1. Volume 12, Issue 4, Pages 817-828 (October 2003)
Activin Receptor-like Kinase (ALK)1 Is an Antagonistic Mediator of Lateral TGFβ/ALK5 Signaling 
Marie-José Goumans, Gudrun Valdimarsdottir, Susumu Itoh, Franck Lebrin, Jonas Larsson, Christine Mummery, Stefan Karlsson, Peter ten Dijke 
Molecular Cell 
Volume 12, Issue 4, Pages (October 2003)
DOI: /S (03)

2. Figure 1
MEECs Lacking ALK5 Are Deficient in TGFβ/ALK1-Induced Smad1/5 Phosphorylation and Transcriptional Response
(A) TGFβ-induced Smad2 and Smad1/5 phosphorylation in wild-type and ALK5−/− MEECs. MEECs were stimulated with 1 ng/ml TGFβ3 for 1 hr and subjected to Western blot analysis using pSmad1/5 or pSmad2 antibodies. Asterisk indicates nonspecific band.
(B–D) Effect of TGFβ on (SBE)4-luc reporter (B), BRE-luc reporter (C), and (CAGA)12-luc (D) in ALK5−/− and wt MEECs. Ability of caALK1 and caALK5 to activate these reporters in MEECs is shown as control.
(E and F) Antisense oligonucleotide (ASO)-mediated knockdown of ALK5 expression inhibits TGFβ-induced Smad2 and Smad1/5 phosphorylation. After loading MEECs with ASO (E), or sense oligonucleotides (SO) (F) for ALK1 or ALK5, the cells were stimulated without or with 1 ng/ml TGFβ3 for 1 hr and subjected to Western blot analysis using the indicated antibodies. PS1 ctrl, cell lysate from COS-7 cells transfected with caALK1 and Smad1; PS2 ctrl, cell lysate from COS-7 cells transfected with caALK5 and Smad2.
(G and H) ASO-mediated knockdown of ALK5 expression inhibits both TGFβ-induced BRE-luc (G) as well as (CAGA)12-luc (H) activity. Transient transfected MEECs with the indicated plasmids (and reporter plasmid) were loaded with ASO, SO, or scrambled oligonucleotides (SCR) and stimulated with TGFβ3.
Molecular Cell  , DOI: ( /S (03) )

3. Figure 2 TGFβ Induces a Heteromeric Complex between ALK1 and ALK5
(A) TGFβ-induced heteromeric complex formation between ALK1 and ALK5 in COS cells. HA-tagged ALK1 and ALK5 were cotransfected with TβR-II into COS cells and treated with TGFβ1 where indicated. Cell lysates were subjected to HA immunoprecipitation followed by Western blotting with ALK5-specific antibody (upper panel). Cell lysates were also directly subjected to Western blotting to check expression of type I receptors (lower panel).
(B and C) ALK5−/− MEECs were infected with caALKs at the indicated multiplicity of infection (moi) and affinity labeled with 125I-TGFβ3, followed by crosslinking 40 hr after infection. Aliquots of cell lysates were first subjected to immunoprecipitation using the indicated antisera followed by SDS-PAGE and autoradiography.
(C) MEECs were transfected with the indicated HA-tagged ALK and RNAi constructs and affinity labeled with 125I-TGFβ3, followed by crosslinking 40 hr after transfection and immunoprecipitated using an anti-HA antibody.
(D and E) ALK5−/− MEECs were transfected with BRE-luc reporter in the absence or presence of various ALK1 or ALK5 (mutant) and chimeric expression constructs. TGFβ3 was added where indicated. ALK1(E)/ALK5(I) is ALK1-extracellular domain and ALK5-intracellular domain; ALK5(E)/ALK1(I) is ALK5-extracellular domain and ALK1 intracellular domain.
(F) ALK5−/− MEECs were transfected with BRE-luc reporter and ALK5(3A) in the presence of SO, ASO, or SCR for ALK1. TGFβ3 was added where indicated.
(G and H) ALK5(3A) does not mediate a (SBE)4-luc activity in epithelial (R4-2) cells lacking functional ALK5 (G) or ALK5−/− MEFs (H). TGFβ3 (1 ng/ml) was added where indicated.
Molecular Cell  , DOI: ( /S (03) )

4. Figure 3
Effect of ALK5 Mutant Defective in Smad Activation on TGFβ-Induced Biological Responses in ALK5−/− MEECs
(A) MEECs were infected for 40 hr with the indicated adenovirus (moi 500). Where indicated, TGFβ3 (1 ng/ml) was added prior to preparing cell lysates. Cell lysates were analyzed with antibodies specific for pSmad1/5, pSmad2, or Id1.
(B and C) MEECs were infected with the indicated viruses and loaded with the indicated s-oligonucleotides (C) 24 hr prior to the proliferation assay. Where indicated, TGFβ3 (0.5 ng/ml) was added to the well. After 3 days, cell numbers were counted using a coulter counter.
(D and E) ALK5−/− MEECs were infected with LacZ, caALK1, caALK5, or ALK5(3A) adenovirus 40 hr and loaded with the indicated s-oligos (E) 4 hr prior to the migration assay. Where indicated, TGFβ3 (0.5 ng/ml) was added to the lower chamber. After 6 hr, the MEECs were stained with crystal violet blue and the number of cells that had migrated through the filter were counted.
Molecular Cell  , DOI: ( /S (03) )

