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