Cripto-1 activates nodal- and ALK4-dependent and -independent signaling pathways in mammary epithelial Cells

Abstract

Cripto-1 (CR-1), an epidermal growth factor-CFC (EGF-CFC) family member, has a demonstrated role in embryogenesis and mammary gland development and is overexpressed in several human tumors. Recently, EGF-CFC proteins were implicated as essential signaling cofactors for Nodal, a transforming growth factor beta family member whose expression has previously been defined as embryo specific. To identify a receptor for CR-1, a human brain cDNA phage display library was screened using CR-1 protein as bait. Phage inserts with identity to ALK4, a type I serine/threonine kinase receptor for Activin, were identified. CR-1 binds to cell surface ALK4 expressed on mammalian epithelial cells in fluorescence-activated cell sorter analysis, as well as by coimmunoprecipitation. Nodal is coexpressed with mouse Cr-1 in the mammary gland, and CR-1 can phosphorylate the transcription factor Smad-2 in EpH-4 mammary epithelial cells only in the presence of Nodal and ALK4. In contrast, CR-1 stimulation of mitogen-activated protein kinase and AKT in these cells is independent of Nodal and ALK4, suggesting that CR-1 may modulate different signaling pathways to mediate its different functional roles.

FIG. 1.

MAPK and AKT activation by CR-1ΔC in EpH-4 cells. Serum-starved EpH-4 cells were stimulated with recombinant CR-1ΔC (200 ng/ml) or EGF (50 ng/ml) protein and analyzed by Western blot (WB) analysis. MW, molecular weight in thousands; C, control.

FIG. 2.

Binding of CR-1 to ALK4 expressed on the cell surface of mammalian epithelial cells. (A) 293 cells transiently transfected with ALK4-HA, ActRIIB-HA, or vector alone were incubated in the presence of CR-1ΔC-Fc recombinant protein or LTβR-Fc control protein. Binding of CR-1ΔC-Fc or LTβR-Fc protein to 293 cells was analyzed by FACS analysis as described in Materials and Methods. (B) 293 cells transiently transfected with ALK4-HA or ActRIIB-HA expression vector were immunoprecipitated (IP) with CR-1ΔC-Fc or LTβR-Fc and analyzed by Western blot analysis using an anti-HA rabbit polyclonal antibody. (C) As control, cell lysates from ALK4-HA- and ActRIIB-HA-transfected 293 cells were analyzed by Western blot analysis using an anti-HA rabbit polyclonal antibody. (D) COS1 cells were transiently transfected with ALK4-Flag and/or CR-1 expression vectors and Flag-tagged proteins were immunoprecipitated with an anti-Flag mouse monoclonal antibody. The immunoprecipitated proteins were analyzed by Western blot (WB) with an anti-CR-1 rabbit polyclonal antibody or an anti-Flag mouse monoclonal antibody. MW, molecular weight in thousands.

FIG. 3.

Binding of CR-1ΔC to the ECD of ALK4 (⧫), ActRIIA (▪), and ActRIIB (▵) in ELISA assay. (A) ECD of ALK4-Fc, ActRIIA-Fc, or ActRIIB-Fc (100 ng/well) was adsorbed to 96-well microtiter plates and incubated with various concentrations of CR-1ΔC recombinant protein (ng/100 μl). ELISA binding of soluble CR-1ΔC to immobilized proteins was detected as described in Materials and Methods. (B) CR-1ΔC (300 ng/well) was adsorbed to 96-well microtiter plates and incubated with various concentrations of the ECD of ALK4-Fc, ActRIIA-Fc, or ActRIIB-Fc recombinant protein (ng/100 μl). ELISA binding of soluble ECD of ALK4-Fc, ActRIIA, or ActRIIB to immobilized CR-1ΔC was detected as described in Materials and Methods. (C) One hundred nanograms of CR-1ΔC aloneor preincubated with different concentrations of ECD of ALK4-Fc, ActRIIA-Fc, or ActRIIB recombinant protein was added to plates that were preadsorbed with ECD of ALK4-Fc. ELISA binding of soluble CR-1ΔC to immobilized ECD of ALK4-Fc was detected as previously described. In panels A and B, the inserts show the K_d of each experiment. OD, optical density.

