Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Regulated Inositol‐Requiring Protein 1‐Dependent Decay as a Mechanism of Corin RNA and Protein Deficiency in Advanced Human Systolic Heart Failure

doi: 10.1161/JAHA.114.001104

Figure Lengend Snippet: TaqMan Probes in Human, Mouse, and Rat Gene Expression Assay

Article Snippet: Human corin protein was measured in left ventricular lysates using the Human Corin Quantikine ELISA Kit (R&D Systems, Minneapolis, MN).

Techniques: Gene Expression, Sequencing

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Regulated Inositol‐Requiring Protein 1‐Dependent Decay as a Mechanism of Corin RNA and Protein Deficiency in Advanced Human Systolic Heart Failure

doi: 10.1161/JAHA.114.001104

Figure Lengend Snippet: Corin mRNA inversely correlates with Atrial natriuretic peptide (ANP )/Brain natriuretic peptide (BNP ) mRNA in chronic, advanced human systolic heart failure. We examined the relationship of corin mRNA to ANP and BNP RNA (A) in the UPENN database in the nonischemic cardiomyopathy (n=120) and nonischemic (n=80) samples. Data are expressed as log 2 normalized RNA abundance (corin on x ‐axis; BNP on y ‐axis.) Corin mRNA was significantly inversely correlated with either ANP or BNP mRNA. The UK heart collection demonstrated a similar significant inverse relationship between corin mRNA and ANP or BNP mRNA (B). In addition, BNP mRNA was significantly positively correlated with GRP78 mRNA, and corin mRNA was significantly inversely correlated with spliced X‐box protein 1 (XBP1).

Article Snippet: Human corin protein was measured in left ventricular lysates using the Human Corin Quantikine ELISA Kit (R&D Systems, Minneapolis, MN).

Techniques:

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Regulated Inositol‐Requiring Protein 1‐Dependent Decay as a Mechanism of Corin RNA and Protein Deficiency in Advanced Human Systolic Heart Failure

doi: 10.1161/JAHA.114.001104

Figure Lengend Snippet: Corin RNA and protein in heart failure compared to nonfailing controls. Using a sample of 100 randomly selected samples of transmural sample hearts from the UPENN collection consisting of left‐ventricular (LV) tissue, we compared Atrial natriuretic peptide (ANP), Brain natriuretic peptide (BNP), and corin gene expression compared to nonfailing controls (n=10) using real‐time polymerase chain reaction. We discovered that corin mRNA was increased in 53 of the hearts compared to nonfailing controls ( high corin mRNA group ) and decreased in 47 hearts ( low corin mRNA group ) (A). The low corin mRNA group , when compared to the high corin mRNA group , demonstrated significantly higher expression of ANP (B) and BNP (C). Corin protein in samples from LV apex was significantly lower in the low corin RNA as compared to the high corin RNA group (D), demonstrating that changes in corin mRNA resulted in expected changes in corin protein. * P< 0.05, ** P< 0.01, *** P <0.001. Error bars are SEM.

Article Snippet: Human corin protein was measured in left ventricular lysates using the Human Corin Quantikine ELISA Kit (R&D Systems, Minneapolis, MN).

Techniques: Gene Expression, Real-time Polymerase Chain Reaction, Expressing

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Regulated Inositol‐Requiring Protein 1‐Dependent Decay as a Mechanism of Corin RNA and Protein Deficiency in Advanced Human Systolic Heart Failure

