Journal: Journal of the American Society of Nephrology

Article Title: Chordin-like 1 and Twisted Gastrulation 1 Regulate BMP Signaling following Kidney Injury

doi: 10.1681/asn.2008070768

Figure Lengend Snippet: Figure 4. CHRDL1 functions as a general BMP-signaling amplifier but specifically antagonizes BMP7 in the presence of TWSG1. (A) P19 embryonal carcinoma cells transfected with the BMP transcriptional reporter pBRE-Luc were incubated overnight with BMP4 (5 ng/ml) or BMP7 (10 ng/ml) and increasing amounts of CHRDL1 (50 to 400 ng/ml) or Chordin (100 to 800 ng/ml). Both CHRDL1 and Chordin amplify BMP4 signaling; however, in contrast to Chordin, which antagonizes BMP7 signaling, CHRDL1 amplifies BMP7 signaling in a dosage-responsive manner. (B) In the presence of TWSG1 (100 to 400 ng/ml), CHRDL1 (200 ng/ml) continues to act as an amplifier of BMP4 signaling unlike Chordin (400 ng/ml), which becomes a potent BMP4 antagonist. In contrast, CHRDL1 amplification of BMP7 signaling is converted to antagonism by addition of TWSG1 in a dosage-dependent manner. (C) In the P19 pBRE-luc reporter assay, the addition of TWSG1, CHRDL1, or Chordin (400 ng/ml) alone does not affect transcriptional activation. (D) CHRDL1 binds BMP7 and TWSG1 only in a trimolecular complex. A co-immunoprecipitation experiment in which CHRDL1 (500 ng/ml) is incubated in the presence of TWSG1 (500 ng/ml) and/or BMP7 (250 ng/ml) demonstrates that CHRDL1 binds TWSG1 and BMP7 only when all three proteins are present. No evidence of binding is detected when TWSG1 or BMP7 alone is incubated with CHRDL1. Recombinant proteins were immunoprecipitated with a goat polyclonal antibody against CHRDL1, and blots of goat IgG serve as loading controls. The last panel, a CHRDL1 immunoblot of an identical immunoprecipitation substituting an irrelevant goat antibody, demonstrates the absence of nonspecific binding by CHRDL1 to either the goat antibody or Protein G beads used in the experiment. The first lane of all blots contains recombinant protein as a positive control. (E) TWSG1 is strongly expressed in tubule epithelia of the mouse and human kidney. (Top) Strong immunohistochemical staining for TWSG1 in tubule epithelia of adult mouse kidney but weak staining in the glomerulus (G) and interstitial cell population (inset). (Right) Negative control using an antibody of the same species specific for macrophage. (Bottom) Immunofluorescent micro- graphs of an adult human kidney showing TWSG1 expression (red) in both proximal tubules (PT), marked green with Lotus lectin, and other tubules. As in the mouse, glomerular expression of TWSG1 is much weaker than seen in tubular epithelia. (Right) Negative control for TWSG1.

Article Snippet: BMP7 was detected with mouse anti- BMP7 (R&D Systems) and a sheep anti-mouse HRP conjugate (Am- ersham).

Techniques: Transfection, Incubation, Amplification, Reporter Assay, Activation Assay, Immunoprecipitation, Binding Assay, Recombinant, Western Blot, Positive Control, Immunohistochemical staining, Staining, Negative Control, Expressing

Journal: Journal of the American Society of Nephrology

Article Title: Chordin-like 1 and Twisted Gastrulation 1 Regulate BMP Signaling following Kidney Injury

