived IGF2 is required for the continuous expansion of the feto-placental microvasculature in late pregnancy. The angiocrine effects of IGF2 on placental microvasculature expansion are mediated, in part, through IGF2R and angiopoietin-Tie2/TEK signaling. Additionally, IGF2 exerts IGF2R-ERK1/2-dependent pro-proliferative and angiogenic effects on primary feto-placental endothelial cells ex vivo. Endothelial and fetus-derived IGF2 also plays an important role in trophoblast morphogenesis, acting through Gcm1 and Synb. Thus, our study reveals a direct role for the imprinted Igf2-Igf2r axis on matching placental development to fetal growth and establishes the principle that hormone-like signals from the fetus play important roles in controlling placental microvasculature and trophoblast morphogenesis.