Multiple Chemical Inducible Tal Effectors for Genome Editing and Transcription Activation

Inducible modulation is often required for precise investigations and manipulations of dynamic biological processes. Transcription activator-like effectors (TALEs) provide a powerful tool for targeted gene editing and transcriptional programming. We designed a series of chemical inducible systems by coupling TALEs with a mutated human estrogen receptor (ER^T2), which renders them 4-hydroxyl-tamoxifen (4-OHT) inducible for access of the genome. Chemical inducible genome editing was achieved <i>via</i> fusing two tandem ER^T2 domains to customized transcription activator-like effector nuclease (TALEN), which we termed “Hybrid Inducible Technology” (HIT-TALEN). Those for transcription activation were vigorously optimized using multiple construct designs. Most efficient drug induction for endogenous gene activation was accomplished with minimal background activity using an optimized inducible TALE based SunTag system (HIT-TALE-SunTag). The HIT-SunTag system is rapid, tunable, selective to 4-OHT over an endogenous ligand, and reversible in drug induced transcriptional activation. Versatile systems developed in this study can be easily applied for editing and transcriptional programming of potentially any genomic loci in a tight and effective chemical inducible fashion

P38 and ERK1/2 MAPKs Act in Opposition to Regulate BMP9-Induced Osteogenic Differentiation of Mesenchymal Progenitor Cells

<div><p>Although previous studies have demonstrated that BMP9 is highly capable of inducing osteogenic differentiation and bone formation, the precise molecular mechanism involved remains to be fully elucidated. In this current study, we explore the possible involvement and detail effects of p38 and ERK1/2 MAPKs on BMP9-indcued osteogenic differentiation of mesenchymal progenitor cell (MPCs). We find that BMP9 simultaneously stimulates the activation of p38 and ERK1/2 in MPCs. BMP9-induced early osteogenic marker, such as alkaline phosphatase (ALP), and late osteogenic markers, such as matrix mineralization and osteocalcin (OC) are inhibited by p38 inhibitor SB203580, whereas enhanced by ERK1/2 inhibitor PD98059. BMP9-induced activation of Runx2 and Smads signaling are reduced by SB203580, and yet increased by PD98059 in MPCs. The <em>in vitro</em> effects of inhibitors are reproduced with adenoviruses expressing siRNA targeted p38 and ERK1/2, respectively. Using mouse calvarial organ culture and subcutaneous MPCs implantation, we find that inhibition of p38 activity leads to significant decrease in BMP9-induced osteogenic differentiation and bone formation, however, blockage of ERK1/2 results in effective increase in BMP9-indcued osteogenic differentiation <em>in vivo</em>. Together, our results reveal that p38 and ERK1/2 MAPKs are activated in BMP9-induced osteogenic differentiation of MPCs. What is most noteworthy, however, is that p38 and ERK1/2 act in opposition to regulate BMP9-induced osteogenic differentiation of MPCs.</p> </div

Inhibition of p38 and ERK1/2 activity leads to opposing effects on BMP9-indcued classical Smads signaling in MPCs.

<p>(<b>A</b>) Western blotting analysis of SB203580 and PD98059 on BMP9-induced Smad1/5/8 phosphorylation. Subconfluent C3H10T1/2 cells were pretreated with SB203580 (25 mM) or PD98059 (10 mM), and then treated with BMP9-CM. At 30 mins post BMP9-CM treatment, total amount and phosphorylated forms of Smad1/5/8 was analyzed by western blotting. (<b>B</b>) Immunofluorescence staining analysis of SB203580 and PD98059 on BMP9-induced nuclear translocation of Smad 1/5/8. Subconfluent C3H10T1/2 cells were pretreated with SB203580 (25 mM) or PD98059 (10 mM), and then treated with BMP9-CM. At 2 hrs post BMP9-CM treatment, nuclear translocation of Smad 1/5/8 by Immunofluenrescence staining. Magnification, ×200 (<b>C</b>) Luciferase reporter analysis of SB203580 and PD98059 on BMP9-induced Smad1/5/8 transcriptional activity. C3H10T1/2 cells were transfected with p12xSBE-luc. Next, cells were pretreated with SB203580 (25 mM) or PD98059 (10 mM), and then treated with BMP9-CM. At 36 hrs, cells were lyzed for luciferase activity assay. Luciferase activity was normalized by total cellular protein concentrations among the samples. “**”, p<0.01 (<i>vs</i>. control groups).</p