doi: 10.1681/asn.2008070768

Figure Lengend Snippet: Figure 5. CHRDL1 reduces BMP7-stimulated Smad activation and ID gene expression in the presence of TWSG1 but has no effect on BMP4 signaling. (A and B) In the presence of TWSG1, CHRDL1 inhibits Smad phosphorylation by BMP7 but not by BMP4. P19 cells were serum starved for 2 h and then incubated with BMPs (10 ng/ml) and antagonists for 2 h before lysis and Western blotting. Blots were probed for phosphorylated Smads 1, 5, and 8 and -tubulin. In lanes 3, 4, and 5, Noggin (200 ng/ml), TWSG1, and CHRDL1 alone (400 ng/ml) were added. In lanes 6 through 9 CHRDL1 (400 ng/ml) was added together with increasing concentrations of TWSG1 (50, 100, 200, and 400 ng/ml). BMPs were incubated at 37 C for 1 h with or without antagonists before application. Note that the arrow points to the band corresponding to pSmad1/5/8. The top band (arrowhead) is a contaminating band specific to P19 lysates and is not present in lysates from MDCK cells (compare C and D). (C and D) The effects of CHRDL1 and TWSG1 on BMP signaling can be reproduced in the MDCK kidney cell line. (E) RT-PCR showing expression of ID1, 2, and 3 genes in HK-2 cells stimulated with BMP4 or 7 (25 ng/ml) for 6 h in the presence of varying concentrations (100 to 400 ng/ml) of CHRDL1 and/or TWSG1. ID expression by HK-2 cells in response to BMP4 is unaffected by the presence of TWSG1. In cells incubated with BMP7, the addition of TWSG1 reduces ID gene expression.

Article Snippet: BMP7 was detected with mouse anti- BMP7 (R&D Systems) and a sheep anti-mouse HRP conjugate (Am- ersham).

Techniques: Activation Assay, Gene Expression, Phospho-proteomics, Incubation, Lysis, Western Blot, Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: Journal of the American Society of Nephrology

Article Title: Chordin-like 1 and Twisted Gastrulation 1 Regulate BMP Signaling following Kidney Injury

doi: 10.1681/asn.2008070768

Figure Lengend Snippet: Figure 6. Overexpression of mouse Chrdl1 in the collecting duct substan- tially reduces BMP signaling in vivo. (A) Diagram of mouse Chrdl1 transgene construct driven by a collecting duct–specific enhancer element from intron 1 of the Bmp7 gene; quantitative PCR assay shows Chrdl1 overexpression in kidneys of two embryonic day 17.5 transgenic embryos (150 and 151) com- pared with wild-type. (B through D) Immunolocalization of phosphorylated Smads (red) in the embryonic day 17.5 wild-type kidney (B), and kidneys from transgenic embryos 150 and 151 (C and D) show that BMP signaling is substantially reduced by expression of the Chrdl1 transgene. Collecting ducts (CD) were localized by staining with the lectin Dolichos Biflorus Agglu- tinin (green), and nuclei were counterstained with DAPI (blue).

Article Snippet: BMP7 was detected with mouse anti- BMP7 (R&D Systems) and a sheep anti-mouse HRP conjugate (Am- ersham).

Techniques: Over Expression, In Vivo, Construct, Real-time Polymerase Chain Reaction, Transgenic Assay, Expressing, Staining

Journal: Biology Open

Article Title: Chemotropic signaling by BMP7 requires selective interaction at a key residue in ActRIIA

doi: 10.1242/bio.042283

Figure Lengend Snippet: Growth cone expression of type II BMP receptor-targeted shRNA vectors in DSC neurons. (A) Representative images of DSC neurons electroporated with the indicated shRNA vectors in dissociated culture labeled for GFP (green), ERM (red) and DAPI (blue). The presence of GFP reflects expression of each of the shRNA vectors in cultured DSC neurons including in the axonal processes (arrows) and growth cones (arrowheads). In the far right column, a Smad4 Δ DSC neuron is shown, treated with 50 ng/ml BMP7 for 30 min, and represents a collapsed growth cone (arrowhead). Scale bar: 50 μm. (B) Western blots of dsRed Δ DSC or AIIA Δ DSC lysates probed with an anti-ActRIIA antibody. Detection of GAPDH provided a loading control. Electroporation of sh-AIIA in DSC cultures decreases ActRIIA protein expression to 58%±7.4% of the expression levels in GFP-enriched, dsRed Δ DSC lysates (mean±s.e.m.; n =4; * P <0.05) (Student's two-tailed t- test).

