Journal: Scientific Reports

Article Title: NG2/ CSPG4 , CD146/ MCAM and VAP1/ AOC3 are regulated by myocardin-related transcription factors in smooth muscle cells

doi: 10.1038/s41598-021-85335-x

Figure Lengend Snippet: The CSPG4 gene is activated by Myocardin-Related Transcription Factors (MRTFs). To examine the transcriptional control of NG2/ CSPG4 we correlated the CSPG4 mRNA versus all other mRNAs ( www.GTExPortal.org ) and calculated the sum of correlation coefficients (R sum ) for all transcripts across 20 human tissues. Panel ( A ) shows the positive extreme of the resulting R sum distribution which has a theoretical maximum of 20 (seen only for CSPG4 itself, not included). Among the 600 (≈1%) most tightly correlating mRNAs we found classical smooth muscle cell (SMC) markers ( TAGLN , MYH11 ) and transcription factors ( SRF , MYOCD , MKL1 , all indicated by different green symbols). Two cell lineage markers ( MCAM and AOC3 , red symbols) were among the mRNAs in the absolute extreme (top 25). Examples of correlations between SRF , MYOCD and MKL1 versus CSPG4 in the transverse colon (N = 274) are shown in panels ( B – D ). P-values and Spearman Rho-coefficients are given in the respective panels. In panel ( E ), adenoviruses were used for overexpression of MRTFs (MYOCD, MRTF-A/ MKL1 , and MRTF-B/ MKL2 ) in primary human coronary artery smooth muscle cells in vitro. The CSPG4 mRNA level was determined by RT-qPCR and compared to that in cells treated with empty virus (ANOVA, followed by Dunnett's Multiple Comparison Test versus Null, N = 6 for all treatments). In this and the following figures showing RT-qPCR data, the relative mRNA level is represented by the official gene symbol in italics. Transcript levels were normalized to 18S as housekeeping gene (Pfaffl equation) and are given as fold change (FC). All statistical comparisons in panel ( E ) of figures 1 through 3 are versus Null as indicated by brackets. Panel ( F ) shows a western blot for NG2/CSPG4 following treatment with control (Null) virus and viruses encoding MRTFs. Membranes were cut horizontally in this, and the following, figures to allow for detection of multiple proteins on the same membrane. Full length blots (as full as possible) are found in Fig. . Panel ( G ) shows summarized data for the western blot experiments (N = 3). The bands migrating at ≥ 250 kDa were included in the analysis. In panel ( H ), cells were transduced with tagged MRTF-A/MKL1 (blue), fixed at 96 h, and stained for NG2/CSPG4 (red) and the intermediate filament synemin/SYNM (green), followed by confocal imaging. Red and green labels are shown in black and white below the colored panels for clarity. Summarized data from two independent experiments with three independent replicates each time (N = 6) is shown in panel ( I ). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, all versus Null.

Article Snippet: We used the following primary antibodies CSPG4 (MAB2029, clone 9.2.27, Millipore), MCAM (SAB5600062, Sigma Aldrich), AOC3 (MAB3957, R&D Systems, and SAB2501957, Sigma-Aldrich), KDM3A (12835-1-AP, Proteintech), H3K9Me2 (4658S, Cell Signaling Technology), Histone H3 (4499S, Cell Signaling Technology), phospho-ERK1/2 (Thr202/Tyr204, 9101S, Cell Signaling Technology), total ERK1/2 (9102S, Cell Signaling Technology), HSP90 (#610418, BD Biosciences), GAPDH (MAB374 from Sigma Aldrich).

Techniques: Over Expression, In Vitro, Quantitative RT-PCR, Virus, Comparison, Western Blot, Membrane, Transduction, Staining, Imaging

Journal: Scientific Reports

Article Title: NG2/ CSPG4 , CD146/ MCAM and VAP1/ AOC3 are regulated by myocardin-related transcription factors in smooth muscle cells

doi: 10.1038/s41598-021-85335-x

Figure Lengend Snippet: AOC3 also resides in the CSPG4 co-expression module and is regulated by MRTFs. Panels ( A ) and ( B ) show correlations between MYOCD and AOC3 in the colon and prostate, respectively. Panels ( C ) and ( D ) show that AOC3 also correlates with CSPG4 and MCAM (ovary). Panel ( E ) shows mRNA levels for AOC3 following overexpression of MRTFs (all statistical comparisons versus Null). Panel ( F ) shows confocal imaging of AOC3 fluorescence in cells transduced with Null virus and with MRTF-A/MKL1-encoding virus. Panel ( G ) shows summarized data from the experiments in ( F ). Panels ( H ) through ( K ) examine if MYOCD is antagonistic with MRTF-A/ MKL1 . No antagonism was noted for the classical target gene ACTA2 ( H ), for CSPG4 ( I ), for MCAM ( J ) or for AOC3 ( K ). *P < 0.05, ***P < 0.001, versus null.

