Integrated multi-omic characterization of congenital heart disease.

The heart, the first organ to develop in the embryo, undergoes complex morphogenesis that when defective results in congenital heart disease (CHD). With current therapies, more than 90% of patients with CHD survive into adulthood, but many suffer premature death from heart failure and non-cardiac causes1. Here, to gain insight into this disease progression, we performed single-nucleus RNA sequencing on 157,273 nuclei from control hearts and hearts from patients with CHD, including those with hypoplastic left heart syndrome (HLHS) and tetralogy of Fallot, two common forms of cyanotic CHD lesions, as well as dilated and hypertrophic cardiomyopathies. We observed CHD-specific cell states in cardiomyocytes, which showed evidence of insulin resistance and increased expression of genes associated with FOXO signalling and CRIM1. Cardiac fibroblasts in HLHS were enriched in a low-Hippo and high-YAP cell state characteristic of activated cardiac fibroblasts. Imaging mass cytometry uncovered a spatially resolved perivascular microenvironment consistent with an immunodeficient state in CHD. Peripheral immune cell profiling suggested deficient monocytic immunity in CHD, in agreement with the predilection in CHD to infection and cancer2. Our comprehensive phenotyping of CHD provides a roadmap towards future personalized treatments for CHD.

Conserved NIMA kinases regulate multiple steps of endocytic trafficking.

Human NIMA-related kinases have primarily been studied for their roles in cell cycle progression (NEK1/2/6/7/9), checkpoint-DNA-damage control (NEK1/2/4/5/10/11), and ciliogenesis (NEK1/4/8). We previously showed that Caenorhabditis elegans NEKL-2 (NEK8/9 homolog) and NEKL-3 (NEK6/7 homolog) regulate apical clathrin-mediated endocytosis (CME) in the worm epidermis and are essential for molting. Here we show that NEKL-2 and NEKL-3 also have distinct roles in controlling endosome function and morphology. Specifically, loss of NEKL-2 led to enlarged early endosomes with long tubular extensions but showed minimal effects on other compartments. In contrast, NEKL-3 depletion caused pronounced defects in early, late, and recycling endosomes. Consistently, NEKL-2 was strongly localized to early endosomes, whereas NEKL-3 was localized to multiple endosomal compartments. Loss of NEKLs also led to variable defects in the recycling of two resident cargoes of the trans-Golgi network (TGN), MIG-14/Wntless and TGN-38/TGN38, which were missorted to lysosomes after NEKL depletion. In addition, defects were observed in the uptake of clathrin-dependent (SMA-6/Type I BMP receptor) and independent cargoes (DAF-4/Type II BMP receptor) from the basolateral surface of epidermal cells after NEKL-2 or NEKL-3 depletion. Complementary studies in human cell lines further showed that siRNA knockdown of the NEKL-3 orthologs NEK6 and NEK7 led to missorting of the mannose 6-phosphate receptor from endosomes. Moreover, in multiple human cell types, depletion of NEK6 or NEK7 disrupted both early and recycling endosomal compartments, including the presence of excess tubulation within recycling endosomes, a defect also observed after NEKL-3 depletion in worms. Thus, NIMA family kinases carry out multiple functions during endocytosis in both worms and humans, consistent with our previous observation that human NEKL-3 orthologs can rescue molting and trafficking defects in C. elegans nekl-3 mutants. Our findings suggest that trafficking defects could underlie some of the proposed roles for NEK kinases in human disease.

The mouse retinal pigment epithelium mounts an innate immune defense response following retinal detachment.