FIG. 4.

3TP dual-luciferase reporter assay in K562 cells transiently expressing ALK4-Flag, ALK4-2-Flag, CR-1, and/or Nodal. (A) Human K562 erythroleukemia cells were transfected with 0.5 μg of different plasmid DNAs together with Renilla luciferase control reporter vector (pRL-TK). Twenty-four hours after transfection, the cells were lysed and luciferase activity was measured using the Dual-Luciferase Reporter Assay system (Promega). M1/M2, plasmid DNA/control reporter vector ratio; RLU, relative light units. (B) Coimmunoprecipitation of dominant-negative ALK4-2-Flag with CR-1 in COS1 cells. COS1 cells transiently transfected with ALK4-2-Flag and/or CR-1 cDNAs were immunoprecipitated (IP) with an anti-Flag mouse monoclonal antibody and analyzed by Western blot (WB) using an anti-CR-1 rabbit polyclonal antibody or an anti-Flag mouse monoclonal antibody. MW, molecular weight in thousands; C, control.

FIG. 5.

RT-PCR for Cr-1, Nodal, ALK4, and ActRIIB in different stages of mouse mammary development. RT-PCR was performed using specific primers as described in Materials and Methods.

FIG. 6.

RT-PCR and Western blot analysis for Nodal in EpH-4 WT and Eph-4 N cells. (A) RT-PCR with specific Nodal primers (313 bp) in EpH-4 WT and EpH-4 N cells. (B) Western blot analysis using a rabbit polyclonal anti-Nodal antibody in the culture medium of EpH-4 WT and EpH-4 N cells. MW, molecular weight in thousands.

FIG. 7.

Smad-2 phosphorylation by CR-1ΔC in EpH-4 WT and EpH-4 N cells. Serum-starved EpH-4 WT (A) and EpH-4 N (B) cells were stimulated for different times with CR-1ΔC (400 ng/ml) or Activin A (50 ng/ml) recombinant protein and analyzed by Western blot (WB) analysis. MW, molecular weight in thousands; C, control.

FIG. 8.

Smad-2, MAPK, and AKT phosphorylation by CR-1ΔC in EpH-4 WT and EpH-4 N cells transiently transfected with a dominant-negative ALK4-2-Flag receptor. EpH-4 WT and EpH-4 N cells transiently transfected with ALK4-2-Flag or pCI-neo plasmid were stimulated with CR-1ΔC, EGF, or Activin A recombinant protein and cell lysates were assessed by Western blot (WB) analysis. (A) EpH-4 N cells transfected with pCI-neo (4 μg) (lanes 1, 2, and 4) and ALK4-2 (4 μg) were stimulated with Activin A (50 ng/ml) and CR-1ΔC (400 ng/ml) and analyzed by Western blot analysis. (B and C) EpH-4 WT or EpH-4 N cells transfected with pCI-neo (6 μg) (lanes 1, 2, and 4) and ALK4-2 (6, 4, 2, and 1 μg) were stimulated with EGF (50 ng/ml) and CR-1ΔC (200 ng/ml) and analyzed by Western blot analysis. MW, molecular weight in thousands; C, control.

FIG. 9.

MAPK and AKT phosphorylation by CR-1ΔC in MC3T3-E1 cells. (A) RT-PCR using specific primers for ALK4 (435 bp) in EpH-4 WT and MC3T3-E1 cells. A 100-bp DNA marker is shown in the first lane. (B and C) Serum-starved MC3T3-E1 cells were stimulated with CR-1ΔC (200 and 400 ng/ml) or EGF (50 ng/ml) recombinant protein and analyzed by Western blot (WB) analysis. MW, molecular weight in thousands; C, control.