Article Snippet: We used the following primary antibodies CSPG4 (MAB2029, clone 9.2.27, Millipore), MCAM (SAB5600062, Sigma Aldrich), AOC3 (MAB3957, R&D Systems, and SAB2501957, Sigma-Aldrich), KDM3A (12835-1-AP, Proteintech), H3K9Me2 (4658S, Cell Signaling Technology), Histone H3 (4499S, Cell Signaling Technology), phospho-ERK1/2 (Thr202/Tyr204, 9101S, Cell Signaling Technology), total ERK1/2 (9102S, Cell Signaling Technology), HSP90 (#610418, BD Biosciences), GAPDH (MAB374 from Sigma Aldrich).

Techniques: Expressing, Over Expression, Imaging, Fluorescence, Transduction, Virus

Journal: Scientific Reports

Article Title: NG2/ CSPG4 , CD146/ MCAM and VAP1/ AOC3 are regulated by myocardin-related transcription factors in smooth muscle cells

doi: 10.1038/s41598-021-85335-x

Figure Lengend Snippet: The MCAM and AOC3 gene loci have SRF binding motifs that confer responsiveness to MRTF-SRF signaling. Panel ( A ) shows graphical representations of the CSPG4 , MCAM and AOC3 gene loci with known (red arrows, ENCODE data accessible via the UCSC genome browser) and predicted (lighter red/pink arrows) binding sites for MRTF-SRF . Gene loci and binding sites are not drawn to scale. Panel ( B ) shows effects of SRF silencing (by 43 ± 4%, P < 0.0001, N = 6), using a short hairpin construct, on the indicated mRNA levels measured by RT-qPCR. Panel ( C ) shows reporter assays for MCAM (S1-S3 plasmid and S2-S4 plasmid, see panel A ) and AOC3 . We used HEK 293 cells for transfection of the reporter plasmids in ( C ), because these cells were more readily transfected compared to human coronary artery smooth muscle cells used elsewhere. Panels ( D ) and ( E ) show time-courses of mRNA induction following overexpression of myocardin (N = 3 for all time points). CSPG4 , MCAM and AOC3 were all increased at least as fast as the direct target gene ACTA2 . Panel ( F ) shows that MRTF-A/MKL1 increases the mRNA level of KDM3A, and that short hairpin silencing of KDM3A (shKDM3A) antagonizes this effect (N = 5–6). Panel ( G ) shows RT-qPCR for MCAM , AOC3 , and CSPG4 in MRTF-A/MKL1-transduced cells in the absence and presence of shKDM3A (N = 5–6). Panel ( H ) shows western blots for cells transduced with null, MKL1, and MKL1 plus shKDM3A viruses. The bar graph at the bottom shows the quantitative analysis for MCAM (vs. HSP90). Quantitative analysis of the KDM3A protein level similarly showed it to be significantly increased by MRTF-A transduction and reduced by KDM3A silencing (not shown). Panel ( I ) shows the effect of MRTF-A/MKL1 on transcript levels of three transcription factors ( SOX10 , ASCL1 , and OLIG2 ) that control CSPG4 expression in brain glial cells. None of these transcription factors were significantly increased, while the positive controls ( ACTA2 , CSPG4 ) were increased in the same samples (N = 6).

Article Snippet: We used the following primary antibodies CSPG4 (MAB2029, clone 9.2.27, Millipore), MCAM (SAB5600062, Sigma Aldrich), AOC3 (MAB3957, R&D Systems, and SAB2501957, Sigma-Aldrich), KDM3A (12835-1-AP, Proteintech), H3K9Me2 (4658S, Cell Signaling Technology), Histone H3 (4499S, Cell Signaling Technology), phospho-ERK1/2 (Thr202/Tyr204, 9101S, Cell Signaling Technology), total ERK1/2 (9102S, Cell Signaling Technology), HSP90 (#610418, BD Biosciences), GAPDH (MAB374 from Sigma Aldrich).