The retinal pigment epithelium (RPE) maintains photoreceptor viability and function, completes the visual cycle, and forms the outer blood-retinal barrier (oBRB). Loss of RPE function gives rise to several monogenic retinal dystrophies and contributes to age-related macular degeneration. Retinal detachment (RD) causes separation of the neurosensory retina from the underlying RPE, disrupting the functional and metabolic relationships between these layers. Although the retinal response to RD is highly studied, little is known about how the RPE responds to loss of this interaction. RNA sequencing (RNA-Seq) was used to compare normal and detached RPE in the C57BL6/J mouse. The naïve mouse RPE transcriptome was compared to previously published RPE signature gene lists and from the union of these 14 genes (Bmp4, Crim1, Degs1, Gja1, Itgav, Mfap3l, Pdpn, Ptgds, Rbp1, Rnf13, Rpe65, Slc4a2, Sulf1 and Ttr) representing a core signature gene set applicable across rodent and human RPE was derived. Gene ontology enrichment analysis (GOEA) of the mouse RPE transcriptome identified expected RPE features and functions, such as pigmentation, phagocytosis, lysosomal and proteasomal degradation of proteins, and barrier function. Differentially expressed genes (DEG) at 1 and 7 days post retinal detachment (dprd) were defined as mRNA with a significant (padj≤0.05) fold change (FC) of 0.67 ≥ FC ≥ 1.5 in detached versus naïve RPE. The RPE transcriptome exhibited dramatic changes at 1 dprd, with 2297 DEG identified. The KEGG pathways and biological process GO groups related to innate immune responses were significantly enriched. Lipocalin 2 (Lcn2) and several chemokines were upregulated, while numerous genes related to RPE functions, such as pigment synthesis, visual cycle, phagocytosis, and tight junctions were downregulated at 1 dprd. The response was largely transient, with only 18 significant DEG identified at 7 dprd, including upregulation of complement gene C4b. Validation studies confirmed RNA-Seq results. Thus, the RPE quickly downregulates cell-specific functions and mounts an innate immune defense response following RD. Our data demonstrate that the RPE contributes to the inflammatory response to RD and may play a role in attraction of immune cells to the subretinal space.

BMP heterodimers signal via distinct type I receptor class functions.

Heterodimeric TGF-ß ligands outperform homodimers in a variety of developmental, cell culture, and therapeutic contexts; however, the mechanisms underlying this increased potency remain uncharacterized. Here, we use dorsal-ventral axial patterning of the zebrafish embryo to interrogate the BMP2/7 heterodimer signaling mechanism. We demonstrate that differential interactions with BMP antagonists do not account for the reduced signaling ability of homodimers. Instead, we find that while overexpressed BMP2 homodimers can signal, they require two nonredundant type I receptors, one from the Acvr1 subfamily and one from the Bmpr1 subfamily. This implies that all BMP signaling within the zebrafish gastrula, even BMP2 homodimer signaling, requires Acvr1. This is particularly surprising as BMP2 homodimers do not bind Acvr1 in vitro. Furthermore, we find that the roles of the two type I receptors are subfunctionalized within the heterodimer signaling complex, with the kinase activity of Acvr1 being essential, while that of Bmpr1 is not. These results suggest that the potency of the Bmp2/7 heterodimer arises from the ability to recruit both Acvr1 and Bmpr1 into the same signaling complex.

Cationic proteins from eosinophils bind bone morphogenetic protein receptors promoting vascular calcification and atherogenesis.

AIMS: Blood eosinophil count and eosinophil cationic protein (ECP) concentration are risk factors of cardiovascular diseases. This study tested whether and how eosinophils and ECP contribute to vascular calcification and atherogenesis. METHODS AND RESULTS: Immunostaining revealed eosinophil accumulation in human and mouse atherosclerotic lesions. Eosinophil deficiency in ΔdblGATA mice slowed atherogenesis with increased lesion smooth muscle cell (SMC) content and reduced calcification. This protection in ΔdblGATA mice was muted when mice received donor eosinophils from wild-type (WT), Il4-/-, and Il13-/- mice or mouse eosinophil-associated-ribonuclease-1 (mEar1), a murine homologue of ECP. Eosinophils or mEar1 but not interleukin (IL) 4 or IL13 increased the calcification of SMC from WT mice but not those from Runt-related transcription factor-2 (Runx2) knockout mice. Immunoblot analyses showed that eosinophils and mEar1 activated Smad-1/5/8 but did not affect Smad-2/3 activation or expression of bone morphogenetic protein receptors (BMPR-1A/1B/2) or transforming growth factor (TGF)-ß receptors (TGFBR1/2) in SMC from WT and Runx2 knockout mice. Immunoprecipitation showed that mEar1 formed immune complexes with BMPR-1A/1B but not TGFBR1/2. Immunofluorescence double-staining, ligand binding, and Scatchard plot analysis demonstrated that mEar1 bound to BMPR-1A and BMPR-1B with similar affinity. Likewise, human ECP and eosinophil-derived neurotoxin (EDN) also bound to BMPR-1A/1B on human vascular SMC and promoted SMC osteogenic differentiation. In a cohort of 5864 men from the Danish Cardiovascular Screening trial and its subpopulation of 394 participants, blood eosinophil counts and ECP levels correlated with the calcification scores of different arterial segments from coronary arteries to iliac arteries. CONCLUSION: Eosinophils release cationic proteins that can promote SMC calcification and atherogenesis using the BMPR-1A/1B-Smad-1/5/8-Runx2 signalling pathway.