Opposing effects of p38 and ERK1/2 on BMP9-induced ALP activity of MPCs.

<p>(<b>A</b>) Inhibition of BMP9-induced ALP activity by p38 inhibitor SB203580 in C3H10T1/2 cells. C3H10T1/2 cells were infected with Ad-BMP9 or Ad-GFP (MOI = 5), followed by treatment with varying concentrations (0, 2, 5 and 10 µM) of SB203580. ALP activity was quantitative measured at day 7. Each assay condition was carried out in triplicate in at least two independent batches. “**”, p<0.01 (<i>vs</i>. control groups); “*”, p<0.05 (<i>vs</i>. control groups). (<b>B</b>) Enhancement of BMP9-induced ALP activity by ERK1/2 inhibitor PD98059 in C3H10T1/2 cells. C3H10T1/2 cells were infected with Ad-BMP9 or Ad-GFP (MOI = 5), followed by treatment with varying concentrations (0, 10, 25 and 50 µM) of PD98059. ALP activity was quantitative measured at 7 days. Each assay condition was carried out in triplicate in at least two independent batches. “**”, p<0.01; “*”, p<0.05 (<i>vs</i>. control groups). (<b>C</b>) Opposing effects of SB203580 and PD98059 on BMP9-induced ALP activity in MEFs. MEFs were infected with Ad-BMP9 or Ad-GFP (MOI = 5), followed by treatment with fixed concentrations of SB203580 (25 µM) or PD98059 (10 µM). ALP activity was quantitative measured at 5 days. Each assay condition was carried out in triplicate in at least two independent batches. “**”, p<0.01 (<i>vs</i>. control groups). (<b>D</b>) Opposing effects of SB203580 and PD98059 on BMP9-induced ALP activity in C2C12 cells. C2C12 cells were infected with Ad-BMP9 or Ad-GFP (MOI = 5), followed by treatment with fixed concentrations of SB203580 (25 µM) or PD98059 (10 µM). ALP activity was quantitative measured at 3 days. Each assay condition was carried out in triplicate in at least two independent batches. “**”, p<0.01 (<i>vs</i>. control groups).</p

Opposing effects of p38 and ERK1/2 on BMP9-induced late osteogenic differentiation and Runx2 activation in MPCs.

