TRAF4/6 Is Needed for CD44 Cleavage and Migration via RAC1 Activation

Abstract

The hyaluronan receptor CD44 can undergo proteolytic cleavage in two steps, leading to the release of its intracellular domain; this domain is translocated to the nucleus, where it affects the transcription of target genes. We report that CD44 cleavage in A549 lung cancer cells and other cells is promoted by transforming growth factor-beta (TGFβ) in a manner that is dependent on ubiquitin ligase tumor necrosis factor receptor-associated factor 4 or 6 (TRAF4 or TRAF6, respectively). Stem-like A549 cells grown in spheres displayed increased TRAF4-dependent expression of CD44 variant isoforms, CD44 cleavage, and hyaluronan synthesis. Mechanistically, TRAF4 activated the small GTPase RAC1. CD44-dependent migration of A549 cells was inhibited by siRNA-mediated knockdown of TRAF4, which was rescued by the transfection of a constitutively active RAC1 mutant. Our findings support the notion that TRAF4/6 mediates pro-tumorigenic effects of CD44, and suggests that inhibitors of CD44 signaling via TRAF4/6 and RAC1 may be beneficial in the treatment of tumor patients.

Keywords: CD44; RAC1; TRAF; hyaluronan; migration; stemness.

Figure 1

Transforming growth factor β (TGFβ) induces ectodomain and intramembranous cleavage of CD44 in a dose- and time-dependent manners. (a,b) Immunoblot analysis of full-length (FL) and cleavage products (transmembrane and intracellular domain (TM-ICD) and ICD) of CD44 in A549 cells after treatment or no treatment with TGFβ (5 ng/mL) for the indicated time periods; knockdown of CD44 by siRNA was performed to confirm the specificity of observed bands (b). (c) Immunoblotting of FL CD44, as well as the TM-ICD and ICD of CD44 after 24 h of treatment with different concentrations of TGFβ. (d), A549 cells treated or not with TGFβ (5 ng/ml), DAPT (40 μM), MG132 and CQ for 24 h, or TPA (80 nM) for 1 h, were subjected to nuclear/cytoplasmic fractionation and subjected to SDS gel electrophoresis in 15% or 10% polyacrylamide gels, followed by immunoblotting for FL, ICD of CD44, lamin, and tubulin. Quantification of CD44-ICD bands was performed via ImageJ after normalizing to CD44 FL (e), Immunoblot analysis of FL CD44, as well as the TM-ICD and ICD of CD44 in U251MG cells treated or not treated with TGFβ for 24 h, or 12-O-tetradecanoylphorbol-13-acetate (TPA) (80 nM) for 1 h. The cultures were treated with the γ-secretase inhibitor DAPT (40 μM)), the proteasomal inhibitor MG132, and the lysosomal inhibitor chloroquine (CQ) for 24 h; tubulin or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used as loading controls in the same immunoblots. Arrowheads indicate CD44 ICD-like products.

Figure 2

CD44 cleavage increases in stem-like A549 cells grown in spheres. (a) Phase contrast microscopy of A549 grown in adherent (Adh) or low-attachment conditions (Non-adh) for 96 h. Zeiss Axiovision was employed to take the micrographs (bars = 100 μm). (b–d) A549 cells were cultured under adherent conditions or as spheres. Adherent cells were stimulated or not with TGFβ for 24 h. The mRNA levels of transcription factors NANOG, OCT4, and SOX2 (b), as well as CD44v isoforms (c) and CD44s (d) were determined by real-time qPCR and normalized to TBP mRNA levels. (e) Lysates from adherent A549 cells, stimulated or not with TGFβ for 24 h, and non-adherent A549 cells were subjected to SDS-PAGE using 8% and 15% polyacrylamide gels, followed by immunoblotting for CD44s and CD44v, CD44 TM-ICD, and CD44 ICD; GAPDH and tubulin were used as loading controls. (f) The mRNA levels of hyaluronan synthase (HAS1, HAS2, HAS3) and hyaluronidase (HYAL1, HYAL2, TMEM2, KIAA1199) family members were determined by real-time qPCR and normalized to TBP. (g), Hyaluronan levels in the culture medium of adherent and non-adherent A549 cells were evaluated by an enzyme-linked immunosorbent assay (ELISA)-like assay and normalized against protein amounts; values are expressed as fold difference. Asterisks illustrate significant differences between the different conditions compared to the respective adherent ones: * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 3