FKBP12 inhibits hepcidin expression by modulating BMP receptors interaction and ligand responsiveness in hepatocytes.

The expression of the iron regulatory hormone hepcidin in hepatocytes is regulated by the BMP-SMAD pathway through the type I receptors ALK2 and ALK3, the type II receptors ACVR2A and BMPR2, and the ligands BMP2 and BMP6. We previously identified the immunophilin FKBP12 as a new hepcidin inhibitor that acts by blocking ALK2. Both the physiologic ALK2 ligand BMP6 and the immunosuppressive drug Tacrolimus (TAC) displace FKBP12 from ALK2 and activate the signaling. However, the molecular mechanism whereby FKBP12 regulates BMP-SMAD pathway activity and thus hepcidin expression remains unclear. Here, we show that FKBP12 acts by modulating BMP receptor interactions and ligand responsiveness. We first demonstrate that in primary murine hepatocytes TAC regulates hepcidin expression exclusively via FKBP12. Downregulation of the BMP receptors reveals that ALK2, to a lesser extent ALK3, and ACVR2A are required for hepcidin upregulation in response to both BMP6 and TAC. Mechanistically, TAC and BMP6 increase ALK2 homo-oligomerization and ALK2-ALK3 hetero-oligomerization and the interaction between ALK2 and the type II receptors. By acting on the same receptors, TAC and BMP6 cooperate in BMP pathway activation and hepcidin expression both in vitro and in vivo. Interestingly, the activation state of ALK3 modulates its interaction with FKBP12, which may explain the cell-specific activity of FKBP12. Overall, our results identify the mechanism whereby FKBP12 regulates the BMP-SMAD pathway and hepcidin expression in hepatocytes, and suggest that FKBP12-ALK2 interaction is a potential pharmacologic target in disorders caused by defective BMP-SMAD signaling and characterized by low hepcidin and high BMP6 expression.

Silencing of long non-coding RNA SDCBP2-AS1/microRNA-656-3p/CRIM1 axis promotes ferroptosis of lung cancer cells.

Long non-coding RNAs (lncRNAs) play central roles in lung cancer progression by acting as competing endogenous RNAs (ceRNAs). This study aimed to explore the roles of lncRNA SDCBP2-AS1 in lung cancer and the molecular mechanism. The expression of SDCBP2-AS1, microRNA (miR)-656-3p, and cysteine-rich transmembrane BMP regulator 1 (CRIM1) was measured using quantitative real-time polymerase chain reaction. Ferroptosis was evaluated by analyzing cell death, ferrous content, reactive oxygen species (ROS) level, and protein levels of ferroptosis markers. The binding relationship was assessed using a dual-luciferase reporter assay. We observed that SDCBP2-AS1 was highly expressed in lung cancer cells. Knockdown of SDCBP2-AS1 promoted ferroptosis of lung cancer cells. SDCBP2-AS1 is a sponge of miR-656-3p, which directly targets CRIM1. Rescue experiments confirmed that SDCBP2-AS1 regulates ferroptosis by miR-656-3p, and overexpression of CRIM1 abrogated the effects of miR-656-3p on ferroptosis. In conclusion, depletion of SDCBP2-AS1 promoted lung cancer cell ferroptosis via the miR-656-3p/CRIM1 axis.

Axin2-CreERT2 mediated knockout of bone morphogenetic protein receptor type 1A caused abnormal secondary dentine and altered cell fate of Axin2-expressing odontogenic cells.