doi: 10.1161/JAHA.114.001104

Figure Lengend Snippet: Thapsigargin (TG)‐induced ER stress causes and time‐dependent corin mRNA decay in neonatal rat cardiomyocytes. Neonatal rat cardiomyocytes were plated in 6‐well plates. Twenty‐four hours later, cells were cultured in serum‐free DMEM‐M199 (4:1) with and without 0.1 μmol/L TG for 6, 16, 24, and 30 hours, separately. Total RNA was extracted and reverse‐transcribed into cDNA and gene expression of the following genes was assessed by RT‐PCR (A through F): (A) GRP78; (B) XBP1 splicing; (C) CHOP; (D) corin; (E) GRP94; and (F) ATF4. Compared with non‐TG‐treated controls, cells treated with TG demonstrated increased ER stress as evidenced by significant increases in GRP78, GRP94, ATF4, and CHOP mRNA, with a corresponding significant increase in XBP1 splicing based on the increase in the spliced form (intron removed) XBP1s mRNA; * P< 0.05, ** P< 0.01. Corin mRNA demonstrated a significant, time‐dependent decrease in the cells treated with TG. We also analyzed XBP splicing using PCR, restriction digestion with Pst1, and gel electrophoresis. Fragments of XBP1 were amplified and resolved in 2% agarose gel. PCR products were not digested by Pst I (G). Lane 1: DNA ladder; Lane 2: TG untreated; Lane 3: TG treated. XBP1 fragment: Spliced=450 bp; unspliced=476 bp. PCR products were digested by Pst I (H). Lane 1: DNA ladder; Lane 2: TG untreated; Lane 3: TG treated. Spliced rat XBP1 fragment was 450 bp, while unspliced rat XBP1 appears as 2 fragments of 186 and 290 bp due to the presence of the Pst I restriction site, which is contained in the intronic segment that is removed by IRE1 endonuclease activity. Error bars represent SEM. ATF6 indicates activating transcription factor‐6; ER, endoplasmic reticulum; IRE1, inositol‐requiring protein 1; RT‐PCR, real‐time polymerase chain reaction; CHOP, C/EBP homology protein; DMEM, Dulbecco's Modified Eagle's medium; XBP1, X‐box protein 1.

Article Snippet: Human corin protein was measured in left ventricular lysates using the Human Corin Quantikine ELISA Kit (R&D Systems, Minneapolis, MN).

Techniques: Cell Culture, Reverse Transcription, Gene Expression, Reverse Transcription Polymerase Chain Reaction, Nucleic Acid Electrophoresis, Amplification, Agarose Gel Electrophoresis, Activity Assay, Real-time Polymerase Chain Reaction, Modification

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Regulated Inositol‐Requiring Protein 1‐Dependent Decay as a Mechanism of Corin RNA and Protein Deficiency in Advanced Human Systolic Heart Failure

doi: 10.1161/JAHA.114.001104

Figure Lengend Snippet: Thapsigargin (TG)‐induced ER stress and time‐dependent corin mRNA decay in adult mouse atrial cardiomyocytes (HL‐1) cells. HL‐1 cells were plated in 6‐well plates. Twenty‐four hours later, cells were cultured in serum‐free Claycomb medium with and without thapsigargin (TG) 0.1 μmol/L for 6, 16, 24, 30 hours, separately. Total RNA was extracted and reverse‐transcribed into cDNA and mRNA abundance of the following genes was assessed by RT‐PCR ( A through F): (A) GRP78; (B) XBP1 splicing; (C) CHOP; (D) corin; (E) GRP94; (F) ATF4. Compared with non‐TG‐treated HL‐1 control cells, HL‐1 cells treated with TG demonstrated increased ER stress as evidenced by significant increases in GRP78, GRP94, ATF4 and CHOP mRNA, with a corresponding significant increase in XBP1 splicing based on the increase in the spliced form (intron removed) XBP1s mRNA; * P< 0.05, ** P< 0.01. Corin mRNA demonstrated a significant, time‐dependent decrease in the TG‐treated cells. Error bars represent standard error of the mean. ATF indicates activating transcription factor; ER, endoplasmic reticulum; HL‐1, adult murine atrial myocytes; RT‐PCR, real‐time polymerase chain reaction; XBP1, X‐box protein 1.

Article Snippet: Human corin protein was measured in left ventricular lysates using the Human Corin Quantikine ELISA Kit (R&D Systems, Minneapolis, MN).

Techniques: Cell Culture, Reverse Transcription, Reverse Transcription Polymerase Chain Reaction, Control, Real-time Polymerase Chain Reaction

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Regulated Inositol‐Requiring Protein 1‐Dependent Decay as a Mechanism of Corin RNA and Protein Deficiency in Advanced Human Systolic Heart Failure

