Fig 2: Transgenic mice overexpressing Heparanase showed increased BMP6/SMAD signaling.(A) evaluation of BMP6 mRNA in the liver of WT and TG-HPA mice (B) evaluation of Id1 mRNA, in relation to Hprt1. (C) Western blot of pSMAD5, of total SMAD5 and of GAPDH as housekeeping for normalization in densitometry quantification. The values are expressed as fold change of wild type mice.

Fig 3: PARP1 ADP-ribosylates, whereas PARG de-ADP-ribosylates Smad1 and Smad5. A, in vitro ADP-ribosylation assay of Smad1, Smad5, Smad4, and Smad3. GST-Smad proteins were incubated with 32P-β-NAD+ and recombinant PARP1. After glutathione-agarose pulldown, ADP-ribosylated GST-Smad1/5/4/3 were imaged by autoradiography. The radioactive protein bands of PARP1 and GST-Smads are marked. The lower panel shows GST-Smad proteins stained with Coomassie Brilliant Blue after SDS-PAGE. M, molecular size marker. A representative autoradiogram of four assays is shown. Molecular size markers in kDa are also marked. B, in vitro de-PARylation of GST-Smad1 and GST-Smad5. PARG or vehicle were incubated with equal amounts of GST-Smad1/5, 32P-β-NAD+, and recombinant PARP1 for 30 min at 37 °C. ADP-ribosylated proteins were imaged by autoradiography. The radioactive protein bands of PARP1 and GST-Smads are marked. The lower panel shows total GST proteins stained with Coomassie Brilliant Blue. M, molecular size marker. A representative autoradiogram of five assays is shown. Molecular size markers in kDa are also marked. C, immunoblot of endogenous PARP1 from HEK293T cell extracts bound to the indicated GST-Smad1 MH1 domain mutants. TCL shows the levels of endogenous PARP1. Total GST-Smad1 mutant proteins used for immunoblotting of endogenous PARP1 are stained with Coomassie Brilliant Blue in the middle panel. The Smad1 sequence motif that was mutated (red letters) and that represents a genuine ADP-ribosylation target sequence is shown in the bottom panel. A representative immunoblot of three repeats is shown. Molecular size markers in kDa are also marked. D, in vitro ADP-ribosylation assay of GST-Smad1-MH1 domain mutants. Control GST, beads, WT-Smad1-MH1 domain, and three mutants (as shown in C) were incubated with 32P-β-NAD+ and recombinant PARP1. ADP-ribosylated proteins were imaged via autoradiography. The radioactive protein bands of PARP1 and GST-Smad1-MH1 are marked. Total GST proteins were checked by Coomassie Brilliant Blue staining. Lane 1/3 WT indicates a reaction where one-third of the GST-Smad1-MH1 protein was used compared with the WT lanes. A representative autoradiogram of two assays is shown. Molecular size markers in kDa are also marked. E, immunoblot of recombinant PARP1 (20 ng) bound to the indicated GST-Smad1 MH1 domain mutants. The experiment is a repeat of the ribosylation assay of Fig. 8D, except that only cold β-NAD+ was used during incubation, followed by pulldown and immunoblotting. On the side, increasing amounts of recombinant PARP1 along with TCL from HEK293T cells show the levels of recombinant PARP1 used in the assay relative to endogenous PARP1. Total GST-Smad1 mutant proteins checked by Coomassie Brilliant Blue staining, used for immunoblotting of recombinant PARP1. A representative immunoblot of two repeats is shown. Molecular size markers in kDa are also marked. F, molecular model adapted to a detail from the crystal structure of two Smad3 MH1 domains bound to the Smad-binding DNA element (PDB code 1mhd). Shown is a ribbon diagram of the whole Smad3 MH1 domain with colored amino acids and the acceptor glutamate (red) and lysine (blue) residues drawn as stick and ball structures on the bottom side of the surface of the regulatory α-helix of one Smad3 MH1 subunit (white arrow). The β-hairpin that contacts DNA is also indicated (white arrow). WB, Western blotting.

Fig 4: PARP1 and PARG modulate Smad1 activity during BMP signaling. A model shows the BMP ligand activating cell surface receptors II and I, with the latter phosphorylating Smad1, Smad5, and Smad8, facilitating oligomerization with Smad4 to enter the nucleus through a nuclear pore. The nuclear Smad complex associates with transcription factors, co-activators or co-repressors on chromatin to regulate target genes such as ID1, SMAD6, and SMAD7. The ADP-ribosylating enzyme PARP1 interacts with the nuclear Smad complex. With β-NAD+ as a donor, PARP1 attaches ADP-ribose chains (of n size, [ADPr]n) on itself (not shown) and on to the Smad complex to assist its dissociation from DNA or inhibit association with DNA (Smad proteins away from DNA). PARG that has affinity toward ADP-ribose chain promotes de-ADP-ribosylation resulting in mono-ADP-ribose chains on the Smad complex (not verified experimentally and not shown for simplicity) and a pool (n) of ADP-ribose, thus facilitating enhanced binding of the Smad complex to DNA. For simplicity, the figure emphasizes Smad1 ADP-ribosylation. The extracellular matrix (ECM), cytoplasmic, and nuclear cell compartments are highlighted.

Fig 5: Smad5 KO induces specific transcriptional profiling changes related to metabolic homeostasis. (A, B) Functional annotation of up or downregulated gene categories in Smad5 KO and LDN193189-treated hES cells identified by GO.