AIM: Inducing odontogenic differentiation and tubular dentine formation is extremely important in dentine repair and tooth regeneration. Bone morphogenic proteins (BMPs) signalling plays a critical role in dentine development and tertiary dentine formation, whilst how BMPR1A-mediated signalling affects odontoblastic differentiation of Axin2-expressing (Axin2+ ) odontogenic cells and tubular dentine formation remains largely unknown. This study aims to reveal the cellular and molecular mechanisms involved in the formation of secondary dentine. METHODOLOGY: Axin2lacZ/+ mice harvested at post-natal 21 (P21) were used to map Axin2+ mesenchymal cells. Axin2CreERT2/+ ; R26RtdTomato/+ mice and Axin2CreERT2/+ ; R26RDTA/+ ; R26RtdTomato/+ mice were generated to observe the tempo-spatial distribution pattern of Axin2-lineage cells and the effect of ablation of Axin2+ cells on dentinogenesis, respectively. A loss-of-function model was established with Axin2CreERT2/+ ; Bmpr1afl/fl ; R26RtdTomato/+ (cKO) mice to study the role of BMP signalling in regulating Axin2+ cells. Micro-computed tomography, histologic and immunostainings, and other approaches were used to examine biological functions, including dentine formation, mineralization and cell differentiation in cKO mice. RESULTS: The results showed rich expression of Axin2 in odontoblasts at P21. Lineage tracing assay confirmed the wide distribution of Axin2 lineage cells in odontoblast layer and dental pulp during secondary dentine formation (P23 to P56), suggesting that Axin2+ cells are important cell source of primary odontoblasts. Ablation of Axin2+ cells (DTA mice) significantly impaired secondary dentine formation characterized with notably reduced dentine thickness (Mean of control: 54.11 µm, Mean of DTA: 27.79 µm, p = .0101). Furthermore, malformed osteo-dentine replaced the tubular secondary dentine in the absence of Bmpr1a with irregular cell morphology, abnormal cellular process formation and lack of cell-cell tight conjunction. Remarkably increased expression of osteogenic markers like Runx2 and DMP1 was detected, whilst DSP expression was observed in a dispersed manner, indicating an impaired odontogenic cell fate and failure in producing tubular dentine in cKO mice. CONCLUSIONS: Axin2+ cells are a critical population of primary odontoblasts which contribute to tubular secondary dentine formation, and BMP signalling pathway plays a vital role in maintaining the odontogenic fate of Axin2+ cells.

Competition between type I activin and BMP receptors for binding to ACVR2A regulates signaling to distinct Smad pathways.

BACKGROUND: Activins and bone morphogenetic proteins (BMPs) play critical, sometimes opposing roles, in multiple physiological and pathological processes and diseases. They signal to distinct Smad branches; activins signal mainly to Smad2/3, while BMPs activate mainly Smad1/5/8. This gives rise to the possibility that competition between the different type I receptors through which activin and BMP signal for common type II receptors can provide a mechanism for fine-tuning the cellular response to activin/BMP stimuli. Among the transforming growth factor-ß superfamily type II receptors, ACVR2A/B are highly promiscuous, due to their ability to interact with different type I receptors (e.g., ALK4 vs. ALK2/3/6) and with their respective ligands [activin A (ActA) vs. BMP9/2]. However, studies on complex formation between these full-length receptors situated at the plasma membrane, and especially on the potential competition between the different activin and BMP type I receptors for a common activin type II receptor, were lacking. RESULTS: We employed a combination of IgG-mediated patching-immobilization of several type I receptors in the absence or presence of ligands with fluorescence recovery after photobleaching (FRAP) measurements on the lateral diffusion of an activin type II receptor, ACVR2A, to demonstrate the principle of competition between type I receptors for ACVR2. Our results show that ACVR2A can form stable heteromeric complexes with ALK4 (an activin type I receptor), as well as with several BMP type I receptors (ALK2/3/6). Of note, ALK4 and the BMP type I receptors competed for binding ACVR2A. To assess the implications of this competition for signaling output, we first validated that in our cell model system (U2OS cells), ACVR2/ALK4 transduce ActA signaling to Smad2/3, while BMP9 signaling to Smad1/5/8 employ ACVR2/ALK2 or ACVR2/ALK3. By combining ligand stimulation with overexpression of a competing type I receptor, we showed that differential complex formation of distinct type I receptors with a common type II receptor balances the signaling to the two Smad branches. CONCLUSIONS: Different type I receptors that signal to distinct Smad pathways (Smad2/3 vs. Smad1/5/8) compete for binding to common activin type II receptors. This provides a novel mechanism to balance signaling between Smad2/3 and Smad1/5/8.

Heterodimer-heterotetramer formation mediates enhanced sensor activity in a biophysical model for BMP signaling.