Article Snippet: Recombinant BMP7 was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, shRNA, Labeling, Cell Culture, Western Blot, Control, Electroporation, Two Tailed Test

Journal: Biology Open

Article Title: Chemotropic signaling by BMP7 requires selective interaction at a key residue in ActRIIA

doi: 10.1242/bio.042283

Figure Lengend Snippet: BMP7-evoked growth cone collapse in DSC neurons in the presence of control and type II BMP receptor shRNA vectors. (A) The percentage of growth cone area decrease [GCAD=(100–{[(GC area in the presence of BMP7–control GC area)/control GC area]×100}); mean±s.e.m.] in dsRed Δ DSC neurons stimulated by 50 ng/ml BMP7 for 30 min is not affected by expression of the non-specific shRNA vector, sh-dsRed (GCAD=48.3%±3.3%) when compared with untransfected, GFP − neurons (GCAD=49.4%±4.8%) in the same culture ( n =3). (B) Growth cone area decrease (mean±s.e.m.) in response to 50 ng/ml BMP7 was measured for shRNA-transfected GFP + neurons only. Expression of pLL3.7, the shRNA parent vector, (GCAD=48.2%±3.5%; n =3) or knockdown of ActRIIB (AIIB Δ DSC, GCAD=54.4%±3.3%; n =3) or Smad4 (Smad4 Δ DSC, GCAD=42.6%±7.2%; n =3) did not interfere with BMP7-evoked growth cone collapse. In contrast, BMP7-evoked growth cone collapse was significantly inhibited in AIIA Δ DSC (GCAD=15.7%±4.4%; n =4, ** P <0.005) and BRII Δ DSC cultures (GCAD=11.3%±1.4%; n =3, ***P <0.001) (Student's two-tailed t- test).

Article Snippet: Recombinant BMP7 was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Control, shRNA, Expressing, Plasmid Preparation, Transfection, Knockdown, Two Tailed Test

Journal: Biology Open

Article Title: Chemotropic signaling by BMP7 requires selective interaction at a key residue in ActRIIA

doi: 10.1242/bio.042283

Figure Lengend Snippet: Site-directed mutagenesis of K76 in ActRIIA does not impede variant receptor expression or BMP7-stimulated Smad1/5/8 phosphorylation. (A) Amino acid sequences of mouse ActRIIA in the region of the K76 (red) and V3 (blue) mutations and mouse ActRIIB in the region of the E75 (green) mutation. Numbering corresponds to the amino acid number: for the K76 and E75 mutations according to PDB ID: 1LX5 and for the V3 mutations according to NCBI accession no.: NP_031422.3 ( Fig. S3 ). The mutations at position 76 in ActRIIA replace lysine (K) with either alanine (A) or glutamic acid (E). The mutation at position 75 in ActRIIB replaces glutamic acid with lysine. The mutations at positions 431 to 433 do not replace the three valine (V) residues in the amino acid sequence but rather alter the nucleotide sequence to create an ActRIIA cDNA that is resistant to sh-AIIA while maintaining the amino acid sequence. (B) Western blots of whole-cell C2C12 lysates, transfected with pcDNA3, ActRIIA V3, ActRIIA V3 K76A or ActRIIA V3 K76E, were probed with an anti-ActRIIA antibody. Detection of GAPDH provided a loading control. (C) Quantification of western blots of transfected whole-cell C2C12 lysates incubated with or without 50 ng/ml BMP7 probed with a phospho-specific Smad1/5/8 antibody. Detection of total Smad1 provided a loading control. Densitometric analysis (mean±s.e.m.; n =2) shows an increase in response to BMP7 in cells expressing ActRIIA V3 (104% over control), ActRIIA V3 K76A (191% over control) and ActRIIA V3 K76E (179% over control).