<p>(<b>A</b>) Opposing effects of p38 and ERK1/2 on BMP9-induced matrix mineralization of MPCs. C3H10T1/2 cells and MEFs were infected with Ad-BMP9 or Ad-GFP, and then treated with SB203580 (25 mM) or PD98059 (10 mM). At day 21, cells were subjected to Alizarin Red S staining. Magnification, ×200. (<b>B</b>) qPCR analysis of SB203580 and PD98059 on BMP9-induced osteocalcin expression at gene level. C3H10T1/2 cells were infected with Ad-BMP9 or Ad-GFP, and then treated with SB203580 (25 mM) or PD98059 (10 mM). At day 9 and day 11, osteocalcin expression was detected by qPCR. All samples were normalized using endogenous levels of β-actin. “**”, p<0.01 (<i>vs</i>. control groups). (<b>C</b>) Immunocytochemical staining analysis of SB203580 and PD98059 on BMP9-induced osteocalcin expression at protein level. C3H10T1/2 cells were infected with Ad-BMP9 or Ad-GFP (MOI = 5), and then treated with SB203580 (25 mM) or PD98059 (10 mM). At day 12, cells were fixed and subjected to immunocytochemical staining analysis. Magnification, ×200. (<b>D</b>) Opposing effects of p38 and ERK1/2 on BMP9-induced Runx2 transcriptional activity. C3H10T1/2 cells were transfected with OC promoter containing Runx2-responsive element reporter, p6xOSE-Luc. Next, cells were infected with Ad-BMP9 in the presence of SB203580 (25 mM) or PD98059 (10 mM). At 36 hrs post infection, cells were lyzed for luciferase activity assay. Each assay condition was carried out in triplicate in at least two independent batches. Luciferase activity was normalized by total cellular protein concentrations among the samples. “**”, p<0.01 (<i>vs</i>. control groups). (<b>E</b>) qPCR analysis of SB203580 and PD98059 on BMP9-induced Runx2 expression at gene level. C3H10T1/2 cells were infected with Ad-BMP9 or Ad-GFP, and then treated with SB203580 (25 mM) or PD98059 (10 mM). At 24 and 48 hrs post treatment, Runx2 gene expression was detected by qPCR. Each assay condition was carried out in triplicate. All samples were normalized by endogenous level of β-actin. “**”, p<0.01 (<i>vs</i>. control groups). (<b>F</b>) Western blotting analysis of SB203580 and PD98059 on BMP9-induced Runx2 expression at protein level. C3H10T1/2 cells were infected with Ad-BMP9 or Ad-GFP, and then treated with SB203580 (25 mM) or PD98059 (10 mM). At 48 hrs post treatment, Runx2 protein expression was detected by western blotting. β-actin was used to demonstrate equal loading of all samples.</p

BMP9 induces phosphorylation/activation of p38 and ERK1/2 in MPCs.

<p>(<b>A</b>). Western blotting analysis of BMP9-induced phosphorylation of Samd1/5/8, p38 and ERK1/2 in C3H10T1/2 cells. C3H10T1/2 cells were infected with Ad-BMP9 or Ad-GFP (MOI = 5), at 24 hrs, total amount and phosphorylated forms of Smad1/5/8, p38 and ERK1/2 was analyzed by western blotting. β-actin was used to demonstrate equal loading of all samples. (<b>B</b>) Western blotting analysis of BMP9-induced phosphorylation of p38 and ERK1/2 in MEFs. MEFs were infected with Ad-BMP9 or Ad-GFP (MOI = 5), at 24 hrs, total amount and phosphorylated forms of p38 and ERK1/2 was analyzed by western blotting. (<b>C</b>) Western blotting analysis of BMP9-induced phosphorylation of p38 and ERK1/2 in C2C12 cells. C2C12 cell were infected with Ad-BMP9 or Ad-GFP (MOI = 5), at 24 hrs, total amount and phosphorylated forms of p38 and ERK1/2 was analyzed by western blotting. (<b>D</b>) Western blotting analysis of BMP9 conditioned medium (BMP9-CM)-induced phosphorylation of p38 and ERK1/2 in C3H10T1/2 cells. C3H10T1/2 cells were treated with BMP9-CM, total amount and phosphorylated forms of p38 and ERK1/2 was analyzed at indicated time points by western blotting.</p

Knockdown of p38 and ERK1/2 leads to opposing effects on BMP9-indcued osteogenic differentiation of MPCs.