Numerous stages of organismal development rely on the cellular interpretation of gradients of secreted morphogens including members of the Bone Morphogenetic Protein (BMP) family through transmembrane receptors. Early gradients of BMPs drive dorsal/ventral patterning throughout the animal kingdom in both vertebrates and invertebrates. Growing evidence in Drosophila, zebrafish, murine and other systems suggests that BMP ligand heterodimers are the primary BMP signaling ligand, even in systems in which mixtures of BMP homodimers and heterodimers are present. Signaling by heterodimers occurs through a hetero-tetrameric receptor complex comprising of two distinct type one BMP receptors and two type II receptors. To understand the system dynamics and determine whether kinetic assembly of heterodimer-heterotetramer BMP complexes is favored, as compared to other plausible BMP ligand-receptor configurations, we developed a kinetic model for BMP tetramer formation based on current measurements for binding rates and affinities. We find that contrary to a common hypothesis, heterodimer-heterotetramer formation is not kinetically favored over the formation of homodimer-tetramer complexes under physiological conditions of receptor and ligand concentrations and therefore other mechanisms, potentially including differential kinase activities of the formed heterotetramer complexes, must be the cause of heterodimer-heterotetramer signaling primacy. Further, although BMP complex assembly favors homodimer and homomeric complex formation over a wide range of parameters, ignoring these signals and instead relying on the heterodimer improves the range of morphogen interpretation in a broad set of conditions, suggesting a performance advantage for heterodimer signaling in patterning multiple cell types in a gradient.

MicroRNA26 attenuates vascular smooth muscle maturation via endothelial BMP signalling.

As small regulatory transcripts, microRNAs (miRs) act as genetic 'fine tuners' of posttranscriptional events, and as genetic switches to promote phenotypic switching. The miR miR26a targets the BMP signalling effector, smad1. We show that loss of miR26a leads to hemorrhage (a loss of vascular stability) in vivo, suggesting altered vascular differentiation. Reduction in miR26a levels increases smad1 mRNA and phospho-Smad1 (pSmad1) levels. We show that increasing BMP signalling by overexpression of smad1 also leads to hemorrhage. Normalization of Smad1 levels through double knockdown of miR26a and smad1 rescues hemorrhage, suggesting a direct relationship between miR26a, smad1 and vascular stability. Using an in vivo BMP genetic reporter and pSmad1 staining, we show that the effect of miR26a on smooth muscle differentiation is non-autonomous; BMP signalling is active in embryonic endothelial cells, but not in smooth muscle cells. Nonetheless, increased BMP signalling due to loss of miR26a results in an increase in acta2-expressing smooth muscle cell numbers and promotes a differentiated smooth muscle morphology. Similarly, forced expression of smad1 in endothelial cells leads to an increase in smooth muscle cell number and coverage. Furthermore, smooth muscle phenotypes caused by inhibition of the BMP pathway are rescued by loss of miR26a. Taken together, our data suggest that miR26a modulates BMP signalling in endothelial cells and indirectly promotes a differentiated smooth muscle phenotype. Our data highlights how crosstalk from BMP-responsive endothelium to smooth muscle is important for smooth muscle differentiation.

Potential receptors in Fenneropenaeus merguiensis ovary and role of saxophone, the bone morphogenetic protein receptor, in ovarian development.

Receptors, which play an initial role in signaling pathways in several physiological processes, including reproduction, are among the several molecular factors that control ovarian development in organisms. This study aimed to identify and study receptors potentially involved in controlling the reproductive process of female banana shrimp, Fenneropenaeus merguiensis. Ovarian transcriptomes derived from 4 developmental stages were generated by RNA sequencing. A total of 53,763 transcripts were obtained from the de novo assembled transcriptome, and 663 genes were identified as receptors. Among them, 185 receptors were differentially expressed during ovarian development. Fifteen of these differentially expressed receptors showed distinct expression patterns that were validated by RT-qPCR. Bone morphogenetic protein receptors (BMPR) and their signaling genes were investigated for their roles in shrimp vitellogenesis. The expressions of F. merguiensis saxophone (FmSax), a BMP type I receptor, and BMP type II receptor (FmBMPRII) as well as FmMad, FmMed, and FmSMAD3 were significantly altered during ovarian development. RNA interference was used to investigate the role of FmSax in vitellogenesis. The result indicated that the expression of vitellogenin (Vg) was significantly reduced in both ovary and hepatopancreas of FmSax-knockdown shrimp compared to control shrimp. Furthermore, in FmSax-silencing shrimp, FmBMPRII, FmMad, and FmMed expressions were decreased as well as Vg expression. These findings suggest that FmSax positively regulates Vg synthesis via the BMP signaling pathway.

Age-Related Low Bone Mineral Density in C57BL/6 Mice Is Reflective of Aberrant Bone Morphogenetic Protein-2 Signaling Observed in Human Patients Diagnosed with Osteoporosis.