Article Snippet: Recombinant BMP7 was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Mutagenesis, Variant Assay, Expressing, Phospho-proteomics, Sequencing, Western Blot, Transfection, Control, Incubation

Journal: Biology Open

Article Title: Chemotropic signaling by BMP7 requires selective interaction at a key residue in ActRIIA

doi: 10.1242/bio.042283

Figure Lengend Snippet: ActRIIA V3 K76 receptor variants cannot rescue sh-AIIA -mediated inhibition of BMP7-stimulated chemotaxis. (A) Chemotaxis in response to 50 ng/mL BMP7 (CI={[(no. BMP7 treated cells in filter pores)–(no. control cells in filter pores)]/(no. control cells in filter pores)}×100; mean±s.e.m.). Chemotaxis indices for dsRed Δ WEHI cells co-expressing pcDNA3 (lane 1, CI=90.9±6.8), ActRIIA V3 (lane 3, CI=92.3±13.2), ActRIIA V3 K76A (lane 5, CI=19.8±2.15) and ActRIIA V3 K76E (lane 7, CI=−4.5±9.5) ( n =2). Chemotaxis indices for AIIA Δ WEHI cells co-expressing pcDNA3 (lane 2, CI=−3.1±4.4), ActRIIA V3 (lane 4, CI=83.8±7.7), ActRIIA V3 K76A (lane 6, CI=−13.3±10) and ActRIIA V3 K75E (lane 8, CI=−13.4±1.6). BMP7-evoked chemotaxis in dsRed Δ WEHI cells co-expressing pcDNA3 differs significantly from chemotaxis in dsRed Δ WEHI cells co-expressing ActRIIA V3 K76A and ActRIIA V3 K76E and in AIIA Δ WEHI cells co-expressing pcDNA3 and ActRIIA V3 K76A (* P <0.02, n =2) (Student's two-tailed t -test). The difference in BMP7-evoked chemotaxis between dsRed Δ WEHI cells co-expressing pcDNA3 and AIIA Δ WEHI cells co-expressing ActRIIA V3 K76E is also significant (** P <0.005, n =2) (Student's two-tailed t -test). (B) Western blots of dsRed Δ WEHI or AIIA Δ WEHI cell lysates co-expressing pcDNA3 were probed for ActRIIA expression. Detection of GAPDH provided a loading control. Electroporation of sh-AIIA in WEHI 274.1 cells decreases ActRIIA protein expression to 36%±5.1% of the expression levels in dsRed Δ WEHI lysates (mean±s.e.m.; n =4; ** P <0.005, Student's two-tailed t- test).

Article Snippet: Recombinant BMP7 was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Inhibition, Chemotaxis Assay, Control, Expressing, Two Tailed Test, Western Blot, Electroporation

Journal: Biology Open

Article Title: Chemotropic signaling by BMP7 requires selective interaction at a key residue in ActRIIA

doi: 10.1242/bio.042283

Figure Lengend Snippet: Expression of ActRIIA V3 K76 variant receptors does not inhibit BMP7-evoked Smad1/5/8 phosphorylation. (A) Western blots of dsRed Δ WEHI or AIIA Δ WEHI lysates co-expressing ActRIIA V3 K76A incubated with or without 50 ng/ml BMP7 for 30 min and probed for pSmad1/5/8. Detection of tSmad provided a loading control. BMP7 stimulated robust increases (mean±s.e.m.) in the levels of pSmad1/5/8 in dsRed Δ WEHI (195% over control, n =2) or AIIA Δ WEHI (113% over control, n =2; * P <0.05, Student's two-tailed t -test) cells co-expressing ActRIIA V3 K76A. (B) Quantification of western blots (mean±s.e.m.) of dsRed Δ WEHI or AIIA Δ WEHI lysates co-expressing pcDNA3, ActRIIA V3, ActRIIA V3 K76A and ActRIIA V3 K76E incubated in the presence of 50 ng/ml BMP7 for 30 min and probed for pSmad1/5/8. Detection of tSmad provided a loading control. Levels of pSmad1/5/8 were normalized to pSmad1/5/8 levels in unstimulated cells. All conditions demonstrated increases in response to BMP7 stimulation compared with control cells. The data for the unstimulated controls are not shown. There was no significant difference in the level of pSmad1/5/8 in dsRed Δ WEHI lysates compared with pSmad1/5/8 levels in AIIA Δ WEHI cells for any of the transfection conditions.