doi: 10.1161/JAHA.114.001104

Figure Lengend Snippet: IRE1‐gene overexpression. Neonatal murine cardiomyocytes were plated in 6‐well plates with complete medium 1 day before infection. On the second day, cells were infected with IRE1‐overexpressed lentivirus containing 8 μg/mL polybrene and incubated overnight. After that, the cells were fed with fresh complete medium. Cells were harvested at 72 hours after transduction and the total RNA was extracted. Quantitative polymerase chain reaction was conducted using specific TaqMan mRNA probes: (A) IRE1 alpha; (B) Corin; (C) Spliced XBP1; (D) GRP78; (E) CHOP; (F) ATF4 mRNA were quantified by normalization to 18S ribosomal RNA. IRE1‐overexpression activated IRE1 endoribonuclease activity, resulting in a significant increase in XBP1s and a parallel significant decrease in corin mRNA. Note: Compared with mock group, * P< 0.05; ** P< 0.01; *** P< 0.001. Mock: cells transduced with empty lentivirus vehicle; IRE1‐: cells transduced with IRE1‐overexpressed lentivirus. Error bars represent standard error of the mean. ATF indicates activating transcription factor; CHOP, ; IRE1, inositol‐requiring protein 1; XBP1, X‐box protein 1.

Article Snippet: Human corin protein was measured in left ventricular lysates using the Human Corin Quantikine ELISA Kit (R&D Systems, Minneapolis, MN).

Techniques: Over Expression, Infection, Incubation, Transduction, Real-time Polymerase Chain Reaction, Activity Assay

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Regulated Inositol‐Requiring Protein 1‐Dependent Decay as a Mechanism of Corin RNA and Protein Deficiency in Advanced Human Systolic Heart Failure

doi: 10.1161/JAHA.114.001104

Figure Lengend Snippet: IRE1 shRNA knockdown attenuates corin mRNA decay. HL‐1 cells were transfected with 2 μg mammalian expression plasmid containing IRE1 shRNA or nontargeted negative control (Negative Ctr), separately. Cells were exposed to thapsigargin 0.1 μmol/L for 12 hours. Thereafter, cellular total RNA was isolated and quantitative polymerase chain reaction was conducted using specific TaqMan probes: (A) IRE1 alpha; (B) Corin; (C) Spliced XBP1; (D) GRP78; (E) CHOP; (F) ATF4 mRNA were quantified by normalization to 18S ribosomal RNA. Cells with IRE1 shRNA knockdown prior to exposure to thapsigargin (12 hours) demonstrated less XBP1 splicing and increased corin mRNA values compared to cells transfected with control plasmid and also exposed to 12 hours thapsigargin. * P< 0.05 and ** P< 0.01. Error bars represent SEM. ATF indicates activating transcription factor; CHOP, C/EBP homology protein; HL‐1, adult murine atrial myocytes; IRE1, inositol‐requiring protein 1; XBP1, X‐box protein 1.

Article Snippet: Human corin protein was measured in left ventricular lysates using the Human Corin Quantikine ELISA Kit (R&D Systems, Minneapolis, MN).

Techniques: shRNA, Knockdown, Transfection, Expressing, Plasmid Preparation, Negative Control, Isolation, Real-time Polymerase Chain Reaction, Control

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Regulated Inositol‐Requiring Protein 1‐Dependent Decay as a Mechanism of Corin RNA and Protein Deficiency in Advanced Human Systolic Heart Failure

doi: 10.1161/JAHA.114.001104

Figure Lengend Snippet: Effect of actinomycin D prior to thapsigargin (TG). HL‐1 cells were incubated with or without actinomycin D at a concentration of 1 μg/μL 3 hours prior to administration of TG (0.1 μmol/L) for 12 hours. Cells were harvested and the relative abundance of corin mRNA and XBP1s (normalized to 18S rRNA) was compared to cells not receiving TG. We compared the changes in corin and XBP1s in cells not treated (A) or treated with actinomycin D (B) prior to the administration of TG to the medium. We compared relative mRNA abundance (normalized to 18S ribosomal RNA) of corin and XBP1s 12 hours after TG as compared to baseline. As demonstrated in (B), the decrease in corin mRNA and corresponding increase in XBP1s ( P <0.001), a marker of IRE1 endoribonuclease activity, was similar in cells pretreated with actinomycin D as compared to cells not pretreated with actinomycin D (B) prior to the induction of ER stress with TG. Baseline XBP1s was higher in cells not pretreated with actinomycin D. The error bars represent SEM for the relative mRNA expression. ( P <0.001 for baseline compared to 12 hours). Error bars represent SEM. ER indicates endoplasmic reticulum; HL‐1, adult murine atrial myocytes; IRE1, inositol‐requiring protein 1; XBP1s, spliced X‐box protein 1.

Article Snippet: Human corin protein was measured in left ventricular lysates using the Human Corin Quantikine ELISA Kit (R&D Systems, Minneapolis, MN).