Osteoporosis (OP) is a bone disorder characterized by decreased bone mineral density (BMD). Bone Morphogenetic Protein-2 (BMP-2) injections are used to promote bone formation in OP patients. However, patients are unresponsive to BMP-2 while displaying an upregulation of BMP Receptor Type 1a (BMPRIa) and protein kinase CK2α (CK2α). A synthetically produced peptide named casein kinase 2.3 (CK2.3) utilizes the BMP-signaling pathway as it enhances osteogenesis of primary osteoblasts isolated from OP patients, whereas BMP-2 does not. Although shown in OP patients, there is currently no reliable mouse model to study BMP-2 and CK2.3 signaling. In this publication, we show that BMPRIa was required for CK2.3-mediated osteogenesis in C2C12 cells with a CRISPR-Cas9-mediated gene knockout for BMPRIa. We utilized the C57BL/6 (B6) mouse strain as an aging-model to study aberrant BMP-2 signaling, demonstrating that, like OP patients, in 15 and 20-month mice, BMP-2 did not increase bone growth and displayed upregulated BMPRIa and CK2α protein expression. Furthermore, CK2.3 enhanced osteogenesis and decreased osteoclastogenesis in all age groups, whereas BMP-2 only increased mineralization in 6-month mice while increasing osteoclast formation in all age groups. These data demonstrated that aging B6 mice were a reliable model and mimicked data obtained from OP patients.

USP4 inhibits SMAD4 monoubiquitination and promotes activin and BMP signaling.

SMAD4 is a common intracellular effector for TGF-ß family cytokines, but the mechanism by which its activity is dynamically regulated is unclear. We demonstrated that ubiquitin-specific protease (USP) 4 strongly induces activin/BMP signaling by removing the inhibitory monoubiquitination from SMAD4. This modification was triggered by the recruitment of the E3 ligase, SMURF2, to SMAD4 following ligand-induced regulatory (R)-SMAD-SMAD4 complex formation. Whereas the interaction of the negative regulator c-SKI inhibits SMAD4 monoubiquitination, the ligand stimulates the recruitment of SMURF2 to the c-SKI-SMAD2 complex and triggers c-SKI ubiquitination and degradation. Thus, SMURF2 has a role in termination and initiation of TGF-ß family signaling. An increase in monoubiquitinated SMAD4 in USP4-depleted mouse embryonic stem cells (mESCs) decreased both the BMP- and activin-induced changes in the embryonic stem cell fate. USP4 sustained SMAD4 activity during activin- and BMP-mediated morphogenic events in early zebrafish embryos. Moreover, zebrafish depleted of USP4 exhibited defective cell migration and slower coordinated cell movement known as epiboly, both of which could be rescued by SMAD4. Therefore, USP4 is a critical determinant of SMAD4 activity.

CircCRIM1 promotes ovarian cancer progression by working as ceRNAs of CRIM1 and targeting miR-383-5p/ZEB2 axis.

BACKGROUND: Ovarian cancer is the leading cause of death in patients with gynecologic cancer, and circular RNAs (circRNAs) are involved in cancer progression. However, there are limited studies on the roles of circRNAs in ovarian cancer. METHODS: We designed divergent and convergent primers, used sanger sequencing and RNase R digestion to verify the source of circCRIM1. We detected the expression of circCRIM1 and its parental gene cysteine rich transmembrane BMP regulator 1 (CRIM1) in ovarian cancer and normal ovarian samples via qRT-PCR. MTT viability assay, apoptosis assay, wound healing assay and invasion assay were used to investigate the function of circCRIM1 and CRIM1 in ovarian cancer cell lines OVCAR3 and CAOV3. Mice xenografts experiment was performed. Bioinformatics predicted the microRNAs that bond with circCRIM1 and CRIM1, and dual luciferase reporter system confirmed it. Rescue experiments of microRNAs mimics transfection on the basis of circCRIM1 over-expression were carried out to uncover the mechanism by which circCRIM1 played cancer-promoting roles in ovarian cancer. RESULTS: CircCRIM1 was derived from CRIM1 by back-splicing. CircCRIM1 and CRIM1 had higher expression in ovarian cancer than in normal ovarian tissues, and both of them promoted ovarian cancer progression in vitro. In vivo circCRIM1 promoted the growth of tumors. CircCRIM1 and CRIM1 had a positive correlation relationship in the same cohort of ovarian cancer tissues. Bioinformatics predicted and dual luciferase assay confirmed circCRIM1 and CRIM1 bond with miR-145-5p, and circCRIM1 bond with miR-383-5p additionally. CircCRIM1 positively affected the expression of CRIM1. After circCRIM1 was over-expressed, miR-145-5p mimics transfection reversed the expression of CRIM1. Western blot discovered circCRIM1 positively affected the expression of zinc finger E-box binding homeobox 2 (ZEB2). Rescue experiments found miR-383-5p mimics reversed ZEB2 expression and the cancer-promoting effects of circCRIM1. CONCLUSIONS: CircCRIM1 bond with miR-145-5p to work as competing endogenous RNA (ceRNA) of CRIM1, and circCRIM1 bond with miR-383-5p to improve the expression of ZEB2 in ovarian cancer. CircCRIM1 and CRIM1 promoted the ovarian cancer progression and supplied a novel insight into the researches of ovarian cancer.