5. Figure 4
ALK5 Kinase Activity Is Important for Optimal Activation of the ALK1 Pathway
(A) Effect of SB on ALK5 or TβRII kinase activity. COS cells, transfected with the indicated plasmids, were lysed, and immunoprecipitated with an anti-HA antibody, followed by an in vitro kinase assay.
(B) After incubation with 5 μM SB for 16 hr, MEECs were incubated without or with TGFβ3 (1 ng/ml) for 1 hr and subjected to Western blot analysis using pSmad1/5 or pSmad2 antibodies. Incubation of the blot with an actin antibody was performed as a control for equal loading.
(C) ALK5−/− MEECs were transfected with (SBE)4-luc reporter in the absence or presence of various ALK1 or ALK5 expression constructs. TGFβ3 (1 ng/ml) and 5 μM SB were added where indicated.
(D and E) Effect of SB on TGFβ-, caALK1-, or caALK5-induced transcriptional activation of BRE-luc or (CAGA)12-luc. The cells were incubated with 5 μM SB for 12 hr and without or with TGFβ3 (1 ng/ml) for an additional 6 hr.
(F) caALK5 can activate the caALK1 kinase activity. MEECs were infected with the indicated adenovirusses. 40 hr after infection, the cells were lysed and immunoprecipitated with an anti-ALK1 antibody followed by in vitro kinase assay.
(G) caALK5 can potentiate caALK1-induced Smad1/5 phosphorylation. MEECs and R4-2 cells were infected with the indicated adenoviruses. 40 hr after infection, the cells were lysed and subjected to Western blot analysis using pSmad1/5 or pSmad2 antibodies. Incubation of the blot with and anti-HA or actin antibody was performed as a control for infection and equal loading.
(H) MEECs were transfected with BRE-luc reporter in combination with caALK1 or caALK5, and luciferase activity was measured.
Molecular Cell  , DOI: ( /S (03) )

6. Figure 5
TβRII Is Required for TGFβ/ALK5- and TGFβ/ALK1-Induced Smad Phosphorylation
(A) MEECs were infected with adenovirus expressing LacZ, dnTβRII using an moi of hr after infection, the cells were stimulated without or with 5 ng/ml TGFβ3 or BMP6 (50 ng/ml) for 1 hr and subjected to Western blot analysis using the indicated antibodies.
(B) TβRII−/− MEECs or wt MEECs were stimulated with TGFβ3 (1 ng/ml) and subjected to Western blot analysis using the indicated antibodies. The blot was incubated with actin antibodies as control for equal loading.
(C) ALK5−/− MEECs, mock transfected or transfected with caALK5 in the absence or presence of dnTβRII, were nontreated or treated with TGFβ3 and luciferase activity was measured.
(D) ALK5−/− MEECs were transfected with the indicated plasmids and either nontreated or treated with TGFβ3 (1 ng/ml) for 18 hr and luciferase activity was measured.
(E) R4-2 cells were infected with indicated adenoviruses expressing various receptors ort LacZ using an moi of 500 and affinity labeled with 125I-TGFβ3 40 hr after infection and subjected to immunoprecipitation using α-ALK1 specific antiserum.
(F) Cell lysates from (E) were analyzed for Smad1/5 phosphorylation and expression of the different ALKs by Western blotting using α-PS1 or α-HA, respectively.
(G) ALK5−/− MEECs, mock transfected or transfected with the indicated receptors, were nontreated or treated with TGFβ3 and luciferase activity was measured. Plotted is the average of induction of three independent experiments.
Molecular Cell  , DOI: ( /S (03) )

7. Figure 6 Inhibitory Effect of ALK1 on TGFβ/ALK5 Signaling
(A) Inhibitory effect of ALK1/Smad5 pathway on TGFβ/ALK5-induced (CAGA)12-luc response. MEECs were transfected with (CAGA)12-luc and the indicated constructs. TGFβ3 (1 ng/ml) was added where indicated, and luciferase acitivity was measured.
(B) Effect of caALK1 on TGFβ/ALK5-induced (CAGA)12-luc response in HepG2 cells.
(C) Effect of caALK1 on caALK5-induced (CAGA)12-luc response in MEEC cells.
(D and E) siRNAi-mediated knockdown of ALK1 potentiates TGFβ/ALK5 activity. MEECs were transfected with (CAGA)12-luc and the indicated constructs. TGFβ3 (1 ng/ml) was added where indicated. To demonstrate the knockdown of ALK1, RT-PCR was performed for the detection of ALK1 mRNA on the transfected cells. Actin expression was measured as an internal control.
(F) Inhibitory effect of ALK1 on TGFβ/ALK5-induced (CAGA)12-luc response in MEECs in the absence or presence of overexpressed TβRII or Smad4. MEECs were transfected and luciferase activity was measured.
(G) MEECs were infected with adenovirus expressing LacZ or caALK1, stimulated with 1 ng/ml TGFβ3 for 1 hr, and subjected to Western blot analysis using the indicated antibodies.
(H) MEECs were transfected with (CAGA)12-luc reporter in combination with caALK1 and/or Smad3, and luciferase activity was measured.
Molecular Cell  , DOI: ( /S (03) )

8. Figure 7 Schematic Model for Activation of TGFβ Receptor in ECs
TGFβ first binds to TβRII, which subsequently recruits ALK5. ALK5 is phosphorylated and activated by TβRII kinase. ALK5 can recruit ALK1 into the complex, and for its activation both ALK5 and TβRII kinase are required. The stoichiometry between receptor components is unknown; ALK1 and ALK5 may form heterodimers with two TβRIIs. Activated ALK1 and activated ALK5 induce phosphorylation of Smad1/5 and Smad2/3, respectively. ALK1 and ALK5 have opposite effects on EC migration and proliferation. In addition, ALK1 directly inhibits ALK5/Smad3 signaling. The ratio between ALK1 and ALK5 expression will determine whether TGFβ will induce activation or quiescence of the endothelium.
Molecular Cell  , DOI: ( /S (03) )