Fig 2: Modulation of CD44 expression controls response to TGM1. (A) SMAD2 phosphorylation of murine NM18 cells with intact (Control) or deleted (KO) CD44 gene expression in response to TGF β and TGM1. Cells were stimulated with 0.5 ng/mL TGF-β or 10 ng/mL TGM1 and lysates were analyzed by SDS-PAGE and Western blotting with anti-CD44, anti-pSMAD2 and anti-GAPDH. Two replicate clones of 2 KO guides were tested. Mol.wt markers are shown in kDa on the left-hand side. (B) Response of NM18-CAGA-mCHERRYd2 transcriptional reporter cells with CD44 KO induced by 0.5 ng/mL TGF-β and 10 ng/mL TGM1. (C and D) Response of HEK293-CAGA-mCHERRYd2 transcriptional reporter cells stably transduced with doxycycline-inducible murine (C) or human (D) CD44, stimulated with TGF-β or the indicated doses of TGM1. (E) Schematic of mouse and human CD44 domain swap constructs. ECD, Extracellular Domains; TM, Transmembrane; ICD, Intracellular Domains. The sequences used are mouse CD44, NP_001034240.1; human CD44, NP_001001391.1. Note that the TM domain is fully conserved between mouse and human. (F) Response of HEK293T-CAGA-mCherry cells stably transduced with doxycycline-inducible mouse and human CD44 domain constructs. (G) Effect of human (hCD44-Fc) or mouse (mCD44-Fc) recombinant CD44-Fc fusion proteins on the response of NIH3T3-CAGA-dynGFP cells to TGM1 (Left) or TGFβ (Right). Ligands were preincubated with control Fc or CD44-Fc for 30 min before adding to cells and fluorescence measured by IncuCyte with Incucyte software.

Fig 3: CD44 mediates cell surface binding and enhances Foxp3 induction by TGM1. (A and B) Costaining of EL-4 (A) and MFB-F11 cells (B) with anti-CD44 and Alexa-Flour-488 labeled TGM1 full-length (D1/2/3/4/5), D1/2/3, or D4/5 constructs. (C) Binding of anti-CD44, or Alexa-Flour-488 labeled TGM1 full-length (D1/2/3/4/5), D1/2/3, or D4/5 constructs to MFB-F11 cells without or with CD44 deletion, with control unstained cells also depicted. (D) Deletion of CD44 in MFB-F11 abolishes the enhancement effect of TGM1 D4/5. (E and F) CD4+ splenic T cells from CD44-deficient mice show reduced Foxp3 induction in response to full-length TGM, following coculture with anti-CD3 and IL-2. Statistical comparisons between Foxp3 induction in CD44-sufficient C57BL/6 mice and CD44-deficient animals are shown, *P < 0.05. **P < 0.01, ***P < 0.001 by unpaired t test corrected for multiple comparisons. (G and H) Absence of CD44 on CD4+ splenic T cells has no effect on TGF-β signaling stimulated by TGM1 D1/2/3 (E), or TGF-β (F) under the same conditions.

Fig 4: Identification of CD44 as coreceptor for TGM1 signaling. (A) Ability of TGM1 (10 ng/mL) and TGF-β (1 ng/mL) to activate SMAD signaling in four cell lines, 771 (murine B cell lymphoma), EL4 (murine thymoma), NM18 (mouse epithelial cells), and HepG2 (human hepatoma), assessed by Western blotting with anti-phospho-Smad2 antibodies after 1 h stimulation in full medium. (B) Binding of 125I-TGM1 to ALK5 (TβRI) and TβRII, but not other TGF-β/BMP type I and type II family receptors. EL4 cells were incubated with radio-labeled ligand for 3 h on ice, then crosslinked with BS3/DSS (15 min), lysed, and incubated overnight with receptor specific antibodies; finally, complexes were isolated with protein A beads (1 h, 4 °C), separated by SDS-PAGE, and detected by autoradiography. (C) Identification of ~80 kDa coreceptor cross-linked to 125I-D4/5 on the cell surface of 771, EL, and NM18 cells, but not HepG2 cells, as detected by SDS-PAGE and autoradiography. (D) Mass spectrometric identification of CD44 from NM18 cells exposed to biotinylated TGM1 D4/5 and coprecipitated with neutravidin beads. Fold change is calculated compared to control cells that have undergone the same procedure, but without the addition of the TGM1-D4/5-biotin. (E) Immunoprecipitation of iodinated full-length TGM1, and D4/5, but not D1/2/3/4 or D5 alone, with anti-CD44 antibody. Intact cells were first incubated with radiolabeled ligands, crosslinked and washed, and thereafter, cell lysates are prepared, as the input into immunoprecipitation protocols. (F) Coprecipitation of TGF-β receptor I (ALK5) and TGFβRII from MFB-F11 cells by biotinylated D1/2/3, and CD44 by biotinylated D4/5, with Streptavidin beads and analysis by SDS-PAGE and Western blot with anti-TβRI, anti-TβRII and anti-CD44 as indicated. For panels A, B, C, and F, Mol.wt markers are shown in kDa on the left-hand side.