A high level of lncFGD5-AS1 inhibits epithelial-to-Mesenchymal transition by regulating the miR-196a-5p/SMAD6/BMP axis in gastric Cancer.

BACKGROUND: Long non-coding RNA (lncRNA) was a vital factor in the progression and initiation of human cancers. This study found a new lncRNA, FGD5-AS1, which can inhibit EMT process, proliferation, and metastasis in vitro and in vivo. METHODS: qRT-PCR was employed to test the expression of lncFGD5-AS1 in 30 gastric cancer patients' cancer tissue and para-cancer tissue. Overexpressed lncFGD5-AS1 cells shown sharply decrease of proliferation, migration, and epithelial-mesenchymal transition (EMT). miR-196a-5p/SMAD6 was confirmed as downstream molecular mechanism of lncFGD5-AS1 by expression correlation analysis and mechanism experiments. In vivo study illustrated overexpression of lncFGD5-AS1 suppression tumor growth. RESULTS: LncFGD5-AS1 served as a ceRNA of miR-196a-5p to release its inhibition on SMAD6, a conventional inhibitor on the BMP pathway. Comparing with normal gastric cancer cells, FGD5-AS1 overexpressed group had fewer migration cells, lower cell viability, and lower EMT transformation rate. Meanwhile, xenografts nude mice injecting with overexpressed-FGD5-AS1 cells also shown smaller tumor weight and volume. CONCLUSION: In conclusion, this research supported the first evidence that FGD5-AS1 suppressed proliferation and metastasis in gastric cancer by regulating miR-196a-5p/SMAD6/BMP axis and suggested a potential therapeutic candidate for gastric cancer.

ESCRT-III-associated proteins and spastin inhibit protrudin-dependent polarised membrane traffic.

Mutations in the gene encoding the microtubule severing ATPase spastin are the most frequent cause of hereditary spastic paraplegia, a genetic condition characterised by length-dependent axonal degeneration. Here, we show that HeLa cells lacking spastin and embryonic fibroblasts from a spastin knock-in mouse model become highly polarised and develop cellular protrusions. In HeLa cells, this phenotype was rescued by wild-type spastin, but not by forms unable to sever microtubules or interact with endosomal ESCRT-III proteins. Cells lacking the spastin-interacting ESCRT-III-associated proteins IST1 or CHMP1B also developed protrusions. The protrusion phenotype required protrudin, a RAB-interacting protein that interacts with spastin and localises to ER-endosome contact sites, where it promotes KIF5-dependent endosomal motility to protrusions. Consistent with this, the protrusion phenotype in cells lacking spastin also required KIF5. Lack or mutation of spastin resulted in functional consequences for receptor traffic of a pathway implicated in HSP, as Bone Morphogenetic Protein receptor distribution became polarised. Our results, therefore, identify a novel role for ESCRT-III proteins and spastin in regulating polarised membrane traffic.

BMP Receptor Inhibition Enhances Tissue Repair in Endoglin Heterozygous Mice.

Hereditary hemorrhagic telangiectasia type 1 (HHT1) is a severe vascular disorder caused by mutations in the TGFß/BMP co-receptor endoglin. Endoglin haploinsufficiency results in vascular malformations and impaired neoangiogenesis. Furthermore, HHT1 patients display an impaired immune response. To date it is not fully understood how endoglin haploinsufficient immune cells contribute to HHT1 pathology. Therefore, we investigated the immune response during tissue repair in Eng+/- mice, a model for HHT1. Eng+/- mice exhibited prolonged infiltration of macrophages after experimentally induced myocardial infarction. Moreover, there was an increased number of inflammatory M1-like macrophages (Ly6Chigh/CD206-) at the expense of reparative M2-like macrophages (Ly6Clow/CD206+). Interestingly, HHT1 patients also showed an increased number of inflammatory macrophages. In vitro analysis revealed that TGFß-induced differentiation of Eng+/- monocytes into M2-like macrophages was blunted. Inhibiting BMP signaling by treating monocytes with LDN-193189 normalized their differentiation. Finally, LDN treatment improved heart function after MI and enhanced vascularization in both wild type and Eng+/- mice. The beneficial effect of LDN was also observed in the hind limb ischemia model. While blood flow recovery was hampered in vehicle-treated animals, LDN treatment improved tissue perfusion recovery in Eng+/- mice. In conclusion, BMPR kinase inhibition restored HHT1 macrophage imbalance in vitro and improved tissue repair after ischemic injury in Eng+/- mice.