Techniques: Binding Assay, Construct, Quantitative RT-PCR, Plasmid Preparation, Transfection, Over Expression, Western Blot, Transduction, Expressing

Journal: Scientific Reports

Article Title: NG2/ CSPG4 , CD146/ MCAM and VAP1/ AOC3 are regulated by myocardin-related transcription factors in smooth muscle cells

doi: 10.1038/s41598-021-85335-x

Figure Lengend Snippet: SMC differentiation, actin dynamics, and loss of Ternary Complex Factors (TCFs) all affect AOC3 , MCAM, and CSPG4 expression. In panel ( A ), human coronary artery smooth muscle cells were incubated with either growth supplement (GS) or differentiation supplement (DS) for 72 h and the mRNA levels of AOC3 , MCAM , and CSPG4 were determined by RT-qPCR (N = 6). In panels ( B ) and ( C ), the effects of Latrunculin B (LatB), which depolymerizes actin, were tested using two different protocols ( B 24 h static, and C 20 h with drug + 4 h washout in cycles for 96 h, N > 6). Transcript levels were assayed using RT-qPCR. All cells in ( B ) and ( C ) were transduced with MRTF-A/ MKL1 . Panel ( D ) (72 h treatment) and panel ( E ) (time-course) show effects of the MRTF-SRF inhibitor CCG-1423. Panel ( F ) compares transcript levels in freshly isolated mouse caudal arteries with mouse caudal arteries that were organ cultured in the presence of vehicle (DMSO) or CCG-1423 (96 h). In panel ( G ), the levels of Aoc3 , Mcam , and Cspg4 were determined by RT-qPCR in mouse embryonic fibroblasts (MEFs) from wild type (WT) mice, and in MEFs from mice that lack three Ternary Complex Factors (Elk1, Elk3, and Elk4). *P < 0.05, **P < 0.01, ***P < 0.001, versus the respective controls.

Article Snippet: We used the following primary antibodies CSPG4 (MAB2029, clone 9.2.27, Millipore), MCAM (SAB5600062, Sigma Aldrich), AOC3 (MAB3957, R&D Systems, and SAB2501957, Sigma-Aldrich), KDM3A (12835-1-AP, Proteintech), H3K9Me2 (4658S, Cell Signaling Technology), Histone H3 (4499S, Cell Signaling Technology), phospho-ERK1/2 (Thr202/Tyr204, 9101S, Cell Signaling Technology), total ERK1/2 (9102S, Cell Signaling Technology), HSP90 (#610418, BD Biosciences), GAPDH (MAB374 from Sigma Aldrich).

Techniques: Expressing, Incubation, Quantitative RT-PCR, Transduction, Isolation, Cell Culture

Journal: Scientific Reports

Article Title: NG2/ CSPG4 , CD146/ MCAM and VAP1/ AOC3 are regulated by myocardin-related transcription factors in smooth muscle cells

doi: 10.1038/s41598-021-85335-x

Figure Lengend Snippet: Co-expression of CD146/ MCAM , NG2/ CSPG4 , and VAP1/ AOC3 in endothelial cells and pericytes in the human brain. Human brain specimen stained with antibodies versus MCAM ( A ) and CSPG4 ( B ) showed co-expression in pericytes and endothelial cells (overlay in C ). MCAM expression in the human brain was restricted to vascular structures ( D ). Panel ( E ) shows four examples of immunohistochemical staining for AOC3 (brown) in the human brain from the Human Protein Atlas (HPA) . Endothelial cells and pericytes (arrows) in capillaries and larger vessels were positive. Panel ( F ) shows a tentative model for regulation of MCAM , CSPG4 and AOC3 by MRTFs in pericytes (and endothelial cells) at the human blood–brain barrier. In the illustration, MRTF refers to the three myocardin-related transcription factors MYOCD, MRTF-A/MKL1, and MRTF-B/MKL2. Upstream activators of MRTFs were not examined herein, but some possibilities are given, such as sphingosine-1-phospate (S1P) and transforming growth factor β (TGFβ). Panel ( G ) shows RT-qPCR data for two validated markers of pericytes, RGS5 and PDGFRB (N = 6, 8 days of transduction) in control conditions and after overexpression of MYOCD. Panel ( H ) shows time-course data for the RGS5 transcript on overexpression of MYOCD and MRTF-A/MKL1, respectively (N = 4 for all times). Null data was generated for each time and used for statistical comparisons but was omitted from the graph for clarity. Panel ( I ) shows staining for TINAGL1 in human cerebral microvessels (from the HPA).

Article Snippet: We used the following primary antibodies CSPG4 (MAB2029, clone 9.2.27, Millipore), MCAM (SAB5600062, Sigma Aldrich), AOC3 (MAB3957, R&D Systems, and SAB2501957, Sigma-Aldrich), KDM3A (12835-1-AP, Proteintech), H3K9Me2 (4658S, Cell Signaling Technology), Histone H3 (4499S, Cell Signaling Technology), phospho-ERK1/2 (Thr202/Tyr204, 9101S, Cell Signaling Technology), total ERK1/2 (9102S, Cell Signaling Technology), HSP90 (#610418, BD Biosciences), GAPDH (MAB374 from Sigma Aldrich).

Techniques: Expressing, Staining, Immunohistochemical staining, Quantitative RT-PCR, Transduction, Over Expression, Generated