Techniques: Incubation, Concentration Assay, Marker, Activity Assay, Expressing

Journal: Journal of the American Heart Association

Article Title: Association Between Genetically Determined Serum Corin and the Risk of Stroke in Chinese Adults: A Mendelian Randomization Study

doi: 10.1161/jaha.124.035858

Figure Lengend Snippet: Figure 1. Flowchart illustrating the study design and selection of participants. Leveraging the Gusu cohort, a 1-sample Mendelian randomization study was designed to test the causal relationship between corin and stroke, with serum corin as the E, SNPs at the coding gene of corin as the IV, and stroke as the O. Calculated GRS was also considered as the IV. GRS indicates genetic risk score; E, exposure, IV, instrumental variable; O, outcome; and SNP, single-nucleotide polymorphism.

Article Snippet: We used a quantikine human corin immunoassay (R&D Systems, Inc, Minneapolis, MN; Catalog: DCRN00;) to test soluble corin levels in serum.

Techniques: Selection

Journal: Journal of the American Heart Association

Article Title: Association Between Genetically Determined Serum Corin and the Risk of Stroke in Chinese Adults: A Mendelian Randomization Study

doi: 10.1161/jaha.124.035858

Figure Lengend Snippet: Figure 2. Scatter plot of results from the IVW MR analysis for individual SNPs and pooled estimates. The fixed-effect IVW analysis is based on the unadjusted, age and sex-adjusted, and multivariate-adjusted SNP-corin and SNP-stroke associations. Abscissa values indicate the SD change in log2-transformed corin per allele. Ordinate values indicate the logarithm of the HR of stroke per allele. HR indicates hazard ratio; IVW, inverse variance–weighted; MR, Mendelian randomization; and SNPs, single-nucleotide polymorphisms.

Article Snippet: We used a quantikine human corin immunoassay (R&D Systems, Inc, Minneapolis, MN; Catalog: DCRN00;) to test soluble corin levels in serum.

Techniques: Transformation Assay

Journal: Journal of the American Heart Association

Article Title: Association Between Genetically Determined Serum Corin and the Risk of Stroke in Chinese Adults: A Mendelian Randomization Study

doi: 10.1161/jaha.124.035858

Figure Lengend Snippet: Figure 3. Forest plot of the results of the leave-one-out sensitivity analysis. The HRs indicate the pooled estimates from the fixed-effect IVW analysis based on the unadjusted, age- and sex-adjusted, and multivariate-adjusted SNP-corin and SNP-stroke associations, where each SNP was iteratively removed from the instruments. HR indicates hazard ratio; IVW, inverse variance–weighted; and SNPs, single-nucleotide polymorphisms.

Article Snippet: We used a quantikine human corin immunoassay (R&D Systems, Inc, Minneapolis, MN; Catalog: DCRN00;) to test soluble corin levels in serum.

Techniques:

Journal: Journal of the American Heart Association

Article Title: Association Between Genetically Determined Serum Corin and the Risk of Stroke in Chinese Adults: A Mendelian Randomization Study

doi: 10.1161/jaha.124.035858

Figure Lengend Snippet: Figure 4. Forest plot of the results from the IVW MR analysis using summarized SNP-stroke associations. The ORs indicate the pooled estimates from the fixed-effect IVW analysis based on the multivariate- adjusted SNP-corin associations in our original data and summarized SNP-stroke associations in external data. CES indicates cardioembolic stroke; IS, ischemic stroke; IVW, inverse variance–weighted; LAS, large artery stroke; OR, odds ratio; SNP, single-nucleotide polymorphism; and SVS, small-vessel stroke.

Article Snippet: We used a quantikine human corin immunoassay (R&D Systems, Inc, Minneapolis, MN; Catalog: DCRN00;) to test soluble corin levels in serum.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Systematic transcriptome profiling of hPSC-derived osteoblasts unveils CORIN's mastery in governing osteogenesis through CEBPD modulation