Article Snippet: Recombinant BMP7 was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Variant Assay, Phospho-proteomics, Western Blot, Incubation, Control, Two Tailed Test, Transfection

Journal: Biology Open

Article Title: Chemotropic signaling by BMP7 requires selective interaction at a key residue in ActRIIA

doi: 10.1242/bio.042283

Figure Lengend Snippet: BMP7-evoked Akt phosphorylation is inhibited by loss of ActRIIA expression or expression of ActRIIA V3 K76A. (A,B) Quantification (mean±s.e.m.; n =2) of western blots of dsRed Δ WEHI and AIIA Δ WEHI lysates co-expressing the indicated cDNA expression constructs incubated with or without 50 ng/ml BMP7 for 30 min and probed for pAkt. Detection of tAkt provided a loading control. pAkt levels for each transfection condition were normalized to pAkt levels in the respective unstimulated cells. (A) BMP7 stimulated increases in the levels of pAkt in dsRed Δ WEHI cells co-expressing pcDNA3 (84%±19% over control; * P <0.05, Student's two-tailed t -test) or ActRIIA V3 (55%±17% over control). (B) BMP7 stimulated increases in the levels of pAkt only in dsRed Δ WEHI co-expressing ActRIIA V3 (55%±17% over control, lane 2). Increases in pAkt over control levels were not observed in any other condition in response to BMP7 including in dsRed Δ WEHI co-expressing ActRIIA V3 K76A (16%±7.5% over control, lane 4), AIIA Δ WEHI co-expressing ActRIIA V3 (1.8%±4.7% over control, lane 6) or AIIA Δ WEHI co-expressing ActRIIA V3 K76A (−3.8%±6.2% over control, lane 8).

Article Snippet: Recombinant BMP7 was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Phospho-proteomics, Expressing, Western Blot, Construct, Incubation, Control, Transfection, Two Tailed Test

Journal: Biology Open

Article Title: Chemotropic signaling by BMP7 requires selective interaction at a key residue in ActRIIA

doi: 10.1242/bio.042283

Figure Lengend Snippet: Signaling and receptor binding interactions of chemotropic BMPs. (A) Model of chemotropic BMP signaling indicating the potential asymmetric recruitment of ActRIIA (red) and BMPR2 (yellow) into the tetrameric receptor complex. A type I BMP receptor pair is represented as a preformed complex (PFC, gray subunits), though it is unclear whether BMP7 selectively engages PFCs containing ActRIIA and BMPR2 or recruits individual receptors into a complex with type I BMP receptor subunits. BMP7 stimulates Smad- (S) and PI3K-dependent (P) downstream signaling linked to type I and type II BMP receptors, respectively. (B) Spacefill representation of the crystal structure of a BMP7 dimer (blue) bound to the ActRIIA ECD (red) generated from Protein Data Bank ID, 1LX5 using NGL Viewer . Enlarged area indicates amino acid positions for BMP7 R48 and E60 as well as ActRIIA K76. (C–F) Representations of wild-type and mutated amino acids in BMP-ActRIIA interactions and the associated chemotropic and Smad activity. (C) BMP7 R48 is predicted to associate with ActRIIA K76 and BMP7-evoked chemotropic activity requires interaction with ActRIIA. (D) BMP6 Q48 is not predicted to associate with ActRIIA K76 and BMP6 does not stimulate chemotropic activity. (E) BMP6 Q48R demonstrates potent chemotropic activity and would potentially interact with ActRIIA K76. (F) Mutation of K76 in ActRIIA to alanine (A76) is predicted to disrupt the interaction with BMP7 R48. The presence of ActRIIA K76A blocks BMP7-evoked chemotropic activity. Mutations at this site do not have any effect on BMP-stimulated Smad phosphorylation.

Article Snippet: Recombinant BMP7 was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Binding Assay, Generated, Activity Assay, Mutagenesis, Phospho-proteomics