Cellular and extracellular matrix of bone, with principles of synthesis and dependency of mineral deposition on cell membrane transport.

Bone differs from other connective tissues; it is isolated by a layer of osteoblasts that are connected by tight and gap junctions. This allows bone to create dense lamellar type I collagen, control pH, mineral deposition, and regulate water content forming a compact and strong structure. New woven bone formed after degradation of mineralized cartilage is rapidly degraded and resynthesized to impart structural order for local bone strength. Ossification is regulated by thickness of bone units and by patterning via bone morphogenetic receptors including activin, other bone morphogenetic protein receptors, transforming growth factor-ß receptors, all part of a receptor superfamily. This superfamily interacts with receptors for additional signals in bone differentiation. Important features of the osteoblast environment were established using recent tools including osteoblast differentiation in vitro. Osteoblasts deposit matrix protein, over 90% type I collagen, in lamellae with orientation alternating parallel or orthogonal to the main stress axis of the bone. Into this organic matrix, mineral is deposited as hydroxyapatite. Mineral matrix matures from amorphous to crystalline hydroxyapatite. This process includes at least two-phase changes of the calcium-phosphate mineral as well as intermediates involving tropocollagen fibrils to form the bone composite. Beginning with initiation of mineral deposition, there is uncertainty regarding cardinal processes, but the driving force is not merely exceeding the calcium-phosphate solubility product. It occurs behind a epithelial-like layer of osteoblasts, which generate phosphate and remove protons liberated during calcium-phosphate salt deposition. The forming bone matrix is discontinuous from the general extracellular fluid. Required adjustment of ionic concentrations and water removal from bone matrix are important details remaining to be addressed.

The functions of ocu-miR-205 in regulating hair follicle development in Rex rabbits.

BACKGROUND: Hair follicles are an appendage of the vertebrate epithelium in the skin that arise from the embryonic ectoderm and regenerate cyclically during adulthood. Dermal papilla cells (DPCs) are the key dermal component of the hair follicle that directly regulate hair follicle development, growth and regeneration. According to recent studies, miRNAs play an important role in regulating hair follicle morphogenesis and the proliferation, differentiation and apoptosis of hair follicle stem cells. RESULTS: The miRNA expression profile of the DPCs from Rex rabbits with different hair densities revealed 240 differentially expressed miRNAs (|log2(HD/LD)| > 1.00 and Q-value≤0.001). Among them, ocu-miR-205-5p was expressed at higher levels in DPCs from rabbits with low hair densities (LD) than in rabbits with high hair densities (HD), and it was expressed at high levels in the skin tissue from Rex rabbits (P < 0.05). Notably, ocu-miR-205 increased cell proliferation and the cell apoptosis rate, altered the progression of the cell cycle (P < 0.05), and modulated the expression of genes involved in the PI3K/Akt, Wnt, Notch and BMP signalling pathways in DPCs and skin tissue from Rex rabbits. It also inhibited the phosphorylation of the CTNNB1 and GSK-3ß proteins, decreased the level of the noggin (NOG) protein, and increased the level of phosphorylated Akt (P < 0.05). A significant change in the primary follicle density was not observed (P > 0.05), but the secondary follicle density and total follicle density (P < 0.05) were altered upon interference with ocu-miR-205-5p expression, and the secondary/primary ratio (S/P) in the ocu-miR-205-5p interfered expression group increased 14 days after the injection (P < 0.05). CONCLUSIONS: In the present study, ocu-miR-205 promoted the apoptosis of DPCs, altered the expression of genes and proteins involved in the PI3K/Akt, Wnt, Notch and BMP signalling pathways in DPCs and skin from Rex rabbits, promoted the transition of hair follicles from the growth phase to the regression and resting phase, and altered the hair density of Rex rabbits.