doi: 10.1016/j.jbc.2024.107494

Figure Lengend Snippet: Systemat ic transcriptome analyses of hPSC-derived cells reveal enriched membrane proteins in human osteoblasts. A , left panel: Cluster analysis of the gene expression patterns during osteogenesis. The horizontal axis represents the stages of osteoblast development: MSCs, preosteoblasts (Pre), maturing osteoblasts (Mat), and osteoblasts (OBs). The vertical axis represents FPKM values using 2 as the logarithmic base of a gene at different developmental stages. The red lines indicate the change in gene expression level between samples. Right panel: GO_BP pathway analysis of 12 gene expression clusters showed the enrichment of genes involved in different biological processes as indicated. B , left panel: diagrammatic depiction of stages of osteogenesis. Right panel: RNA-seq analysis of hPSC-derived osteoblasts revealed the enriched expression of AMIGO2, CDH13, CDH2, PVR, and SDC1 in MSCs and the enriched expression of CORIN, IL1R1, CD82, APOD, TGFBR2, ADAMTSL4, SERPING1, MME1, VCAM1, and LEPR in osteoblasts. C , heatmap showing the differential expression of identified membrane proteins during osteogenesis. The enriched membrane proteins in osteoblasts are in Clusters 3 and 6. The enriched membrane proteins in MSCs are in Cluster 12. D , RT-qPCR validated the upregulation/downregulation of the identified membrane proteins in multiple hPSC-derived osteoblasts as biological repeats (WT-1H, WT-1G, WT-1J, WT-F6, and WT-F37) compared to MSCs. MSCs were derived from hPSCs using the bFGF/PDGF-AB method. E , RT-qPCR demonstrated the upregulation/downregulation of the identified membrane proteins in H1, WT-F6, and WT-F37 hPSC-derived osteoblasts compared to MSCs. MSCs were derived from hPSCs using the SB-431542/7.5%CO 2 method. (n = 3, mean ± S.D.). F , RT-qPCR demonstrated the upregulation/downregulation of the identified membrane proteins in osteoblasts differentiated from ad-HMSCs and bm-HMSCs. (n = 3, mean ± S.D.). G , RT-qPCR demonstrated the upregulation/downregulation of the identified membrane proteins in hFOB-differentiated osteoblasts. (n = 3, mean ± S.D.). ad-HMSC, adipose tissue-derived human MSC; CD, clusters of differentiation; FPKM, Fragments Per Kilobase per Million mapped fragments; GO_BP, Gene Ontology biological process; HMSC, human MSC; hPSC, human pluripotent stem cells; MSC, mesenchymal stem cell; RT-qPCR, real-time quantitative PCR; SDC1, syndecan-1; TGF-β transforming growth factor-β.

Article Snippet: To analyze the expression of surface receptors CORIN and SDC1, the detached cells were stained with human CORIN antibody (R&D System, AF2209) and PE-conjugated human SDC1 (BD Bioscience, 552026).

Techniques: Derivative Assay, Membrane, Gene Expression, RNA Sequencing, Expressing, Quantitative Proteomics, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Systematic transcriptome profiling of hPSC-derived osteoblasts unveils CORIN's mastery in governing osteogenesis through CEBPD modulation

doi: 10.1016/j.jbc.2024.107494

Figure Lengend Snippet: Functional studies on CORIN and SDC1 in osteogenesis. A , flow cytometry analysis of CORIN and SDC1 expression in hESC H9-derived MSCs and osteoblasts (OBs). B , IHC staining of CORIN and SDC1 expression in human femur bone tissue using anti-human CORIN and anti-human SDC1 antibodies. Goat and mouse IgG were utilized as a negative control. The scale bar represents 100 μm. C , RT-qPCR confirmed the impairment of osteoblast lineage genes COL1A1, PTH1R1, BGLAP, and RUNX2 in CORIN-depleted or SDC1-overexpressed hESC H1-derived osteoblasts. (n = 3, mean ± S.D.). D , ARS staining indicated that depletion of CORIN as well as overexpression of SDC1 in osteoblasts led to impaired bone mineral production. The scale bar represents 100 μm. E , diagrammatic depiction of preparation of GelMA discs. Hybridization of gelatin and methacrylic anhydride was followed by UV cross-linking to make the GelMA discs. F , morphology of GelMA discs, which had a diameter of 4 mm and a thickness of 1 mm. G , scanning electron microscope (SEM) image showing the surface and hollow structures of the GelMA discs. The scale bar represents 400 μm. H and I , left : 3D μCT images of GelMA discs loaded with CORIN-depleted or SDC1-overexpressed hESC H1-derived osteoblasts compared to control osteoblasts at 3 weeks and 6 weeks culture in osteoblastic differentiation media. Right : bone mineral density and BV/TV (%) analyses of GelMA discs loaded with CORIN-depleted ( upper ), or SDC1-overexpressed ( lower ) hESC H1-derived osteoblasts. The scale bar represents 1 mm. The difference between two groups was compared by the two-tailed unpaired Student’s t test or ANOVA to calculate the p -value. (n = 3, mean ± S.D.). ARS, Alizarin Red S; BV/TV, bone volume/total tissue volume; GelMA, gelatin methacrylate; hESC, human embryonic stem cell; IHC, immunohistochemistry; MSC, mesenchymal stem cell; RT-qPCR, real-time quantitative PCR; SDC1, syndecan-1; μCT, microcomputed tomography.