Tumor necrosis factor receptor associated factor (TRAF) family members mediate TGFβ-induced CD44 cleavage. (a) A549 cells were transfected with non-targeting siRNA (siCtrl) or siRNAs against TRAF6 or TRAF4, and stimulated or not with TGFβ for up to 24 h, followed by immunoblotting for CD44 FL, TM-ICD, TRAF6, and TRAF4; tubulin and GAPDH served as loading controls. CD44 TM-ICD bands were quantified using the ImageJ software and normalized to either tubulin or GAPDH; values are depicted as fold difference; lysates were run at either 10% or 15% polyacrylamide gels, as depicted. (b), A549 cells were transfected with control siRNA or siRNAs targeting TRAF4 or CD44, and subsequently treated or not with TGFβ (5 ng/mL) for 24 h. Relative mRNA levels of CCND1 and MMP2 were quantified by real-time qPCR and normalized to TBP. (c), A549 cells were co-transfected with scrambled control siRNA or siRNAs against TRAF4, a β-gal reporter, and a TPA-responsive element (TRE) luciferase reporter, after which luciferase activity was measured and normalized to β-gal activity. All graph bars are shown as the average ± standard error of the mean (SEM), based on at least three independent experiments, unless mentioned otherwise. Asterisks illustrate significant differences between the conditions indicated with lines; * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 4

TRAF4, but not TRAF6, is necessary for expression of CD44 and HAS2 in A549 cells grown in spheres. A549 cells were transfected with control siRNA or siRNAs targeting TRAF4 or TRAF6, and were thereafter seeded in non-adherent conditions for 96 h. (a), Cells were collected, and samples were resolved in 8% SDS-PAGE, followed by immunoblotting for CD44s and v isoforms. CD44 bands were quantified by the software ImageJ and normalized to tubulin levels and expressed as fold difference. (b–c) mRNA expression of CD44, HAS2, and TMEM2 (b), as well as TRAF4 and TRAF6 (c) were determined by real-time qPCR and normalized to TBP mRNA. (d) Hyaluronan levels in the culture medium of cells in low-attachment conditions was quantified by an ELISA-like assay; the results are given a fold difference after normalization to total protein levels of the collected cells. All graph bars are shown as the average ± SEM based from at least three independent experiments. Asterisks illustrate significant differences compared to the siCtrl condition: * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 5

TRAF4 is required for TGFβ-induced RAC1 activity and for CD44 cleavage. (a) A549 cells were treated with TGFβ (5 ng/mL) for the indicated time periods, and RAC1 activity was assessed after pull-down with GST-PAK1, followed by immunoblotting for RAC1 and GST-PAK1; in addition, whole cell lysate was immunoblotted for RAC1. (b), A549 cells were transfected with control siRNA or TRAF4 siRNA, and treated with TGFβ (5 ng/mL) for 2 h. RAC1 activity was determined as in panel a. (c,d), A549 cells were transfected with either empty vector, different amounts of RAC1 dominant-negative mutant (MYC-RAC1 N17) (c), or MYC-tagged RAC1 constitutively active mutant (MYC-RAC1 L61) (d), followed by treatment with or without TGFβ (5 ng/mL) for 24 h. Cell lysates were subjected to immunoblotting for MYC, FL CD44 and CD44 TM-ICD. GAPDH was used as loading control. Cells transfected with non-targeting siRNA or siRNAs against TRAF4 were also analyzed (d). CD44 TM-ICD bands were quantified using the ImageJ software and normalized to GAPDH; values are shown as fold-difference.

Figure 6

Knockdown of TRAF4 diminishes cell motility, which is rescued by constitutively active RAC1. A549 cells were transfected with control siRNA or siRNAs targeting TRAF4 or CD44 and co-transfected with either empty vector or the constitutively active RAC1 L61 mutant. Cell migration was evaluated using a cell culture wound healing assay. Pictures of the cell cultures were taken at 0 h and at 24 h. The results are presented as a percentage of wound closure and illustrate the average values ± SEM out of three independent experiments. Asterisks illustrate significant differences between the conditions indicated with lines: * p < 0.05, ** p < 0.01.

Figure 7

Schematic illustration of TGFβ-induced activation of TRAF4/6 and RAC1, leading to CD44 cleavage, enhanced migration, and altered gene regulation. Binding of TGFβ to its type II receptor (TβII) recruits and activates the type I receptor (TβRI), activating TRAF4/6 and RAC1, leading to the activation of MMPs, resulting in the formation of CD44 TM-ICD; after further processing by γ-secretase, CD44 ICD is translocated to the nucleus, where it regulates transcription and promotes cell migration.