<p>(<b>A</b>) Effective knockdown of p38 and ERK1/2 expression by RNAi. C3H10T1/2 cells were infected with AdR-si-p38 or AdR-si-ERK1/2. At 24 hrs, total amount forms of p38 and ERK1/2 were analyzed by western blotting. NC, negative control. (<b>B</b>) Effect of p38 and ERK1/2 knockdown on BMP9-induced ALP activity. Subconfluent C3H10T1/2 cells were infected with AdR-si-p38 or AdR-si-ERK1/2, and then treated with BMP9-CM. ALP activity was quantitatively assessed at the indicated time points. “*”, p<0.05 (<i>vs</i>. control groups). (<b>C</b>) Effect of p38 and ERK1/2 knockdown on BMP9-induced matrix mineralization. C3H10T1/2 cells were Infected with AdR-si-p38 or AdR-si-ERK1/2, and then treated with BMP9-CM. At 21 days, cells were subjected to Alizarin Red S staining. Magnification, ×200. (<b>D</b>) Effect of p38 and ERK1/2 knockdown on BMP9-induced OC protein expression. C3H10T1/2 cells were infected with AdR-si-p38 or AdR-si-ERK1/2, and then treated with BMP9-CM. At day 12, cells were fixed and subjected to Immunocytochemical stain. Magnification, ×200. (<b>E</b>) Effect of p38 and ERK1/2 knockdown on BMP9-induced Smad1/5/8 phosphorylation. C3H10T1/2 cells were infected with AdR-si-p38 or AdR-si-ERK1/2, and then treated with BMP9-CM. At 30 min post BMP9-CM treatment, total amount and phosphorylated forms of Smad1/5/8 was analyzed by western blotting.</p

Opposing effects of p38 and ERK1/2 on BMP9-induced ALP activity is correlated with the change of cell proliferation.

<p>(<b>A</b>) Effect SB203580 and PD98059 on BMP9-induced ALP activity in C3H10T1/2 cells. Subconfluent C3H10T1/2 cells were infected with Ad-BMP9 (MOI = 5) or Ad-GFP (MOI = 5), followed by treatment with SB203580 (25 µM) or PD98059 (10 µM), ALP activity was quantitative measured at the indicated time points. Each assay condition was carried out in triplicate in at least two independent batches. (<b>B</b>) Effect of SB203580 and PD98059 on BMP9-induced cell proliferation in C3H10T1/2 cells. Subconfluent C3H10T1/2 cells were infected with Ad-BMP9 (MOI = 5) or Ad-GFP (MOI = 5), followed by treatment with SB203580 (25 µM) or PD98059 (10 µM), cell proliferation was monitored by MTT assay at the indicated time points. Each assay condition was carried out in triplicate in at least two independent batches.</p

Opposing effects of p38 and ERK1/2 on BMP9-induced new bone formation in calvarial organ culture.

<p>(<b>A</b>) Mouse calvariae was infected with Ad-BMP9, and then treated with SB203580 (25 µM) and PD98059 (10 µM). At 4 days post infection, calvariae was placed into fresh media and return to incubator for another 3 day until day 7. The amount of original bone remaining and new bone was assessed by H&E staining. Magnification, ×600. (<b>B</b>) measurement of new bone width using Image Pro Plus. “*”, p<0.05 (<i>vs.</i> BMP9).</p

Knockdown of p38 and ERK1/2 leads to opposing effects on BMP9-indcued ectopic bone formation.

<p>(<b>A</b>) Co-infection efficiency of Ad-BMP9 and AdR-si-p38 (or AdR-si-ERK1/2). C3H10T1/2 cells were co-infected with Ad-BMP9 and AdR-si-p38 (or AdR-si-ERK1/2). At 24 hrs post co-infection, the co-infection efficiency was determined under a fluorescence microscope. (<b>B</b>) Macrographic images of ectopic bone mass. (<b>C</b>) Total volume of retrieved samples. “**”, p<0.01 (<i>vs.</i> control groups) (<b>D</b>) Histological stains of retrieved samples. Retrieved tissues were decalcified, fixed in 10% formalin overnight, and embedded in paraffin. Serial sections of the embedded specimens were stained with H&E, Masson’s Trichrome stain and Alcian Blue stain. BM, Bone Matrix; Ch, Chondrocyte; UMPCs, Undifferentiated mesenchymal progenitor cells; CM, Cartilage Matrix. Magnification, ×150.</p