Article Snippet: To analyze the expression of surface receptors CORIN and SDC1, the detached cells were stained with human CORIN antibody (R&D System, AF2209) and PE-conjugated human SDC1 (BD Bioscience, 552026).

Techniques: Functional Assay, Flow Cytometry, Expressing, Derivative Assay, Immunohistochemistry, Negative Control, Quantitative RT-PCR, Staining, Over Expression, Hybridization, Microscopy, Control, Two Tailed Test, Real-time Polymerase Chain Reaction, Tomography

Journal: The Journal of Biological Chemistry

Article Title: Systematic transcriptome profiling of hPSC-derived osteoblasts unveils CORIN's mastery in governing osteogenesis through CEBPD modulation

doi: 10.1016/j.jbc.2024.107494

Figure Lengend Snippet: Systematic analyses of the CORIN/SDC1-regulated transcriptome. A , volcano plots for DEGs in CORIN-depleted ( left panel) or SDC1-overexpressed ( right panel) hESC H9-derived osteoblasts compared with control osteoblasts. DEGs were selected with the parameters p < 0.05 and log 2 (fold-change) > 1. Vertical dashed lines demarcate the 2-fold change cutoff. Blue and dark blue dots represent genes significantly downregulated ≤2-fold or >2-fold, respectively; yellow and orange dots represent genes significantly upregulated ≤2-fold or >2-fold, respectively; gray dots represent genes without significantly differential expression following depletion or CORIN or overexpression of SDC1. B , Venn diagram depicting the number of genes considered to be DEGs in CORIN-knockdown and SDC1-overexpressed hESC H9-derived osteoblasts. The intersection represents commonly upregulated ( upper panel) or downregulated ( lower panel) genes in both the shCORIN and SDC1 groups. C , Mouse Gene Atlas analyses by Enrichr indicated that downregulated genes in CORIN-depleted and SDC1-overexpressed osteoblasts are significantly enriched in the later stages of mouse osteoblast differentiation (days 14 and 21). D , GSEA GO_BP analyses ( left panel) demonstrated that genes significantly downregulated in CORIN-depleted and SDC1-overexpressed hESC H9-derived osteoblasts were involved in skeletal system development ( right upper panel) and collagen fibril organization ( right lower panel). Gene sets enriched in control cells are shown in dark blue (FDR q value < 0.25; normalized p value < 0.05), while white indicates a nonsignificant difference. E , BioPlanet pathway analyses demonstrate that genes significantly downregulated in CORIN-depleted and SDC1-overexpressed hESC H9-derived osteoblasts are involved in TGF-β regulation of extracellular matrix, extracellular matrix organization, and ECM receptor interactions. Gene sets enriched in control cells are shown in blue (FDR q value < 0.25; normalized p value < 0.05), while white indicates a nonsignificant difference. F , BioPlanet pathway analyses demonstrate that genes significantly upregulated in CORIN-depleted and SDC1-overexpressed hESC H9-derived osteoblasts are overrepresented for genes in cellular functions such as EGFR1 and cholesterol biosynthesis. ECM, extracellular matrix; EGFR, epidermal growth factor receptor; FDR, false discovery rate; hESC, human embryonic stem cell; GO_BP, Gene Ontology biological process; GSEA, gene set enrichment analysis; SDC1, syndecan-1; TGF-β transforming growth factor-β.

Article Snippet: To analyze the expression of surface receptors CORIN and SDC1, the detached cells were stained with human CORIN antibody (R&D System, AF2209) and PE-conjugated human SDC1 (BD Bioscience, 552026).

Techniques: Derivative Assay, Control, Quantitative Proteomics, Over Expression, Knockdown

Journal: The Journal of Biological Chemistry

Article Title: Systematic transcriptome profiling of hPSC-derived osteoblasts unveils CORIN's mastery in governing osteogenesis through CEBPD modulation

doi: 10.1016/j.jbc.2024.107494

Figure Lengend Snippet: CORIN/SDC1 governs the determination of osteoblast identities via CEBPD. A , ChEA analysis of DEGs in hESC H9-derived osteoblasts upon CORIN depletion as well as SDC1 overexpression. Genes enriched in control cells are shown in blue , and genes enriched in osteoblasts with CORIN depletion or SDC1 overexpression are shown in orange . The color intensity of the circle indicates the number of targets by a given transcription factor in the dataset, while the circle size represents the significance of the interaction. B and C , immunoblotting of CEBPD in CORIN-depleted ( B ) and SDC-overexpressed ( C ) hESC H9-derived osteoblasts. RT-qPCR confirmed the efficacy of shCORIN and SDC1 in modifying gene expression. (n = 3, mean ± S.D.). D , immunoblotting showed an increase in p38 MAPK phosphorylation and subsequent CORIN and CEBPD expression in hESC H9-derived osteoblasts compared to MSCs. E , immunoblotting showed inhibition of p38 MAPK by p38 MAPK inhibitor SB202190 (200 nM) and an associated downregulation of CEBPD expression. F , ARS staining of hESC H9-derived differentiated osteoblasts treated with p38 MAPK inhibitor SB202190 (200 nM) compared with control osteoblasts and MSCs. The scale bar represents 5 mm. G , immunoblotting showed a decrease in p38 MAPK phosphorylation upon CORIN knockdown ( upper panel) or SDC1 overexpression ( lower panel) in H1 hESC-derived osteoblasts. H , IHC staining showed the expression of CEBPD in human and mouse femur bone tissues. Rabbit IgG served as a control. I , scRNA-seq analysis of human osteoblasts reveals the expression of CEBPD in a distinct osteoblast population. Left panel: three osteoblast clusters (preosteoblasts [Pre-OBs], osteoblasts [OBs], and undetermined osteoblasts [Undetermined OBs]) are defined, colored, and visualized by UMAP using 5329 osteoblasts. Middle panel: logarithm-normalized expression showed CEBPD is highly expressed in the three osteoblast clusters. Right panel: distributions of CEBPD expression within the three osteoblast clusters showed the highest CEBPD expression in the undetermined osteoblast cluster. J , ARS staining confirmed that depletion of CEBPD impairs bone mineralization in hESC H9-derived osteoblasts. The scale bar represents 100 μm. K , RT-qPCR reveals the impairment of osteoblast lineage genes COL1A1, PTH1R1, BGLAP, and RUNX2 in CEBPD-depleted hESC H9-derived osteoblasts. (n = 3, mean ± S.D.). L , left panels: 3D μCT images of GelMA discs loaded with CEBPD-depleted hESC H9-derived osteoblasts (sgCEBPD-1) at 3 weeks and 6 weeks culture in osteoblast differentiation media. Right panels: bone mineral density and BV/TV (%) analyses of GelMA discs loaded with CEBPD-depleted hESC H9-derived osteoblasts (sgCEBPD). The scale bar represents 1 mm. The difference between two groups was compared by the two-tailed unpaired Student’s t test or ANOVA to calculate the p -value. (n = 3, mean ± S.D.). ARS, Alizarin Red S; BV/TV, bone volume/total tissue volume; CEBPD, CCAAT enhancer binding protein delta; ChEA, ChIP enrichment analysis; DEG, differentially expressed gene; GelMA, gelatin methacrylate; hESC, human embryonic stem cell; IHC, immunohistochemistry; MAPK, mitogen-activated protein kinase; MSC, mesenchymal stem cell; RT-qPCR, real-time quantitative PCR; scRNA-seq, single-cell RNA-seq; SDC1, syndecan-1; μCT, microcomputed tomography; UMAP, uniform manifold approximation and projection.

Article Snippet: To analyze the expression of surface receptors CORIN and SDC1, the detached cells were stained with human CORIN antibody (R&D System, AF2209) and PE-conjugated human SDC1 (BD Bioscience, 552026).

Techniques: Derivative Assay, Over Expression, Control, Western Blot, Quantitative RT-PCR, Gene Expression, Phospho-proteomics, Expressing, Inhibition, Staining, Knockdown, Immunohistochemistry, Two Tailed Test, Binding Assay, Real-time Polymerase Chain Reaction, RNA Sequencing, Tomography